Traditional Chinese medicine composition with glucolipid metabolism regulating effect and preparation method and application thereof

Abstract

The invention belongs to the technical field of traditional Chinese medicine compositions, and particularly relates to a traditional Chinese medicine composition with a glycolipid metabolism regulating effect and a preparation method and application thereof. The traditional Chinese medicine composition with the glucolipid metabolism regulating effect is prepared from the following raw materials in parts by weight: 10 to 15 parts of herba cepbalanoplosis segeti, 7 to 12 parts of fructus phyllanthi, 10 to 15 parts of fructus citri sarcodactylis and 15 to 20 parts of semen cassiae. The traditional Chinese medicine composition disclosed by the invention can be used for carrying out comprehensive and integrated prevention and control and overall regulation on glycolipid metabolic diseases, is good in treatment effect, is natural medicines, and has few side effects.

Description

Technical Field

[0001] The present invention belongs to the technical field of Chinese medicine compositions, and in particular relates to a Chinese medicine composition having the function of regulating sugar and lipid metabolism, and a preparation method and application thereof. Background Art

[0002] Glucose and lipid metabolic diseases (GLMD) include dysglycemia, dyslipidemia, non-alcoholic fatty liver disease, overweight, hypertension, atherosclerotic cardiovascular and cerebrovascular diseases, etc., with a high incidence rate, which is a global problem. Its core pathological mechanisms are insulin resistance, oxidative stress, neuroendocrine disorders, inflammation and intestinal flora disorders. In GLMD, various inflammatory factors and immune cells play an important role in related tissues and organs such as blood vessels, pancreatic islets, liver, and adipocytes, causing inflammatory damage, and forming a multi-organ crosstalk network through inflammatory mediators and signaling pathways, affecting the development of the disease.

[0003] Diseases such as abnormal glucose metabolism, abnormal lipid metabolism, hypertension and non-alcoholic fatty liver disease are often co-morbid or concurrent, and they affect each other and are closely related. At present, the strategy of single disease diagnosis and treatment is mostly adopted for glucose and lipid metabolic diseases, and the comprehensive compliance rate of blood sugar, blood lipids, blood pressure, etc. is low. Traditional Chinese medicine emphasizes the holistic concept and conducts comprehensive and integrated prevention and control of diseases. In terms of treatment, Chinese medicine has the advantages of overall regulation and few side effects. Many Chinese medicines have significant effects in treating glucose and lipid metabolic disorders through inflammatory targets. Compared with drugs targeting inflammatory mediators, Chinese medicine acts on multiple downstream inflammatory mediators through multiple signaling pathways at the same time, showing more aspects of regulatory effects, and the effect is mild and safe, which has good prospects for the development of GLMD drugs. However, most of the current research on glucose and lipid metabolism is single, with complex ingredients and low drug utilization rate.

[0004] For example, Chinese patent CN104073416B discloses a ginseng health wine, which is made of 35-proof liquor and the following Chinese medicinal materials: ginseng, Sterculia lychnophora, Poria, peach kernel, thistle, white hyacinth bean flower, longan meat, Ophiopogon japonicus, tortoise shell, Alpinia oxyphylla, lotus leaf, radish seed, cassia seed, cinnamon bark, Phyllanthus emblica, bergamot, seabuckthorn, Atractylodes macrocephala, Terminalia chebula, red peony root, Glehnia littoralis, Fritillaria cirrhosa, Scrophularia ningpoensis, Radix Rehmanniae, Citron, Aloe, Longifolia leaf, Imperatae chinensis, Peucedanum purpurogenum, Smilax china. The health wine of the invention uses pure Chinese medicine as raw materials, and multiple Chinese medicines are organically combined and easily absorbed by the human body. It has the effects of tonifying the middle qi, warming the spleen and kidney, nourishing health, enhancing immunity, strengthening the stomach and digestion, strengthening physical fitness, and improving sleep quality; the preparation method is simple, convenient to take, safe and non-toxic and non-side effects, and good taste. Although the patent discloses Phyllanthus emblica, Citron, Cassia seed and Cirsium cyrtonema, the relationship between the monarch, minister, assistant and envoy is completely different from that of the present invention, the functions they play are also different, and the ingredients are complex.

[0005] Another example is Chinese patent CN118662593A, which discloses a Chinese medicine composition and use for improving sugar and lipid metabolism, body fat level and non-alcoholic fatty liver disease. The technical solution of the invention is a Chinese medicine composition for improving sugar and lipid metabolism, body fat level and non-alcoholic fatty liver disease, including the following ingredients: ginseng, malt, Poria cocos, Buza leaves, Prunella vulgaris and Sophora japonica. The Chinese medicine composition of the invention has the effects of soothing the liver, regulating qi and strengthening the spleen, clearing the liver, purging fire and dispersing knots, and is used for weight loss, lipid reduction, blood sugar reduction, improving body fat rate, treating liver steatosis, inflammation, and improving liver fibrosis. It can also prevent the occurrence of cardiovascular and cerebrovascular diseases induced by sugar and lipid metabolism disorders through lipid reduction, anti-inflammatory, and anti-oxidation. Although the invention and the present invention have similar effects, the technical solution adopted is completely different from the present invention.

