CN101606955B - Tamariskoid spikemoss herb effective part for reducing blood lipid, extraction method and application thereof, and extraction method of amentoflavone - Google Patents

Abstract

The invention discloses a tamariskoid spikemoss herb effective part for reducing blood lipid, and an extraction method and application thereof. The effective part is an ethyl acetate part obtained by extracting tamariskoid spikemoss herb by acetone water, concentrating and extracting by ethyl acetate. The tamariskoid spikemoss herb effective part for reducing blood lipid is obtained by extracting from lipid solubility part of tamariskoid spikemoss herb and the tamariskoid spikemoss herb is distributed widely in nature, so that the raw material is easy to obtain and low in cost. In addition, the extraction method has simple technological process and easy operation. Experiment shows that the amentoflavone obtained by further extracting the tamariskoid spikemoss herb effective part for reducing blood lipid has good function of reducing blood lipid. Experiment in vivo shows that the blood lipid level can be reduced by reducing serum total cholesterol and low-density lipoprotein cholesterol of hyperlipidemic mouse. The amentoflavone has higher extraction rate, and the once extraction rate can reach as high as 30 to 40 percent.

Description

The effective parts of Selaginella chinensis for lowering blood fat, its extraction method and application, and the extraction method of abiflavonoids from Selaginella

technical field

The present invention relates to a method for extracting the effective part for lowering blood fat from the fat-soluble part of Selaginella chinensis - ethyl acetate part, and also relates to the extraction method and application of the effective part; in addition, it also relates to the effective part of ethyl acetate through The method for further extracting and preparing the flavonoids.

Background technique

Hyperlipaemia (Hyperlipaemia, HLP) mainly refers to serum total cholesterol (TC) or triglyceride (TG) levels are too high and/or serum high-density lipoprotein-cholesterol (HDL-C) levels are too low. Hyperlipidemia is a common and frequently-occurring disease in middle-aged and elderly people. With the improvement of people's living standards and changes in living habits, the incidence of this disease has increased significantly, and the age of onset has also advanced. Atherosclerosis caused by hyperlipidemia is the main cause of coronary heart disease, hypertension and cerebrovascular disease, which seriously endangers human life and health. At present, although Western medicines have certain curative effects on regulating blood lipids, they have side effects of varying degrees, and their targets are single. Western medicine statins are currently recognized as the most effective and widely used drugs for lipid-lowering, but they have side effects such as liver and muscle toxicity. In recent years, TCM blood lipid regulation has become a research focus due to its advantages of good curative effect and few side effects.

Selaginella is a plant of the genus Selaginella (Selaginellaceae) of the fern family Selaginellaceae, and there are about 700 species, which are widely distributed all over the world. There are about 50 species in my country, which abound in all parts of the country. The 2005 edition of "The Pharmacopoeia of the People's Republic of China" recorded two varieties: Selaginella tamariscina (Beauv.) Spring; mat-packed Selaginella tamariscina (Hook.et Grev.) Maxim. Selaginella is a traditional Chinese medicinal material in my country. It has been recorded as early as in "The Classic", and it is listed as a top grade. The whole herb of Selaginella is used as medicine, and Selaginella is a folk traditional Chinese medicinal material with a long history of medicinal use. Since traditional medicines are mostly taken by decoction, modern pharmacological research also uses decoction as a whole, and it is found that decoction has good pharmacological activities in anti-tumor, immunity, and hemostasis. However, there are few studies on its fat-soluble parts, especially the research on lowering blood lipids has not been reported.

Amentoflavone, also known as Amentoflavone, is a kind of biflavone compound isolated and purified from natural plants, with a molecular formula of C ₃₀ H ₁₈ O ₁₀ and a molecular weight of 538. It has a wide range of plant sources and has obvious pharmacological activity. It exists in many kinds of Chinese herbal medicines such as Ginkgo biloba and Hypericum perforatum, and the content of biflavonoids in Selaginella spica (including Selaginella, Selaginella mat, Selaginella chinensis and Selaginella jiangnan, etc.) is much higher than other Chinese herbal medicines . It has been reported in the literature that the amiflavones of Suichata spiconiae have antiviral, anti-inflammatory, anti-epileptic, nerve cell protection, and Ca ²⁺ release-promoting activities. At present, the process of extracting the biflavonoids of Suicha spiconiae is relatively cumbersome, and the cost is relatively high.

Contents of the invention

The object of the present invention is to provide an effective part of Selaginella chinensis for lowering blood fat obtained by extracting the fat-soluble part of Selaginella chinensis, ie the ethyl acetate part.

At the same time, the purpose of the present invention is also to provide a method for extracting the effective part of Selaginella chinensis for reducing blood lipid.

