Composition of abelmoschus manihot general flavone and vitamin B12 and application of composition in preparation of medicine for treating nephropathy

Abstract

The invention discloses a composition of abelmoschus manihot general flavone and vitamin B12 and application of the composition in preparation of a medicine for treating nephropathy, and belongs to the field of pharmacy, and the pharmaceutical composition comprises the following components: abelmoschus manihot general flavone and VB12. Compared with the prior art, the composition disclosed by the invention has an obvious nephropathy treatment effect and relatively good safety.

Description

Technical Field

[0001] The invention belongs to the pharmaceutical field, and specifically relates to a pharmaceutical composition and application thereof in preparing a drug for treating kidney disease. Background Art

[0002] Medicinal Hibiscus flower is the dried corolla of Abelmoschus manihot (L.) Medic, a plant of the Malvaceae family. It tastes sweet and cold, and enters the kidney and bladder meridians. It has the effects of clearing dampness and heat, reducing swelling and detoxifying, and is used for carbuncle swelling, toxicity, and burns caused by water or fire. This medicine was first recorded in "Jiayou Materia Medica", which records: Hibiscus flower is used to treat all kinds of malignant sores and pus that have not healed for a long time, and it can be applied as powder. "Compendium of Materia Medica" says: Hibiscus flower, its flower smell is sweet, cold, slippery, and non-toxic. It can treat all kinds of malignant sores and pus that have not healed for a long time, and it can be cured by applying it as powder, which is an important medicine for sores.

[0003] Flavonoids are one of the main components of hibiscus flower, and also one of its pharmacologically active ingredients. Hibiscus capsules have been on the market for many years, and their main active ingredient is hibiscus flower extract. Studies have confirmed that the flavonoids in hibiscus flower extract not only have a significant therapeutic effect on sores, but also have a significant protective effect on ischemic damage to the heart, brain, tissues, etc. In addition, many literature reports show that hibiscus flower total flavonoids may have good effects in anti-inflammatory, antipyretic and analgesic, protecting heart and brain ischemic damage, lowering blood sugar, and antiviral. For example, patent CN201210082553.7 discloses a total flavonoid extract of hibiscus flower, containing flavonoids with a weight ratio of (11-16) to (2.5-6) ​​to (4-6.5) of quercetin-3'-glucoside, quercetin-3'-glucoside, and isoquercetin, which can be used to prepare nephritis. Patent CN200610097615.6 discloses a total flavonoid extract of hibiscus flower, 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-acacia glycoside, 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 of hibiscus flower can be used to prepare drugs for treating nephritis, the flavonoid components are clear in qualitative and quantitative terms, and the therapeutic effect is reliable.

[0004] Although bioflavonoids have various therapeutic effects, there are many types of flavonoids, which can currently be 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 effect on ocular blood flow, and when flavonoids are dehydrogenated to flavanones, significant improvement in ocular blood flow is obtained. In addition, compounds that increase blood flow also lead to a significant increase in the recovery of retinal function 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 macular degeneration caused by high myopia. Among them, CNV secondary to AMD is the most common and has become one of the main causes of irreversible visual impairment in the elderly. Insufficient blood flow is also a factor affecting visual fatigue. Increasing eye blood flow or acting on eye smooth muscles can help to restore visual fatigue.

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

[0007] Epidermal growth factor receptor antagonists (EGFRIs) are the most widely used anti-tumor drugs in clinical practice in recent years. They are favored by doctors and patients for their outstanding clinical efficacy. However, their adverse reactions are diverse, and rash (mainly acne / acne-like rash) is the most common one. Acne / acne-like rash not only affects the patient's quality of life, but also may cause the treatment to be unable to proceed normally, seriously affecting the treatment effect of tumors. Acne / acne-like rash limits the application of EGFRIs to a certain extent. Western medicine often uses antibiotics, topical hormones and other methods for treatment, but the effect is not very obvious.

[0008] Skin wounds are a common clinical symptom. Conventional skin wound treatments include skin grafting and artificial substitute coverage. Skin ulcers are common and frequently occurring clinical diseases. They refer to local skin tissue defects caused by various reasons. Wounds are exposed and prone to infection. Chronic skin ulcers, in particular, cannot heal for a long time or are prone to recurrence. Treatment is difficult, seriously affecting the quality of life of patients and bringing a certain social burden. More safe, effective, and economical treatment methods and drug research and development are urgently needed in clinical practice.

[0009] Epidemiological surveys show that the incidence of diabetes is increasing year by year, which is closely related to changes in lifestyle and the aging of society. In addition, 15% of diabetic patients will eventually develop long-term skin ulcers. How to promote the rapid healing of diabetic ulcer wounds, reduce the disability rate, and protect limb function is an important topic and hot topic in today's medical research. An animal model of chronic difficult-to-heal wounds under diabetic conditions can be established by artificially creating sterile wounds on the backs of experimental diabetic rats, so as to carry out research on drugs that promote wound healing.

[0010] The extraction methods of hibiscus flower reported in the literature include ethanol reflux extraction, ultrasonic extraction, and room temperature immersion extraction, but the composition of hibiscus flower extracts is complex, the content of effective ingredients varies greatly, the extraction transfer rate is low, the extraction energy consumption is high, and the amount of solvent used is large. Therefore, it is necessary to explore a hibiscus flower therapeutic drug and its extraction process that can be used to treat kidney disease, especially diabetic nephropathy, and can also treat multiple diseases such as eye diseases.

[0011] The applicant counted the adverse reactions of Huangkui Capsules and found that in a recent year, a total of 194 cases of adverse reactions to Huangkui Capsules were received. According to the statistics of adverse drug reactions, nausea, itching, rash, vomiting, diarrhea, upper abdominal distension and discomfort occurred more than 10 times, all of which are known adverse reactions. Recently, new ADR manifestations have appeared, such as 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 Huangkui Capsules damage liver cells. Therefore, it is clinically significant to develop a new hibiscus flower flavonoid extract to reduce side effects and be used to treat diseases such as ulcers.

[0012] Vitamin B12 (VB12) is one of the B vitamins, CAS number: 13408-78-1, is a kind of complex organic compound containing cobalt, molecular formula C 63 H 88 CoN 14 O 14 P. The molecular structure is a nucleotide composed of a corrin ring with a cobalt ion as the center and 5,6-dimethylbenzimidazole as the base. The effects and functions of vitamin B12 mainly include promoting the development and maturation of red blood cells, preventing pernicious anemia, increasing the utilization of folic acid, and promoting the metabolism of carbohydrates, fats and proteins. At present, there is no clear research on the therapeutic effect of VB12 on kidney disease. For example, Chen Huanzhen et al. explored the relationship between serum folic acid and VB12 and early diabetic nephropathy. The results showed that folic acid may be related to early diabetic nephropathy, while VB12 did not show a correlation.

[0013] There is no detailed report on the research of flavonoids extract from Hibiscus tiliaceus and VB12 in kidney disease. Summary of the invention

[0014] The present invention provides a pharmaceutical composition and its application in the preparation of a drug for treating kidney disease. By means of chemical component separation and accompanying pharmacological activity tracking, an effective component for treating kidney disease is obtained, and by a dose-effect relationship study, an optimal ratio of total flavonoids of hibiscus flower and VB12 is obtained. It is found that under this ratio, VB12 can cooperate with total flavonoids of hibiscus flower to further improve the therapeutic effect on kidney disease.

[0015] At the same time, the hibiscus flower total flavonoids or the pharmaceutical composition of the present invention has lower side effects, thus providing a better therapeutic drug for patients.

[0016] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0017] In a first aspect, the present invention provides a pharmaceutical composition comprising the following components: total flavonoids from hibiscus flowers and VB12.

[0018] The total flavonoids of hibiscus flower in the present invention comprises at least one of hibiscus flower extract, hibiscus total flavonoids extract and hibiscus flower flavonoids effective parts.

[0019] The mass ratio of the total flavonoids of hibiscus flower to VB12 in the present invention is 6500-95000:1, preferably 6550-8550:1, 74000-94000:1. As a preferred embodiment of the present invention, the mass ratio of the total flavonoids of hibiscus flower to VB12 is 7550:1, 84000:1.

[0020] The total flavonoids of hibiscus flower of the present invention include 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;

[0021] 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;

[0022] 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;

[0023] 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;

[0024] 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;

[0025] 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;

[0026] 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;

[0027] 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.

[0028] Specifically, the mass ratio of quercetin-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 quercetin-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-acacia glycoside, wherein the mass ratio of gossypol-8-O-β-D-glucuronide to quercetin-3-O-acacia glycoside is 10:0.1-2.5, preferably 10:0.1-0.9, more preferably 10:0.15-0.5.

[0029] In other preferred embodiments, the total flavonoids of hibiscus flower include flavonoid components in the following mass ratio: the mass ratio of gossypol-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;

[0030] 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;

[0031] 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;

[0032] 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;

[0033] 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;

[0034] 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;

[0035] 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;

[0036] 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;

[0037] 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;

[0038] 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.

[0039] Further, 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; further, it contains quercetin-3-O-acacia glycoside, wherein the mass ratio of gossypol-8-O-β-D-glucuronide to quercetin-3-O-acacia glycoside is 10:0.1-2.5, preferably 10:0.1-0.9, and preferably 10:0.15-0.5.

[0040] In other preferred embodiments, the total flavonoids of hibiscus flower are hibiscus flower extract.

[0041] The hibiscus flower extract comprises the following flavonoid components in a mass ratio: the mass ratio of gossypol-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;

[0042] 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;

[0043] 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;

[0044] 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;

[0045] 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;

[0046] 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;

[0047] 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;

[0048] 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;

[0049] 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;

[0050] 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, or 10:6-15:5-13:1.0-1.8:4.0-5.9:0.6-0.99.

[0051] Further, 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; further, it contains quercetin-3-O-acacia glycoside, wherein the mass ratio of gossypol-8-O-β-D-glucuronide to quercetin-3-O-acacia glycoside is 10:0.1-2.5, preferably 10:0.1-0.9, and preferably 10:0.15-0.5.

[0052] In other preferred embodiments, the total flavonoids of hibiscus flower are the effective part of hibiscus flower flavonoids, and the effective part of hibiscus flower flavonoids includes flavonoid components in the following mass ratios: the mass ratios of isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin: gossypol-8-O-β-D-glucuronide: hyperoside are: 0.8-1.2: 0.05-1.6: 0.2-4.6: 0.05-1.2: 0.2-3.5: 0.25-3.6.

[0053] Specifically, the flavonoid effective part of hibiscus flower includes 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.

[0054] Specifically, the effective flavonoid part of hibiscus flower includes flavonoid components in the following mass ratio: the mass ratio of isoquercetin:quercetin:quercetin-3'-O-glucoside: myricetin: gossypol-8-O-β-D-glucuronide: hyperoside 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.

[0055] Specifically, the effective flavonoid part of hibiscus flower includes flavonoid components in the following mass ratio: the mass ratio of isoquercetin:quercetin:quercetin-3'-O-glucoside: myricetin: gossypol-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.

[0056] Specifically, the effective flavonoid part of hibiscus flower includes flavonoid components in the following mass ratio: the mass ratio of isoquercetin:quercetin:quercetin-3'-O-glucoside: myricetin: gossypol-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.

[0057] More specifically, the mass ratio of the isoquercetin is 1.2 or 1.0.

[0058] More specifically, the hibiscus flower flavonoid effective part 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.

[0059] In the pharmaceutical composition of the present invention, the total content of quercetin, quercetin-3'-O-glucoside, myricetin, gossypol-8-O-β-D-glucuronide, isoquercetin and hyperoside contained in the total flavonoids of hibiscus flower 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 13-2 5%, preferably 14-25%, or 12.6-23%, or 13-20%; further, the content of quercetin is more than 0.7%, preferably more than 1.0%, preferably 1.0-12%, preferably 1.5-12%, or 1.0%-10%, or 1.5%-5%; further, the content of quercetin-8-O-β-D-glucuronide is 8.5-34%, preferably 12-34%, or 8.5-30%, or 12%-23%.

[0060] In other preferred embodiments, the total flavonoids of hibiscus flower contain quercetin, quercetin-3'-O-glucoside, myricetin, gossypol-8-O-β-D-glucuronide, isoquercetin, rutin and hyperoside with a total content of more than 55%, preferably more than 60%, 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%.

[0061] In other preferred embodiments, the Hibiscus tiliaceus flower extract comprises the following components by mass content: 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, 12.0-19.8% isoquercetin, 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; Uridine 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%.

[0062] Specifically, the hibiscus flower also contains 0.01-1.0% rutin by mass, 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 0.08-2.5% quercetin-3-O-acacia glycoside, preferably 0.1-1.5%, further 0.1-0.9%, further 0.1-0.8%, further 0.1-0.5%.

[0063] Specifically, the mass content of flavonoid components in the hibiscus flower extract is above 55%, preferably above 60%, preferably 65-90%, and preferably 69-82%.

[0064] In a second aspect, the present invention provides a method for preparing the above-mentioned total flavonoids of hibiscus flower, comprising the following steps: (1) extracting hibiscus flower or the medicinal part of hibiscus 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 of hibiscus flower flavonoids or a hibiscus flower extract; 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 extractant used for the extraction in step (2) is n-butanol, petroleum ether or ethyl acetate, the extraction method is continuous countercurrent extraction, the solid-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.

[0066] Specifically, step (2) further comprises subjecting the extract to activated carbon adsorption, alcohol precipitation or acid precipitation treatment before extraction.

[0067] 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 4-12 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.

[0068] In some preferred embodiments, the method for preparing the total flavonoids of hibiscus flower 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 out the supernatant; (3) eluting the supernatant with a polyamide resin to obtain the effective part of hibiscus flower flavonoids or hibiscus flower extract; the preferred extraction method is percolation.

[0069] Specifically, 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 amount of the clarifier in step (2) is 4-8% of that of hibiscus flower or hibiscus medicinal part; 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.

[0070] In some preferred embodiments, the preparation method of the total flavonoids of hibiscus flower 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 out the 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 of hibiscus flower flavonoids or hibiscus flower extract; the preferred extraction method is reflux.

[0071] Specifically, 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.

[0072] In a fourth aspect, the present invention provides the use of the above-mentioned pharmaceutical composition in the preparation of a drug for treating kidney disease; preferably, the kidney disease includes nephritis; further preferably, the kidney disease is chronic glomerulonephropathy, diabetic nephropathy, diabetic nephropathy with renal fibrosis and / or nephritis.

[0073] In a fifth aspect, the present invention provides the use of total flavonoids from hibiscus flowers in the preparation of a drug for treating kidney disease in combination with VB12.

[0074] Preferably, the hibiscus flower total flavonoids are the hibiscus flower total flavonoids in the aforementioned pharmaceutical composition.

[0075] Preferably, the renal disease includes nephritis; further preferably, the renal disease is chronic glomerular nephropathy, diabetic nephropathy, diabetic nephropathy with renal fibrosis and / or nephritis.

[0076] In a sixth aspect, a combination of hibiscus flower total flavonoids and VB12 is preferably directly mixed together or combined together after being packaged separately. The hibiscus flower total flavonoids are the hibiscus flower total flavonoids in the aforementioned pharmaceutical composition.

[0077] In a seventh aspect, the present invention provides a hibiscus flower total flavonoids cream, which contains (i) the above-mentioned pharmaceutical composition, and (ii) pharmaceutically acceptable excipients; preferably, the cream also includes (iii) water.

[0078] Wherein, 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.

[0079] Wherein, the Tween accounts for 8-16% of the total mass of the auxiliary materials and water, preferably 10-14%, preferably 12%.

[0080] 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%, preferably 1.2%.

[0081] 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%.

[0082] Wherein, 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%.

[0083] 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%, preferably 4.5%.

[0084] 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%.

[0085] 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%.

[0086] Wherein, the glyceryl monostearate accounts for 8.6-9.4% of the total mass of the auxiliary materials and water, preferably 8.8-9.2%, preferably 9%.

[0087] 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%.

[0088] Wherein, the white vaseline accounts for 6.8-7.6% of the total mass of the auxiliary materials and water, preferably 7-7.4%, preferably 7.2%.

[0089] 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%.

[0090] Among them, 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%.

[0091] The hibiscus flower flavonoid effective part (hibiscus flower extract) of the present invention not only detects the seven components of hyperoside, rutin, isoquercetin, hibifolin, myricetin, quercetin-3'-o-glucoside, and quercetin, but also converts the peak area of ​​quercetin-3-O-acacia glycoside content of the solid sample prepared by the embodiment of the present invention into content according to the proportion, and 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-acacia glycoside*fixed volume / sample weight (after water reduction), and the content is not higher than 2.0%, further not higher than 0.9%, and preferably 0.1%-0.7%. The total content of the detected multiple flavonoid components is above 70%, further above 75%, and further 80%-90%.

[0092] Beneficial effects:

[0093] (1) The pharmaceutical composition of the present invention can effectively treat kidney diseases such as membranous glomerulonephritis, postoperative acute renal injury or immunoglobulin A nephropathy, idiopathic focal segmental glomerulosclerosis, chronic glomerulonephropathy, diabetic nephropathy, diabetic nephropathy with renal fibrosis and / or nephritis through the combination of total flavonoids of hibiscus flowers and VB12, especially having a good therapeutic effect on chronic glomerulonephropathy.

[0094] (2) The total flavonoids of hibiscus flowers provided by the present invention have a good preventive and therapeutic effect on skin diseases, such as dermatitis and acne.