[0006] Therefore, it is urgent to study a Chinese medicine composition with simple ingredients and lipid-lowering, anti-inflammatory and antioxidant effects. Summary of the invention

[0007] Based on the deficiencies of the prior art, the present invention aims to provide a Chinese medicine composition having the function of regulating glucose and lipid metabolism, and its preparation method and application. The Chinese medicine composition provided by the present invention has simple ingredients, small toxic and side effects, and the interaction between drugs can better play the role of regulating glucose and lipid metabolism.

[0008] The raw material effects of the present invention are:

[0009] Thistle is the above-ground part of the Asteraceae plant, which is cool in nature, sweet and bitter in taste. It enters the heart and liver meridians. It has the effects of cooling blood and stopping bleeding, removing blood stasis and reducing swelling. It is a blood-cooling and hemostatic drug under the category of hemostatic drugs. The total flavonoids of thistle can reduce blood sugar, cholesterol, triglyceride and low-density lipoprotein levels, thereby improving the body's blood sugar and lipid metabolism disorders.

[0010] Phyllanthus emblica is the mature fruit of the Euphorbiaceae plant Phyllanthus emblica, which is cool in nature and tastes sweet, sour and astringent. It enters the lung meridian and stomach meridian, and has the effects of clearing heat and cooling blood, promoting digestion and strengthening the stomach, promoting body fluid and relieving cough. Phyllanthus emblica is rich in phenolic compounds such as gallic acid and ascorbic acid, which can scavenge free radicals and exert antioxidant effects. It is clinically used to lower blood lipids and anti-oxidation.

[0011] Buddha's hand is the fruit of the Rutaceae plant, which is warm in nature and tastes pungent, bitter and sour. It belongs to the lung meridian, spleen meridian and liver meridian, and has the effects of soothing the liver and regulating qi, harmonizing the stomach and relieving pain, and drying dampness and resolving phlegm. It is used to treat liver and stomach qi stagnation, chest and flank pain, stomach fullness, loss of appetite and vomiting, cough and excessive phlegm, etc. In recent years, with the continuous deepening of research on the chemical composition and pharmacological effects of Buddha's hand, its anti-cancer, antioxidant, blood pressure lowering and anti-aging effects have gradually become known to the public.

[0012] Cassia seed is the dried and mature seed of Cassia obtusifolia or Cassia tora of the Leguminosae family, named for its function of improving eyesight; it is slightly cold in nature, sweet, bitter and salty in taste, enters the liver, large intestine and kidney meridians, and has the functions of clearing the liver and improving eyesight, and moistening the intestines and promoting defecation. Scholars at home and abroad have conducted numerous studies on the chemical constituents of cassia seed and found that it contains various chemical constituents and exhibits various pharmacological activities, such as lowering blood pressure, regulating lipid metabolism, protecting the liver and improving eyesight, antibacterial, antioxidant, etc., and is mainly used to treat eye diseases, constipation, hyperlipidemia, hypertension and diabetes.

[0013] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0014] On the one hand, the present invention provides a traditional Chinese medicine composition with the function of regulating glucose and lipid metabolism, which is composed of the following raw materials in parts by weight: 10-15 parts of Cirsium setosum, 7-12 parts of Phyllanthus emblica, 10-15 parts of Citrus medica var. sarcodactylis and 15-20 parts of cassia seed.

[0015] More preferably, the traditional Chinese medicine composition with the function of regulating glucose and lipid metabolism is composed of the following raw materials in parts by weight: 12-14 parts of Cirsium setosum, 9-11 parts of Phyllanthus emblica, 10-12 parts of Citrus medica var. sarcodactylis and 15-18 parts of cassia seed.

[0016] Further preferably, the traditional Chinese medicine composition with the function of regulating glucose and lipid metabolism is composed of the following raw materials in parts by weight: 12-13 parts of Cirsium setosum, 9-10 parts of Phyllanthus emblica, 10-11 parts of Citrus medica var. sarcodactylis, 15-16 parts of cassia seed.

[0017] Most preferably, the traditional Chinese medicine composition with the function of regulating glucose and lipid metabolism is composed of the following raw materials in parts by weight: 12 parts of Cirsium setosum, 9 parts of Phyllanthus emblica, 10 parts of Citrus medica var. sarcodactylis and 15 parts of cassia seed.

