Application of medicine composition in preparation of osteoporosis medicine

Abstract

The invention relates to an application of a pharmaceutical composition in preparation of a medicine for treating gout or osteoporosis, belongs to the field of pharmacy, and has an obvious effect of preventing or treating gout or osteoporosis and better safety.

Description

Application of a pharmaceutical composition in the preparation of osteoporosis drugs Technical Field

[0001] The invention belongs to the field of pharmaceuticals, and particularly relates to application of a pharmaceutical composition in preparing a medicine for promoting wound healing. Background Art

[0002] Medicinal hollyhock flowers are the dried corollas of Abelmoschus manihot (L.) Medicinal, a plant of the Malvaceae family. They have a sweet and cold flavor and enter the kidney and bladder meridians. They clear dampness and heat, reduce swelling and detoxify, and are used for carbuncles, swellings, and burns caused by water or fire. This medicine was first mentioned in the Jiayou Materia Medica, which states: "Hibiscus flowers are effective for treating persistent sores and pus. Applying a powdered powder to the affected area can cure persistent sores and pus." The Compendium of Materia Medica states: "Hibiscus flowers have a sweet, cold, slippery flavor and are non-toxic. Applying a powdered powder to the affected area can cure persistent sores and pus, making it an essential remedy for sores."

[0003] Flavonoids are one of the main components of hibiscus flowers and also one of their pharmacologically active ingredients. Hibiscus capsules have been on the market for many years, and their main active ingredient is hibiscus flower extract. Research has confirmed that the flavonoids in hibiscus flower extracts not only have a significant therapeutic effect on sores but also have a significant protective effect against ischemic damage to the heart, brain, and tissues. Furthermore, numerous literature reports indicate that total flavonoids from hibiscus flowers may have significant effects in anti-inflammatory, antipyretic and analgesic effects, protection against ischemic damage to the heart and brain, hypoglycemic effects, and antiviral effects. For example, patent CN201210082553.7 discloses a total flavonoid extract from hibiscus flowers containing quercetin-3'-glucoside, quercetin-3'-glucoside, and isoquercetin in a weight ratio of (11-16):(2.5-6):(4-6.5), which can be used to prepare flavonoids for treating nephritis. Patent CN200610097615.6 discloses a total flavonoid extract from hibiscus australis flowers, which has a total flavonoid content of 50-90% by weight. The flavonoid components contained in the extract are: 1.0-5.0% quercetin-3-acaciaside, 8-24.0% hyperoside, 7.0-20.0% isoquercetin, 5.0-15.0% quercetin-3'-glucoside, 3.0-10.0% gossypol-3'-glucoside, 0.5-5.0% myricetin, 0.5-5.0% gossypol, 2.0-8.0% quercetin, and several other flavonoid components. The total flavonoid extract from hibiscus australis flowers can be used to prepare a drug for treating nephritis. The flavonoid components are clearly defined in terms of quality and quantity, and the therapeutic effect is reliable.

[0004] Although bioflavonoids have various therapeutic effects, there are many types of flavonoids, which can currently be mainly divided into 7 different subtypes, and different flavonoid composition will lead to different therapeutic effects. For example, although the literature reports that bioflavonoids can effectively prevent or treat diabetic retinopathy, compounds with five hydroxyl groups, such as quercetin, have a negative impact on ocular blood flow. When flavonoids are dehydrogenated to flavanones, significant improvement in ocular blood flow is achieved. In addition, compounds that increase blood flow also lead to a significant increase in retinal function recovery after ischemic injury. How to develop such compounds to effectively treat eye diseases such as macular degeneration, visual fatigue and cataracts, and even improve the effect on diabetic retinopathy, is an important research task.

[0005] Choroidal neovascularization (CNV) is seen in many fundus diseases, such as age-related macular degeneration (AMD), central exudative chorioretinitis, and maculopathy caused by high myopia. CNV secondary to AMD is the most common and has become one of the leading causes of irreversible visual impairment in the elderly. Insufficient blood flow is also a factor in visual fatigue. Increasing ocular blood flow or acting on ocular smooth muscle can help alleviate visual fatigue.

[0006] Glucose reabsorption in the proximal renal tubules is mediated by the sodium-glucose cotransporter (SGLT) 1 and SGLT2 transporters, with approximately 90% of glucose reabsorption mediated by SGLT2 and the remaining 10% by SGLT1. SGLT2 inhibitors selectively bind to the SGLT2 receptor, inhibiting renal tubular glucose reabsorption, thereby lowering the renal glucose threshold and increasing urinary glucose excretion, achieving significant hypoglycemic effects. Recent clinical studies have found that SGLT2 inhibitors have a renal protective effect in non-diabetic chronic kidney disease (CKD). SGLT2 inhibitors can reduce the expression of collagen and fibronectin by decreasing TGFβ-1, PAI1, STAT 1, and MMP 7, as well as inhibiting the AGEs-RAGE axis, thereby reducing extracellular matrix accumulation and alleviating the progression of renal fibrosis in DN. In addition to regulating tubuloglomerular feedback, reducing proteinuria progression, and promoting anti-inflammatory and anti-fibrotic effects, SGLT2 inhibitors can also reduce serum uric acid (SUA) in diabetic patients. For example, compared with the control group, SGLT2 inhibitors (empagliflozin, canagliflozin, dapagliflozin, togliflozin, lupagliflozin, and empagliflozin) significantly reduced serum uric acid levels. SGLT2 inhibitors can be beneficial in the prevention and treatment of heart failure by reducing plasma volume, lowering preload and postload, improving myocardial energy metabolism, and improving myocardial remodeling.

[0007] Epidermal growth factor receptor inhibitors (EGFRs) are the most widely used anti-tumor drugs in recent years, highly favored by physicians and patients for their outstanding clinical efficacy. However, adverse reactions vary, with rash (primarily acne / acneform rashes) being the most common. Acne / acneform rashes not only affect patients' quality of life but can also disrupt treatment, seriously compromising the effectiveness of tumor therapy. Acne / acneform rashes have, to a certain extent, limited the use of EGFRs. Western medicine often uses antibiotics and topical steroids for treatment, but these are ineffective.

[0008] Skin wounds are a common clinical symptom. Conventional skin wound treatments include skin grafting and artificial replacement coverage. Skin ulcers are common and frequently occurring clinical conditions. They refer to localized skin tissue defects caused by various reasons. The exposed wounds are susceptible to infection. Chronic skin ulcers, in particular, cannot heal for a long time or are prone to recurrence. Treatment is difficult, seriously affecting patients' quality of life and bringing a certain social burden. Clinically, there is an urgent need for the research and development of more safe, effective, and economical treatments and drugs.

[0009] Epidemiological studies show that the incidence of diabetes in my country is increasing annually, a trend closely linked to changing lifestyles and an aging population. Furthermore, 15% of diabetic patients will eventually develop long-lasting skin ulcers. Promoting rapid wound healing, reducing disability, and preserving limb function are key research topics and hot topics in current medical research. By artificially creating sterile wounds on the backs of experimental diabetic rats, an animal model of chronic, non-healing wounds in diabetes can be established, enabling research on drugs that promote wound healing.

[0010] Reported extraction methods for hibiscus flowers include ethanol reflux extraction, ultrasonic extraction, and room-temperature immersion extraction. However, hibiscus flower extracts have complex compositions, large variations in the content of active ingredients, low extraction transfer rates, high extraction energy consumption, and large solvent usage. Therefore, it is necessary to develop a hibiscus flower therapeutic agent and its extraction process that can be used to treat multiple diseases, including kidney disease, particularly diabetic nephropathy, and ocular diseases.

[0011] The applicant collected data on adverse reactions to Hibiscus capsules and found that in a recent year, a total of 194 adverse reactions to Hibiscus capsules were received. According to the statistics of adverse drug reactions, nausea, itching, rash, vomiting, diarrhea, upper abdominal distension and discomfort, etc., occurred more than 10 times, all of which are known adverse reactions. Recently, new ADR manifestations include dizziness, headache, and decreased appetite. In addition, among the serious adverse reactions received, one case involved liver cell damage, which may be a signal that Hibiscus capsules damage liver cells. Therefore, it is clinically significant to develop a new Hibiscus flower flavonoid extract to reduce side effects and be used as a drug for treating diseases such as ulcers.

[0012] Summary of the Invention

[0013] To address the above-mentioned deficiencies, the present invention provides an effective fraction of flavonoids from hibiscus flowers and its use. The present invention prepares a high-purity effective fraction of flavonoids from hibiscus flowers through an extraction-coupling resin method. It has been found to have enhanced wound healing, treatment, and / or prevention effects on skin diseases, as well as improved activity in treating kidney diseases, such as diabetic nephropathy, contrast-induced renal injury, or lupus erythematosus nephritis. It also has therapeutic effects on eye diseases. Furthermore, the effective fraction, extract, or composition of flavonoids from hibiscus flowers described herein has reduced side effects, providing a better therapeutic agent for patients.

[0014] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0015] In a first aspect, the present invention provides a use of a pharmaceutical composition in the preparation of a medicament for treating and / or preventing gout or osteoporosis, wherein the pharmaceutical composition comprises a flavonoid component in the following mass ratio: gossypolamine-8-O-β-D-glucuronide: hyperoside: isoquercetin: myricetin: quercetin-3'-O-glucoside in a mass ratio of 10:3.0-20:2.0-18:0.2-6.0:1-20;

[0016] Preferably, the mass ratio of linalool-8-O-β-D-glucuronide: hyperoside: isoquercetin: myricetin: quercetin-3'-O-glucoside is 10: 3.0-20: 2.0-3.9: 0.2-6.0: 1-20;

[0017] or the mass ratio of gossypol-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside is 10:3.0-20:4.0-18:0.2-6.0:1-20;

[0018] or the mass ratio of gossypol-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside is 10:3.0-20:2.0-3.9:0.2-1.8:1-20;

[0019] or the mass ratio of gossypol-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside is 10:3.0-20:2.0-3.9:1.9-6.0:1-20;

[0020] or the mass ratio of gossypolamine-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside is 10:3.0-20:2.0-18:0.2-6.0:1-5.9;

[0021] or the mass ratio of gossypol-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside is 10:3.0-20:2.0-18:0.2-6.0:6.0-20;

[0022] Alternatively, the mass ratio of gossypol-8-O-β-D-glucuronide: hyperoside: isoquercetin: myricetin: quercetin-3'-O-glucoside is 10:-3.0-20:4.0-18:1.9-6.0:6.0-20.

[0023] Specifically, the mass ratio of lintol-8-O-β-D-glucuronide: hyperoside: isoquercetin: myricetin: quercetin-3'-O-glucoside is 10:4.0-17:4.50-16:1.9-5.5:7.0-16; preferably 10:6.0-15.6:5.0-13:2.0-5.0:8.0-14; more preferably 10:6.0-15:5.0-13:2.0-5.0:8.0-14; more preferably 10:6.5-14:5.7-11.2:2.2-3.1:8.7-11.4; further, containing quercetin, wherein the mass ratio of lintol-8-O-β-D-glucuronide to quercetin is 10:0.1-10.0, preferably 1 0:0.1-0.9 or 10:1.0-10.0, more preferably 10:1.0-6.0, more preferably 10:1-5.6, most preferably 10:1.5-5.0; further, containing rutin, wherein the mass ratio of gossypol-8-O-β-D-glucuronide to rutin is 10:0.05-0.6, preferably 10:0.1-0.5, more preferably 10:0.1-0.45, most preferably 10:0.1-0.3; further, containing quercetin-3-O-sophoroside, wherein the mass ratio of gossypol-8-O-β-D-glucuronide to quercetin-3-O-sophoroside is 10:0.1-2.5, preferably 10:0.1-0.9, more preferably 10:0.15-0.5.

[0024] In a second aspect, the present invention further provides a use of a pharmaceutical composition in the preparation of a medicament for gout or osteoporosis, wherein the pharmaceutical composition comprises the following flavonoid components in a mass ratio: gossypol-8-O-β-D-glucuronide: hyperoside: isoquercetin: myricetin: quercetin-3'-O-glucoside: quercetin in a mass ratio of 10:3.0-20:2.0-18:0.2-6.0:1-20:0.1-10.0;

[0025] Preferably, the mass ratio of linalool-8-O-β-D-glucuronide: hyperoside: isoquercetin: myricetin: quercetin-3'-O-glucoside: quercetin is 10: 3.0-20: 2.0-3.9: 0.2-6.0: 1-20: 0.1-10.0;

[0026] or the mass ratio of linalool-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside:quercetin is 10:3.0-20:4.0-18:0.2-6.0:1-20:0.1-10.0;

[0027] or the mass ratio of linalool-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside:quercetin is 10:3.0-20:2.0-3.9:0.2-1.8:1-20:0.1-10.0;

[0028] or the mass ratio of gossypolamine-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside:quercetin is 10:3.0-20:2.0-3.9:1.9-6.0:1-20:0.1-10.0;

[0029] or the mass ratio of gossypolamine-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside:quercetin is 10:3.0-20:2.0-18:0.2-6.0:1-5.9:0.1-10.0;

[0030] or the mass ratio of gossypolamine-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside:quercetin is 10:3.0-20:2.0-18:0.2-6.0:6.0-20:0.1-10.0;

[0031] or the mass ratio of linalool-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside:quercetin is 10:3.0-20:2.0-18:0.2-6.0:1-20:0.1-0.9;

[0032] or the mass ratio of gossypolamine-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside:quercetin is 10:3.0-20:2.0-18:0.2-6.0:1-20:1.0-10.0;

[0033] Alternatively, the mass ratio of linalool-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside:quercetin is 10:6-15:5-13:1.0-2.9:4.0-9.5:0.6-6.0, preferably 10:6-15:5-13:1.0-1.8:4.0-5.9:0.6-0.99.

[0034] Furthermore, it contains rutin, wherein the mass ratio of gossypol-8-O-β-D-glucuronide to rutin is 10:0.05-0.6, preferably 10:0.1-0.5, more preferably 10:0.1-0.45, and most preferably 10:0.1-0.3; furthermore, it contains quercetin-3-O-sophoroside, wherein the mass ratio of gossypol-8-O-β-D-glucuronide to quercetin-3-O-sophoroside is 10:0.1-2.5, preferably 10:0.1-0.9, and preferably 10:0.15-0.5.

[0035] Wherein, the composition is hibiscus flower extract.

[0036] In a third aspect, the present invention provides a use of a Hibiscus truncatum flower extract in the preparation of a medicament for gout or osteoporosis, wherein the extract comprises flavonoid components in the following mass ratio: gossypol-8-O-β-D-glucuronide: hyperoside: isoquercetin: myricetin: quercetin-3'-O-glucoside: quercetin in a mass ratio of 10:3.0-20:2.0-18:0.2-6.0:1-20:0.1-10.0;

[0037] Preferably, the mass ratio of linalool-8-O-β-D-glucuronide: hyperoside: isoquercetin: myricetin: quercetin-3'-O-glucoside: quercetin is 10: 3.0-20: 2.0-3.9: 0.2-6.0: 1-20: 0.1-10.0;

[0038] or the mass ratio of linalool-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside:quercetin is 10:3.0-20:4.0-18:0.2-6.0:1-20:0.1-10.0;

[0039] or the mass ratio of linalool-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside:quercetin is 10:3.0-20:2.0-3.9:0.2-1.8:1-20:0.1-10.0;

[0040] or the mass ratio of gossypolamine-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside:quercetin is 10:3.0-20:2.0-3.9:1.9-6.0:1-20:0.1-10.0;

[0041] or the mass ratio of gossypolamine-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside:quercetin is 10:3.0-20:2.0-18:0.2-6.0:1-5.9:0.1-10.0;

[0042] or the mass ratio of gossypolamine-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside:quercetin is 10:3.0-20:2.0-18:0.2-6.0:6.0-20:0.1-10.0;

[0043] or the mass ratio of linalool-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside:quercetin is 10:3.0-20:2.0-18:0.2-6.0:1-20:0.1-0.9;

[0044] or the mass ratio of gossypolamine-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside:quercetin is 10:3.0-20:2.0-18:0.2-6.0:1-20:1.0-10.0;

[0045] Alternatively, the mass ratio of linalool-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside:quercetin is 10:6-15:5-13:1.0-2.9:4.0-9.5:0.6-6.0, or 10:6-15:5-13:1.0-1.8:4.0-5.9:0.6-0.99.

[0046] Furthermore, it contains rutin, wherein the mass ratio of gossypol-8-O-β-D-glucuronide to rutin is 10:0.05-0.6, preferably 10:0.1-0.5, more preferably 10:0.1-0.45, and most preferably 10:0.1-0.3; furthermore, it contains quercetin-3-O-sophoroside, wherein the mass ratio of gossypol-8-O-β-D-glucuronide to quercetin-3-O-sophoroside is 10:0.1-2.5, preferably 10:0.1-0.9, and preferably 10:0.15-0.5.

[0047] In a fourth aspect, the present invention provides an application of a flavonoid effective part in the preparation of a drug for gout or osteoporosis, wherein the flavonoid effective part comprises flavonoid components in the following mass ratio: isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin: gossypolamine-8-O-β-D-glucuronide: hyperoside, the mass ratio of which is: 0.8-1.2: 0.05-1.6: 0.2-4.6: 0.05-1.2: 0.2-3.5: 0.25-3.6.

[0048] Specifically, the effective flavonoid fraction of hibiscus flower includes the following flavonoid components in the following mass ratios: isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin: gossypol-8-O-β-D-glucuronide: hyperoside in a mass ratio of: 0.8-1.2: 0.1-1.2: 0.4-3.1: 0.10-0.9: 0.4-3.1: 0.5-3.0; further 0.8-1.2 : 0.16-1.0: 0.6-2.4: 0.14-0.75: 0.6-2.5: 0.6-2.4; further 0.8-1.2: 0.2-0.8: 0.8-2.1: 0.18-0.6: 0.8-2.0: 0.7-2.0; further 0.8-1.2: 0.2-0.7: 0.8-1.6: 0.2-0.5: 0.8-1.8: 0.8-1.4.

[0049] Specifically, the effective flavonoid part of the hibiscus flower includes flavonoid components in the following mass ratio: isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin: gossypolamine-8-O-β-D-glucuronide: hyperoside, the mass ratio of which is: 0.8-1.2: 0.2-0.6: 1.0-1.2: 0.2-0.4: 0.9-1.7: 1.0-1.3.

[0050] Specifically, the effective flavonoid part of the hibiscus flower includes flavonoid components in the following mass ratio: the mass ratio of isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin: gossypolamine-8-O-β-D-glucuronide: hyperoside is: 0.8-1.2: 0.09-1.6: 0.2-2.0: 0.05-1.2: 0.5-3.0: 0.25-3.6.

[0051] Specifically, the effective flavonoid part of the hibiscus flower includes flavonoid components in the following mass ratio: the mass ratio of isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin: gossypolamine-8-O-β-D-glucuronide: hyperoside is: 0.8-1.2: 0.12-0.7: 0.8-1.6: 0.2-0.5: 0.85-1.7: 1.0-1.4.

[0052] More specifically, the mass ratio of the isoquercetin is 1.2 or 1.0.

[0053] More specifically, the flavonoid effective fraction of hibiscus flower further contains rutin, and the mass ratio of isoquercetin to rutin is 1:0.001-0.08, preferably 1:0.005-0.06, preferably 1:0.006-0.05, preferably 1:0.007-0.04, preferably 1:0.008-0.04, or preferably 1:0.009-0.04; further, it contains quercetin-3-O-sophoroside, and the mass ratio of isoquercetin to quercetin-3-O-sophoroside is 1:0.01-0.25, preferably 1:0.01-0.09, more preferably 1:0.015-0.065, and most preferably 1:0.015-0.05.

[0054] Wherein, the flavonoid effective part is the flavonoid effective part of hibiscus flower.

[0055] In the first to fourth aspects above, the total content of quercetin, quercetin-3'-O-glucoside, myricetin, gossypol-8-O-β-D-glucuronide, isoquercetin and hyperoside contained in the composition, hibiscus flower flavonoid extract or effective fraction is 55% or more, preferably 60% or more, preferably 65-85%, preferably 69-82%; further, the content of quercetin-3'-o-glucoside is 12.1-25%, preferably 12.6-25%, preferably 69-82%. The content of quercetin is preferably 13-25%, preferably 14-25%, or 12.6-23%, or 13-20%; further, the content of quercetin is 0.7% or more, preferably 1.0% or more, preferably 1.0-12%, preferably 1.5-12%, or 1.0%-10%, or 1.5%-5%; further, the content of linalool-8-O-β-D-glucuronide is 8.5-34%, preferably 12-34%, or 8.5-30%, or 12%-23%.

[0056] In the first to fourth aspects above, the composition, extract or effective fraction contains quercetin, quercetin-3'-O-glucoside, myricetin, gossypol-8-O-β-D-glucuronide, isoquercetin, rutin and hyperoside in a total content of 55% or more, preferably 60% or more, preferably 65-85% or 66-84%, preferably 69-82% or 69-90%; further, the content of quercetin-3'-o-glucoside is 12.1-25%, preferably 12.6-23%, preferably 13-20%.

[0057] In a fifth aspect, the present invention provides a use of a Hibiscus truncatum flower extract in the preparation of a drug for promoting wound healing or treating and / or preventing skin diseases, the extract containing flavonoids, wherein the extract includes the following components by mass: 8-30% hyperoside, 10-24% isoquercetin, 8.5-34% gossypol-8-O-β-D-glucuronide, 3.0-4.9% or 5.1-7.0% myricetin, 14-25% quercetin-3'-o-glucoside, 1.6-12% quercetin; or 8-26% hyperoside, 12.0-1 9.8%, gossypol-8-O-β-D-glucuronide 8.5-30%, myricetin 3.0-4.9% or 5.1-6.0%, quercetin-3'-o-glucoside 14-25%, quercetin 1.6-4.9%; or hyperoside 11-22%, isoquercetin 12.0-17%, gossypol-8-O-β-D-glucuronide 12-23%, myricetin 1.6-4.9% or 5.1-9.0%, quercetin-3'-o-glucoside 13-22%, quercetin 1.4-8% or 1.4-7.8%.

[0058] Specifically, the extract also contains rutin with a mass content of 0.01-1.0%, further 0.05-0.95%, further 0.09-0.95%, further 0.1-0.95%, or 0.05-0.8%, further 0.09-0.8%, further 0.1-0.6%; further, it contains quercetin-3-O-sophoraside 0.08-2.5%, preferably 0.1-1.5%, further 0.1-0.9%, further 0.1-0.8%, and further 0.1-0.5%.

[0059] Specifically, the mass content of flavonoids in the hibiscus flower extract is above 55%, preferably above 60%, preferably 65-90%, and preferably 69-82%.

[0060] In a sixth aspect, the present invention provides a method for preparing the above-mentioned pharmaceutical composition, the above-mentioned effective part or the above-mentioned hibiscus flower extract, the method comprising the following steps: (1) extracting hibiscus flower or the medicinal part of hibiscus flower with ethanol to obtain an extract; (2) concentrating the extract and then extracting it to obtain an extract; (3) removing the solvent from the extract and eluting it with a macroporous resin to obtain the hibiscus flower flavonoid effective part or extract; the preferred extraction method is percolation.

[0061] Specifically, the amount of ethanol used in step (1) is 10-25 times that of hibiscus flower or medicinal part of hibiscus, and the ethanol is a 60-95% ethanol solution; the extractant used for the extraction in step (2) is n-butanol, petroleum ether or ethyl acetate, the extraction method is continuous countercurrent extraction, the material-liquid ratio of the extraction is 0.8-4:1, and the extraction level of the extraction is 1-5; the macroporous resin model in step (3) is D101, HPD100 or AB-8.

