KR102268670B1 - Pharmaceutical compositions for preventing or improving obesity and overweight, and method for manufacturing the same - Google Patents

Abstract

The pharmaceutical composition for preventing or improving obesity and overweight according to an embodiment of the present invention contains an extract of a mixed herbal medicine including sagebrush, gardenia, hwangbaek and ephedra as an active ingredient.

Description

A pharmaceutical composition for preventing or improving obesity and overweight, and a method for manufacturing the same {PHARMACEUTICAL COMPOSITIONS FOR PREVENTING OR IMPROVING OBESITY AND OVERWEIGHT, AND METHOD FOR MANUFACTURING THE SAME}

Provided are a pharmaceutical composition for preventing or improving obesity and overweight, and a method for preparing the same.

Recently, due to changes in lifestyle, changes in diet, or stress, overweight and obesity are increasing, and it has become a social problem. Overweight refers to a case in which the body mass index (BMI, Body Mass Index, a measure of obesity divided by the weight squared by the height) is 25 or more and less than 30, and obesity refers to a case in which the body mass index is 30 or more.

Obesity and overweight are not caused by a single cause, but by several factors such as genetic, environmental, and psychological factors, so it may be difficult to treat them with any one method.

Currently, methods for treating obesity include diet control, lifestyle modification such as exercise, drug treatment, and surgical treatment. Although active efforts to correct lifestyle should precede drug or surgical treatment, it may not be easy to correct lifestyle, and various side effects may appear in the case of surgical treatment.

Therefore, there is a need to develop a pharmaceutical composition capable of improving or preventing obesity and overweight, which can reliably obtain the effect of improving obesity and overweight without side effects, and can be conveniently applied without the need for special efforts.

Conventionally, an obesity treatment composition using ephedra is known, and as a related prior literature, there is Korean Patent No. 10-1297918 regarding an obesity treatment composition using medicinal materials such as ephedra, banha, rhubarb, and banggi.

A pharmaceutical composition according to an embodiment of the present invention is for preventing or improving obesity and overweight.

The pharmaceutical composition according to an embodiment of the present invention is to minimize side effects that may occur due to ephedra.

The pharmaceutical composition according to an embodiment of the present invention is for reducing body weight and reducing the amount of food.

The pharmaceutical composition according to an embodiment of the present invention is for reducing the weight of liver tissue and adipose tissue.

The pharmaceutical composition according to an embodiment of the present invention is for reducing the size of fat droplets in liver tissue and adipose tissue, inhibiting the differentiation of fat, and promoting lipolysis.

In addition to the above problems, the embodiment according to the present invention may be used to achieve other problems not specifically mentioned.

The pharmaceutical composition for preventing or improving obesity and overweight according to an embodiment of the present invention contains an extract of a mixed herbal medicine including sagebrush, gardenia, hwangbaek and ephedra as an active ingredient.

In the mixed herbal medicine, 2 to 8 parts by weight of tofu, 1 to 5 parts by weight of gardenia, 1 to 8 parts by weight of hwangbaek, and 1 to 8 parts by weight of ephedra.

In the mixed herbal medicine, 4 to 6 parts by weight of tofu, 2 to 3 parts by weight of gardenia, 3 to 6 parts by weight of hwangbaek, and 5 to 7 parts by weight of ephedra.

The pharmaceutical composition may be formulated in a dosage form selected from the group consisting of injections, granules, tablets, pills, capsules, gels, syrups, suspensions, emulsions, or solutions.

The pharmaceutical composition for preventing or improving obesity and overweight according to an embodiment of the present invention includes the pharmaceutical composition according to the embodiment.

The method for producing a pharmaceutical composition for preventing or improving obesity and overweight according to an embodiment of the present invention comprises the steps of preparing a mixed herbal medicine containing shiitake, gardenia, hwangbaek and ephedra, after mixing water with the mixed herbal medicine, 80 to 140 It includes the step of hot water extraction at ℃ for 2-3 hours, and the step of filtering the hot water extracted extract with a membrane filter.

In the mixed herbal medicine, 4 to 6 parts by weight of tofu, 2 to 3 parts by weight of gardenia, 3 to 6 parts by weight of hwangbaek, and 5 to 7 parts by weight of ephedra.

Pharmaceutical composition according to an embodiment of the present invention can prevent or improve obesity and overweight, can minimize the side effects that may occur due to ephedra, can reduce the weight, can reduce the amount of food, , can reduce the weight of liver tissue and adipose tissue, reduce the size of fat droplets in liver tissue and adipose tissue, inhibit the differentiation of fat in liver tissue and adipose tissue, It can promote the breakdown of fat in tissues.

