Seven-component yang-warming traditional Chinese medicine composition for treating obesity and application of seven-component yang-warming traditional Chinese medicine composition

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to application of a seven-ingredient yang-warming traditional Chinese medicine composition in treating obesity. The seven-ingredient yang-warming traditional Chinese medicine composition provided by the invention has a remarkable treatment effect on obesity in a caenorhabditis elegans obesity pathological model, can remarkably reduce fat storage in caenorhabditis elegans, and has the potential of treating obesity.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to application of a seven-flavor yang-warming traditional Chinese medicine composition in preparing a medicine for treating obesity. Background Art

[0002] Obesity is the excessive accumulation of body fat, often accompanied by overweight. In 1997, the World Health Organization (WHO) announced that obesity is one of the world's leading health problems. [1] . The harm of obesity is very extensive. In terms of metabolism, it is easy to cause diseases such as dyslipidemia and hyperglycemia, increase the risk of diabetes and cardiovascular diseases, lead to atherosclerosis, and affect the heart's blood supply function. From the perspective of bones and joints, it will increase the load on joints, promote the formation of osteoarthritis, and make people unable to move. Furthermore, obesity is also related to the increased incidence of certain cancers, such as endometrial cancer in women, which seriously threatens life, health and quality of life. [2] .

[0003] Obesity treatment includes a variety of methods such as diet control, exercise, surgery and drugs. Although diet control and exercise are very important for improving obesity, the treatment effect is not ideal because most obese patients find it difficult to adhere to them. Surgery has contraindications and greater risks and is not a conventional treatment method. Drugs will still be the main method for treating obesity in the future. The Western medicines currently used for weight loss are mainly simvastatin, orlistat, phentermine, phentermine / topiramate sustained-release tablets, lorcaserin, naltrexone / bupropion sustained-release tablets and liraglutide [3] Although this type of weight loss drug can effectively relieve obesity, the side effects of the drug itself cannot be ignored. For example, it can cause patients to suffer from insomnia, headaches, anxiety, sensory dullness, gastrointestinal side effects, and reduced absorption of fat-soluble vitamins. [4] Therefore, there is an urgent need to screen and discover highly effective and low-toxic anti-obesity drugs. Traditional Chinese medicine compositions have the characteristics of multiple active components and multiple targets, so screening anti-obesity drugs from traditional Chinese medicine is very promising.

[0004] C. elegans is a highly advantageous pathological model. It is easy to culture and has strong fertility. 60-80% of its genes have been identified as human gene homologs, and the corresponding genes of many human disease-causing genes also exist in C. elegans. [5] For example, in the case of obesity, the genes responsible for fat regulation in C. elegans are homologous to important genes that regulate fat metabolism in humans, and the fat in C. elegans is easily quantified by Nile red labeling, so C. elegans is an extremely advantageous obesity pathology model.

[0005] Qiwei Wenyang Capsule is a compound Chinese medicine, which is composed of deer antler glue, deer antler frost, salt dodder seed, prepared rehmannia root, cypress seed, poria cocos, and psoralea corylifolia. Its main functions are to nourish the kidney and warm the yang, and to nourish the essence and blood. In previous studies, it has been found to have new anti-inflammatory and analgesic effects. [6] .

[0006] In the process of evaluating the efficacy of the seven-flavor warming yang Chinese medicine composition, the present invention unexpectedly discovered that the seven-flavor warming yang Chinese medicine composition showed significant therapeutic effects in the Caenorhabditis elegans obesity pathological model, can significantly reduce fat storage in the body of Caenorhabditis elegans, has the potential to treat obesity, and can be used to prepare drugs for the treatment of obesity.

[0007] References

[0008] [1] Xiang Hongding. Obesity and metabolic syndrome: current situation in China[J]. Modern Rehabilitation, 2001, (13): 12-14.

[0009] [2] Zhang Zhifu, Wang Zhenhua. Harms of obesity[J]. Chinese Community Physician (Medical Specialty), 2012, 14(20): 346.

[0010] [3] Wushouertukezi. Effects of cherry plum fruit polyphenols on lipid metabolism in high-fat diet obese mice[D]. Urumqi: Xinjiang Normal University, 2021.

[0011] [4]Apovian CM, Aronne LJ, Bessesen DH, et al. Pharmacological Management of Obesity: AnEndocrine Society Clinical Practice Guideline[J]. JClin Endocrinol Metab, 2015(2):342-362.

[0012] [5]Kaletta T,Hengartner M O.Finding function in novel targets:C.elegans as a model organism[J].Nature reviews Drug discovery,2006,5(5):387-399.

[0013] [6] Lü Dong, Du Yajuan, Ma Yingchun, et al. Experimental study on analgesic and anti-inflammatory effects of Qiwei Wenyang Capsule [J]. Chinese Journal of Rational Drug Use, 2017, 14(12): 59-61. Summary of the invention

[0014] In view of the above technical problems, the present invention discloses an application of a Chinese medicine composition in preparing a drug for treating obesity, which specifically includes the following contents:

[0015] In a first aspect, the present invention provides a use of a seven-flavor warming yang Chinese medicine composition in the preparation of a drug for treating obesity.

[0016] Preferably, the active ingredients in the seven-flavor warming yang traditional Chinese medicine composition are composed of deer antler glue, deer antler frost, cypress seed, salt dodder seed, rehmannia root, poria, and psoralea corylifolia.