[0095] (3) The total flavonoids of hibiscus flowers described in the present invention have the effect of treating various nephritis, such as diabetic nephropathy, contrast-induced renal injury or lupus erythematosus nephritis, and also have 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 total flavonoids of hibiscus flowers described in the present invention also have the effect of treating pulmonary fibrosis.

[0096] (4) The total flavonoids of hibiscus flowers provided by the present invention can effectively treat skin diseases at a lower dosage.

[0097] (5) The total flavonoids of hibiscus flowers described in the present invention have lower side effects and provide a better treatment method for patients.

[0098] (6) The preparation method of the present invention has simple process, short production cycle, mild treatment conditions, low energy consumption and good separation effect of the seven flavonoid components of hibiscus flower. The raw materials and reagents used are widely available, low in cost, and easy to realize industrial large-scale production.

[0099] (7) The total flavonoids of hibiscus flower provided by the present invention have the effect of promoting wound healing, and have obvious effects of promoting wound healing and treating diabetic ulcers and have good safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0100] Figure 1 This is the dissolution process of 5-ASA and ZHT.

[0101] Figure 2 The effect of ZHT on DSS-induced colon atrophy; A is the colon appearance picture of each group of mice, B is the statistical chart of colon length of each group of mice, and the 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.

[0102] Figure 3 The effect of ZHT on DSS-induced colonic tissue pathological changes; A is the H&E staining picture of the colon tissue of each group of mice, under 5x objective magnification, Scale bar = 500μm; under 20x objective magnification, Scale bar = 100μm, B is the statistical chart of the colon tissue inflammation score of each group of mice. 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.

[0103] Figure 4 These are the pathological images of each group in the oral ulcer efficacy experiment, among which a is the pathological image of the blank group, b is the pathological image of the model group, c is the pathological image of the alcohol extract group, d is the pathological image of the water extract group, and e is the pathological image of the total flavonoid extract group.

[0104] Figure 5 Flow chart for preparation, drug administration, and material collection of acne model.

[0105] Figure 6 Flowchart for construction and dosing of atopic dermatitis model.

[0106] Figure 7 The effects of ZHT and HKY on the appearance of DNFB-induced atopic dermatitis model.

[0107] Figure 8 The effects of ZHT and HKY on the skin pathological changes in the DNFB-induced atopic dermatitis model, where the representative images are 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.

[0108] Fig. 9 A is a representative graph of the skin wound area of ​​rats in each group at different time points; A is a representative graph of the skin wound area of ​​rats in each group at different time points, B is a graph of the skin wound healing curve of rats in each group, *P<0.05, **P<0.01 vs. Model.

[0109] Fig.10 HE staining results of rat skin wound tissue and quantitative graph of epidermal thickness; A is the HE staining results of rat wound and edge tissue, B is the quantitative graph of epidermal thickness of rat skin wound in each group, **P<0.01, vs. Model; ##, P<0.01, vs. Control.

[0110] Fig.11 These are the Masson staining results of rat skin wound tissue and the quantitative graph of collagen deposition; A is the Masson staining results of rat wound and edge tissue, B is the quantitative graph of collagen deposition in rat skin wounds of each group, N=6, *P<0.05, **P<0.01vs.Model; ##P<0.01, vs.Control.

[0111] Fig.12 The changes of CD31 and VEGF mRNA levels in rat skin wound tissue; A shows the changes of CD31 mRNA levels in rat skin wound tissue after 9 days of treatment, and B shows the changes of VEGF mRNA levels in rat skin wound tissue after 9 days of treatment.

[0112] Fig.13 These are the results of CD31 immunofluorescence staining of rat skin wound tissue and the quantitative graph of fluorescence intensity; A is the CD31 immunofluorescence staining of rat skin tissue after 9 days of treatment, the scale bar is 250μm; B is the quantitative graph of CD31 fluorescence intensity, N=6, #P<0.05vs.control.

[0113] Fig.14 Changes in VEGF protein levels in rat skin wound tissue; Changes in VEGF protein levels in rat skin wound tissue 9 days after modeling, N=6, **P<0.05vs model.

[0114] Fig.15 is the urine creatinine content in the urine of rats with adriamycin nephritis model.

[0115] Fig.16 is the urine protein content in the urine of rats with adriamycin nephritis model.

[0116] Fig.17 The urine protein content in the urine of diabetic kidney model mice, #P<0.05vs.Model; *P<0.05, vs.Normal.

[0117] Fig.18 The urine protein content in the urine of mice with adriamycin nephritis model, #P<0.05vs.Model; *P<0.05, vs.Normal.

[0118] Fig.19 The urinary creatinine content in the urine of mice with adriamycin nephritis model, #P<0.05vs.Model; *P<0.05, vs.Normal.

[0119] Fig. 20 The results of UACR values ​​before grouping and the fourth week of administration, before grouping: DKD (n=13), HK (n=13), HK+VB (n=13), HT (n=14), HT+VB (n=13); the fourth week of administration: DKD (n=4), HK (n=4), HK+VB (n=4), HT (n=4), HT+VB (n=4), in the figure, compared with the DKD group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0120] Fig.21 This is the result of HE staining (200×) of kidney tissue of db / db mice. DETAILED DESCRIPTION

[0121] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional unless otherwise specified, and the experimental methods for which specific conditions are not specified in the examples are generally conventional. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified. The percentage content in the present invention is only mass percentage.

[0122] The hibiscus flowers in the following embodiments are dried hibiscus corollas, and unless otherwise specified, are from the same batch of hibiscus flowers.

[0123] The contents of 7 components of the flavonoid effective parts of Hibiscus tiliaceus prepared in each embodiment were determined. These 7 components are: hyperoside, rutin, isoquercetin, quercetin-8-O-β-D-glucuronide, myricetin, quercetin-3'-o-glucoside, and quercetin. During the preparation of the effective part samples, the solution before the effective part was dried was also tested for the content of each component, and the sample content before and after drying had a certain change. The detection method uses the UPLC method reported in "One Test and Multiple Evaluation Method for Determining 7 Components in Hibiscus tiliaceus Flowers" ("Journal of Pharmaceutical Analysis", Issue 12, 2013, 2082-2087) to determine the content of the components therein.

[0124] The material-liquid ratio of the extraction (i.e., extractant: extracting liquid) refers to the volume ratio.

[0125] Unless otherwise specified, the Huangkui Capsules described in the following examples are all Huangkui Capsules produced and sold by Suzhong Pharmaceutical Group Co., Ltd.

[0126] Example 1 Preparation of the effective fraction of flavonoids from Hibiscus tiliaceus flowers

[0127] 100g of hibiscus flower was crushed into coarse powder, and 15 times of 60% ethanol was used for percolation extraction to obtain hibiscus flower extract; ethanol was removed from the hibiscus flower extract, diluted with water, and adsorbed on activated carbon, and then continuously countercurrent extracted with n-butanol, the solid-liquid ratio of the extraction (i.e., extractant: extracting solution) was 1.5:1, the injection speed of the extraction was 40mL / min, the speed of the extraction machine was 40Hz, the extraction level of the extraction was 2, the extract was decompressed and the solvent was recovered at 40°C, and then treated with D101 macroporous resin, and the treatment process of the macroporous resin was as follows: the diameter-to-height ratio of the macroporous resin was 1:6, the concentration of the loading solution (aqueous solution) was 0.15g crude drug / mL, the loading solution volume was 7BV, the sample was loaded at a flow rate of 2BV / h, 6BV pure water and 3BV 5% ethanol at a flow rate of 2BV / h for impurity removal, 4BV60% ethanol at a flow rate of 2BV / h for elution 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 2.23g of total solids of hibiscus flower flavonoids effective parts were obtained. The total of the seven components was 1769mg, accounting for 79.32% of the total solids.

[0128] Table 1. Sample content determination data

[0129]

[0130] Example 2 Preparation of the effective fraction of flavonoids from Hibiscus tiliaceus flowers

[0131] Example 2-1

[0132] 100g of hibiscus flower was crushed into coarse powder, and 70% ethanol was used for percolation extraction to obtain hibiscus flower extract; ethanol was removed from the hibiscus flower extract, diluted with water, and continuously countercurrently extracted with ethyl acetate, the solid-liquid ratio of the extraction (i.e., extractant: extracting solution) was 2.5:1, the injection speed of the extraction was 80mL / min, the speed of the extraction machine was 40Hz, the extraction level of the extraction was 4, the extract was reduced pressure at 40°C to recover the solvent, and then 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 1:5, the concentration of the sample solution was 0.15g crude drug / mL, the volume of the sample solution was 6BV, the sample was loaded at a flow rate of 2BV / h, 6BV of pure water and 3BV of 5% ethanol were used for impurity removal at a flow rate of 2BV / h, 4BV 60% ethanol was eluted at a flow rate of 1.5 BV / h to obtain an eluate, and then the eluate was decompressed at 60°C to recover ethanol to obtain a hibiscus flower flavonoid extract, and the content was tested. According to the content determination results, the contents of the seven components were adjusted by addition method, so that the contents of each component were as shown in the following table, and the total solid content of the hibiscus flower flavonoid effective part was 2.99. The content of quercetin-3-O-acacia glycoside was 0.28%.

[0133] Table 2. Sample content determination data

[0134]

[0135] Example 2-A Preparation of the effective fraction of flavonoids from Hibiscus tiliaceus flowers

[0136] According to the method of Example 2-1 above, hibiscus flower was magnified 10 times, and 2 batches of hibiscus flower flavonoid effective parts were prepared to obtain samples with content determination data of samples in Table 3. The content of quercetin-3-O-acacia glycoside was 0.20-0.50%.

[0137] Table 3. Sample content determination data

[0138]

[0139] Among them, 2-A1 contains 0.24% quercetin-3-O-acacia glycoside content converted by peak area, and the proportion of each component is shown in Table 4 below.

[0140] Table 4. Content ratio data between the components of the samples

[0141]

[0142] Example 2-A3

[0143] Take hyperoside, isoquercetin, hibifolin, myricetin, quercetin-3'-o-glucoside, quercetin, and quercetin 3-O-acacia glycoside as raw materials, and mix them according to the ratio of Example 2-A1 (quercetin 3-O-acacia glycoside replaces rutin, and the ratio is adjusted to 0.02) to prepare total flavonoids from hibiscus flower as shown in Table 5.

[0144] Table 5.

[0145]

[0146] Example 2-A4

[0147] Take hyperoside, isoquercetin, hibifolin, myricetin, quercetin-3'-o-glucoside, quercetin, and quercetin 3-O-acacia glycoside as raw materials, mix them according to the ratio of Example 2-A1 (quercetin 3-O-acacia glycoside replaces rutin, and the ratio is adjusted to 0.05) to prepare total flavonoids from hibiscus flower.

[0148] Example 2-B

[0149] In order to obtain an effective part of hibiscus flower with uniform quality, the content relationship between the ingredients in the medicinal material and the extract was studied according to the content results of the extracts of multiple batches of Example 2-1. Three batches of hibiscus flowers with different sources and large differences in ingredients were mixed according to the software calculation method. 2000 g of the mixed hibiscus flowers were extracted by 18 times 70% ethanol percolation to obtain hibiscus flower extract, ethanol was removed, water was diluted, and continuous countercurrent extraction was performed with ethyl acetate. The solid-liquid ratio of the extraction (i.e., extractant: extract) was 3:1. The extraction injection speed is 120mL / min, the extraction machine speed is 60Hz, the extraction level is 4, the extract is reduced pressure at 50°C to recover the solvent, then diluted with water, centrifuged, and the supernatant is treated with D101 macroporous resin. The treatment process of the macroporous resin is as follows: the macroporous resin diameter-to-height ratio 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 3BV 5% ethanol are used for impurity removal at a flow rate of 2BV / h, and 4BV 60% ethanol was eluted at a flow rate of 2BV / h to obtain an eluate, which was concentrated and dried under reduced pressure at 60°C to obtain a total solid content containing 0.22% rutin, 20.58% hyperoside, 17.81 isoquercetin, 16.07% hibifolin, 5.15% myricetin, 17.37% quercetin-3'-o-glucoside, 1.77% quercetin, and 0.43% quercetin-3-O-acacia glycoside peak area. The mass ratios of the components are shown in Table 6 below.

[0150] Table 6. Content ratio data between the components of the samples

[0151]

[0152] Example 3 Preparation of the effective fraction of flavonoids from Hibiscus tiliaceus flowers

[0153] Example 3-1

[0154] 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: extracting solution) was 3:1, the injection speed of the extraction was 100mL / min, the speed of the extraction machine was 40Hz, the extraction level of the extraction 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 diameter-to-height ratio of the macroporous resin was 1:8, the concentration of the sample solution was 0.15g crude drug / mL, the volume of the sample solution 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 60% ethanol was eluted at a flow rate of 3BV / h to obtain an eluate, and then the eluate was decompressed at 60°C to recover ethanol to obtain a hibiscus flower flavonoid extract, and the content was tested. According to the content determination results, the contents of the seven components were adjusted by addition method, so that the contents of each component were as shown in Table 7 below, and 3.18g of total solid content of hibiscus flower flavonoid effective parts was obtained. The content of quercetin-3-O-sophoroside was 0.20-0.50%.

[0155] Table 7. Sample content determination data

[0156]

[0157]

[0158] Example 3-2

[0159] According to the preparation method of Example 3-1, 500 g of Hibiscus hibiscus flower medicinal material was used for repeated experiments, and the content data of 2 batches were obtained as shown in Table 8. The content of quercetin-3-O-acacia glycoside was 0.30-0.80%. Where A represents "each component / total solids (%)", and B represents "each component / isoquercetin".

[0160] Table 8. Content determination data of samples

[0161]

[0162] Example 4 Preparation of the effective fraction of flavonoids from Hibiscus tiliaceus flowers

[0163] Example 4-1

[0164] 100g of hibiscus flower 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 countercurrently extracted with n-butanol, the material-liquid ratio of the extraction (i.e., extractant: extracting solution) was 3:1, the injection speed of the extraction was 120mL / min, the speed of the extraction machine was 60Hz, the extraction level 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 diameter-to-height ratio of the macroporous resin was 1:6, the concentration of the sample solution was 0.15g crude drug / mL, the volume of the sample solution was 7BV, the sample was loaded at a flow rate of 2BV / h, 6BV of pure water and 3BV of 5% ethanol were used for impurity removal at a flow rate of 2BV / h, 4BV The eluate was eluted with 60% ethanol at a flow rate of 2BV / h to obtain an eluate, and then the eluate was decompressed at 60°C to recover the ethanol to obtain a hibiscus flower extract. The content was tested, and the contents of the seven components were adjusted by addition method according to the determination results, so that the contents of each component were as shown in Table 9, and 2.05 g of total solids of the effective part of flavonoids in hibiscus flower were obtained.

[0165] Table 9. Sample content determination data

[0166]

[0167] Example 4-2

[0168] The flower of Hibiscus tiliaceus was extracted by diafiltration 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 added was 4% B component and 2% A component respectively. The water bath temperature was 60°C, the water bath insulation time was 60min, centrifugation, filtration, dilution, and treatment 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 pure water and 5BV 80% ethanol to obtain an eluate, which was concentrated and dried under reduced pressure at 60°C. The content was tested, and the contents of the seven components were adjusted by addition method according to the content determination results, so that the contents of each component were as shown in Table 10 below, and the total solids of the effective part of flavonoids of Hibiscus tiliaceus was obtained, and the content of quercetin-3-O-acacia glycoside was 0.39%.

[0169] Table 10. Content determination data of samples

[0170]

[0171] Example 5 Preparation of the effective fraction of flavonoids from Hibiscus tiliaceus flowers

[0172] Example 5-1

[0173] 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 countercurrently extracted with n-butanol, the solid-liquid ratio of the extraction (i.e., extractant: extracting solution) was 2.5:1, the injection speed of the extraction was 80mL / min, the speed of the extraction machine was 50Hz, the extraction level 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 diameter-to-height ratio of the macroporous resin was 1:6, the concentration of the sample solution was 0.2g crude drug / mL, the volume of the sample solution 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, 3BV The eluate was eluted with 60% ethanol at a flow rate of 2BV / h, and the eluate was decompressed at 60°C to recover the ethanol to obtain a hibiscus flower extract. The content was tested, and the contents of seven components were adjusted by addition method according to the determination results, as shown in Table 11, to obtain 2.86g of total solids of flavonoids effective parts of hibiscus flower.

[0174] Example 5-2

[0175] The flower of Hibiscus tiliaceus is extracted by reflux with 15 times 70% ethanol, the ethanol is removed from the extract to 0.5-1.0 g crude drug / ml, 10% hydrochloric acid solution is added to adjust the pH value to 2.0-3.0, refrigerated, filtered, washed, the precipitate is taken, dissolved in water, and continuously countercurrent extracted with ethyl acetate, the material-liquid ratio of the extraction (i.e., the extractant: the extract) is 3:1, the extraction level is 4, the extract is reduced pressure at 50°C to recover the solvent, diluted with water, and treated with polyamide resin, the resin treatment process is: the resin diameter-height ratio is 1:6, the sample solution concentration is 0.15-0.5 g crude drug / mL, the sample solution volume is 6BV, 4BV pure water and 4BV 70% ethanol are used to elute to obtain the eluent, concentrated, and dried under reduced pressure at 60°C to obtain the quercetin-3-O-acacia glycoside content of 0.47%.