[0062] Specifically, step (2) further includes subjecting the extract to activated carbon adsorption, alcohol precipitation or acid precipitation treatment before extraction.

[0063] Specifically, the elution process of the macroporous resin described in step (3) is as follows: the diameter-to-height ratio of the macroporous resin is 1:4-1:9, the loading solution concentration is 0.10-0.30 g crude drug / mL, the loading solution volume is 4-12BV, the sample is loaded at a flow rate of 1-4BV / h, 0.5-8BV pure water and 1-5BV 3-15% ethanol are used for impurity removal at a flow rate of 0.5-4BV / h, and 2-8BV 50-80% ethanol is used for elution at a flow rate of 1-5BV / h.

[0064] In a seventh aspect, the present invention provides a method for preparing the above-mentioned effective part or the above-mentioned hibiscus flower extract, wherein the method comprises the following steps: the preparation method comprises the following steps: (1) extracting hibiscus flower or the medicinal part of hibiscus flower with ethanol to obtain an extract; (2) adding a clarifier to the extract, treating it in a water bath, and filtering to remove the supernatant; (3) eluting the supernatant with a polyamide resin to obtain the effective part or extract of flavonoids from hibiscus flower; the preferred extraction method is percolation.

[0065] Specifically, the amount of ethanol used in step (1) is 10-25 times that of hibiscus flower or medicinal part of hibiscus, and the ethanol is a 60-95% ethanol solution; the amount of the clarifier used in step (2) is 4-8% of that of hibiscus flower or medicinal part of hibiscus; the water bath temperature is 50-70°C, and the water bath time is 30-90min; the resin diameter-to-height ratio in step (3) is 1:4-1:9, the sample solution concentration is 0.10-0.60g crude drug / mL, the sample solution volume is 4-12BV, and elution is performed with 4-8BV pure water and 4-8BV 60-95% ethanol.

[0066] In an eighth aspect, the present invention provides a method for preparing the above-mentioned effective part or the above-mentioned hibiscus flower extract, which comprises the following steps: (1) extracting hibiscus flower or the medicinal part of hibiscus flower with ethanol to obtain an extract; (2) adjusting the pH value of the extract to 2.0-3.0, refrigerating, filtering to obtain a precipitate, and dissolving it in water; (3) extracting the dissolved solution to obtain an extract; (4) removing the solvent from the extract and eluting it with a polyamide resin to obtain the effective part or extract of flavonoids from hibiscus flower; the preferred extraction method is reflux.

[0067] Specifically, the amount of ethanol used in step (1) is 10-25 times that of hibiscus flower or medicinal part of hibiscus, and the ethanol is a 60-95% ethanol solution; the pH regulator in step (2) is hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid, citric acid, tartaric acid or maleic acid; the extractant used for extraction in step (3) is n-butanol, petroleum ether or ethyl acetate, the extraction method is continuous countercurrent extraction, the material-liquid ratio of the extraction is 0.8-4:1, and the extraction level of the extraction is 1-5; the resin diameter-to-height ratio in step (4) is 1:4-1:9, the sample solution concentration is 0.10-0.60g crude drug / mL, the sample solution volume is 4-12BV, and elution is carried out with 4-8BV pure water and 4-8BV 60-95% ethanol.

[0068] In a ninth aspect, the present invention provides a hibiscus flower total flavonoids cream, which contains (i) the pharmaceutical composition in the above application, the hibiscus flower extract in the above application, the effective part or extract in the above application, or the hibiscus extract in the above application, and (ii) pharmaceutically acceptable excipients; preferably, the cream also includes (iii) water.

[0069] Wherein, the excipient is selected from any one or a combination of Tween, sodium lauryl sulfate, glycerol, ethanol, isopropyl myristate, methylparaben, azone, glyceryl monostearate, palmitic acid, and white petrolatum; preferably, the Tween is Tween 80.

[0070] Wherein, the Tween accounts for 8-16% of the total mass of the auxiliary materials and water, preferably 10-14%, preferably 12%.

[0071] Wherein, the sodium lauryl sulfate accounts for 0.8-1.6% of the total mass of the auxiliary materials and water, preferably 1-1.4%, and preferably 1.2%.

[0072] Wherein, the glycerol accounts for 1.4-2.2% of the total mass of the auxiliary materials and water, preferably 1.6-2.0%, preferably 1.8%.

[0073] Wherein, the ethanol accounts for 9.1-9.9% of the total mass of the auxiliary materials and water, preferably 9.3-9.7%, and preferably 9.5%.

[0074] Wherein, the isopropyl myristate accounts for 4.1-4.9% of the total mass of the auxiliary materials and water, preferably 4.3-4.7%, and preferably 4.5%.

[0075] Wherein, the methylparaben accounts for 0.08-0.16% of the total mass of the auxiliary materials and water, preferably 0.1-0.14%, preferably 0.12%.

[0076] Wherein, the azone accounts for 0.7-2.5% of the total mass of the auxiliary materials and water, preferably 0.9-2.3%, preferably 2.1%.

[0077] Wherein, the glyceryl monostearate accounts for 8.6-9.4% of the total mass of the excipients and water, preferably 8.8-9.2%, preferably 9%.

[0078] Wherein, the palmitic acid accounts for 10.4-11.2% of the total mass of the auxiliary materials and water, preferably 10.6-11%, preferably 10.8%.

[0079] Wherein, the white vaseline accounts for 6.8-7.6% of the total mass of the excipients and water, preferably 7-7.4%, preferably 7.2%.

[0080] The water accounts for 37.78-45.78% of the total mass of the auxiliary materials and water, preferably 39.78-43.78%, and preferably 41.78%.

[0081] Wherein, component (i) is 0.01-2.5% of the total mass of the auxiliary materials and water, preferably 0.1-2.4%, preferably 0.5-2%, preferably 0.8-1.7%, preferably 0.8-1.4%, preferably 0.9-1.1%, preferably 1%.

[0082] The flavonoid effective part of hibiscus flower (hibiscus flower extract) of the present invention is tested for seven components, namely hyperoside, rutin, isoquercetin, hibifolin-8-O-β-D-glucuronide, myricetin, quercetin-3'-O-glucoside, and quercetin. During the testing process, the peak area of ​​the solid sample prepared in the embodiment of the present invention is also converted into the content of quercetin-3-O-sophoroside according to the proportion. The content conversion calculation method is = peak area of ​​hyperoside reference substance / concentration of hyperoside reference substance*correction factor*peak area of ​​quercetin-3-O-sophoroside*fixed volume / sample weight (after water deduction). The content is calculated to be no more than 2.0%, further no more than 0.9%, and preferably 0.1%-0.7%. The total content of the multiple flavonoid components tested is above 70%, further above 75%, and further between 80% and 90%. Beneficial effects:

[0083] (1) The effective fraction or extract of flavonoids from hibiscus flowers provided by the present invention has a good preventive and therapeutic effect on skin diseases, such as dermatitis and acne.

[0084] (2) The effective fraction or extract of flavonoids from hibiscus flowers of the present invention has the effect of treating various nephritis, such as diabetic nephropathy, contrast-induced renal injury or lupus erythematosus nephritis, and also has the effect of treating eye diseases, especially various diseases related to inhibiting blood flow and inhibiting ocular blood vessels, such as age-related macular degeneration, central exudative chorioretinitis and macular lesions caused by high myopia, etc. In particular, the effective fraction or extract of flavonoids from hibiscus flowers of the present invention also has the effect of treating pulmonary fibrosis.

[0085] (3) The total flavonoids of Astragalus membranaceus provided by the present invention can effectively treat skin diseases at a lower dosage.

[0086] (4) The effective part, extract or composition of flavonoids from hibiscus flowers of the present invention has relatively low side effects, thus providing a better treatment method for patients.

[0087] (5) The preparation method of the present invention has a simple process, a short production cycle, mild processing conditions, low energy consumption, and good separation effect of the seven flavonoid components of hibiscus flowers. The raw materials and reagents used are widely available, low in cost, and can be easily industrialized and mass-produced.

[0088] (6) The effective part or extract of flavonoids from hibiscus flowers provided by the present invention has the effect of promoting wound healing, has obvious effects of promoting wound healing and treating diabetic ulcers and has good safety.

[0089] (7) The effective fraction or extract of flavonoids from hibiscus flowers provided by the present invention has a significant therapeutic effect on gout or osteoporosis, and the symptoms of gout include arthritis.

[0090] (8) The effective part or extract of flavonoids from hibiscus flowers provided by the present invention has obvious therapeutic effects on diabetic foot, atopic dermatitis, acne, osteoporosis, myocarditis, heart failure, coronary heart disease, reperfusion injury, atherosclerosis, anticoagulation, thrombosis, postpartum depression, metabolic regulation of weight loss, analgesia, purpura nephritis, angiographic kidney injury, mechanical kidney injury, gynecological inflammation, andrological inflammation, urinary system inflammation, and other cardiovascular and cerebrovascular diseases such as hypertension, heart and brain damage, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figure 1 shows the dissolution process of 5-ASA and ZHT.

[0092] Figure 2 shows the effect of ZHT on DSS-induced colon atrophy; (A) Images of the colon appearance of mice in each group, (B) Statistical graph of the colon length of mice in each group. Data are presented as mean ± standard error. N = 8. **, P < 0.01, vs. Control. # , P < 0.05, vs. DSS, analyzed using one-way ANOVA and Dunnett's multiple comparison test.

[0093] Figure 3 shows the effect of ZHT on DSS-induced colonic histopathological changes; (A) H&E staining of colonic tissues of mice in each group, 5x objective magnification, scale bar = 500 μm; 20x objective magnification, scale bar = 100 μm; (B) Statistical graph of colonic inflammation scores of mice in each group. Data are expressed as mean ± standard error. N = 7. **, P < 0.01, vs. Control. ## , P < 0.01, vs. DSS, analyzed using one-way ANOVA and Dunnett's multiple comparison test.

[0094] Figure 4 is the pathological images of each group in the oral ulcer efficacy experiment.

[0095] Figure 5 is a flow chart of acne model preparation, drug administration, and material collection.

[0096] Figure 6 is a flowchart of atopic dermatitis model construction and drug administration.

[0097] FIG7 shows the effects of ZHT and HKY on the appearance of the DNFB-induced atopic dermatitis model.

[0098] FIG8 shows the effects of ZHT and HKY on the pathological changes of the skin in the DNFB-induced atopic dermatitis model, including representative images of HE staining of the skin tissue of mice in each group. The blue line indicates the thickness of the epidermis, and the scale bar = 100 μm.

[0099] Figure 9 shows representative images of skin wound areas in rats in each group at different time points; (A) Representative images of skin wound areas in rats in each group at different time points, (B) Skin wound healing curves in rats in each group. *P<0.05, **P<0.01 vs. Model.

[0100] Figure 10 shows HE staining results of rat skin wound tissue and quantitative images of epidermal thickness; (A) HE staining results of rat wound and edge tissue, (B) Quantification of epidermal thickness of rat skin wounds in each group. **P < 0.01, vs. Model; ##, P < 0.01, vs. Control.

[0101] Figure 11 shows Masson staining results and quantitative images of collagen deposition in rat skin wound tissue; (A) Masson staining results of rat wound and peripheral tissue; (B) Quantitative images of collagen deposition in rat skin wounds in each group. N = 6, *P < 0.05, **P < 0.01 vs. Model; ##P < 0.01 vs. Control.

[0102] Figure 12 shows the changes in CD31 and VEGF mRNA levels in rat skin wound tissue; (A) changes in CD31 mRNA levels in rat skin wound tissue after 9 days of treatment, (B) changes in VEGF mRNA levels in rat skin wound tissue after 9 days of treatment.

[0103] Figure 13 shows the results of CD31 immunofluorescence staining of rat skin wound tissue and the quantitative fluorescence intensity graph; (A) CD31 immunofluorescence staining of rat skin tissue after 9 days of treatment, scale bar is 250 μm; (B) Quantitative fluorescence intensity graph of CD31, N=6, #P<0.05 vs. control.

[0104] Figure 14 shows the changes in VEGF protein levels in rat skin wound tissues; changes in VEGF protein levels in rat skin wound tissues 9 days after modeling, N=6, **P<0.05 vs model.

[0105] FIG15 shows the effects of different concentrations of ZHT on HE staining of bone tissue in OVX rats. DETAILED DESCRIPTION

[0106] The present invention will be further described in detail below with reference to specific examples. The following examples are not intended to limit the present invention but are merely intended to illustrate the present invention. The experimental methods used in the following examples are generally based on conventional conditions unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0107] The hibiscus flowers in the following examples are dried corollas of hibiscus, and unless otherwise specified, are from the same batch of hibiscus flowers.

[0108] The contents of seven components of the flavonoid effective fraction of Hibiscus tiliaceus flower prepared in each embodiment were determined. These seven components are: hyperoside, rutin, isoquercetin, quercetin-8-O-β-D-glucuronide, myricetin, quercetin-3'-o-glucoside, and quercetin. During the preparation of the effective fraction samples, the solution of the effective fraction before drying was also tested for the content of each component. The sample content changed to a certain extent before and after drying. The detection method adopted the UPLC method reported in "One-Test-Multiple-Evaluation Method for Determination of Seven Components in Hibiscus tiliaceus Flower" (Journal of Pharmaceutical Analysis, Issue 12, 2013, 2082-2087) to determine the content of the components therein.

[0109] The material-liquid ratio of the extraction (i.e., extractant: extracting liquid) refers to the volume ratio.

[0110] Unless otherwise specified, the Huangkui capsules described in the following examples are all Huangkui capsules produced and sold by Suzhong Pharmaceutical Group Co., Ltd.

[0111] Example 1 Preparation of the Effective Fraction of Flavonoids from Hibiscus tiliaceus Flowers

[0112] 100g of hibiscus flower was crushed into coarse powder and extracted with 15 times 60% ethanol to obtain hibiscus flower extract; ethanol was removed from the hibiscus flower extract, diluted with water, adsorbed on activated carbon, and then continuously countercurrent extracted with n-butanol, the material-liquid ratio of the extraction (i.e., extractant: extract) was 1.5:1, the extraction injection speed was 40mL / min, the extraction machine speed was 40Hz, the extraction stage was 2, the extract was decompressed and the solvent was recovered at 40°C, and then treated with D101 macroporous resin, the treatment process of the macroporous resin was as follows: the macroporous resin diameter-to-height ratio was 1:6, the sample solution (aqueous solution) concentration was 0.15g crude drug / mL, the sample solution volume was 7BV, the sample was loaded at a flow rate of 2BV / h, 6BV pure water and 3BV 5% ethanol were used at a flow rate of 2BV / h to remove impurities, and 4BV 60% ethanol was eluted at a flow rate of 2BV / h to obtain an eluate, and then the eluate was decompressed at 50°C to recover ethanol to obtain a hibiscus flower flavonoid extract. The content was tested by adjusting the contents of the seven components by addition method according to the content determination results, so that the contents of each component were as shown in the following table, and the total solid content of the effective part of flavonoids in hibiscus flower was 2.23g, and the total of the seven components was 1769mg, accounting for 79.32% of the total solids.

[0113] Table 1 Content determination data of samples

[0114] Example 2 Preparation of the Effective Fraction of Flavonoids from Hibiscus tiliaceus Flowers

[0115] Example 2-1

[0116] 100g of hibiscus flower was crushed into coarse powder, and extracted by percolation with 18 times 70% ethanol to obtain hibiscus flower extract; ethanol was removed from the hibiscus flower extract, diluted with water, and continuously countercurrent extracted with ethyl acetate, the material-liquid ratio of the extraction (i.e., extractant: extract) was 2.5:1, the extraction injection speed was 80mL / min, the extraction machine speed was 40Hz, the extraction stage was 4, the extract was reduced pressure and solvent was recovered at 40°C, and then treated with D101 macroporous resin, the treatment process of the macroporous resin was as follows: the macroporous resin diameter-to-height ratio was 1:5, the sample solution concentration was 0.15g crude drug / mL, the sample solution volume was 6BV, the sample was loaded at a flow rate of 2BV / h, 6BV pure water and 3BV 5% ethanol were used for impurity removal at a flow rate of 2BV / h, and 4BV Elution was performed with 60% ethanol at a flow rate of 1.5 BV / h to obtain an eluate. The eluate was then decompressed and ethanol was recovered at 60°C to obtain a hibiscus flower flavonoid extract. The extract was then assayed and the contents of the seven components were adjusted by addition based on the assay results, resulting in an effective fraction of hibiscus flower flavonoids containing 2.99% total solids. The quercetin-3-O-sophoroside content was 0.28%.

[0117] Table 2-1 Content determination data of samples

[0118] Example 2-A Preparation of the Effective Fraction of Flavonoids from Hibiscus tiliaceus Flowers

[0119] According to the method of Example 2-1 above, the hibiscus flower was magnified 10 times to prepare two batches of hibiscus flower flavonoid effective fractions, and the samples with the content determination data of Table 2-2 were obtained. The quercetin-3-O-sophoroside content was 0.20-0.50%.

[0120] Table 2-2 Content determination data of samples

[0121] Among them, 2-A1 contains 0.24% quercetin-3-O-sophoroside calculated by peak area, and the proportions of the components are shown in Table 2-3 below.

[0122] Table 2-3 Content ratio data between the components of the samples

[0123] Example 2-A3

[0124] Hyperoside, isoquercetin, hibifolin, myricetin, quercetin-3'-o-glucoside, quercetin, and quercetin-3-O-sophoroside raw materials were mixed according to the ratio of Example 2-A1 (quercetin-3-O-sophoroside was used to replace rutin, and the ratio was adjusted to 0.02) as follows to prepare a total flavonoid composition.

[0125] Table 2-4

[0126] Example 2-A4

[0127] Hyperoside, isoquercetin, hibifolin, myricetin, quercetin-3'-o-glucoside, quercetin, and quercetin 3-O-acaciaside were mixed according to the ratios of Example 2-A1 (quercetin 3-O-acaciaside was used to replace rutin, and the ratio was adjusted to 0.05) to prepare a total flavonoid composition.

[0128] Example 2-B

[0129] To obtain an effective part of hibiscus flower that meets uniformity requirements, the content relationship between the medicinal material and the extract was studied based on the content results of the various components of the extracts in Example 2-1. Three batches of hibiscus flowers from different sources with significantly different components were mixed according to software calculations. 2000 g of the mixed hibiscus flowers were percolated with 18 times 70% ethanol to obtain a hibiscus flower extract. The ethanol was removed, the extract was diluted with water, and continuous countercurrent extraction was performed with ethyl acetate. The material-to-liquid ratio (i.e., extractant:extractant) was 3:1. The extraction injection speed is 120mL / min, the extraction machine speed is 60Hz, the extraction stage is 4, the extract is reduced pressure at 50℃ to recover the solvent, diluted with water, centrifuged, and the supernatant is treated with D101 macroporous resin. The treatment process of the macroporous resin is as follows: the diameter-to-height ratio of the macroporous resin is 1:6, the sample concentration is 0.15g crude drug / mL, the sample volume is 7BV, the sample is loaded at a flow rate of 2BV / h, 6BV pure water and 3BV5% ethanol are used for impurity removal at a flow rate of 2BV / h, and 4BV Elution was performed with 60% ethanol at a flow rate of 2 BV / h to obtain an eluate, which was concentrated and dried under reduced pressure at 60°C to obtain a total solids content containing 0.22% rutin, 20.58% hyperoside, 17.81% isoquercetin, 16.07% hibifolin, 5.15% myricetin, 17.37% quercetin-3'-o-glucoside, and 1.77% quercetin. The peak area of ​​quercetin-3-O-sophoroside was 0.43%. The mass ratios of the components are shown in Tables 2-5 below.

[0130] Table 2-5 Content ratio data between the components of the samples

[0131] Example 3 Preparation of the Effective Fraction of Flavonoids from Hibiscus tiliaceus Flowers

[0132] Example 3-1

[0133] 100g of hibiscus flower was crushed into coarse powder, and extracted by percolation with 18 times 60% ethanol to obtain hibiscus flower extract; ethanol was removed from the hibiscus flower extract, diluted with water, and continuously countercurrent extracted with ethyl acetate, the material-liquid ratio of the extraction (i.e., extractant: extract) was 3:1, the extraction injection speed was 100mL / min, the extraction machine speed was 40Hz, the extraction stage was 5, the extract was decompressed and the solvent was recovered at 40°C, and then treated with D101 macroporous resin, the treatment process of the macroporous resin was as follows: the macroporous resin diameter-to-height ratio was 1:8, the sample solution concentration was 0.15g crude drug / mL, the sample solution volume was 8BV, the sample was loaded at a flow rate of 2BV / h, 6BV pure water and 3BV 5% ethanol were used for impurity removal at a flow rate of 2BV / h, and 4BV Elution was performed with 60% ethanol at a flow rate of 3 BV / h to obtain an eluate, which was then decompressed and ethanol recovered at 60°C to obtain a hibiscus flower flavonoid extract. The contents of the seven components were adjusted by addition based on the assay results, resulting in a total solid content of 3.18 g of the hibiscus flower flavonoid active fraction. The quercetin-3-O-sophoroside content was 0.20-0.50%.

[0134] Table 3-1 Sample content determination data

[0135] Example 3-2

[0136] Following the preparation method of Example 3-1, the experiment was repeated using 500 g of Hibiscus hibiscus flower medicinal material. The content data for two batches are shown in Table 3-2 below. The quercetin-3-O-sophoroside content was 0.30-0.80%. Where A represents "components / total solids (%)" and B represents "components / isoquercetin."

[0137] Table 3-2

[0138] Example 4 Preparation of the Effective Fraction of Flavonoids from Abelmoschus odoratus Flowers

[0139] Example 4-1

[0140] 100g of hibiscus flower was extracted by percolation with 18 times of 70% ethanol to obtain hibiscus flower extract; ethanol was removed from the hibiscus flower extract, diluted with water, and continuously countercurrent extracted with n-butanol, the material-liquid ratio of the extraction (i.e., extractant: extract) was 3:1, the extraction injection speed was 120mL / min, the extraction machine speed was 60Hz, the extraction stage was 5, the extract was decompressed and the solvent was recovered at 50°C, and then treated with D101 macroporous resin, the treatment process of the macroporous resin was as follows: the macroporous resin diameter-to-height ratio was 1:6, the sample solution concentration was 0.15g crude drug / mL, the sample solution volume was 7BV, the sample was loaded at a flow rate of 2BV / h, 6BV pure water and 3BV 5% ethanol were used for impurity removal at a flow rate of 2BV / h, and 4BV The extract was eluted with 60% ethanol at a flow rate of 2 BV / h to obtain an eluate, which was then decompressed at 60°C to recover the ethanol and obtain a hibiscus flower extract. The content was tested and the contents of the seven components were adjusted by addition according to the determination results, so that the contents of each component were as shown in the table below, thereby obtaining 2.05 g of the total solids of the effective part of flavonoids from hibiscus flowers.

[0141] Table 4-1 Sample content determination data

[0142] Example 4-2

[0143] Hibiscus tiliaceus flower was extracted by percolation with 18 times 70% ethanol, and the ethanol was removed from the extract to 0.5-1.0 g crude drug / ml. The amount of ZTC1+1-II clarifier was 4% of component B and 2% of component A, respectively. The water bath temperature was 60°C and the water bath was kept warm for 60 minutes. The extract was centrifuged, filtered, diluted, and treated with polyamide resin (80-100 mesh). The resin treatment process was as follows: the resin diameter-to-height ratio was 1:7, the sample solution concentration was 0.15-0.5 g crude drug / mL, the sample solution volume was 8BV, and the eluate was eluted with 5BV of pure water and 5BV of 80% ethanol to obtain an eluate. The eluate was concentrated and dried under reduced pressure at 60°C. The content was tested. According to the content determination results, the contents of seven components were adjusted by addition method so that the contents of each component were as shown in the following table. The total solids of the effective flavonoid fraction of Hibiscus tiliaceus flower were obtained, and the content of quercetin-3-O-sophoroside was 0.39%.