1 is a graph showing the results of comparing the average dietary amount of a high-fat diet-induced obese mouse and a mouse orally administered with the samples according to Examples 1 to 4 together with the high-fat diet administration.
Figure 2a is a graph showing changes in body weight for each week of a normal weight mouse, a high-fat diet-induced obese mouse, and a mouse orally administered with the samples according to Examples 1 to 4 together with the high-fat diet administration, FIG. It is a graph showing the result of comparing the weight.
Figure 3a is a graph showing the results of comparing the weight of the liver tissue of the high-fat diet-induced obese mouse and the mice orally administered the samples according to Examples 1 to 4 together with the high-fat diet administration, Figure 3b is the weight of the adipose tissue It is a graph showing the comparison result.
Figure 4a is a graph showing the AST level of the serum of a normal weight mouse, a high-fat diet-induced obese mouse, and a mouse orally administered with the samples according to Examples 1 to 4 together with the high-fat diet administration, Figure 4b is a graph showing the ALT level; It is a graph, and FIG. 4c is a graph showing creatine levels.
5A is a graph showing the serum triglyceride levels of normal-weight mice, high-fat diet-induced obese mice, and mice orally administered with the samples according to Examples 1 to 4 together with high-fat diet administration, and FIG. 5b is total cholesterol level. is a graph showing the, Figure 5c is a graph showing the LDL cholesterol level, Figure 5d is a graph showing the glucose level.
Figure 6a is a photograph showing the histological changes in liver tissue of a normal-weight mouse, a high-fat diet-induced obese mouse, and a mouse orally administered with the samples according to Examples 1 to 4 together with the high-fat diet administration, Figure 6b is adipose tissue; A photograph showing histological changes in
7a is a graph comparing the expression level of PPARγ in adipose tissue of normal-weight mice, high-fat diet-induced obese mice, and mice orally administered with the samples according to Examples 1 to 4 together with high-fat diet administration, FIG. 7b is a graph comparing the expression level of C/EBPa in adipose tissue, FIG. 7c is a graph comparing the expression level of PPARγ in liver tissue, and FIG. 7d is a graph comparing the expression level of C/EBPa in liver tissue It is a graph.
8A is a graph comparing the expression levels of ATGL in adipose tissue of normal-weight mice, high-fat diet-induced obese mice, and mice orally administered with the samples according to Examples 1 to 4 together with high-fat diet administration, FIG. 8b is a graph comparing the expression level of HSL in adipose tissue, FIG. 8C is a graph comparing the expression level of ATGL in liver tissue, and FIG. 8D is a graph comparing the expression level of HSL in liver tissue.

With reference to the accompanying drawings, the embodiments of the present invention will be described in detail so that those of ordinary skill in the art to which the present invention pertains can easily implement them. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. In order to clearly explain the present invention in the drawings, parts irrelevant to the description are omitted, and the same reference numerals are used for the same or similar components throughout the specification. In addition, in the case of a well-known known technology, a detailed description thereof will be omitted.

Throughout the specification, when a part "includes" a certain element, it means that other elements may be further included, rather than excluding other elements, unless otherwise stated.

Throughout the specification, "administration" means providing a given pharmaceutical composition according to the present invention to a subject by any suitable method.

A pharmaceutical composition for preventing or improving obesity and overweight according to embodiments contains an extract of a mixed herbal medicine including two shiitake, gardenia, hwangbaek and ephedra as an active ingredient.

The pharmaceutical composition can reduce body weight, can reduce the amount of food, can reduce the weight of liver tissue and adipose tissue, can reduce the size of fat droplets in liver tissue and adipose tissue, liver tissue And it can inhibit the differentiation of fat in adipose tissue, and can promote lipolysis in liver tissue and adipose tissue, thereby exhibiting excellent efficacy in preventing or improving obesity and overweight.

In addition, the pharmaceutical compositions according to the embodiments are harmless to the human body based on natural products and have excellent safety, side effects can be minimized by including a plurality of materials that do not show toxicity, and are safe even when taken for a long time for preventive purposes can be used

In mixed herbal medicine, Dusi (豆, Glycine Semen Preparata) is fermented by boiling the seeds of soybeans (Glycine max Merrill) of the legume family, which can be used for wind and wind, relieves skin, sweats, and burns the heart. It is a medicinal agent that can remove the symptoms of nausea, has detoxification effects, and aids digestion.

In addition, when used in conjunction with gardenia, yellowtail and ephedra, it can exhibit excellent efficacy in improving obesity and overweight, and side effects that can occur due to ephedra are insomnia, palpitation, arrhythmia, hypertension, heartburn, bleeding, etc. can alleviate or eliminate the symptoms of

Gardenia (Gardenia jasminoides) is a mature fruit of a gardenia tree belonging to the Marcus family. It can lower the heat of the heart and lungs, relieve inflammatory diseases by reducing the heat of inflammation, and have diuretic and hemostatic properties, etc. shows the pharmacological action of Gardenia can be used for treatment or prevention of inflammatory diseases such as conjunctivitis, diabetes, hepatitis, jaundice, hematemesis, and the like.

In addition, the gardenia extract can exhibit an excellent anti-obesity effect, including crocetine as an active ingredient, and when used in combination with doushi, ephedra and hwangbaek, the effect of inhibiting obesity and overweight can be further improved, It can greatly alleviate the side effects that can occur from ephedra.