[0018] On the other hand, the present invention also provides a preparation method of the traditional Chinese medicine composition with the function of regulating glucose and lipid metabolism, including the following steps:

[0019] (1) Weigh the formula amounts of Cirsium setosum, Phyllanthus emblica, Citrus medica var. sarcodactylis and cassia seed respectively, mix them, then add an ethanol solution to the mixture of the four drugs, heat under reflux for extraction, and filter to obtain a filtrate and filter residue.

[0020] (2) Concentrate the filtrate obtained in step (1) to obtain the traditional Chinese medicine composition.

[0021] Preferably, the volume concentration of ethanol in step (1) is 65%-75%, and more preferably 70%.

[0022] Preferably, the material-liquid ratio of the total mass of the four drugs and ethanol in step (1) is: 1-3 g: 4-10 mL;

[0023] Further preferably, the ratio of the total mass of the four drugs described in step (1) to the liquid material of ethanol is: 1 - 2 g : 5 - 8 mL;

[0024] Even more preferably, the ratio of the total mass of the four drugs described in step (1) to the liquid material of ethanol is: 1 g : 5 mL.

[0025] Preferably, the temperature of the heating and reflux extraction described in step (1) is 65 - 70 °C, the number of extraction times is 2 - 4 times, preferably 2 times; each extraction is 2 - 4 h, preferably 2 h.

[0026] Preferably, the concentration in step (2) is concentrated to 70% - 90% of the solid content, preferably 88%.

[0027] Secondly, the present invention also provides the application of the above traditional Chinese medicine composition in the preparation of drugs for regulating glycolipid metabolism.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) The traditional Chinese medicine composition of the present invention can comprehensively and integrally prevent and control glycolipid metabolic diseases, and has an overall regulation effect with good treatment effect.

[0030] (2) Western medicine treatment of glycolipid metabolic diseases is often accompanied by many irreversible toxic and side effects, while the traditional Chinese medicine composition prepared by the present invention is all natural drugs with fewer side effects. Description of the Drawings

[0031] Figure 1 Effect of the traditional Chinese medicine composition prepared in Example 1 (TZDXF) on the proliferation of RAW264.7 cells (compared with the blank group (0 mg / mL), **P < 0.01, *P < 0.05, n = 3);

[0032] Figure 2 Effect of the traditional Chinese medicine composition prepared in Example 1 (TZDXF) on TNF-α, IL-1β, and IL-6 in LPS-induced RAW264.7 cells (A is IL-6, B is TNF-α, C is IL-1β; compared with the Control group, ## P < 0.01, # P < 0.05; compared with the Model group, **P < 0.01, *P < 0.05, n = 4);

[0033] Figure 3 Effect of the traditional Chinese medicine composition prepared in Example 1 (TZDXF) on the acetic acid writhing reaction of mice (compared with the Control group, **P < 0.01, *P < 0.05, n = 8);

[0034] Figure 4Effect of the traditional Chinese medicine composition prepared in Example 1 (TZDXF) on the body weight of golden hamsters over time (compared with the Control group, # P < 0.05, ## P < 0.01, compared with the Model group, *P < 0.05, **P < 0.01, n = 10);

[0035] Figure 5 Effect of the traditional Chinese medicine composition prepared in Example 1 (TZDXF) on the weight gain rate of golden hamsters (compared with the Control group, # P < 0.05; compared with the Model group, **P < 0.01, n = 10);

[0036] Figure 6 Effect of the traditional Chinese medicine composition prepared in Example 1 on the blood lipids of hamsters with metabolic inflammation model (A is TG, B is TC, C is LDL-C, compared with the Control group, # P < 0.05, ## P < 0.01, compared with the Model group, *P < 0.05, **P < 0.01, n = 10);

[0037] Figure 7 Effect of the traditional Chinese medicine composition prepared in Example 1 on the liver morphology, liver weight and liver index changes of hamsters with metabolic inflammation model (A. Liver morphology; B. Liver weight; C. Liver index, compared with the Control group, ## P < 0.01, compared with the Model group, **P < 0.01, *P < 0.05, n = 10);

[0038] Figure 8 Effect of the traditional Chinese medicine composition prepared in Example 1 on the fasting blood glucose changes of hamsters with metabolic inflammation model (compared with the Control group, ## P < 0.01, compared with the Model group, **P < 0.01, *P < 0.05, n = 3);

[0039] Figure 9 Effect of the traditional Chinese medicine composition prepared in Example 1 on the liver pathology of hamsters with metabolic inflammation model (microscope magnification 200×, scale bar = 100 μm);

[0040] Figure 10 Effect of the traditional Chinese medicine composition prepared in Example 1 on serum TNF-α, CRP, IL-6 and MCP-1 of hamsters with metabolic inflammation model (compared with the Control group, ## P < 0.01, compared with the Model group, **P < 0.01, n = 8);