[0144] Table 4-2

[0145] Example 5 Preparation of the Effective Fraction of Flavonoids from Abelmoschus odoratus Flowers

[0146] Example 5-1

[0147] 100g of hibiscus flower was extracted by percolation with 15 times of 70% ethanol to obtain hibiscus flower extract; ethanol was removed from the hibiscus flower extract, diluted with water, and continuously countercurrent extracted with n-butanol, the material-liquid ratio of the extraction (i.e., extractant: extract) was 2.5:1, the extraction injection speed was 80mL / min, the extraction machine speed was 50Hz, the extraction stage was 4, the extract was decompressed and the solvent was recovered at 50°C, and then treated with D101 macroporous resin, the treatment process of the macroporous resin was as follows: the macroporous resin diameter-to-height ratio was 1:6, the sample solution concentration was 0.2g crude drug / mL, the sample solution volume was 6BV, the sample was loaded at a flow rate of 2BV / h, 1BV of pure water and 4BV of 5% ethanol were used for impurity removal at a flow rate of 2BV / h, and 3BV The extract was eluted with 60% ethanol at a flow rate of 2 BV / h to obtain an eluate, which was then decompressed at 60°C to recover the ethanol to obtain a hibiscus flower extract. The content of the extract was tested, and the contents of seven components were adjusted by addition method according to the determination results to obtain 2.86 g of total solids of the effective part of flavonoids from hibiscus flowers.

[0148] Example 5-2

[0149] Hibiscus tiliaceus flowers are reflux-extracted with 15-fold 70% ethanol. The ethanol is removed from the extract to 0.5-1.0 g of crude drug / mL. The pH is adjusted to 2.0-3.0 by adding 10% hydrochloric acid solution. The extract is refrigerated, filtered, washed, and the precipitate is dissolved in water. The extract is then continuously countercurrent-extracted with ethyl acetate at a solid-liquid ratio (i.e., extractant:extractant) of 3:1 and four extraction stages. The extract is then decompressed at 50°C to recover the solvent, diluted with water, and treated with a polyamide resin. The resin treatment process is as follows: a resin diameter-to-height ratio of 1:6, a sample solution concentration of 0.15-0.5 g of crude drug / mL, and a sample solution volume of 6 BV. The eluate is eluted with 4 BV of pure water and 4 BV of 70% ethanol, concentrated, and dried under reduced pressure at 60°C to obtain the product. The quercetin-3-O-sophoroside content is 0.47%.

[0150] Table 5

[0151] Example 6 Preparation of the Effective Fraction of Flavonoids from Abelmoschus odoratus Flowers

[0152] 100g of hibiscus flower was extracted by percolation with 16 times of 60% ethanol to obtain hibiscus flower extract; ethanol was removed from the hibiscus flower extract, diluted with water, and continuously countercurrent extracted with ethyl acetate, the material-liquid ratio of the extraction (i.e., extractant: extract) was 2:1, the extraction injection speed was 100mL / min, the extraction machine speed was 40Hz, the extraction stage was 4, the extract was reduced pressure and solvent was recovered at 50°C, and then treated with D101 macroporous resin, the treatment process of the macroporous resin was as follows: the macroporous resin diameter-to-height ratio was 1:6, the sample solution concentration was 0.3g crude drug / mL, the sample solution volume was 3BV, the sample was loaded at a flow rate of 2BV / h, 3BV pure water and 3BV 5% ethanol were used for impurity removal at a flow rate of 2BV / h, and 4BV The eluate was eluted with 70% ethanol at a flow rate of 2 BV / h to obtain an eluate, and the content was tested. Seven specific ingredients were added to adjust the contents of the seven ingredients so that the contents of each ingredient were as shown in the following table. The eluate was then decompressed and ethanol was recovered at 60°C to obtain a hibiscus flower extract. The content was tested, and the contents of the seven ingredients were adjusted by addition according to the determination results. The addition amount did not exceed 10% of the total weight, so that the contents of each ingredient were as shown in the following table, thereby obtaining 3.16 g of total solids of the effective part of flavonoids from hibiscus flowers.

[0153] Table 6 Content determination data of samples

[0154] Example 7 Preparation of the Effective Fraction of Flavonoids from Hibiscus tiliaceus Flowers (Comparative Example)

[0155] Example 7-1

[0156] 500 g of hibiscus flower was crushed into coarse powder and extracted by percolation with 20 times of 70% ethanol to obtain hibiscus flower extract; after reducing pressure to recover the solvent, the extract was treated with D101 macroporous resin. The treatment process of the macroporous resin was as follows: the diameter-to-height ratio of the macroporous resin was 1:4, the loading solution concentration was 0.15 g of crude drug / mL, the loading solution volume was 6 BV, the sample was loaded at a flow rate of 2 BV / h, 7 BV of pure water and 4 BV of 5% ethanol were used at a flow rate of 2 BV / h for impurity removal, and 4 BV of 60% ethanol were used for elution at a flow rate of 1.5 BV / h to obtain an eluate. After reducing pressure to recover ethanol from the eluate at 60° C., 16.12 g of total solids of hibiscus flower was obtained. The contents of each component are shown in the following table.

[0157] Table 7-1 Sample content determination data

[0158] Example 7-2

[0159] 100 g of hibiscus flower was crushed into coarse powder, and the powder was extracted by percolation with 18 times 70% ethanol to obtain a hibiscus flower extract; after the solvent was recovered under reduced pressure, the extract was treated with D101 macroporous resin, and the treatment process of the macroporous resin was as follows: the macroporous resin diameter-to-height ratio was 1:5, the loading solution concentration was 0.15 g crude drug / mL, the loading solution volume was 6 BV, the sample was loaded at a flow rate of 2 BV / h, 6 BV of pure water and 3 BV of 5% ethanol were used at a flow rate of 2 BV / h for impurity removal, and 4 BV of 60% ethanol was used at a flow rate of 1.5 BV / h to obtain an eluate, and the eluate was decompressed and ethanol was recovered at 60°C to obtain 3.56 g of hibiscus flower total solids.

[0160] Table 7-2 Sample content determination data

[0161] Example 8 Preparation of the Effective Fraction of Flavonoids from Hibiscus arvensis Flowers (Comparative Example)

[0162] 100g of hibiscus flower was crushed into coarse powder, and extracted by percolation with 18 times 70% ethanol to obtain hibiscus flower extract; ethanol was removed from the hibiscus flower extract, diluted with water, and continuously countercurrent extracted with ethyl acetate, the material-liquid ratio of the extraction (i.e., extractant: extract) was 2.5:1, the extraction injection speed was 80mL / min, the extraction machine speed was 40Hz, the extraction stage was 4, the extract was decompressed and the solvent was recovered at 40°C, and then treated with D101 macroporous resin, the treatment process of the macroporous resin was as follows: the macroporous resin diameter-to-height ratio was 1:5, the sample solution concentration was 0.15g crude drug / ml, the sample solution volume was 6BV, the sample was loaded at a flow rate of 2BV / h, 6BV pure water and 3BV 5% ethanol were used for impurity removal at a flow rate of 2BV / h, and 4BV The eluate was eluted with 60% ethanol at a flow rate of 1.5 BV / h to obtain an eluate, and the content was tested. Then, rutin and hyperoside were added to the eluate to make the contents of each component as shown in the following table. Then, ethanol was recovered under reduced pressure at 60° C. to obtain 4.28 g of total solids of hibiscus flowers.

[0163] Table 8 Content determination data of samples

[0164] Example 9

[0165] Example 9-1 Preparation of the Effective Fraction of Flavonoids from Abelmoschus truncatus Flowers

[0166] 25 kg of hibiscus flower was crushed into coarse powder, and the extract was extracted by percolation with 18 times 70% ethanol to obtain hibiscus flower extract; ethanol was removed from the hibiscus flower extract, diluted with water, and continuously countercurrent extracted with ethyl acetate, the material-liquid ratio of the extraction (i.e., extractant: extract) was 2.5:1, the extraction stage was 4, the extract was reduced pressure and the solvent was recovered at 40°C, and then treated with D101 macroporous resin, the treatment process of the macroporous resin was as follows: the macroporous resin diameter-to-height ratio was 1:5, the sample solution concentration was 0.15 g crude drug / mL, the sample solution volume was 3BV, the sample was loaded at a flow rate of 1.5BV / h, 6BV pure water and 3BV Impurities were removed using 10% ethanol at a flow rate of 2 BV / h, and elution was performed using 4 BV of 60% ethanol at a flow rate of 1.5 BV / h to obtain an eluate. The eluate was then decompressed and the ethanol was recovered at 60°C to obtain the total solids of hibiscus flower. Seven specific ingredients were added and adjusted (the content of the added ingredients did not exceed 20% of the total weight) to obtain the total solids of the effective flavonoid fraction of hibiscus flower as shown in the table below. The quercetin-3-O-sophoroside content was 0.20-0.50%.

[0167] Table 9-1 Sample content determination data

[0168] Example 9-2 Preparation of the Effective Fraction of Flavonoids from Abelmoschus truncatus Flowers

[0169] Reference Example 2-B, 30kg of hibiscus flower mixture was crushed to a coarse powder and extracted with 18 times 70% ethanol percolation to obtain a hibiscus flower extract; the hibiscus flower extract was removed from the ethanol, diluted with water, and continuously countercurrent extracted with ethyl acetate. The solid-liquid ratio of the extraction (ie, the extractant: the extract) was 2.5:1, the extraction injection rate was 80mL / min, the extraction speed of the extraction machine was 40Hz, the extraction stage was 4, and the extract was recovered under reduced pressure at 40°C. The solvent was then treated with D101 macroporous resin. The treatment process of the macroporous resin was as follows: the macroporous resin diameter-to-height ratio was 1:5, the sample concentration was 0.15g crude drug / mL, the sample volume was 5BV, the sample was loaded at a flow rate of 1.5BV / h, 3BV of pure water and 4BV5% ethanol were removed at a flow rate of 2BV / h, and 5BV Elution was performed with 60% ethanol at a flow rate of 1.5 BV / h to obtain an eluate, which was then decompressed at 65°C to recover the ethanol to obtain the total solids of hibiscus flowers. The product was vacuum-dried and pulverized, and the contents (%) of the seven flavonoid components were detected. The results were as follows: the content of quercetin-3-O-sophoroside was 0.20-0.50%.

[0170] Table 9-2 Content determination data of samples

[0171] Example 11 Preparation of eye drops containing the effective fraction of flavonoids from hibiscus flowers

[0172] Preparation process: The effective fraction prepared according to any of the methods in Examples 1-7 is dissolved in water for injection by stirring, and the volume is made up to 100 L. The pH is adjusted to 7, filtered, filled, and sterilized to obtain the eye drops. The pH adjuster used is sodium hydroxide and / or hydrochloric acid.

[0173] Example 12 Pharmacological Experiment on Diabetic Kidney Model

[0174] Example 12-1

[0175] Animal model: Streptozotocin (STZ) + high-fat diet diabetic kidney model, C57BL / 6 mice, 20±10g, SPF grade, male.

[0176] Administration: Oral administration.

[0177] Experimental modeling: After three days of adaptive feeding, the mice were fed a high-fat, high-sugar diet (laboratory-made, formula: lard: sucrose: egg yolk: basal diet = 18:20:3:59) for eight consecutive weeks. They were then intraperitoneally injected with 100 mg / kg of STZ sodium citrate solution for seven consecutive days. Blood glucose levels were measured via the tail vein on the eighth day. A blood glucose level greater than 16.7 mmol / L was considered a successful diabetic model. A blood glucose level greater than 13.8 mmol / L, along with the presence of proteinuria and renal dysfunction, indicated successful modeling.

[0178] Experimental groups included: normal group, model group, positive drug group (dapagliflozin tablets, 45.5 mg / kg), sample group prepared according to the method of Example 2-1 (62.4 mg / kg), sample group prepared according to the method of Example 6 (62.4 mg / kg), and sample group prepared according to the method of Example 8 (62.4 mg / kg). A total of 6 groups, 15 mice in each group, were tested for urine protein and urine creatinine after 4 weeks of administration, and daily food intake and mouse status were calculated. The test results are shown in Table 12-1 below.

[0179] Table 12-1

[0180] Note: *Compared with the normal control group, P < 0.05; #Compared with the model control group, P < 0.05.

[0181] All groups in the examples had a reducing effect on urine protein, and the sample group in Example 2-1 significantly reduced urine protein and urine protein / urine creatinine (ACR). The observation results of the mouse state showed that the mice in the sample group in Example 2-1 had significantly fewer diarrhea and abdominal distension than those in the other groups.

[0182] Example 12-2

[0183] Animal models: db / db mice, 16 weeks old, C57BL / 6 mice, 16 weeks old.

[0184] Administration: Oral administration.

[0185] Experimental modeling: After 17 weeks of breeding, db / db mice were tested for urine protein. If urine protein and renal function abnormalities were present, the model was considered successful and used for subsequent experiments.

[0186] Experimental groups: C57BL / 6 mice were divided into a normal group, and db / db mice were divided into a model group, a positive drug group (dapagliflozin tablets, 45.5 mg / kg), a sample group prepared by the method of Example 3-1 (62.4 mg / kg), a sample group prepared by the method of Example 9-1 (62.4 mg / kg), a sample group prepared by the method of Example 2-A3 (62.4 mg / kg), and a sample group of Example 7-1 (62.4 mg / kg), with 15 mice in each group. After 4 weeks of administration, urine protein and urine creatinine, daily food intake, and the condition of the mice were observed. The test results are shown in Table 12-2 below.

[0187] Table 12-2

[0188] Note: *Compared with the normal control group, P < 0.05; #Compared with the model control group, P < 0.05.

[0189] All groups in the examples had a reducing effect on urine protein, and each sample group could reduce urine protein and urine protein / creatinine (ACR). The results of mouse condition observation showed that the number of mice with diarrhea and abdominal distension in the sample groups of Example 3-1 and Example 9-1, and the sample group of Example 2-A3 was significantly less than that in the other groups.

[0190] Example 13 Effects of flavonoids on ocular blood flow and choroidal neovascularization

[0191] Experimental animals: rats, body weight 150-180 g, SPF grade, half male and half female.

[0192] Administration: Oral administration.

[0193] Experimental modeling: Healthy male Brown-Norway rats were selected and anesthetized with intramuscular injection of ketamine (50 mg / kg). Mydriasis was performed with a mixture of 0.5% tropicamide and phenylephrine. Krypton yellow laser (Coyne, USA) (wavelength 568 nm, parameters: spot diameter 100 μm, exposure time 0.1 s, power 150-200 mW) was used. Under a 120D special front lens, 8 points were photocoagulated between the large retinal blood vessels around the optic disc at a distance of 2-4 disc diameters. The qualified photocoagulation points were those with small bubbles produced after the Bruch's membrane was broken. The rats with retinal, choroidal or vitreous hemorrhage were excluded.

[0194] Drug grouping and dosage: normal group, model group, positive control group (vertebofen 1.35 mg / kg), Example 2-1 sample group (69.9 mg / kg), Example 3-1 sample group (87.36 mg / kg), Example 7-2 sample group (87.36 mg / kg), Example 9-1 sample group (87.36 mg / kg), Example 9-2 sample group (87.36 mg / kg), 15 mice per group. After 4 weeks of administration, the choroidal blood flow inhibition rate and choroidal neovascularization area were measured. The test results are shown in Table 13 below.

[0195] Table 13

[0196] Note: *Compared with the normal control group, P < 0.01; #Compared with the model control group, P < 0.05.

[0197] Conclusion: The samples in each example have an inhibitory effect on the choroidal blood flow inhibition rate and the choroidal angiogenesis area. The sample groups of Example 2-1, Example 3-1, Example 9-1 and Example 9-2 can significantly reduce choroidal angiogenesis.

[0198] Example 15 Effects of flavonoids on lupus erythematosus nephritis

[0199] Twenty-six MRL / lpr male mice (lupus nephritis mice) were selected as the observation group, with a body weight of 18-22 g, 13 weeks of age, purchased from Nanjing Junke Bioengineering Co., Ltd., and 24 male C57BL / 6 mice were selected as the normal group, with a body weight of 18-22 g, 13 weeks of age, purchased from Wuhan Hualianke Biotechnology Co., Ltd.

[0200] Drug grouping and dosage: normal group, MRL / lpr mouse model group, positive control drug group (dexamethasone 1 mg / kg), Example 2-B sample group (69.9 mg / kg), Example 3-1 sample group (87.36 mg / kg), Example 7-1 sample group (87.36 mg / kg), Example 5-2 sample group (87.36 mg / kg), Example 8 sample group (87.36 mg / kg), 15 mice in each group, and administration for 4 weeks.

[0201] Experimental Indicators: After dosing, urine was collected for 24 hours for protein testing. Following urine collection, all mice had their eyeballs removed and blood collected. Serum was centrifuged and used for antinuclear antibody testing. Simultaneously, kidneys were removed, quickly frozen in liquid nitrogen, and stored in a -80 freezer for renal immunoassays. The test results are shown in Table 15 below.

[0202] Table 15

[0203] Note: *Compared with the normal group, P < 0.01; #Compared with the model group, P < 0.05.

[0204] Conclusion: The urine protein, serum antinuclear antibody level, IgG and C3 deposition in the renal tissue of the lupus nephritis mice in the model group were significantly higher than those in the other drug-administered groups; while the sample group of Example 5-2, the sample group of Example 2-B, the sample of Example 3-1, the sample of Example 8 and the sample of Example 7-1 were all able to improve the urine protein, serum antinuclear antibody level, IgG and C3 deposition in the renal tissue, and the samples of Example 2-B, Example 3-1 and Example 5-2 had better effects.

[0205] Example 16 Toxic effects of flavonoids

[0206] Experimental animals: rats, body weight 150-180 g, SPF grade, half male and half female.

[0207] Administration: Oral administration.

[0208] Drug grouping and dosage: normal group, Huangkui capsule group (extract 8g / kg), Example 2-A1 sample group (5g / kg), Example 9-2 sample group (8g / kg), 30 rats in each group, once a day for 12 consecutive weeks, the dosage is calculated based on the extract.

[0209] Experimental results: After the administration, the kidney weight, visceral body and visceral brain coefficient of the Huangkui Capsule group increased compared with the normal group. Pathology showed that the Huangkui Capsule group had moderate renal tubular hypertrophy, while the Example 9-2 sample group and the Example 2-A1 group did not show renal tubular hypertrophy in pathological examination. The test results are shown in Table 16 below.

[0210] Table 16

[0211] Note: *P<0.05 compared with the normal group.

[0212] Example 17 Effects of the Effective Fractions of Total Flavonoids on Idiopathic Pulmonary Fibrosis

[0213] Preparation of experimental sample 1: Take hyperoside, isoquercetin, hibifolin, myricetin, quercetin-3'-o-glucoside, quercetin, and quercetin 3-O-sophoraside as raw materials, weigh each active ingredient according to the feeding ratio in Table 17-1, mix, and prepare a pharmaceutical composition.

[0214] Table 17-1

[0215] Experimental animals: Kunming mice, half male and half female, weighing 18-22 g, clean grade.

[0216] Animal grouping: The mice were randomly divided into a normal group, a model group, a control drug group (rosiglitazone 5 mg / kg), an Example 2-A3 sample group (70 mg / kg), an Example 9-2 sample group (70 mg / kg), a Huangkui capsule sample group (180 mg / kg as the extract), and an experimental sample group 1 (62.4 mg / kg), with 10 mice in each group.

[0217] Experimental modeling: After three days of adaptive feeding, experimental mice were anesthetized with 4% chloral hydrate (0.01 ml / g) via intraperitoneal injection. The mice were placed in a supine position, routinely disinfected, and a midline cervical incision was performed. The trachea was exposed by blunt dissection. Bleomycin (5 mg / kg) was slowly injected through the interstitial space of the tracheal cartilage rings in the model, control, and treatment groups. An equal volume of normal saline was injected into the normal control group. Immediately after injection, the mice were rotated upright for 3-5 minutes to evenly distribute the drug solution in both lungs. The skin was then sutured and the suture sites were disinfected. After awakening, the mice were transferred to a clean observation room for feeding.

[0218] Dosage: The drug was administered starting from the second day of modeling. The drug-treated group was given the above-mentioned dose once a day. The normal group and the model group were given the same volume of distilled water (10 ml / kg) in the same way for 28 consecutive days.

[0219] Lung tissue pathology staining: After administration, the right lung was removed and fixed, embedded, sectioned, and stained with HE according to conventional pathology methods. The degree of alveolitis and pulmonary fibrosis was divided into four grades.

[0220] The experimental results are shown in Table 17-2 below. The results show that the degree of alveolitis and pulmonary fibrosis in the lung tissue of mice in the model group was significantly aggravated, showing typical parenchymal lesions of pulmonary fibrosis. After 28 days of administration, the lung coefficient of mice in each dose group was significantly reduced, and the degree of alveolitis and pulmonary fibrosis in the lung tissue was significantly alleviated. The effective fraction of the total flavonoids of the present invention can significantly reduce the degree of alveolitis and pulmonary fibrosis in mice with pulmonary fibrosis, has a protective effect on the lungs of mice with pulmonary fibrosis, and can alleviate the damage caused by fibrotic lung tissue.

[0221] Table 17-2

[0222] Note: *Compared with the normal group, P < 0.01; #Compared with the model group, P < 0.05.

[0223] Example 18 Study on the Mechanism of Action of SGLT2

[0224] Sodium-glucose contransporter 2 (SLC5A2, also known as SGLT2) is a gene that encodes a sodium-glucose cotransporter that reabsorbs approximately 90% of glucose filtered through the glomerulus. SLC5A2 is expressed exclusively on the inner brush border of the renal proximal tubule epithelial cells and is considered a marker of the proximal tubule. Its primary function is to recycle filtered sodium and glucose. Inhibiting SLC5A2 protein expression in the kidneys of patients with diabetes and diabetic nephropathy can excrete sodium, excrete water, and lower blood sugar, thereby protecting the kidneys of patients with diabetes and diabetic nephropathy.

[0225] Animal model: db / db mice, 18 weeks old.

[0226] Administration: Oral administration.

[0227] Experimental Grouping: db / db mice with UACR > 200 mg / g were divided into three groups: diabetic nephropathy (DKD), Huangkui capsule group (DKD + HK, 0.84 g / kg / d), and total flavonoid group (DKD + HT, Example 2-A2 sample, 0.0755 g / kg / d). The Huangkui capsule and total flavonoid groups were administered for four weeks (28 days). Urinary microalbumin (UACR) in db / db mice was measured by enzyme-linked immunosorbent assay (ELISA), SLC5A2 mRNA expression was measured by reverse transcription polymerase chain reaction (RT-PCR), and SLC5A2 protein expression was measured by immunohistochemistry (IHC).