[0176] Table 11. Sample content determination data

[0177]

[0178] Example 6 Preparation of the effective fraction of flavonoids from Hibiscus tiliaceus flowers

[0179] 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 countercurrently extracted with ethyl acetate, the solid-liquid ratio of the extraction (i.e., extractant: extracting solution) was 2:1, the injection speed of the extraction was 100mL / min, the speed of the extraction machine was 40Hz, the extraction level was 4, the extract was reduced pressure at 50°C to recover the solvent, and then 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 1:6, the concentration of the sample solution was 0.3g crude drug / mL, the volume of the sample solution was 3BV, the sample was loaded at a flow rate of 2BV / h, 3BV of pure water and 3BV of 5% ethanol were used for impurity removal at a flow rate of 2BV / h, 4BV The eluate was eluted with 70% ethanol at a flow rate of 2BV / 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 Table 12 below. The eluate was then decompressed and ethanol was recovered at 60°C to obtain a hibiscus flower extract, which was tested for content. The contents of the seven ingredients were adjusted by addition method according to the content determination results, and the added amount did not exceed 10% of the total weight, so that the contents of each ingredient were as shown in the following table, and 3.16 g of total solids of flavonoids effective parts of hibiscus flower were obtained.

[0180] Table 12. Sample content determination data

[0181]

[0182] Example 7 Preparation of the effective fraction of flavonoids from Hibiscus tiliaceus flowers

[0183] Example 7-1

[0184] 500 g of hibiscus flower was crushed into coarse powder, and extracted by percolation with 20 times 70% ethanol to obtain hibiscus flower extract; after reducing pressure to recover the solvent, it was treated with D101 macroporous resin, and 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 crude drug / mL, the loading solution volume was 6BV, the sample was loaded at a flow rate of 2BV / h, 7BV of pure water and 4BV of 5% ethanol were used at a flow rate of 2BV / h for impurity removal, 4BV of 60% ethanol was used at a flow rate of 1.5BV / h to obtain an eluate, and the eluate was reduced pressure at 60°C to recover ethanol to obtain 16.12 g of hibiscus flower total solids, and the contents of each component thereof are shown in Table 13 below.

[0185] Table 13. Sample content determination data

[0186]

[0187] Example 7-2

[0188] 100 g of hibiscus flower was crushed into coarse powder, and extracted by percolation with 18 times 70% ethanol to obtain hibiscus flower extract; after reducing pressure to recover the solvent, it was treated with D101 macroporous resin, and the treatment process of the macroporous resin was as follows: the diameter-to-height ratio of the macroporous resin was 1:5, the loading solution concentration was 0.15 g crude drug / mL, the loading solution volume was 6BV, the sample was loaded at a flow rate of 2BV / h, 6BV of pure water and 3BV of 5% ethanol were used at a flow rate of 2BV / h for impurity removal, 4BV of 60% ethanol was used at a flow rate of 1.5BV / h for elution to obtain an eluate, and the eluate was reduced pressure at 60°C to recover ethanol to obtain 3.56 g of hibiscus flower total solids, and the contents of each component thereof are shown in Table 14 below.

[0189] Table 14. Sample content determination data

[0190]

[0191] Example 8 Preparation of the Effective Fraction of Flavonoids from Hibiscus tiliaceus Flowers (Comparative Example)

[0192] 100g of hibiscus flower was crushed into coarse powder, and 70% ethanol was used for percolation extraction to obtain hibiscus flower extract; ethanol was removed from the hibiscus flower extract, diluted with water, and continuously countercurrently extracted with ethyl acetate, the solid-liquid ratio of the extraction (i.e., extractant: extracting solution) was 2.5:1, the injection speed of the extraction was 80mL / min, the speed of the extraction machine was 40Hz, the extraction level of the extraction was 4, the extract was reduced pressure at 40°C to recover the solvent, and then 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 1:5, the concentration of the sample solution was 0.15g crude drug / ml, the volume of the sample solution was 6BV, the sample was loaded at a flow rate of 2BV / h, 6BV of pure water and 3BV of 5% ethanol were used for impurity removal at a flow rate of 2BV / h, 4BV 60% ethanol was used for elution 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 Table 15. Then, ethanol was recovered under reduced pressure at 60°C to obtain 4.28 g of total solids of hibiscus flowers.

[0193] Table 15. Sample content determination data

[0194]

[0195] Example 9

[0196] Example 9-1 Preparation of the effective fraction of flavonoids from Hibiscus tiliaceus flowers

[0197] 25 kg of hibiscus flower was crushed into coarse powder, and 18 times of 70% ethanol was used for percolation extraction to obtain hibiscus flower extract; ethanol was removed from the hibiscus flower extract, diluted with water, and continuously countercurrently extracted with ethyl acetate, the solid-liquid ratio of the extraction (i.e., extractant: extract) was 2.5:1, the extraction level was 4, the extract was reduced pressure at 40°C to recover the solvent, 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 loading solution concentration was 0.15 g crude drug / mL, the loading solution volume was 3BV, the sample was loaded at a flow rate of 1.5BV / h, 6BV pure water and 3BV 10% ethanol was used for impurity removal at a flow rate of 2BV / h, and 4BV60% ethanol was used for elution at a flow rate of 1.5BV / h to obtain an eluate, and the eluate was decompressed at 60°C to recover ethanol to obtain the total solids of hibiscus flower. By adding 7 specific ingredients, the 7 ingredients were adjusted (the content of the added ingredients did not exceed 20% of the total weight) so that the content of each ingredient was as shown in Table 16 below, and the total solids of the effective part of flavonoids of hibiscus flower were obtained. The content of quercetin-3-O-acacia glycoside was 0.20-0.50%.

[0198] Table 16. Sample content determination data

[0199]

[0200] Example 9-2 Preparation of the effective fraction of flavonoids from Hibiscus tiliaceus flowers

[0201] Reference Example 2-B, 30kg of hibiscus flower mixture 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 solid-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 level was 4, the extract was reduced pressure at 40°C to recover the solvent, and then treated with D101 macroporous resin, the treatment process of the macroporous resin was: 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 5BV, the sample was loaded at a flow rate of 1.5BV / h, 3BV pure water and 4BV5% ethanol were used at a flow rate of 2BV / h for impurity removal, and 5BV The eluate was eluted with 60% ethanol at a flow rate of 1.5 BV / h, and the eluate was decompressed at 65°C to recover the ethanol to obtain the total solids of hibiscus flowers, which was vacuum dried and crushed. The contents of flavonoids (%) of the seven components were detected. The results are shown in Table 17, wherein the content of quercetin-3-O-acacia glycoside is 0.20-0.50%.

[0202] Table 17. Sample content determination data

[0203]

[0204] Example 10 Preparation of eye drops containing the effective fraction of flavonoids from hibiscus flowers

[0205] Preparation process: The effective part prepared by any method of Examples 1-7 is added with injection water, stirred and dissolved, added to 100L, and the pH is adjusted to 7, filtered, filled, and sterilized to obtain eye drops. The pH adjuster used is sodium hydroxide and / or hydrochloric acid.

[0206] Example 11 Pharmacological Experiment on Diabetic Kidney Model

[0207] Example 11-1

[0208] Animal model: Streptozotocin (STZ) + high-fat diet diabetic kidney model, C57BL / 6 mice, 20±10g, SPF grade, male.

[0209] Administration method: intragastric administration.

[0210] Experimental modeling: After 3 days of adaptive feeding, the experimental mice were fed with a high-fat and high-sugar diet (self-made in the laboratory, formula: lard: sucrose: egg yolk: basic feed = 18:20:3:59) for 8 consecutive weeks, and then began to be injected with STZ sodium citrate solution at a dose of 100 mg / kg intraperitoneally for 7 consecutive days. The blood glucose level of the mice was tested through the tail vein on the 8th day, and the blood glucose level greater than 16.7 mmol / L was used as the standard for the diabetic model. The modeling was successful when the blood glucose was greater than 13.8 mmol / L, urine protein and renal function abnormalities occurred.

[0211] Experimental groups: normal group, model group, positive drug group (dapagliflozin tablets, 45.5 mg / kg), sample group prepared by the method of Example 2-1 (62.4 mg / kg), sample group prepared by the method of Example 6 (62.4 mg / kg), sample group prepared by the method of Example 8 (62.4 mg / kg), a total of 6 groups, 15 mice in each group, urine protein and urine creatinine were detected after 4 weeks of administration, and the daily food intake and mouse status were calculated. The test results are shown in Table 18 below.

[0212] Table 18.

[0213] Group Urine protein / mg Urine protein / creatinine Diarrhea / Number Abdominal distension / number Normal group 19.37±13.28 0.24±0.08 0 0 Model Group <![CDATA[58.36±36.31 * ]]> <![CDATA[2.82±1.12 * ]]> 0 0 Positive drug group <![CDATA[17.51±12.34 # ]]> <![CDATA[1.04±1.09 # ]]> 0 0 Example 8 Sample Group 39.62±18.67 <![CDATA[1.81±0.37 # ]]> 3 2 Example 2-1 Sample Group <![CDATA[20.15±15.67 # ]]> <![CDATA[1.04±2.21 # ]]> 1 0 Example 6 Sample Group 29.74±17.78 <![CDATA[1.11±0.98 # ]]> 4 3

[0214] Note: *Compared with the normal control group, P < 0.05; #Compared with the model control group, P < 0.05.

[0215] Each group of the examples had a reducing effect on urine protein, and the sample group of Example 2-1 could significantly reduce urine protein and urine protein / urine creatinine (ACR). The observation results of mouse status showed that the number of mice with diarrhea and abdominal distension in the sample group of Example 2-1 was significantly less than that in other groups.

[0216] Example 11-2

[0217] Animal models: db / db mice, 16 weeks old, C57BL / 6 mice, 16 weeks old.

[0218] Administration method: intragastric administration.

[0219] Experimental modeling: After 17 weeks of breeding, urine protein was detected in db / db mice. If urine protein and renal function abnormalities were found, the model was successful and used for subsequent experiments.

[0220] Experimental groups: C57BL / 6 mice were divided into a normal group, 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), 15 mice in each group, urine protein and urine creatinine and daily food intake were detected after 4 weeks of administration, and the state of the mice was observed. The test results are shown in Table 19 below.

[0221] Table 19.

[0222] Group Urine protein / mg Urine protein / creatinine Diarrhea / Number Abdominal distension / number Normal group 17.21±10.07 0.29±0.16 0 0 Model Group <![CDATA[61.97±32.17 * ]]> <![CDATA[2.68±1.03 * ]]> 0 0 Positive drug group <![CDATA[15.09±10.27 # ]]> <![CDATA[1.17±1.26 # ]]> 0 0 Example 3-1 Sample Group 17.28±14.54 <![CDATA[1.29±1.21 # ]]> 1 0 Example 9-1 Sample Group <![CDATA[16.91±13.32 # ]]> <![CDATA[1.17±1.91 # ]]> 2 0 Example 2-A3 Sample Group <![CDATA[19.38±14.67 # ]]> <![CDATA[1.31±1.28 # ]]> 1 1 Example 7-1 Sample Group 36.42±14.23 <![CDATA[1.45±1.29 # ]]> 5 3

[0223] Note: *Compared with the normal control group, P < 0.05; #Compared with the model control group, P < 0.05.

[0224] Each group of the examples had a reducing effect on urine protein, and each sample group could reduce urine protein and urine protein / urine creatinine (ACR). The observation results of mouse status showed that the 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 were significantly less than those in other groups.

[0225] Example 12 Effects of total flavonoids from hibiscus flowers on ocular blood flow and choroidal neovascularization

[0226] Experimental animals: rats, body weight 150-180g, SPF grade, half male and half female.

[0227] Administration method: intragastric administration.

[0228] Experimental modeling: Healthy male Brown-Norway rats were selected and anesthetized by intramuscular injection of ketamine (50 mg / kg). The mydriasis was dilated with a mixture of 0.5% tropicamide and phenylephrine. Krypton yellow laser (Coyle, 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. Those with retinal, choroidal or vitreous hemorrhage were excluded.

[0229] Drug grouping and dosage: normal group, model group, positive control drug group (1.35 mg / kg of vertebofen), sample group of Example 2-1 (69.9 mg / kg), sample group of Example 3-1 (87.36 mg / kg), sample group of Example 7-2 (87.36 mg / kg), sample group of Example 9-1 (87.36 mg / kg), sample group of Example 9-2 (87.36 mg / kg), 15 mice in each group, choroidal blood flow inhibition rate and choroidal angiogenesis area were detected after 4 weeks of administration. The test results are shown in Table 20 below.

[0230] Table 20.

[0231] Group Choroidal blood flow inhibition rate <![CDATA[Choroidal neovascularization area / μm 2 > Normal group 0 0 Model Group <![CDATA[39.78±10.45 * ]]> <![CDATA[34.67±1.78 * ]]> Positive drug control group <![CDATA[87.98±11.23 # ]]> <![CDATA[19.80±1.23 # ]]> Example 2-1 Sample Group <![CDATA[80.67±13.78 # ]]> <![CDATA[23.24±1.33 # ]]> Example 3-1 Sample Group <![CDATA[83.76±14.68 # ]]> <![CDATA[22.89±1.34 # ]]> Example 7-2 Sample Group <![CDATA[61.98±16.21 # ]]> <![CDATA[28.78±1.45 # ]]> Example 9-1 Sample Group <![CDATA[84.19±15.92 # ]]> <![CDATA[24.57±1.39 # ]]> Example 9-2 Sample Group <![CDATA[85.27±11.34 # ]]> <![CDATA[23.79±1.41 # ]]>

[0232] Note: *Compared with the normal control group, P < 0.01; #Compared with the model control group, P < 0.05.

[0233] 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.

[0234] Example 13 Effects of total flavonoids from hibiscus flowers on lupus nephritis

[0235] 26 MRL / lpr male mice (lupus nephritis mice) were selected as the observation group, with a body weight of 18-22g, 13 weeks of age, purchased from Nanjing Junke Biotechnology Co., Ltd., and 24 male C57BL / 6 mice were selected as the normal group, with a body weight of 18-22g, 13 weeks of age, purchased from Wuhan Hualianke Biotechnology Co., Ltd.

[0236] 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.

[0237] Experimental indicators: After the administration, urine was collected for 24 hours for the detection of urine protein. After the urine was collected, the eyeballs of all mice were removed to obtain blood, and serum was obtained by centrifugation for antinuclear antibody detection. At the same time, the kidneys were removed, quickly frozen in liquid nitrogen, and transferred to a negative 80 refrigerator for storage for kidney immune detection. The test results are shown in Table 21 below.

[0238] Table 21.

[0239]

[0240]

[0241] Note: *Compared with the normal group, P < 0.01; #Compared with the model group, P < 0.05.

[0242] Conclusion: The urine protein, serum antinuclear antibody level, IgG and C3 deposition in renal tissue of lupus nephritis mice in the model group were significantly higher than those in the drug-treated 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 able to improve the urine protein, serum antinuclear antibody level, IgG and C3 deposition in renal tissue, and the samples of Example 2-B, Example 3-1 and Example 5-2 had better effects.

[0243] Example 14 Toxicological effects of total flavonoids from hibiscus flowers

[0244] Experimental animals: rats, body weight 150-180g, SPF grade, half male and half female.

[0245] Administration: intragastric administration.

[0246] 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 mice in each group, once a day for 12 consecutive weeks, the dosage is calculated based on the extract.

[0247] Experimental results: After the administration, the kidney weight, visceral body and visceral brain coefficient of the Huangkui Capsule group were increased compared with the normal group. Pathology showed that the Huangkui Capsule group had moderate renal tubular hypertrophy, while pathological examination of the Example 9-2 sample group and the Example 2-A1 group showed no renal tubular hypertrophy. The test results are shown in Table 22 below.

[0248] Table 22.

[0249]

[0250] Note: Compared with the normal group, *P<0.05.

[0251] Example 15 Effects of effective fractions of total flavonoids on idiopathic pulmonary fibrosis

[0252] Preparation of experimental sample 1: Take hyperoside, isoquercetin, hibifolin, myricetin, quercetin-3'-o-glucoside, quercetin, and quercetin 3-O-acacia glycoside raw materials, weigh each active ingredient according to the feeding ratio in Table 23, mix them, and prepare the total flavonoids of Hibiscus tiliaceus flower.

[0253] Table 23.

[0254]

[0255] Experimental animals: Kunming mice, half male and half female, weighing 18-22 grams, clean grade.

[0256] 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.

[0257] Experimental modeling: After the experimental mice were adaptively fed for 3 days, they were anesthetized by intraperitoneal injection of 4% chloral hydrate (0.01 ml / g). They were placed in a supine fixed position, routinely disinfected, and a midline cervical incision was performed. The trachea was exposed by blunt separation. The model group, control drug group, and treatment group were slowly injected with bleomycin (5 mg / kg) through puncture in the gap between the tracheal cartilage rings. The normal control group was injected with an equal volume of saline. Immediately after the injection, the mice were rotated upright for 3-5 minutes to evenly distribute the drug solution in both lungs. Then the skin was sutured and the suture was disinfected. After awakening, the mice were sent to a clean observation room for feeding.

[0258] Dosage method: Dosage began on the second day of modeling. The drug-treated group was given the above 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.

[0259] Pathological section staining of lung tissue: After the administration, the right lung was taken and fixed, embedded, sectioned, and HE stained according to conventional pathological methods. The degree of alveolitis and pulmonary fibrosis was divided into four grades.

[0260] The experimental results are shown in Table 24 below. The experimental results show that the degree of alveolitis and pulmonary fibrosis in the lung tissue of the model group mice was significantly aggravated, showing typical pulmonary fibrosis parenchymal lesions. After 28 days of administration, the lung coefficient of mice in each dosage group was significantly reduced, and the degree of alveolitis and pulmonary fibrosis in the lung tissue was significantly alleviated. The effective fraction of 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 slow down the damage caused by fibrosis to the lung tissue of the other party.