[0041] Figure 11Effect of the Traditional Chinese Medicine Composition Prepared in Example 1 on Inflammatory Factors and Oxidative Stress in the Liver of Hamsters with Metabolic Inflammation (compared with the Control group, ## P < 0.01, compared with the Model group, **P < 0.01, n = 6). Detailed implementation manners

[0042] The technical solution of the present invention will be clearly and completely described below in specific implementation manners. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention. The materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0043] Cirsium setosum and Citrus medica var. sarcodactylis were uniformly purchased from the Institute of Traditional Chinese Medicine of Guangdong Pharmaceutical University; Phyllanthus emblica was purchased from Beijing Tongrentang, and the origin was Yunnan; Cassia obtusifolia was purchased from Baihetang Pharmacy, Suishi Village, Xiaoguwei Street, University Town, Guangzhou, and the batch number was 0090956.

[0044] Example 1 A traditional Chinese medicine composition with the effect of regulating glucose and lipid metabolism

[0045] It is composed of the following raw materials:

[0046] 12 parts of Cirsium setosum, 9 parts of Phyllanthus emblica, 10 parts of Citrus medica var. sarcodactylis, and 15 parts of Cassia obtusifolia.

[0047] It is prepared according to the following method:

[0048] (1) Weigh the formula amounts of Cirsium setosum, Phyllanthus emblica, Citrus medica var. sarcodactylis, and Cassia obtusifolia respectively, mix them, then add an ethanol solution with a volume concentration of 70%, heat and reflux for extraction twice at 70 °C, extract for 2 hours each time, and filter to obtain a filtrate and a filter residue.

[0049] (2) Concentrate the filtrate obtained in step (1) to a solid content of 88% to obtain the traditional Chinese medicine composition.

[0050] Example 2 A traditional Chinese medicine composition with the effect of regulating glucose and lipid metabolism

[0051] It is composed of the following raw materials:

[0052] 10 parts of Cirsium setosum, 12 parts of Phyllanthus emblica, 15 parts of Citrus medica var. sarcodactylis, and 20 parts of Cassia obtusifolia.

[0053] It is prepared according to the following method:

[0054] (1) Weigh the formula amounts of Cirsium setosum, Phyllanthus emblica, Citrus medica var. sarcodactylis, and Cassia obtusifolia separately, mix them, then add an ethanol solution with a volume concentration of 75%, heat under reflux for extraction 4 times at 70 °C, extract for 2 hours each time, filter to obtain a filtrate and a filter residue.

[0055] (2) Concentrate the filtrate obtained in step (1) to a solid content of 71% to obtain the described traditional Chinese medicine composition.

[0056] Example 3 A traditional Chinese medicine composition with the function of regulating glucose and lipid metabolism

[0057] It is composed of the following raw materials:

[0058] 15 parts of Cirsium setosum, 7 parts of Phyllanthus emblica, 12 parts of Citrus medica var. sarcodactylis, and 18 parts of Cassia obtusifolia.

[0059] It is prepared according to the following method:

[0060] (1) Weigh the formula amounts of Cirsium setosum, Phyllanthus emblica, Citrus medica var. sarcodactylis, and Cassia obtusifolia separately, mix them, then add an ethanol solution with a volume concentration of 75%, heat under reflux for extraction 3 times at 65 °C, extract for 3 hours each time, filter to obtain a filtrate and a filter residue.

[0061] (2) Concentrate the filtrate obtained in step (1) to a solid content of 78% to obtain the described traditional Chinese medicine composition.

[0062] Example 4 A traditional Chinese medicine composition with the function of regulating glucose and lipid metabolism

[0063] It is composed of the following raw materials:

[0064] 14 parts of Cirsium setosum, 11 parts of Phyllanthus emblica, 11 parts of Citrus medica var. sarcodactylis, and 16 parts of Cassia obtusifolia.

[0065] (1) Weigh the formula amounts of Cirsium setosum, Phyllanthus emblica, Citrus medica var. sarcodactylis, and Cassia obtusifolia separately, mix them, then add an ethanol solution with a concentration of 70%, heat under reflux for extraction 2 times at 70 °C, extract for 4 hours each time, filter to obtain a filtrate and a filter residue.

[0066] (2) Concentrate the filtrate obtained in step (1) to a solid content of 78% to obtain the described traditional Chinese medicine composition.

[0067] Comparative Example 1 A traditional Chinese medicine composition with the function of regulating glucose and lipid metabolism

[0068] It is composed of the following raw materials:

[0069] Compared with Example 1, the difference is only that 9 parts of Phyllanthus emblica are replaced with 9 parts of Cirsium setosum, that is: 21 parts of Cirsium setosum, 10 parts of Citrus medica var. sarcodactylis, and 15 parts of Cassia obtusifolia.

