Pigeon pea extract, active compound of pigeon pea extract and application of pigeon pea extract and active compound in preparation of lipid-lowering, sugar-lowering and weight-losing medicines or health-care products

Abstract

The invention discloses a pigeonpea extract and an active compound thereof, and application of the pigeonpea extract and the active compound in preparation of medicines or health care products for lowering lipid, reducing blood sugar and losing weight, the pigeonpea extract and the active compound thereof have a remarkable lipid lowering effect, and can remarkably reduce adipogenesis and lipolysis of 3T3-L1 cells; the pigeon pea extract and the active compound can remarkably reduce the expression quantity of SREBP-1c, PPAR gamma and C / EBP alpha, and signal molecules are important targets of some diabetes and weight-reducing medicines besides regulating synthesis and metabolism of fat, so that the pigeon pea extract and the active compound thereof can be possibly deduced to have application potential in the aspects of sugar control and weight reduction; the pigeonpea extract and the active compounds thereof are derived from natural plants, and the active compounds CAB and CAC are natural small molecule compounds, are low in toxicity and few in side effects, and have good development and application prospects.

Description

Technical field:

[0001] The invention belongs to the field of medical technology, and specifically relates to a pigeon pea extract and active compounds thereof and applications thereof in preparing lipid-lowering, blood sugar-lowering and weight-loss medicines or health products. Background technology:

[0002] Obesity is caused by a long-term imbalance between energy intake and energy expenditure. Currently, obesity has become a health problem that has attracted global attention. It is closely related to a variety of diseases such as dyslipidemia, hyperglycemia, hypertension, coronary heart disease and diabetes. Obesity is mainly related to adipose tissue hyperplasia, which is the result of increased adipogenesis and cytoplasmic triglyceride deposition. Adipogenesis reflects the process of differentiation of preadipocytes into fully mature adipocytes, which is actively regulated by various adipogenesis-related factors and adipogenic transcription factors, such as the peroxisome proliferator-activated receptor family (PPARs). Among them, PPARγ in PPARs is considered to be a major regulator involved in adipocyte differentiation, adipogenesis and glucose metabolism. Therefore, regulating the PPARγ pathway is an important target for preventing obesity.

[0003] For decades, many traditional anti-obesity drugs have been widely used. However, as the limitations and harmful side effects (such as abdominal pain and hypoglycemia) of these drugs are gradually exposed, it is a general trend to find new drugs that inhibit lipogenesis from natural products. Pigeon pea (Cajanus cajan (L.) Millsp.) is a plant of Leguminosae, the fifth largest soybean crop in the world, with a long history of edible and medicinal use, widely distributed in my country, low price of medicinal materials, and relatively mature extraction process. Existing studies have shown that pigeon pea extract can be used to prepare drugs for the treatment of osteoporosis and anti-tumor, but its application in anti-obesity is rarely reported. Summary of the invention:

[0004] The present invention provides a pigeon pea extract and its active compounds and use thereof in the preparation of lipid-lowering, blood sugar-lowering and weight-loss drugs or health products, providing a theoretical basis and technical support for accelerating the development of new lipid-lowering drugs and weight control therapies.

[0005] The present invention is achieved through the following technical solutions:

[0006] The first object of the present invention is to provide pigeon pea ketone B and pigeon pea ketone C.

[0007] The structural formulas of the described caryophyllene B (CAB) and caryophyllene C (CAC) are as follows:

[0008]

[0009] The second object of the present invention is to provide the use of pigeon pea extract B, pigeon pea extract C or their pharmaceutically acceptable salts or stereoisomers or prodrug molecules and pigeon pea extract in the preparation of lipid-lowering, blood sugar-lowering and weight-loss drugs or health products.

[0010] Preferably, the pigeon pea extract includes pigeon pea root extract, pigeon pea stem extract, and pigeon pea leaf extract.

[0011] More preferably, the pigeon pea extract is pigeon pea leaf extract.

[0012] Preferably, the preparation method of the pigeon pea extract comprises the following steps: taking dried pigeon pea roots, pigeon pea stems or pigeon pea leaves, crushing them and placing them in an ethanol aqueous solution for soaking and extraction, filtering and collecting the filtrate after soaking, and performing reduced pressure distillation to obtain a pigeon pea ethanol extract, suspending the ethanol extract in water, extracting with n-hexane and ethyl acetate, and concentrating the ethyl acetate portion by reduced pressure distillation to obtain an ethyl acetate extract, which is the pigeon pea extract.

[0013] Preferably, the solid-liquid ratio of the crushed pigeon pea roots, pigeon pea stems or pigeon pea leaves to the ethanol aqueous solution is 3g / 10ml.

[0014] Preferably, the concentration of the ethanol aqueous solution is 95% (v / v).

[0015] Preferably, the soaking extraction is performed three times, with the first soaking for 72 hours. After the soaking is completed, the filtrate is filtered and collected. The filter residue is soaked again for 48 hours, and the filtrate is collected after filtering. The filter residue is soaked for the third time for 24 hours, and the filtrate is filtered and collected after soaking. The filtrates after the three soaking and filtering are combined and distilled under reduced pressure.

