Quinoa peel saponin compound as well as preparation method and application thereof

Abstract

The invention provides a saponin compound separated and purified from chenopodium quinoa bran. Meanwhile, the invention also provides a preparation method of the compound. The compound is found to have certain physiological activity and can be used as some medicines.

Description

Technical Field

[0001] The present invention relates to the field of natural compounds. Background Art

[0004] Quinoa husk is rich in saponins, flavonoids, phenolic acids, alkaloids and other chemical components. These components give quinoa husk a variety of biological activities. Studies have shown that quinoa husk extract can effectively inhibit the activity of α-glucosidase, thereby slowing down the digestion and absorption of carbohydrates and helping to control blood sugar levels. This discovery provides new ideas for the development of natural hypoglycemic foods, health products and medicines. The flavonoids and phenolic acid compounds in quinoa husk have significant antioxidant capacity, which can remove free radicals in the body and reduce the damage of oxidative stress to cells, thereby protecting cell health and delaying aging. The saponin compounds in quinoa husk have inhibitory effects on a variety of bacteria and fungi, and can be used to develop natural antibacterial agents, which are widely used in food preservation, medicine and other fields. Phenolic acid compounds have health functions such as lowering blood sugar and blood lipids.

[0005] Quinoa husk is a natural resource rich in various beneficial ingredients, has significant pharmacological activity and broad application prospects. The purpose of the present invention is to provide a new product based on quinoa husk, by extracting, separating and purifying the active ingredients in quinoa husk, developing products with specific pharmacological activity, so as to meet market demand and promote the development of human health. Summary of the invention

[0006] The present invention intends to provide a natural compound with good pharmaceutical activity, and a preparation method and use thereof.

[0007] Specifically, the present invention provides a compound as shown in Formula 2:

[0008]

[0010] The present invention also provides a method for preparing the compound shown in Formula 2, comprising the following steps:

[0011] (1) 70-80% ethanol extract of quinoa peel is eluted with macroporous resin, and water and 20-100% ethanol are used in sequence to elute, and the 100% ethanol elution fraction is collected;

[0012] (2) Elution using a silica gel column, using chloroform:methanol 100:0 and 95:5 in sequence, and collecting the elution portion of chloroform:methanol 95:5;

[0013] (3) using MCI gel column chromatography, using 70-80% methanol aqueous solution for gradient elution, combining the eluates from the same parts, and obtaining four components in sequence;

[0014] (4) taking the second fraction, eluting it with methanol in a gel column, and combining the eluates from the same part to obtain three fractions in sequence;

[0015] (5) The first component was taken and eluted by preparative HPLC using 75-80% methanol aqueous solution as the mobile phase to obtain three compounds in sequence. The second compound was taken in sequence to obtain the compound of formula 2.

[0016] The preparation method of the ethanol extract can be conventionally used, including but not limited to:

[0017] 1. Extraction method, which is one of the most basic and commonly used extraction methods. The specific steps include:

[0018] Material preparation: Pre-process the plant samples (such as fresh leaves, hay, etc.) by washing, drying, and crushing.

[0019] Extraction: Add the crushed plant sample to 95% ethanol or other ethanol solution of appropriate concentration in a certain ratio (such as 1:10, W / V) for soaking. Stirring or constant temperature heating can be used during the soaking process to improve the extraction efficiency.

[0020] Filtration and concentration: After soaking, the plant residues are removed by filtration to obtain an ethanol extract. Subsequently, the extract is concentrated to the desired concentration by methods such as reduced pressure concentration to obtain an ethanol extract in the form of an extract or powder.

[0021] 2. Ultrasound-assisted extraction

[0022] Ultrasonic assisted extraction uses the cavitation effect, thermal effect and mechanical effect of ultrasound to accelerate the rupture of plant cell walls and the penetration of solvents, thereby improving extraction efficiency. The specific steps are similar to the single solvent extraction method, but ultrasonic treatment is added during the extraction process.