[0070] It is prepared according to the following method:

[0071] (1) Weigh the large thistle, fingered citron, and cassia seed in the formula respectively and mix them. Then add an ethanol solution with a volume concentration of 70%, and heat and reflux for extraction twice at 70°C, with each extraction lasting for 2 hours. Filter to obtain the filtrate and the residue.

[0072] (2) Concentrate the filtrate obtained in step (1) to a solid content of 88% to obtain the described traditional Chinese medicine composition.

[0073] Comparative Example 2 A traditional Chinese medicine composition with the effect of regulating glucose and lipid metabolism

[0074] It consists of the following raw materials:

[0075] Compared with Example 1, the difference is only that cassia seed is replaced with hawthorn, that is: 12 parts of Cirsium setosum, 9 parts of Phyllanthus emblica, 10 parts of fingered citron, and 15 parts of hawthorn.

[0076] It is prepared according to the following method:

[0077] (1) Weigh the Cirsium setosum, Phyllanthus emblica, fingered citron, and hawthorn in the formula respectively and mix them. Then add an ethanol solution with a volume concentration of 70%, and heat and reflux for extraction twice at 70°C, with each extraction lasting for 2 hours. Filter to obtain the filtrate and the residue.

[0078] (2) Concentrate the filtrate obtained in step (1) to a solid content of 88% to obtain the described traditional Chinese medicine composition.

[0079] Comparative Example 3 A traditional Chinese medicine composition with the effect of regulating glucose and lipid metabolism

[0080] It consists of the following raw materials:

[0081] Compared with Example 1, the difference is: adjust Cirsium setosum to 5 parts, Phyllanthus emblica to 16 parts, fingered citron to 7 parts, and cassia seed to 18 parts.

[0082] It is prepared according to the following method:

[0083] (1) Weigh the Cirsium setosum, Phyllanthus emblica, fingered citron, and cassia seed in the formula respectively and mix them. Then add an ethanol solution with a volume concentration of 70%, and heat and reflux for extraction twice at 70°C, with each extraction lasting for 2 hours. Filter to obtain the filtrate and the residue.

[0084] (2) Concentrate the filtrate obtained in step (1) to a solid content of 71% to obtain the described traditional Chinese medicine composition.

[0085] Effect experiment

[0086] Experimental reagents and consumables are shown in Table 1 specifically.

[0087] Table 1

[0088]

[0089]

[0090] 1. Free radical scavenging experiment - Study on the antioxidant effect of traditional Chinese medicine composition

[0091] The DPPH method and ABTS method were used to explore the free radical scavenging effects of Examples 1 - 4 and Comparative Examples 1 - 3, and comprehensively evaluate the antioxidant activities of Examples 1 - 4 and Comparative Examples 1 - 3.

[0092] 1.1 Detection method

[0093] The detection kits were used to detect the scavenging rates of DPPH and ABTS free radicals. First, the sample solutions were prepared. The low (1mg / mL), medium (4mg / mL), and high-dose (8mg / mL) groups of the formula and the low (0.25mg / mL), medium (0.5mg / mL), and high-dose (1mg / mL) groups of positive control vitamin C were set, and 3 parallel duplicate wells were set. The specific operation steps are as follows:

[0094] Table 2 Sampling and detection steps of DPPH kit

[0095]

[0096] DPPH free radical scavenging rate (%) = [(1 - (A determination - A sample) ÷ A blank) × 100]%

[0097] Table 3 Sampling and detection steps of ABTS kit

[0098]

[0099] ABTS free radical scavenging rate (%) = [(1 - (A determination - A sample) ÷ A blank) × 100]%

[0100] The experimental results are shown in Tables 4 and 5.

[0101] Table 4

[0102]

[0103] Table 5

[0104]

[0105] 2. Cell experiment - Study on the in vitro anti-inflammatory effect of traditional Chinese medicine composition

[0106] For in vitro anti-inflammatory research, a lipopolysaccharide (100ng / mL)-induced inflammatory model of mouse macrophages RAW264.7 was established. The CCK-8 detection method was used to screen the administration concentrations, and the Elisa method was used to detect the expression levels of inflammatory factors IL-1β, TNF-α, and IL-6.

[0107] 2.1 Cell culture and model establishment

[0108] Drug administration for cell model establishment: The experiment was divided into the following groups: (1) Blank control group (Control): Cells were treated with DMEM. (2) Example 1 group: Cells were treated with different concentrations of Example 1 for a certain period of time, and then 100 ng / mL LPS was added and co - acted for 24 h. (3) Model group (Model): Cells were treated with 100 ng / mL LPS for 24 h (LPS group).

[0109] 2.2 Determination of cell viability by CCK - 8 method

[0110] A CCK - 8 kit was used to determine the cytotoxicity of the traditional Chinese medicine composition, and the drug - administration concentration was screened according to the cell survival rate.