[0016] Preferably, the health care product is a functional health care product that is beneficial to obese patients or a functional health care product that is beneficial to diabetic patients.

[0017] Preferably, medically acceptable excipients are added to the medicine.

[0018] The third object of the present invention is to provide a lipid-lowering, blood sugar-lowering and weight-loss medicine or health product containing the above-mentioned pigeon pea ketone B, pigeon pea ketone C or their pharmaceutically acceptable salts or stereoisomers or their prodrug molecules and pigeon pea extract.

[0019] The fourth object of the present invention is to provide a method for preparing pigeon pea ketone B and pigeon pea ketone C, which are prepared and separated from pigeon pea leaves. Preferably, the specific steps are as follows:

[0020] 1) taking dried pigeon pea leaves, crushing them and putting them into an ethanol aqueous solution for soaking and extraction, filtering and collecting the filtrate after soaking, and performing reduced pressure distillation to obtain a pigeon pea ethanol extract, suspending the ethanol extract in water, extracting with n-hexane and ethyl acetate, and concentrating the ethyl acetate part by reduced pressure distillation to obtain an ethyl acetate part extract;

[0021] 2) The ethyl acetate extract was mixed with 60-100 mesh silica gel and subjected to normal phase silica gel column chromatography, with n-hexane / ethyl acetate (v / v 100:0→0:1) as the mobile phase for gradient elution. Based on the TLC results, the components with similar results were combined and dried by spin drying to obtain 11 components F. 1-11 Component F 10 After TLC detection, with n-hexane-ethyl acetate 3:1 as the developing solvent and vanillin concentrated sulfuric acid as the color developing agent, multiple spots such as red, yellow, dark green and purple-gray cone tails were visible; methanol / water system (40%→100%, v / v) and methanol / acetone system (v / v 20:1→0:1) were used for reverse phase silica gel RP C 18 Gradient elution, according to the TLC results, the components with similar results were combined and dried, and a total of 9 components F were obtained. 10-1 -F 10-9 , ; F 10-3 With n-hexane-ethyl acetate 3:1 as the developing solvent and vanillin concentrated sulfuric acid as the color developing agent, red, yellow, dark green and other spots can be seen; F was obtained by Sephadex LH-20 gel column chromatography (CHCl3 / MeOH v / v 1:1). 10-3-1 -F 10-3-7 A total of 7 components; F 10-3-5 After TLC detection, three spots were visible under 254nm ultraviolet light with n-hexane-ethyl acetate 3:1 as the developing solvent, and two spots of yellow and dark green were visible with vanillin concentrated sulfuric acid as the color developing agent. F was purified by normal phase silica gel column with n-hexane-ethyl acetate 3:1 (v / v) system. 10-3-5-1 -F 10-3-5-3 ; F 10-3-5-3 After TLC detection, a dark green spot (Rf = 0.36) was observed with n-hexane-ethyl acetate 3:1 as the developing solvent and concentrated sulfuric acid of vanillin as the color developing agent. The product was further purified on RP-HPLC with 50% acetonitrile-water (v / v) as the mobile phase at a flow rate of 2 mL / min at t R =19.5min peak, compound caesalpinone B was obtained;

[0022] F 10-4After TLC detection, a circular spot and a conical tailing spot were visible under 254nm ultraviolet light with n-hexane-ethyl acetate 2:1 as the developing solvent, and a distinct purple-grey spot (Rf=0.38) was visible with concentrated sulfuric acid of vanillin as the color developing agent; after TLC detection with n-hexane-ethyl acetate 2:1 as the developing agent, purple-grey spots were displayed after the color development with concentrated sulfuric acid of vanillin and no other miscellaneous spots were used as indicators, and the product was purified by Sephadex LH-20 gel column chromatography (CHCl3 / MeOH v / v 1:3) and normal phase silica gel column chromatography (n-hexane / ethyl acetate v / v 3:1), and then eluted by RP-HPLC with 30% acetonitrile-water (v / v) as the mobile phase at a flow rate of 2mL / min at t R =20.5min peak was obtained, and compound caesalpinone C was obtained.

[0023] The beneficial effects of the present invention are as follows:

[0024] 1. The present invention has found that the pigeon pea extract and its active compounds pigeon pea ketone B and pigeon pea ketone C have significant lipid-lowering effects. The pigeon pea extract and its active compounds pigeon pea ketone B and pigeon pea ketone C can significantly reduce the lipogenesis and lipolysis of 3T3-L1 cells, and therefore have good development and application prospects in the preparation of lipid-lowering drugs.

[0025] 2. The pigeon pea extract of the present invention is derived from natural plants, and the active compounds pigeon pea ketone B and pigeon pea ketone C are natural small molecule compounds with low toxicity and few side effects. Description of the drawings:

[0026] Figure 1 The cytotoxicity of the compounds CAB and CAC in Example 2 on 3T3-L1 preadipocytes.