[0023] 3. Reflux extraction method

[0024] For some plant materials that are difficult to extract or require higher extraction efficiency, the reflux extraction method can be used. This method boils the ethanol solution by heating to generate steam, which condenses and flows back into the extraction container to form a cycle. This cyclic heating method can continuously increase the extraction temperature and promote the dissolution of active ingredients in plant cells.

[0025] 4. Microwave-assisted extraction

[0026] Microwave-assisted extraction uses the rapid heating characteristics of microwaves to make plant materials reach high temperatures in a short period of time, thereby accelerating the dissolution of active ingredients. This method has the advantages of short extraction time and high efficiency, but it is necessary to control the heating temperature and time to avoid the destruction of active ingredients.

[0027] MCI gel column is a small pore resin gel column (polystyrene-based reverse phase resin filler).

[0028] Macroporous adsorption resin is a kind of polymer adsorption resin with macroporous structure and no exchange groups. This kind of resin has good macroporous network structure and large specific surface area, and can selectively physically adsorb organic matter in aqueous solution.

[0029] The "combining of eluates from the same part" in the present invention can be achieved by conventional detection methods, such as thin layer chromatography, HPLC, etc. If thin layer chromatography is used, as long as the spots shown in the eluates are in the same position, the eluates can be combined.

[0030] Furthermore, in step (1), a 75% ethanol extract of quinoa husk is used; and the macroporous resin is a D101 macroporous resin.

[0031] D101 macroporous adsorption resin is a styrene-type non-polar copolymer, which is mainly prepared by suspension copolymerization of styrene and divinylbenzene monomers, with vinylbenzene added as a crosslinking agent, toluene, xylene, etc. as porogens, and these components are crosslinked and polymerized to form a porous skeleton structure. The resin is generally white spherical particles with a particle size of 20-60 mesh, a smooth surface, and high strength and stability.

[0032] Gel column is a chromatographic column that uses gel particles as the stationary phase. It is widely used in the separation and purification of substances in the fields of biochemistry, medicine, food, etc. LH-20 gel is a commercial model of dextran gel, which is obtained by hydroxypropylation. The particle diameter of LH-20 gel is usually 18 to 111um, which is lipophilic and suitable for a variety of organic solvents. The glucose part in its structure is combined with hydroxypropyl to form an ether bond, which enhances the stability and separation effect of the gel in organic solvents.

[0033] The separation mechanism of LH-20 gel mainly includes gel filtration and reverse phase distribution:

[0034] Gel filtration: When the mixture solution passes through the gel column, smaller molecules can enter the pores inside the gel particles, while larger molecules can only flow outside the particles. Due to the screening effect of the pores, molecules of different sizes are separated according to their molecular size. In the LH-20 gel, large molecular compounds have small resistance and move quickly, and flow out first; small molecular compounds have large resistance and move slowly, and flow out later.

[0035] Reverse phase distribution: In reverse phase solvents, LH-20 gel also performs reverse phase distribution on compounds. At this time, compounds with high polarity are weakly retained and are eluted first; compounds with low polarity are strongly retained and are eluted later. This reverse phase distribution further improves the separation effect of LH-20 gel.

[0036] Furthermore, the gel column in step (4) is LH-20.

[0037] Furthermore, in step (5), the mobile phase is 78% methanol aqueous solution.

[0038] The present invention also provides use of the compound of formula 2 in preparing α-glucosidase inhibitors.

[0039] α-Glucosidase, also known as glucosyltransferase, is one of the common items in clinical biochemical examinations. It is a type of starch hydrolyzing enzyme that mainly acts outside the cell. α-Glucosidase inhibitors are a class of drugs that inhibit the activity of α-glucosidase in the brush border of the small intestinal mucosa, thereby delaying the decomposition of carbohydrates (such as starch, sucrose, etc.) into monosaccharides (such as glucose) and absorption by the small intestine, thereby achieving the goal of lowering postprandial blood sugar. This type of drug plays an important role in the treatment of diabetes, especially for patients with elevated postprandial blood sugar. Commonly used α-glucosidase inhibitors include acarbose, voglibose, etc.