[0228] Experimental results:

[0229] Compared with the DKD group, there were no significant differences in pre-group body weight (DKD (N=5), HK (N=5), HT (N=6), body weight in the first and fourth weeks of administration) between the HK (Huangkui capsule) and HT (total flavonoids) groups (DKD (N=5), HK (N=5), HT (N=6). There were no significant differences in blood glucose levels (DKD (N=5), HK (N=5), HT (N=6) in the first and fourth weeks of administration). There were no significant differences in UACR values ​​(DKD (N=5), HK (N=5), HT (N=6) in the first week of administration. In the fourth week of administration, UACR values ​​were significantly lower in the HT group compared with the DKD group (*P<0.05). There were no significant differences in mean kidney function (DKD (N=5), HK (N=5), HT (N=6) in the fourth week of administration). Renal index (DKD, N=5), HK, N=5, and HT) (N=6) increased significantly in the fourth week of treatment (*P<0.05). In the fourth week of treatment, some glomeruli in the DKD group showed lobulated structures, with significantly thickened basement membranes, significant glomerular capillary compression, and mesangial cell proliferation and matrix expansion. In the HK group, glomerular basement membrane thickening was significantly improved, glomerular capillaries were intact, and mesangial cell and matrix proliferation was significantly reduced. In the HT group, glomerular basement membrane thinning was significant, with no glomerular proliferation, no proliferation of glomerular capillary interstitium, and no proliferation of mesangial cells and matrix. IHC-P quantification results at the fourth week of treatment showed that SLC5A2 protein expression levels were significantly decreased in the HK (N=5) and HT (N=6) groups compared with the DKD group (N=5). RT-PCR results validated the immunohistochemistry results at week 4 of drug administration, showing that SLC5A2 mRNA expression levels were significantly decreased in the Huang Kui HK group (N=5) and the HT group (N=6) compared with the DKD group (N=5). At week 4 of drug administration, the ratio of SLC5A2 IHC-P quantified expression to glomerular number was significantly decreased in the Huang Kui HK group (N=5) and the HT group (N=6) compared with the DKD group (N=5), consistent with the IHC-P quantification results.

[0230] Therefore, Huangkui capsules and total flavonoids can effectively reduce microalbuminuria in db / db mice. Huangkui capsules and total flavonoids can inhibit the expression of SLC5A2 protein on the proximal tubule cell membrane, reduce the expression of SLC5A2 protein in the kidneys of DKD mice, and effectively inhibit the activity of SLC5A2 protein, thereby effectively reducing the reabsorption of glucose in the kidneys of DKD mice.

[0231] Example 19 Preparation of total flavonoids extract from hibiscus flowers

[0232] Referring to the preparation method of Example 3-1 above, the experiment was repeated with 5000 g of hibiscus flowers, and the content (unit: %) of two batches was shown in the following table: quercetin-3-O-sophoroside content was 0.30-0.80%.

[0233] Table 19

[0234] Example 20 Animal Experiment on Ulcerative Colitis

[0235] 1. Experimental Materials

[0236] 1.1 Experimental animals

[0237] SPF male C57BL / 6J mice, 6–8 weeks old, weighing 22–25 g, were provided by the Center for Comparative Medicine of Yangzhou University.

[0238] 1.2 Reagents and drugs

[0239] Table 20-1

[0240] 2. Experimental Methods

[0241] 2.1 Animal husbandry and management

[0242] The experimental mice were housed in an SPF-grade animal room throughout the experiment. The light-to-dark ratio was 1:1, the room temperature was controlled at 23±2°C, and the humidity was controlled at 55%. The mice were able to eat and drink freely. The bedding was changed every three days to ensure that the mice were in a dry and clean environment.

[0243] 2.2 Drug preparation

[0244] All drugs were dissolved according to the steps in Figure 1: ①: Prepare 0.5% CMC-Na the day before dosing: Slowly add 0.5g of CMC-Na powder to 100mL of 70°C pure water, stir thoroughly, and let stand at room temperature overnight. The next day, sonicate until a homogeneous 0.5% CMC-Na solution is obtained before preparing the drug solutions. ②: Weigh 12.5mg, 37.5mg, and 75mg of ZHT and 100mg of 5-ASA, respectively, and add 0.1mL of DMSO to prepare DMSO stock solutions. ③: Add 0.1mL of Tween-80 to each stock solution in step ②. ④: Add 4.8mL of 0.5% CMC-Na solution to each solution in step ③. Prepare 2.5mg / mL, 7.5mg / mL, and 15mg / mL ZHT suspensions and 20mg / mL 5-ASA suspensions, respectively.

[0245] Preparation method of 3% DSS solution: Weigh 3g of DSS powder and fully dissolve it in 100mL of pure water to obtain 3% DSS solution.

[0246] 2. Preparation of 3DSS-induced ulcerative colitis (UC) model in mice

[0247] After 5 days of adaptive feeding, the mice were randomly divided into 6 groups, each containing two cages and 4 mice per cage. These groups included a blank control group (Control), a sunflower flavonoid extract control group (150 mg / kg / day ZHT), a model group (DSS), a model group plus 5-aminosalicylic acid group (DSS + 200 mg / kg / day 5-ASA), and a model group plus ZHT (DSS + 75 mg / kg / day ZHT).

[0248] As shown in the following method, the mouse ulcerative colitis model was induced by free drinking of 3% DSS for 7 consecutive days. The 3% DSS solution (80 mL) was replaced every 2 days for each cage of 4 mice to ensure the effectiveness of the DSS solution. The mice in the blank control group were free to drink pure water, and the water was replaced every 2 days for each cage of 4 mice.

[0249] Different doses of ZHT or the positive therapeutic drug 5-ASA were administered orally at a dose of 0.1 mL / 10 g bw daily between 9:00 and 10:00 AM for 7 consecutive days. The blank and model groups received the same dose of a blank vehicle solution (containing 2% DMSO, 2% Tween 80, and 0.5% CMC-Na). Disease activity index (DAI) scores were assessed daily before dosing. On day 8, mice were euthanized, and blood and colon tissue were collected.

[0250] 2.4 Stool consistency / fecal occult blood test and DAI score

[0251] Before dosing daily, mice were weighed and feces collected for fecal occult blood testing. Disease severity was assessed using the DAI score based on weight loss, stool consistency, and fecal occult blood, with each score ranging from 0 to 4. The scoring criteria are shown in Table 20-2. For fecal occult blood testing, a small amount of feces was smeared onto the center of a glass slide (pre-ignited to minimize color variation). Three drops of 10 / L acetone sulfate solution and three drops of 3% hydrogen peroxide solution were added, mixed thoroughly, and the results were immediately observed and scored according to Table 20-3.

[0252] Table 20-2 DAI scoring rules

[0253] Table 20-3 Fecal Occult Blood Test Scoring Table

[0254] 2.5 Specimen Collection

[0255] After the last dose, mice in each group were fasted for 24 hours and then sacrificed by cervical dislocation. The abdominal cavity of the mice was cut open to expose the colon. The entire colon tissue of the mice was measured and its length was recorded. The intestinal contents were flushed in freshly prepared pre-chilled saline. A 1 cm segment of the colon near the rectum was cut and fixed overnight in 4% paraformaldehyde for paraffin section preparation and subsequent pathological examination. The remaining tissue was quickly frozen in liquid nitrogen in the middle section and stored at -80°C for subsequent experimental use. Blood samples were allowed to stand at room temperature for 2-3 hours, then centrifuged at 3500 rpm for 15 minutes. Serum was collected and quickly stored at -80°C for subsequent testing.

[0256] 2.6 Determination of myeloperoxidase (MPO) content in colon tissue

[0257] MPO is a functional marker and activation marker of neutrophils. Under pH 6.0, with H2O2 and 3,3′-dimethoxybenzidine hydrochloride as substrates, MPO can catalyze the substrates to generate an orange product. The amount of the product generated is measured by colorimetry at 460 nm, thereby calculating the MPO content.

[0258] The specific measurement steps are as follows:

[0259] 1) Sample pretreatment: Accurately weigh the tissue and add homogenization medium at a weight-to-volume ratio of 1:9. Grind in a tissue grinder at 60 Hz for 1 min, repeat five times, until no precipitate is visible to the naked eye, to prepare a 10% tissue homogenate.

[0260] 2) Prepare the reaction system: Transfer 0.09 mL of 10% tissue homogenate or serum sample to two new 2 mL EP tubes (control tube and assay tube) and add the corresponding reagents in the kit according to Table 20-4 for reaction.

[0261] 3) Absorbance measurement: 150 μL of the reaction product was placed in a 96-well plate and the absorbance was measured at 460 nm using a microplate reader.

[0262] 4) Calculate the MPO content according to the following formula:

[0263] Where W is the sample volume; for tissue homogenate, W (g) = homogenate concentration × sampling volume (mL).

[0264] Table 20-4 MPO content determination operation table

[0265] 2.7 H&E staining

[0266] The colon was fixed in 4% paraformaldehyde for 24 hours and then embedded in paraffin. Serial transverse sections (10 μm) were made on the embedded colon tissue and then stained with H&E. The H&E staining steps were as follows:

[0267] 1) Dewaxing: Dewaxing with xylene I for 10 min, and dewaxing with xylene II for 5 min;

[0268] 2) Ethanol soaking: Soak in anhydrous ethanol twice, then soak in 95%, 90%, and 85% ethanol once, each soaking for 1 minute, and finally wash with water for 2 minutes;

[0269] 3) Staining: Stain with hematoxylin for 5 minutes, then wash with tap water for 1 minute;

[0270] 4) Differentiation: Differentiate with 1% hydrochloric acid alcohol for 20 seconds, then wash with tap water for 1 minute;

[0271] 5) Anti-blueing: Anti-blue with 1% dilute ammonia for 30 seconds, then wash with tap water for 1 minute;

[0272] 6) Eosin staining: stain for 5 minutes, then wash with tap water for 30 seconds;

[0273] 7) Ethanol dehydration: Dehydration was performed with different ethanol contents in the following order: 85% ethanol dehydration for 20 seconds, 90% ethanol dehydration for 30 seconds, 95% ethanol I dehydration for 1 minute, 95% ethanol II dehydration for 1 minute, anhydrous ethanol I dehydration for 2 minutes, and anhydrous ethanol II dehydration for 2 minutes;

[0274] 8) Transparency: xylene I treatment for 2 min, xylene II treatment for 2 min, xylene III treatment for 2 min;

[0275] 9) Finally, the slides were sealed with neutral gum and scanned using a digital pathology slide scanner (NanoZoomer 2.0RS);

[0276] 10) Pathological examination: The degree of colon damage was scored based on pathological indicators such as the degree of colon tissue lesions, inflammatory cell infiltration, and crypt cell damage. The specific scoring criteria are shown in Table 20-5.

[0277] Table 20-5 Inflammation Score Table

[0278] 2.8 Real-time fluorescence quantitative PCR

[0279] Place the colon tissue in a grinding tube, add grinding beads and 400 μL Trizol reagent, and grind continuously at 60 Hz in a tissue grinder for 1 minute. Repeat 5 times until no precipitate is visible to the naked eye. Collect the supernatant, centrifuge at 12,000 rpm for 10 minutes, and transfer the supernatant to a new 1.5 mL enzyme-free tube. Extract with chloroform-isopropanol overnight and centrifuge. Discard the supernatant and wash away the ions attached to the RNA with 75% ethanol. After drying, dissolve in DEPC water and aspirate 2 μL for quantification using a Nano-100 micro-spectrophotometer. A 260 / A 280 The RNA concentration should be between 1.8 and 2.0. Adjust the RNA concentration to 1 μg using enzyme-free water. Add 3 μL of 5×gDNA wiper mix and heat at 42°C for 2 minutes to remove genomic DNA. Then, add 5 μL of 4×qRT Super Mix II to create a 20 μL system. Perform reverse transcription in a PCR instrument at 25°C for 2 minutes, 50°C for 30 minutes, and 85°C for 5 minutes. Store the cDNA at -80°C until needed.

[0280] According to 1μL cDNA + 10μL Real-time fluorescence quantitative PCR reaction was performed using a system of Green+0.8μL Primers+8.2μL ddH2O. Comparative Ct(2 -ΔΔCt ) method to calculate relative mRNA expression, and the expression level of each gene was corrected by the expression level of GAPDH. The primers used are shown in Table 20-6.

[0281] Table 20-6 Primer sequences used in PCR

[0282] 2.8 Determination of TNF-α, IL-1β, and IL-6 Contents

[0283] According to the operating procedures of the ELISA kit, the levels of TNF-α, IL-1β and IL-6 in colon tissue were detected.

[0284] 2.9 Statistical Analysis

[0285] Statistical analysis was performed using GraphPad Prism (Ver 6.0). All data were expressed as mean ± standard error (Mean ± SEM). Changes in body weight and DAI scores were analyzed using two-way ANOVA, and other data were analyzed using one-way ANOVA. Dunnett's multiple test was used to analyze the significance of differences between the groups. When P < 0.05, the differences between the groups were statistically significant.

[0286] 3. Experimental Results

[0287] 3.1 Effects of different doses of total flavonoids from Astragalus membranaceus on the DAI score of mice

[0288] The DAI score was calculated by adding the three indicators in Table 20-2 and taking the average. As shown in Table 20-7, the DAI scores of mice in the DSS model group increased significantly starting on day 3, with a statistically significant difference compared to the control group (P < 0.01). Three mice developed diarrhea on day 6, and seven on day 7. The DAI score in the DSS+ZHT (75 mg / kg / day) group showed a more gradual increase, with statistically significant differences in DAI scores compared to the DSS model group on days 4, 5, 6, and 7 (P < 0.01). No mice developed diarrhea during the experiment. The active therapeutic drug 5-ASA (200 mg / kg / day) alleviated DSS-induced colitis symptoms and reduced DAI scores. Starting on day 4, the difference was significant compared to the DSS model group (P < 0.01). No mice developed diarrhea during the experiment. During the experiment, there was no significant difference between the normal group and the control group, and ZHT 150 mg / kg / day had no effect on the weight of mice. One mouse developed diarrhea symptoms on the 6th day.

[0289] Table 20-7 DAI scores of mice in each group

[0290] Note: Data are expressed as mean ± standard error. N = 8. *, P < 0.05, **, P < 0.01, vs. Control; # ,P<0.05, ## , P < 0.01, vs. DSS, analyzed using two-way ANOVA and Dunnett's multiple comparison test.

[0291] 3.2 Effects of different doses of total flavonoids from Astragalus membranaceus on the colon length of mice

[0292] As shown in Figure 2 and Table 20-8, the colon length of the control group was 7.34±0.08 cm, while the colon of mice in the DSS model group atrophied to 4.85±0.18 cm, a significant difference compared to the control group (P<0.01). The colon length of the ZHT (150 mg / kg / day) group (7.43±0.13 cm) was close to that of the control group. ZHT (75 mg / kg / day) significantly improved DSS-induced colon atrophy, with the DSS+ZHT (75 mg / kg / day) group showing a statistically significant difference compared to the DSS model group (P<0.05). The positive drug 5-ASA (200 mg / kg / day) also improved DSS-induced colon atrophy, with a statistically significant difference compared to the control group (P<0.05).

[0293] Table 20-8 Statistics of colon length of mice in each group

[0294] Note: Data are expressed as mean ± standard error. N = 8. **, P < 0.01, vs. Control; #, P < 0.05, vs. DSS, analyzed using one-way ANOVA and Dunnett's multiple comparison test.

[0295] 3.3 Effects of different doses of total flavonoids from Astragalus membranaceus on the myeloperoxidase (MPO) content in mouse colon tissue

[0296] The MPO content in the colon tissue of the control group was 0.30±0.04U / g, and the MPO content in the colon tissue of the DSS model group mice increased to 1.04±0.10U / g. Compared with the control group, the difference was statistically significant (P<0.01); 75mg / kg / day ZHT had an inhibitory effect on the DSS-induced increase in MPO content in the colon, and the inhibition rate of 75mg / kg / day ZHT was 92.8±19.4%, and the difference was statistically significant (P<0.01); the positive therapeutic drug 5-ASA (200mg / kg / day) significantly inhibited the DSS-induced increase in MPO content in the colon, with an inhibition rate of 88.7±9.9% (P<0.01).

[0297] 3.4 Effects of different doses of total flavonoids from Astragalus membranaceus on pathological changes in mouse colon tissue

[0298] Experimental results showed (Figure 3) that the colon tissue structure of mice in the control and ZHT (150 mg / kg / day) groups was normal, with little inflammatory cell infiltration, and intact crypt cells and epidermal cells. In the DSS model group, the colon tissue of mice showed a significant increase in inflammatory cells, with a complete loss of crypt cells and epidermal cells, and the extent of lesions exceeded 80%. Compared with the control group, the pathological score was significantly different (P < 0.01). The colon tissue of the DSS+ZHT (75 mg / kg / day) group showed significant improvement, and the pathological score of the DSS+ZHT (75 mg / kg / day) group was significantly different from that of the DSS model group (P < 0.01). The positive therapeutic drug 5-ASA (200 mg / kg / day) significantly improved the DSS-induced colon tissue pathological changes, and the pathological score was significantly different from that of the DSS model group (P < 0.01).

[0299] 3.5 Effects of total flavonoids from Astragalus membranaceus on DSS-induced IL-1β, IL-2, IL-4, IL-6, TNF-α, and IFN-γ mRNA expression levels

[0300] Experimental results showed that the expression of IL-1β mRNA in the colon tissue of mice in the DSS model group increased by 7.36±0.79 times that of the control group (P<0.01). ZHT (150mg / kg / day) alone increased IL-1β mRNA expression in the colon tissue of mice to 2.21±0.24 times that of the control group, but the difference was not significant compared with the control group (P>0.05). ZHT (75mg / kg / day) inhibited the DSS-induced increase in IL-1β mRNA expression by 81.9±5.0% (P<0.01). The positive therapeutic drug 5-ASA (200mg / kg / day) inhibited DSS-induced IL-1β mRNA expression by 91.6±3.6% (P<0.01).

[0301] The results showed that the expression of IL-2 mRNA in the colon tissue of mice in the DSS model group was significantly increased to 6.65±0.56 times that of the control group (P<0.01). ZHT (150 mg / kg / day) alone increased the expression of IL-2 mRNA in the colon tissue of mice to 1.74±0.15 times that of the control group, but the difference was not statistically significant (P>0.05). ZHT (75 mg / kg / day) inhibited the DSS-induced increase in IL-2 mRNA expression by 78.7±4.5%, which was significantly different from the DSS model group (P<0.01). The positive therapeutic drug 5-ASA (200 mg / kg / day) significantly inhibited the DSS-induced increase in IL-2 mRNA expression in the colon tissue by 94.2±2.8% (P<0.01).

[0302] The results showed that the expression of IL-4 mRNA in the colon tissue of mice in the DSS model group was significantly increased to 5.78±1.22 times that of the control group (P<0.01). In the group treated with ZHT alone at 150 mg / kg / day, the expression of IL-4 mRNA in the colon tissue was 1.07±0.16 times that of the control group, with no significant difference compared to the control group (P>0.05). ZHT at 75 mg / kg / day inhibited the DSS-induced upregulation of IL-4 mRNA expression by 78.65±7.18%, a significant difference compared to the DSS model group (P<0.01). The positive therapeutic drug 5-ASA (200 mg / kg / day) significantly inhibited the DSS-induced upregulation of IL-4 transcription by 69.15±6.17% (P<0.01).

[0303] Experimental results showed that IL-6 mRNA expression in the colon tissue of mice in the DSS model group increased significantly to 13.53±2.34 times that of the control group (P<0.01). Compared with the control group, there was no significant change in IL-6 mRNA expression in the colon tissue of mice in the ZHT (150mg / kg / day) group. ZHT (75mg / kg / day) inhibited DSS-induced IL-6 transcriptional upregulation by 79.4±6.5%, a highly significant difference compared with the DSS model group (P<0.01). The positive therapeutic drug 5-ASA (200mg / kg / day) inhibited DSS-induced IL-6 transcriptional upregulation by 91.5±6.2% (P<0.01).

[0304] Experimental results showed that TNF-α mRNA expression in the colon tissue of mice in the DSS model group was significantly increased to 7.82±1.68 times that of the control group (P<0.01). ZHT (150mg / kg / day) increased TNF-α mRNA expression in the colon tissue of mice to 3.29±1.09 times that of the control group, but the difference was not statistically significant (P>0.05). ZHT (75mg / kg / day) inhibited DSS-induced TNF-α transcriptional upregulation by 88.5±7.1%. The DSS+ZHT (75mg / kg / day) group showed a significant difference compared with the DSS model group (P<0.01). The positive treatment drug 5-ASA (200mg / kg / day) inhibited DSS-induced TNF-α transcriptional upregulation by 87.4±7.2% (P<0.01).

[0305] Experimental results showed that IFN-γ mRNA expression in the colon tissue of mice in the DSS model group was significantly increased to 5.60±0.50 times that of the control group (P<0.01). Compared with the control group, there was no significant change in IFN-γ mRNA expression in the colon tissue of mice in the ZHT (150mg / kg / day) group. ZHT (75mg / kg / day) inhibited DSS-induced IFN-γ transcriptional upregulation by 74.2±6.0%, with a highly significant difference between the DSS+ZHT (75mg / kg / day) group and the DSS model group (P<0.01). The positive therapeutic drug 5-ASA (200mg / kg / day) inhibited DSS-induced IFN-γ transcriptional upregulation by 83.0±4.2% (P<0.01).

[0306] 4. Experimental Conclusions and Discussion

[0307] In this example, a DSS-induced mouse ulcerative colitis model was used, and 5-aminosalicylic acid was used as a positive control drug to investigate the therapeutic effect of 75 mg / kg / day of total flavonoids from sunflower seed in treating colitis. The results showed that the ZHT (75 mg / kg / day) group significantly protected mice from DSS-induced colitis symptoms such as weight loss, diarrhea, and bloody stools; significantly improved DSS-induced histopathological changes such as colon atrophy, crypt structure damage, and tissue inflammatory infiltration; and reduced DSS-induced increases in tissue MPO levels and inflammatory factor expression, demonstrating a good therapeutic effect on colitis that was comparable to that of the positive therapeutic drug 5-aminosalicylic acid.

[0308] In summary, ZHT has a certain therapeutic effect on colitis. At 75 mg / kg / day, its effect is comparable to that of the positive drug 5-ASA and it has a good safety profile.

[0309] Example 21 Oral Ulcer Medicinal Efficacy Experiment

[0310] 1 Experimental Materials

[0311] 1.1 Animals: SPF male SD rats, weighing 250-300 g, were purchased from Shanghai JST Laboratory Animal Co., Ltd.

[0312] 1.2 Drugs: Guilin Watermelon Frost, Guilin Sanjin Pharmaceutical Co., Ltd.; ethanol extract of hibiscus flower (dry powder, prepared by refluxing and extracting hibiscus flower with 95% ethanol three times, concentrating and drying); water extract of hibiscus flower (thick paste, prepared by refluxing and extracting hibiscus flower water three times, concentrating and drying), total flavonoids extract of hibiscus flower (prepared in Example 19, batch number 19-2).

[0313] 1.3 Reagents: Phenol (analytical grade), Nanjing Chemical Reagent Co., Ltd.; Chloral hydrate (analytical grade), Sinopharm Chemical Reagent Co., Ltd.

[0314] 2 Experimental methods

[0315] 2.1 Modeling: SPF male Sprague-Dawley rats were randomly divided into groups of 9-11 rats each. These groups included a blank (normal) group, a model group, a hibiscus flower ethanol extract group, a hibiscus flower water extract group, a hibiscus flower total flavonoid extract group, and a watermelon frost group. All rats were anesthetized with an intraperitoneal injection of chloral hydrate (400 mg / kg). A glass tube with a 4 mm inner diameter was placed at the bottom of the tube. For the model group and rats in each treatment group, 90% phenol solution was added dropwise to the tube until the cotton ball was just soaked. The cotton ball was then placed flat on the buccal mucosa approximately 1 mm from the left and right corners of the mouth of the rat for 30 seconds. White lesions approximately 4-5 mm in diameter were observed in this area. The blank group was treated with double-distilled water instead of phenol and subjected to the same treatment.