[0017] Preferably, the seven-flavor warming yang Chinese medicine composition is composed of the following raw materials in parts by weight: 83.3 parts of deer antler glue, 83.3 parts of deer antler frost, 83.3 parts of cypress seeds, 83.3 parts of salt dodder seeds, 83.3 parts of rehmannia root, 41.7 parts of poria, and 41.7 parts of psoralea corylifolia.

[0018] Preferably, the seven-flavor warming yang Chinese medicine composition is a water extract of a combination of seven-flavor warming yang Chinese medicines.

[0019] Preferably, the preparation process of the seven-flavor warming yang Chinese medicine composition comprises the following steps:

[0020] S1: Weigh antler glue, antler cream, cypress seed, salt dodder seed, rehmannia root, poria, and psoralea corylifolia according to weight;

[0021] S2: Add 10 times the amount of water to soak, heat to reflux, and centrifuge to collect the supernatant solution;

[0022] S3: Add 8 times the amount of water to the drug residue precipitate after centrifugation in S2, heat and reflux, and centrifuge to collect the supernatant drug solution;

[0023] S4: The supernatant solutions in S2 and S3 are combined and concentrated, the concentrated solution is freeze-dried, and the freeze-dried powder is collected.

[0024] Preferably, S2 is: adding 10 times the amount of water, soaking for 2 hours, heating and reflux in a condensation reflux device for 2 hours, centrifuging at 12000 rpm for 10 minutes, and collecting the supernatant.

[0025] Preferably, S3 is: adding 8 times the amount of water to the medicinal residue precipitate after centrifugation in S2, heating and reflux in a condensation reflux device for 2 hours, centrifuging at 12000 rpm for 10 minutes, and collecting the supernatant medicinal liquid.

[0026] Preferably, S4 comprises: combining the supernatant solutions in S2 and S3, and concentrating the solution using a rotary evaporator, then freezing the concentrated solution in a -80°C refrigerator for 24 hours, placing it in a freeze dryer for freeze drying, collecting the freeze-dried powder, and storing it at -20°C.

[0027] Preferably, the seven-flavor Yang-warming Chinese medicine composition is added with pharmaceutically acceptable excipients to form any pharmaceutically acceptable dosage form.

[0028] Preferably, the dosage forms include powders, granules, powders, pills, tablets, capsules, pills, and oral liquids.

[0029] Beneficial effects of the present invention:

[0030] The present invention conducts experiments on a Caenorhabditis elegans obesity pathological model, and the results show that: (1) the water extract of the seven-flavor warming yang Chinese medicine composition has a significant therapeutic effect on the Caenorhabditis elegans obesity pathological model; (2) compared with each single herb and other single herb combinations, the water extract of the seven-flavor warming yang Chinese medicine composition has a more significant therapeutic effect on the Caenorhabditis elegans obesity pathological model; (3) the water extract of the seven-flavor warming yang Chinese medicine composition has a higher safety.

[0031] The following specific embodiments are provided to implement the technical solutions of the present invention, but the protection scope of the present invention is not limited to the following embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The therapeutic effect of the water extract of the seven-flavor warming yang Chinese medicine composition on the obese model nematodes; wherein, N is the normal group; Control is the obese model control group; P is the positive drug L-carnitine group (100μM); 0.01 is the group of the water extract of the seven-flavor warming yang Chinese medicine composition at 0.01mg / mL (raw drug amount 0.026mg / mL); 0.1 is the group of the water extract of the seven-flavor warming yang Chinese medicine composition at 0.1mg / mL (raw drug amount 0.256mg / mL); 1 is the group of the water extract of the seven-flavor warming yang Chinese medicine composition at 1mg / mL (raw drug amount 2.556mg / mL);

[0033] Figure 2 Comparison of the therapeutic effects of the water extract of the seven-flavor warming yang Chinese medicine composition and each single medicine on the obese model nematodes; wherein, N is the normal group; Control is the obese model control group; P is the positive drug L-carnitine group (100μM); M is the 0.1mg / mL (raw drug amount 0.256mg / mL) water extract of the seven-flavor warming yang Chinese medicine composition group; A is the raw drug amount 0.043mg / mL antler glue group; B is the raw drug amount 0.043mg / mL antler frost group; C is the raw drug amount 0.043mg / mL Rehmannia root group; D is the raw drug amount 0.043mg / mL salt dodder group; E is the raw drug amount 0.043mg / mL Platycladus orientalis group; F is the raw drug amount 0.021mg / mL Poria group; G is the raw drug amount 0.021mg / mL Psoralea corylifolia group;

[0034] Figure 3Comparison of the therapeutic effects of the water extract of the seven-flavor warming yang Chinese medicine composition and other single-flavor medicinal compositions on the obese model nematodes; wherein N is the normal group; Control is the obese model control group; P is the positive drug L-carnitine group (100μM); M is the 0.1mg / mL (raw drug amount 0.256mg / mL) water extract group of the seven-flavor warming yang Chinese medicine composition; I is the raw drug amount 0.107mg / mL of Rehmannia glutinosa, Cuscuta australis, and Poria group The compound group; J is a crude drug amount of 0.150 mg / mL of Rehmannia glutinosa, salt dodder seed, Poria cocos, and antler glue; K is a crude drug amount of 0.150 mg / mL of Rehmannia glutinosa, salt dodder seed, Poria cocos, and antler frost; O is a crude drug amount of 0.150 mg / mL of Rehmannia glutinosa, salt dodder seed, Poria cocos, and Semen Platycladi; Q is a crude drug amount of 0.128 mg / mL of Rehmannia glutinosa, salt dodder seed, Poria cocos, and Psoralea corylifolia;