In addition, the object of the present invention is also to provide an application of the effective fraction of Selaginella chinensis for lowering blood fat.

The purpose of the present invention is also to provide a method for further extracting the effective parts of Selaginella chinensis for reducing blood lipids to prepare spikenin flavonoids.

In order to achieve the above object, the technical solution of the present invention adopts an effective part of Selaginella for reducing blood lipid, which is the ethyl acetate part obtained by extracting Selaginella with acetone water, concentrating and extracting with ethyl acetate.

The technical scheme of the present invention also lies in the adoption of a method for extracting effective parts of Selaginella chinensis for lowering blood lipids, comprising the following steps: extracting the whole Selaginella herb with a mixed solution of acetone and water at room temperature for 2-4 times, each time 6 After -9 days, after filtration, the combined filtrates are decompressed, the pressure is 0.1-0.15Mpa, the solvent is recovered to obtain a concentrated extract, the concentrated extract is dispersed in water, and extracted 2-4 times with ethyl acetate to obtain Selaginella The effective part for lowering blood fat is the ethyl acetate part.

The volume ratio of the acetone to water is acetone:water=(6-8):(2-4).

Furthermore, the technical solution of the present invention also adopts a method for extracting the biflavone of Selaginella, which is obtained by further extracting the effective parts of Selaginella chinensis for lowering blood fat, and specifically includes the following steps: the whole herb of Selaginella chinensis is mixed with acetone and water The mixed solution is leached 2-4 times at room temperature, each time for 6-9 days, and then filtered, the combined filtrate is reduced in pressure, the pressure is 0.1-0.15Mpa, the solvent is recovered to obtain a concentrated extract, and the concentrated extract is dispersed In water, extract 2-4 times with ethyl acetate to obtain the effective part of Selaginella chinensis for lowering blood fat, that is, the ethyl acetate part; carry out silica gel atmospheric pressure column chromatography on the prepared ethyl acetate part, and use petroleum ether-acetone according to volume Gradient elution at a ratio of 10:1-7:3, TLC detection of the combined fraction containing spikyflavones, and elution with acetone on Sephadex LH-20 column chromatography to obtain spicantone flavonoids.

The silica gel is 200-300 mesh.

In addition, the technical solution of the present invention also adopts the application of the effective part of Selaginella chinensis for lowering blood fat.

The effective part of Selaginella for lowering blood fat of the present invention is extracted from the fat-soluble part of Selaginella, and Selaginella is widely distributed in nature, which makes the raw material of the present invention easy to obtain and low in cost. In addition, the extraction method of the present invention has a simple process and is easy to operate. Experiments show that: the biflavone obtained by further extracting the effective parts of Selaginella cypress of the present invention for lowering blood fat has a good effect of lowering blood fat. In vivo experiments have shown that it can reduce serum total cholesterol and low-density lipoprotein cholesterol in mice with hyperlipidemia to reduce blood lipid levels. The biflavone of the invention has a higher extraction rate, and the extraction rate at one time can be as high as 30%-40%.

The monomer compound further prepared by adopting the effective part of Selaginella chinensis for lowering blood lipid-ethyl acetate part of the present invention is identified as abiflavone, and its structural formula is:

Amentoflavone

Description of drawings

Fig. 1 is the standard curve figure of Suohuashan biflavone standard substance quality and peak area;

Fig. 2 is the chromatogram of sample content determination.

Detailed ways

Example 1

This embodiment is the preparation method of the effective part of Selaginella chinensis for lowering blood fat - the ethyl acetate part. The specific steps are as follows: take the whole dry Selaginella herb and cold-soak 3 times with acetone and water at a volume ratio of 7:3, each time 7 day, filter, combine the filtrates to recover the solvent under reduced pressure (the pressure is 0.1Mpa) to obtain a concentrated extract, disperse the obtained concentrated extract in water, and extract three times with ethyl acetate to obtain the ethyl acetate part.

Example 2

The present embodiment is the extraction method of abiflavone of spikenard, and concrete steps are as follows: take the ethyl acetate part that embodiment 1 makes, carry out silica gel normal pressure column (200-300 mesh) chromatography, sherwood oil-acetone by volume ratio is 10:1, 8:2 and 7:3 for gradient elution (5 column volumes for each gradient elution, the speed is 5ml/sec), TLC detection combined with the fraction containing spikeniflavone, in Sephadex LH-20 column chromatography Elution is carried out with acetone to obtain the pure product of abiflavone.