[0261] Table 24.

[0262] Group Alveolitis degree Degree of fibrosis Normal group 0.43±0.58 0.29±0.38 Model Group <![CDATA[1.69±0.66 * ]]> <![CDATA[1.87±0.69 * ]]> Control group <![CDATA[1.03±0.49 # ]]> <![CDATA[1.15±0.81 # ]]> Example 2-A3 Sample Group <![CDATA[1.23±0.55 # ]]> <![CDATA[1.20±0.67 # ]]> Example 9-2 Sample Group <![CDATA[1.19±0.68 # ]]> <![CDATA[1.17±0.90 # ]]> Huangkui Capsule Group <![CDATA[1.48±0.62 # ]]> <![CDATA[1.69±0.63 # ]]> Experimental sample 1 group <![CDATA[1.33±0.74 # ]]> <![CDATA[1.48±0.54 # ]]>

[0263] Note: *Compared with the normal group, P < 0.01; #Compared with the model group, P < 0.05.

[0264] Example 16 Study on the mechanism of action of SGLT2

[0265] Sodium-glucose contransporter2 (SLC5A2, also known as SGLT2) is a gene that encodes a sodium-glucose co-transporter and can reabsorb more than 90% of glucose filtered by the glomerulus. SLC5A2 is only expressed in the brush border of the epithelial cell membrane of the proximal tubules of the kidney and is considered a marker of the proximal tubules. Its main function is to recycle filtered sodium and glucose. In the renal tissue of patients with diabetes and diabetic nephropathy, inhibiting the expression of SLC5A2 protein can play a role in excreting sodium, excreting water and lowering blood sugar, thereby protecting the renal tissue of diabetes and diabetic patients.

[0266] Animal model: db / db mice, 18 weeks old.

[0267] Administration method: intragastric administration.

[0268] Experimental grouping: db / db mice were divided into three groups when UACR>200mg / g, diabetic nephropathy group (DKD), Huangkui capsule group (DKD+HK, 0.84g / kg / d) and Huangkui flower total flavonoids group (DKD+HT, Example 2-A2 sample, 0.0755g / kg / d), Huangkui capsule group and Huangkui flower total flavonoids group were administered for four weeks (28d). Enzyme-linked immunosorbent assay (ELISA) method was used to measure the trace albumin (UACR) in the urine of db / db mice, Reverse Transcription Polymerase Chain Reaction (RT-PCR) method was used to detect the expression level of SLC5A2 mRNA, and ImmunoHistoChemistry (IHC) method was used to detect the expression level of SLC5A2 protein.

[0269] Experimental results:

[0270] Compared with the DKD group, the weight of the HK group (Huangkui capsule group) and the HT group (total flavonoids group) before grouping DKD (N=5), HK (N=5), HT (N=6), the weight of the first week of administration and the weight of the fourth week of administration DKD (N=5), HK (N=5), HT (N=6) had no significant difference. There was no significant difference in blood glucose in the first week of administration and the fourth week of administration DKD (N=5), HK (N=5), HT (N=6). There was no significant difference in UACR value in the first week of administration DKD (N=5), HK (N=5), HT (N=6). Compared with the DKD group in the fourth week of administration, the UACR value of the HT group was significantly reduced, *P<0.05, with a significant difference. There was no significant difference in the mean kidney value in the fourth week of administration DKD (N=5), HK (N=5), HT (N=6); Renal index DKD (N=5), HK (N=5), HT (N=6) in the fourth week of administration; *P<0.05, the renal index increased significantly, with significant difference. In the fourth week of administration, some glomeruli in the DKD group were lobed, the basement membrane was significantly thickened, the glomerular capillaries were significantly compressed, the mesangial cells in the mesangial area proliferated, and the mesangial matrix was significantly expanded; in the HK group, the thickening of the glomerular basement membrane was significantly improved, the glomerular capillaries were intact, and the proliferation of mesangial cells and mesangial matrix was significantly improved; in the HT group, the glomerular basement membrane was significantly thinned, the glomeruli did not proliferate, the glomerular capillary interstitium did not proliferate, and the proliferation of mesangial cells and mesangial matrix did not proliferate. The IHC-P quantification results in the fourth week of administration showed that compared with the DKD group (N=5), the SLC5A2 protein expression level in the Huangkui HK group (N=5) and the HT group (N=6) was significantly reduced. The RT-PCR results at the fourth week of administration verified the immunohistochemistry results and showed that compared with the DKD group (N=5), the SLC5A2 mRNA expression level in the Huangkui HK group (N=5) and the HT group (N=6) was significantly reduced. Compared with the DKD group (N=5), the ratio of the SLC5A2 IHC-P quantified expression to the number of glomeruli in the Huangkui HK group (N=5) and the HT group (N=6) was significantly reduced at the fourth week of administration, which was consistent with the IHC-P quantification results.

[0271] Therefore, Huangkui capsule and total flavonoids can effectively reduce microalbuminuria in db / db mice. Huangkui capsule 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 by the kidneys of DKD mice.

[0272] Example 17 Preparation of total flavonoids extract from hibiscus flowers

[0273] Referring to the preparation method of Example 3-1 above, 5000 g of Hibiscus hibiscus flower was used for repeated experiments, and the content (unit: %) data of two batches were obtained as shown in the following Table 25. The content of quercetin-3-O-acacia glycoside was 0.30-0.80%.

[0274] Table 25.

[0275]

[0276] Example 18 Animal Experiment on Ulcerative Colitis

[0277] 1. Experimental Materials

[0278] 1.1 Experimental animals

[0279] SPF male C57BL / 6J mice, 6-8 weeks old, weighing 22-25 g, were provided by the Center for Comparative Medicine of Yangzhou University.

[0280] 1.2 Reagents and drugs

[0281] Table 26.

[0282]

[0283]

[0284] 2. Experimental Methods

[0285] 2.1 Animal husbandry and management

[0286] The experimental mice were kept in an SPF-grade animal room throughout the experiment. The light-to-dark time ratio was 1:1, the room temperature was controlled at 23±2℃, the humidity was controlled at 55%, and 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.

[0287] 2.2 Drug preparation

[0288] All medicines are Figure 1 Steps for dissolution: ①: Prepare 0.5% CMC-Na one day before administration: slowly add 0.5g CMC-Na powder to 100mL 70℃ pure water, stir evenly, and let stand at room temperature overnight. The next day, ultrasonicate before preparing the drug solution until a homogeneous 0.5% CMC-Na solution is obtained. ②: Weigh 12.5mg, 37.5mg, 75mg ZHT and 100mg 5-ASA respectively, and add 0.1mL DMSO to prepare DMSO mother solution. ③: Add 0.1mL Tween-80 to each mother solution in ②. ④: Add 4.8mL 0.5% CMC-Na solution to each solution in ③. Prepare 2.5mg / mL, 7.5mg / mL and 15mg / mL ZHT suspension and 20mg / mL 5-ASA suspension respectively.

[0289] Preparation method of 3% DSS solution: weigh 3g DSS powder and fully dissolve it in 100mL pure water to obtain 3% DSS solution.

[0290] 2.3DSS-induced ulcerative colitis (UC) model in mice

[0291] After 5 days of adaptive feeding, the mice were randomly divided into 6 groups, each with two cages and 4 mice in each cage. They were blank control group (Control), sunflower total flavonoids extract control group (150 mg / kg / day ZHT), model group (DSS), model + 5-aminosalicylic acid group (DSS + 200 mg / kg / day 5-ASA), model + ZHT group (DSS + 75 mg / kg / day ZHT).

[0292] 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, which was replaced every 2 days for each cage of 4 mice.

[0293] Different doses of ZHT or positive therapeutic drug 5-ASA were given by gavage at 0.1mL / 10g bw every day from 9:00 to 10:00 am, and the blank group and model group were given the same dose of blank vehicle solution (containing 2% DMSO, 2% Tween 80 and 0.5% CMC-Na) for 7 consecutive days. Disease activity index (DAI) was scored before administration every day; on the 8th day, the mice were euthanized and blood and colon tissue were collected.

[0294] 2.4 Stool consistency / fecal occult blood test and DAI score

[0295] The weight of the mice was recorded before daily administration, and feces were collected for fecal occult blood test. The severity of the disease was scored by DAI based on three indicators: weight loss, fecal viscosity, and fecal occult blood. Each indicator corresponds to 0-4 points. The scoring criteria are shown in Table 27. Fecal occult blood test: Take a small amount of feces and smear it in the middle of the glass slide (the glass slide was pre-burned to reduce color error), add 3 drops of 10 / L acetone sulfate acetic acid solution and 3 drops of 3% hydrogen peroxide solution, mix well, observe the results immediately, and score according to Table 28.

[0296] Table 27. DAI scoring details

[0297]

[0298]

[0299] Table 28. Fecal occult blood test scoring table

[0300]

[0301] 2.5 Specimen Collection

[0302] After the last administration, mice in each group were fasted for 24 hours and killed by cervical dislocation. The abdominal cavity of the mouse was cut open to expose the colon of the mouse. All the colon tissues of the mouse were measured and their lengths were recorded. They were placed in freshly prepared pre-cooled saline for intestinal content flushing. The 1 cm proximal rectal colon was cut and fixed in 4% paraformaldehyde overnight 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 experiments. The blood sample was left at room temperature for 2-3 hours, then centrifuged at 3500 rpm for 15 minutes, and the serum was taken and quickly stored at -80 ° C for subsequent testing.

[0303] 2.6 Determination of myeloperoxidase (MPO) content in colon tissue

[0304] MPO is a functional marker and activation marker of neutrophils. Under the condition of pH 6.0, with H2O2 and 3,3′-dimethoxybenzidine hydrochloride as substrates, MPO can catalyze the substrate to generate an orange-yellow product. The amount of the product generated is measured by colorimetry at 460nm, thereby calculating the MPO content.

[0305] The specific measurement steps are as follows:

[0306] 1) Sample pretreatment: Accurately weigh the tissue, add homogenate medium at a weight-to-volume ratio of 1:9, grind in a tissue grinder at a frequency of 60 Hz for 1 min, repeat 5 times until no precipitation is visible to the naked eye, and prepare a 10% tissue homogenate;

[0307] 2) Prepare the reaction system: Take 0.09 mL of 10% tissue homogenate or serum sample to be tested into two new 2 mL EP tubes (control tube and test tube), and add the corresponding reagents in the kit according to Table 29 for reaction;

[0308] 3) Determine absorbance: Take 150 μL of the reaction product in a 96-well plate and measure the absorbance at 460 nm using an enzyme reader;

[0309] 4) Calculate the MPO content according to the following formula:

[0310]

[0311] Where W is the sample volume; for tissue homogenate, W (g) = homogenate concentration × sampling volume (mL).

[0312] Table 29. MPO content determination operation table

[0313]

[0314] 2.7 H&E staining

[0315] The colon was fixed with 4% paraformaldehyde for 24 hours and then routinely embedded in paraffin. The embedded colon tissue was serially sectioned (10 μm) and then stained with H&E. The steps of H&E staining were as follows:

[0316] 1) Dewaxing: dewaxing in xylene I for 10 min, dewaxing in xylene II for 5 min;

[0317] 2) Ethanol soaking: soak in anhydrous ethanol twice, then soak in 95%, 90%, and 85% ethanol once, each time for 1 min, and finally wash with water for 2 min;

[0318] 3) Staining: Stain with hematoxylin for 5 min, then wash with tap water for 1 min;

[0319] 4) Differentiation: Differentiate with 1% hydrochloric acid alcohol for 20 seconds, then wash with tap water for 1 minute;

[0320] 5) Anti-blueing: Anti-blueing with 1% dilute ammonia for 30 seconds, then washing with tap water for 1 minute;

[0321] 6) Eosin staining: stain for 5 minutes, then wash with tap water for 30 seconds;

[0322] 7) Ethanol dehydration: Dehydration was performed with different contents of ethanol in the following order: 85% ethanol dehydration for 20 s, 90% ethanol dehydration for 30 s, 95% ethanol I dehydration for 1 min, 95% ethanol II dehydration for 1 min, anhydrous ethanol I dehydration for 2 min, and anhydrous ethanol II dehydration for 2 min;

[0323] 8) Transparency: xylene I treatment for 2 min, xylene II treatment for 2 min, xylene III treatment for 2 min;

[0324] 9) Finally, the slides were sealed with neutral gum and scanned with a digital pathology slide scanner (NanoZoomer 2.0RS);

[0325] 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 30.

[0326] Table 30. Inflammation score table

[0327]

[0328] 2.8 Real-time fluorescence quantitative PCR

[0329] Place the colon tissue in a grinding tube, add grinding beads and 400 μL Trizol reagent, and grind continuously for 1 min at a frequency of 60 Hz in a tissue grinder. Repeat 5 times until no precipitation is visible to the naked eye. Collect the supernatant, centrifuge at 12,000 rpm for 10 min, and transfer the supernatant to a new 1.5 mL enzyme-free tube. Extract with chloroform-isopropanol overnight and centrifuge, discard the supernatant, wash off the ions attached to the RNA with 75% ethanol, dry and dissolve in DEPC water, and take 2 μL for quantification with Nano-100 micro-spectrophotometer. A 260 / A 280 It should be between 1.8 and 2.0. Use enzyme-free water to adjust the RNA concentration to 1μg, add 3μL of 5×gDNA wiperMix and heat at 42℃ for 2min, remove genomic DNA, then add 5μL of 4×qRT Super MixⅡ to achieve a 20μL system, perform reverse transcription reaction at 25℃-2min, 50℃-30min, and 85℃-5min in a PCR instrument, and store cDNA at -80℃ for later use.

[0330] According to 1μL cDNA + 10μL Green + 0.8μL Primers + 8.2μL ddH2O system for real-time fluorescence quantitative PCR reaction, using Comparative Ct (2 -ΔΔCt ) method to calculate the 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 31.

[0331] Table 31. Primer sequences used for PCR

[0332]

[0333] 2.8 Determination of TNF-α, IL-1β and IL-6 Content

[0334] According to the operating procedures of the ELISA kit, the contents of TNF-α, IL-1β and IL-6 in colon tissue were detected.

[0335] 2.9 Statistical analysis

[0336] GraphPad Prism (Ver 6.0) was used for statistical analysis. All data were expressed as mean ± standard error (Mean ± SEM). Two-way ANOVA was used to analyze weight changes and DAI score changes, and One-way ANOVA was used to analyze 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.

[0337] 3. Experimental results

[0338] 3.1 Effects of different doses of total flavonoids from Astragalus membranaceus on DAI scores of mice

[0339] The DAI score was calculated by adding the three index values ​​in Table 27 and taking the average value. As shown in Table 32, the DAI score of mice in the DSS model group increased significantly from the 3rd day, and the difference was statistically significant compared with the Control group (P<0.01). Three mice had diarrhea on the 6th day of the experiment, and 7 mice had diarrhea on the 7th day. The DAI score of the DSS+ZHT (75 mg / kg / day) group showed a relatively gentle growth trend. On the 4th, 5th, 6th, and 7th days, the DAI score was statistically significant compared with the DSS model group (P<0.01). No mice had diarrhea during the experiment. The positive treatment drug 5-ASA (200 mg / kg / day) relieved the symptoms of DSS-induced colitis and reduced the DAI score. Starting from the 4th day, the difference was significant compared with the DSS model group (P<0.01). No mice had diarrhea during the experiment. During the experiment, there was no significant difference between the normal group and the control group in the ZHT 150 mg / kg / day group, and it had no effect on the weight of mice. One mouse had diarrhea symptoms on the 6th day.

[0340] Table 32. DAI scores of mice in each group

[0341]

[0342] 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.

[0343] 3.2 Effects of different doses of total flavonoids from Astragalus membranaceus on colon length in mice

[0344] like Figure 2As shown in Table 33, the colon length of the Control group was 7.34±0.08cm, and the colon of the mice in the DSS model group atrophied to 4.85±0.18cm, which was significantly different from the Control group (P<0.01); the colon length of the ZHT (150mg / kg / day) group (7.43±0.13cm) was close to that of the Control group. 75mg / kg / day ZHT significantly improved the colon atrophy induced by DSS, and the difference between the DSS+ZHT (75mg / kg / day) group and the DSS model group was statistically significant (P<0.05); the positive drug 5-ASA (200mg / kg / day) also improved the colon atrophy induced by DSS, and the difference was statistically significant compared with the Control group (P<0.05).

[0345] Table 33. Statistics of colon length of mice in each group

[0346]

[0347] 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.

[0348] 3.3 Effects of different doses of total flavonoids from Astragalus membranaceus on the content of myeloperoxidase (MPO) in mouse colon tissue

[0349] 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, and the difference was statistically significant compared with the Control group (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, and the inhibition rate was 88.7±9.9% (P<0.01).

[0350] 3.4 Effects of different doses of total flavonoids from Astragalus membranaceus on pathological changes in mouse colon tissue

[0351] The experimental results show that ( Figure 3), the colon tissue structure of mice in the Control group and ZHT (150mg / kg / day) group was normal, with little inflammatory cell infiltration, and the crypt cells and epidermal cells were intact. In the DSS model group, the number of inflammatory cells in the colon tissue of mice increased significantly, the crypt cells and epidermal cells were completely missing, and the range of lesions was more than 80%. Compared with the Control group, the pathological score was significantly different (P<0.01); the colon tissue of the DSS+ZHT (75mg / kg / day) group was significantly improved, and the pathological score of the DSS+ZHT (75mg / kg / day) group was significantly different from that of the DSS model group (P<0.01). The positive therapeutic drug 5-ASA (200mg / 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).