[0027] Figure 2 The effects of the compounds CAB and CAC on lipid accumulation, TG and FFA release in Example 2, (a) cells were stained with DAPI / BODIPY and photographed with a Bio Tek Cytation 5 cell ion imager, scale bar 1000 μm; (b) the inhibitory effect of CAB and CAC on total lipids was analyzed using Gen5Image+3.10; (c) the inhibitory effect of CAB and CAC on TG; (d) the inhibitory effect of CAB and CAC on FFA; each value is expressed as mean ± standard deviation (n = 3), ### P<0.001, compared with the results of the preadipocyte (UD) group; *** P < 0.001, ** P < 0.01 and * P<0.05, compared with the results of the adipocyte group without compound treatment (Control).

[0028] Figure 3 The effects of the compounds CAB and CAC in Example 3 on the mRNA expressions of HSL, ATGL, PPARγ and C / EBPα; each value is expressed as mean ± standard deviation (n = 3), *** P < 0.001, ** P < 0.01, compared with the adipocyte group without compound treatment (Control); ns, not statistically significant.

[0029] Figure 4 The effects of the compounds CAB and CAC in Example 3 on the mRNA expressions of ACC, FAS and SREBP-1c; each value is expressed as mean ± standard deviation (n = 3); *** P < 0.001, ** P < 0.01, compared with the adipocyte group without compound treatment (Control); ns, not statistically significant.

[0030] Figure 5 The possible binding modes of the ligand and PPARγ in Example 3, (left) ligand interaction, (right) secondary structure.

[0031] Figure 6 The effects of the compounds CAB and CAC in Example 3 on the accumulation of lipids in cells after ROS treatment were shown. The cells were stained with DAPI / BODIPY and photographed with a Bio Tek Cytation 5 cell ion imager. The scale bar is 1000 μm. (a) Staining of the preadipocyte (UD) group, the induced control group (Control) and the ROS group; (b) Staining of the CAB-treated group; (c) Staining of the CAC-treated group; (d) The inhibitory effects of CAB and CAC on total lipids were analyzed using Gen5 Image+3.10; each value is expressed as mean ± standard deviation (n = 3); ### P<0.001, compared with the results of the preadipocyte (UD) group; *** P<0.001 compared with the results of ROS group.

[0032] Figure 7 The pigeon pea leaf raw material for preparing the pigeon pea extract in Example 1. Specific implementation method:

[0033] It will be appreciated by those skilled in the art that the disclosed techniques in the following examples represent techniques that the inventors have found to work well in the practice of the present invention. However, many changes may be made in the disclosed specific embodiments and still achieve the same or similar results without departing from the spirit and scope of the present invention.

[0034] Example 1 Preparation of Pigeon Bean Extract and Its Active Compounds

[0035] 1. Plant Materials

[0036] The research material, pigeon pea, was collected from Honghe Prefecture, Yunnan Province and Limu Mountain, Qiongzhong County, Hainan Province, and was identified as pigeon pea [Cajanus cajan (L.) Millsp] by Professor Deng Yunfei of the South China Botanical Garden.

[0037] 2. Extraction and separation

[0038] 2.1 Preparation of pigeon pea extract

[0039] Take 30kg of dried pigeon pea leaves, crush them, and put them into 95% (v / v) ethanol aqueous solution according to the solid-liquid ratio of 3g / 10ml for three soaking extractions, the first soaking is 72h, and the filtrate is collected after filtering. The filter residue is soaked again for 48h according to the above steps, and the filtrate is collected after filtering. The filter residue is soaked for the third time, the soaking time is 24h, and the filtrate is collected after soaking. After the filtrate is combined, it is distilled under reduced pressure to obtain an ethanol extract, the ethanol extract is suspended in water, extracted with n-hexane and ethyl acetate, and the ethyl acetate part is concentrated by reduced pressure distillation to obtain the ethyl acetate part extract, which is the pigeon pea extract.

[0040] 2.2 Separation of caesalpinone B and caesalpinone C

[0041] The ethyl acetate extract of pigeon pea was mixed with 60-100 mesh silica gel in a volume ratio of 1:1. After the solvent evaporated, it was subjected to normal phase silica gel column chromatography with n-hexane / ethyl acetate (v / v 100:0→0:1) as the mobile phase for gradient elution. According to the TLC results, the components with similar results were combined and dried by spin drying to obtain 11 components F in total. 1-11 .