[0035] Figure 4 Effect of the aqueous extract of the seven-flavor warming yang Chinese medicine composition on the body length of nematodes; wherein, Control is the blank control group; 0.01 is the group of the aqueous extract of the seven-flavor warming yang Chinese medicine composition with a concentration of 0.01 mg / mL (raw drug amount 0.026 mg / mL); 0.1 is the group of the aqueous extract of the seven-flavor warming yang Chinese medicine composition with a concentration of 0.1 mg / mL (raw drug amount 0.256 mg / mL); 1 is the group of the aqueous extract of the seven-flavor warming yang Chinese medicine composition with a concentration of 1 mg / mL (raw drug amount 2.556 mg / mL). DETAILED DESCRIPTION

[0036] Example 1 Preparation method of water extract of seven-flavor warming yang Chinese medicine composition

[0037] Weigh 83.3 parts of antler glue, 83.3 parts of antler cream, 83.3 parts of cypress seeds, 83.3 parts of salt dodder seeds, 83.3 parts of Rehmannia glutinosa, 41.7 parts of Poria cocos, and 41.7 parts of psoralea corylifolia by weight. Add 10 times the amount of water, soak for 2 hours, heat and reflux in a condensation reflux device for 2 hours, centrifuge at 12000rpm for 10 minutes, take the supernatant solution, and set aside. Add 8 times the amount of water to the centrifuged residue precipitate, heat and reflux in a condensation reflux device for 2 hours, centrifuge at 12000rpm for 10 minutes, take the supernatant solution, and set aside. Combine the two supernatant solutions, and use a rotary evaporator to concentrate the solution, then freeze the concentrated solution in a -80℃ refrigerator for 24 hours, place it in a freeze dryer for freeze drying, collect the freeze-dried powder, store it at -20℃, and set aside.

[0038] Example 2 Therapeutic effect of the aqueous extract of the seven-flavor warming yang Chinese medicine composition on obese nematodes

[0039] 1. Biomaterials:

[0040] (1) The wild-type strain N2 of Caenorhabditis elegans was purchased from Caenorhabditis Genetics Center (CGC);

[0041] (2) Escherichia coli OP50 (uracil leaky mutant), purchased from Caenorhabditis Genetics Center (CGC), was used as food for C. elegans.

[0042] 2. Reagents

[0043] (1) The composition and preparation of solid NGM (Nematode Growth Medium) culture medium (taking 1000 mL as an example) are shown in Table 1:

[0044] Table 1 Solid NGM medium

[0045] Element content NaCl 3.00g <![CDATA[K 2 HPO 4 ]]> 2.34g <![CDATA[KH 2 AFTER 4 ]]> 17.23g Peptone 2.50g Agar 17.00g <![CDATA[Supplementary H 2 O to]]> 1000mL

[0046] After the solid NGM medium was prepared, it was sterilized at 121°C under high pressure for 20 min. 1 mL of 5 mg / mL cholesterol and 1 M MgSO 4 1 mL, 1M CaCl 2 1mL, shake well, and pour into sterilized 9cm culture plates while hot, about 20mL / plate. Let stand and wait for the culture medium to solidify, and set aside.

[0047] (2) The composition and preparation of solid high-glucose NGM (Nematode Growth Media) medium (taking 100 mL as an example) are shown in Table 2:

[0048] Table 2 Solid high glucose NGM medium

[0049]

[0050]

[0051] After the solid NGM medium was prepared, it was sterilized at 121°C under high pressure for 20 min. 0.1 mL of 5 mg / mL cholesterol and 1 M MgSO 4 0.1 mL, 1 M CaCl 2 0.1mL, 10mL of 20% (w / v) glucose, shake well, pour into sterilized 6cm culture plates while hot, about 15mL / plate. Let stand and wait for the culture medium to solidify, and set aside.

[0052] (3) The formula of M9 liquid is shown in Table 3:

[0053] Table 3M9 liquid formula

[0054] Element content <![CDATA[Na 2 HPO 4 ]]> 6.00g <![CDATA[KH 2 AFTER 4 ]]> 3.00g NaCl 5.00g <![CDATA[1M MgSO 4 ]]> 1.00mL <![CDATA[Supplementary H 2 O to]]> 1000mL

[0055] (4) Preparation of lysis solution: 6.4% NaClO solution and 1 M NaOH solution were mixed in a volume ratio of 1:1.

[0056] 3. Preparation of high-sugar NGM plates containing the aqueous extract of the seven-flavor warming yang Chinese medicine composition

[0057] Preparation of the aqueous extract of the seven-flavor warming yang Chinese medicine composition: 0.1 g of the freeze-dried powder of the aqueous extract of the seven-flavor warming yang Chinese medicine composition is added with water to make up to 10 mL, filtered and sterilized, and set aside.

[0058] Prepare high-glucose NGM plates containing the water extract of the seven-flavor warming yang Chinese medicine composition: take a solution of the water extract of the seven-flavor warming yang Chinese medicine composition with an appropriate dilution, add it to the high-glucose NGM culture medium, and pour it onto the plates.

[0059] The final concentration of the water extract of the seven-flavor warming yang Chinese medicine composition is 10 times, 100 times, and 1000 times of the initially prepared medicinal solution. The culture medium is left to stand and wait for solidification. Escherichia coli OP50 is evenly spread on the culture medium as food for nematodes.