[0352] 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

[0353] The experimental results showed that the mRNA expression of IL-1β in the colon tissue of mice in the DSS model group increased to 7.36±0.79 times that of the Control group (P<0.01); the administration of 150mg / kg / day ZHT alone increased the mRNA expression of IL-1β 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). 75mg / kg / day ZHT could inhibit the upregulation of IL-1β mRNA expression induced by DSS, with an inhibition rate of 81.9±5.0% (P<0.01); the positive therapeutic drug 5-ASA (200mg / kg / day) inhibited the expression of IL-1β mRNA induced by DSS, with an inhibition rate of 91.6±3.6% (P<0.01).

[0354] The experimental results showed that the mRNA expression of IL-2 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 (150mg / kg / day) alone increased the mRNA expression of IL-2 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). 75mg / kg / day ZHT inhibited 78.7±4.5% of the increase in IL-2 mRNA expression induced by DSS, which was significantly different from the DSS model group (P<0.01); the positive therapeutic drug 5-ASA (200mg / kg / day) significantly inhibited the increase in IL-2 mRNA expression in colon tissue induced by DSS, with an inhibition rate of 94.2±2.8% (P<0.01).

[0355] The experimental results showed that the mRNA expression of IL-4 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); the mRNA expression of IL-4 in the colon tissue of mice in the group given 150mg / kg / day ZHT alone was 1.07±0.16 times that of the Control group, and there was no significant difference compared with the Control group (P>0.05). 75mg / kg / day ZHT inhibited the DSS-induced upregulation of IL-4 mRNA expression, with an inhibition rate of 78.65±7.18%, which was significantly different from the DSS model group (P<0.01); the positive therapeutic drug 5-ASA (200mg / kg / day) significantly inhibited the DSS-induced upregulation of IL-4 transcription, with an inhibition rate of 69.15±6.17% (P<0.01).

[0356] The experimental results showed that the mRNA expression of IL-6 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); there was no significant change in the mRNA expression of IL-6 in the colon tissue of mice in the ZHT (150mg / kg / day) group compared with the Control group. 75mg / kg / day ZHT inhibited 79.4±6.5% of the DSS-induced IL-6 transcriptional upregulation, which was significantly different from the DSS model group (P<0.01); the positive therapeutic drug 5-ASA (200mg / kg / day) inhibited the DSS-induced IL-6 transcriptional upregulation, with an inhibition rate of 91.5±6.2% (P<0.01).

[0357] The experimental results showed that the mRNA expression of TNF-α 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); 150mg / kg / day ZHT increased the mRNA expression of TNF-α 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). 75mg / kg / day ZHT inhibited 88.5±7.1% of the TNF-α transcriptional upregulation induced by DSS, and the difference between the DSS+ZHT (75mg / kg / day) group and the DSS model group was significant (P<0.01). The positive treatment drug 5-ASA (200mg / kg / day) group inhibited the DSS-induced TNF-α transcriptional upregulation, with an inhibition rate of 87.4±7.2% (P<0.01).

[0358] The experimental results showed that the mRNA expression of IFN-γ 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); there was no significant change in the expression of IFN-γ mRNA in the colon tissue of mice in the ZHT (150mg / kg / day) group compared with the Control group. 75mg / kg / day ZHT inhibited 74.2±6.0% of the DSS-induced IFN-γ transcriptional upregulation, and the difference between the DSS+ZHT (75mg / kg / day) group and the DSS model group was extremely significant (P<0.01). The positive therapeutic drug 5-ASA (200mg / kg / day) inhibited the DSS-induced IFN-γ transcriptional upregulation, with an inhibition rate of 83.0±4.2% (P<0.01).

[0359] 4. Experimental conclusions and discussion

[0360] 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 ZHT in treating colitis. The results showed that the ZHT (75 mg / kg / day) group significantly protected the DSS-induced colitis symptoms of mice, such as weight loss, diarrhea, and bloody stools; significantly improved DSS-induced colon atrophy, crypt structure damage, and tissue inflammatory infiltration and other histopathological changes; and reduced DSS-induced increases in tissue MPO levels and the expression of inflammatory factors, showing a good therapeutic effect on colitis, which was comparable to the effect of the positive therapeutic drug 5-aminosalicylic acid.

[0361] 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 its safety is relatively good.

[0362] Example 19 Oral ulcer efficacy experiment

[0363] 1 Experimental Materials

[0364] 1.1 Animals: SPF male SD rats, 250-300 g, purchased from Shanghai Jiesijie Experimental Animal Co., Ltd.

[0365] 1.2 Drugs: Guilin Watermelon Frost, Guilin Sanjin Pharmaceutical Co., Ltd.; ethanol extract of hibiscus flower (dry powder, prepared by refluxing extraction of hibiscus flower with 95% ethanol for three times, concentrating and drying); water extract of hibiscus flower (thick paste, prepared by refluxing extraction of hibiscus flower water for three times, concentrating and drying), total flavonoids extract of hibiscus flower (prepared in Example 17, batch number 9-2).

[0366] 1.3 Reagents: Phenol (analytical grade), Nanjing Chemical Reagent Co., Ltd.; Chloral hydrate (analytical grade), Sinopharm Chemical Reagent Co., Ltd.

[0367] 2 Experimental methods

[0368] 2.1 Modeling: SPF male SD rats were randomly divided into groups, with 9-11 rats in each group. They were blank group (normal group), model group, hibiscus flower ethanol extract group, hibiscus flower water extract group, hibiscus flower total flavonoids extract group, and watermelon frost group. All rats were anesthetized by intraperitoneal injection of chloral hydrate (400 mg / kg), and a glass tube with an inner diameter of 4 mm at the lower end was taken, and a small cotton ball was placed at its lower end. For the model group and rats in each drug-treated group, 90% phenol solution was dripped into the glass tube until the small cotton ball was just soaked, and the lower end was placed flat on the buccal mucosa about 1 mm from the left and right corners of the mouth of the rat for 30 seconds. White lesions with a diameter of about 4-5 mm were observed in this area. The blank group was treated in the same way with double distilled water instead of phenol.

[0369] 2.2 Administration: Each drug was prepared into a suspension with double distilled water, and evenly applied to the oral mucosal wound after modeling, 4 times a day. The dosage of each drug was as follows: watermelon frost, 20 mg / wound / day; 10 mg / wound / day of hibiscus flower alcohol extract; 8.75 mg / wound / day of hibiscus flower water extract, 1.05 mg / wound / day of hibiscus flower total flavonoids extract. The drug was administered for 5 consecutive days, and the ulcer area and ulcer healing were observed before and after administration. On the 6th day, the ulcer surface was photographed after anesthesia with chloral hydrate (400 mg / kg). After the rats were killed by cervical vertebrae dislocation, the bilateral buccal membrane tissues of the rats were removed. 7-9 samples were randomly selected from each group and immersed in 4% formaldehyde (prepared with saline) for fixation, routine sampling, dehydration, and paraffin embedding. The sections were stained with HE and observed under an optical microscope. The remaining buccal membrane tissues were frozen in a -70℃ refrigerator for use to measure other inflammatory indicators.

[0370] 2.3 Data statistics: Data are expressed as mean ± SD. The significant difference between the model group and the normal group (blank control group) was tested by Student's-t test; the significant difference between the model group and the other drug-treated groups was analyzed by One-way ANOVA test, and the significant difference between the two groups was further tested by Newman-Keuls multiple comparison test.

[0371] 3 Results

[0372] 3.1 Visual observation and evaluation

[0373] The surface of the oral mucosa of the rats in the blank group was smooth, uniform in color, and without blood spots. After phenol stimulation, the local mucosa turned white quickly, edema was visible after 2 hours, inflammatory exudation, bleeding, and local ulceration were visible within 1-5 days, and the surface was covered with a yellow-white pseudomembrane and fell off. On the 6th day, except for the blank group, the wound surface of rats in each group was sunken; the vascular dilation of the wound surface of rats in the 50% model group was more obvious, and yellow pus spots were visible in one rat. The results are shown in Table 34 (each rat had two wound surfaces in the mouth).

[0374] Table 34. Statistics of the degree of phenol buccal ulcer in rats

[0375]

[0376] 0: no ulcer, normal oral mucosa;

[0377] Ⅰ: There is ulcer depression, no obvious bleeding or vascular dilation, no obvious swelling, and no obvious pseudomembrane;

[0378] II: There are ulcers and pits, but less blood spots;

[0379] III: There are ulcer depressions, obvious blood spots, and slight swelling.

[0380] 3.2 Pathological changes

[0381] The following items were examined under an optical microscope: (1) whether the mucosal epithelial cells showed degeneration, necrosis, erosion, ulceration, or inflammatory cell infiltration; and (2) whether the submucosal tissue showed congestion, edema, or inflammatory cell infiltration.

[0382] Scoring criteria for lesion severity: The lesions are scored from 0.5 to 4 points from mild to severe, and the scoring criteria are: 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; no lesions are scored as 0 points. The length of the ulcer is measured with a "microscope side micrometer" under a 40x light microscope (eyepiece 4×, objective lens 10×). The length of the ulcer is scored as 1 point if it is less than 5mm / 40X, and 2 points if it is greater than 5mm / 40X ​​or 5mm / 40X. Add all the scores and calculate the average score of lesions for each group of animals. The higher the score, the more severe the disease.

[0383] The statistical results of pathological changes in each group are shown in Table 35, and the pathological images are shown in Figure 4 , Figure 4 In b, the short black arrow shows skin necrosis and shedding to form an ulcer (without epidermal coverage), the long black arrow shows inflammatory cells infiltrating the dermis, the green arrow shows vascular dilation and congestion, the green arrow shows fibroblasts, and the green star shows the thin layer of stratified squamous epithelium on the skin surface with mild keratinization on the surface.

[0384] Table 35. Scoring results of pathological changes in oral mucosa of rats

[0385]

[0386]

[0387] Note: ## P<0.01 vs blank group; *P<0.05, **P<0.01 vs model group.

[0388] Example 20 Pharmacodynamic evaluation of total flavonoids from Astragalus membranaceus in treating skin diseases

[0389] Example 20-1 Pharmacodynamic evaluation of total flavonoids from Astragalus membranaceus in the treatment of acne

[0390] 1. Experimental purpose:

[0391] To investigate the therapeutic effect of topical administration of total flavonoids from Hibiscus truncatula (ZHT) on acne induced by 100% oleic acid and Propionibacterium acnes (P. acnes), and to compare the efficacy with that of positive drugs benzoyl peroxide / clindamycin.

[0392] 2. Experimental Materials

[0393] 2.1. Experimental animals

[0394] 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).

[0395] Reagents and medicines

[0396] Total flavonoids from Astragalus membranaceus (ZHT-1) were prepared according to the method of Example 17, also known as "Total flavonoids extract from Astragalus membranaceus", batch number 19-1. Total flavonoids from Astragalus membranaceus (ZHT-2) were prepared according to the method of Example 6.

[0397] 2.3. Preparation of experimental drugs

[0398] Preparation method of ZHT cream: 1% total flavonoids cream of Astragalus membranaceus is prepared according to the prescription in Table 36.

[0399] Table 36. ZHT cream formulation

[0400]

[0401] 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 evenly dispersed.

[0402] Oil phase: Weigh the prescribed amount of glyceryl monostearate, palmitic acid and white vaseline into a beaker, and heat in a water bath at 80°C to dissolve until liquid.

[0403] Slowly add the water phase into the oil phase while stirring. After all the water phase is added, take it out from the water bath and stir at room temperature until it cools into a cream.

[0404] Preparation method of reinforced Clostridial medium (RCM): weigh 19.0 g of reinforced Clostridial medium powder into a 500 mL conical flask, add 500 mL of distilled water, heat and stir, divide into portions, and sterilize at 121°C for 15 minutes for use.

[0405] Culture method of P. acnes: Add 4 mL of RCM culture medium and 1 mL of P. acnes bacterial solution into a sterile test tube and mix well by blowing. Wrap the sterile container with sealing film and place it vertically into an anaerobic culture bag containing an anaerobic gas-producing bag. Culture it in a 37°C electric constant temperature incubator and subculture it every 2 days.

[0406] 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, 6×107 CFU / mL concentration of bacterial suspension, measure its OD 600nm Between 0.63 and 0.65.)

[0407] 2.3 Construction and treatment of rat acne model

[0408] Rat acne model establishment and drug efficacy evaluation methods Figure 5 As shown, 100% oleic acid combined with P. acnes was used to establish an acne model, and 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.

[0409] Rats, half male and half female, 6 in each group, were housed in separate cages. After one week of adaptive feeding, 100% oleic acid combined with P.acnes were used to induce acne model in rats other than the blank group. The right auricle of rats was used as a carrier to construct the acne model, and the left ear was not treated. 100% oleic acid was applied once on the inside of the right ear of the rats at 9 am every day, 0.5 mL each time, and the blank group was applied with an equal amount of saline; oleic acid was applied every other day, and the prepared P.acnes injection was injected into the inside of the right ear of the rats 1 hour later, 50 μL each time; the blank group was injected with saline.

[0410] On the 8th day of modeling, the rats were regrouped to ensure that the severity of the model in each group of rats was basically the same, and 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 had one cage for each female and one cage for each male rat, with three rats in each cage. Starting from the afternoon of the 8th day after grouping, 50 mg of the drug to be tested was applied to the ears every afternoon.

[0411] 2.4 Auricular swelling measurement and symptom scoring

[0412] 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 graph of the change of the rat's right auricle thickness over time was drawn.

[0413] The right auricle of the rats was photographed and scored for symptoms before modeling on the morning of the 1st, 3rd, 7th, 11th, 15th, and 17th day. The severity of the disease was scored based on four indicators: redness, swelling, desquamation, papules, and cysts. Each indicator corresponded to 0-4 points, and the scores of the four indicators were added together to get the total score. According to the scoring standard: 0 points, no symptoms; 1 point, mild; 2 points, moderate; 3 points, severe; 4 points, extremely severe.

[0414] 2.5 Specimen Collection

[0415] The rats were killed on the 19th day, and the right auricles of the rats were completely cut off. 1 / 4 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.

[0416] 2.6 H&E staining and pathological examination

[0417] The rat ear tissue was fixed with 4% paraformaldehyde for 24 hours, embedded in paraffin and sliced, and then stained with H&E. The pathological changes of the rat acne model were evaluated according to the histological criteria.

[0418] The specific scoring criteria are shown in Table 37.

[0419] Table 37. Acne pathological histological scoring standard

[0420]

[0421] 2.8 Determination of protein contents of IL-1β, IL-6, TNF-α and MCP-1 in rat ear tissue

[0422] According to the operating procedures recommended by the ELISA kit manufacturer, the contents of IL-1β, IL-6, TNF-α and MCP-1 in various ear tissues were detected.

[0423] 3. Experimental results

[0424] 3.1 Effects of ZHT on the appearance and thickness of the auricle in rats with acne

[0425] During the entire experiment, the inner side 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.

[0426] The right auricle of the rats in the Model group showed obvious symptoms of redness, swelling and desquamation on the 7th day of modeling, and the symptoms of redness, swelling and desquamation continued to worsen with the progress of modeling. 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. From the 6th day, it was significantly higher than that of the Veh group, and the difference was statistically significant (P<0.01) (Table 38). The skin lesion symptom score showed that the degree of skin lesions gradually worsened after modeling, and from the 3rd day, it was significantly different from that of the Veh group (P<0.01) (Table 39).

[0427] 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. The thickening of the ear caused by modeling was gradually delayed after treatment with BPO hydrogel. 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 38). 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 39).

[0428] After applying 1% ZHT-1 and 1% ZHT-2 creams, the thickening of the ears caused by modeling was significantly reduced on the 12th day, which was significantly different from the Model group (P<0.01) (Table 22-3); the application of 1% ZHT-2 improved the desquamation caused by modeling on the 15th day, and the skin lesion symptom score was significantly reduced compared with the Model group (P<0.01), but it could not further improve the redness and papules caused by modeling, so there was no significant difference in the skin lesion symptom score compared with the Model group at other time points (P>0.05). The application of 1.0% ZHT-1 alleviated the acne-like symptoms such as redness, desquamation and papules caused by modeling. On the 11th, 15th and 17th days, the symptom score of the Model+1.0% ZHT group was significantly lower than that of the Model group (P<0.01) (Table 39).

[0429] Table 38. Changes in thickness of right auricle of rats in each group (unit: mm)

[0430]

[0431] 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.

[0432] Table 39. Symptom scores of right auricle skin lesions in each group of rats

[0433]

[0434] 3.2 Effect of ZHT on ear pathological morphology in rats with acne

[0435] There was no obvious abnormality in the ear tissue of rats in the Veh group. 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 seen in the dermis.

[0436] 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 follicle openings and infundibulum were filled with keratinized substances. The keratin plugs were heavily blocked and enlarged into a kettle 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).

[0437] The thickness of the epidermis 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 the infundibulum was less, the sebaceous glands were normal in morphology, the dermis was not edematous, and the degree of inflammatory cell infiltration was alleviated 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).

[0438] 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).

[0439] Applying 1% ZHT-2 and 1.0% ZHT-1 can improve the excessive thickening of the epidermis and stratum corneum, alleviate dermal edema and reduce inflammatory cell infiltration in a dose-dependent manner. The pathological scores of each group were 3.33±0.33 and 3.33±0.33, respectively.