[0042] Component F 10 (39.8 g) was detected by TLC, with n-hexane-ethyl acetate 3:1 as the developing solvent and vanillin concentrated sulfuric acid as the color developing agent, and multiple spots such as red, yellow, dark green and purple-gray cone tails were visible. The product was purified by reverse phase silica gel RP C using methanol / water system (40%→100%, v / v) and methanol / acetone system (v / v 20:1→0:1). 18 Gradient elution, according to the TLC results, the components with similar results were combined and dried, and a total of 9 components F were obtained. 10-1 -F 10-9 ,. F 10-3 (0.8 g) was developed with n-hexane-ethyl acetate 3:1 and concentrated vanillin sulfuric acid as a color developer, and red, yellow, dark green and other spots were visible. F was obtained by Sephadex LH-20 gel column chromatography (CHCl3 / MeOH v / v 1:1). 10-3-1 -F 10-3-7 There are 7 components in total. 10-3-5(54.3 mg) was detected by TLC. With n-hexane-ethyl acetate 3:1 as the developing solvent, three spots were visible under 254 nm ultraviolet light. With vanillin concentrated sulfuric acid as the color developing agent, two spots, yellow and dark green, were visible. F was purified by normal phase silica gel column with n-hexane-ethyl acetate 3:1 (v / v) system. 10-3-5-1 -F 10-3-5-3 . F 10-3-5-3 (15.9 mg) was detected by TLC, using n-hexane-ethyl acetate 3:1 as the developing solvent and concentrated sulfuric acid of vanillin as the color developing agent, and a dark green spot (Rf=0.36) was observed. 18 ) was further purified using 50% acetonitrile-water (v / v) as the mobile phase at a flow rate of 2 mL / min. R =19.5min peak, compound Cajanotone B (CAB) (3.0 mg) was obtained. 10-4 (0.5 g) was detected by TLC, with n-hexane-ethyl acetate 2:1 as the developing solvent, and a circular spot and a conical tailing spot were visible under 254 nm ultraviolet light. With vanillin concentrated sulfuric acid as the color developing agent, an obvious purple-gray spot (Rf = 0.38) was visible. With n-hexane-ethyl acetate 2:1 as the developing solvent, the purple-gray spot after vanillin concentrated sulfuric acid development and no other miscellaneous spots were used as indicators, and it was purified by dextran Sephadex LH-20 gel column chromatography (CHCl3 / MeOH v / v 1:3), normal phase silica gel column chromatography (n-hexane / ethyl acetate v / v 3:1), and then RP-HPLC (YMC-Pack C) with 30% acetonitrile-water (v / v) as the mobile phase 18 ) with a flow rate of 2 mL / min and at t R =20.5min peak was obtained, and compound Cajanotone C (CAC) (3.0 mg) was obtained.

[0043] 3. Spectral data of main compounds

[0044] Table 1 Proton spectrum (500MHz) and carbon spectrum (125MHz) data of compounds CAB and CAC

[0045]

[0046]

[0047] Note: The unit of chemical shift is ppm; the solvent used for the compounds in the table is deuterated chloroform.

[0048] Cajanotone B (CAB), molecular formula: C 19 H 20O3; yellow powder. UV(MeOH)λ max / nm(logε):202(2.97),238(2.35),280(1.87);IRν max :3354, 2926, 1596, 1448, 1211, 1139, 1013, 752, 686, 552cm -1 ; 1 H(500MHz) and 13 C (125MHz) NMR data, see Table 1; ESIMS m / z 314 [M+H] + ; HRESIMS m / z 319.1307[M+Na] + (C 19 H 20 NaO3, calculated value is 319.1310).

[0049] Cajanotone C (CAC), molecular formula: C 20 H 22 O4; yellow powder. UV(MeOH)λ max / nm(logε):202(3.30),238(2.69),282(2.12);IRν max :3325, 2947, 2837, 1456, 1018, 689, 592, 574, 565, 556, 529cm -1 ; 1 H(500MHz) and 13 C (125MHz) NMR data, see Table 1; ESIMS m / z 327 [M+H] + ; HRESIMS m / z 327.1590[M+H] + (for C 20 H 23 O4, calculated value: 327.1596).

[0050] The structural formulas of caesalpinone B and caesalpinone C are shown below:

[0051]

[0052] Example 2: Pigeonolone B and Pigeonolone C have significant lipid-lowering activity

[0053] 1. Experimental Materials

[0054] CCK-8 kit was purchased from Glpbio, USA; firefly luciferase reporter gene detection kit (catalog number: RG005) was purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; NCS newborn calf serum was purchased from Hangzhou Sijiqing Bioengineering Research Institute; insulin (catalog number: I2643) and 0.5mM isobutylmethylxanthine (catalog number: I5879) were purchased from Sigma platform; dexamethasone (catalog number: B25793) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; DMEM culture medium was purchased from Gibco, USA; fetal bovine serum (Fetal bovine serum, FBS) was purchased from Hangzhou Sijiqing Bioengineering Research Institute; trypsin was purchased from Amersco, USA; Penicillin / Streptomycin (Penicillin / Streptomycin, P / S) was purchased from Hyclone, USA; phosphate buffer saline (Phosphate buffer saline, PBS) was purchased from Wuhan Boster Company; experimental water was provided by the pure water and ultrapure water system of Millipore, USA, and all chemical reagents were analytical grade.