[0111] 2.3 Determination of cytokines by Elisa method

[0112] The supernatant of centrifuged cells was taken, and an Elisa kit was used to detect the contents of TNF - α, IL - 6, and IL - 1β.

[0113] 3. Animal experiment - Study on the anti - acute - inflammation effect of traditional Chinese medicine combination in vivo

[0114] The in - vivo inflammation study was carried out from two aspects: acute inflammation and chronic inflammation. Animal experiment models including the acetic - acid writhing method and xylene - induced auricular swelling in mice were used to study the anti - inflammatory and analgesic activities of Example 1.

[0115] 3.1 Experimental animals

[0116] Kunming mice were purchased from Zhuhai Besttone Biotechnology Co., Ltd., with the certificate number SCXK(Yue)2020 - 0051, weighing 25 ± 10 g, male, SPF - level, and 80 mice. They were adaptively fed for 1 week before the formal experiment.

[0117] 3.2 Experimental drugs

[0118] The low - dose group of Example 1 (0.473 g / mL), the medium - dose group of Example 1 (0.945 g / mL), the high - dose group of Example 1 (1.89 g / mL), the blank control group, and the positive - drug aspirin group (1 mg / mL) were set up, with 8 mice in each group (n = 8).

[0119] 3.3 Acetic - acid writhing method

[0120] Forty healthy male Kunming mice weighing 25±10 g (about 6-8 weeks old) were selected, allowed to eat and drink freely. All were given drugs by gavage; the blank group was gavaged with CMC-Na, and the other groups were gavaged with drugs at corresponding concentrations. According to the drug administration methods of each group, drugs were continuously administered for 3 days, once a day. One hour after the last drug administration, 0.6% glacial acetic acid (10 mL / kg) was intraperitoneally injected (prepared freshly before use to reduce experimental error), and the number of writhing times of each group of mice within 15 minutes was recorded. The specific behavioral manifestations of writhing were: concave abdomen, hind limbs extended backward, and hips twisted and elevated left and right. The analgesic rate was calculated according to the following formula.

[0121] Analgesic rate = (mean value of blank group - mean value of drug administration group) / mean value of blank group × 100%

[0122] Table 6

[0123]

[0124]

[0125] 3.4 Xylene-induced auricle swelling in mice

[0126] Forty healthy male SPF-grade Kunming mice weighing 25±10 g were selected. After one week of adaptive feeding in an SPF-grade animal room, they were divided into the low-dose group of Example 1, the medium-dose group of Example 1, the high-dose group of Example 1, the low-dose group of Comparative Example 1, the medium-dose group of Comparative Example 1, the high-dose group of Comparative Example 1, the blank control group, and the positive drug aspirin group. The drug administration concentrations of each group are shown in Table 6. After 0.5 h of drug administration, 0.05 mL / rat of xylene was evenly and slowly applied to the right ear of the mice, and an equal amount of distilled water was applied to the left ear. After 2 h, the mice were sacrificed, and the left and right ear pieces were removed along the auricle baseline. Round ear pieces were punched at the same position on the left and right ears with a 6-mm hand-held punch and weighed. The difference in weight between the left and right ears was the swelling degree; the inhibition rate was calculated according to the following formula, and the results are shown in Table 7.

[0127] Inhibition rate (%) = (average ear swelling degree of control group - ear swelling degree of drug administration group) / average ear swelling degree of control group × 100%

[0128] Table 7

[0129] Group Dosing concentration (g / kg) Ear swelling degree (mg) Inhibition rate (%) Blank control group — 5.0±1.07 — Example 1 - Low dose group 0.473 3.0±1.33** 40 Example 1 - Medium dose group 0.945 2.33±0.82** 53 Example 1 - High dose group 1.89 3.88±1.51 23 Aspirin group 0.01 1.43±0.53** 71 Comparative example 1 - Low dose group 0.473 4.8±1 5 Comparative example 1 - Medium dose group 0.945 4±1.1 20 Comparative example 1 - High dose group 1.89 3.5±1.4* 30

[0130] Note: Compared with the blank control group, *P < 0.05, **P < 0.01, n = 8.

[0131] 4. Animal experiment - Study on the effect of treating glycolipid metabolic diseases

[0132] Research on anti-glycolipid metabolic diseases. A metabolic inflammation model was established in golden hamsters induced by a high-fat diet. By detecting the body weight changes of hamsters, blood glucose and lipid metabolism, observing liver pathological changes, and detecting serum and liver metabolic inflammation-related indicators CRP, TNF-α, IL-6, MCP-1, SOD, MDA, the regulatory effect of Example 1 on metabolic inflammation was explored.