Hwangbaek (黃栢, Phellodendri Cortex) is the dried bark of a rhododendron tree belonging to the rutaceae family. It can be used by peeling the bark from the tree trunk to remove the bark (rough bark) or cutting it and drying it in the sun. Yellow white is known to have sterilizing power against Escherichia coli, typhus bacillus, and cholera bacillus, and antibacterial activity against Gram-positive bacteria and gonococcus. , dysentery, ringworm, etc. In addition, Hwangbaek contains active ingredients such as flavonoids and alkaloids, and thus exhibits UV-blocking effects, antibacterial, anti-inflammatory, and antiviral effects on the skin.

In addition, hwangbaek exhibits the effect of suppressing appetite, and when used in combination with turmeric, gardenia and ephedra, the improvement effect of obesity and overweight can be further improved, and side effects that may occur due to ephedra can be alleviated or eliminated. .

Ephedrae Herba (麻黃, Ephedrae Herba) is a medicinal product made using Ephedra sinica Stapf (Ephedra sinica Stapf) of the Ephedra family or the soft stem of plants and animals. , Pyungcheon (平喘) and expectorant (鎭咳祛痰) action, double regulating action of raising and lowering blood pressure, diuretic action, kidney function improvement action, anti-inflammatory action, allergy improvement action, etc. are known. It can be applied to mechanical infection, pneumonia, bronchitis, bronchial asthma, pertussis, hypotension, heart disease, skin mucous membrane disease, nephritis, edema, joint muscle disease, diarrhea, etc.

In addition, although ephedra is disclosed as a conventional drug for improving obesity and overweight, ephedra contains ephedrine alkaloids, which are sympathetic nerve stimulants, so side effects such as insomnia, palpitations, arrhythmias, hypertension, heartburn, and bleeding may appear. Because there is, when ephedra is used alone or mixed with drugs that cannot manage the side effects of ephedra, the above-mentioned side effects may appear.

The pharmaceutical composition according to the embodiments is a composition comprising two shii, gardenia and hwangbaek together with ephedra, and symptoms such as insomnia, palpitations, arrhythmias, hypertension, heartburn, and bleeding can be minimized. In addition, the pharmaceutical composition may further improve the preventive or ameliorating effect on obesity and overweight by including all of ephedra and dusi, gardenia, and hwangbaek.

Since the pharmaceutical composition exhibits a synergistic or synergy effect as a whole, compared to the case where one or more of the components are excluded, including the case where each of the constituent substances is used alone, dooshi, gardenia, When yellow white and ephedra are used together, the maximum effect can be exhibited.

In the mixed herbal medicine, about 2 to 8 parts by weight of tofu, about 1 to 5 parts by weight of gardenia, about 1 to 8 parts by weight of hwangbaek, and about 1 to 8 parts by weight of ephedra, within this range, those who are obese and overweight The preventive or ameliorating effect may be further improved, and the effect of alleviating side effects that may occur due to ephedra may be further improved.

For example, in the mixed herbal medicine, when two shii is less than about 2 parts by weight, gardenia is less than about 1 part by weight, or yellow white is less than about 1 part by weight, the effect of mitigating side effects that may occur due to ephedra may be reduced. In addition, in the mixed herbal medicine, when ephedra is less than about 1 part by weight, the effect of inhibiting the differentiation of fat in human tissues and decomposing fat can be reduced, and when ephedra exceeds about 8 parts by weight, insomnia, palpitations, arrhythmias, Side effects such as high blood pressure and bleeding may occur. In addition, when the gardenia exceeds about 5 parts by weight, symptoms such as digestive symptoms, sleepiness, and chills may occur, and when yellow white powder exceeds about 8 parts by weight, symptoms such as diarrhea, digestive disorders, and nausea may occur due to digestive side effects. have.

More preferably, in the mixed herbal medicine, tofu may be about 4 to 6 parts by weight, gardenia about 2 to 3 parts by weight, hwangbaek about 3 to 6 parts by weight, and ephedra may be about 5 to 7 parts by weight, within this range In the prevention or improvement effect for obesity and overweight can be maximized, and the effect of alleviating side effects that may occur due to ephedra can be further improved.

Meanwhile, the pharmaceutical composition may include other pharmaceutically active ingredients other than the above-mentioned ingredients, or may be used in combination with a pharmaceutical composition including other active ingredients.

The method for preparing a pharmaceutical composition for preventing or improving obesity and overweight according to an embodiment comprises the steps of preparing a mixed herbal medicine containing spruce, gardenia, hwangbaek and ephedra, mixing water with the mixed herbal medicine, and then hot water extraction, and hot water and filtering the extracted extract with a membrane filter.

A step of preparing a mixed herbal medicine containing two shii, gardenia, hwangbaek and ephedra and hot water extraction are performed

Hot water extraction, for example, may be carried out at 80 ~ 140 ℃ for 2-3 hours, within this range, the extraction can be effectively made without destruction or deterioration of the extract components.

Next, the step of filtering the hot water extracted extract with a membrane filter is performed. In this case, the average pore size of the membrane filter may be about 0.5 to 0.7 μm, and impurities may be effectively removed within this range, and an extract may be effectively obtained.