[0055] 2. Cell Culture and Differentiation

[0056] 3T3-L1 preadipocytes are from the Shanghai Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, and are cultured in a high-glucose DMEM medium containing 10% NCS and 1% P / S, at 37°C and 5% CO2. DMEM medium containing 10% fetal bovine serum (FBS), 1% P / S, 10 μg / mL insulin, 0.5 mM isobutylmethylxanthine and 1 μM dexamethasone is used to induce 3T3-L1 preadipocyte differentiation (day 0). After 48 hours (day 2), the differentiation medium is removed and replaced with DMEM containing 10% FBS and 10 μg / mL insulin. On the 4th day, the DMEM medium containing 10% FBS is replaced. Subsequently, the medium is replaced with DMEM containing 10% FBS and 1% P / S on days 6, 8 and 10, respectively.

[0057] 3. Cytotoxicity Assay

[0058] The CCK-8 kit (Cell Counting Kit-8) was used to detect the cytotoxicity of compounds CAB and CAC on 3T3-L1, and the experiment was completed according to the instructions of the kit. Briefly, the cells were first digested with trypsin, and the appropriate cell concentration was adjusted with a culture medium containing 10% FBS and inoculated into a 96-well plate, and cultured in a 37°C, 5% CO2 incubator for 24 hours. The culture medium was discarded, and the compounds CAB, CAC and rosiglitazone (ROS) (concentrations of 5, 10, 20, and 30 μM, respectively) diluted continuously with a culture medium containing 2% FBS were added and cultured for 48 hours; the adipocyte group not treated with the compound was used as the control group (Control). The culture medium was discarded, 10% CCK-8 reagent was added and placed in the dark for about 45 minutes, the absorbance at 450nm was detected and the CC was calculated. 50 The results are shown in Figure 1 .

[0059] 4. Cell fluorescence staining experiment

[0060] Lipid accumulation is the main hallmark of adipogenesis in 3T3-L1 adipocytes. To investigate whether compounds CAB and CAC would inhibit adipocyte differentiation, different concentrations (5-20 μM) of compounds CAB and CAC were added to the culture medium on day 0. After culture and differentiation (the same steps as 2. Cell culture and differentiation), 3T3-L1 adipocytes were stained with DAPI / BODIPY on day 10 to evaluate the accumulation of lipid droplets. The adipocyte group not treated with the compound was used as the control group (Control), and the undifferentiated preadipocytes were used as the blank group (UD).

[0061] The staining steps were as follows: mature 3T3-L1 adipocytes were washed with phosphate buffered saline (PBS) and then fixed with 4% paraformaldehyde for 20 minutes at room temperature. After three rinses with PBS, they were stained with BODIPY for 20 minutes, then stained with DAPI for 10 minutes, and finally rinsed with PBS (3×5 minutes). The stained adipocytes were observed using a Bio Tek Cytation 5 ion imager (Agilent, California, USA), and image statistics and analysis of total lipids were performed using Gen5 Image+3.10.

[0062] 5. Triglyceride content detection

[0063] On day 0, different concentrations (5-20 μM) of compounds CAB and CAC were added to the culture medium. After culture and differentiation (same as 2. Cell culture and differentiation), on day 11, a triglyceride (TG) assay kit (Nanjing Jiancheng Bioengineering Institute, catalog number: A110-1-1) was used to determine the triglyceride content in the culture supernatant according to the kit instructions; the adipocyte group not treated with the compound was used as the control (Control).

[0064] 6. Free fatty acid content detection

[0065] On day 0, different concentrations (5-20 μM) of compounds CAB and CAC were added to the culture medium. After culture and differentiation (same as 2. Cell culture and differentiation), on day 11, an FFA assay kit (Nanjing Jiancheng Bioengineering Institute, catalog number: A042-2-1) was used to measure the content of free fatty acid (FFA) released into the culture medium according to the kit instructions; the adipocyte group not treated with the compound was used as the control (Control).

[0066] The results of total lipid, triglyceride and free fatty acid content are shown in Figure 2 .

[0067] 7. Experimental results and analysis

[0068] In order to detect the cytotoxicity of CAB and CAC on 3T3-L1 adipocytes, cell viability was determined using the Cell Counting Kit-8. The results showed that CAB and CAC had no significant cytotoxicity to 3T3-L1 adipocytes at the test concentrations (5, 10, 20, 30 μM) used in this study. Figure 1 ).

[0069] like Figure 2 As shown in a, lipid droplets were almost invisible in the undifferentiated group, while the mature adipocytes induced to differentiate in the control group were highly stained with the lipophilic fluorescent dye BODIPY, showing obvious lipid accumulation. In contrast, oil droplet formation was reduced in the CAB and CAC groups, and lipid droplets were reduced in a compound dose-dependent manner. Compared with CAB, CAC can more effectively inhibit the differentiation and lipid accumulation of 3T3-L1 preadipocytes. When 3T3-L1 cells were treated with CAC at concentrations of 10μM and 20μM, total lipids were reduced to 54.5% and 43.4% of the control group, respectively ( Figure 2 b).