[0133] 4.1 Experimental animals

[0134] A total of 60 SPF-grade male Syrian golden hamsters (LVG Hamster) were purchased from Vital River Laboratory Animal Technology Co., Ltd., with the certificate number SCXK(Beijing)2021-0011. The quarantine time for hamsters was 5 days, and they were adaptively fed for 1 week before the experiment began. During the experiment, they were housed on the third floor of the Experimental Animal Center of Guangdong Pharmaceutical University (experimental number SPF2022447), in an SPF-grade animal house. Breeding conditions: 3 golden hamsters per small cage. Feed provided: The animal center provided maintenance feed for mice and rats at 3.4 kcal / g (Beijing Keao Xieli Feed Co., Ltd.), and high-fat and high-cholesterol feed D190820 (Dyets, USA).

[0135] 4.2 Experimental drugs

[0136] Blank control: Normal saline

[0137] Positive control: Simvastatin, provided by Shandong Lukang Pharmaceutical Co., Ltd., batch number 1033466

[0138] 4.3 Animal grouping and modeling

[0139] Golden hamsters were fed a high-fat diet to construct a hyperlipidemia model. They were randomly grouped according to body weight; 60 hamsters were divided into 6 groups (n = 10), namely the blank group (Control), the model group (Model), the low-dose group of Example 1 (TZDXF-L 0.284 g / mL), the medium-dose group of Example 1 (TZDXF-M 0.567 g / mL), the high-dose group of Example 1 (TZDXF-H 1.135 g / mL), and the positive drug control group of simvastatin (Simvastatin 12 mg / kg), with 10 in each group, a total of 6 groups. The modeling was carried out for 2 weeks, and after the modeling was stable, continuous regular drug administration was carried out for 2 weeks; both the model group, the traditional Chinese medicine composition treatment group, and the positive drug control group received a high-fat diet-induced hyperlipidemia model. The drug administration groups were treated with the corresponding concentration of the drug at 10 mL / kg while on a high-fat diet, and the blank group and the model group were gavaged with normal saline.

[0140] 4.4 Determination of body weight and weight gain rate of hamsters with hyperlipidemia model

[0141] The body weight recorded before modeling was used as the initial body weight, and the body weight after 2 weeks of modeling with a high-fat diet was the successful modeling body weight. After the administration ended, the animals were fasted but allowed to drink water for 8 hours, and their body weight after administration was measured before sacrifice. The weight gain rate was calculated based on this, as follows: Figure 5 As shown below:

[0142] Weight gain rate = [(body weight after administration - body weight at successful modeling) / body weight at successful modeling] × 100%

[0143] 4.5 Determination of liver weight and liver index of hamsters in the metabolic inflammation model

[0144] The hamsters sacrificed by taking blood from the abdominal aorta were dissected, and their livers were taken. The liver weight was weighed and the liver index was calculated. The calculation formula is as follows:

[0145] Liver index = liver weight (g) / body weight (g) × 100%

[0146] 4.6 Detection of fasting blood glucose and blood lipids using kits

[0147] Kits were used to detect the levels of four blood lipid items TC, TG, LDL-C, HDL-C and fasting blood glucose GLU in the blood of hamsters.

[0148] 4.7 Detection of pathological damage of liver tissue by H&E staining

[0149] The liver tissue was dehydrated, embedded, sectioned and stained with H&E to observe the pathological damage of the liver tissue.

[0150] 4.8 Determination of related factors in hamsters in the metabolic inflammation model by Elisa method

[0151] Elisa detection kits were used to detect the contents of CRP, TNF-α, IL-6 and MCP-1 in the serum of hamsters; at the same time, Elisa detection kits were used to detect the contents of CRP, TNF-α, IL-6, MCP-1, SOD and MDA in the homogenate of hamster liver tissue.

[0152] 5. Conclusion

[0153] As can be seen from Table 4 and Table 5, the composition in the present invention has good antioxidant activity, and the scavenging effects on DPPH and ABTS free radicals are much higher than those of the comparative examples.

[0154] The results of co-culturing RAW264.7 cells with different concentrations of Example 1 are as follows Figure 1 As shown; when the culture concentration was 300 μg / mL, the cell viability was significantly reduced (P<0.01). Therefore, three concentrations of 50, 100, and 200 μg / mL were selected as the low, medium, and high administration concentrations for subsequent experiments.

[0155] FromFigure 2 It can be seen that the composition in the present invention has an improving effect on the inflammatory response of RAW264.7 cells induced by LPS modeling. By reducing the levels of inflammatory factors TNF-α, IL-1β, and IL-6, especially the high dose in Example 1 has a significant difference in reducing IL-6 (P < 0.05), and the low, medium, and high dose groups have significant differences in reducing TNF-α (P < 0.05); the medium and high doses have significant differences in reducing IL-1β (P < 0.01), showing a good anti-inflammatory effect in vitro.