Thereafter, the filtered extract may be freeze-dried and prepared in powder form, but is not limited thereto and may have various formulations.

The pharmaceutical composition according to the embodiment may be used as a medicament for preventing or improving obesity and overweight, and depending on the desired method, oral or parenteral (eg, intravenous, subcutaneous, intraperitoneal, or topical application) may be used in patients by any means. Although there is no particular limitation on the dosage form, it may be determined according to the age, sex, or severity of the disease of the patient.

Here, the solid preparation for oral administration may be in the form of powders, granules, tablets, capsules, soft capsules, pills, and the like. In addition, the liquid formulation for oral use may be in the form of a suspension, an oral solution, an emulsion, a syrup, a gel, an aerosol, and the like. Formulations for parenteral administration may be external preparations such as powders, granules, tablets, capsules, sterilized aqueous solutions, solutions, non-aqueous solutions, suspensions, emulsions, syrups, suppositories, aerosols, etc., or sterilized according to a conventional method, respectively. It can be formulated and used in the form of an injection. The preferred dosage of the composition varies depending on the absorption of the active ingredient in the body, the inactivation rate and excretion rate, the age, sex and condition of the patient, and the severity of the disease to be treated, but may be appropriately selected by those skilled in the art.

The pharmaceutical composition according to the embodiment may further include a conventionally used excipient, disintegrant, sweetener, lubricant, flavoring agent, and the like. For example, excipients include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol , starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone ) may be used. As a disintegrant, for example, sodium starch glycolate, crospovidone, alginic acid, carboxymethyl cellulose calcium, carboxymethyl cellulose sodium ), chitosan, guar gum, low-substituted hydroxypropyl cellulose, magnesium aluminum silicate, etc. may be used.

The pharmaceutical composition may further include a pharmaceutically acceptable additive. In this case, the pharmaceutically acceptable additive is, for example, starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose ( lactose), mannitol, arabic gum, hydroxypropyl cellulose, sodium starch glycolate, carnauba wax, stearic acid, magnesium stearate, aluminum stearate ( aluminum stearate), calcium stearate, sucrose, syrup, dextrose, sorbitol, talc, etc., but are not limited thereto. Pharmaceutically acceptable additives may be included in an amount of from about 0.1 to about 90 parts by weight based on the entire pharmaceutical composition.

The pharmaceutical composition may be prepared by further including one or more pharmaceutically acceptable carriers for administration. Here, the pharmaceutically acceptable carrier may be saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components may be mixed.

Hereinafter, the present invention will be described in more detail by way of Examples and Experimental Examples, but the present invention is not limited thereto.

Experimental example 1 - measure weight loss

27 subjects with a body mass index (BMI, Body Mass Index) of 23 or more took the pharmaceutical composition for preventing or improving obesity and overweight according to the example, and the change in body weight according to the dose was measured, and the results are shown in Table 1 and As shown in Table 2, information on whether side effects occurred due to taking was collected.

Of the 27 subjects, 6 were male and 21 were female, and the mean age was 42.56±11.11 years old, and the average weight before administration was 71.07±11.58 kg. Also, among 27 subjects, 5 (18.5%) had hypertension and 2 (7.4%) had hyperlipidemia.

No gender age duration of use
(Work) before taking
weight
(Kg) after taking
weight
(Kg) weight
decrease
(Kg) weight
decrease rate
(%) One female 24 60 77.8 68.1 9.7 12.47 2 female 34 60 83.8 75 8.8 10.50 3 female 39 75 94.1 85.5 8.6 9.14 4 female 42 60 69 61.4 7.6 11.01 5 male 32 45 78 70.5 7.5 9.62 6 female 55 45 66.3 60 6.3 9.50 7 female 43 60 82 76 6 7.32 8 male 43 75 79 73 6 7.59 9 male 62 45 77 71 6 7.79 10 male 58 30 89.7 84.4 5.3 5.91 11 female 37 30 70 65 5 7.14 12 female 28 75 56.5 52 4.5 7.96 13 female 32 30 59.3 55 4.3 7.25 14 female 51 30 76 72 4 5.26 15 male 20 30 82.2 78.2 4 4.87 16 female 50 15 62 58.6 3.4 5.48 17 female 63 15 71.4 68.2 3.2 4.48 18 female 53 45 62 59 3 4.84 19 female 40 30 71 68 3 4.23 20 male 58 45 81.3 79 2.3 2.83 21 female 37 30 85.8 83.6 2.2 2.56 22 female 35 30 53.7 51.5 2.2 4.10 23 female 42 30 56 54 2 3.57 24 female 35 15 50.5 48.5 2 3.96 25 female 53 30 63.2 61.3 1.9 3.01 26 female 41 15 56.5 55 1.5 2.65 27 female 42 15 65 64 One 1.54

duration of use
(Work) weight before taking
(Kg) weight after taking
(Kg) weight loss
(Kg) 45 days or more 75.56 ± 9.95 69.21 ± 9.16 6.36 ± 2.17 30 days or less 67.48 ± 11.56 64.48 ± 10.93 3.00 ± 1.26

Referring to Tables 1 and 2, the average weight after taking was 66.59 ± 10.45 kg, which was 4.49 ± 2.40 kg, and 15 persons with a weight loss of 5% or more were 55.6%. Among the subjects, 10 (83.3%) of 12 subjects who took the sample for 45 days or more lost 5% or more of their weight, while 5 (33.3%) of 15 subjects who took the sample for 30 days or less lost 5% or more of their body weight. Judging from this reduction, it can be seen that the longer the period of taking, the more significant the weight loss effect is.

