CN113116955A

CN113116955A - Pharmaceutical composition containing plant extracts and application thereof

Info

Publication number: CN113116955A
Application number: CN202110496793.0A
Authority: CN
Inventors: 吴亮
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2021-02-24
Filing date: 2021-05-07
Publication date: 2021-07-16
Anticipated expiration: 2041-05-07
Also published as: CN113116955B

Abstract

The invention discloses a pharmaceutical composition for preventing, treating and improving metabolic diseases, fibrotic diseases and liver diseases. At least two active ingredients: one or more of Coleus forskohlii extract, forskolin, and isoforskolin; (II) pentacyclic triterpenoid, and one or more plant extracts containing the pentacyclic triterpenoid. Compared with the two components which are used independently, the pharmaceutical composition provided by the invention can play a remarkable synergistic effect on the prevention and treatment of metabolic diseases, fibrotic diseases and liver diseases.

Description

Pharmaceutical composition containing plant extracts and application thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to a pharmaceutical composition containing plant extracts and application thereof in preparing medicines for preventing, treating and improving metabolic diseases, fibrotic diseases and liver diseases.

Background

Nonalcoholic fatty liver disease (NAFLD) and its subtype nonalcoholic steatohepatitis (NASH) are a leading cause of morbidity and mortality in liver-related diseases on a global scale. NAFLD affects up to one third of the world's population and may increase cardiovascular metabolic risk, which is characterized by insulin resistance and is closely associated with type 2 diabetes and obesity. NAFLD has hallmarks of pathological ectopic fat accumulation with mild chronic inflammatory states. Nonalcoholic steatohepatitis (NASH) is the next stage of NAFLD and is estimated to have a prevalence in the general population of 3-5%. It is associated with visceral fat accumulation and metabolic syndrome, of which metabolic stress, inflammation and fibrosis are the main pathogenic mechanisms. Patients with NASH are at much higher risk of developing clinically significant and progressive liver fibrosis, cirrhosis and hepatocellular carcinoma.

Liver fibrosis is a chronic liver disease that develops after a long-term liver injury. During hepatic fibrosis, parenchymal cells undergo a fundamental remodeling characterized by a progressive accumulation of fibrillar extracellular matrix associated with regeneration of parenchymal cells. The liver cells undergo continuous and repeated necrosis or inflammation in the liver fibrosis process, massive fibroplasia is accompanied by relative or absolute deficiency of fiber degradation, extracellular matrix is deposited in the liver in a large amount and finally develops into liver cirrhosis, so that the normal liver function is lost, and the liver failure and death can be caused in severe cases.

Coleus forskohlii (Willd.) Briq, also known as Coleus forskohlii, belongs to the family Labiatae, and is a perennial root, semifleshy herb, of the genus Coleus, with distribution in India, Srilanca, Nepal, Plumbum, and tropical Africa. The Coleus forskohlii extract is water extract or alcohol extract of Coleus forskohlii, the chemical components of the extract mainly include terpenoids, flavones, sterol, volatile oil, etc., and the index active component is Forskolin (Forskolin). Forskolin is one of the strongest known activators of adenylate cyclase, and can directly excite adenylate cyclase, increase the concentration of cyclic adenosine monophosphate in various tissue cells, and participate in the regulation of various cell functions.

Centella asiatica (L.) Urban, also known as Centella asiatica, pennisetum asiaticum, and corm Eleocharitis, is a whole plant of Centella asiatica of Umbelliferae. Centella asiatica is native to india and is now widely distributed in tropical and subtropical regions of the world. The herba Centellae extract is water extract or alcohol extract of herba Centellae, has brown yellow to white fine powder appearance and slightly bitter taste, contains various pentacyclic triterpenes, and contains asiaticoside, madecassoside, asiatic acid and madecassic acid as main ingredients. Long-term studies have indicated that centella asiatica extracts exhibit a broad spectrum of biological activities, most importantly antioxidant, anti-inflammatory, antibacterial and anticancer activities. Because the centella asiatica extract has the effects of resisting aging, resisting inflammation and the like, the centella asiatica extract is widely used in cosmetics for repairing scars, removing acnes and whitening skin.

Fructus Ligustri Lucidi is dry mature fruit of Ligustrum lucidum Ait (Ligustrum lucidum Ait.) belonging to Oleaceae. The glossy privet fruit is neutral in nature, sweet and bitter in taste, enters liver and kidney channels, and has the effects of nourishing yin, prolonging life, tonifying liver and kidney, clearing heat, improving eyesight, blackening hair and beard and the like. The fructus Ligustri Lucidi extract is water or ethanol extract of fructus Ligustri Lucidi, and has main chemical components such as pentacyclic triterpenes, iridoid glycosides, flavones, phenethyl alcohol, amino acids, polysaccharides, volatile oil, anthocyanins, etc. Researches in recent years find that the glossy privet fruit and chemical components thereof have stronger antioxidant activity, can effectively remove free radicals, and have various biological effects of sedation, anti-inflammation, antibiosis, diabetes resistance, ulcer resistance and the like.