[0316] 2.2 Administration: Each drug was prepared into a suspension in double-distilled water and evenly applied to the oral mucosal wound surface after modeling, four times daily. The dosage of each drug was as follows: watermelon frost, 20 mg / wound surface / day; hibiscus flower ethanol extract, 10 mg / wound surface / day; hibiscus flower aqueous extract, 8.75 mg / wound surface / day; and hibiscus flower total flavonoid extract, 1.05 mg / wound surface / day. Administration was continued for five consecutive days. The ulcer area and ulcer healing were observed before and after administration. On the sixth day, the ulcer surface was photographed after anesthesia with chloral hydrate (400 mg / kg). After sacrifice by cervical dislocation, bilateral buccal tissue was removed from each rat. Seven to nine samples were randomly selected from each group, fixed in 4% formaldehyde (prepared in saline), harvested, dehydrated, and embedded in paraffin according to standard procedures. Sections were stained with hematoxylin and eosin (HE) and examined under a light microscope. The remaining buccal tissue was frozen at -70°C until further evaluation for other inflammatory markers.

[0317] 2.3 Data Statistics: Data are presented as mean ± SD. The significant differences between the model group and the normal group (blank control group) were tested using the Student's t test. The significant differences between the model group and the other treatment groups were analyzed using a one-way ANOVA test, and further analyzed using the Newman-Keuls multiple comparison test.

[0318] 3 Results

[0319] 3.1 Visual observation and evaluation

[0320] The oral mucosa of rats in the blank group had a smooth, uniform color, and no blood spots or stains. After phenol stimulation, the local mucosa rapidly blanched, and edema became visible after 2 hours. Within 1-5 days, inflammatory exudate, bleeding, and localized ulceration were observed, covered by a yellowish-white pseudomembrane that subsequently fell off. On day 6, all rats except the blank group showed concave wounds. 50% of the rats in the model group had significantly dilated blood vessels in their wounds, and one rat had yellow pus spots. The results are shown in Table 21-1 (each rat had two oral wounds).

[0321] Table 21-1 Statistics of the degree of phenol buccal ulcers in rats

[0322] 0: no ulcer, normal oral mucosa;

[0323] Ⅰ: There is ulcer concave, no obvious bleeding or vascular dilation, no obvious swelling, and no obvious pseudomembrane;

[0324] II: There are ulcers and pits, but fewer blood spots;

[0325] III: There are ulcer depressions, obvious blood spots, and slight swelling.

[0326] 3.2 Pathological changes

[0327] The following items were examined under an optical microscope: (1) whether the mucosal epithelial cells showed degeneration, necrosis, erosion, ulceration, or inflammatory cell infiltration; (2) whether the submucosal tissue showed congestion, edema, or inflammatory cell infiltration.

[0328] Scoring criteria for lesion severity: Lesions are scored from mild to severe as 0.5-4 points, with the scoring criteria being: mild or very mild lesions are scored as 0.5 points; mild lesions are scored as 1 point; moderate lesions are scored as 2 points; severe lesions are scored as 3 points; very severe lesions are scored as 4 points; and no lesions are scored as 0 points. The length of the ulcer is measured using a "microscope side micrometer" under a 40x light microscope (eyepiece 4×, objective lens 10×). Ulcers <5mm / 40X ​​are scored as 1 point, >5mm / 40X ​​or 5mm / 40X ​​are scored as 2 points. Add up all the scores and calculate the average lesion score for each group of animals. The higher the score, the more severe the disease.

[0329] The statistical results of pathological changes in each group are shown in Table 21-2, and the pathological images are shown in Figure 4. In Figure 4b, the short black arrows indicate ulcers formed by skin necrosis and shedding (without epidermal coverage), the long black arrows indicate inflammatory cells infiltrating the dermis, the green arrows indicate dilated and congested blood vessels, the green arrows indicate fibroblasts, and the green stars indicate the thin layer of stratified squamous epithelium on the surface of the skin with mild keratinization on the surface.

[0330] Table 21-2 Scoring results of pathological changes in oral mucosa of rats

[0331] Note: ## P<0.01 vs blank group; *P<0.05, **P<0.01 vs model group.

[0332] Example 22 Pharmacodynamic evaluation of total flavonoids from Astragalus membranaceus in treating skin diseases

[0333] Example 22-1 Pharmacodynamic evaluation of total flavonoids from Astragalus membranaceus in the treatment of acne

[0334] 1. Experimental purpose:

[0335] To investigate the therapeutic effect of topical administration of total flavonoids from Hibiscus truncatum (ZHT) on acne induced by 100% oleic acid and Propionibacterium acnes (P. acnes), and to compare the efficacy with that of the positive drug benzoyl peroxide / clindamycin.

[0336] 2. Experimental Materials

[0337] Experimental animals

[0338] SPF-grade SD rats, half male and half female, were housed in separate cages, 6-8 weeks old, weighing 140-150 g, and were provided by the Medical Experimental Animal Center of Nanjing Medical University (Experimental Animal Production License No.: SCXK(Su)2021-0011).

[0339] Reagents and drugs

[0340] Total flavonoids from sunflower seeds (ZHT-1) were prepared according to the method of Example 19 and are also referred to as "total flavonoids extract from sunflower seeds", batch number 19-1. Total flavonoids from sunflower seeds (ZHT-2) were prepared according to the method of Example 6.

[0341] 2.3. Preparation of experimental drugs

[0342] Preparation method of ZHT cream: 1% total flavonoids cream of Astragalus membranaceus is prepared according to the prescription in Table 22-1.

[0343] Table 22-1: ZHT cream preparation prescription.

[0344] Aqueous phase: Weigh the prescribed amount of ethanol, add ZHT, stir to dissolve, add Tween 80, sodium lauryl sulfate, glycerol, isopropyl myristate, methylparaben, azone, and purified water into the beaker in sequence, and stir until uniformly dispersed.

[0345] Oil phase: Weigh the prescribed amount of glyceryl monostearate, palmitic acid, and white petrolatum into a beaker and heat in an 80°C water bath to dissolve until liquid.

[0346] Slowly add the water phase to the oil phase while stirring. After all the water phase is added, remove it from the water bath and stir at room temperature until it cools to form a cream.

[0347] Preparation method of reinforced Clostridial culture medium (RCM): weigh 19.0 g of reinforced Clostridial culture medium powder into a 500 mL conical flask, add 500 mL of distilled water, heat and stir, divide into portions, and sterilize under high pressure at 121°C for 15 minutes before use.

[0348] Culture method of P. acnes: Add 4 mL of RCM culture medium and 1 mL of P. acnes bacterial solution to a sterile test tube and mix well by pipetting. Wrap the sterile container with sealing film and place it vertically in an anaerobic culture bag containing an anaerobic gas-producing bag. Place it in a 37°C electric constant temperature incubator for culture and subculture once every 2 days.

[0349] Preparation of P. acnes injection: Prepare immediately before use. Dilute the bacterial solution to a concentration of 6×10 7 CFU / mL, and the P. acnes injection solution was obtained (the McFarland turbidity of the bacterial solution was about 2McFarland, and after diluting 10 times with normal saline, 6×10 7CFU / mL concentration of bacterial suspension, measure its OD 600nm Between 0.63 and 0.65.)

[0350] 2.3 Construction and treatment of rat acne model

[0351] [Corrected 26.11.2024 according to Rule 91] The methods for establishing a rat acne model and evaluating drug efficacy are shown in Figure 5. The acne model was established using 100% oleic acid combined with P. acnes. ZHT cream was applied on the 8th day for treatment, and 2.5% benzoyl peroxide / 1% clindamycin (BPO) hydrogel was used as a positive control.

[0352] Rats, half male and half female, with six rats per group, were housed in separate cages. After one week of adaptive feeding, the rats other than the control group were treated with 100% oleic acid combined with P. acnes to induce an acne model. The right auricle was used as a carrier for the acne model, while the left ear remained untreated. 100% oleic acid (0.5 mL) was applied to the inner side of the right ear of the rats at 9:00 AM daily. The control group received an equal amount of saline. Every other day, 1 hour after the oleic acid application, the prepared P. acnes injection solution (50 μL) was injected into the inner side of the right ear of the rats. The control group received saline.

[0353] On the eighth day of modeling, the rats were regrouped to ensure that the severity of the model was essentially the same within each group. The groups were divided into a blank control group (Veh), a model group (Model), a model + benzoyl peroxide / clindamycin group (Model+BPO), a model + 1% ZHT-1 group (Model+1% ZHT-1), and a model + 1% ZHT-2 group (Model+1% ZHT-2). Each group contained three male and female rats in one cage. Starting in the afternoon of the eighth day after grouping, 50 mg of the test drug was applied to the ears daily.

[0354] 2.4 Auricular swelling measurement and symptom scoring

[0355] The thickness of the rats' right auricle was measured with a vernier caliper before modeling on the morning of the 0th, 3rd, 6th, 9th, 12th, 15th, and 18th day. Three different measurement sites were selected for each rat, and the average value was taken to ensure the accuracy and objectivity of the measurement. A curve of the change of the rats' right auricle thickness over time was drawn.

[0356] On the mornings of the 1st, 3rd, 7th, 11th, 15th, and 17th days, before modeling, the right auricles of the rats were photographed and symptom scores were scored. Disease severity was assessed based on four indicators: redness, swelling, scaling, papules, and cysts. Each indicator was scored on a scale of 0 to 4, and the total score was calculated by summing the scores of the four indicators. The scoring criteria were: 0, no symptoms; 1, mild; 2, moderate; 3, severe; and 4, extremely severe.

[0357] 2.5 Specimen Collection

[0358] Rats were killed on the 19th day, and their right auricles were completely removed. One-quarter of the ear tissue was fixed in 4% paraformaldehyde overnight for paraffin section preparation and pathological examination. The remaining ear tissue was quickly frozen in liquid nitrogen and stored at -80°C for subsequent ELISA and qPCR detection.

[0359] 2.6 H&E staining and pathological examination

[0360] The rat ear tissue was fixed with 4% paraformaldehyde for 24 hours, embedded in paraffin, and then sectioned. H&E staining was performed, and the pathological changes of the rat acne model were evaluated according to the histological criteria.

[0361] The specific scoring criteria are shown in Table 22-2.

[0362] Table 22-2 Acne pathological histological scoring standard

[0363] 2.8 Determination of IL-1β, IL-6, TNF-α, and MCP-1 Protein Content in Rat Ear Tissue

[0364] According to the operating procedures recommended by the ELISA kit manufacturer, the levels of IL-1β, IL-6, TNF-α and MCP-1 in various ear tissues were detected.

[0365] 3. Experimental Results

[0366] 3.1 Effects of ZHT on the appearance and thickness of the auricle in rats with acne

[0367] During the entire experiment, the inner surface of the right auricle of the rats in the Veh group remained smooth and flat, without symptoms such as redness, swelling, desquamation, papules, and cysts, and the thickness of the right auricle remained basically unchanged.

[0368] The right auricle of the rats in the Model group showed obvious symptoms of redness, swelling and desquamation on the 7th day after modeling, and the symptoms of redness, swelling and desquamation continued to worsen as the modeling progressed. In the later stage, the surface of the right auricle became uneven and accompanied by cysts. The thickness of the right auricle of the rats increased significantly after modeling. Starting from the 6th day, it was significantly higher than that of the Veh group, and the difference was statistically significant (P<0.01) (Table 22-3). The skin lesion symptom score showed that the degree of skin lesions gradually worsened after modeling, and starting from the 3rd day, it was significantly different from that of the Veh group (P<0.01) (Table 22-4).

[0369] The application of BPO hydrogel improved the redness, swelling, desquamation and cysts caused by modeling. The surface of the right auricle of rats in the Model+BPO group was also smoother and flatter. After treatment with BPO hydrogel, the thickening of the ear caused by modeling was gradually delayed. On the 18th day, the thickness of the right auricle of rats in the Model+BPO group was significantly reduced compared with that in the Model group (P<0.01) (Table 22-3). The skin lesion symptom score showed that the application of BPO hydrogel had a certain therapeutic effect on the acne-like symptoms caused by modeling. Starting from the 11th day, there was a significant difference compared with the Model group (P<0.01) (Table 22-4).

[0370] Application of 1% ZHT-1 and 1% ZHT-2 creams significantly reduced modeling-induced ear thickening on day 12, with significant differences compared to the Model group (P < 0.01) (Table 22-3). Application of 1% ZHT-2 improved modeling-induced desquamation on day 15, with a significant decrease in skin lesion symptom scores compared to the Model group (P < 0.01), but failed to further improve modeling-induced redness, swelling, and papules. Therefore, there were no significant differences in skin lesion symptom scores compared to the Model group at other time points (P > 0.05). Application of 1.0% ZHT-1 alleviated modeling-induced acne-like symptoms such as redness, swelling, desquamation, and papules. On days 11, 15, and 17, the Model + 1.0% ZHT group had significantly lower symptom scores compared to the Model group (P < 0.01) (Table 22-4).

[0371] Table 22-3 Changes in thickness of right auricle of rats in each group (unit: mm)

[0372] Data are expressed as mean ± standard error. N = 6. **, P < 0.01, vs. Veh; ## ,P<0.01,vs.Model, analyzed using two-way ANOVA and Dunnett's multiple comparison test.

[0373] Table 22-4 Symptom scores of right auricle skin lesions in rats in each group

[0374] 3.2 Effects of ZHT on the pathological morphology of the ears of rats with acne

[0375] The ear tissues of rats in the Veh group showed no obvious abnormalities. The thickness of the epidermis was measured to be 12.56±2.39μm. The boundaries between the hair follicles, epidermis, and dermis were clearly visible, and inflammatory cell infiltration was occasionally observed in the dermis.

[0376] The thickness of the epidermis of the ear tissue of rats in the Model group was 171.73±17.18μm, which was significantly increased compared with that in the Veh group (P<0.01). The epidermis and stratum corneum were excessively thickened, and inflammatory cells were seen in the stratum corneum. The hair follicles were dilated, and the follicular openings and infundibulum were filled with keratinized substances. The keratin plugs were severely blocked and enlarged into a pot shape. The sebaceous glands were atrophied, and the dermis was edematous with a large amount of inflammatory infiltration. The pathological score was 3.83±0.17 points, which was significantly different from that in the Veh group (P<0.01).

[0377] The epidermal thickness of the ear tissue of rats in the Model+BPO group was 104.90±7.35μm, which was significantly lower than that in the Model group (P<0.01); the stratum corneum was slightly thickened, the keratinized material in the hair follicle orifice and infundibulum was less, the sebaceous gland morphology was normal, the dermis was not edematous, and the degree of inflammatory cell infiltration was reduced compared with that in the Model group. The pathological score was 2.67±0.33 points, which was significantly lower than that in the Model group (P<0.01).

[0378] The epidermal thickness of rat ear tissue in the Model+1% ZHT-2 group and the Model+1.0% ZHT-1 group were 133.36±13.17μm and 128.08±8.83μm, respectively. 1.0% ZHT-1 could significantly reduce the epidermal thickening caused by modeling (P<0.05).

[0379] Applying 1% ZHT-2 and 1.0% ZHT-1 can improve the excessive thickening of the epidermis and stratum corneum, reduce edema of the dermis and reduce inflammatory cell infiltration in a dose-dependent manner. The pathological scores of each group were 3.33±0.33 points and 3.33±0.33 points, respectively.

[0380] Effects of ZHT on the mRNA expression levels of Mcp-1, Il-1β, Il-6, and Tnf-α in ear tissues of acne rats

[0381] The mRNA expression of Mcp-1 in the ear tissue of rats in the Model group increased to 3.11±0.30 times that of the Veh group (P<0.01); compared with the Model group, the application of BPO hydrogel could significantly improve the model-induced upregulation of Mcp-1 mRNA expression, with an improvement rate of 70.9±14.9% (P<0.01 vs. Model); the application of 1% ZHT-1 cream could improve the model-induced upregulation of Mcp-1 mRNA expression, with an improvement rate of 25.5±16.0%, but the difference was not statistically significant (P>0.05 vs. Model); and the application of 1% ZHT-2 cream had an inhibitory effect on the model-induced upregulation of Mcp-1 mRNA expression.

[0382] The mRNA expression of Il-1β in the ear tissue of rats in the Model group increased to 14.23±2.24 times that of the Veh group, and the difference was statistically significant (P<0.01); compared with the Model group, the application of BPO hydrogel could improve the modeling-induced upregulation of Il-1β mRNA expression, with an improvement rate of 76.6±6.9% (P<0.05vs.Model); the application of 1% ZHT-2 and 1% ZHT-1 creams could improve the modeling-induced upregulation of Il-1β mRNA expression, with improvement rates of 18.5±15.3% (P>0.05vs.Model) and 58.4±6.5% (P<0.01vs.Model), respectively.

[0383] The mRNA expression of Il-6 in the ear tissue of rats in the Model group was significantly increased to 4.62±0.50 times that of the Veh group, and the difference was statistically significant (P<0.01); the application of BPO hydrogel, 1% ZHT-2, and 1% ZHT-1 cream reduced the modeling-induced upregulation of Il-6 mRNA expression, with improvement rates of 60.0±13.5% (P<0.05vs.Model), 46.2±5.4% (P<0.05vs.Model), and 61.1±20.3% (P<0.05vs.Model), respectively.

[0384] The mRNA expression of Tnf-α in the ear tissue of rats in the Model group was significantly increased to 3.98±0.52 times that of the Veh group (P<0.01); the application of BPO hydrogel could improve the modeling-induced upregulation of Tnf-α mRNA expression, with an improvement rate of 71.2±10.0% (P<0.05 vs. Model); the application of 1% ZHT-2 and 1% ZHT-1 creams could inhibit the modeling-induced upregulation of Tnf-α mRNA expression, with improvement rates of 18.2±12.9% (P>0.05 vs. Model) and 58.3±15.0% (P<0.05 vs. Model), respectively.

[0385] Effects of ZHT on the levels of IL-1β, IL-6, TNF-α, and MCP-1 in ear tissues of rats with acne

[0386] The IL-1β protein content in the ear tissue of the Veh group was 175.2±29.5 pg / mL, and the IL-1β protein content in the ear tissue of the rats in the Model group was 270.1±18.9 pg / mL, which was 1.54±0.11 times that of the Veh group (P<0.05); after administration of BPO, 1% ZHT-2 and 1% ZHT-1, the IL-1β protein content in the ears of rats was 224.0±20.0 pg / mL, 194.4±20.5 pg / mL and 179.7±25.8 pg / mL, which improved the increase of IL-1β protein content induced by modeling by 48.5±21.1% (P>0.05 vs. Model), 79.8±21.6% (P<0.05) vs. Model and 95.2±27.2% (P<0.05 vs. Model), respectively.

[0387] The IL-6 protein content in the ear tissue of the Veh group was 93.7±20.4 pg / mL, and the IL-6 protein content in the ear tissue of rats in the Model group was 266.4±15.2 pg / mL, which was 2.84±0.16 times higher than that of the Veh group, and the difference was extremely significant (P<0.01); after administration of BPO hydrogel, the IL-6 protein content in the rat ear tissue was 189.0±10.8 pg / mL, which inhibited the increase of IL-6 protein content in the rat ear tissue induced by modeling by 44.8±6.3% (P<0.01 vs. Model). The protein contents of IL-6 in the ear tissue of rats after administration of 1% ZHT-2 and 1% ZHT-1 were 237.2±21.4 pg / mL and 235.2±25.6 pg / mL, respectively. Each group improved the increase of IL-6 protein content in the ear tissue of rats induced by modeling by 16.9±12.4% (P>0.05vs.Model) and 18.1±14.8% (P>0.05vs.Model), respectively.

[0388] The TNF-α protein content in the ear tissue of the Veh group was 103.7±23.7 pg / mL, and the TNF-α protein content in the ear tissue of the rats in the Model group was 423.2±37.0 pg / mL, which was 4.08±0.58 times higher than that of the Veh group, and the difference was extremely significant (P<0.01). The protein contents of TNF-α in the ear tissue of rats after ZHT-1 were 286.2±46.1pg / mL, 224.9±49.1pg / mL, and 221.5±60.3pg / mL, respectively. Compared with the Model group, the increase of TNF-α protein content in the ear tissue of rats induced by modeling was improved by 62.1±15.4% (P<0.01vs.Model), 42.9±14.4% (P<0.05vs.Model), and 63.1±18.9% (P<0.01vs.Model), respectively.

[0389] Example 22-2 Pharmacodynamic evaluation of total flavonoids from Astragalus membranaceus in the treatment of atopic dermatitis

[0390] 1. Research content and objectives:

[0391] The therapeutic effects of different doses of total flavonoids from Sunflower Hibiscus (ZHT-1) and Sunflower Hibiscus HKY on DNFB-induced atopic dermatitis (AD) were investigated and compared with the positive drug mometasone (Mome) to determine the therapeutic effects of ZHT and HKY on AD.

[0392] 2. Experimental Materials

[0393] 2.1 Experimental animals

[0394] SPF Balb / c mice, 7–8 weeks old and weighing 18–20 g, were purchased from the Center for Comparative Medicine, Yangzhou University [Laboratory Animal Production License No. SCXK(Su)2017-0007]. They were housed at 23 ± 2°C, 55% humidity, and a 1:1 light-to-dark ratio with free access to food and water.

[0395] 2.2 Drugs and main reagents

[0396] 2.3 Experimental instruments

[0397] 3. Experimental Methods

[0398] 3.1 Animal grouping and atopic dermatitis model construction

[0399] After 5 days of adaptive feeding, 80 clean-grade Balb / c mice were randomly divided into 10 groups: a blank control group (Sham), a DNFB modeling group (DNFB), a positive drug mometasone furoate group (Mome), a 0.5% sunflower flavonoids (ZHT) cream group, a 0.5% sunflower flavonoids HKY (HKY) cream group, and a group receiving 100 mg / kg SZHT by oral gavage. Hair was removed with rosin wax wax. On the morning of day 1, each mouse was treated with 50 μL of 1.0% 2,4-dinitrofluorobenzene (DNFB) (in an acetone:olive oil mixture (acetone:olive oil = 3:1)) at the back. On the mornings of days 4, 6, 8, and 10, 50 μL of 0.5% (W / V) DNFB was applied. The control group was treated with an equal amount of solvent control (acetone:olive oil = 3:1). Animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and in compliance with animal ethics standards. The atopic dermatitis model is shown in Figure 6.

[0400] 3.2 Administration and Dosage

[0401] Starting on day 5, 100 mg of different doses of ZHT cream and HKY cream were accurately weighed each afternoon using an electronic analytical balance and evenly applied to the dorsal modeling area. The control group received 100 mg of blank cream, while the 100 mg / kg SZHT group was administered via gavage. The specific modeling and dosing procedures are shown in Figure 6.

[0402] 3.2.1 Preparation of ZHT Cream and HKY Cream

[0403] The prescriptions of ZHT cream and HKY cream are shown in Table 22-3-1.

[0404] Table 22-3-1 Total flavonoids cream of sunflower and sunflower HKY prescription

[0405] Cream preparation process:

[0406] Aqueous phase: Weigh the prescribed amount of ethanol, add ZHT / HKY, stir to dissolve, add Tween 80, sodium lauryl sulfate, glycerol, isopropyl myristate, methylparaben, azone, and purified water into the beaker in sequence, and stir until uniformly dispersed.

[0407] Oil phase: Weigh the prescribed amount of glyceryl monostearate, palmitic acid, and white petrolatum into a beaker and heat in an 80°C water bath to dissolve until liquid.

[0408] Slowly add the water phase to the oil phase while stirring. After all the water phase is added, remove it from the water bath and stir at room temperature until it cools to form a cream.