[0060] 4. Implementation steps

[0061] (1) Cultivation of nematodes

[0062] The nematodes were plated on solid NGM plates coated with E. coli OP50 and then cultured in an incubator at 20°C. When the nematodes grew into adults, they were synchronized.

[0063] (2) Synchronization of nematodes

[0064] Select NGM culture medium containing a large number of adult worms and some nematode eggs that have already hatched, wash the nematodes off the culture medium with M9 solution, transfer to a centrifuge tube, let the nematodes settle freely to the bottom of the tube, and discard the supernatant. Add lysis solution to the centrifuge tube according to the amount of nematodes, oscillate on a vortex for 5-7 minutes, stop vortexing when all the nematodes are broken, and divide into 1.5mL centrifuge tubes, and wash the nematode eggs three times with M9 solution.

[0065] (3) Establishment of Caenorhabditis elegans obesity pathology model

[0066] The synchronized nematodes were cultured on high-sugar NGM medium containing 2% (w / v) glucose, so that the content of nematodes per plate was about 600, and cultured in a 20°C biochemical incubator for 48 hours. If the Nile red staining results showed that the fluorescence intensity of the high-sugar induced nematode obesity model was significantly higher than that of the nematodes cultured without 2% (w / v) high-sugar NGM medium, the obesity model was successfully established.

[0067] (4) Experimental group settings

[0068] Normal group (N): C. elegans were cultured using normal NGM medium without 2% (w / v) glucose;

[0069] Obesity model control group (Control): C. elegans were cultured in high-glucose NGM medium containing 2% (w / v) glucose;

[0070] Positive drug L-carnitine (100 μM) group (P): C. elegans were cultured in high-glucose NGM medium containing 100 μM L-carnitine and 2% (w / v) glucose;

[0071] 0.01, 0.1, 1 mg / mL (the amount of crude drug is 0.026, 0.256, 2.556 mg / mL, respectively) of the water extract of the seven-flavor warming yang Chinese medicine composition group: the nematodes were cultured in a high-glucose NGM medium containing 0.01, 0.1, 1 mg / mL (the amount of crude drug is 0.026, 0.256, 2.556 mg / mL, respectively) of the water extract of the seven-flavor warming yang Chinese medicine composition and 2% (w / v) glucose;

[0072] (5) Therapeutic effect of the aqueous extract of seven-flavor warming Chinese medicine composition on the Caenorhabditis elegans obesity pathological model

[0073] Nematode drug treatment: The incubated L1 nematodes were transferred to high-sugar NGM plates coated with OP50 and mixed with different concentrations of the aqueous extract of the seven-yang warming Chinese medicine composition. 600 nematodes were plated on each plate. After culturing at 20°C for 48 hours, the nematodes were washed with M9 and transferred to a centrifuge tube.

[0074] Lipid staining: 100 μL of 40% isopropanol was added to the nematode sediment and incubated at room temperature for 3 min; the nematodes were centrifuged at 800 rpm for 1 min and the supernatant was removed. 600 μL of Nile Red working solution was added to each sample in the dark, and the tube was inverted three times to mix thoroughly; the samples were rotated in the dark at room temperature for 2 h; after incubation, the nematodes were centrifuged at 800 rpm for 1 min and the supernatant was removed; 600 μL of M9 buffer was added to the sample and incubated in the dark for 30 min to remove excess Nile Red stain; the sample was centrifuged at 800 rpm for 1 min and about 50 μL of supernatant was removed. Red fluorescence photography was performed using TRITC on an Olympus inverted fluorescence microscope (IX73+DP74). 30 fluorescence images were randomly taken for each group, and the experiment was repeated 3 times.

[0075] Data processing and statistical analysis: The photos were processed using image-J software. High fluorescence intensity indicates high fat storage in the nematode, and low fluorescence intensity indicates low fat storage in the nematode.

[0076] 5. Experimental results

[0077] like Figure 1 As shown, the obesity model control group significantly increased the fat accumulation in C. elegans N2 compared with the normal group, indicating that the obesity model was successfully constructed. The positive drug L-carnitine (100 μM) significantly reduced the fat accumulation in C. elegans N2 compared with the obesity model control group. The 0.01, 0.1, and 1 mg / mL (raw drug amount 0.026, 0.256, and 2.556 mg / mL) seven-flavor warming yang Chinese medicine composition water extract groups significantly reduced the fat accumulation in C. elegans N2 compared with the obesity model control group. This shows that the seven-flavor warming yang Chinese medicine composition water extract has a significant therapeutic effect on the Caenorhabditis elegans obesity pathological model.

[0078] Example 3 Comparison of the therapeutic effects of the aqueous extract of the seven-flavor warming Chinese medicine composition and each single herb on obese nematodes

[0079] 1. Preparation of the extracts of each single herb in the seven-herb warming yang Chinese medicine composition

[0080] 0.1 mg / mL (raw drug amount 0.256 mg / mL) Seven-flavor warming yang Chinese medicine composition water extract group (M): prepared as shown in Example 1

[0081] Drug group A (deer antler glue water extract with a crude drug amount of 0.043 mg / mL): 2g of deer antler glue, add 10 times the amount of water, soak for 2h, heat and reflux for 2h, centrifuge at 12000rpm for 10min, add 8 times the amount of water to the drug residue, heat and reflux for 2h, centrifuge at 12000rpm for 10min. Combine the two drug solutions, make up to 50mL, filter and sterilize, and set aside. Dilute to 0.430mg / mL mother solution when using, and dilute 10 times when preparing drug-containing high-sugar NGM culture medium.