White birch (Betula platyphylla Suk), a plant of the genus Betula of the family betulaceae, deciduous tree, has white, smooth, paper-like bark that can be peeled off in layers. The white birch bark belongs to traditional Chinese medicinal materials, and the traditional Chinese medicine has bitter taste and mild nature, and has the effects of clearing heat and removing toxicity, relieving cough and reducing sputum, and promoting diuresis. The birch bark contains large amount of pentosan, pentacyclic triterpenes and sterol fatty acid ester, and has strong biological activity and low toxicity.

At present, there is no report that the combination of the extract of Coleus forskohlii and the plant extract containing pentacyclic triterpenoid compounds is applied to the prevention and treatment of metabolic diseases, fibrotic diseases and liver diseases.

Disclosure of Invention

In order to solve the above technical problems, the present invention provides a pharmaceutical composition comprising at least the following two types of active ingredients:

one or more of Coleus forskohlii extract, forskolin, and isoforskolin; (II) pentacyclic triterpenoid, and one or more plant extracts containing the pentacyclic triterpenoid.

According to embodiments of the invention, in some embodiments, the composition comprises at least two of the following classes of active ingredients: firstly, coleus forskohlii extract; (II) plant extracts containing pentacyclic triterpenoid; in some embodiments, the composition comprises at least two of the following active ingredients: firstly, coleus forskohlii extract; (II) pentacyclic triterpenoid; in some embodiments, the composition comprises at least two of the following active ingredients: one or two of forskolin and isoforskolin; (II) plant extracts containing pentacyclic triterpenoid; in some embodiments, the composition comprises at least two of the following active ingredients: one or two of forskolin and isoforskolin; (II) pentacyclic triterpenoid;

according to an embodiment of the invention, the plant extract containing pentacyclic triterpenoid comprises extracts of centella asiatica, ligustrum lucidum, bearberry, liquorice, birch, lupin, hawthorn, banaba; preferably, the plant extract containing pentacyclic triterpenoid is selected from the group consisting of: centella asiatica extract, ligustrum lucidum extract, and birch bark extract.

According to an embodiment of the present invention, the coleus forskohlii extract, centella asiatica extract, ligustrum lucidum extract, and birch bark extract may be selected from a raw material meal of a traditional Chinese medicine (commercially available product); the coarse powder is water/alcohol extract of Coleus forskohlii, herba Centellae, fructus Ligustri Lucidi, and cortex Betulae Pendulae. The coarse powder is water/ethanol extract of Coleus forskohlii, herba Centellae, fructus Ligustri Lucidi, and cortex Betulae Pendulae. In some embodiments, the coarse powder is an aqueous extract of coleus forskohlii, centella asiatica, fructus ligustri lucidi, birch bark; in some embodiments, the coarse powder is a 50-98% ethanol extract of coleus forskohlii, centella asiatica, ligustrum lucidum, birch bark, such as 50% ethanol, 55% ethanol, 60% ethanol, 65% ethanol, 70% ethanol, 75% ethanol, 80% ethanol, 85% ethanol, 90% ethanol, 95% ethanol extract.

The preparation process of the coarse powder comprises the steps of adding a certain proportion of water, ethanol or water-ethanol mixed liquor (ethanol) into the raw materials to be extracted of coleus forskohlii, centella asiatica, glossy privet fruit and white birch bark, extracting, then concentrating, and finally drying to obtain solid powder, wherein the extraction process can comprise the steps of soaking and hot reflux; the drying can be selected from spray drying, oven drying and the like, and the product can be further screened after being dried.

According to an embodiment of the present invention, in the preparation process, the raw material is first pulverized, 5-15 times of water or 50-98% ethanol, such as 50% ethanol, 55% ethanol, 60% ethanol, 65% ethanol, 70% ethanol, 75% ethanol, 80% ethanol, 85% ethanol, 90% ethanol, 95% ethanol is added, and the extraction is performed under reflux for 1-5 times, preferably 1-3 times, each time for 0.5-4 hours, such as 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 hours. The "5 to 15 times" can be understood as that 5 to 15L of the solvent is added to 1kg of the raw material, and further 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times and 14 times can be selected. According to an embodiment of the present invention, said coleus forskohlii extract is preferably selected from the group consisting of an overground dried extract of coleus forskohlii, preferably with alcohol; the Coleus forskohlii extract mainly contains forskolin and isoforskolin, preferably, the Coleus forskohlii extract contains more than or equal to 8% of forskolin (preferably more than or equal to 10% of forskolin), more preferably, the Coleus forskohlii extract also contains more than or equal to 2% of isoforskolin (preferably more than or equal to 3% of forskolin).

According to the embodiment of the present invention, the fructus ligustri lucidi extract is preferably extracted from fructus ligustri lucidi, and the main active ingredients of the fructus ligustri lucidi extract comprise oleanolic acid, acetyl oleanolic acid, ursolic acid, acetic acid ursolic acid; preferably, the glossy privet fruit extract contains more than or equal to 8 percent of oleanolic acid (preferably more than or equal to 10 percent of oleanolic acid).

