Preparation method of spirostanol saponin and application of spirostanol saponin in aspect of losing weight

Abstract

The invention discloses a method for extracting, separating and purifying spirostanol saponin compounds from small root garlic, namely, 3-O-{O-beta-D-glucopyranosyl-(1-> 2)-O-[beta-D-glucopyranosyl-(1-> 3)]-O-beta-D-glucopyranosyl-(1-> 4)-beta-D-glucopyranosyl}, and an application of the spirostanol saponin compounds in the aspect of preparing weight-losing products. Fresh small root garlic is extracted with water, an extracting solution is adsorbed by macroporous adsorption resin and eluted by an ethanol solution, the obtained extract is separated and purified through silica gel column chromatography and preparative high performance liquid chromatography, and the obtained extract has a remarkable inhibition effect on lipase and can be used for preparing food, health food and medicine with the weight losing effect.

Description

Technical Field

[0001] The invention relates to a preparation method for extracting and separating spirosteroidal saponin monomer compounds from garlic root and application thereof in weight loss, belonging to the research field of natural drug chemistry. Background Art

[0002] Allium macrostemon Bge. is a perennial herbaceous plant of the genus Allium in the Liliaceae family. It is one of the basal plants of the Chinese medicine Allii macrostemonis bulbus. Allium macrostemonis bulbus is widely distributed in the northern and northeastern regions of my country. It is rich in resources and is a typical medicinal and edible plant. Its dried bulb is the commonly used Chinese medicine Allii macrostemonis bulbus. Allii macrostemonis bulbus was first recorded in the Shennong's Herbal Classic and is listed as a top-grade herbal medicine. It has the effects of promoting yang and dispersing nodules, promoting qi and removing stagnation. It is widely used in the clinical treatment of chest obstruction and heart failure.

[0003] Allium tuberosum contains a variety of active ingredients, including steroidal saponin compounds, alkaloid compounds, polysaccharide compounds, volatile oils, fatty acids, amino acids and other chemical components. Among them, steroidal saponin is the main active ingredient of allium tuberosum, mainly including furostanols, spirostanols, isospirostanols and deformed spirostanols.

[0004] The chemical components in garlic and Allium macrostemon have pharmacological effects such as inhibiting platelet aggregation, antibacterial and analgesic, antispasmodic and antiasthmatic, anti-cancer, anti-oxidant, lowering blood sugar and blood lipids, and preventing and treating atherosclerosis. Allium macrostemon is often used in combination with other medicinal materials as a traditional Chinese medicine to treat heart disease, heart failure, myocardial infarction, hyperlipidemia, irritable bowel syndrome, endocrine disorders and other diseases.

[0005] Obesity has always been an important health issue that has received widespread attention worldwide. It is often closely related to some diseases, such as cardiovascular and cerebrovascular diseases, diabetes, gout, musculoskeletal diseases and certain cancers. Reducing the absorption of lipids from dietary fat is one of the strategies for preventing and treating obesity. Pancreatic lipase is an enzyme responsible for breaking down fat in the human digestive tract. Finding pancreatic lipase inhibitors is one of the important means to develop weight loss products. There are two major types of weight loss drugs on the market, pancreatic lipase inhibitors and appetite suppressants that act on the central nervous system. Orlistat is currently a widely used pancreatic lipase inhibitor, which can inhibit the activity of pancreatic lipase and then inhibit the decomposition and absorption of fat in food to achieve weight loss effects. Although orlistat has a good weight loss effect, it is not suitable for long-term use because of certain side effects. Appetite suppressant weight loss drugs are also restricted due to adverse reactions in the nervous system. Therefore, it is still of great significance to continue to develop new weight loss drugs with good efficacy and few side effects. Summary of the invention

[0006] The invention discloses a method for extracting and separating a spirosteroidal saponin monomer compound from garlic root, and studies the application of the monomer in weight loss.

[0007] The specific technical solutions and research results of the present invention are as follows:

[0008] 1. Extraction, separation and structural identification

[0009] Take fresh garlic cloves, wash them, crush them with a wall-breaking machine, add water, stir and extract, centrifuge, pass the filtrate through a macroporous adsorption resin column for adsorption, elute with 85% ethanol solution, and recover the solvent of the ethanol eluate under reduced pressure to obtain an extract. The extract was subjected to a first silica gel column chromatography with a mixed solution of ethyl acetate-ethanol-water in a volume ratio of 11:0.4:0.24 to obtain component A containing 3-O-{O-β-D-glucopyranosyl-(1→2)-O-[β-D-glucopyranosyl-(1→3)]-O-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside} (hereinafter referred to as spirosteroid saponin M). Component A was subjected to a second silica gel column chromatography with a mixed solution of dichloromethane-methanol-water in a volume ratio of 21:3:0.3 as the mobile phase to obtain component B containing spirosteroid saponin M. Finally, an amide chromatography column using ultrapure silica gel bonded with neutral amide as a filler and acetonitrile-water (89:11, V / V) as the mobile phase was used to separate and purify the spirosteroid saponin M monomer by preparative high performance liquid chromatography. The obtained spirosteroidal saponin M monomer compound was detected by HPLC-ELSD (HPLC chromatographic conditions: chromatograph: Agilent 1200HPLC; chromatographic column: AlphasilXD-Amide (5μm, 100A, 4.6×250mm); mobile phase: acetonitrile-water (89:11, V / V); flow rate: 1.0mL / min; column temperature: 30°C; injection volume: 20μL), and its purity was 96.5%. The NMR data of the isolated spirosteroidal saponin M monomer compound were compared with the NMR data of 3-O-{O-β-D-glucopyranosyl-(1→2)-O-[β-D-glucopyranosyl-(1→3)]-O-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside} reported in the literature. The two were consistent, and spirosteroidal saponin M was finally determined to be 3-O-{O-β-D-glucopyranosyl-(1→2)-O-[β-D-glucopyranosyl-(1→3)]-O-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside}.