[0040] Associations between α-glucosidase inhibitors and other diseases include, but are not limited to:

[0041] 1. Diabetes

[0042] α-Glucosidase inhibitors are one of the important drugs for the treatment of diabetes, especially for patients with elevated blood sugar after meals. They delay the absorption of carbohydrates, effectively reduce postprandial blood sugar peaks, and reduce blood sugar fluctuations, thereby helping to control diabetes.

[0043] Mechanism of action: By inhibiting α-glucosidase, it slows down the decomposition of polysaccharides such as starch into glucose, thereby reducing the absorption of glucose in the small intestine and achieving the effect of lowering blood sugar.

[0044] 2. Cardiovascular complications

[0045] Studies have shown that α-glucosidase inhibitors can not only lower blood sugar, but also reduce the risk of cardiovascular complications in diabetic patients. This may be related to their multiple mechanisms, such as improving insulin resistance, reducing blood sugar fluctuations, and lowering blood lipid levels.

[0046] Mechanism of action: By reducing blood sugar fluctuations and insulin resistance, α-glucosidase inhibitors help protect vascular endothelial cells, reduce inflammation and oxidative stress, and thus reduce the incidence of cardiovascular complications.

[0047] Based on the above mechanism, the present invention also provides the use of the compound of formula 2 in the preparation of a method for treating diabetes and diabetic cardiovascular complications. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Separation flow chart DETAILED DESCRIPTION

[0049] Experimental instruments

[0050] Bruker DRX-500MHz nuclear magnetic resonance spectrometer (Bruker, Germany); LC-IT-TOF mass spectrometer (Shimadzu, Japan); Waters 2535 semi-preparative liquid chromatograph (Waters, USA); Agilent 1260 high performance liquid chromatograph (Agilent, USA); Büchi rotary evaporator (Büchi, Switzerland); SHB-3 circulating multi-purpose vacuum pump (Yuhua Instrument Co., Ltd., Gongyi); ZF-1 three-purpose UV instrument (Shanghai Jingke Industrial Co., Ltd.); column chromatography silica gel (80-100, 200-300 mesh, Qingdao Kangyexin Pharmaceutical Silica Gel Desiccant Co., Ltd.); GF254 silica gel plate (50-100nm, Linyi Haixiang Chemical Co., Ltd.); Sephadex LH-20 (Amersham, Sweden); Biosciences Company), preparative reverse phase chromatography column (250mm×20mm, 10μm, Daisogel Company, Japan); BUCHI medium pressure preparative chromatograph; RP-C18 (40~63μm, Merck Company, Germany); macroporous resin LXT-28 (Xi’an Lanxiao Technology New Materials Co., Ltd.); microporous resin MCI (Mitsubishi Chemical Company, Japan); chromatography grade methanol (Shanghai Xingke High Purity Solvent Co., Ltd.), TQ-1 automatic extraction equipment.

[0051] Source of experimental materials

[0052] Quinoa husk, obtained by dry peeling and polishing process (purchased from Beijing Tong Ren Tang Health Pharmaceutical (Qinghai) Co., Ltd.)

[0053] Example 1 Preparation of Quinoa Peel Ethanol Extract

[0054] The quinoa peel sample was crushed with a grinder, and then soaked in 75% v / v ethanol with a volume of 5 times the sample volume, and the extract was collected and concentrated by a rotary evaporator. The extraction was repeated twice according to the above method to obtain a crude quinoa peel extract.

[0055] Example 2 Preparation of Quinoa Peel Ethanol Extract

[0056] After the quinoa peel sample was crushed by a grinder, 80% v / v ethanol with a volume of 10 times the sample was used to ultrasonically extract the plant sample, and the ultrasonic extract was collected and concentrated by a rotary evaporator. The extraction was repeated twice according to the above method to obtain a crude quinoa peel extract.