According to an embodiment of the invention, the major active principle of the extract of centella asiatica comprises asiaticoside, madecassoside, asiatic acid, madecassic acid. Preferably, the centella asiatica extract contains more than or equal to 8% of asiaticoside; further, the centella asiatica extract contains more than or equal to 8% of madecassoside (preferably more than or equal to 10% of madecassoside); more preferably, the centella asiatica extract contains more than or equal to 20% of total asiaticosides (preferably more than or equal to 25% of total asiaticosides).

According to an embodiment of the present invention, the main active ingredients of the birch bark extract comprise betulinic acid and betulin. Preferably, the birch bark extract contains betulinic acid more than or equal to 8%; further, the birch bark extract contains betulin (more than or equal to 8% (preferably betulin) more than or equal to 10%.

According to an embodiment of the present invention, the coleus forskohlii extract, centella asiatica extract, ligustrum lucidum extract, and birch bark extract may be purchased from conventional commercially available products, for example, from west ampere wanfang biotechnology limited, west ampere scott biotechnology limited, west ampere sure biotechnology limited, and the like. The Coleus forskohlii extract, herba Centellae extract, fructus Ligustri Lucidi extract, and cortex Betulae Pendulae extract can be selected from 5:1, 10:1, 15:1, 20:1, etc. According to an embodiment of the invention, the pentacyclic triterpenoid is selected from one or more compounds of the oleanane type, ursane type, lupane type and friedelane type. Preferably, the pentacyclic triterpenoid is selected from one or more compounds of oleanane type, ursane type and lupane type. The oleanane-type pentacyclic triterpenoid is selected from oleanolic acid and acetyl oleanolic acid; the ursolic acid, acetylursolic acid, asiatic acid and its analogs are selected from asiatic acid, madecassic acid, asiaticoside and madecassoside; the lupane pentacyclic triterpene compound is selected from betulinic acid and betulin.

According to an embodiment of the invention, the weight ratio of the first component to the second component is 1:0.01 to 100, preferably 1:0.1 to 100, more preferably 1:0.1 to 50, most preferably 1:0.1 to 20, e.g. may be selected from 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1: 10.

According to embodiments of the present invention, in some embodiments, the pharmaceutical composition comprises only the first type of ingredient and the second type of ingredient as active ingredients, in other embodiments the pharmaceutical composition further comprises an additional type of active ingredient.

According to an embodiment of the invention, the pharmaceutical composition further comprises pharmaceutically acceptable excipients. The auxiliary materials comprise excipient, adhesive, disintegrating agent, emulsifier, preservative, suspending agent, aromatic, pH regulator, flocculating agent, deflocculating agent, surfactant, filler, lubricant, thickening agent, humectant, plasticizer, bacteriostatic agent, coating material, foaming agent, defoaming agent, coating agent, isotonic conditioning agent and stabilizing agent.

According to an embodiment of the present invention, the pharmaceutical composition may be prepared in a suitable pharmaceutical preparation form, which may be administered orally or parenterally.

The invention also provides a preparation containing the pharmaceutical composition, the dosage form of the preparation is selected from oral preparations such as tablets, effervescent tablets, atomizing agents, gels, granules, pills, capsules, dripping pills, suspensions and injections, and the preparation is preferably an oral preparation.

Further, the composition of the invention accounts for 1-99%, preferably 1-60% of the total mass of the pharmaceutical preparation.

The invention further provides application of the pharmaceutical composition in preparing medicines for preventing, treating and improving metabolic diseases, fibrotic diseases and liver diseases.

According to an embodiment of the present invention, the metabolic, fibrotic and liver diseases are selected from hyperglycemia, hypertriglyceridemia, hypercholesterolemia, diabetes, obesity, fatty liver, liver injury, liver cirrhosis, chronic hepatitis, liver cancer, bile obstruction, gallstones, cardiovascular diseases, atherosclerosis, liver fibrosis, kidney fibrosis, lung fibrosis (e.g., chronic obstructive COPD, cystic fibrosis CF), skin fibrosis (e.g., scleroderma, nephrogenic fibrotic skin disease, mixed connective tissue disease, scleromyxedema, scleredema and eosinophilic fasciitis), cardiac fibrosis and the like; preferably, the metabolic, fibrotic and liver diseases are selected from obesity, non-alcoholic fatty liver disease, liver injury (including chronic liver injury), liver fibrosis.

After the drug combination is used together, the drug combination of the invention has obvious synergistic effect on both an animal fatty liver model and an animal hepatic fibrosis model, and the synergistic effect is embodied in inhibiting weight rise, reducing fat accumulation in liver, reducing AST and ALT, improving liver function, reducing liver collagen content and improving liver fibrosis degree.

Advantageous effects

Compared with the two components which are used independently, the pharmaceutical composition provided by the invention can play a remarkable synergistic effect on the prevention and treatment of metabolic diseases, fibrosis diseases and liver diseases. For example, experiments prove that the pharmaceutical composition can remarkably inhibit the body weight of a non-alcoholic fatty liver disease model mouse induced by high-fat diet from increasing, reduce the lipid accumulation and triglyceride level in the liver and other internal organs, and reduce the serum Free Fatty Acid (FFA) and Total Cholesterol (TC) level. In the experiment of the mouse with chronic liver injury/hepatic fibrosis induced by carbon tetrachloride, the pharmaceutical composition can reduce the collagen content in the liver, the arrangement of liver cells is neat, the contents of glutamic-oxaloacetic transaminase (AST) and glutamic-pyruvic transaminase (ALT) in the liver are reduced, the liver function can be obviously improved, the liver injury is reduced, and the liver fibrosis is inhibited.