[0082] Drug group B (antler frost water extract with a crude drug dosage of 0.043 mg / mL): 2g of antler frost, add 10 times the amount of water, soak for 2h, heat to reflux for 2h, centrifuge at 12000rpm for 10min, add 8 times the amount of water to the residue, heat to reflux for 2h, centrifuge at 12000rpm for 10min. Combine the two drug solutions, make up to 50mL, filter and sterilize, and set aside. Dilute to 0.430mg / mL mother solution when using, and dilute 10 times when preparing drug-containing high-sugar NGM culture medium.

[0083] Drug group C (raw drug amount of 0.043 mg / mL Rehmannia glutinosa water extract): 2g Rehmannia glutinosa, add 10 times the amount of water, soak for 2h, heat and reflux for 2h, centrifuge at 12000rpm for 10min, add 8 times the amount of water to the residue, heat and reflux for 2h, centrifuge at 12000rpm for 10min. Combine the two drug solutions, make up to 50mL, filter and sterilize, and set aside. Dilute to 0.430mg / mL mother solution when using, and dilute 10 times when preparing drug-containing high-sugar NGM culture medium.

[0084] Drug group D (water extract of salt dodder with a crude drug amount of 0.043 mg / mL): 2g of salt dodder, add 10 times the amount of water, soak for 2h, heat and reflux for 2h, centrifuge at 12000rpm for 10min, add 8 times the amount of water to the residue, heat and reflux for 2h, centrifuge at 12000rpm for 10min. Combine the two drug solutions, make up to 50mL, filter and sterilize, and set aside. Dilute to 0.430mg / mL mother solution when using, and dilute 10 times when preparing drug-containing high-sugar NGM culture medium.

[0085] Drug group E (water extract of cypress seeds with a crude drug amount of 0.043 mg / mL): 2g of cypress seeds, add 10 times the amount of water, soak for 2h, heat and reflux for 2h, centrifuge at 12000rpm for 10min, add 8 times the amount of water to the residue, heat and reflux for 2h, centrifuge at 12000rpm for 10min. Combine the two drug solutions, make up to 50mL, filter and sterilize, and set aside. Dilute to 0.430mg / mL mother solution when using, and dilute 10 times when preparing drug-containing high-sugar NGM culture medium.

[0086] Drug group F (poria water extract with a crude drug amount of 0.021 mg / mL): 2g of Poria, add 10 times the amount of water, soak for 2h, heat and reflux for 2h, centrifuge at 12000rpm for 10min, add 8 times the amount of water to the drug residue, heat and reflux for 2h, centrifuge at 12000rpm for 10min. Combine the two drug solutions, make up to 50mL, filter and sterilize, and set aside. Dilute to 0.210mg / mL mother solution when using, and dilute 10 times when preparing drug-containing high-sugar NGM culture medium.

[0087] G drug group (psoralea corylifolia water extract with a crude drug amount of 0.021 mg / mL): 2g of psoralea corylifolia, add 10 times the amount of water, soak for 2h, heat and reflux for 2h, centrifuge at 12000rpm for 10min, add 8 times the amount of water to the drug residue, heat and reflux for 2h, centrifuge at 12000rpm for 10min. Combine the two drug solutions, make up to 50mL, filter and sterilize, and set aside. Dilute to 0.210mg / mL mother solution when using, and dilute 10 times when preparing drug-containing high-sugar NGM culture medium.

[0088] 2. Preparation of drug-containing tablets for each drug group

[0089] Normal group (N): C. elegans were cultured using normal NGM medium without 2% (w / v) glucose;

[0090] Obesity model control group (Control): C. elegans were cultured in high-glucose NGM medium containing 2% (w / v) glucose;

[0091] Positive drug L-carnitine (100 μM) group (P): C. elegans were cultured in high-glucose NGM medium containing 100 μM L-carnitine and 2% (w / v) glucose;

[0092] 0.1 mg / mL water extract of seven-flavor warming yang Chinese medicine composition group (M): C. elegans were cultured in a high-glucose NGM medium containing 0.1 mg / mL (raw drug amount 0.256 mg / mL) water extract of seven-flavor warming yang Chinese medicine composition and 2% (w / v) glucose;

[0093] Drug group A: C. elegans was cultured using the deer antler glue aqueous extract prepared in the previous step (raw drug amount 0.043 mg / mL) and high-glucose NGM medium containing 2% (w / v) glucose;

[0094] Drug group B: C. elegans was cultured using the antler frost water extract prepared in the previous step (raw drug amount 0.043 mg / mL) and high-glucose NGM medium containing 2% (w / v) glucose;

[0095] Drug group C: C. elegans were cultured using the Rehmannia glutinosa aqueous extract prepared in the previous step (raw drug amount 0.043 mg / mL) and high-glucose NGM medium containing 2% (w / v) glucose;

[0096] Drug group D: C. elegans was cultured using the water extract of Cuscuta salina prepared in the previous step (raw drug amount 0.043 mg / mL) and high-glucose NGM medium containing 2% (w / v) glucose;

[0097] Drug group E: C. elegans were cultured using the water extract of Semen Platycladi prepared in the previous step (raw drug amount 0.043 mg / mL) and high-glucose NGM medium containing 2% (w / v) glucose;

[0098] Drug group F: C. elegans was cultured using the Poria cocos water extract prepared in the previous step (raw drug amount 0.021 mg / mL) and high-glucose NGM medium containing 2% (w / v) glucose;

[0099] Drug group G: C. elegans were cultured using the aqueous extract of Psoralea corylifolia prepared in the previous step (raw drug amount 0.021 mg / mL) and high-glucose NGM medium containing 2% (w / v) glucose;

[0100] 3. Experimental Procedure

[0101] The specific implementation steps are the same as those in Example 2.