[0070] This inference was further confirmed by triglyceride content analysis. The triglyceride content in 3T3-L1 adipocytes treated with compounds CAB and CAC was significantly lower than that in the control group. In addition, the inhibitory effect of the compounds on triglyceride accumulation during adipogenesis was dose-dependent, with the triglyceride levels in the 10μM CAC and 20μM CAC treatment groups falling to 55.6% and 48.8% of the control group, respectively ( Figure 2 c).

[0071] Compared with mature adipocytes in the control group, free fatty acids (FFA) secreted into the culture medium by the CAB and CAC treatment groups were significantly reduced. After treatment with CAC at doses of 5μM, 10μM, and 20μM, free fatty acid levels were significantly reduced: 30.1%, 22.8%, and 17.5% of the control group, respectively, while CAB showed a similar but weaker effect, indicating that the CAB and CAC compounds inhibited lipolysis ( Figure 2 d).

[0072] Example 3 Study on the mechanism of lipid-lowering activity of caesalpinol B and caesalpinol C

[0073] 1. Molecular docking

[0074] First, the protein structure of PPARγ (PDBID: 1FM6) was downloaded from the PDB database (https: / / www.rcsb.org / ). The drawn CAB and CAC structures were saved in the "SDF" format using ChemOffice 2021 software. First, we confirmed the skeleton of the conserved region after removing irrelevant ligands in the template chain and aligning the target sequence with the template sequence. Multiple transition models were constructed by arranging and combining candidate loop regions and side chain rotamers, and these models were initially energy minimized using the AMBER 10:EHT force field and implicit solvent model, and then evaluated using the GB / VI score. After further energy optimization, the top-ranked model was selected as the final model. After preparing the protein and ligand, docking began, and the Molecular Operating Environment (MOE 2019.0102) software was applied for docking. The docking process followed the induced fit mode, in which the active pocket side chains can be automatically adjusted according to the ligand conformation. The weight for restricting side chain rotation was set to 10. Finally, the binding modes and interactions between the compounds and target proteins were analyzed using Pymol software (www.pymol.org).

[0075] 2. Real-time Quantitative PCR Analysis

[0076] The cell culture medium was removed and washed twice with PBS, and the cells of each group cultured as in Example 2 were collected with a cell scraper, and total RNA was extracted using RNAEx ZOL reagent according to the manufacturer's specifications. The RNA yield was determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA), and the integrity of the RNA was assessed by ethidium bromide-stained agarose gel electrophoresis.

[0077] Quantification was performed using a two-step reaction process: reverse transcription (RT) and PCR. Each RT reaction included 0.5 μg RNA, 2 μL 5× TransScript All-in-one SuperMix for qPCR, and 0.5 μL gDNA Remover in a total volume of 10 μL. PCR System 9700 (Applied Biosystems, USA) was performed at 42°C for 15 minutes and 85°C for 5 seconds. Then 10 μL of the RT reaction mixture was diluted 10-fold with nuclease-free water and stored at -20°C.

[0078] use Real-time PCR was performed using a 480Ⅱ Real-time PCR instrument (Roche, Switzerland). The PCR reaction mixture was 10 μL, including 1 μL cDNA, 5 μL 2×PerfectStartTM Green qPCR SuperMix, 0.2 μL forward primer, 0.2 μL reverse primer, and 3.6 μL nuclease-free water. The reaction was incubated at 94°C for 30 seconds in a 384-well optical plate (Roche, Switzerland), followed by 45 cycles of 94°C for 5 seconds and 60°C for 30 seconds. After the PCR cycle, a melting curve analysis was performed to verify the specifications of the expected PCR product. The primer sequences were designed based on the mRNA sequences obtained from the NCBI database (Table 2) and synthesized by Qingke Biotechnology. The expression level of mRNA was based on ACTB and 2 -ΔΔCt Method calculation.

[0079] Table 2 Primer sequences used for qPCR

[0080]

[0081] 3. Experimental results and analysis

[0082] To determine the molecular mechanism by which CAB and CAC inhibit adipogenesis, the effects of these two compounds on the expression of genes involved in lipid biosynthesis and degradation were examined. Figure 3As shown, the results of qRT-PCR analysis showed that CAC inhibited the mRNA expression of HSL and ATGL. In particular, for hormone-sensitive lipase HSL, the inhibition rates of CAC at 5μM, 10μM and 20μM concentrations were 56.1%, 65.5% and 69.3%, respectively. CAB treatment also reduced the expression of HSL and ATGL, but its effect was weaker than that of CAC. However, even at the highest concentration of 20μM compound treatment, CAB and CAC seemed to have little effect on the expression of ACC and FAS ( Figure 3 ).