Drawings

FIG. 1 is a graph of the effect of a pharmaceutical composition on weight change in a non-alcoholic fatty liver disease model mouse;

FIG. 2 is a graph of the effect of a pharmaceutical composition on liver weight ratio in a non-alcoholic fatty liver model mouse;

FIG. 3 is a graph showing the effect of a pharmaceutical composition on the quality of white fat in the groin of a non-alcoholic fatty liver disease model mouse;

FIG. 4 is a graph of oil-red-O staining of a liver slice of a non-alcoholic fatty liver model mouse by a pharmaceutical composition;

FIG. 5 is a graph showing the effect of a pharmaceutical composition on the percentage of the area of red oil-O staining of liver slices of a non-alcoholic fatty liver model mouse;

FIG. 6 is the effect of the pharmaceutical composition on liver weight ratio of liver fibrosis model mice;

FIG. 7 shows the effect of the pharmaceutical composition on the content of PC III in liver of a liver fibrosis model mouse;

FIG. 8 is a H & E staining chart of liver slices of a liver fibrosis model mouse by using the pharmaceutical composition;

FIG. 9 is a sirius red staining chart of the pharmaceutical composition on liver slices of a liver fibrosis model mouse;

FIG. 10 shows the effect of the pharmaceutical composition on the ratio of sirius red-stained area of liver slices of a liver fibrosis model mouse.

Detailed Description

The technical solution of the present invention will be further described in detail with reference to specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.

Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.

Note: statistical analysis GraphPad Prism 8.0 statistical software was used for analysis processing, and t-Test was used for statistical analysis. Compared with model group<0.05 the difference between the groups was statistically significant, P < 0.01, P < 0.001, P < 0.0001. Joint index (CI) calculation method: the determination was carried out by the Burgi method, and CI was obtained by the following equation: q ═ E_a/2+b/2/E_a(E_a>E_bWhen is equal to E) or q ═ E_a/2+b/2/E_b(E_a<E_bTime). In the formula E_a、E_bAnd E_a/2+b/2Is the effect of combining half dose of the A-medicine group, the B-medicine group and the two medicines respectively. q. q.s<1, the two medicines are combined to generate antagonism; q is 1, which indicates that the two drugs are combined to generate the addition effect; q. q.s>1, the synergistic effect of the two drugs is demonstrated. In short, the half dose combination group had a stronger effect than the single group with a better effect, indicating a synergistic effect.

Experimental materials:

(1) coleus forskohlii extract:

conventional commercial products (aerial parts of alcoholic extract, with different specifications of 10-98%) can be used. The sample of the embodiment of the invention is purchased from West Anwanfang biological technology limited company, and the specific product information is as follows: powdered, the extraction part is bark, the extraction solvent is alcohol, and the specification is marked as 10: 1. Laboratory assay components (HPLC method): the main active ingredients of the medicine are forskolin 12.2 +/-1 percent and isoforskolin 3.2 +/-1 percent; the homemade amplification extraction process comprises the following steps: taking the overground part of coleus forskohlii which is qualified by inspection, crushing and sieving, heating and refluxing for 1-3 times (each time is 1-3 hours) by using ethanol with the volume concentration of 8-12 times of 85-95%, combining extracting solutions, decompressing, concentrating and drying to obtain solid powder, determining that the obtained solid coarse powder contains more than or equal to 10% of active ingredient forskolin and more than or equal to 3% of isoforskolin, wherein the qualified sample can be used as a qualified sample with the content, and experiments show that the qualified sample and a commercially available raw material have basically the same medicinal effect.

(2) And (3) glossy privet fruit extract:

conventional commercial products, typically brown-yellow powders, can be used, screened through a 100 mesh screen, having a specification of 5:1,10: 1,20: 1, sample of the present invention example was purchased from west ampere wanfang biotechnology limited, 10:1 standard, extract source: dried ripe fruit of Ligustrum lucidum ait of Oleaceae; laboratory assay components (HPLC method): the main active chemical components comprise: oleanolic acid, acetyl oleanolic acid (about 8%), ursolic acid acetate, etc. The homemade amplification extraction process 1: the method comprises the steps of taking glossy privet fruit as a raw material, crushing and sieving, heating and refluxing the glossy privet fruit with ethanol with the volume concentration of 5-10 times and 40-98% for 2-3 times (each time is 1-3 hours), combining extracting solutions, carrying out reduced pressure concentration and drying to obtain solid powder, determining that the obtained solid coarse powder contains the active ingredient oleanolic acid which is more than or equal to 8%, wherein the obtained solid coarse powder can be used as a qualified sample to be used when the content of the product in each batch meets the requirement, and experiments show that the qualified sample prepared by the process has the basically same efficacy as the commercially available raw. The homemade amplifying extraction process 2: the method comprises the steps of taking glossy privet fruit as a raw material, crushing and sieving, heating and refluxing the glossy privet fruit with 10-15 times of purified water for 2-3 times (each time is 0.5-2 hours), combining extracting solutions, decompressing, concentrating and drying to obtain solid powder, determining that the obtained solid coarse powder contains oleanolic acid serving as an active ingredient which is more than or equal to 8%, enabling the obtained solid coarse powder to serve as a qualified sample to be used when the content of the product meets the requirement, and obtaining the qualified sample which has the basically same efficacy effect with the commercially available raw material through experiments.