[0010] Table 1 13C-NMR (150MHZ) nuclear magnetic data (δ, ppm, C5D5N) of spirosteroidal saponin M

[0011]

[0012]

[0013] 2. Inhibitory effect of spirosterol saponin M on pancreatic lipase activity

[0014] The present invention adopts an in vitro model experiment (p-nitrophenol method) to investigate the inhibitory effect of the spirosteroidal saponin M monomer compound isolated from fresh garlic root on pancreatic lipase activity. p-Nitrophenyl palmitate (pNPP) can be hydrolyzed and converted into p-nitrophenol (pNP) under the action of lipase, and p-nitrophenol (pNP) has an absorbance value at 405nm and has high sensitivity.

[0015] 2.1 Experimental instruments, reagents and samples

[0016] Table 2.1 Experimental instruments

[0017]

[0018] Table 2.2 Reagents and samples

[0019]

[0020] 2.2 Experimental methods

[0021] 2.2.1 Solution preparation

[0022] 2.2.1.1 Preparation of PBS buffer solution with pH = 8.0

[0023] Weigh 1.95 g Na2HPO4·12H2O and add appropriate amount of water to dissolve it, make up to volume in a 250 mL volumetric flask and set aside.

[0024] Weigh 4.48 g NaH2PO4·H2O and add appropriate amount of water to dissolve it, make up to volume in a 250 mL volumetric flask and set aside.

[0025] Take 6.0 mL of the above-mentioned Na2HPO4·12H2O aqueous solution and 94 mL of NaH2PO4·H2O aqueous solution and mix them thoroughly to obtain a PBS buffer solution with a pH of 8.0.

[0026] 2.2.1.2 Preparation of lipase (PPL) solution

[0027] Weigh 12.5 mg of lipase, dissolve it in a small amount of the above-prepared buffer solution with pH=8.0, and make up to volume in a 5 mL volumetric flask with the buffer solution. Let it stand and then centrifuge, and take the supernatant for later use.

[0028] 2.2.1.3 Preparation of p-nitrophenyl palmitate (pNPP) solution

[0029] Weigh 1.0 mg of p-nitrophenyl palmitate, add an appropriate amount of isopropanol to dissolve it, and make up to volume in a 5 mL volumetric flask with isopropanol. Store it in a refrigerator away from light for later use.

[0030] 2.2.1.4 Preparation of sample solution

[0031] Weigh 5 mg of spirosterol saponin M, add an appropriate amount of DMSO reagent to dissolve it, and make up to volume in a 1 mL volumetric flask with DMSO to obtain a sample solution for later use.

[0032] 2.2.1.5 Absorbance determination

[0033] Determination of A1: According to the dosage shown in Table 2.3, add a certain amount of PBS solution, lipase solution and sample solution to the reaction tube, mix thoroughly, incubate at 37°C for 10 minutes, add substrate pNPP, react at 37°C for 15 minutes again, take out, use a pipette to transfer 200μL of reaction solution to a 96-well plate, and use an ELISA reader to measure the absorbance value A1 at 405nm at 37°C. Measure A0, B1 and B0 separately in the same way, and measure each absorbance nine times in parallel and record.

[0034] Table 2.3 Dosage of each solution in the reaction system

[0035]

[0036] 2.2.1.6 Calculation of inhibition rate

[0037] Inhibition rate (%) = [1-(B1-B0) / (A1-A0)]*100%

[0038] A1 is the absorbance value of the reaction system when pNPP and PPL are added but no sample is added;

[0039] A0 is the absorbance value of the reaction system when pNPP is added but PPL and sample are not added;

[0040] B1 is the absorbance value of the reaction system when pNPP, PPL and sample are added;

[0041] B0 is the absorbance value of the reaction system with pNPP and sample added but without PPL. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 .Inhibition curve of pancreatic lipase activity of spirosterol saponin M

[0043] Figure 2 .13C-NMR spectrum of spirosterol saponin M

[0044] Figure 3 .DEPT90,135 of spirosterol saponin M

[0045] Figure 4 .Spirosteroidal saponin M 1 H-NMR graph

[0046] Figure 5 .Structural formula of spirosterol saponin M DETAILED DESCRIPTION

[0047] The preferred embodiments of the present invention are described below. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0048] Example 1

[0049] Take 5 kg of fresh garlic roots, clean them and crush them with a wall breaking machine, add distilled water for extraction, combine the extracts, centrifuge, pass the filtrate through a D101 macroporous adsorption resin column, elute with 85% ethanol, concentrate the eluate under reduced pressure, and dry to obtain 160 g of extract.