In addition, in all 27 subjects, side effects caused by ephedra such as insomnia, palpitations, arrhythmias, hypertension, heartburn, and bleeding were almost nonexistent.

From this, it can be seen that the pharmaceutical composition according to the embodiment has an excellent effect on the improvement of overweight and obesity, and the side effects that may occur due to ephedra are minimized.

Example 1 - Sample 1 preparation

About 60 g of turmeric, about 30 g of gardenia, about 15 g of hwangbaek, and about 45 g of ephedra are measured and mixed to prepare a mixed herbal medicine. Put the mixed herbal medicine in a non-woven fabric, mix it with about 6800 cc of water, and then heat it with a hot water heater (daylight/pressurized) at about 110 ° C for about 2 hours and 30 minutes to extract hot water. Then, the hot water extract is filtered through a membrane filter having a pore size of about 0.5 μm, and the filtrate obtained after filtration is freeze-dried to obtain Sample 1 in the form of a dry powder.

Example 2 - Preparation of sample 2

Sample 2 was prepared in the same manner as in Example 1, except that about 60 g of turmeric, about 30 g of gardenia, about 45 g of hwangbaek, and about 45 g of ephedra were mixed to prepare a mixed herbal medicine.

Example 3 - Preparation of sample 3

Sample 3 was prepared in the same manner as in Example 1, except that about 90 g of turmeric, about 45 g of gardenia, about 45 g of hwangbaek, and about 90 g of ephedra were mixed to prepare a mixed herbal medicine.

Example 4 - Preparation of sample 4

Sample 4 was prepared in the same manner as in Example 1, except that about 60 g of two shiitake, about 30 g of gardenia, about 90 g of hwangbaek, and about 90 g of ephedra were mixed to prepare a mixed herbal medicine.

The following Experimental Examples 2 to 9 are high-fat diet induction prepared with reference to the report of Wang et al. (Wang CY and Liao JK., A Mouse Model of Diet-Induced Obesity and Insulin Resistance, Methods Mol Biol, 2012; 821: 421-433) This was performed using obese mice.

For high-fat diet-induced obese mice, 42 6-week-old C57BL/6J male mice (n=7/group) were purchased from Daehan Biolink (Negative, Chungbuk) and adapted for 1 week, followed by a high-fat diet (60 kcal% fat diet: Research Diets, D12492i) were administered in a free-fed mode for a total of 4 weeks to induced obesity. While the high-fat diet was performed for 4 weeks, the amount of food and body weight were measured once a week.

Thereafter, the samples according to Examples 1 to 4 were orally administered (100 mg/kg/day) along with the administration of the high-fat diet described above for an additional 4 weeks. Even during these additional 4 weeks, the amount of food and body weight were measured once a week.

Then, blood, adipose tissue, liver tissue, etc. of the mouse were collected and analyzed.

In Experimental Examples 2 to 9, ND denotes a mouse that did not receive a high-fat diet and sample administration, HFD denotes a mouse that was fed a high-fat diet in a free diet for 8 weeks, and HFD+1 denotes a high-fat diet in a free diet. Means a mouse administered with Sample 1 according to Example 1 for 4 weeks after progressing in the method for 4 weeks, and HFD+2 is Sample 2 according to Example 2 after proceeding with a high-fat diet as a free diet for 4 weeks means a mouse administered for 4 weeks, HFD+3 means a mouse administered a high-fat diet in a free diet for 4 weeks, and then sample 3 according to Example 3 was administered for 4 weeks, and HFD+4 is It refers to a mouse administered with sample 4 according to Example 4 for 4 weeks after the high-fat diet was performed as a free diet for 4 weeks. In addition, 7 mice were used in each of the ND, HFD, HFD+1, HFD+2, HFD+3, and HFD+4 groups.

The composition of the general diet, not the high-fat diet applied to the ND, and the composition of the high-fat diet are shown in Table 3 below. The general diet is corn starch, which generates most of the calories, and the high-fat diet contains a large amount of lard.

Furtherance general diet high fat diet Casein 200.0 265.0 L-Cystine 3.0 4.0 corn starch 397.486 - Maltodextrin 132.0 160.0 Sucrose 100.0 90.0 Lard - 310.0 Soybean oil 70.0 30.0 Cellulose 50.0 65.5 Mineral mix 35.0 48.0 Calcium phasphate, dibasic - 3.4 Vitamin mix 10.0 21.0 Choline bitartrate 2.5 3.0

Experimental example 2 - amount of food Measure

During the 8-week experimental period, the amount of food was measured once per week, and the results are shown in FIG. 1 .