(3) Centella asiatica extract:

conventional commercially available products can be adopted, and the sample of the embodiment of the invention is purchased from West Anwanfang biotechnology limited, 10:1 specification, and the main active ingredients comprise madecassoside, asiaticoside, asiatic acid and madecassic acid; the homemade amplification extraction process comprises the following steps: pulverizing herba Centellae, sieving, soaking in 5-10 times of 70-90% ethanol, extracting under reflux for 1-3 times (2-3 hr each time), concentrating the extractive solution under reduced pressure, and drying to obtain solid powder. HPLC detection shows that the obtained herba Centellae extract coarse powder contains 8% +/-1% of asiaticoside, the content of madecassoside is 10% +/-2%, and the total asiaticoside content is not less than 25%. The products of each batch meeting the content can be used as qualified samples to be used, and the qualified samples have basically the same drug effect with the commercially available raw materials through experiments.

(4) Birch bark extract:

conventional commercial products, typically brown-yellow powders, may be used, and the samples of the examples of the invention are available from sienna inscription biotechnology limited, 10:1 specification, extract source: bark of Betula birch belonging to family Betulaceae; laboratory assay components (HPLC method): the main active chemical components comprise: betulin (not less than 8%), betulinic acid (about 10%), etc. The homemade amplification extraction process comprises the following steps: pulverizing cortex Betulae Pendulae, sieving, soaking in 8-12 times of 65-85% ethanol, extracting under reflux for 1-3 times (2 hr each time), concentrating the extractive solution under reduced pressure, and drying to obtain solid powder. HPLC detection shows that the obtained coarse powder of cortex Betulae Pendulae extract contains betulin 8% and betulinic acid 10% or more. The products of each batch meeting the content can be used as qualified samples to be used, and the qualified samples have basically the same drug effect with the commercially available raw materials through experiments.

Example 1 evaluation experiment of therapeutic effects of the pharmaceutical composition of the present invention on fatty liver mice

1. Establishment of non-alcoholic fatty liver model mouse

100 SPF male C57BL/6 mice, 8 weeks old, 18-20g in weight, purchased from Yangzhou university, were acclimatized in SPF animal houses for one week, maintained at room temperature of 24 + -2 deg.C, humidity controlled between 40% -60%, and maintained at 12 hours light/dark daily. After one week of adaptive feeding, 80 mice with uniform weight and obesity were averagely divided into 8 groups, which were respectively a model group, a coleus forskohlii extract group, a centella asiatica extract group, a glossy privet fruit extract group, a white birch bark extract group, a coleus forskohlii extract + a snowgrass extract group, a coleus forskohlii extract + a glossy privet fruit extract group, and a coleus forskohlii extract + a white birch bark extract group. Feeding with high fat feed (containing 35% fat, 26% carbohydrate, 26% protein), continuously feeding for 12 weeks to establish obesity and non-alcoholic fatty liver model, and performing continuous gavage administration while modeling for 12 weeks.

2. Treatment experiment of non-alcoholic fatty liver disease model mouse

Preparing an extract suspension: weighing a certain amount of Coleus forskohlii extract, herba Centellae extract, fructus Ligustri Lucidi extract, and cortex Betulae Pendulae extract each time, adding into 0.5% CMC-Na solution, mixing with vortex instrument, and making into 50mg/ml CMC-Na suspension. Mixing the prepared Coleus forskohlii extract suspension with herba Centellae extract, fructus Ligustri Lucidi extract, and cortex Betulae Pendulae extract suspension at equal volume to obtain suspension containing 25mg/ml composition.

Administration dose:

the administration mode comprises the following steps: the administration is carried out by intragastric administration, the solvent is 0.5% CMC-Na solution, and the administration is carried out once a day and continuously for 12 weeks.

Sampling and sample retention: after the last administration, the mice are fasted for 12 hours without water prohibition, the eyeballs are picked after weighing, blood is taken out, the whole blood is kept still for 2 hours at 3500rpm, the blood serum is separated after centrifugation for 15 minutes, and the precipitated blood cells are kept and stored in a refrigerator at the temperature of minus 80 ℃ for standby. After sacrifice of the mice by decapitation, the livers were dissected and weighed. Storing the liver of the left outer leaf into 4% paraformaldehyde, using the liver as a frozen section, performing oil red O staining, and quantifying; and preserving the other part of the liver in liquid nitrogen, quickly freezing and transferring to a refrigerator at the temperature of-80 ℃ for later use. The inguinal fat of the right thigh of the mouse was taken, weighed, and stored in liquid nitrogen.