[0102] 4. Experimental Results

[0103] The experimental results show that: Figure 2As shown, compared with the obesity model control group, 0.1 mg / mL (raw drug amount 0.256 mg / mL) seven-flavor warming yang Chinese medicine composition water extract group (M), antler glue (raw drug amount 0.043 mg / mL) group (A), antler frost (raw drug amount 0.043 mg / mL) group (B), prepared rehmannia root (raw drug amount 0.043 mg / mL) group (C), salt dodder seed (raw drug amount 0.043 mg / mL) group (D) can significantly reduce the fat accumulation in the body of Caenorhabditis elegans N2 (p < 0.001) , the Platycladus orientalis (raw drug amount 0.043 mg / mL) group (E) and the Psoralea corylifolia (raw drug amount 0.021 mg / mL) group (G) were able to reduce the fat accumulation in the body of Caenorhabditis elegans N2 (p < 0.05), while the Poria cocos (raw drug amount 0.021 mg / mL) group (F) had no significant effect on the fat accumulation in the nematodes; and the 0.1 mg / mL (raw drug amount 0.256 mg / mL) water extract group of the seven-flavor warming yang Chinese medicine composition (M) had a better therapeutic effect on the Caenorhabditis elegans obesity pathological model than any single drug.

[0104] Example 4 Comparison of the therapeutic effects of the aqueous extract of the seven-flavor warming yang Chinese medicine composition and other single-flavor medicinal compositions on obese nematodes 1. Preparation of aqueous extracts of each single-flavor medicinal composition of the seven-flavor warming yang Chinese medicine composition

[0105] The present invention designs a comparative test based on the retrieved compositions with fat-reducing effects, further proving that the seven-flavor warming yang Chinese medicine composition has a more significant fat-reducing effect.

[0106] 0.1 mg / mL (raw drug amount 0.256 mg / mL) Seven-flavor warming yang Chinese medicine composition water extract group (M): prepared as shown in Example 1

[0107] I drug group (raw drug amount 0.107mg / mL Rehmannia glutinosa + salt dodder seed + Poria water extract): Rehmannia glutinosa 1g, salt dodder seed 1g, Poria 0.5g, add 10 times the amount of water, soak for 2h, heat and reflux for 2h, centrifuge at 12000rpm for 10min, add 8 times the amount of water to the residue, heat and reflux for 2h, centrifuge at 12000rpm for 10min. Combine the 2 medicinal solutions, set the volume to 50mL, filter and sterilize, and set aside. Dilute to 1.070mg / mL mother liquor when used, and dilute 10 times when preparing drug-containing high-sugar NGM culture medium.

[0108] Drug group J (raw drug amount 0.150mg / mL Rehmannia glutinosa + Cuscuta australis + Poria cocos + antler glue water extract): Rehmannia glutinosa 0.6g, Cuscuta australis 0.6g, Poria cocos 0.3g, antler glue 0.6g, add 10 times the amount of water, soak for 2h, heat and reflux for 2h, centrifuge at 12000rpm for 10min, add 8 times the amount of water to the residue, heat and reflux for 2h, centrifuge at 12000rpm for 10min. Combine the two drug solutions, make up to 50mL, filter and sterilize, and set aside. Dilute to 1.500mg / mL mother liquor when used, and dilute 10 times when preparing drug-containing high-sugar NGM culture medium.

[0109] Drug group K (raw drug amount 0.150mg / mL Rehmannia glutinosa + Cuscuta australis + Poria cocos + antler frost water extract): 0.6g Rehmannia glutinosa, 0.6g Cuscuta australis, 0.3g Poria cocos, 0.6g antler frost, add 10 times the amount of water, soak for 2h, heat and reflux for 2h, centrifuge at 12000rpm for 10min, add 8 times the amount of water to the residue, heat and reflux for 2h, centrifuge at 12000rpm for 10min. Combine the two drug solutions, make up to 50mL, filter and sterilize, and set aside. Dilute to 1.500mg / mL mother liquor when used, and dilute 10 times when preparing drug-containing high-sugar NGM culture medium.

[0110] O drug group (raw drug amount 0.150mg / mL Rehmannia glutinosa + Cuscuta australis + Poria cocos + Platycladus orientalis water extract): 0.6g Rehmannia glutinosa, 0.6g Cuscuta australis, 0.3g Poria cocos, 0.6g Platycladus orientalis, add 10 times the amount of water, soak for 2h, heat and reflux for 2h, centrifuge at 12000rpm for 10min, add 8 times the amount of water to the residue, heat and reflux for 2h, centrifuge at 12000rpm for 10min. Combine the two drug solutions, make up to 50mL, filter and sterilize, and set aside. Dilute to 1.500mg / mL mother liquor when using, and dilute 10 times when preparing drug-containing high-sugar NGM culture medium.