[0050] 100 g of the extract was subjected to the first silica gel column chromatography using a mixed solution of ethyl acetate-ethanol-water in a volume ratio of 11:0.4:0.24 as the mobile phase, and TLC detection was performed to obtain component A containing spirosteroidal saponin M. Component A was subjected to a second silica gel column chromatography using a mixed solution of dichloromethane-methanol-water in a volume ratio of 21:3:0.3 as the mobile phase, and TLC detection was performed to obtain component B containing spirosteroidal saponin M. Finally, component B was subjected to amide chromatography column (filler is ultrapure silica gel bonded with neutral amide) using acetonitrile-water (89:11, V / V) as the mobile phase, and was separated and purified by preparative high performance liquid chromatography to obtain 12.5 mg of spirosteroidal saponin M monomer.

[0051] The purity of the separated spirosterol saponin M monomer was 96.5% as determined by HPLC-ELSD.

[0052] Example 2

[0053] The inhibitory effect of spirosterol saponin M on pancreatic lipase activity was determined according to the experimental method given in 2.2.

[0054]

[0055]

[0056] The experimental results showed that spirosteroid saponin M monomer had a significant inhibitory effect on pancreatic lipase activity in a concentration-dependent manner. 50 The value is 28.678μg / ml, and the pancreatic lipase activity inhibition curve is as follows Figure 1 shown.

[0057] In summary, the present invention discloses a preparation method for separating and extracting spirosterol saponin 3-O-{O-β-D-glucopyranosyl-(1→2)-O-[β-D-glucopyranosyl-(1→3)]-O-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside} monomer from garlic root, finds the inhibitory effect of 3-O-{O-β-D-glucopyranosyl-(1→2)-O-[β-D-glucopyranosyl-(1→3)]-O-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside} on pancreatic lipase activity, and points out the application prospect of spirosterol saponin 3-O-{O-β-D-glucopyranosyl-(1→2)-O-[β-D-glucopyranosyl-(1→3)]-O-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside} in preparing foods, health foods and medicines with weight loss effects. It should be explained here that for those skilled in the art, referring to the research process and embodiments disclosed in the present invention, they can still improve and modify the technical solutions recorded in the present invention and the aforementioned embodiments, or replace some of the technical features therein by equivalents. It should be emphasized here that any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A spirosterol saponin, namely 3-O-{O-β-D-glucopyranosyl-(1→2)-O-[β-D-glucopyranosyl-(1→3)]-O-β-D- glucopyranosyl-(1→4)-β-D-galactopyranoside}, characterized in that For the preparation of 3-O-{O-β -D-glucopyranosyl-(1→2)-O-[β-D-glucopyranosyl-(1→3)]-O-β-D-glucopyranosyl-(1→4)-β -D-galactopyranoside} is made from fresh garlic cloves.

2. The preparation method according to claim 1, characterized in that Fresh garlic root was used as raw material, extracted with water, the extract was adsorbed by macroporous adsorption resin, eluted with organic solvent, the eluate was recovered under reduced pressure, and dried to obtain a product containing 3-O-{O-β-D-glucopyranosyl-(1→2)-O-[β The crude extract of fresh garlic root, including β-D-glucopyranosyl-(1→3)]-O-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside}, was separated and purified by silica gel column chromatography and preparative high performance liquid chromatography to obtain 3-O-{O-β-D-glucopyranosyl-(1→2)-O-[β-D-glucopyranosyl-(1→3)]-O-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside}.

3. The preparation method according to claim 2, characterized in that The macroporous adsorption resin is selected from one of AB-8, D101, D4042 and HP-20 or a mixture of two or more of them. The stationary phase of the preparative high performance liquid chromatography is ultrapure silica gel bonded with neutral amide.

4. The preparation method according to claim 2, characterized in that The macroporous adsorption resin eluent is an aqueous solution of an organic solvent with a volume ratio concentration of 20-100%, and the organic solvent is selected from ethanol, methanol, acetone, or a mixed solvent of two or more thereof.

5. The preparation method according to claim 2, characterized in that The mobile phase of the silica gel column chromatography is a mixed solution of ethyl acetate-ethanol-water with a volume ratio of 25:1:0.1 to 3:1:0.1 or a mixed solution of dichloromethane-methanol-water with a volume ratio of 40:7.5:1 to 12:3:0.

4.

6. The method according to claim 2, characterized in that The mobile phase for preparative HPLC was acetonitrile-water (89:11, V / V).

7. Application of 3-O-{O-β-D-glucopyranosyl-(1→2)-O-[β-D-glucopyranosyl-(1→3)]-O-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside} in the preparation of foods, health foods and medicines with weight loss effects.

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