Index detection: serum Free Fatty Acid (FFA) and Total Cholesterol (TC) levels were measured with a blood biochemical analyzer. The liver tissue remaining in the liquid nitrogen was disrupted with a homogenizer and centrifuged, and the supernatant was collected and TG content was measured with a TG kit, and the operation was performed with reference to the instructions.

3. Results of the experiment

(1) Influence of the pharmaceutical composition on weight change of non-alcoholic fatty liver disease model mice

The results are shown in figure 1, the weight of the body of the mouse fed with the high-fat feed is obviously increased, the weight of the mouse in the model group is the heaviest at the 12 th week of administration, the weight of the mouse administered with each extract is slightly less than that of the mouse in the model group, the combination of the coleus forskohlii extract and the plant extract containing the pentacyclic triterpenoid compounds can obviously inhibit the weight increase, and the CI value of the weight reduction rate is more than 1, wherein the effects of the coleus forskohlii extract and the glossy privet fruit extract are the best, which indicates that the combination of the coleus forskohlii extract and the plant extract containing the pentacyclic triterpenoid compounds has a synergistic weight-reducing effect.

(2) Influence of the pharmaceutical composition on liver weight ratio of non-alcoholic fatty liver model mouse

Non-alcoholic fatty liver is mainly formed by excessive accumulation of fat in the liver, and thus the liver-to-weight ratio (liver weight/body weight) increases. The results are shown in fig. 2, compared with the liver weight ratio of a model group mouse, the liver weight ratio of the mouse administrated by using various extracts alone is slightly smaller, only using the centella asiatica extract has significance, the combined use of the coleus forskohlii extract and the plant extract containing the pentacyclic triterpenoid compounds can obviously reduce the liver weight ratio, and the liver index reduction rate CI values are all larger than 1, wherein the combined use effect of the coleus forskohlii extract and the glossy privet fruit extract is the best, and the effect of treating fatty liver is shown by combining the coleus forskohlii extract and the plant extract containing the pentacyclic triterpenoid compounds.

(3) Influence of the pharmaceutical composition on visceral fat of non-alcoholic fatty liver disease model mouse

Visceral fat is distributed on the surface of each visceral organ, wherein inguinal fat is the most typical and convenient material, the content of the inguinal fat can reflect the content of the visceral fat to a certain extent, and the ratio of the content of the visceral fat to the body weight is called the fat-weight ratio. The result is shown in fig. 3, compared with the lipid weight ratio of a model group mouse, the centella asiatica extract has limited effect, other single-use extract groups have certain effect, the coleus forskohlii extract and the plant extract containing the pentacyclic triterpenoid compound can be used together to obviously reduce the lipid weight ratio, and the CI value of the lipid-lowering weight ratio efficiency is more than 1, wherein the combined effect of the coleus forskohlii extract and the glossy privet fruit extract is optimal, which indicates that the combined effect of the coleus forskohlii extract and the plant extract containing the pentacyclic triterpenoid compound has the effect of reducing visceral fat.

(4) Influence of the pharmaceutical composition on serum FFA and TC levels of nonalcoholic fatty liver model mice

The concentration of free fatty acids in serum is related to lipid metabolism, carbohydrate metabolism, and endocrine function, and the concentration of free fatty acids is increased due to diseases such as diabetes, liver dysfunction, and obesity. The liver is the main organ for synthesizing and storing cholesterol, the serum concentration of the liver can be used as an index of lipid metabolism, when obesity causes fatty liver, the content of total cholesterol in serum can be increased, and the high total cholesterol can cause atherosclerosis and heart diseases of patients. The results are shown in table 1, compared with serum free fatty acid and total cholesterol levels of mice in a model group, the single extract group has certain effects, the combination of the coleus forskohlii extract and the plant extract containing the pentacyclic triterpenoid can obviously reduce serum FFA and TC levels, and the serum FFA and TC rate CI values are both more than 1, wherein the combination of the coleus forskohlii extract and the glossy privet fruit extract has the best effect, and the combination of the coleus forskohlii extract and the plant extract containing the pentacyclic triterpenoid has the effects of improving glycolipid metabolism and improving liver function.

Table 1. effect of the pharmaceutical composition of the invention on serum FFA, TC levels in NASH mice (mmol/L,

)

(5) influence of the pharmaceutical composition on the triglyceride level of liver tissue of non-alcoholic fatty liver model mouse

Triglyceride is a storage form of fat in liver, the content of triglyceride in non-alcoholic fatty liver cell is increased, Triglyceride (TG) detection is carried out on liver tissue homogenate of a non-alcoholic fatty liver mouse, and the result is shown in Table 2, the content of TG in the mouse in a model group is the highest, compared with the model group, the content of TG in the liver of the mouse can be reduced by singly using various extracts, but the TG level can be obviously reduced after the coleus forskohlii extract and a plant extract containing pentacyclic triterpenoid compounds are combined, the CI value of the TG reducing rate is more than 1, and the effect of the coleus forskohlii extract and the combined glossy privet fruit extract is the best.