Referring to Figure 1, compared with the average food amount of HFD, the average food amount of the mice administered the samples according to Examples 1 to 3 (HFD+1, HFD+2, HFD+3) is at a similar level, It can be seen that the mice administered the sample according to Example 4 (HFD+4) significantly decreased the amount of food.

Experimental example 3 - Measure weight change

Body weight was measured once per week during the 8-week experimental period, and the results are shown in FIGS. 2A and 2B.

Referring to FIGS. 2A and 2B , after a total of 8 weeks of experimentation, the average body weight for each group was ND 26.74 ± 0.32 g, HFD 48.88 ± 0.66 g, HFD+1 44.63 ± 0.75 g, HFD+2 45.25 ± 1.05 g , HFD+3 44.75 ± 0.52 g, HFD+4 43.13 ± 0.43 g, HFD in all mice administered the samples according to Examples 1 to 4 (HFD+1, HFD+2, HFD+3, HFD+4) It can be seen that there was a significant weight loss effect compared to

From this, it can be confirmed that the excellent weight loss effect of the pharmaceutical compositions according to the Examples.

Experimental example 4 - Tissue Weighing

After the 8-week experimental period, the weights of liver and adipose tissue were measured, and the results are shown in FIGS. 3A and 3B.

Referring to Figure 3a, the average weight of liver tissue in each group was ND 1.35 ± 0.08 g, HFD 2.75 ± 0.13 g, HFD+1 2.04 ± 0.20 g, HFD+2 2.12 ± 0.17 g, HFD+3 1.70 ± 0.11 g , HFD+4 was 1.82 ± 0.11 g. From this, in all of the mice (HFD+1, HFD+2, HFD+3, HFD+4) administered with the samples according to Examples 1 to 4, compared to HFD, the effect of reducing the weight of liver tissue increased due to fat was excellent it can be seen that In HFD+3 administered with the sample according to Example 3, the liver tissue weight reduction effect was the most excellent.

Referring to Figure 3b, the average weight of adipose tissue in each group is ND 0.27 ± 0.04 g, HFD 2.72 ± 0.11 g, HFD+1 2.57 ± 0.14 g, HFD+2 2.65 ± 0.13 g, HFD+3 2.47 ± 0.86 g , HFD+4 was 2.44 ± 0.09 g. From this, in all mice (HFD+1, HFD+2, HFD+3, HFD+4) administered with the samples according to Examples 1 to 4, compared to HFD, the effect of reducing the weight of adipose tissue is excellent. can figure out In HFD+4 administered with the sample according to Example 4, the adipose tissue weight reduction effect was the most excellent.

Experimental example 5 - Hepatotoxicity and renal toxicity evaluation

After the 8-week experimental period, serum was collected and the levels of AST (aspartate aminotransferase), ALT (alanine aminotransaminase), and renal toxicity markers, creatine, were measured, and the results are shown in FIGS. 4a to 4c. .

Here, AST is an enzyme present in hepatocytes, cardiac muscle cells, skeletal muscle cells, etc., and when the cells are necrotic or destroyed, they are leaked into the blood and the concentration increases, and ALT is an enzyme intensively present in hepatocytes, and when the liver cells are damaged Since the concentration increases and is usually produced specifically only in the liver, it can be an important factor in determining liver function (Kwo PY, Cohen SM, Lim JK. ACG Clinical Guideline: Evaluation of Abnormal Liver Chemistries. Am J Gastroenterol. 2017). Jan;112(1):18-35). Creatine is used as a marker of renal toxicity, and obesity is recognized as a risk factor for kidney damage (Lin TY, Liu JS, Hung SC. Obesity and risk of end-stage renal disease in patients with chronic kidney disease: a cohort study. Am J Clin Nutr. 2018 Oct 13. doi: 10.1093/ajcn/nqy200)

4a to 4c, compared to the high-fat diet administration, HFD significantly increased AST and ALT by about 2.85 times and about 7.04 times, respectively, compared to ND, mice administered the sample according to Example 4 (HFD+4) ), the AST level was significantly decreased, and the mice administered the sample according to Example 2 (HFD+2), the mice administered the sample according to Example 3 (HFD+3) and the sample according to Example 4 were administered It can be seen that the ALT level was significantly decreased in one mouse (HFD+4). In addition, in the case of creatine, compared to HFD, the levels were significantly decreased in HFD+1, HFD+2, HFD+3 and HFD+4, and in particular, it can be seen that the greatest decrease was observed in HFD+3 and HFD+4.

From this, it can be seen that the pharmaceutical composition according to the embodiments protects or improves liver function and renal function.

Experimental example 6 - serum triglycerides, cholesterol and of glucose evaluation

After the 8-week experimental period, serum was collected and the levels of triglycerides, total cholesterol, LDL cholesterol and glucose were measured, and the results are shown in FIGS. 5A to 5D .