[0440] Effect of ZHT on the mRNA expression levels of Mcp-1, Il-1β, Il-6 and Tnf-α in ear tissues of acne rats

[0441] 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 can significantly improve the modeling-induced up-regulation of Mcp-1 mRNA expression, with an improvement rate of 70.9±14.9% (P<0.01vs.Model); the application of 1% ZHT-1 cream can improve the modeling-induced up-regulation of Mcp-1 mRNA expression, with an improvement rate of 25.5±16.0%, but the difference was not statistically significant (P>0.05vs.Model); and the application of 1% ZHT-2 cream has an inhibitory effect on the modeling-induced up-regulation of Mcp-1 mRNA expression.

[0442] 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 up-regulation 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 up-regulation 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.

[0443] 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 upregulation of Il-6 mRNA expression induced by modeling, and the improvement rates were 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.

[0444] 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 up-regulation of Tnf-α mRNA expression, with an improvement rate of 71.2±10.0% (P<0.05vs.Model); the application of 1% ZHT-2 and 1% ZHT-1 creams could inhibit the modeling-induced up-regulation of Tnf-α mRNA expression, with improvement rates of 18.2±12.9% (P>0.05vs.Model) and 58.3±15.0% (P<0.05vs.Model), respectively.

[0445] Effects of 3.5ZHT on the contents of IL-1β, IL-6, TNF-α and MCP-1 in ear tissues of rats with acne

[0446] 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.

[0447] 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 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 ear tissue of rats 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±21.4pg / mL and 235.2±25.6pg / 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.

[0448] The TNF-α protein content in the ear tissue of the Veh group was 103.7±23.7pg / mL, and the TNF-α protein content in the ear tissue of the Model group was 423.2±37.0pg / mL, which was 4.08±0.58 times 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.

[0449] Example 20-2 Pharmacodynamic evaluation of total flavonoids from Astragalus membranaceus in the treatment of atopic dermatitis

[0450] 1. Research content and purpose:

[0451] The therapeutic effects of different doses of total flavonoids from Huang Kui (ZHT-1) and Huang Kui 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.

[0452] 2. Experimental Materials

[0453] 2.1 Experimental animals

[0454] SPF Balb / c mice were purchased from the Comparative Medicine Center of Yangzhou University [Laboratory Animal Production License No.: SCXK (Su) 2017-0007], 7-8 weeks old, weighing 18-20 g. They were kept in an environment with a room temperature of 23±2°C, a humidity of 55%, and a light-to-dark time ratio of 1:1, and the mice were free to eat and drink.

[0455] 2.2 Drugs and main reagents

[0456]

[0457] 2.3 Experimental instruments

[0458]

[0459] 3. Experimental Methods

[0460] 3.1 Animal grouping and atopic dermatitis model construction

[0461] After 5 days of adaptive feeding, 80 clean-grade Balb / c mice were randomly divided into 10 groups, including blank control group (Sham), DNFB modeling group (DNFB), positive drug mometasone furoate group (Mome), 0.5% group of total flavonoids of sunflower (ZHT) cream, 0.5% group of sunflower HKY (HKY) cream, and 100 mg / kg SZHT oral administration group. Rosin wax wax depilation was performed, and each mouse was smeared with 50 μL of 1.0% [W / V, solvent is acetone olive oil mixture (acetone: olive oil = 3:1)] 2,4-dinitrofluorobenzene (DNFB) on the back in the morning of the first day, and 0.5% (W / V) DNFB was applied on the morning of the 4th, 6th, 8th, and 10th days, 50 μL each time, and the control group was smeared with the same amount of solvent control (acetone: olive oil = 3:1). Animal experiments followed the guidelines for the care and use of experimental animals and were in accordance with animal ethics standards. Construction of atopic dermatitis model Figure 6 shown.

[0462] 3.2 Administration and dosage

[0463] Starting from the 5th day, 100 mg of different doses of ZHT cream and HKY cream were accurately weighed with an electronic analytical balance every afternoon and evenly applied to the back modeling area. The control group was given 100 mg of blank cream; the 100 mg / kg SZHT group was given by gavage. Figure 6 shown.

[0464] 3.2.1 Preparation of ZHT cream and HKY cream

[0465] The prescriptions of ZHT cream and HKY cream are shown in Table 40.

[0466] Table 40. Total flavonoids cream of Huang Kui and prescription of Huang Kui HKY

[0467]

[0468] Cream preparation process:

[0469] 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 evenly dispersed.

[0470] Oil phase: Weigh the prescribed amount of glyceryl monostearate, palmitic acid and white vaseline into a beaker, and heat in a water bath at 80°C to dissolve until liquid.

[0471] Slowly add the water phase into the oil phase while stirring. After all the water phase is added, take it out from the water bath and stir at room temperature until it cools into a cream.

[0472] 3.2.2 Preparation of SZHT suspension

[0473] Preparation of 0.5% CMC-Na: One day before administration, slowly add 0.5g CMC-Na powder to 100mL 70℃ pure water, stir evenly, and let stand at room temperature overnight. The next day, accurately weigh 50mg ZHT and add 0.1mL DMSO to prepare DMSO mother solution. Add 0.1mL Tween-80 and 9.8mL ultrasonically homogenized 0.5% CMC-Na solution to the ZHT mother solution to obtain a 5mg / mL ZHT suspension.

[0474] 3.3 Atopic dermatitis severity score (AD Score)

[0475] On the 4th, 7th, 9th and 11th days of modeling, the back skin of each mouse was photographed and recorded, and the severity of dermatitis appearance was evaluated by four indicators: ① erythema / hemorrhage ② dryness ③ exudation / scab ④ edema. Each indicator was scored from 0 to 3 points, and the scores of the four indicators were added together to obtain the total score. The AD Score scoring criteria are as follows: 0, no symptoms; 1, mild symptoms; 2, moderate symptoms; 3, severe symptoms.

[0476] 3.5 HE staining

[0477] Paraffin sections were routinely dewaxed to water, and the sections were placed in hematoxylin dye, stained for 5 minutes, and then rinsed with tap water. The sections were then placed in hydrochloric acid ethanol, differentiated for 30 seconds, soaked in tap water for 15 minutes, and then placed in eosin dye and stained for 2 minutes. Routine dehydration, transparency, and neutral resin sealing were used. Photos were taken using a digital pathology section scanner, and the thickness of the epidermis was counted.

[0478] 3.10 Statistical analysis

[0479] GraphPad Prism 6.0 software was used for statistical analysis and drawing of data, and all data were expressed as mean ± standard error (mean ± SEM). Two-way ANOVA was used to analyze dermatitis scores, and one-way ANOVA was used to analyze other data. Dunnett's multiple test was used to analyze the significance of differences between groups. When P < 0.05, the differences between groups were significant and statistically significant.

[0480] 4. Experimental Results

[0481] Effects of ZHT and HKY on the appearance of DNFB-induced atopic dermatitis model

[0482] like Figure 7As shown, during the whole experiment, the back skin of the blank control group (Sham) was always smooth and flat, without exudation, scabs, erythema and bleeding. After repeated stimulation of DNFB, from the 4th day, the DNFB group mice showed obvious symptoms such as scabs, erythema and edema, 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).

[0483] 4.3 Effects of ZHT and HKY on skin pathological changes in DNFB-induced atopic dermatitis model.

[0484] The results of HE staining of skin tissue sections in each group are as follows Figure 8 As shown in the figure, 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 obviously 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, compared with the DNFB group, 0.5% ZHT reduced the thickening by 34.7±5.0%, which was significantly different (P<0.05, N=6), which was consistent with the dermatitis score results; however, 0.5% HKY had no significant improvement 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).

[0485] Example 21 Pharmacodynamic evaluation of total flavonoids from Astragalus membranaceus in promoting wound healing

[0486] 1. Experimental Materials

[0487] 1.1 Experimental animals

[0488] SPF SD rats, 6-8 weeks old, 150-200g.

[0489] 1.2 Reagents and drugs

[0490] Table 41.

[0491]

[0492] 2. Experimental Methods

[0493] 2.1 Animal husbandry and management

[0494] SD rats (purchased from Nanjing Medical University, license number: SCXK (Su) 2021-0001) were kept in an SPF 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 were free to eat and drink. The litter was changed every three days during the high-sugar and high-fat diet feeding stage, and the litter was changed once a day during the STZ injection stage to establish the diabetes model. After the full-thickness skin resection model was established, the litter 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).

[0495] 2.2 Administration and Dosage

[0496] An electronic analytical balance was used to weigh 100 mg of blank cream, 0.5%, 1%, and 2% total flavonoids cream from Astragalus membranaceus, and Deshijie hydrocolloid, respectively, and applied evenly on the surface of the ulcer wound twice a day. The groups and the drugs applied in each group are as follows:

[0497] Blank control group (Control): no treatment

[0498] Model group: applied a blank cream without total flavonoids from Astragalus membranaceus;

[0499] Positive group: Apply Dewex hydrocolloid;

[0500] Low-0.5% group: 0.5% total flavonoids from Astragalus membranaceus cream was applied;

[0501] Middle-1% group: Apply 1% total flavonoids ointment of Astragalus membranaceus;

[0502] High-2% group: Apply 2% total flavonoids cream from Astragalus membranaceus;

[0503] 2.3 Reagent preparation

[0504] STZ solution preparation: ① Preparation of citric acid buffer: Add 2.1g of citric acid to 100mL of distilled water to prepare solution A; add 2.94g of sodium citrate to 100mL of distilled water to prepare solution B. ② When using, mix A and B in proportion and adjust the pH to 4.2-4.5, which is the citric acid buffer for preparing STZ. ③ Before injection, dissolve STZ in citric acid buffer at a concentration of 1%. Protect from light and refrigerate at 4℃ for later use.

[0505] Preparation of 4% chloral hydrate: Weigh 4g chloral hydrate, add appropriate amount of physiological saline to dissolve, and dilute to 100mL. Protect from light and refrigerate at 4℃ for later use.

[0506] 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 different concentrations were made up with water.

[0507] 2.4 Establishment of diabetic ulcer rat model

[0508] 2.4.1 STZ-induced diabetic rat model

[0509] After one week of adaptive feeding, 70 healthy male SD rats (SPF grade, 4-6 weeks old, weighing 150-200g) were randomly divided into cages, 5 rats per cage, and fed with high-sugar and high-fat feed as the diabetes model group. Ten normal rats were selected, 5 rats per cage, and fed with ordinary feed as the blank control group. Fasting blood glucose (FBG) and body weight were measured for 60 SD rats. After four weeks of feeding, streptozotocin (35mg / kg) was injected intraperitoneally according to body weight in the diabetes model group rats, and the blank control group was injected with an equal amount of citric acid buffer according to body weight. STZ solution and citric acid buffer were injected into the left lower abdomen of the rats according to fasting body weight, and the injection was completed within 10min. All rats were fasted for 6h before administration, and tail vein blood was collected 72h after injection to measure fasting blood glucose. The fasting blood glucose value was ≥16.7mmol / L and the body weight was not significantly reduced. The diabetes model was successfully constructed. Some rats with blood sugar levels between 5-8mmol / L were injected with STZ (35mg / kg) again; some rats with blood sugar levels between 8-10mmol / L were injected with STZ (25mg / kg) again; some rats with blood sugar levels between 10-16mmol / L were injected with STZ (20mg / kg). 72 hours after the injection, tail vein blood was collected to measure fasting blood sugar. If the fasting blood sugar value was ≥16.7mmol / L, the diabetes model was considered to be successfully established. If the fasting blood sugar value was <16.7mmol / L, STZ was injected again according to body weight until the fasting blood sugar value was ≥16.7mmol / L, which was considered to be a successful preparation of the diabetes model. It took 12 days to inject STZ to induce the diabetic animal model in SD rats in this experiment. After all the models were established, they were fed with a high-sugar and high-fat diet for one week to stabilize the model.

[0510] 2.4.2 Establishment of full-thickness skin excision wound model in diabetic rats

[0511] After the diabetic rats were stably molded, the rats were anesthetized by intraperitoneal injection of 4% chloral hydrate according to their body weight (2 mg / kg). After the rats were completely anesthetized, the backs of the rats were shaved with an electric shaver. After shaving, medical cotton swabs were dipped in iodine and applied to the backs of the rats for 4-5 times for disinfection. A sterile 8-mm Biopsy Punch was used to print a circular area with a diameter of 8 mm in the middle of the back of the rats. The skin in the middle of the circular area was clamped with sterile tweezers, and the skin was excised along the circular mark outlined by the Biopsy Punch with sterile iris scissors to create a circular full-thickness skin excision open wound with a diameter of 8 mm, deep to the muscles, avoiding arterial and venous blood vessels. Immediately afterwards, the wound was continuously applied with iodine for 4-5 times, and appropriate pressure was given 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 their backs were shaved, and no full-thickness skin excision open wound was established. The rats that died during modeling were eliminated, 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 (blank cream), positive drug group (Deshijie hydrocolloid), low-dose group (0.5% cream of total flavonoids of Asparagus cochinchinensis), medium-dose group (1% cream of total flavonoids of Asparagus cochinchinensis), and high-dose group (2% cream of total flavonoids of Asparagus cochinchinensis). After the full-thickness skin excision wound model was established, drug treatment was given immediately, and photos were taken and recorded on the 0th day (before drug application), 3rd day, 7th day, and 9th day after treatment, and the wound area was measured using ImageJ software.

[0512] 2.5 Overall evaluation of type 2 diabetic ulcer model

[0513] 1. Observe general symptoms, i.e., whether there are changes in polydipsia, polyphagia, polyuria, poor mental state, dull and yellow fur, hair loss, and a significant decrease in activity;

[0514] 2. Fasting blood glucose measured by tail vein blood sampling was 16.7-33.3mmol / L;

[0515] 3. Typical appearance of full-thickness skin defect: ulcer, redness and swelling. The ulcer area is used as an indicator for statistics. The calculation formula is as follows: wound area (%) = unhealed wound area / original wound area × 100%.

[0516] 2.6 HE staining

[0517] The skin tissue was attached to the filter paper, fixed vertically in 4% paraformaldehyde solution for 48 hours, and then embedded in paraffin wax. The embedded skin tissue was sliced ​​into full-thickness sections (5 μm) in a row, with 2 sections placed on each slide, and then HE staining was performed. The specific steps are as follows:

[0518] 1) Dewaxing: dewaxing in xylene I for 10 min, dewaxing in xylene II for 5 min;

[0519] 2) Ethanol soaking: soak in anhydrous ethanol twice, then soak in 95%, 90%, and 85% ethanol once, each time for 1 min, and finally wash with water for 2 min;

[0520] 3) Staining: Stain with hematoxylin for 5 min, then wash with tap water for 1 min;

[0521] 4) Differentiation: Differentiate with 1% hydrochloric acid alcohol for 20 seconds, then wash with tap water for 1 minute;

[0522] 5) Anti-blueing: Anti-blueing with 1% dilute ammonia for 30 seconds, then washing with tap water for 1 minute;

[0523] 6) Eosin staining: stain for 5 minutes, then wash with tap water for 30 seconds;

[0524] 7) Ethanol dehydration: Dehydration was performed with different contents of ethanol in the following order: 85% ethanol dehydration for 20 s, 90% ethanol dehydration for 30 s, 95% ethanol I dehydration for 1 min, 95% ethanol II dehydration for 1 min, anhydrous ethanol I dehydration for 2 min, and anhydrous ethanol II dehydration for 2 min;

[0525] 8) Transparency: xylene I treatment for 2 min, xylene II treatment for 2 min, xylene III treatment for 2 min;

[0526] 9) Finally, the slides were sealed with neutral gum and scanned with a digital pathology slide scanner (NanoZoomer 2.0RS).

[0527] 2.7 Masson staining

[0528] The 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 aqueous solution for 5 minutes, stained with 2% brilliant green dye solution for 5 minutes, and rinsed with 1% glacial acetic acid aqueous solution for 1 minute. Subsequently, they were dehydrated with 95% ethanol and anhydrous ethanol, transparentized in xylene, and sealed with neutral gum. The sections were scanned and imaged using a digital pathology section scanner (NanoZoomer 2.0RS).

[0529] 2.8 Immunofluorescence of vascular marker CD31

[0530] Take skin tissue, fix it with 4% paraformaldehyde overnight, embed it in paraffin, and make skin paraffin sections. Put the paraffin sections in a 60℃ oven, bake for 20 minutes, then soak in xylene I for 15 minutes, xylene II for 15 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 95% alcohol for 5 minutes, 85% alcohol for 5 minutes, 75% alcohol for 5 minutes, and then rinse with distilled water and PBS for 5 minutes respectively. Place the sections in a staining jar filled with antigen retrieval buffer and perform antigen heat retrieval in a pressure cooker. Rinse the pressure cooker with running water, cool it to room temperature, shake off the excess liquid, add 5% BSA, and block at room temperature for 1 hour. Then, shake off the blocking solution, add diluted primary antibody (1:400), and place the sections in a humidified box for overnight incubation at 4℃. The next day, the primary antibody was removed, and the sections were rinsed with PBS for 3 times, 5 min each time, followed by the addition of diluted fluorescent secondary antibody (1:1000), incubated at room temperature in the dark for 1 h, the secondary antibody was removed, and the sections were rinsed with PBS for 3 times, 5 min each time. After drying the sections, anti-fluorescence quencher containing Hoechst was added, and the sections were observed and photographed under a fluorescence microscope.