Structure Identification:

Compound spicantone, EIMS m/z: 538 (M ⁺ ), 270, 121; ¹ H-NMR (DMSO-d ₆ ) δ: 13.10 (1H, s, 5-OH), 13.00 (1H, s, 5″-OH), 6.83 (1H, s, H-3), 6.21 (1H, d, J=2.0Hz, H-6), 6.48 (1H, d, J=2.0Hz, H-8), 8.03 (1H, s, H-2'), 7.17 (1H, d, J=9.3Hz, H-5'), 8.01 (1H, d, J=9.3Hz, H-6'), 6.80 (1H, s , H-3″), 6.42 (1H, s, H-6″), 7.59 (2H, d, J=8.8Hz, H-2″′, 6″′), 6.74 (2H, d, J=8.8 Hz, H-3″′, 5″′). ¹³ C-NMR (DMSO-d ₆ ) δ: 163.8 (C-2), 102.9 (C-3), 181.7 (C-4), 161.5 (C- 5), 98.9(C-6), 164.1(C-7), 94.0(C-8), 157.4(C-9), 103.7(C-10), 121.0(C-1132.3(C-1′), 127.8(C-2′), 120.0(C-4′), 116.2(C-5′), 131.4(C-6′), 163.7(C-2″), 102.6(C-3″), 182.1( C-4″), 160.5 (C-5″), 98.6 (C-6″), 161.9 (C-7″), 104.0 (C-8″), 154.5 (C-9″), 103.7 (C- 10″), 121.4 (C-1″′), 128.2 (C-2″′), 115.8 (C-3″′), 161.0 (C-4″′), 115.8 (C-5″′), 128.2 (C-6"').

Example 3

This example is an application example of the effective part of Selaginella chinensis for lowering blood lipid——ethyl acetate part and abiflavone of spikenard in reducing blood lipid.

In vivo lipid-lowering antioxidant test:

Animals: Kunming mice, all males with a fasting body weight of 23±2 g. Provided by Henan Medical Animal Experiment Center.

Materials and reagents: The effective part of Selaginella chinensis for lowering blood lipids—ethyl acetate part, and abiflavonoids of Selaginella were prepared and provided by the Institute of Natural Medicine, Henan University. Semen Cassiae Jiangzhi Tablets, Jilin Jichun Pharmaceutical Co., Ltd. Batch number: 070501. Superoxide dismutase (SOD) and malondialdehyde (MDA) assay kits were provided by Nanjing Jiancheng Bioengineering Institute. Total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) assay kits were provided by Beijing Zhongsheng Bioengineering High-Tech Co., Ltd.

experiment method:

Preparation of high-fat emulsion: 40g of lard, 20g of cholesterol, 1g of propylthiouracil, 2g of bile salt (made into a 10% solution), 2ml of propylene glycol and 801ml of Tween, add water to 100ml, and supercharge for 30min.

Animal grouping and treatment: Take 50 mice, with an average body weight of 23±2g, and divide them into normal control group, high-fat model group, and drug treatment group (including the group of two flavonoids administered with spicanthus and the effective part of Selaginella chinensis for reducing blood lipids) according to the experimental requirements. ——Ethyl acetate site administration group), positive control group (Semen Cassiae Jiangzhi Tablets). Animals in the normal control group were given distilled water by ig (gavage) in the morning, and 0.5% CMC-Na by ig in the afternoon. The high-fat model group was given high-fat emulsion (0.2ml/10g d) by ig every morning, and 0.5% CMC-Na by ig in the afternoon. -Na, the administration group was given high-fat emulsion (0.2ml/10g·d) in the morning ig respectively, and in the afternoon ig administration, the dosage is shown in Table 2. After 20 days, each group of mice fasted for 12h. Blood was collected from the venous plexus, and the serum was separated by centrifugation for the determination of blood lipids, SOD and MDA.

Table 1 Animal grouping and treatment

Index determination method: TC, TG, LDL-C, HDL-C, MDA, SOD were measured according to the kit instructions.

Data processing: The experimental data expressed as x±s, and the experimental statistics were analyzed by one-way analysis of variance using spss10.0 statistical software.

result:

The effective parts of Selaginella for lowering blood lipids—the ethyl acetate part and the compound spikeflavone on the blood lipids of mice with hyperlipidemia, the results are shown in Table 2. Compared with the high-fat model control group: the effective part of Selaginella chinensis for lowering blood lipids - ethyl acetate part, the compound spike flavonoids, TC and LDL-C in plasma were all reduced, the data difference was significant, and HDL-C was significantly increased , TG had no significant effect; positive control group plasma TC, LDL-C were significantly reduced. Compared with the positive drug group: the effective part of Selaginella chinensis for lowering blood lipid - ethyl acetate part administration group and the compound spike flavonoid group can more significantly increase the level of HDL-C in plasma.