[0156] It is known from Figure 3 It can be seen that the composition of Example 1 can significantly reduce the number of writhing responses. Among them, the number of writhing in the low, medium, high dose groups and the positive control group is significantly reduced, with statistical differences (P < 0.05 or P < 0.01); as can be seen from Table 6, the analgesic effect of the composition is significant, and the analgesic effect of the high dose group is better than that of the positive group, showing a good anti-inflammatory and analgesic effect.

[0157] As can be seen from Table 7, the composition of Example 1 can significantly reduce the ear swelling degree. Among them, the swelling degrees of the low and medium dose groups and the positive control group are significantly reduced, with statistical differences (P < 0.01); it shows a good anti-inflammatory effect, and the anti-inflammatory effect is better than that of the comparative example.

[0158] It is known from Figure 4 - 11 It can be seen that the formula of the present invention shows significant curative effects, and various indicators of hyperlipidemia model hamsters are significantly improved. The composition of Example 1 can significantly reduce the body weight and weight gain rate of the model group, reduce the fasting blood glucose and the levels of TC, TG, and LDL-C, and the liver weight and liver index are also significantly reduced. Histopathological observation of the liver tissue shows that the composition of Example 1 can improve liver lesions, reduce fat vacuoles and inflammatory infiltration. The results of Elisa determination show that the composition of Example 1 can reduce the expression levels of C-reactive protein (CRP), tumor necrosis factor TNF-α, interleukin IL-6, and monocyte chemoattractant protein MCP-1 in serum and liver (P < 0.05).

[0159] The composition of Example 1 can also significantly increase the content of superoxide dismutase (SOD) in the liver tissue of hamsters (P < 0.01), while reducing the level of malondialdehyde (MDA) (P < 0.05). SOD is an important antioxidant enzyme that can scavenge free radicals in the body and reduce oxidative stress damage; while MDA is a product of oxidative stress reaction, and an increase in its content indicates that cells have been oxidatively damaged. Therefore, the composition of Example 1 further confirms its role in reducing oxidative stress reaction by increasing the SOD content and reducing the MDA level.

[0160] From the results of the improvement of metabolic inflammation by the composition prepared in the embodiments of the present invention, it can be concluded that the composition provided by the present invention can prevent and treat glycolipid metabolism diseases by improving metabolic inflammation. This discovery provides new ideas and methods for the treatment of glycolipid metabolic diseases and is expected to provide useful references for future clinical research.

Claims

1. A Chinese medicine composition having the function of regulating sugar and lipid metabolism, characterized in that: The invention is composed of the following raw materials by weight: 10-15 parts of thistle, 7-12 parts of emblica, 10-15 parts of bergamot and 15-20 parts of cassia seed.

2. The Chinese medicine composition having the effect of regulating sugar and lipid metabolism according to claim 1, characterized in that: The invention is composed of the following raw materials by weight: 12-14 parts of thistle, 9-11 parts of emblica fruit, 10-12 parts of bergamot and 15-18 parts of cassia seed.

3. The Chinese medicine composition having the effect of regulating sugar and lipid metabolism according to claim 1, characterized in that: The invention is composed of the following raw materials by weight: 12 parts of thistle, 9 parts of emblica, 10 parts of bergamot and 15 parts of cassia seed.

4. The method for preparing the Chinese medicine composition having the function of regulating glucose and lipid metabolism according to any one of claims 1 to 3, characterized in that: The steps include: (1) Weighing a formula amount of thistle, emblica, citron, and cassia seed to mix to obtain a mixture; then adding an ethanol solution to the mixture, heating and refluxing for extraction, filtering, and obtaining a filtrate and a filter residue; (2) Concentrating the filtrate obtained in step (1) to obtain the Chinese medicine composition.

5. The preparation method according to claim 4, characterized in that: The volume concentration of the ethanol in step (1) is 65%-75%.

6. The preparation method according to claim 4, characterized in that: The solid-liquid ratio of the mixture in step (1) to ethanol is: 1-3 g: 4-10 mL.

7. The preparation method according to claim 6, characterized in that: The solid-liquid ratio of the mixture in step (1) to ethanol is: 1g:5mL.

8. The preparation method according to claim 4, characterized in that: The temperature of the heating reflux extraction in step (1) is 65-70° C., and the number of times of the heating reflux extraction is 2-4 times.

9. The preparation method according to claim 4, characterized in that: The concentration in step (2) is to concentrate to 70%-90% of the solid content.

10. Use of the Chinese medicinal composition with the effect of regulating glucose and lipid metabolism as claimed in any one of claims 1 to 3 or the Chinese medicinal composition with the effect of regulating glucose and lipid metabolism obtained by the preparation method as claimed in any one of claims 4 to 9 in the preparation of drugs for regulating glucose and lipid metabolism.

Images