[0531] 2.9 Real-time fluorescence quantitative PCR

[0532] Place the skin tissue in a grinding tube, add grinding beads and 800 μL Trizol reagent, and grind it in a tissue grinder at a frequency of 60 Hz for 1 min. Repeat 5 times until no precipitation is visible to the naked eye. Collect the supernatant, centrifuge at 12,000 rpm for 10 min, and transfer the supernatant to a new 1.5 mL enzyme-free tube. Extract with chloroform-isopropanol overnight and centrifuge, discard the supernatant, wash the ions attached to the RNA with 75% ethanol, dry it, add DEPC water to dissolve it, and draw 2 μL for quantification with Nano-100 micro-spectrophotometer. During the detection, A260 / A280 should be between 1.8-2.0. Use enzyme-free water to adjust the RNA concentration to 1 μg, add 3 μL of 5×gDNA wiperMix and heat at 42°C for 2 min to remove genomic DNA, then add 5 μL of 4×qRT Super MixⅡ to achieve a 20 μL system, perform reverse transcription reaction at 25°C-2 min, 50°C-30 min, and 85°C-5 min in a PCR instrument, and store cDNA at -80°C for later use.

[0533] 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 42.

[0534] Table 42. Primer sequences used for PCR

[0535]

[0536] 2.10 Determination of vascular endothelial growth factor (VEGF) content

[0537] According to the operating procedures of the ELISA kit, the content of VEGF in skin tissue was detected.

[0538] 2.11 Statistical analysis

[0539] GraphPad Prism (Ver 6.0) was used for statistical analysis. All data were expressed as mean ± standard error (Mean ± SEM). The wound healing rate data were analyzed by Two-way ANOVA, and other data were analyzed by One-way ANOVA. 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.

[0540] 3. Experimental results

[0541] 3.1 Effects of different concentrations of total flavonoids cream from Astragalus membranaceus on skin wound area in rats

[0542] 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 of diabetic rats was constructed, and the wound healing of each group of rats 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.

[0543] Fig. 9 A in the middle is a representative diagram of the skin wound area of ​​rats in each group at different time points. Before drug administration (24h after injury), fibrin clots ("scabs") appeared at the skin wounds of each group, and there was a small amount of exudate, indicating that there was an inflammatory reaction at the wound at this time; on the third day after drug administration, the scabs of the wounds in each drug administration group began to harden, and the scabs became dry. The dry scabs can play an antibacterial role, thereby protecting the wound to a certain extent, and provide a temporary extracellular matrix for the proliferation and migration of keratinocytes, promoting wound re-epithelialization; the wound area of ​​the model group was 99.48±8.76% of the original wound area. Compared with the drug administration group and the model group, the wound area of ​​the rats in the Deshijie hydrocolloid group began to shrink, and the wound area was 64.52±7.04% of the initial wound area. The wound areas of the 0.5%, 1%, and 2% sunflower total flavonoids cream groups were 106.80±9.37%, 91.41±5.51%, and 98.38±8.74% of the initial wound area, respectively. Fig. 9As shown in B. On the 7th day after administration, the hard scabs in each group began to fall off. The wound area of ​​the model group was 47.62±10.68% of the initial wound area, and the wound areas of 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. Among them, the 1% cream group promoted wound healing most significantly, while the wound area of ​​the Deshijie hydrocolloid group was 17.86±4.68% of the initial wound area, and its effect on promoting wound healing was better than that of the 1% sunflower flavonoids cream group. On the ninth day after administration, the wound area of ​​the model group was 12.05±4.03% of the original wound area; the wound areas of 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 of ​​the Deshijie hydrocolloid group was 8.03±4.70% of the initial wound area. Overall, the wound healing of the positive drug Deshijie hydrocolloid group was obvious, with significant differences compared with the model at 3 days and 7 days, and the 1% sunflower flavonoids cream group showed that the treatment was basically cured.

[0544] 3.2 Analysis of HE staining results

[0545] In order to further evaluate the effect of different concentrations of total flavonoids cream of Asparagus cochinchinensis on promoting diabetic wound healing, we performed HE staining on rat skin tissue 9 days after modeling to observe the changes in the tissue microscopic level. HE staining is one of the commonly used paraffin section staining methods. It uses hematoxylin, eosin and other dyes for staining, so it is also called hematoxylin-eosin staining. Hematoxylin stain is an alkaline dye that can dye the cell nucleus purple-blue, eosin dyes the components in the extracellular matrix and cytoplasm pink, and other structures show different shades 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 structure of the tissue sample, which can be used to observe the number and distribution of fibroblasts, capillaries, inflammatory cells, etc. during wound healing.

[0546] like Fig.10As shown in A, the results of tissue section microscopy showed that the skin tissue of the normal group rats showed a complete keratinocyte layer, skin appendages, collagen fibers cross-linked and arranged in parallel bundles, and a small number of fibroblasts and inflammatory cells. In contrast, the skin tissue of the model group rats was loosely arranged, and keratinocytes began to proliferate, but had not yet migrated to the center of the wound. The overall skin structure was disordered, the new epidermis was thin, and the new granulation tissue was less. Inflammatory cells and fibroblasts migrated to the wound, among which the number of inflammatory cells was relatively large, indicating that the model group rats were mainly in the first stage of wound healing: the inflammatory stage. The keratinocyte layer at the wound of the different concentrations of the total flavonoids cream group and the Deshijie hydrocolloid group had proliferated and migrated to the center of the wound. The collagen fibers in the granulation tissue under the keratinocyte layer were closely arranged, and more fibroblasts were seen growing. New capillaries were scattered and visible, the new epidermis was thick, and the inflammatory cells were relatively small, indicating that the skin wounds of the rats in each drug-treated group had entered the 2nd and 3rd stages of wound healing: the proliferation and remodeling stage. Fig.10 Middle B is a quantitative statistical graph of rat epidermal thickness. The blank control group epidermal thickness was 26.83±2.65μm, the model group epidermal thickness increased significantly to 87.72±1.95μm (P<0.01vs. control group), the Dejie hydrocolloid group epidermal thickness was 112.70±7.58μm, the 0.5% sunflower total flavonoids cream group, the 1% sunflower total flavonoids cream group, and the 2% sunflower total flavonoids cream group epidermal thickness 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% sunflower total flavonoids cream group, the 2% sunflower total flavonoids cream group, and the Dejie hydrocolloid group was significantly higher than that of the model group, indicating that the degree of re-epithelialization of the wound was higher than that of the model group, and it can effectively promote the healing of full-thickness skin wounds in rats.

[0547] 3.3 Analysis of Masson staining results

[0548] 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.

[0549] Fig.11 In the figure A, we can see 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 different concentrations of sunflower total flavonoids cream groups has different degrees of deepening. Among them, the 1% sunflower total flavonoids cream group has a larger blue area, darker color, and more uniform distribution than other groups. Fig.11B is a quantitative statistical chart of 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%. 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 difference analysis showed that there was a significant difference between the Dejie hydrocolloid group and the model group (P<0.05). Among the groups treated with different concentrations of sunflower total flavonoids cream, the 1% sunflower total flavonoids cream group had the highest collagen content, which was significantly different from the model group (P<0.01).

[0550] 3.4 Changes in CD31 and VEGF mRNA levels

[0551] Under normal conditions, blood vessels are stable and fully perfused, delivering sufficient nutrients and oxygen to tissues. Once tissue damage occurs, local ischemia and hypoxia occur, and a large number of angiogenic factors are produced at the wound site, thereby promoting the growth of microvessels. In order to determine the changes in the vascular marker CD31 and angiogenic factor (VEGF) at the gene level, RT-qPCR was performed to detect the expression of CD31 and VEGF mRNA on the 9th day after administration.

[0552] Fig.12 Figure A 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 total flavonoids cream groups was 0.10±0.01, 0.21±0.02, and 0.12±0.02, respectively. Among them, the 1% sunflower total flavonoids cream enhanced the expression of CD31 mRNA most significantly, with statistical differences (P<0.01), and there were no statistical differences in the other groups. Fig.12 Figure B shows that the expression of VegfmRNA decreased after modeling, from 0.25±0.07 to 0.14±0.04. After drug administration, the expression of VEGF mRNA increased to varying degrees. 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.

[0553] 3.5 Analysis of immunofluorescence staining results of skin tissue

[0554] 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 very intuitively and objectively obtain the distribution, density and content of CD31 in the new tissue, and then analyze whether its microcirculation is successfully established. Using the mean fluorescence intensity as an indicator, multiple fields of view of different sections were selected for photography to evaluate the angiogenesis of different groups.

[0555] like Fig.13 As shown in A, DAPI (blue) was used to locate the cell nucleus, and anti-CD31 antibody (red) was used to locate the vascular endothelial cells, and the clear distribution of CD31 in the tissue was obtained. The number of new blood vessels in the model group was small, and CD31 was weakly positive, with a relative fluorescence intensity of 41.63±7.46% of the control group (P<0.05). The CD31 expression in the Dejie hydrocolloid group was higher than that in the model group, with a relative fluorescence intensity of 73.75±18.13% of the control group, but there was no statistical difference. The CD31 expression in the different concentrations of the total flavonoids cream of Asparagus cochinchinensis was higher than that in the model group. The relative fluorescence intensity of the 0.5%, 1%, and 2% total flavonoids cream of Asparagus cochinchinensis was 49.49±13.50%, 70.92±9.53%, and 47.69±8.99% of the control group, respectively, but there was no statistical difference.

[0556] 3.6 Changes in vascular endothelial growth factor (VEGF) protein levels

[0557] The main reason why diabetic wounds are difficult to heal is the insufficient microcirculatory perfusion caused by high blood sugar and persistent inflammatory response. 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, accelerates 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 angiogenesis of vascular endothelial cells, promote the proliferation of new capillaries in ulcer wounds, and stimulate the growth of granulation tissue, thereby promoting the healing of wounds.

[0558] like Fig.14As shown, 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.

[0559] 4. Experimental conclusion

[0560] In this experiment, STZ-induced diabetes model was used, and a full-thickness skin excision model of rats was constructed using a sterile 8-mm Biopsy Punch. Dejie hydrocolloid was used as the positive control drug, and blank cream was used as the blank control drug of the model group to investigate the therapeutic effect of total flavonoids from Astragalus membranaceus on rats with diabetic ulcers.

[0561] The results showed that total flavonoids from Astragalus membranaceus can accelerate wound re-epithelialization, promote collagen fiber deposition, increase the expression of VEGF protein levels, and promote the healing of full-thickness skin wounds in diabetic rats. In addition, total flavonoids from Astragalus membranaceus can also promote the formation of new blood vessels by increasing the expression of CD31, thereby accelerating the healing process of diabetic ulcer wounds. In summary, total flavonoids from Astragalus membranaceus have a certain effect in treating diabetic ulcers.

[0562] Example 22 Postoperative acute kidney injury experiment

[0563] Animal model: 90 SPF male SD rats, 4-6 weeks old, weighing 260-300 g, provided by Nantong University. Animal license number: SCXK (Su) 2019-0001. Raised in the SPF animal laboratory of Jiangsu Suzhong Pharmaceutical Group Biopharmaceutical Co., Ltd., experimental animal use license number: SYXK (Su) 2022-0057. Rats were fed for one week of environmental adaptation before the formal start of the experiment, and were given regular pellet feed, free access to food and water, room temperature 20-24 ° C, humidity about 45%-65%, 12h light and 12h dark cycle.

[0564] Administration: oral administration, except for tacrolimus intraperitoneal injection. The Hibiscus hibiscus flower extract was prepared according to the method of Example 9-1.

[0565] Experimental modeling: Rat amputation trauma model: 3% sodium amobarbital 80 mg / kg body weight was intraperitoneally injected, and the rats were fixed in the supine position after anesthesia. First, the skin was cut 1-2 cm in the left groin, and the superficial abdominal artery and vein and other branches were separated and ligated. Then, the left femoral artery and vein were freed and ligated, and the left hind limb of the rat was cut off 1.2-1.4 cm above the knee joint, and the bleeding was stopped by suture to establish the rat left hind limb amputation model.

[0566] Experimental groups: normal group, model group, tacrolimus group (0.1 mg / kg), hibiscus flower extract low, medium and high groups, the dosages were 135 mg / kg, 270 mg / kg and 540 mg / kg respectively, 6 groups in total, 15 mice in each group, blood and kidney tissue were collected 3 days after administration to detect kidney injury molecule 1 (KIM-1). The test results are shown in Table 43 below.

[0567] Table 43.

[0568] Group Plasma KIM-1 (pg / ml) Kidney KIM-1 Normal group 69.32±23.18 0.012±0.002 Model group (72 hours after amputation) <![CDATA[2278.36±326.32 * ]]> <![CDATA[2.822±0.132 * ]]> Tacrolimus group <![CDATA[717.41±132.64 # ]]> <![CDATA[0.64±0.09 # ]]> Hibiscus tiliaceus flower extract low dose group <![CDATA[1039.72±128.87 # ]]> <![CDATA[0.81±0.17 # ]]> Hibiscus tiliaceus flower extract medium dose group <![CDATA[820.25±125.57 # ]]> <![CDATA[0.64±0.21 # ]]> Hibiscus tiliaceus flower extract high dose group <![CDATA[728.74±127.38 # ]]> <![CDATA[0.51±0.18 # ]]>

[0569] Note: *Compared with the normal control group, P < 0.05; #Compared with the model control group, P < 0.05

[0570] As can be seen from the above table, each group of hibiscus flower extracts had a significant reducing effect on plasma KIM-1 and kidney tissue KIM-1.

[0571] Example 23 Immunoglobulin A (IgA) nephropathy experiment

[0572] Animal model: 90 SPF male SD rats, 4-6 weeks old, weighing 220-240 g, provided by Nantong University. Animal license number: SCXK (Su) 2019-0001. Raised in the SPF animal laboratory of Jiangsu Suzhong Pharmaceutical Group Biopharmaceutical Co., Ltd., experimental animal use license number: SYXK (Su) 2022-0057. Before the formal start of the experiment, the rats were fed with a one-week environmental adaptation diet, given regular pellet feed, free access to food and water, room temperature 20-24 ° C, humidity about 45%-65%, 12h light and 12h dark cycle.

[0573] Administration method: intragastric administration, wherein the hibiscus flower extract is prepared according to Example 2-A2.

[0574] Experimental modeling: After SD rats were adaptively fed for 1 week, 50 rats were selected to establish the IgA nephropathy model. The immunogen bovine serum albumin (BSA) was given at 400 mg / kg for the first 6 weeks and 600 mg / kg for the last 4 weeks; subcutaneous injection (0.1 ml carbon tetrachloride + 0.5 ml castor oil) was given once a week for 10 weeks, and lipopolysaccharides (LPS) were used in combination (LPS 0.05 mg was injected into the tail vein at the 6th and 8th weeks, respectively). The deposition of IgA in the renal mesangial area was used as the modeling condition.

[0575] Experimental groups: normal group, model group, losartan potassium group (4.5 mg / kg), Hibiscus hibiscus flower extract low, medium and high groups, the dosages were 135 mg / kg, 270 mg / kg and 540 mg / kg respectively, a total of 6 groups, 15 in each group, urine protein and urine creatinine were detected after 4 weeks of administration. The test results are shown in Table 44 below.

[0576] Table 44.

[0577]

[0578]

[0579] Note: *Compared with the normal control group, P < 0.05; #Compared with the model control group, P < 0.05

[0580] As can be seen from the above table, each group of hibiscus flower extracts had a significant reducing effect on urine protein and PCR.

[0581] Example 24MN Membranous Nephropathy (Membranous Glomerulonephritis)

[0582] Animal model: 90 SPF male SD rats, 4-6 weeks old, weighing 180-220 g, provided by Nantong University. Animal license number: SCXK (Su) 2019-0001. Raised in the SPF animal laboratory of Jiangsu Suzhong Pharmaceutical Group Biopharmaceutical Co., Ltd., experimental animal use license number: SYXK (Su) 2017-0059. Before the formal start of the experiment, the rats were fed with a one-week environmental adaptation diet, given regular pellet feed, free access to food and water, room temperature 20-24 ° C, humidity about 45%-65%, 12 h light and 12 h dark cycle.

[0583] Administration method: intragastric administration, wherein the Hibiscus sibiricum flower extract is prepared according to the method of Example 9-1.

[0584] Experimental modeling: After SD rats were adaptively fed for 1 week, 75 rats were taken to establish a membranous nephropathy model. Pre-immunization: First, 1 mg C-BSA was dissolved in 0.5 mL 0.9% NaCl solution, and then 0.5 mL of Freund's incomplete adjuvant was added thereto. After complete emulsification, 1 mL of emulsifier was injected into the rats (injection site: underarms of both forelimbs and both groins, injection method: subcutaneous, injection frequency: once every other day, 3 times a week), pre-immunization for 1 week; formal immunization: After the pre-immunization, each rat was injected with C-BSA (16 mg / kg) through the tail vein every other day, 3 times a week, for a total of 15 times. After the modeling was completed, the 24h urine protein content of the rats was measured, and the modeling was successful if it exceeded 20 mg. All 40 rats in the study were successfully modeled, and 8 rats were set as normal controls and given an equal volume of saline in the same way.

[0585] Experimental groups: normal group, model group, losartan potassium group (4.5 mg / kg), Hibiscus hibiscus flower extract low, medium and high groups, the dosages were 135 mg / kg, 270 mg / kg and 540 mg / kg respectively, a total of 6 groups, 15 in each group, urine protein and urine creatinine were detected after 4 weeks of administration. The test results are shown in Table 45 below.