[0111] Q drug group (raw drug amount 0.128mg / mL Rehmannia glutinosa + Cuscuta australis + Poria cocos + Psoralea corylifolia water extract): 0.6g Rehmannia glutinosa, 0.6g Cuscuta australis, 0.3g Poria cocos, 0.3g Psoralea corylifolia, add 10 times the amount of water, soak for 2h, heat to reflux for 2h, centrifuge at 12000rpm for 10min, add 8 times the amount of water to the residue, heat to reflux for 2h, centrifuge at 12000rpm for 10min. Combine the two drug solutions, make up to 50mL, filter and sterilize, and set aside. Dilute to 1.500mg / mL mother liquor when used, and dilute 10 times when preparing drug-containing high-sugar NGM culture medium.

[0112] 2. Preparation of drug-containing tablets for each drug group

[0113] Normal group (N): C. elegans were cultured using normal NGM medium without 2% (w / v) glucose;

[0114] Obesity model control group (Control): C. elegans were cultured in high-glucose NGM medium containing 2% (w / v) glucose;

[0115] Positive drug L-carnitine (100 μM) group (P): C. elegans were cultured in high-glucose NGM medium containing 100 μM L-carnitine and 2% (w / v) glucose;

[0116] 0.1 mg / mL water extract of seven-flavor warming yang Chinese medicine composition group (M): C. elegans were cultured in a high-glucose NGM medium containing 0.1 mg / mL (raw drug amount 0.256 mg / mL) water extract of seven-flavor warming yang Chinese medicine composition and 2% (w / v) glucose;

[0117] Drug group I: C. elegans were cultured using the aqueous extract of Rehmannia glutinosa + Cuscuta australis + Poria cocos (raw drug amount 0.107 mg / mL) prepared in the previous step and a high-glucose NGM medium containing 2% (w / v) glucose;

[0118] Drug group J: C. elegans were cultured using the aqueous extract of Rehmannia glutinosa + Cuscuta australis + Poria cocos + antler glue prepared in the previous step (raw drug amount 0.150 mg / mL) and high-glucose NGM medium containing 2% (w / v) glucose;

[0119] Drug group K: C. elegans were cultured using the aqueous extract of Rehmannia glutinosa + Cuscuta australis + Poria cocos + Psoralea corylifolia prepared in the previous step (raw drug amount 0.150 mg / mL) and high-glucose NGM medium containing 2% (w / v) glucose;

[0120] O drug group: C. elegans were cultured with the aqueous extract of Rehmannia glutinosa + Cuscuta australis + Poria cocos + Semen Platycladi prepared in the previous step (raw drug amount 0.150 mg / mL) and high-glucose NGM medium containing 2% (w / v) glucose;

[0121] Q drug group: C. elegans were cultured using the aqueous extract of Rehmannia glutinosa + Cuscuta australis + Poria cocos + Psoralea corylifolia prepared in the previous step (raw drug amount 0.128 mg / mL) and high-glucose NGM medium containing 2% (w / v) glucose;

[0122] 2. Experimental Procedure

[0123] Other experimental steps are the same as in Example 2.

[0124] 3. Experimental results

[0125] The experimental results are as follows Figure 3As shown, compared with the obesity model control group, 0.1 mg / mL (raw drug amount 0.256 mg / mL) seven-flavor warming yang Chinese medicine composition water extract group (M), positive control L-carnitine (100 μM) group (P), cooked rehmannia root + salt dodder seed + Poria cocos + antler frost (raw drug amount 0.150 mg / mL) group (K), cooked rehmannia root + salt dodder seed + Poria cocos + psoralea corylifolia (raw drug amount 0.128 mg / mL) group (Q) all significantly reduced fat accumulation in the Caenorhabditis elegans obesity model, while cooked rehmannia root + salt dodder seed + Poria cocos (raw drug amount 0.107 mg / mL) group (I), cooked rehmannia root + salt dodder seed + Poria cocos (raw drug amount 0.150 mg / mL) group (K), cooked rehmannia root + salt dodder seed + Poria cocos + psoralea corylifolia (raw drug amount 0.128 mg / mL) group (Q) The group of Radix Rehmanniae + salt dodder seed + Poria cocos + antler glue (raw drug amount 0.150 mg / mL) (J) and the group of Rehmanniae glutinosae + salt dodder seed + Poria cocos + arborvitae seed (raw drug amount 0.150 mg / mL) (O) did not significantly affect the fat accumulation in the nematodes; and the group of 0.1 mg / mL (raw drug amount 0.256 mg / mL) water extract of the seven-flavor warming yang Chinese medicine composition (M) had a significantly better lipid-lowering effect than the group of Rehmanniae glutinosae + salt dodder seed + Poria cocos + antler frost (raw drug amount 0.150 mg / mL) (K) and the group of Rehmanniae glutinosae + salt dodder seed + Poria cocos + psoralea corylifolia (raw drug amount 0.128 mg / mL) (Q).

[0126] It can be seen that the combination of Cuscuta chinensis + Rehmannia glutinosa + Poria cocos does not have a fat-reducing effect, and even the addition of antler glue, which has a fat-reducing effect, does not improve the fat-reducing effect. On this basis, the addition of Psoralea corylifolia and Platycladus orientalis seeds, which have almost no fat-reducing effect, unexpectedly makes the seven-flavor warming yang Chinese medicine combination have a more significant fat-reducing effect than single herbs and publicly available Chinese medicine combinations.