[0409] 3.2.2 Preparation of SZHT suspension

[0410] Preparation of 0.5% CMC-Na: One day before dosing, slowly add 0.5g of CMC-Na powder to 100mL of 70°C pure water, stir thoroughly, and let stand at room temperature overnight. The next day, accurately weigh 50mg of ZHT and add 0.1mL of DMSO to prepare a DMSO stock solution. To this ZHT stock solution, add 0.1mL of Tween-80 and 9.8mL of a 0.5% CMC-Na solution, which has been sonicated to a homogeneous consistency, to obtain a 5mg / mL ZHT suspension.

[0411] 3.3 Atopic Dermatitis Severity Score (AD Score)

[0412] On days 4, 7, 9, and 11 of modeling, the dorsal skin of each mouse was photographed and the severity of dermatitis was assessed using four indicators: ① erythema / hemorrhage ② dryness ③ exudation / scabbing ④ edema. Each indicator was scored on a scale of 0 to 3, and the sum of the scores for the four indicators was used to obtain a total score. The AD Score is as follows: 0, no symptoms; 1, mild symptoms; 2, moderate symptoms; 3, severe symptoms.

[0413] 3.5 HE staining

[0414] Paraffin sections were routinely dewaxed to water, stained in hematoxylin for 5 minutes, and then rinsed with tap water. Sections were then placed in hydrochloric acid-ethanol for 30 seconds, soaked in tap water for 15 minutes, and then stained in eosin for 2 minutes. Sections were routinely dehydrated, transparentized, and mounted with neutral resin. Images were taken using a digital pathology scanner, and epidermal thickness was calculated.

[0415] 3.10 Statistical Analysis

[0416] GraphPad Prism 6.0 software was used for statistical analysis and graphing. All data are presented as mean ± standard error (SEM). Dermatitis scores were analyzed using two-way ANOVA, and other data were analyzed using one-way ANOVA. Dunnett's multiple-test was used to analyze the significance of differences between groups. When P < 0.05, the differences between groups were considered statistically significant.

[0417] 4. Experimental Results

[0418] 4.1 Effects of ZHT and HKY on the appearance of DNFB-induced atopic dermatitis model

[0419] As shown in Figure 7, during the entire experiment, the back skin of the blank control group (Sham) was always smooth and flat, without symptoms such as exudation, scabs, erythema and bleeding. After repeated stimulation of DNFB, starting from the 4th day, the scabs, erythema and edema of the mice in the DNFB group were obvious, and their dermatitis score (AD Score) was significantly different from that of the Sham group (P < 0.01, N = 8), indicating that the AD model was successfully established; the positive drug mometasone furoate (Mome) can gradually improve the AD-like symptoms caused by DNFB, and there is a significant difference between the Mome group and the DNFB group in the dermatitis score on the 11th day (P < 0.05, N = 8); applying 0.5% ZHT can significantly alleviate AD-like symptoms, and the difference between the 0.5% ZHT group and the DNFB group in the dermatitis score on the 11th day is extremely significant (P < 0.01, N = 8); oral administration of 100 mg / kg SZHT did not show any effect on alleviating AD-like symptoms (P>0.05, N=8); 0.5% HKY could alleviate AD-like symptoms to a certain extent, but there was no significant difference in dermatitis scores between the 0.5% HKY group and the DNFB group (P>0.05, N=8).

[0420] 4.3 Effects of ZHT and HKY on skin pathological changes in DNFB-induced atopic dermatitis model.

[0421] The results of HE staining of skin tissue sections of each group are shown in Figure 8. There was no obvious change in the back skin tissue of the Sham group. The structure of each layer of the skin was intact, the boundary between the dermis and the epidermis was clear, there was no obvious inflammatory cell infiltration, and the epidermal thickness (shown by the blue line) was 37.18±8.93μm. The skin tissue of the DNFB group showed obvious hyperkeratosis and acanthosis, and the epidermal thickness was 134.28±22.46μm, which was significantly increased compared with the Sham group (P<0.01, N=6), which was consistent with the pathological characteristics of atopic dermatitis skin tissue. After applying Mome treatment, it was significantly observed that the degree of epidermal thickening was reduced, and the epidermal thickness was 46.09±14.29μm (P<0.01, N=6, compared with the DNFB group). The skin acanthosis of the 0.5% ZHT group was improved, and the epidermal thickness was 100.59±10.95μm, which was significantly increased compared with the DNFB group. ZHT reduced the thickening by 34.7±5.0%, which was a significant difference (P<0.05, N=6), which was consistent with the dermatitis score results; however, 0.5% HKY had no significant improvement effect on DNFB-induced AD-like pathological changes, and its epidermal thickness was not significantly different from that of the DNFB group (P>0.05, N=6).

[0422] Example 23 Pharmacodynamic evaluation of total flavonoids from Astragalus membranaceus in promoting wound healing

[0423] 1. Experimental Materials

[0424] 1.1 Experimental animals

[0425] SPF SD rats, 6-8 weeks old, weighing 150-200 g.

[0426] 1.2 Reagents and drugs

[0427] Table 23-1

[0428] 2. Experimental Methods

[0429] 2.1 Animal husbandry and management

[0430] SD rats (purchased from Nanjing Medical University, license number: SCXK (Su) 2021-0001) were kept in an SPF-grade animal room throughout the whole process, with a light-to-dark time ratio of 1:1, room temperature controlled at 23 ± 2 ° C, humidity controlled at 55%, and rats had free access to food and water. The bedding was changed every three days during the high-sugar and high-fat diet feeding stage. During the STZ injection stage of establishing the diabetic model, the bedding was changed once a day. After the full-thickness skin resection model was established, the bedding was changed every half a day to ensure that the rats were in a dry and clean environment. All animal experimental protocols were approved by the Experimental Animal Care and Use Committee of China Pharmaceutical University (License No.: SYXK (Su) 2018-0019).

[0431] 2.2 Administration and Dosage

[0432] 100 mg of blank cream, 0.5%, 1%, and 2% total flavonoids cream from Astragalus membranaceus, and Deshijie hydrocolloid were weighed on an electronic analytical balance and evenly applied to the ulcer wound surface twice daily. The groups and the medications applied to each group are as follows:

[0433] Blank control group (Control): no treatment

[0434] Model group: applied a blank cream without total flavonoids from Astragalus membranaceus;

[0435] Positive group: Apply Dewet-Clean hydrocolloid;

[0436] Low-dose group (Low-0.5%): 0.5% total flavonoids from Astragalus membranaceus cream was applied;

[0437] Middle-1% group: Apply 1% total flavonoids from Astragalus membranaceus cream;

[0438] High-2% group: applied 2% total flavonoids from Astragalus membranaceus cream;

[0439] 2.3 Reagent preparation

[0440] STZ solution preparation: ① To prepare the citric acid buffer solution, add 2.1g of citric acid to 100mL of distilled water to make Solution A. Add 2.94g of sodium citrate to 100mL of distilled water to make Solution B. ② Before use, mix Solution A and Solution B in the appropriate proportions and adjust the pH to 4.2-4.5. This will create the STZ citric acid buffer solution. ③ Before injection, dissolve the STZ in the citric acid buffer solution at a concentration of 1%. Store in a dark place at 4°C.

[0441] To prepare 4% chloral hydrate: Weigh 4g of chloral hydrate and dissolve it in saline. Dilute to 100mL. Refrigerate at 4°C in a dark place until ready to use.

[0442] Preparation of different concentrations of total flavonoids from Astragalus membranaceus cream: 0.5%, 1% and 2% total flavonoids from Astragalus membranaceus cream were prepared according to the prescription in Table 22-1 of Example 22, and the different concentrations were made up with water.

[0443] 2.4 Establishment of diabetic ulcer rat model

[0444] 2.4.1 STZ-induced diabetic rat model

[0445] After one week of adaptive feeding, 70 healthy male Sprague-Dawley rats (SPF, 4-6 weeks old, weighing 150-200g) were randomly divided into cages, 5 rats per cage, and fed a high-sugar, high-fat diet to serve as the diabetes model group. Ten normal rats, 5 rats per cage, were fed a standard diet to serve as the blank control group. Fasting blood glucose (FBG) and body weight were measured in all 60 Sprague-Dawley rats. After four weeks of feeding, the diabetic model group rats received a single intraperitoneal injection of streptozotocin (35 mg / kg) based on body weight, while the blank control group received a single intraperitoneal injection of an equal volume of citrate buffer based on body weight. STZ solution and citrate buffer were injected into the left lower abdomen based on fasting body weight within 10 minutes. All rats were deprived of food and water for 6 hours before administration. Blood was collected from the tail vein 72 hours after injection for fasting blood glucose measurement. Successful diabetic models were considered established in rats with a fasting blood glucose value ≥16.7 mmol / L and no significant weight loss. When the blood glucose level of some rats was between 5-8mmol / L, STZ (35mg / kg) was injected again; when the blood glucose level of some rats was between 8-10mmol / L, STZ (25mg / kg) was injected again; when the blood glucose level of some rats was between 10-16mmol / L, STZ (20mg / kg) was injected. 72 hours after the injection, blood was collected from the tail vein to measure fasting blood glucose. If the fasting blood glucose value was ≥16.7mmol / L, the diabetic model was considered to be successfully established. If the fasting blood glucose value was <16.7mmol / L, STZ was injected again according to body weight until the fasting blood glucose value was ≥16.7mmol / L, which was considered to be a successful diabetic model. It took 12 days to establish the diabetic animal model of SD rats by injecting STZ in this experiment. After all the rats were established, they were fed with a high-sugar and high-fat diet for one week to stabilize the model.

[0446] 2.4.2 Establishment of a full-thickness skin excision wound model in diabetic rats

[0447] After the diabetic rats were stabilized into the model, they were anesthetized by intraperitoneal injection of 4% chloral hydrate (2 mg / kg) according to their body weight. After the rats were fully anesthetized, the rats' backs were shaved with an electric shaver. After shaving, a medical cotton swab dipped in iodine was used to continuously apply the rats' backs 4-5 times for disinfection. A sterile 8-mm Biopsy Punch was used to print an 8 mm diameter circular area in the middle of the rat's back. The skin in the middle of the circular area was pinched with sterile tweezers, and the skin was excised along the circular mark outlined by the Biopsy Punch with sterile iris scissors. A circular full-thickness skin excision wound with a diameter of 8 mm was created, deep into the muscle, avoiding the arteries and veins. The wound was then immediately applied with iodine 4-5 times, and appropriate pressure was applied to fully stop bleeding. Rats in the blank control group were only injected with an equal dose of chloral hydrate according to their body weight and shaved on the back, without creating a full-thickness skin excision wound. Rats that died during modeling were excluded, and the diabetic rats that were successfully modeled, awake, and in good condition were randomly divided into the following groups according to the random number table method: model group (application of blank cream), positive drug group (application of Deshijie hydrocolloid), low-dose group (application of 0.5% total flavonoids cream of Asparagus corylifolia), medium-dose group (application of 1% total flavonoids cream of Asparagus corylifolia), and high-dose group (application of 2% total flavonoids cream of Asparagus corylifolia). After the full-thickness skin excision wound model was established, drug treatment was immediately given, and photos were taken and recorded on day 0 (before application and drug administration), day 3, day 7, and day 9 after treatment. The wound area was measured using ImageJ software.

[0448] Overall evaluation of type 2.5 diabetic ulcer model

[0449] 1. Observe general symptoms, i.e., whether there are changes in polydipsia, polyphagia, polyuria, poor mental state, dull and yellow fur, hair loss, or a significant decrease in activity;

[0450] 2. Fasting blood glucose measured by tail vein blood sampling is 16.7-33.3mmol / L;

[0451] 3. Typical appearance of full-thickness skin defect: ulcer, redness and swelling. The ulcer area is used as the indicator for statistical calculation. The calculation formula is as follows: wound area (%) = unhealed wound area / original wound area × 100%.

[0452] 2.6 HE staining

[0453] The skin tissue was attached to a filter paper sheet and fixed vertically in 4% paraformaldehyde solution for 48 hours before conventional paraffin embedding. Serial full-thickness sections (5 μm) were performed on the embedded skin tissue, with two sections placed on each slide. The sections were then stained with HE using the following steps:

[0454] 1) Dewaxing: Dewaxing with xylene I for 10 min and dewaxing with xylene II for 5 min;

[0455] 2) Ethanol soaking: Soak in anhydrous ethanol twice, then soak in 95%, 90%, and 85% ethanol once, each soaking for 1 minute, and finally wash with water for 2 minutes;

[0456] 3) Staining: Stain with hematoxylin for 5 minutes, then wash with tap water for 1 minute;

[0457] 4) Differentiation: Differentiate with 1% hydrochloric acid alcohol for 20 seconds, then wash with tap water for 1 minute;

[0458] 5) Anti-blueing: Anti-blue with 1% dilute ammonia for 30 seconds, then wash with tap water for 1 minute;

[0459] 6) Eosin staining: stain for 5 minutes, then wash with tap water for 30 seconds;

[0460] 7) Ethanol dehydration: Dehydration was performed with different ethanol contents in the following order: 85% ethanol dehydration for 20 seconds, 90% ethanol dehydration for 30 seconds, 95% ethanol I dehydration for 1 minute, 95% ethanol II dehydration for 1 minute, anhydrous ethanol I dehydration for 2 minutes, and anhydrous ethanol II dehydration for 2 minutes;

[0461] 8) Transparency: xylene I treatment for 2 min, xylene II treatment for 2 min, xylene III treatment for 2 min;

[0462] 9) Finally, the slides were sealed with neutral gum and scanned using a digital pathology slide scanner (NanoZoomer 2.0RS).

[0463] 2.7 Masson staining

[0464] Paraffin sections were dewaxed to water, stained with Weigert's hematoxylin solution for 10 minutes, differentiated with 1% hydrochloric acid-ethanol for 1 minute, rinsed with distilled water for 25 minutes, stained with Ponceau acid fuchsin solution for 10 minutes, differentiated with 1% phosphomolybdic acid solution for 5 minutes, stained with 2% brilliant green solution for 5 minutes, and rinsed with 1% glacial acetic acid solution for 1 minute. Subsequently, sections were dehydrated with 95% ethanol and anhydrous ethanol, cleared in xylene, and mounted with neutral gum. Sections were scanned and imaged using a digital pathology slide scanner (NanoZoomer 2.0RS).

[0465] 2.8 Immunofluorescence of the vascular marker CD31

[0466] Skin tissue was fixed overnight with 4% paraformaldehyde and embedded in paraffin to prepare paraffin sections. Paraffin sections were placed in a 60°C oven for 20 minutes. The sections were then immersed in xylene I for 15 minutes, xylene II for 15 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 95% ethanol for 5 minutes, 85% ethanol for 5 minutes, and 75% ethanol for 5 minutes. The sections were then rinsed in distilled water and PBS for 5 minutes each. The sections were placed in a staining jar filled with antigen retrieval buffer and heat-retrieved in an autoclave. The autoclave was rinsed with running water. After cooling to room temperature, the excess liquid was removed and 5% BSA was added dropwise for blocking at room temperature for 1 hour. The blocking solution was then removed and the diluted primary antibody (1:400) was added dropwise. The sections were then incubated in a humidified chamber at 4°C overnight. The next day, the primary antibody was removed and the sections were rinsed three times with PBS for 5 minutes each. A diluted fluorescent secondary antibody (1:1000) was then added dropwise. After incubation at room temperature in the dark for 1 hour, the secondary antibody was removed and the sections were rinsed three times with PBS for 5 minutes each. After drying the sections, a Hoechst-containing anti-fluorescence quencher was added dropwise. The sections were observed and photographed under a fluorescence microscope.

[0467] 2.9 Real-time fluorescence quantitative PCR

[0468] Place skin tissue in a grinding tube, add grinding beads and 800 μL of Trizol reagent, and grind in a tissue grinder at 60 Hz for 1 minute. Repeat five times until no precipitate is visible. Collect the supernatant, centrifuge at 12,000 rpm for 10 minutes, and transfer to a fresh 1.5 mL tube without enzyme. Extract with chloroform-isopropanol overnight, centrifuge, discard the supernatant, and wash with 75% ethanol to remove ions attached to the RNA. After drying, dissolve in DEPC-treated water. Aspirate 2 μL for quantification using a Nano-100 micro-spectrophotometer. The A260 / A280 ratio should be between 1.8 and 2.0. Adjust the RNA concentration to 1 μg using enzyme-free water. Add 3 μL of 5×gDNA wiper mix and heat at 42°C for 2 min to remove genomic DNA. Then, add 5 μL of 4×qRT Super Mix II to achieve a 20 μL system. Perform reverse transcription in a PCR instrument at 25°C for 2 min, 50°C for 30 min, and 85°C for 5 min. Store cDNA at -80°C until use.

[0469] According to 1μL cDNA + 10μL Green+0.8μL Primers+8.2μL ddH2O system was used for real-time fluorescence quantitative PCR reaction. -ΔCt Methods: mRNA expression was calculated, and the expression level of each gene was corrected by the expression level of GAPDH. The primers used are shown in Table 23-2.

[0470] Table 23-2 Primer sequences used in PCR

[0471] 2.10 Determination of vascular endothelial growth factor (VEGF) content

[0472] According to the operating procedures of the ELISA kit, the content of VEGF in skin tissue was detected.

[0473] 2.11 Statistical Analysis

[0474] Statistical analysis was performed using GraphPad Prism (Ver 6.0). All data were expressed as mean ± standard error (SEM). Two-way ANOVA was used to analyze the wound healing rate data, and one-way ANOVA was used to analyze the other data. Dunnett's multiple test was used to analyze the significance of the differences between the groups. When P < 0.05, the differences between the groups were statistically significant.

[0475] 3. Experimental Results

[0476] 3.1 Effects of different concentrations of total flavonoids cream from Astragalus membranaceus on the wound area of ​​rat skin

[0477] In order to investigate the effect of different concentrations of total flavonoids cream from Astragalus membranaceus on promoting wound healing, a full-thickness skin wound model was constructed in diabetic rats. The wound healing status of rats in each group was observed. Photos were taken before application of medication on days 0, 3, 7, and 9, and the wound changes were recorded. The wound area was calculated and statistical analysis was performed.

[0478] Figure 9A is a representative image of the skin wound area of ​​rats in each group at different time points. Before administration (24 hours after injury), fibrin clots ("scabs") appeared at the skin wounds of each group, and a small amount of exudate was present, indicating that there was an inflammatory reaction at the wounds at this time. On the third day after administration, the scabs of the wounds in each group began to harden and the scabs became dry. The dry scabs can play an antibacterial role, thereby playing a certain protective role on the wounds and providing a temporary extracellular matrix for the proliferation and migration of keratinocytes, promoting The wounds in the model group were 99.48±8.76% of the original wound area. Compared with the drug-treated and model groups, the wounds in the Deshijie hydrocolloid group began to shrink, with the wound area being 64.52±7.04% of the initial wound area. The wound areas in the 0.5%, 1%, and 2% sunflower flavonoids cream groups were 106.80±9.37%, 91.41±5.51%, and 98.38±8.74% of the initial wound area, respectively (Figure 9B). On day 7 after administration, the hard crusts in each group began to fall off. The wound area in the model group was 47.62±10.68% of the initial wound area, and the wound areas in the 0.5%, 1%, and 2% sunflower flavonoids cream groups were 49.65±8.79%, 27.38±6.61%, and 36.54±5.72% of their initial wound areas, respectively. The 1% cream group showed the most significant improvement in wound healing, while the wound area in the Deshijie hydrocolloid group was 17.86±4.68% of the initial wound area, demonstrating superior wound healing compared to the 1% sunflower flavonoids cream group. Nine days after administration, the wound area in the model group was 12.05±4.03% of the initial wound area; the wound areas in the 0.5%, 1%, and 2% sunflower flavonoids cream groups were 15.98±5.63%, 10.04±5.64%, and 10.60±3.14% of the initial wound area, respectively; and the wound area in the Deshijie hydrocolloid group was 8.03±4.70% of the initial wound area. Overall, wound healing was evident in the positive drug Deshijie hydrocolloid group, with significant differences compared to the model group at both days 3 and 7. The 1% sunflower flavonoids cream group demonstrated near-complete healing.

[0479] 3.2 Analysis of HE staining results

[0480] To further evaluate the effect of different concentrations of total flavonoids cream from Asparagus cochinchinensis on promoting diabetic wound healing, we performed HE staining on rat skin tissue 9 days after modeling to observe changes at the microscopic level of the tissue. HE staining is one of the commonly used paraffin section staining methods. It uses dyes such as hematoxylin and eosin for staining, so it is also called hematoxylin-eosin staining. Hematoxylin stain is a basic dye that stains cell nuclei purple-blue, while eosin stains the components of the extracellular matrix and cytoplasm pink. Other structures show different tones and combinations of these colors. In addition, the overall color pattern of the stained tissue shows the overall layout and distribution of cells and provides an overall overview of the tissue sample structure, which can be used to observe the number and distribution of fibroblasts, capillaries, inflammatory cells, and other conditions during wound healing.

[0481] As shown in Figure 10A, microscopic examination of tissue sections revealed that the skin tissue of rats in the normal group showed an intact keratinocyte layer, skin appendages, and collagen fibers arranged in parallel bundles, with a small number of fibroblasts and inflammatory cells. In contrast, the skin tissue of rats in the model group was loosely arranged, with keratinocytes beginning to proliferate but not yet migrating to the wound center. The overall skin structure was disordered, the new epidermis was thin, and the new granulation tissue was sparse. Inflammatory cells and fibroblasts migrated to the wound, with a relatively large number of inflammatory cells, indicating that the rats in the model group were primarily in the first stage of wound healing: the inflammatory stage. In contrast, the keratinocyte layer in the wounds of the rats in the groups treated with different concentrations of total flavonoids from Asparagus radiata cream and the Deshijie hydrocolloid group had already proliferated and migrated to the wound center. The collagen fibers in the granulation tissue beneath the keratinocyte layer were tightly arranged, with a large number of fibroblasts growing, scattered new capillaries, a thick new epidermis, and relatively few inflammatory cells, indicating that the skin wounds of the rats in each treatment group had entered the second and third stages of wound healing: the proliferation and remodeling stage. Figure 10B is a quantitative statistical graph of rat epidermal thickness. The epidermal thickness of the blank control group was 26.83±2.65μm, and the epidermal thickness of the model group increased significantly to 87.72±1.95μm (P<0.01vs. control group). The epidermal thickness of the group given Dejie hydrocolloid was 112.70±7.58μm, and the epidermal thickness of the 0.5% total flavonoids from Astragalus membranaceus cream group, the 1% total flavonoids from Astragalus membranaceus cream group, and the 2% total flavonoids from Astragalus membranaceus cream group were 99.97±3.06μm, 121.00±2.35μm, and 121.10±5.64μm, respectively. The results showed that the epidermal thickness of rats in the 1% total flavonoids from Astragalus membranaceus cream group, the 2% total flavonoids from Astragalus membranaceus cream group, and the Dejie hydrocolloid group was significantly increased compared to the model group, indicating that the degree of re-epithelialization of the wound was higher than that of the model group, and that it could effectively promote the healing of full-thickness skin wounds in rats.

[0482] 3.3 Analysis of Masson staining results

[0483] Masson staining is a commonly used staining method in histology. It can dye collagen fibers blue and keratin and muscle fibers red. It is used to evaluate the formation of collagen fibers in the wound surface.