Table 2. effect of the pharmaceutical composition of the invention on hepatic TG levels in NASH mice (mmol/L,

)

to show fat in the tissue, staining is often done with oil red O, the red stained area represents a lipid droplet in the liver, and the fat level in the liver can be quantified. The results are shown in fig. 4 and fig. 5, the red dyeing area of the model group is the largest, the lipid accumulation is the largest, compared with the model group, the liver lipid content can be reduced by singly using various extracts, but the liver lipid content can be more obviously reduced after the coleus forskohlii extract and the plant extract containing the pentacyclic triterpenoid are combined, wherein the effect of combining the coleus forskohlii extract and the glossy privet fruit extract is the best, and the effect of treating the fatty liver is shown when the coleus forskohlii extract and the plant extract containing the pentacyclic triterpenoid are combined.

Example 2 evaluation of therapeutic Effect of the pharmaceutical composition of the present invention on hepatic fibrosis mice

1. Establishment of hepatic fibrosis model mouse

SPF male C57BL/6 mice, 8 weeks old, 18-20g in weight, purchased from Yangzhou university, were acclimatized in SPF animal houses for one week, maintained at room temperature of 24 + -2 deg.C and humidity of 40% -60%, and maintained at light/dark conditions for 12 hours per day. The method comprises the following steps of averagely dividing 80C 57 mice into 8 groups, wherein each group comprises 10 mice, namely a model group, a coleus forskohlii extract group, a centella asiatica extract group, a glossy privet fruit extract group, a white birch bark group, a coleus forskohlii extract + snowgrass extract group, a coleus forskohlii extract + glossy privet fruit extract group, and a coleus forskohlii extract + white birch bark extract group. Intraperitoneal injection of 5% carbon tetrachloride (CCl) into all mice every thursday and every weekday₄) The injection volume of the olive oil solution is calculated according to 20ml/kg and lasts for 8 weeks, so that a hepatic fibrosis model caused by chronic liver injury is formed, and continuous intragastric administration is carried out for 12 weeks while modeling is carried out.

2. Therapeutic experiment of hepatic fibrosis model mouse

Administration dose:

the administration mode comprises the following steps: the administration is carried out by intragastric administration, the solvent is 0.5% CMC-Na solution, and the administration is carried out once a day and continuously for 8 weeks.

Sampling and sample preservation: weighing the mouse 24 hours after the last molding, picking eyeballs and taking blood, standing the whole blood for 2 hours, 3500rpm, centrifuging for 15 minutes, separating serum, retaining the precipitated blood cells, and storing in a refrigerator at-80 ℃ for later use. After sacrifice of the mice by decapitation, the livers were dissected and weighed. Storing the left outer leaf liver in 4% paraformaldehyde, taking the left outer leaf liver as a paraffin section, staining with hematoxylin/eosin (H & E) and sirius red, and grading; and preserving the other part of the liver in liquid nitrogen, quickly freezing and transferring to a refrigerator at the temperature of-80 ℃ for later use.

Sirius red dyeing fibrosis calculation standard: the ratio of the total area occupied by the red stained area was quantified using Image J software.

Index detection: serum AST and ALT levels were measured using a blood biochemical analyzer. The liver tissue remaining in the liquid nitrogen was disrupted with a homogenizer and centrifuged, and the supernatant was collected and assayed for procollagen type III (PCIII) content with a procollagen type III ELISA kit, according to the kit instructions.

3. Results of the experiment

(1) The influence of the pharmaceutical composition on the liver weight ratio of the hepatic fibrosis model mouse

In the case of hepatic fibrosis, connective tissue in the liver abnormally proliferates, and thus the liver weight ratio (liver weight/body weight) increases. The results are shown in fig. 6, compared with the liver weight ratio of a model group mouse, the liver weight ratio can be reduced to a certain extent by using various extracts singly, the liver weight ratio can be obviously reduced after the coleus forskohlii extract and the plant extract containing the pentacyclic triterpenoid compounds are combined, the liver index reduction rate CI values are all more than 1, the combined effect of the coleus forskohlii extract and the glossy privet fruit extract is the best, and the effect of treating hepatic fibrosis is shown by combining the coleus forskohlii extract and the plant extract containing the pentacyclic triterpenoid compounds.

(2) The influence of the pharmaceutical composition on the content of procollagen type III (PCIII) in liver of a liver fibrosis model mouse

The PC III is closely related to the activity degree of the formation of hepatic fibrosis, and the PC III detection is carried out on liver tissue homogenate of a hepatic fibrosis mouse, the result is shown in figure 7, the content of the PC III in the mouse in a model group is highest, compared with the model group, the content of the PC III in the mouse can be reduced by singly using various extracts, the administration group of the coleus forskohlii extract is most obvious, the PC III level can be more obviously reduced after the coleus forskohlii extract is combined with a plant extract containing pentacyclic triterpenes, and the liver PC III rate reduction CI value is more than 1, wherein the combined effect of the coleus forskohlii extract and the glossy privet fruit extract is optimal, which shows that the combined effect of the coleus forskohlii extract and the plant extract containing pentacyclic triterpenes has the effect of treating the.