[0127] Example 5 Effect of the aqueous extract of the seven-flavor warming yang Chinese medicine composition on the body length of nematodes

[0128] 1. Experimental group settings:

[0129] Blank control group (Control): C. elegans cultured in normal NGM medium

[0130] 0.01, 0.1, 1 mg / mL (the raw drug amounts are 0.026, 0.256, 2.556 mg / mL, respectively) of the water extract of the seven-flavor warming yang Chinese herbal composition group: Nematodes were cultured using NGM medium containing 0.01, 0.1, 1 mg / mL (the raw drug amounts are 0.026, 0.256, 2.556 mg / mL, respectively) of the water extract of the seven-flavor warming yang Chinese herbal composition.

[0131] 2. Experimental Procedure

[0132] (1) Nematode drug treatment: The incubated L1 nematodes were transferred to NGM plates coated with OP50 and mixed with different concentrations of the aqueous extract of the seven warming yang Chinese medicine composition. 600 nematodes were plated on each plate. After culturing at 20°C for 48 h, the nematodes were washed with M9 and transferred to a centrifuge tube.

[0133] (2) Measurement of nematode body length: Each group of nematodes was photographed using the bright field of an Olympus inverted fluorescence microscope (IX73+DP74). Thirty pictures were randomly taken for each group, and the experiment was repeated three times. The pictures were processed using image-J software, and the nematode body length was measured using ROIManager.

[0134] 3. Experimental results

[0135] The results are as follows Figure 4 As shown, within the concentration range of 0.01 mg / mL to 1 mg / mL (crude drug amount 0.026 to 2.556 mg / mL), the water extract of the seven-flavor warming yang Chinese medicine composition had no obvious effect on the body length of the wild-type nematode N2, which indicates that within the concentration range of 0.01 mg / mL to 1 mg / mL (crude drug amount 0.026 to 2.556 mg / mL), the water extract of the seven-flavor warming yang Chinese medicine composition has good safety.

[0136] The results of the above examples show that the water extract of the seven-flavor warming yang Chinese medicine composition can significantly reduce the amount of fat stored in nematodes, and the water extract of the seven-flavor warming yang Chinese medicine composition has a significant therapeutic effect on the Caenorhabditis elegans obesity pathological model, and has good safety. It is suggested that the water extract of the seven-flavor warming yang Chinese medicine composition has the potential to treat obesity, can be used in the preparation of drugs for treating obesity, and can also be used in the preparation of health products for preventing and assisting the treatment of obesity. It is expected to be developed into a new generation of anti-obesity drugs or weight loss health products.

Claims

1. Application of a seven-flavor warming yang Chinese medicine composition in the preparation of a drug for treating obesity.

2. The use according to claim 1, characterized in that The active components of the seven-flavor yang-warming Chinese medicine composition are composed of deer antler glue, deer antler frost, cypress seed, salt dodder seed, rehmannia root, tuckahoe and psoralea corylifolia.

3. The use according to claim 2, characterized in that The seven-flavor warming yang Chinese medicine composition is composed of the following raw materials in parts by weight: 83.3 parts of deer antler glue, 83.3 parts of deer antler frost, 83.3 parts of cypress seeds, 83.3 parts of salt dodder seeds, 83.3 parts of rehmannia root, 41.7 parts of tuckahoe, and 41.7 parts of psoralea corylifolia.

4. The use according to claim 3, characterized in that The seven-flavor warming yang Chinese medicine composition is a water extract of a combination of seven-flavor warming yang Chinese medicines.

5. The use according to claim 4, characterized in that Preparation process of the seven-flavor yang-warming Chinese medicine composition The following steps are involved: S1: Weigh antler glue, antler cream, cypress seed, salt dodder seed, rehmannia root, poria, and psoralea corylifolia according to weight; S2: Add 10 times the amount of water to soak, heat to reflux, and centrifuge to collect the supernatant solution; S3: Add 8 times the amount of water to the drug residue precipitate after centrifugation in S2, heat and reflux, and centrifuge to collect the supernatant drug solution; S4: The supernatant solutions in S2 and S3 are combined and concentrated, the concentrated solution is freeze-dried, and the freeze-dried powder is collected.

6. The use according to claim 5, characterized in that The S2 is as follows: adding 10 times the amount of water, soaking for 2 hours, heating and reflux in a condensation reflux device for 2 hours, centrifuging at 12000 rpm for 10 minutes, and collecting the supernatant.

7. The use according to claim 5, characterized in that The step S3 is as follows: adding 8 times the amount of water to the medicinal residue precipitate after centrifugation in S2, heating and reflux in a condensation reflux device for 2 hours, centrifuging at 12000 rpm for 10 minutes, and collecting the supernatant medicinal solution.

8. The use according to claim 5, characterized in that The S4 comprises: combining the supernatant solutions in S2 and S3, concentrating the solution using a rotary evaporator, freezing the concentrated solution in a -80°C refrigerator for 24 hours, placing the solution in a freeze dryer for freeze drying, collecting the freeze-dried powder, and storing it at -20°C.

9. The use according to any one of claims 1 to 8, characterized in that: The seven-flavor warming yang traditional Chinese medicine composition is added with pharmaceutically acceptable excipients to prepare any pharmaceutically acceptable dosage form.

10. The use according to claim 9, characterized in that The dosage forms include powders, granules, powders, pills, tablets, capsules, pills, and oral liquids.

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