[0484] Figure 11A shows the collagen deposition in each group after 9 days of treatment. The darker the color, the greater the amount of collagen deposition. It can be seen intuitively from the figure that the blue staining in the model group is lighter, and the blue staining in the Deshijie hydrocolloid group and the groups with different concentrations of sunflower total flavonoids cream has different degrees of deepening. Among them, the blue area of ​​the 1% sunflower total flavonoids cream group is larger than that of the other groups, the color is darker, and the distribution is more uniform. Figure 11B is a quantitative statistical chart of the collagen area percentage. The collagen area percentage of the blank control group was 36.02±2.95%, and the collagen area percentage of the model group decreased significantly to 14.74±1.64% (P<0.01 vs. control group). The collagen area percentage of the Dejie hydrocolloid group was 23.24±1.83%, and the collagen area percentages of the 0.5% sunflower total flavonoids cream group, the 1% sunflower total flavonoids cream group, and the 2% sunflower total flavonoids cream group were 19.33±2.46%, 27.45±1.74%, and 22.76±2.48%, respectively. The results of the difference analysis showed that there was a significant difference between the Dejie hydrocolloid group and the model group (P<0.05). Among the groups receiving different concentrations of sunflower total flavonoids cream, the collagen content of the 1% sunflower total flavonoids cream group was the highest, which was significantly different from the model group (P<0.01).

[0485] 3.4 Changes in CD31 and VEGF mRNA levels

[0486] Under normal conditions, blood vessels are stable and well perfused, delivering ample nutrients and oxygen to tissues. Once tissue is damaged and ischemic and hypoxic, the wound site produces a large amount of pro-angiogenic factors, which promote microvascular growth. To determine changes in the vascular markers CD31 and angiogenic factor (VEGF) at the genetic level, RT-qPCR was performed to measure CD31 and VEGF mRNA expression on day 9 after administration.

[0487] Figure 12A shows that the expression of CD31 mRNA in the modeling group decreased significantly from 0.22±0.03 to 0.10±0.01 (P<0.01). After administration, the expression of CD31 mRNA increased to varying degrees. The expression of CD31 mRNA in the Dejie hydrocolloid group was 0.17±0.02, and the expression of CD31 mRNA in the 0.5%, 1%, and 2% sunflower flavonoids cream groups were 0.10±0.01, 0.21±0.02, and 0.12±0.02, respectively. Among them, the 1% sunflower flavonoids cream enhanced the expression of CD31 mRNA most significantly, with statistical differences (P<0.01). There were no statistical differences in the other groups. Figure 12B shows that the expression of Vegf mRNA decreased from 0.25±0.07 to 0.14±0.04 after modeling. The expression of VEGF mRNA increased to varying degrees after drug administration. Compared with the model group, the expression of VEGF mRNA in the Dejie hydrocolloid group was 0.23±0.08, and the expression of VEGF mRNA in the 0.5%, 1%, and 2% sunflower total flavonoids cream groups were 0.10±0.02, 0.21±0.04, and 0.14±0.04, respectively. However, no significant difference was found between the drug administration group and the model group.

[0488] 3.5 Analysis of skin tissue immunofluorescence staining results

[0489] CD31 is a typical marker of vascular endothelial cells and can be used to measure angiogenesis. Immunohistochemical staining of tissue sections using anti-CD31 antibodies can visually and objectively determine the distribution, density, and content of CD31 within newly formed tissue, thereby analyzing the success of microcirculation establishment. Using mean fluorescence intensity as an indicator, multiple fields of view from different sections were photographed to evaluate angiogenesis in different groups.

[0490] As shown in Figure 13A, DAPI (blue) was used to locate the cell nucleus, and anti-CD31 antibody (red) was used to locate the vascular endothelial cells, which can obtain a clear distribution of CD31 in the tissue. The number of new blood vessels in the wound of the model group was small, and CD31 was weakly positive. The relative fluorescence intensity was 41.63±7.46% of that of the control group (P<0.05). The CD31 expression in the Dejie hydrocolloid group was higher than that in the model group, and the relative fluorescence intensity was 73.75±18.13% of that in the control group, but there was no statistical difference. The CD31 expression in the different concentrations of the total flavonoids cream of sunflower was higher than that in the model group. The relative fluorescence intensities of the 0.5%, 1%, and 2% total flavonoids cream of sunflower were 49.49±13.50%, 70.92±9.53%, and 47.69±8.99% of the control group, respectively, but no statistical difference was shown.

[0491] 3.6 Changes in vascular endothelial growth factor (VEGF) protein levels

[0492] A key reason why diabetic wounds are difficult to heal is the inadequate microcirculatory perfusion caused by hyperglycemia and persistent inflammatory responses. Therefore, the most critical step in the healing process is the reconstruction of new blood vessels. The effective establishment of microcirculation makes the repair process more efficient, accelerating the exchange of nutrients between capillaries and the migration of activated leukocytes. VEGF belongs to a subfamily of growth factors, namely the platelet-derived growth factor family. It is an important signaling protein involved in angiogenesis and vascularization. It can provide a matrix for the migration and formation of vascular endothelial cells, promote the proliferation of new capillaries in ulcer wounds, stimulate the growth of granulation tissue, and thus promote wound healing.

[0493] As shown in Figure 14, the VEGF protein level in the model group decreased compared with the blank group, and increased to varying degrees after administration. The VEGF protein level in the model group was 44.93±3.69pg / ml, the VEGF protein level in the Dejie hydrocolloid group was 114.30±13.11pg / ml, and the VEGF protein levels in the 0.5%, 1%, and 2% sunflower total flavonoids cream groups were 77.74±5.90pg / ml, 98.14±11.53pg / ml, and 99.29±17.35pg / ml, respectively. The data showed that among all the drug-treated groups, the Dejie hydrocolloid group had the most significant increase in VEGF protein level (P<0.01). The effects of the 1% and 2% sunflower flavonoids cream groups were weaker than those of Dejie hydrocolloid, but they were also able to significantly increase the VEGF protein level in skin tissue (P<0.01). The VEGF protein expression level in the 0.5% sunflower flavonoids cream group was increased compared with the model group, but there was no statistical difference.

[0494] 4. Experimental Conclusion

[0495] In this study, an STZ-induced diabetes model was used. A full-thickness skin excision model was constructed in rats using a sterile 8-mm Biopsy Punch. Dejieshi hydrocolloid was used as the positive control drug, and blank cream was used as the blank control drug in the model group to investigate the therapeutic effect of total flavonoids from Astragalus membranaceus on diabetic ulcer rats.

[0496] Results showed that total flavonoids from Astragalus membranaceus accelerated wound re-epithelialization, promoted collagen fiber deposition, and increased VEGF protein expression, promoting full-thickness wound healing in diabetic rats. Furthermore, total flavonoids from Astragalus membranaceus promoted angiogenesis by increasing CD31 expression, thereby accelerating the healing process of diabetic ulcer wounds. In summary, total flavonoids from Astragalus membranaceus have a certain therapeutic effect on diabetic ulcers.

[0497] Example 24 Gouty arthritis experiment

[0498] Modeling: Rats were anesthetized by inhalation of ether. Under sterile conditions, the posterior aspect of the right ankle joint was selected for puncture. The needle was inserted into the ankle joint cavity with the bevel facing forward and upward at a 45° angle to the tibia. 50 μL of PBS was injected into the right ankle joint of the control rats. 50 μL of urate suspension (80 mg / ml) was injected into the joint cavity of the remaining rats. The injection standard was bulging of the contralateral joint capsule. A small amount of erythromycin ointment was then applied to the injection site to prevent infection. One hour after the urate injection, the test drug was administered once daily for one week.

[0499] Group administration: Group administration: randomly divided into control group, ovariectomized group, positive drug group and total flavonoids group (52 mg / kg), with 10 rats in each group. All groups were given 0.1 ml / 10 g body weight of total flavonoids, and the positive drug group was given colchicine 0.1 mg / kg. Among them, the extract of Hibiscus tiliaceus flower was prepared according to the method of Example 9-1.

[0500] One week after administration, joint swelling and inflammatory factor levels (IL-1β, TNF-α) were measured. The results are as follows:

[0501] Table 2 Results of various test indicators

[0502] Compared with the normal group, # P<0.05; compared with the model, * P<0.05

[0503] The experimental results showed that each group of the examples had a reducing effect on the degree of segmental swelling and the levels of inflammatory factors (IL-1β, TNF-α). Compared with the model group, the hibiscus flower extract group and the positive drug group could significantly reduce the degree of segmental swelling and the levels of inflammatory factors (IL-1β, TNF-α). In addition, no gastrointestinal reactions such as diarrhea were observed.

[0504] Example 23 Osteoporosis Experiment

[0505] Modeling: Sixty SPF female Sprague-Dawley rats, 3 months old and weighing 200-220 g, were fed adaptively for one week before the experiment. All rats, except the sham-operated group, were anesthetized with intraperitoneal injection of 10% chloral hydrate and bilaterally ovariectomized to establish a postmenopausal osteoporosis model. The sham-operated group only had an equal amount of adipose tissue surrounding the ovaries removed. Postoperatively, 80,000 units of veterinary penicillin were administered per rat to prevent infection. The corresponding drugs were administered daily by gavage starting one week after surgery for 12 consecutive weeks.

[0506] Grouping and administration: The mice were randomly divided into sham operation group, ovariectomized group, positive drug group and Hibiscus truncatum total flavonoids group (52 mg / kg), with 10 mice in each group. The positive drug group was given estrogen estradiol valerate tablets (0.09 mg / kg). The Hibiscus truncatum flower extract was prepared according to the method of Example 9-1.

[0507] After 12 weeks of administration, serum alkaline phosphatase (ALP), calcium (Ca), and bone metabolism marker osteocalcin (OC) were measured.

[0508] Table 3 Data of relevant indicators

[0509] Compared with the sham operation group, # P<0.05; compared with the ovariectomized group, * P<0.05

[0510] The experimental results showed that each group of the examples had a lowering effect on ALP and OC, and an increasing effect on Ca. Compared with the ovariectomized group, the hibiscus flower extract group and the positive drug group could significantly reduce ALP and OC levels and increase Ca levels. In addition, no gastrointestinal reactions such as drug-related diarrhea were observed.

[0511] Example 25: Experiment on the combined use of total flavonoids from Abelmoschus truncatus flowers and irbesartan in the treatment of diabetic nephropathy

[0512] 1. Experimental Animals

[0513] Db / db (BKS.Cg-Dock7m + / + Leprdb / J) mice were obtained from the Institute of Animal Models, Nanjing University, and were introduced at the age of 10 weeks with a body weight difference of no more than 8 g.

[0514] 1. Animal information

[0515] Table 47 Animal Information Table of Example 22

[0516] 2. Animal Grouping and Dosing

[0517] First, grouping was performed based on blood glucose and body weight. C57BL / KsJ mice were grouped into cages of five, numbered 1, 2, and 3, for a total of three cages. db / db mice were grouped into cages of three, for a total of 45 cages. The mice were numbered 1, 2, and 3, for example, 45-1 represents mouse 1 in cage 45. After a week of acclimation, all mice were released from the quarantine room into the experimental room. Starting at 11 weeks of age, blood glucose and body weight were measured weekly. Starting at 13 weeks of age, mice were placed in metabolic cages and urine was collected. Urine albumin (UACR) levels were measured using the Mouse MAU (microalbuminuria) ELISA Kit, Catalog No. E-EL-M0792C, and urine creatinine (Cr) ELISA Kit, Catalog No. E-EL-0058C. Treatment was initiated when UACR values ​​exceeded 200 mg / g. The db / db mice were grouped three times based on the time of onset of disease: the first, second, and third groups. The group names were WT group (non-diabetic wild-type mice), DN group (type 2 diabetic nephropathy db / db mice), HKC group (Hibiscus flower extract group), EB group (irbesartan group), HKCEB group (Hibiscus flower extract and irbesartan combined group), TFA group (Hibiscus flower total flavonoids group), and TFAEB group (Hibiscus flower total flavonoids and irbesartan combined group), among which the Hibiscus flower total flavonoids were prepared according to the method of Example 9-1.

[0518] 3. Dosage regimen

[0519] Irbesartan is from Sanofi Biotech Co., Ltd.

[0520] The preparation method of hibiscus flower extract is as follows: take 2 kg of hibiscus flower, add 18 times the amount of ethanol, heat and reflux for 1 hour, filter, reduce the pressure of the filtrate to recover ethanol, concentrate, adjust the pH to 6.0, let it stand, remove the upper layer of oil, vacuum dry, and crush to obtain the extract.

[0521] Table 48 Dosage regimen

[0522] 4.db / db mouse metabolic cage data

[0523] Compared with before administration, the changes in blood glucose, body weight, UACR and blood pressure of each group after 4 weeks of administration are shown in FIG15 .

[0524] The results showed that after four weeks of administration, blood glucose, body weight, and UACR values ​​in the diabetic nephropathy group were significantly increased compared to their wild-type littermates. Compared to the diabetic nephropathy group, blood glucose and body weight did not change significantly in any of the treated groups, while UACR values ​​were significantly decreased. Observation of the mice's condition revealed that the TFAEB and TFA groups experienced no diarrhea or abdominal distension, and their overall therapeutic efficacy was significantly superior to that of the other groups.

[0525] This study, through serum metabolomics analysis, found that cortisol is significantly upregulated in the serum of diabetic nephropathy patients. Studies have shown that serum cortisol secretion is associated with the presence of microalbuminuria in patients with T2DM and prediabetes. Elevated cortisol levels in patients with T2DM and prediabetes with high proteinuria levels may be associated with the development of microalbuminuria, even within the normal range (Zhang et al., 2020). This study showed that total flavonoids from hibiscus flowers can regulate the expression of serum cortisol in patients with diabetes. Therefore, total flavonoids from hibiscus flowers may reduce proteinuria in patients with diabetic nephropathy by lowering serum cortisol levels in diabetic patients.

[0526] Example 26 Pharmacodynamic evaluation of total flavonoids from Astragalus membranaceus in treating osteoporosis in ovariectomized rats

[0527] 1. Solution Preparation

[0528] (1) Preparation of 4% chloral hydrate solution

[0529] Weigh 6 g of chloral hydrate and dissolve it in 150 mL of normal saline.

[0530] (2) Preparation of 0.5% sodium carboxymethylcellulose (0.5% CMC-Na) solution

[0531] Slowly add 0.5g of CMC-Na powder to 100mL of 70℃ pure water, stir evenly, and let it stand at room temperature overnight. The next day, sonicate before preparing the drug solution until a homogeneous 0.5% CMC-Na solution is obtained.

[0532] (3) Preparation of drug solution

[0533] Preparation of solvent control aqueous solution for blank control group and model group: 1 mL DMSO + 49 mL 0.5% CMC-Na solution.

[0534] Positive drug group: 134 mg alendronate sodium vitamin D3 tablet powder (70 mg D3 / 300 mg / tablet) + 1 mL DMSO + 4.9 mL 0.5% CMC-Na solution.

[0535] ZHT dosage group: 300 mg ZHT + 1 mL DMSO + 49 mL 0.5% CMC-Na solution.

[0536] The total flavonoids from sunflower seed (ZHT) were prepared using the method of Example 3.2-1.

[0537] (4) Preparation of 4% paraformaldehyde

[0538] Weigh 4 g of paraformaldehyde and dissolve it in 100 mL of normal saline. Dissolve it at 55-60°C in the dark overnight. Adjust the pH to 7.4 and store it at 4°C in the dark.

[0539] 2 Establishment of bilateral ovariectomy-induced osteoporosis model in rats

[0540] Female Sprague-Dawley rats, 8 months old and weighing 450 ± 50 g, were purchased from Jiangsu Qinglongshan Biotechnology Co., Ltd. and randomly divided into the following groups: sham operation group (Sham), model group (OVX), positive drug group (VD3, 6.25 mg / kg / week), and sunflower flavonoids group (60 mg / kg / day), with 8 rats in each group. Before the experiment, the rats were housed in a quiet, light-proof environment for 7 days to acclimate to the environment. The light-to-dark ratio was 1:1, the laboratory temperature was maintained at 23°C, and the humidity was approximately 50%. The rats had free access to food and water.

[0541] On day 0 of the experiment, rats underwent bilateral ovariectomy (OVX) to establish an osteoporosis model. Chloral hydrate anesthetic (0.9 mL / 200 g) was injected intraperitoneally. After complete anesthesia, the rats were disinfected, draped, and a longitudinal incision was made on the back. All rats except the sham-operated group underwent bilateral ovariectomy, in which only an equal amount of adipose tissue surrounding the ovaries was removed. The incisions were then closed with layer-by-layer sutures. Postoperatively, 80,000 units of veterinary penicillin were administered per rat to prevent infection.

[0542] Seven days after surgery, the blank and model groups were gavaged daily with a 10 mL / kg solvent control aqueous solution. The ZHT-dosage group was gavaged daily with a ZHT solution. The positive drug group was gavaged with a VD3 solution every Monday. Rats were fasted for 12 hours before each gavage. Rats were weighed every Monday before dosing.

[0543] After 12 weeks of dosing, the rats were anesthetized, and blood was collected from the abdominal aorta. The blood was then centrifuged at 3,000 rpm for 15 minutes. Serum was then collected for analysis of tartrate-resistant acid phosphatase (TRACP), osteoprotegerin (OPG), osteocalcin (OC), and bone alkaline phosphatase (BALP). Tibias and femurs were collected from the rats; the left femur was used for bone biomechanical analysis, and the right femur was used for pathological sectioning and bone density analysis.

[0544] 2.5 Bone biomechanical testing

[0545] The bone mineral density (BMD) of the left femur of rats was measured using the SkyScan 1176 micro-CT imaging system from Bruker, Germany. The CT images were processed by computer to obtain the BMD value, two-dimensional scans of the femur, and three-dimensional reconstructions of the trabeculae in the proximal femoral metaphysis.

[0546] An Instron E10000 universal material testing machine was used for testing. During the test, the specimens were placed between two supporting crossbars, with the load rod located in the center with a span of 20 mm. All specimens were uniformly loaded at a loading rate of 3 mm / min until the specimens fractured. The maximum bending force and maximum bending deformation of the tested bone tissue samples before fracture were recorded, and the bending stiffness, elastic modulus, and bending stress parameters were calculated.

[0547] 2.6ELISA test

[0548] The levels of tartrate-resistant acid phosphatase (TRACP) and bone alkaline phosphatase (BALP) were detected using ELISA kits from Jiangsu Enzyme Immunity Industry Co., Ltd. The detection was performed according to the instructions. The specific steps are as follows.

[0549] (1) Allow the reagents to equilibrate at room temperature for 30 minutes.

[0550] (2) Set up blank wells, standard wells, and sample wells. Add 50 μL of the standard to the standard well on the enzyme-labeled plate, and add 40 μL of sample diluent and 10 μL of sample to the sample well.

[0551] (3) Add 100 μL of HRP enzyme-labeled reagent to each well except the blank well. Seal the plate and incubate at 37°C for 60 min.

[0552] (4) After incubation, discard the liquid, add 300 μL of washing solution, let it stand for 1 min, shake off the washing solution, pat dry, and repeat this process 5 times.

[0553] (5) Add 50 μL of substrate A and B to each well and incubate at 37°C in the dark for 15 min.

[0554] (6) Add 50 μL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm within 15 minutes.

[0555] The contents of osteoprotegerin (OPG) and osteocalcin (OC) were detected by ELISA kits produced by Wuhan Aibotek Biotechnology Co., Ltd. The detection was performed according to the instructions. The specific steps are as follows.

[0556] (1) Allow the reagents to equilibrate at room temperature for 30 minutes.

[0557] (2) Add 350 μL of 1× washing solution to each well, let it stand for 40 seconds, then discard the washing solution, pat dry, and repeat this operation three times.

[0558] (3) Add 100 μL of sample diluent to the blank wells, add 100 μL of standards of different concentrations to the standard wells, and add 100 μL of sample to the sample wells. Seal the plate and incubate at 37°C for 2 h.

[0559] (4) Discard the liquid in the wells and add 350 μL of 1× washing solution to each well. Let it stand for 40 seconds, then discard the washing solution and pat dry. Repeat this operation three times.

[0560] (5) Add 100 μL of biotinylated antibody to each well, seal the plate, and incubate at 37°C for 1 h.

[0561] (6) Discard the liquid in the wells and add 350 μL of 1× washing solution to each well. Let it stand for 40 seconds, then discard the washing solution and pat dry. Repeat this operation three times.

[0562] (7) Add 100 mL of streptavidin-HRP working solution to each well, seal the plate, and incubate at 37°C for 30 min.

[0563] (8) Discard the liquid in the wells and add 350 μL of 1× washing solution to each well. Let it stand for 40 seconds, then discard the washing solution and pat dry. Repeat this operation three times.

[0564] (9) Add 90 μL of TMB substrate to each well and incubate at 37°C in the dark for 15-20 min.

[0565] (10) Add 50 μL of stop solution to each well and immediately measure the OD value of each well at a wavelength of 450 nm.

[0566] 2.10 H&E staining

[0567] (1) Femoral specimens were fixed with 4% paraformaldehyde at room temperature for 48 h;

[0568] (2) After fixation, the specimen was removed and placed in a 10% EDTA solution for decalcification for 4 weeks, with the decalcification solution changed every other day;

[0569] (3) After decalcification, routine paraffin embedding should be performed, and all specimens should be embedded in the same position as much as possible;

[0570] (4) Paraffin slices were sliced ​​to a thickness of 4 μm. The slices were spread in a 40°C water bath, dried, and then baked at 37°C overnight.

[0571] (5) Dewax the selected paraffin sections in xylene twice, each time for 10 min, rehydrate with a series of ethanol (100%, 95%, 85%, 75%), each gradient for 3 min, and then soak in distilled water for 2 min;

[0572] (6) Stain with hematoxylin solution for 10 min and wash with distilled water to remove floating color;

[0573] (7) Differentiation in differentiation solution for 30 seconds, followed by rinsing in tap water twice, each time for 3 minutes;

[0574] (8) Stain with eosin solution for 1 min, remove excess staining solution, and then dehydrate quickly;

[0575] (9) Soak in a series of ethanol (75%, 85%, 95%, 100%) for 3 minutes each:

[0576] (10) Wash with 100% ethanol for 1 min, clear with xylene twice, 1 min each time, seal with neutral gum, and observe under a microscope.

[0577] 2.11 Statistical Analysis

[0578] GraphPad Prism (Ver 8.0) was used for statistical analysis. All data were expressed as mean ± standard error (Mean ± SEM) and analyzed by One-way ANOVA. Dunnett's multiple test was used to analyze the significance of differences between groups. When P < 0.05, it was considered statistically significant.

[0579] 3 Experimental results

[0580] 3.1 Effects of ZHT on bone morphology in OVX rats

[0581] H&E staining of the femurs of rats in each group showed that the sham group had smaller bone marrow cavities, thicker, fuller, and regular trabeculae, and a clear and complete morphology. Compared with the sham group, the OVX group had a larger number of vacuoles in the bone marrow cavities, with sparsely arranged, disorganized trabeculae and poor connectivity. Treatment with the positive drugs VD3 and ZHT alleviated bone microstructural damage in rats, with an increase in trabeculae and a significant decrease in bone marrow cavities. ZHT had the most significant effect. See Figure 15 for details.

[0582] 3.2 Effects of ZHT on bone biomechanics in OVX rats

[0583] Micro-CT scans of the right distal femur of rats in each group were performed, and two-dimensional (2D) and three-dimensional (3D) reconstructions were performed. The results showed that compared with the sham group, OVX rats had significantly reduced cancellous bone mass and sparse trabeculae. VD3 intervention increased cancellous bone mass in rats, and the trabeculae were denser than those in the OVX group. Compared with the OVX group, the low-dose group did not significantly increase cancellous bone mass or change the density of trabeculae. The medium-dose and high-dose groups showed significant increases in cancellous bone mass and denser trabeculae. Micro-CT scanning was used to calculate the femoral microstructure-related parameters. The BMD results showed that the BMD values ​​of the Sham group were 0.606±0.003 g / cm2 and 0.493±0.026 g / cm2 in the OVX group, which were significantly lower than those of the Sham group (P<0.01); the BMD values ​​of the VD3 group were 0.577±0.017 g / cm2, which were significantly higher than those of the OVX group (P<0.05); and the BMD values ​​of the ZHT group were 0.591±0.036 g / cm2, which were significantly higher than those of the OVX group (P<0.05).