[0083] To further elucidate the mechanism by which CAB and CAC inhibit preadipocyte differentiation, the mRNA expression of several transcription factors involved in adipocyte differentiation was evaluated. After treatment with 5μM, 10μM, and 20μM CAC, the expression of SREBP-1c was 82.6%, 76.4%, and 63.5% of that in the control group, respectively, while the expression of PPARγ was 50.0%, 43.6%, and 39.4% of that in the control group, respectively, and the expression of C / EBPα was 31.9%, 29.2%, and 24.6% of that in the control group, respectively. Figure 4 ). The effect of CAB was similar to that of CAC, but weaker. In conclusion, the mRNA expression of PPARγ and C / EBPα decreased mainly in a dose-dependent manner after CAB and CAC treatment, suggesting that the compounds may inhibit adipogenesis by regulating the expression of key adipose transcription factors.

[0084] Molecular docking was used to predict the possible binding mode of compounds to PPARγ. The results showed that the known PPARγ agonist rosiglitazone (ROS) has a high affinity for PPARγ, with a docking score of -8.09 kcal / mol (Table 3). In the secondary structure, it can be seen that there are multiple intermolecular forces between ROS and PPARγ. ROS forms three hydrogen bonds with the amino acid residues His449, His 323 and Ser 289 of the protein, and has an electrostatic interaction with residue His 323. In addition, the pyridine ring of ROS also participates in the Pi-H conjugation on the hydrogen atoms of residues Ile 34 and Ser 342 ( Figure 5 a).

[0085] The docking results of PPARγ with CAB and CAC showed that the docking scores of the ligand and the receptor were -6.81 and -7.64 kcal / mol, respectively (Table 3). Both CAB and CAC interact with PPARγ through van der Waals forces and electrostatic interactions. However, compared with CAB, CAC has more types of forces with PPARγ and a lower docking score, which may be one of the reasons why the effect of CAC is significantly stronger than that of CAB. As for CAB, the hydrogen atom of the Leu 340 amino acid residue forms a hydrogen bond with the oxygen atom of its C-3 hydroxyl group. There is also a Pi-H conjugation between the b-ring of CAB and the Cys 285 and Tyr 327 residues ( Figure 5 b). In contrast, there are not only hydrogen bonds and Pi-H conjugation between CAC and PPARγ, but also Pi-cation interactions. The hydrogen bond is generated by the dipole interaction between the oxygen atom of Cys 285 and the hydrogen of the hydroxyl group attached to CAC. In addition, the a-ring of CAC generates Pi-H conjugation with Cys 285 and Tyr 327 residues, and its b-ring generates Pi-cation interaction with the amino acid residue Arg 288 ( Figure 5 c).

[0086] Table 3 Docking scores of drug molecules and PPARγ

[0087]

[0088] To further determine the inhibitory effects of CAB and CAC on PPARγ, ROS were added to the differentiation medium and co-cultured with the test compounds at different concentrations (5-20 μM). Figure 6 It can be seen that the formation of lipid droplets in the ROS group increased significantly, indicating that ROS effectively promoted the differentiation of adipocytes. Compared with the control group, the total lipids in the ROS group increased by 138.3%. In contrast, when 3T3-L1 cells were co-treated with CAB and CAC with ROS, respectively, cellular lipid accumulation decreased in a dose-dependent manner. When the dose of CAB and CAC was 10 μM, the total lipids were reduced to 88.8% and 87.2% of the control group, respectively. When the dose of CAB and CAC was increased to 20 μM, the inhibition rates of total lipids were 79.6% and 77.7%, respectively, indicating that CAB and CAC can eliminate the promoting effect of PPARγ agonist ROS on adipogenesis.

[0089] In summary, the pigeon pea extract and its active compounds have significant lipid-lowering effects and can significantly reduce the adipogenesis and lipolysis of 3T3-L1 cells. The pigeon pea extract is derived from natural plants, and its active compounds are natural small molecule compounds with low toxicity and few side effects, which have good development and application prospects.

[0090] The above detailed description is a specific description of one feasible embodiment of the present invention. The embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not deviate from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. Psoralen B or Psoralen C, the structural formula is as follows:

2. Use of pigeon pea ketone B, pigeon pea ketone C or their pharmaceutically acceptable salts or stereoisomers or prodrug molecules and pigeon pea extracts as claimed in claim 1 in the preparation of lipid-lowering, blood sugar-lowering and weight-loss drugs or health products.

3. The use according to claim 2, characterized in that: The pigeon pea extract comprises pigeon pea root extract, pigeon pea stem extract and pigeon pea leaf extract.

4. The use according to claim 3, characterized in that: The pigeon pea extract is a pigeon pea leaf extract.

5. The use according to claim 2, characterized in that: The preparation method of the pigeon pea extract comprises the following steps: Take dried pigeon pea roots, pigeon pea stems or pigeon pea leaves, grind them and put them into ethanol aqueous solution for soaking extraction, filter and collect the filtrate after soaking, and perform vacuum distillation to obtain pigeon pea ethanol extract, suspend the ethanol extract in water, extract with n-hexane and ethyl acetate, and concentrate the ethyl acetate part by vacuum distillation to obtain the ethyl acetate part extract, which is the pigeon pea extract.