5A to 5D, the serum levels of triglycerides (TG) and glucose were significantly increased in HFD, but mice administered the samples according to Examples 1 to 4 (HFD+1, HFD+2, Both HFD+3 and HFD+4) showed a significant decrease. However, it can be seen that there was no inhibitory effect on the increase of total cholesterol and LDL cholesterol.

From this, it can be seen that the pharmaceutical compositions according to the embodiments have the effect of lowering serum triglycerides and glucose levels.

Experimental example 7 - Evaluation of histological changes in liver and adipose tissue

After the 8-week experimental period, liver tissue and adipose tissue were collected and histological changes were observed through hematoxylin-eosin (H&E) staining, and a photograph of the liver tissue is shown in FIG. 6A and a picture of the adipose tissue is shown in FIG. 6B . In this case, hematoxylin, a basic dye, is widely used as a method of confirming the histological structure of a specimen by staining the nucleus in blue-purple, and eosin, an acid dye, in the cytoplasm and intercellular material in pink.

The sequence of H&E staining was performed as follows. Liver tissue and adipose tissue samples fixed in paraffin were sectioned to a thickness of 4 μm, and the sections were washed with xylene to remove paraffin, and then reduced in alcohol. Next, after treating the tissue section in about 150 μl of 0.1 trypsin working solution (composition: trypsin 0.4 ml, calcium 0.01 g, chloride 0.01 g in DW 7 ml) for 15 minutes, block using FBS (fetal bovine serum) (block). Then, for H&E staining, the cut tissue was immersed in hematoxylin solution for 5 minutes, distilled water for 5 minutes, and eosin solution for 30 seconds, dried and fixed, and the slides of the stained tissue samples were Olympus IX71 Research Inverted Phase Microscope was observed using

6a and 6b, Normal means ND, Control (control) means HFD, Sample 1 is HFD+1, Sample 2 is HFD+2, Sample 3 is HFD+3, Sample 4 is HFD+4 means each.

Referring to FIG. 6a , in the case of a mouse (HFD) induced to obesity through a high-fat diet, it can be seen that lipid droplets are formed by the accumulation of triglycerides in the liver tissue, and the samples according to Examples 1 to 4 It can be seen that the fat droplets decreased in all of the administered mice (HFD+1, HFD+2, HFD+3, HFD+4).

Through this, it can be understood that the pharmaceutical compositions according to the embodiments have an effect of reducing the formation of fat droplets in the liver tissue.

Referring to FIG. 6b , it can be seen that the size of lipid droplets increases due to excessive accumulation of triglycerides in adipose tissue in mice (HFD) induced to obesity through a high-fat diet, Examples 1 to 4 It can be seen that fat droplets decreased in all of the mice (HFD+1, HFD+2, HFD+3, HFD+4) administered the sample according to

Through this, it can be understood that the pharmaceutical compositions according to the embodiments have an effect of reducing the size of fat droplets in adipose tissue.

Experimental example 8 - Evaluation of factors related to adipogenesis in tissues

After the 8-week experimental period, liver tissue and adipose tissue were collected to determine the expression levels of PPARγ and C/EBPa, factors related to adipogenesis, and the results are shown in FIGS. 7A to 7D . PPARγ and C/EBPa are transcription factors that play a key role in the differentiation of adipocytes into mature adipocytes (Lefterova MI, Zhang Y, Steger DJ, Schupp M, Schug J, Cristancho A, Feng D, Zhuo D, Stoeckert CJ Jr, Liu XS, Lazar MA. PPARgamma and C/EBP factors orchestrate adipocyte biology via adjacent binding on a genome-wide scale. Genes Dev. 2008 Nov 1:22(21):2941-52).

A western blot technique was used to detect the protein expression of the factor, and the experimental method is as follows.

First, the liver and adipose tissue are chopped and homogenized using the Bullet Blender Homogenization Kit, and the homogenized tissue is dissolved in RIPA buffer and centrifuged at about 13,000 rpm for about 20 minutes at about 4 ℃ to remove insoluble substances. to extract the protein. Then, the total concentration of the extracted protein was measured using the Bradford technique, separated by size through electrophoresis on 8% sodium dodecyl sulfate (SDS) gel, and transferred to a PVDF (Polyvinylidene fluoride) membrane, and the PVDF membrane was moved to room temperature (approx. After blocking in a TBST (mixture of tris-buffered saline (TBS) and Polysorbate 20) solution containing 5% skim milk at 25 ° C.) for about 1 hour, at about 4 ° C., a suitable primary antibody (PPARγ, C/EBPa) was reacted for about 12 hours to prepare a PVDF membrane to which the primary antibody was attached, and the PVDF membrane to which the primary antibody was attached was reacted with the secondary antibody at room temperature for about 1 hour, and then exposed to ECL solution. Fluorescence was confirmed. The chemiluminescence intensity of the protein signal was quantified through ImageJ software.