[0057] Example 3

[0058] The extract prepared in Example 1 was dissolved in water, and allowed to stand for 12 hours after loading to allow the D101 resin to fully adsorb, eluted with water to remove free sugars, and then eluted with EtOH-H2O gradient (20% to 100%, v / v, three column volumes for each concentration) to obtain six components F1-F6 in turn.

[0059] Component F6 (100% ethanol v / v elution portion) was chromatographed on a silica gel column and gradient eluted with CHCl3-MeOH (100:0 to 0:100, v / v). After combining their TLC spectra, five components F6-1 to F6-5 were obtained in sequence.

[0060] Component F6-2 was fractionated into four components F6-2-1 to F6-2-4 by MCI gel column chromatography eluted with MeOH-H2O (70% to 80%). F6-2-2 was separated into three components F6-2-1-1 to F6-2-1-3 by Sephadex LH-20 column eluted with MeOH. F6-2-1-1 was purified by preparative HPLC using 78% MeOH as mobile phase to obtain three compounds, which were named Compound 1, Compound 2, and Compound 3 in sequence.

[0061] The compound of formula 2 was identified as Queretaroic acid 28-O-glucoside.

[0062] Example 4 Results of screening of monomeric compounds for α-glucosidase inhibitory activity

[0063] In this experiment, five fractions were first separated by silica gel column chromatography, and each fraction was separated and purified by MCI, gel column, medium pressure preparation and preparative liquid chromatography. A total of more than 10 monomer compounds were separated, and all of them were pentacyclic triterpenoid saponin compounds after structural identification.

[0064] Table 1 Name, purity and preparation amount of triterpenoid saponins

[0065]

[0066] The structural formula is as follows:

[0067]

[0068] In vitro activity screening was performed, and the results of monomer compound activity screening are shown in Table 2.

[0069] Table 2 Inhibition rate of samples on α-glucosidase activity

[0070]

[0071] The results show that compound 2 Queretaroic acid 28-O-glucoside has better inhibitory activity on α-glucosidase than the positive control acarbose, with an inhibition rate of more than 50%.

Claims

1. The compound shown in formula 2:

2. A method for preparing a compound represented by formula 2, characterized in that: The steps include: (1) 70-80% ethanol extract of quinoa peel is eluted with macroporous resin, and water and 20-100% ethanol are used in sequence to elute, and the 100% ethanol elution fraction is collected; (2) Elution using a silica gel column, using chloroform:methanol 100:0 and 95:5 in sequence, and collecting the elution portion of chloroform:methanol 95:5; (3) using MCI gel column chromatography, using 70-80% methanol aqueous solution for gradient elution, combining the eluates from the same parts, and obtaining four components in sequence; (4) taking the second fraction, eluting it with methanol in a gel column, and combining the eluates from the same part to obtain three fractions in sequence; (5) The first component was taken and eluted by preparative HPLC using 75-80% methanol aqueous solution as the mobile phase to obtain three compounds in sequence. The second compound was taken in sequence to obtain the compound of formula 2.

3. The preparation method according to claim 2, characterized in that: In step (1), 75% ethanol extract of quinoa peel is used; the macroporous resin is D101 macroporous resin.

4. The preparation method according to claim 2, characterized in that: The gel column in step (4) is LH-20.

5. The preparation method according to claim 2, characterized in that: In step (5), the mobile phase is 78% methanol in water.

6. Use of the compound of formula 2 according to claim 1 in the preparation of α-glucosidase inhibitors.

7. Use of the compound of formula 2 according to claim 1 in the preparation of a medicament for improving postprandial blood sugar in patients.

8. Use of the compound of formula 2 according to claim 1 in the preparation of a drug for treating diabetes and diabetic cardiovascular complications.

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