[0584] The maximum bending force and maximum bending deformation of the left femur of rats before fracture were tested using an Instron E10000 universal material testing machine, and parameters such as bending stiffness, elastic modulus and bending stress were calculated.

[0585] The elastic modulus of the femur of rats in each group showed that the elastic modulus of the Sham group was 5786±587.5 MPa, and the elastic modulus of the OVX group was 3269±269.7 MPa, which were significantly lower than those in the Sham group (P<0.05); the elastic modulus of the VD3 group was 6464±404.5 MPa, which was significantly higher than that in the OVX group (P<0.01); the elastic modulus of the ZHT group was 6844±1080 MPa, which was significantly higher than that in the OVX group (P<0.01).

[0586] The femoral bending stress diagrams of rats in each group showed that the bending stress in the Sham group was 236.7±13.06 MPa, and that in the OVX group was 173.2±13.84 MPa, which were significantly lower than those in the Sham group (P<0.01); the bending stress in the VD3 group was 256.0±12.03 MPa, which was significantly higher than that in the OVX group (P<0.01); and the bending stress in the ZHT group was 236.4±14.17 MPa, which was significantly higher than that in the OVX group (P<0.01).

[0587] 3.3 Effects of ZHT on serum biochemical parameters in OVX rats

[0588] The levels of TRACP, a bone turnover marker, in the serum of rats in each group showed that the serum TRACP content in the Sham group was 86.98±1.025 pg / mL, and that in the OVX group was 99.74±2.808 pg / mL, which were significantly higher than those in the Sham group (P<0.01); the serum TRACP content in the VD3 group was 88.58±3.364 pg / mL, which was significantly lower than that in the OVX group (P<0.05); the serum TRACP content in the medium-dose ZHT group was 80.40±3.313 pg / mL, which was significantly lower than that in the OVX group (P<0.01).

[0589] The levels of OPG, a bone turnover marker, in the serum of rats in each group showed that the serum OPG content in the Sham group was 146.4±6.249 pg / mL, and the serum OPG content in the OVX group was 184.9±3.492 pg / mL, which were significantly higher than those in the Sham group (P<0.05); the serum OPG content in the VD3 group was 130.0±8.835 pg / mL, which was significantly lower than that in the OVX group (P<0.01); the serum OPG content in the ZHT group was 118.9±7.219 pg / mL, which was significantly lower than that in the OVX group (P<0.01).

[0590] The results of the levels of bone metabolism marker BALP in the serum of rats in each group showed that the serum BALP content in the Sham group was 1145±29.83 pg / mL, and the serum BALP content in the OVX group was 1316±30.26 pg / mL, which were significantly higher than those in the Sham group (P<0.01); the serum BALP content in the VD3 group was 1146±37.23 pg / mL, which was significantly lower than that in the OVX group (P<0.01); the serum BALP content in the ZHT group was 845.9±40.18 pg / mL, which was significantly lower than that in the OVX group (P<0.01).

[0591] 4 Experimental Conclusions

[0592] This study investigated the therapeutic efficacy of total flavonoids from Asparagus chinensis on bilateral ovariectomy-induced osteoporosis, using alendronate sodium and vitamin D3 tablets (6.25 mg / kg / week) as a positive control. After bilateral ovariectomy, rats lost weight. HE pathological sections revealed significant damage to bone microstructure. Micro-CT revealed a significant decrease in cancellous bone mass, thin and sparse trabeculae in the proximal tibial metaphysis, and significantly reduced bone mineral density (BMD). Bone biomechanical testing revealed a significant decrease in femoral strength and toughness after bilateral ovariectomy, and significant changes in serum levels of bone turnover and bone metabolism markers. These results suggest that a bilateral ovariectomized rat osteoporosis model has been successfully established. A 60 mg / kg / day dose of total flavonoids from Asparagus chinensis ameliorates OVX-induced weight loss and bone morphology in OVX rats, significantly increases the decrease in BMD induced by OVX, significantly enhances femoral strength and toughness, and improves bone turnover and bone metabolism in OVX rats. The 60mg / kg / day total flavonoids from Astragalus membranaceus tested in this study showed good effects on weight loss, changes in bone tissue morphology, decreased bone density, changes in bone biomechanical indicators, bone turnover, and bone metabolism caused by the osteoporosis model in OVX rats, and was superior to the positive therapeutic drug alendronate sodium vitamin D3 tablets in terms of bone turnover and bone metabolism.

[0593] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. Use of a pharmaceutical composition in the preparation of a drug for preventing or treating gout or osteoporosis, characterized in that: The composition comprises the following flavonoid components in the following mass ratio: The mass ratio of gossypol-8-O-β-D-glucuronide: hyperoside: isoquercetin: myricetin: quercetin-3'-O-glucoside is 10: 3.0-20: 2.0-18: 0.2-6.0: 1-20; Preferably, the mass ratio of quercetin-8-O-β-D-glucuronide: hyperoside: isoquercetin: myricetin: quercetin-3'-O-glucoside is 10: 3.0-20: 2.0-3.9: 0.2-6.0: 1-20; or the mass ratio of gossypol-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside is 10:3.0-20:4.0-18:0.2-6.0:1-20; or the mass ratio of quercetin-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside is 10:3.0-20:2.0-3.9:0.2-1.8:1-20; or the mass ratio of gossypol-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside is 10:3.0-20:2.0-3.9:1.9-6.0:1-20; or the mass ratio of gossypol-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside is 10:3.0-20:2.0-18:0.2-6.0:1-5.9; or the mass ratio of gossypol-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside is 10:3.0-20:2.0-18:0.2-6.0:6.0-20; Or the mass ratio of quercetin-8-O-β-D-glucuronide: hyperoside: isoquercetin: myricetin: quercetin-3'-O-glucoside is 10:3.0-20:4.0-18:1.9-6.0:6.0-20.

2. The use according to claim 1, characterized in that: The mass ratio of the cotton linden-8-O-β-D-glucuronide: hyperoside: isoquercetin: myricetin: quercetin-3'-O-glucoside is 10:4.0-17:4.50-16:1.9-5.5:7.0-16; preferably 10:6.0-15.6:5.0-13:2.0-5.0:8.0-14; more preferably 10:6.0-15:5.0-13:2.0-5.0:8.0-14; more preferably 10:6.5-14:5.7-11.2:2.2-3.1:8.7-11.4; Further, it contains quercetin, wherein the mass ratio of quercetin-8-O-β-D-glucuronide to quercetin is 10:0.1-10.0, preferably 10:0.1-0.9 or 10:1.0-10.0, more preferably 10:1.0-6.0, more preferably 10:1-5.6, and most preferably 10:1.5-5.0; Furthermore, it contains rutin, wherein the mass ratio of linalool-8-O-β-D-glucuronide to rutin is 10:0.05-0.6, preferably 10:0.1-0.5, more preferably 10:0.1-0.45, and most preferably 10:0.1-0.3; Further, it contains quercetin-3-O-acacia glycoside, wherein the mass ratio of quercetin-8-O-β-D-glucuronide to quercetin-3-O-acacia glycoside is 10:0.1-2.5, preferably 10:0.1-0.9, and more preferably 10:0.15-0.5; The composition is a hibiscus flower extract.

3. Use of a pharmaceutical composition in the preparation of a drug for gout or osteoporosis, characterized in that: The composition comprises the following flavonoid components in the following mass ratio: The mass ratio of quercetin-8-O-β-D-glucuronide: hyperoside: isoquercetin: myricetin: quercetin-3'-O-glucoside: quercetin is 10: 3.0-20: 2.0-18: 0.2-6.0: 1-20: 0.1-10.0; Preferably, the mass ratio of quercetin-8-O-β-D-glucuronide: hyperoside: isoquercetin: myricetin: quercetin-3'-O-glucoside: quercetin is 10: 3.0-20: 2.0-3.9: 0.2-6.0: 1-20: 0.1-10.0; or the mass ratio of quercetin-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside:quercetin is 10:3.0-20:4.0-18:0.2-6.0:1-20:0.1-10.0; or the mass ratio of quercetin-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside:quercetin is 10:3.0-20:2.0-3.9:0.2-1.8:1-20:0.1-10.0; or the mass ratio of quercetin-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside:quercetin is 10:3.0-20:2.0-3.9:1.9-6.0:1-20:0.1-10.0; or the mass ratio of quercetin-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside:quercetin is 10:3.0-20:2.0-18:0.2-6.0:1-5.9:0.1-10.0; or the mass ratio of quercetin-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside:quercetin is 10:3.0-20:2.0-18:0.2-6.0:6.0-20:0.1-10.0; or the mass ratio of quercetin-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside:quercetin is 10:3.0-20:2.0-18:0.2-6.0:1-20:0.1-0.9; or the mass ratio of quercetin-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside:quercetin is 10:3.0-20:2.0-18:0.2-6.0:1-20:1.0-10.0; Or the mass ratio of quercetin-8-O-β-D-glucuronide: hyperoside: isoquercetin: myricetin: quercetin-3'-O-glucoside: quercetin is 10:6-15:5-13:1.0-2.9:4.0-9.5:0.6-6.0, preferably 10:6-15:5-13:1.0-1.8:4.0-5.9:0.6-0.99; Further, it contains rutin, wherein the mass ratio of linalool-8-O-β-D-glucuronide to rutin is 10:0.05-0.6, preferably 10:0.1-0.5, more preferably 10:0.1-0.45, and most preferably 10:0.1-0.3; Furthermore, it contains quercetin-3-O-acaciaside, wherein the mass ratio of quercetin-8-O-β-D-glucuronide to quercetin-3-O-acaciaside is 10:0.1-2.5, preferably 10:0.1-0.9, and more preferably 10:0.15-0.5; The composition is a hibiscus flower extract.

4. Use of a Hibiscus hibiscus flower extract in the preparation of a medicament for preventing or treating gout or osteoporosis, characterized in that: The extract comprises the following flavonoid components in the following mass ratio: The mass ratio of quercetin-8-O-β-D-glucuronide: hyperoside: isoquercetin: myricetin: quercetin-3'-O-glucoside: quercetin is 10: 3.0-20: 2.0-18: 0.2-6.0: 1-20: 0.1-10.0; Preferably, the mass ratio of quercetin-8-O-β-D-glucuronide: hyperoside: isoquercetin: myricetin: quercetin-3'-O-glucoside: quercetin is 10: 3.0-20: 2.0-3.9: 0.2-6.0: 1-20: 0.1-10.0; or the mass ratio of quercetin-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside:quercetin is 10:3.0-20:4.0-18:0.2-6.0:1-20:0.1-10.0; or the mass ratio of quercetin-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside:quercetin is 10:3.0-20:2.0-3.9:0.2-1.8:1-20:0.1-10.0; or the mass ratio of quercetin-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside:quercetin is 10:3.0-20:2.0-3.9:1.9-6.0:1-20:0.1-10.0; or the mass ratio of quercetin-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside:quercetin is 10:3.0-20:2.0-18:0.2-6.0:1-5.9:0.1-10.0; or the mass ratio of quercetin-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside:quercetin is 10:3.0-20:2.0-18:0.2-6.0:6.0-20:0.1-10.0; or the mass ratio of quercetin-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside:quercetin is 10:3.0-20:2.0-18:0.2-6.0:1-20:0.1-0.9; or the mass ratio of quercetin-8-O-β-D-glucuronide:hyperoside:isoquercetin:myricetin:quercetin-3'-O-glucoside:quercetin is 10:3.0-20:2.0-18:0.2-6.0:1-20:1.0-10.0; Or the mass ratio of quercetin-8-O-β-D-glucuronide: hyperoside: isoquercetin: myricetin: quercetin-3'-O-glucoside: quercetin is 10:6-15:5-13:1.0-2.9:4.0-9.5:0.6-6.0, preferably 10:6-15:5-13:1.0-1.8:4.0-5.9:0.6-0.99; Further, it contains rutin, wherein the mass ratio of linalool-8-O-β-D-glucuronide to rutin is 10:0.05-0.6, preferably 10:0.1-0.5, more preferably 10:0.1-0.45, and most preferably 10:0.1-0.3; Furthermore, it contains quercetin-3-O-acacia glycoside, wherein the mass ratio of quercetin-8-O-β-D-glucuronide to quercetin-3-O-acacia glycoside is 10:0.1-2.5, preferably 10:0.1-0.9, preferably 10:0.15-0.

5.

5. Use of an effective fraction of flavonoids in the preparation of a drug for preventing or treating gout or osteoporosis, characterized in that: The flavonoid effective fraction includes the following flavonoid components in mass ratio: ; preferably: ; More preferably: ; Further preferably: ; More preferably:

6. The use according to claim 5, characterized in that The flavonoid effective fraction includes the following flavonoid fractions in mass ratio: ;or ;or 7. The use according to any one of claims 5-6, characterized in that: The mass ratio of the isoquercetin is 1.2 or 1.

0.

8. The use according to any one of claims 5-6, characterized in that: Containing rutin, the mass ratio of isoquercetin to rutin is 1:0.001-0.08, preferably 1:0.005-0.06, more preferably 1:0.006-0.05, more preferably 1:0.007-0.04, more preferably 1:0.008-0.04, most preferably 1:0.009-0.04; Further, it contains quercetin-3-O-acaciaside, and the mass ratio of isoquercetin to quercetin-3-O-acaciaside is 1:0.01-0.25, preferably 1:0.01-0.09, more preferably 1:0.015-0.065, and most preferably 1:0.015-0.05; The flavonoid effective fraction is the flavonoid effective fraction of hibiscus flower.

9. The use according to any one of claims 2 to 6, characterized in that: The total content of quercetin, quercetin-3'-O-glucoside, myricetin, gossypol-8-O-β-D-glucuronide, isoquercetin and hyperoside contained in the composition, extract or effective part is 55% or more, preferably 60% or more, preferably 65-85%, preferably 69-82%; Further, the content of quercetin-3'-O-glucoside is 12.1-25%, preferably 12.6-25%, preferably 13-25%, preferably 14-25%, or 12.6-23%, or 13-20%; Further, the content of quercetin is 0.7% or more, preferably 1.0% or more, preferably 1.0-12%, preferably 1.5-12%, or 1.0%-10%, or 1.5%-5%; Furthermore, the content of cottonseed lin-8-O-β-D-glucuronide is 8.5-34%, preferably 12-34%, or 8.5-30%, or 12%-23%.

10. The use according to any one of claims 2-3, 7-8, characterized in that: The composition, extract or effective fraction contains quercetin, quercetin-3'-O-glucoside, myricetin, gossypol-8-O-β-D-glucuronide, isoquercetin, rutin and hyperoside in a total content of 55% or more, preferably 60% or more, preferably 65-85% or 66-84%, preferably 69-82% or 69-90%; Furthermore, the content of quercetin-3'-O-glucoside is 12.1-25%, preferably 12.6-23%, preferably 13-20%.

11. Use of an extract of Hibiscus tiliaceus in the preparation of a drug for preventing or treating gout or osteoporosis, wherein the extract contains flavonoid components, characterized in that: The extract includes the following components by mass content: Hyperoside 8-30%, isoquercetin 10-24%, gossypol-8-O-β-D-glucuronide 8.5-34%, myricetin 3.0-4.9% or 5.1-7.0%, quercetin-3'-o-glucoside 14-25%, quercetin 1.6-12%; or hyperoside 8-26%, isoquercetin 12.0-19.8%, gossypol-8-O-β-D-glucuronide 8.5-30%, myricetin 3.0 -4.9% or 5.1-6.0%, quercetin-3'-o-glucoside 14-25%, quercetin 1.6-4.9%; or hyperoside 11-22%, isoquercetin 12.0-17%, cottonwood-8-O-β-D-glucuronide 12-23%, myricetin 1.6-4.9% or 5.1-9.0%, quercetin-3'-o-glucoside 13-22%, quercetin 1.4-8% or 1.4-7.8%.

12. The use according to claim 11, characterized in that: Also includes rutin with a mass content of 0.01-1.0%, further 0.05-0.95%, further 0.09-0.95%, further 0.1-0.95%, or 0.05-0.8%, further 0.09-0.8%, further 0.1-0.6%; Furthermore, the composition contains 0.08-2.5% of quercetin-3-O-acaciaside, preferably 0.1-1.5%, further 0.1-0.9%, further 0.1-0.8%, further 0.1-0.5%.

13. The use according to claim 11, characterized in that: The mass content of flavonoid components in the hibiscus flower extract is above 55%, preferably above 60%, preferably 65-90%, preferably 69-82%.

14. The use according to any one of claims 1 to 13, characterized in that: The gout has arthritis symptoms.

15. A method for preparing the pharmaceutical composition for use according to any one of claims 1 to 3, the hibiscus flower extract for use according to claim 4, the effective fraction or extract for use according to any one of claims 5 to 10, or the hibiscus extract for use according to any one of claims 11 to 13, characterized in that: The method comprises the following steps: (1) extracting hibiscus flower or hibiscus medicinal part with ethanol to obtain an extract; (2) concentrating the extract and extracting it to obtain an extract; (3) removing the solvent from the extract and eluting it with a macroporous resin to obtain an effective part or extract of flavonoids from hibiscus flower; the preferred extraction method is percolation.

16. The preparation method according to claim 15, characterized in that: The amount of ethanol used in step (1) is 10-25 times that of hibiscus flower or hibiscus medicinal part, and the ethanol is a 60-95% ethanol solution; the extractant used for the extraction in step (2) is n-butanol, petroleum ether or ethyl acetate, the extraction method is continuous countercurrent extraction, the material-liquid ratio of the extraction is 0.8-4:1, and the extraction level of the extraction is 1-5; the model of the macroporous resin in step (3) is D101, HPD100 or AB-8.

17. The preparation method according to claim 15, characterized in that: Step (2) further comprises subjecting the extract to activated carbon adsorption, alcohol precipitation or acid precipitation treatment before extraction.

18. The preparation method according to claim 15, characterized in that: The elution process of the macroporous resin is as follows: the diameter-to-height ratio of the macroporous resin is 1:4-1:9, the loading solution concentration is 0.10-0.30 g crude drug / mL, the loading solution volume is 4-12 BV, the sample is loaded at a flow rate of 1-4 BV / h, 0.5-8 BV of pure water and 1-5 BV of 3-15% ethanol are used for impurity removal at a flow rate of 0.5-4 BV / h, and 2-8 BV of 50-80% ethanol are used for elution at a flow rate of 1-5 BV / h.

19. A method for preparing the pharmaceutical composition for use according to any one of claims 1 to 3, the hibiscus flower extract for use according to claim 4, the effective part or extract for use according to any one of claims 5 to 10, or the hibiscus extract for use according to any one of claims 11 to 13, characterized in that: The method comprises the following steps: (1) extracting hibiscus flower or hibiscus medicinal part with ethanol to obtain an extract; (2) adding a clarifying agent to the extract, treating it in a water bath, and filtering out the supernatant; (3) eluting the supernatant with a polyamide resin to obtain the hibiscus flower flavonoid effective part or extract; the preferred extraction method is percolation.

20. The preparation method according to claim 19, characterized in that: The amount of ethanol in step (1) is 10-25 times that of hibiscus flower or hibiscus medicinal part, and the ethanol is a 60-95% ethanol solution; the water bath temperature in step (2) is 50-70°C, and the water bath time is 30-90min; the resin diameter-to-height ratio in step (3) is 1:4-1:9, the loading solution concentration is 0.10-0.60g crude drug / mL, the loading solution volume is 4-12BV, and elution is performed with 4-8BV pure water and 4-8BV 60-95% ethanol.

21. A method for preparing the pharmaceutical composition for use according to any one of claims 1 to 3, the hibiscus flower extract for use according to claim 4, the effective fraction or extract for use according to any one of claims 5 to 10, or the hibiscus extract for use according to any one of claims 11 to 13, characterized in that: The method comprises the following steps: (1) extracting hibiscus flower or hibiscus medicinal part with ethanol to obtain an extract; (2) adjusting the pH value of the extract to 2.0-3.0, refrigerating, filtering to obtain a precipitate, and adding water to dissolve it; (3) extracting the dissolved solution to obtain an extract; (4) removing the solvent from the extract and eluting with a polyamide resin to obtain an effective part or extract of flavonoids from hibiscus flower; and the preferred extraction method is reflux.

22. The preparation method according to claim 21, characterized in that: The amount of ethanol in step (1) is 10-25 times that of hibiscus flower or medicinal part of hibiscus, and the ethanol is a 60-95% ethanol solution; the pH regulator in step (2) is hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid, citric acid, tartaric acid or maleic acid; the extractant used for extraction in step (3) is n-butanol, petroleum ether or ethyl acetate, the extraction method is continuous countercurrent extraction, the material-liquid ratio of the extraction is 0.8-4:1, and the extraction level of the extraction is 1-5; the resin diameter-to-height ratio in step (4) is 1:4-1:9, the sample solution concentration is 0.10-0.60g crude drug / mL, the sample solution volume is 4-12BV, and elution is performed with 4-8BV pure water and 4-8BV 60-95% ethanol.

23. A hibiscus flower total flavonoids cream, characterized in that: The ointment comprises (i) the pharmaceutical composition for use according to any one of claims 1 to 3, the hibiscus flower extract for use according to claim 4, the effective part or extract for use according to any one of claims 5 to 10, or the hibiscus extract for use according to any one of claims 11 to 13, and (ii) pharmaceutically acceptable excipients; preferably, the ointment further comprises (iii) water.

24. The cream according to claim 23, characterized in that The auxiliary material is selected from any one or a combination of Tween, sodium lauryl sulfate, glycerol, ethanol, isopropyl myristate, methylparaben, azone, glyceryl monostearate, palmitic acid, and white vaseline; preferably, the Tween is preferably Tween 80; Preferably, the Tween accounts for 8-16% of the total mass of the excipients and water, preferably 10-14%, preferably 12%; Preferably, the sodium lauryl sulfate accounts for 0.8-1.6% of the total mass of the auxiliary materials and water, preferably 1-1.4%, preferably 1.2%; Preferably, the glycerol accounts for 1.4-2.2% of the total mass of the auxiliary materials and water, preferably 1.6-2.0%, preferably 1.8%; Preferably, the ethanol accounts for 9.1-9.9% of the total mass of the auxiliary material and water, preferably 9.3-9.7%, preferably 9.5%; Preferably, the isopropyl myristate accounts for 4.1-4.9% of the total mass of the excipients and water, preferably 4.3-4.7%, preferably 4.5%; Preferably, the methylparaben accounts for 0.08-0.16% of the total mass of the auxiliary materials and water, preferably 0.1-0.14%, preferably 0.12%; Preferably, the azone accounts for 0.7-2.5%, preferably 0.9-2.3%, preferably 2.1% of the total mass of the auxiliary materials and water; Preferably, the glyceryl monostearate accounts for 8.6-9.4%, preferably 8.8-9.2%, preferably 9% of the total mass of the excipients and water; Preferably, the palmitic acid accounts for 10.4-11.2% of the total mass of the auxiliary materials and water, preferably 10.6-11%, preferably 10.8%; Preferably, the white vaseline accounts for 6.8-7.6% of the total mass of the excipients and water, preferably 7-7.4%, preferably 7.2%; Preferably, the water accounts for 37.78-45.78% of the total mass of the auxiliary materials and water, preferably 39.78-43.78%, preferably 41.78%.

25. The cream according to claim 23, characterized in that Component (i) is 0.01-2.5% of the total mass of the auxiliary materials and water, preferably 0.1-2.4%, preferably 0.5-2%, preferably 0.8-1.7%, preferably 0.8-1.4%, preferably 0.9-1.1%, preferably 1%.

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