(3) The influence of the pharmaceutical composition on the fibrosis degree of liver slices of a liver fibrosis model mouse

The sections were subjected to H & E staining for morphological observation of hepatocytes, and the degree of inflammation and necrosis was evaluated, and the results are shown in FIG. 8. Sirius red can specifically stain the fibrous component, collagen, red, and quantify the fibrosis of liver tissue, the results are shown in fig. 9-10. The liver cells of the model group mice have irregular shapes, a large amount of inflammatory cells infiltrate, the red staining area proportion is the highest, and the liver fibrosis degree is the most serious. Compared with a model group, the mouse liver shape is improved, the red dyeing area proportion is reduced, the coleus forskohlii extract administration group is most obvious, and the coleus forskohlii extract and the plant extract containing the pentacyclic triterpenoid compound are combined to obviously improve the liver cell shape, reduce the red dyeing area of sirius chinensis and improve the hepatic fibrosis, wherein the combined effect of the coleus forskohlii extract and the glossy privet fruit extract is optimal, which shows that the combined effect of the coleus forskohlii extract and the plant extract containing the pentacyclic triterpenoid compound has the effect of treating the hepatic fibrosis.

(4) The influence of the pharmaceutical composition on the serum AST and ALT levels of the hepatic fibrosis model mouse

The AST and ALT detection is carried out on the serum of a hepatic fibrosis mouse, the results are shown in table 3, the AST and ALT content of the mouse in a model group is highest, compared with the model group, the AST and ALT content of the mouse can be reduced by singly using various extracts, the asiatic centella extract administration group is most obvious, the AST and ALT content can be more obviously reduced after the coleus forskohlii extract and the plant extract containing the pentacyclic triterpenoid compounds are combined, the AST and ALT rate CI values are both more than 1, and the combination of the coleus forskohlii extract and the plant extract containing the pentacyclic triterpenoid compounds has the effects of improving the liver function and reducing the chronic liver injury.

Table 3. effect of the pharmaceutical composition of the present invention on serum AST, ALT levels of liver fibrosis mice (U/L,

)

the embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A pharmaceutical composition comprising at least two of the following active ingredients:

one or more of Coleus forskohlii extract, forskolin, and isoforskolin;

(II) pentacyclic triterpenoid, and one or more plant extracts containing the pentacyclic triterpenoid.

2. The pharmaceutical composition of claim 1, wherein the plant extract containing pentacyclic triterpenoid comprises extracts of centella asiatica, ligustrum lucidum, bearberry, glycyrrhiza, birch, lupin, hawthorn, crape myrtle; the pentacyclic triterpenoid is selected from one or more compounds of oleanane type, ursane type, lupane type and friedelane type; preferably, the pentacyclic triterpenoid is selected from oleanane type, ursane type and lupane type; the oleanane-type pentacyclic triterpenoid is selected from oleanolic acid and acetyl oleanolic acid; the ursolic acid, acetylursolic acid, asiatic acid and its analogs are selected from asiatic acid, madecassic acid, asiaticoside and madecassoside; the lupane pentacyclic triterpene compound is selected from betulinic acid and betulin; preferably, the plant extract containing pentacyclic triterpenoid is selected from the group consisting of: centella asiatica extract, ligustrum lucidum extract, and birch bark extract.

3. The pharmaceutical composition of claim 1, wherein: the Coleus forskohlii extract contains forskolin and isoforskolin as main active ingredients.

4. The pharmaceutical composition of claim 2, wherein the main active ingredient of the fructus Ligustri Lucidi extract comprises oleanolic acid, acetyl oleanolic acid, ursolic acid, and ursolic acid acetate.

5. The pharmaceutical composition according to claim 2, characterized in that the main active principle of the extract of centella asiatica comprises asiaticoside, madecassoside, asiatic acid, madecassic acid.

6. The pharmaceutical composition of claim 2, wherein the main active ingredient of the birch bark extract comprises betulinic acid and betulin.

7. The pharmaceutical composition of claim 1, wherein the weight ratio of the first component to the second component is 1:0.01 to 100.

8. Formulation comprising a pharmaceutical composition according to any of claims 1 to 7, characterized in that the formulation is in a dosage form selected from the group consisting of oral formulations such as tablets, effervescent tablets, nebulisers, gels, granules, pills, capsules, dripping pills, suspensions and injections, preferably oral formulations.

9. Use of the pharmaceutical composition of any one of claims 1 to 7 for the preparation of a medicament for the prevention, treatment, amelioration of metabolic diseases, fibrotic diseases and liver diseases.

10. The use according to claim 9, characterized in that the metabolic, fibrotic and liver diseases are selected from hyperglycemia, hypertriglyceridemia, hypercholesterolemia, diabetes, obesity, fatty liver, liver injury, cirrhosis, chronic hepatitis, liver cancer, bile obstruction, gallstones, cardiovascular diseases, atherosclerosis, liver fibrosis, kidney fibrosis, lung fibrosis (e.g. chronic obstructive COPD, cystic fibrosis CF), skin fibrosis (e.g. scleroderma, nephrogenic fibrotic skin disease, mixed connective tissue disease, scleromyxedema, scleredema and eosinophilic fasciitis), cardiac fibrosis and the like; preferably, the metabolic, fibrotic and liver diseases are selected from obesity, non-alcoholic fatty liver disease, liver injury (including chronic liver injury), liver fibrosis.