[0586] Table 45.

[0587] Group Urine protein / mg Urine protein / creatinine (PCR) Normal group 8.47±1.78 0.14±0.18 Model Group <![CDATA[78.26±26.21 * ]]> <![CDATA[2.82±1.12 * ]]> Losartan potassium group <![CDATA[23.11±10.34 # ]]> <![CDATA[1.04±1.09 # ]]> Hibiscus tiliaceus flower extract low dose group <![CDATA[32.42±12.87 # ]]> <![CDATA[1.31±0.37 # ]]> Hibiscus tiliaceus flower extract medium dose group <![CDATA[25.05±10.37 # ]]> <![CDATA[1.09±0.21 # ]]> Hibiscus tiliaceus flower extract high dose group <![CDATA[19.34±11.78 # ]]> <![CDATA[1.04±0.58 # ]]>

[0588] Note: *Compared with the normal control group, P < 0.05; #Compared with the model control group, P < 0.05

[0589] As can be seen from the above table, each group of hibiscus flower extracts had a significant reducing effect on urine protein and PCR.

[0590] Example 25 Idiopathic focal segmental glomerulosclerosis (FSGS)

[0591] Modeling: The FSGS rat model was established by two tail vein injections of doxorubicin: 1 mg / mL doxorubicin solution was injected into the tail vein of the rats, the dosage was 10 mg / kg, and it was repeated once every 4 weeks. The normal group was given the corresponding amount of normal saline.

[0592] Group administration: Group administration: Randomly divided into normal group, model group, positive drug group and Hibiscus truncatum total flavonoids group, 10 in each group, all groups were given total flavonoids (52 mg / kg) at 0.1 ml / 10 g body weight, and the positive drug group was given losartan potassium (4.5 mg / kg). The Hibiscus truncatum flower extract was prepared according to the method of Example 9-1.

[0593] Before administration and 4 weeks after administration, urine was collected for 24 hours, and urine protein concentration was detected by Coomassie Brilliant Blue method. The results are shown in Table 46.

[0594] Table 46. 24-hour urine protein quantitative (mg) measurement data

[0595] Group Before administration 4 weeks of intragastric administration Normal group 6.57±1.37 5.95±1.98 Model Group 47.76±4.67# 46.89±4.32# Positive drug group 46.79±4.83# 21.56±3.81* Total flavonoids from Astragalus membranaceus 45.12±5.72# 22.37±3.84*

[0596] Compared with the normal group, #P<0.05; compared with the model, *P<0.05

[0597] The experimental results in the above table show that each group in the examples has a reducing effect on urine protein. Compared with the model group, the total flavonoids group and the positive drug group can significantly reduce urine protein.

[0598] Example 26: Preparation of Hibiscus tiliaceus Flower Extract

[0599] Take 2 kg of hibiscus flowers, add 18 times the amount of ethanol, heat and reflux for 1 hour, filter, reduce the pressure of the filtrate to recover the ethanol, concentrate, adjust the pH to 6.0, let it stand, remove the upper layer of oil, vacuum dry, and crush to obtain the product.

[0600] Example 28: Therapeutic effect of total flavonoids of Hibiscus tiliaceus flower combined with VB12 on diabetic nephropathy in db / db mice 1 Experimental materials

[0601] 1.1 Drug Name

[0602] Vitamin B12, Shanxi Lifeng Huarui Pharmaceutical Co., Ltd.

[0603] Hibiscus tiliaceus flower total flavonoids HK were prepared according to Example 26.

[0604] Abelmoschus umbellata total flavonoids ZHT were prepared according to the method of Example 9-1.

[0605] 1.2 Drug configuration

[0606] ①VB12 solution (10μg / kg / d): Take out a piece of VB12 (25μg), crush it, dissolve it in deionized water, vortex it thoroughly to mix, and make up to 12.5ml (concentration 2μg / ml, 0.1ml for every 20g body weight).

[0607] ② Hibiscus flower total flavonoids HK (0.84 g / kg / d) group (denoted as HK): Weigh the total flavonoids of Hibiscus flower, add deionized water to the required volume, shake well, prepare once a week and store in a 4°C refrigerator, take out and bring to room temperature before use, and vortex mix thoroughly before administration (concentration 0.168 g / ml, 0.1 ml for every 20 grams of body weight).

[0608] ③ Hibiscus tiliaceus flower total flavonoids HK (0.84 g / kg / d) + VB12 (10 μg / kg / d) group (referred as HK+VB):

[0609] Weigh the required total flavonoids from hibiscus flowers, add the VB12 solution prepared above, shake well, protect from light, and use immediately after preparation (the concentration of total flavonoids from hibiscus flowers is 0.168 g / ml, 0.1 ml for every 20 grams of body weight).

[0610] ④ Hibiscus flower total flavonoids ZHT (75.50 mg / kg / d) group (denoted as ZHT): Weigh the required Hibiscus flower total flavonoids, add deionized water to the required volume, shake well, prepare once a week and store in a 4°C refrigerator, take out and bring to room temperature before use, and vortex mix thoroughly before administration (Hibiscus flower total flavonoids concentration 15.1 mg / ml, 0.1 ml for every 20 grams of body weight).

[0611] ⑤ Abelmoschus flavonoids ZHT group (75.50 mg / kg / d) + VB12 (10 μg / kg / d) group (referred as ZHT+VB):

[0612] Weigh the required total flavonoids of Hibiscus hibiscus, add the VB12 solution prepared above, shake well, protect from light, and use it immediately (the concentration of total flavonoids of Hibiscus hibiscus flowers is 15.1 mg / ml, 0.1 ml is given for every 20 grams of body weight).

[0613] 2. Experimental Animals

[0614] Overview of experimental animals:

[0615]

[0616] The blood glucose level of db / db mice can reach 16.7mmol / L. If the level reaches or exceeds this level for two consecutive days, it is determined to be diabetes.

[0617] After the db / db mice were brought in, they were randomly divided into groups according to their weight, 3 mice per cage, for a total of 34 cages. Blood glucose was measured immediately after the one-week adaptation period. The blood glucose of the animals was measured first, and then the body weight was weighed (blood glucose and body weight were recorded).

[0618] Experimental animal grouping and dosing regimen:

[0619]

[0620] After the adaptation period, urine of all mice was collected for the measurement of UACR (MAU / Cr). Once db / db mice developed hyperglycemia > 16.7mmol / L and UACR ratio > 200mg / g, it was determined that diabetic nephropathy had occurred and treatment was started.

[0621] 3. Routine biochemical index testing and sample collection

[0622] (1) All groups of db / db mice were placed in a metabolic cage once a week (1 day) to collect urine, and the mice's water intake and food intake were recorded. The collected urine was centrifuged at 4°C, 1000 rpm, for 3 min, and the supernatant was stored at -80 degrees. All groups of db / db mice had their blood sugar measured once a week (blood was collected from the tail vein), and their body weight measured once a week.

[0623] (2) Blood was collected from the eye sockets of mice; serum metabolome analysis: Peripheral blood was collected by removing the eyeball, first a drop of blood was dripped for blood smear, then the blood was dripped into an EP tube, gently rotated, and then centrifuged at 2500rpm x 10min, serum was collected into a new tube, and stored in a -80℃ refrigerator. Blood cells were stored at -80℃.

[0624] (3) At 4 weeks of administration, blood was collected from the eyeballs of mice, and sodium pentobarbital was injected and perfused. Samples were collected from the eyeballs, left kidney, right kidney, liver, heart, spleen, and jejunum (with feces).

[0625] ① Kidney: Cut the right kidney into two parts longitudinally, and store one half in paraformaldehyde for HE staining. Cut off 1 / 2 of the other half, that is, 1 / 4 of the kidney, and place it in a cryotube with liquid nitrogen, then transfer it to -80 degrees for Western Blot analysis. Then cut the remaining part into 4-5 small pieces and store it in RNA later (stored in a -80 degree refrigerator). The left kidney is preserved intact, placed in a cryotube with liquid nitrogen, and then transferred to -80 degrees for storage.

[0626] ②Heart: After taking out the heart, put it into a cryotube with liquid nitrogen and then store it in a -80 degree refrigerator.

[0627] ③ Liver: Take the middle lobe, cut the middle lobe into 3 parts, put them into RNA later, store them in paraformaldehyde and cryopreservation tubes in liquid nitrogen, and perform mRNA and lncRNA detection respectively; HE staining and Western Blot detection and analysis.

[0628] ④ Spleen: Divide the spleen tissue into two parts, put one part into liquid nitrogen in a cryopreservation tube, and store the other part in RNAlater, and then transfer to a -80 degree refrigerator for storage.

[0629] ⑤ Intestinal-kidney axis analysis: Jejunum (with feces): The jejunum was obtained by surgical double ligation, each section was 8 cm. All samples were placed in cryopreservation tubes and stored in liquid nitrogen for 5-60 minutes, and then transferred to -80 degrees for intestinal flora detection.

[0630] ⑥Detection of flavonoid chemical composition in small intestine contents (7 main flavonoids).

[0631] ⑦ Detection of flavonoid chemical components in peripheral blood serum (7 main flavonoids).

[0632] ⑧Detection of flavonoid chemical composition in feces (7 main flavonoids).

[0633] ⑨ Detection of urine albumin and creatinine: The ELISA kit was used to detect the values ​​of trace albumin and creatinine in urine in the prevention group and the treatment group, and the ratio (UACR) was calculated.

[0634] ⑩Related cytosolic transmitters and Megalin / Cubilin+VB12 molecular biology validation experiments are planned to use animal and patient kidney tissue specimens.

[0635] 5. Experimental Results

[0636] Fig. 20 The results showed that after the fourth week of administration, the HK+VB group, HT+VB group, and HT group all significantly reduced the UACR values ​​(P<0.0001), and the HK+VB group had the largest reduction in UACR values.

[0637] Fig.21 The staining results showed that: in the DKD group, some glomeruli were lobed, the glomerular capsule was still clear, the glomerular capillaries were compressed and unclear, the basement membrane was obviously thickened, and the mesangial cells and mesangial matrix in the mesangial area were significantly increased. In the drug administration group, the glomerular capsule was still clear, the focal glomerular capillaries were compressed and unclear, the basement membrane was slightly thickened, and the mesangial cells and mesangial matrix in the mesangial area were increased. It is proved that the pharmaceutical composition of the total flavonoids of Abelmoschus hibiscus flower and VB12 of the present invention has a good therapeutic effect on diabetic nephropathy.

[0638] At the same time, the observation results of the mouse state showed that the mice in each group of the total flavonoids combined with VB12 of the present invention had no abdominal distension or diarrhea, indicating that the combination of the total flavonoids of hibiscus flowers and VB12 of the present invention has good safety.

[0639] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A pharmaceutical composition, characterized in that The invention comprises the following components: total flavonoids of hibiscus flower and VB12.

2. The pharmaceutical composition according to claim 1, characterized in that The mass ratio of the total flavonoids of hibiscus flower to VB12 is 6500-95000:1; preferably 6550-8550:1, 74000-94000:1, and further preferably 7550:1, 84000:1; preferably, the mass content of flavonoid components in the total flavonoids of hibiscus flower is more than 55%, more preferably more than 60%, more preferably 65-90%, and most preferably 69-82%.

3. The pharmaceutical composition according to claim 1, characterized in that The total flavonoids of hibiscus flower include the following flavonoid components in 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 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; Further preferably, the mass ratio of the cotton linaloside-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; Furthermore, the total flavonoids of hibiscus flower contain 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, and more preferably 10:1.0-6.

0. More preferably, it is 10:1-5.6, and most preferably, it is 10:1.5-5.0; Furthermore, the total flavonoids of hibiscus flower contain rutin, wherein the mass ratio of safflower-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, the total flavonoids of hibiscus flower contain quercetin-3-O-acacia glycoside, wherein the mass ratio of safflower-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; Alternatively, the total flavonoids of hibiscus flower include flavonoid components in the following mass ratios: 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, the total flavonoids of hibiscus flower contain rutin, wherein the mass ratio of safflower-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, the total flavonoids of hibiscus flower contain quercetin-3-O-acacia glycoside, wherein the mass ratio of safflower-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; Alternatively, the total flavonoids of hibiscus flower include flavonoid components in the following mass ratio: the mass ratio of gossypol-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; Furthermore, the total flavonoids of hibiscus flower contain rutin, wherein the mass ratio of safflower-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, the total flavonoids of hibiscus flower contain quercetin-3-O-acacia glycoside, wherein the mass ratio of safflower-8-O-β-D-glucuronide to quercetin-3-O-acacia glycoside is 10:0.1-2.5, preferably 10:0.1-0.9, and preferably 10:0.15-0.

5.

4. The pharmaceutical composition according to claim 1, characterized in that The total flavonoids of hibiscus flower are effective flavonoid parts of hibiscus flower including flavonoid components in the following mass ratios: ； Preferably: ； More preferably: ； More preferably: ； More preferably: ； Further: ； or ； or Furthermore, the mass ratio of the isoquercetin is 1.2 or 1.0; More preferably, the total flavonoids of hibiscus flower contain rutin, and 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, and most preferably 1:0.009-0.04; Furthermore, the total flavonoids of hibiscus flower contain quercetin-3-O-acacia glycoside, and the mass ratio of isoquercetin to quercetin-3-O-acacia glycoside 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.

5. The pharmaceutical composition according to any one of claims 1 to 8, characterized in that The total content of quercetin, quercetin-3'-O-glucoside, myricetin, gossypol-8-O-β-D-glucuronide, isoquercitrin and hyperoside contained in the total flavonoids of hibiscus flower is more than 55%, preferably more than 60%, 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 gossypol-8-O-β-D-glucuronide is 8.5-34%, preferably 12-34%, or 8.5-30%, or 12%-23%; further, the total content of quercetin, quercetin-3'-O-glucoside, myricetin, gossypol-8-O-β-D-glucuronide, isoquercitrin, rutin and hyperoside is 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%. Furthermore, the total flavonoids of hibiscus flower include 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 1 1-22%, isoquercetin 12.0-17%, cotton lin-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%; further, it 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%.

6. The pharmaceutical composition according to claim 1, characterized in that The method for preparing total flavonoids from hibiscus flower comprises the following steps: (1) extracting hibiscus flower or a medicinal part of hibiscus flower 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 total flavonoids from hibiscus flower; the preferred extraction method is percolation; Preferably, 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 extractant used for the extraction in step (2) is n-butanol, petroleum ether or ethyl acetate, the extraction method is continuous countercurrent extraction, the solid-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; Further preferably, step (2) further comprises, before extraction, subjecting the extract to activated carbon adsorption, alcohol precipitation or acid precipitation treatment; Further preferably, the elution process in step (3) is as follows: the macroporous resin diameter-to-height ratio 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; Alternatively, the method for preparing total flavonoids from hibiscus flower comprises the following steps: (1) extracting hibiscus flower or medicinal part of hibiscus flower 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 total flavonoids from hibiscus flowers; the preferred extraction method is percolation; Preferably, 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 temperature of the water bath in step (2) is 50-70°C, and the water bath time is 30-90min; the elution process in step (3) is as follows: the diameter-to-height ratio of the polyamide resin is 1:4-1:9, the concentration of the sample solution is 0.10-0.60g crude drug / mL, the volume of the sample solution is 4-12BV, and 4-8BV of pure water is used. and 4-8 BV of 60-95% ethanol for elution; or, the preparation method of the total flavonoids of hibiscus flower comprises the following steps: (1) extracting hibiscus flower or the medicinal part of hibiscus 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; (3) extracting the dissolved solution to obtain an extract; (4) removing the solvent from the extract and eluting with a polyamide resin to obtain the total flavonoids of hibiscus flower; the preferred extraction method is reflux; Preferably, 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 adjusting agent used for adjustment 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 elution process in step (4) is: the diameter-to-height ratio of the polyamide resin 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; Alternatively, the method for preparing total flavonoids from hibiscus flowers comprises the following steps: taking hibiscus flowers, adding ethanol to reflux for extraction, filtering, reducing the pressure on the filtrate to recover ethanol, concentrating, adjusting the pH to 6.0, standing, removing the upper layer of grease, and drying to obtain the total flavonoids.

7. Use of the pharmaceutical composition according to any one of claims 1 to 19 in the preparation of a drug for treating kidney disease; preferably, the kidney disease includes nephritis; further preferably, the kidney disease is chronic glomerulonephropathy, diabetic nephropathy, diabetic nephropathy with renal fibrosis and / or nephritis.

8. Use of total flavonoids from hibiscus flowers in the preparation of a drug for treating kidney disease in combination with VB12, characterized in that: The total flavonoids of hibiscus flower are the total flavonoids of hibiscus flower in the pharmaceutical composition according to any one of claims 1 to 19. Preferably, the kidney disease includes nephritis; further preferably, the kidney disease is chronic glomerulonephropathy, diabetic nephropathy, diabetic nephropathy with renal fibrosis and / or nephritis.

9. A combination of total flavonoids from hibiscus flowers and VB12, preferably directly mixed together or combined together after being packaged separately; the total flavonoids from hibiscus flowers are the total flavonoids from hibiscus flowers in the pharmaceutical composition according to any one of claims 1 to 19.

10. Use of total flavonoids from hibiscus flowers in the preparation of a medicine or medicine package for treating kidney disease, especially diabetic nephropathy, in combination with VB12. The total flavonoids from hibiscus flowers are the total flavonoids from hibiscus flowers in the pharmaceutical composition according to any one of claims 1 to 19.

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