7A and 7B , as the differentiation and accumulation of fat occurred in the adipose tissue of mice (HFD) induced to obesity through a high-fat diet, the corresponding factors were about 151% and about 198, respectively, compared to the normal control group (ND). % increased, and it can be seen that the expression of PPARγ and C/EBPa significantly decreased according to samples 1 to 4. Specifically, in the case of PPARγ, HFD+1 decreased by about 86.5%, HFD+2 by about 64.9%, HFD+3 by about 67.8%, and HFD+4 by about 51.6%, and in the case of C/EBPa, HFD+ 1 decreased about 83.8%, HFD+2 decreased about 70.6%, HFD+3 decreased about 43.0%, and HFD+4 decreased about 41.0%.

From this, it can be seen that the pharmaceutical compositions according to the embodiments have the effect of inhibiting the differentiation of fat in adipose tissue.

Referring to FIGS. 7c and 7d , PPARγ and C/EBPa in liver tissue of mice (HFD) induced by high-fat diet were 214% and 140%, respectively, compared to normal-weight mice (ND). , It can be seen that according to samples 1 to 4, the expression of PPARγ and C/EBPa significantly decreased. Specifically, in the case of PPARγ, HFD+1 decreased by about 61.2%, HFD+2 by about 54.6%, HFD+3 by about 51.3%, and HFD+4 by about 49.8%, and in the case of C/EBPa, HFD+ 1 decreased about 57.5%, HFD+2 decreased about 35.9%, HFD+3 decreased about 21.5%, and HFD+4 decreased about 7.3%.

From this, it can be seen that the pharmaceutical compositions according to the embodiments have the effect of inhibiting the differentiation of fat in the liver tissue.

Experimental example 9 - Evaluation of factors related to lipolysis in tissues

After the 8-week experimental period, liver tissue and adipose tissue were collected to confirm the expression levels of Adipose triglyceride lipase (ATGL) and Hormone-sensitive lipase (HSL), which are factors related to lipolysis, and the results are shown in FIGS. 8a to 8d. It was. ATGL and HSL are both lipases and are enzymes that act in the process of decomposing fat (Bolsoni-Lopes A, Alonso-Vale MI. Lipolysis and lipases in white adipose tissue - An update. Arch Endocrinol Metab. 2015 Aug;59(4)) :335-42) (Quiroga AD, Lehner R. Pharmacological intervention of liver triacylglycerol lipolysis: The good, the bad and the ugly. Biochem Pharmacol. 2018 Sep;155:233-241).

The protein expression of the factor was detected using the Western blot technique, and was performed in the same manner as in Experimental Example 8, except that ATGL and HSL were used as antibodies.

8A and 8B , there was no significant difference in the expression of ATGL and HSL in the adipose tissue of HFD+1 and HFD+2, whereas the expression level of ATGL in HFD+3 and HFD+4 was about It showed an increase rate of 143.6% and about 161.9%, and the expression level of HSL showed an increase rate of about 63.6% and about 79.4%, so that adipogenic differentiation in the adipose tissue of mice administered with the samples according to Examples 3 and 4 was induced. confirmed that it was.

Referring to FIGS. 8c and 8d , there was no significant difference in the expression of ATGL and HSL in the liver tissues of HFD+1 and HFD+2, whereas the expression level of ATGL in HFD+3 and HFD+4 was about It showed an increase rate of 46.3% and about 38.8%, and the expression level of HSL increased to about 19.5-fold and about 8.5-fold, indicating that adipogenic differentiation was induced in the liver tissue of mice administered the samples according to Examples 3 and 4. Confirmed.

Although the preferred embodiment of the present invention has been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements by those skilled in the art using the basic concept of the present invention as defined in the following claims are also provided. is within the scope of the

Claims (7)

Contains extracts of mixed herbal medicines consisting of shiitake, gardenia, hwangbaek and ephedra as active ingredients,
Relieves insomnia, palpitations, arrhythmias, high blood pressure, heartburn and bleeding;
In the mixed herbal medicine, the two shii 4 to 6 parts by weight, the gardenia 2 to 3 parts by weight, the hwangbaek 3 to 6 parts by weight, and the ephedra is 5 to 7 parts by weight
A pharmaceutical composition for preventing or improving obesity and overweight.
delete delete In claim 1,
The pharmaceutical composition is an injection, granule, tablet, pill, capsule, gel, syrup, suspension, emulsion, or a pharmaceutical composition for the prevention or improvement of obesity and overweight formulated in a dosage form selected from the group consisting of.
A medicament for preventing or improving obesity and overweight, comprising the pharmaceutical composition of any one of claims 1 or 4.
Preparing a mixed herbal medicine consisting of two shii, gardenia, hwangbaek and ephedra
After mixing water with the mixed herbal medicine, extracting hot water at 80 ~ 140 ° C. for 2 ~ 3 hours, and
Filtering the hot water extracted extract with a membrane filter
including,
Relieves insomnia, palpitations, arrhythmias, high blood pressure, heartburn and bleeding;
In the mixed herbal medicine, the two shii 4 to 6 parts by weight, the gardenia 2 to 3 parts by weight, the hwangbaek 3 to 6 parts by weight, and the ephedra is 5 to 7 parts by weight
Method for preparing a pharmaceutical composition for preventing or improving obesity and overweight.
delete

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