6. The use according to claim 5, characterized in that: The material-liquid ratio of the crushed pigeon pea root, pigeon pea stem or pigeon pea leaf to the ethanol aqueous solution is 3g / 10ml; the volume percentage concentration of the ethanol aqueous solution is 95%.

7. The use according to claim 5, characterized in that: The soaking extraction is performed three times, with the first soaking for 72 hours. After the soaking is completed, the filtrate is filtered and collected. The filter residue is soaked again for 48 hours, and the filtrate is collected after filtering. The filter residue is soaked for the third time for 24 hours, and the filtrate is filtered and collected after soaking. The filtrates after the three soaking and filtering are combined and distilled under reduced pressure.

8. The use according to claim 1, characterized in that: The health care product is a functional health care product beneficial to obese patients or a functional health care product beneficial to diabetic patients; and medically acceptable auxiliary materials are added to the medicine.

9. A lipid-lowering, blood sugar-lowering and weight-loss medicine or health product, characterized in that: Containing pigeon pea ketone B, pigeon pea ketone C or pharmaceutically acceptable salts thereof or stereoisomers thereof or prodrug molecules thereof as claimed in claim 1 and pigeon pea extract.

10. A method for preparing pigeon pea ketone B and pigeon pea ketone C in claim 1, which are prepared and separated from pigeon pea leaves, preferably, the specific steps are as follows: 1) taking dried pigeon pea leaves, crushing them and putting them into an ethanol aqueous solution for soaking and extraction, filtering and collecting the filtrate after soaking, and performing reduced pressure distillation to obtain a pigeon pea ethanol extract, suspending the ethanol extract in water, extracting with n-hexane and ethyl acetate, and concentrating the ethyl acetate part by reduced pressure distillation to obtain an ethyl acetate part extract; 2) The ethyl acetate extract was mixed with 60-100 mesh silica gel and then subjected to normal phase silica gel column chromatography, using n-hexane / ethyl acetate as the mobile phase at a volume ratio of 100:0→0:1 for gradient elution. Based on the TLC results, the components with similar results were combined and dried in a spin-drying process to obtain 11 components F. 1-11 Component F 10 After TLC detection, with n-hexane-ethyl acetate 3:1 as the developing solvent and vanillin concentrated sulfuric acid as the color developing agent, multiple spots such as red, yellow, dark green and purple-gray cone tails were visible; methanol / water system at a volume ratio of 40%→100% and methanol / acetone system at a volume ratio of 20:1→0:1 were used for reverse phase silica gel RP C 18 Gradient elution, according to the TLC results, the components with similar results were combined and dried, and a total of 9 components F were obtained. 10-1 -F 10-9， ; F 10-3 With n-hexane-ethyl acetate 3:1 as the developing solvent and vanillin concentrated sulfuric acid as the color developing agent, red, yellow, dark green and other spots can be seen; F was obtained by Sephadex LH-20 gel column chromatography with chloroform / methanol at a volume ratio of 1:

1. 10-3-1 -F 10-3-7 A total of 7 components; F 10-3-5 After TLC detection, three spots were visible under 254nm ultraviolet light with n-hexane-ethyl acetate 3:1 as the developing solvent, and two spots of yellow and dark green were visible with vanillin concentrated sulfuric acid as the color developer. F was purified by normal phase silica gel column with n-hexane-ethyl acetate system in a volume ratio of 3:

1. 10-3-5-1 -F 10-3-5-3 ; F 10-3-5-3 After TLC detection, dark green spots were observed with Rf = 0.36 using n-hexane-ethyl acetate 3:1 as the developing solvent and concentrated sulfuric acid of vanillin as the color developing agent. It was further purified on RP-HPLC using acetonitrile-water system with a volume ratio of 50% as the mobile phase at a flow rate of 2 mL / min at t R =19.5min peak, compound caesalpinone B was obtained; F 10-4 After TLC detection, with n-hexane-ethyl acetate 2:1 as the developing solvent, a circular spot and a conical tailing spot can be seen under 254nm ultraviolet light irradiation, and with vanillin concentrated sulfuric acid as the color developer, an obvious purple-gray spot can be seen, Rf=0.38; with n-hexane-ethyl acetate 2:1 as the developing solvent, the purple-gray spot after vanillin concentrated sulfuric acid color development and no other miscellaneous spots are used as indicators for TLC detection, and the product is purified by dextran Sephadex LH-20 gel column chromatography with a chloroform / methanol system in a volume ratio of 1:3, and by normal phase silica gel column chromatography with a n-hexane / ethyl acetate system in a volume ratio of 3:1, and then eluted by RP-HPLC with an acetonitrile-water system in a volume ratio of 30% as the mobile phase, the flow rate is 2mL / min, and the product is purified by t R =20.5min peak was obtained, and compound caesalpinone C was obtained.

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