Table 2 The effective parts of Selaginella chinensis for lowering blood lipid——the ethyl acetate part and the effect of spikenin biflavone on the blood lipids of mice with nutritional hyperlipidemia (x±s)

TC TG HDL LDL blank group 3.9651±0.7858 2.7095±0.7724 0.6319±0.1816 1.6215±0.1955 model group 5.9915±1.2886 ^△△△ 0.9419±0.2251 0.4309±0.1565 ^△△ 3.5811±1.3695 ^△△△ positive control 3.7332±0.8868 ^*** 0.8659±0.1771 0.3271±0.1737 0.6276±0.3332 ^*** Abiflavanone 3.8587±1.1729 ^*** 1.1331±0.2522 1.8084±0.2808 ^*** 1.8909±1.1736 ^*** Ethyl acetate site 4.0221±1.0117 ^** 0.9721±0.2663 1.4164±0.4264 ^*** 2.0875±1.0011 ^**

Note: Compared with the model group: ^*** P<0.001, ^** P<0.01, ^* P<0.05

Compared with blank group: ^△△△ P<0.001, ^△△ P<0.01, ^△ P<0.05

From the above results, it can be known that when the amount of abioflavone is 1/2 of the ethyl acetate part, the effects of the two on the blood lipids of hyperlipidemia mice are basically the same. It was found that the content of abiflavone in the ethyl acetate part was 31.64%, so it can be seen that the main active ingredient in the ethyl acetate part is abiflavone. The following is the determination of the content of spikantiflavone in the ethyl acetate fraction.

Determination by HPLC

Instruments and reagents

Instruments: Shimadzu LC-2010 high performance liquid chromatography, UV-2010A detector, autosampler, quaternary low-pressure gradient pump, CLASS-vp chromatography workstation, AL-104 electronic balance (Mettler-Toledo) , KQ3200 ultrasonic cleaner.

Reagents: Methanol (chromatographically pure, manufacturer), high-purity water, phosphoric acid is analytically pure, Suihuashan biflavone standard is self-made by the Institute of Natural Products, with a purity of 98%.

Chromatographic conditions

RP-18 chromatographic column (4.6mm × 250mm, 5 μ m), produced by Merck Company; mobile phase: methanol: 0.1% phosphoric acid aqueous solution (65: 35), flow rate 0.8ml/min; detection wavelength 337nm; column temperature 25 ℃, quantitative method The external standard peak area method.

Establishment of standard curve

Accurately weigh 1.2mg of the standard substance into a 10ml volumetric flask, shake well and set aside. Take a certain amount and filter with a filter (0.45 μm) to obtain a 0.12 mg/ml standard solution, inject 1 μl, 3 μl, 5 μl, 7 μl, and 10 μl respectively, and perform linear regression on the obtained peak area to the quality of biflavones to obtain a standard curve Y= 5E+06X-10436, r=1.000, the linear range is 0.12μg～1.2μg. The results are shown in Table 4, and a standard curve was made according to Table 4, as shown in Figure 1.

Table 4

Standard concentration μg Peak area

0.12 593562

0.36 1793563

0.60 2993563

0.84 4193563

1.20 5993563

Sample determination: Accurately weigh the effective part of 3mg of ethyl acetate into a 10ml volumetric flask, add 8ml of methanol, dissolve it by ultrasonic, dilute to 10ml, filter (0.45μm) sample solution. Parallel 6 copies, the content determination chromatogram is shown in Figure 2, wherein A is the standard product chromatogram, B is the sample chromatogram.

Results: The average content was 31.64%, RSD=0.75%.

The present invention also separately measures the content of the biflavonoids in Selaginella genus Selaginella such as Selaginella, Pad Selaginella, Jiangnan Selaginella, and Chinese Selaginella, and the results are shown in Table 5.

Table 5 The content of abiflavones in different species of Selaginella plants

Finally, it should be noted that: the above embodiments are only used to illustrate, rather than limit the technical solution of the present invention, although the present invention has been described in detail with reference to the above embodiments, those of ordinary skill in the art should understand that: the present invention can still be carried out Any modification or equivalent replacement without departing from the spirit and scope of the present invention shall fall within the scope of the claims of the present invention.

Claims (1)

1. Application of a blood lipid-lowering effective part of Selaginella in the preparation of blood lipid-lowering drug, wherein said effective part of Selaginella for reducing blood fat is ethyl acetate extracted from Selaginella acetone water and then concentrated and extracted with ethyl acetate Parts, specifically: extract the whole Selaginella herb with a mixture of acetone and water for 2-4 times at room temperature, each time for 6-9 days, then filter, combine the filtrate to recover the solvent under reduced pressure to obtain a concentrated extract, Disperse the concentrated extract in water, extract 2-4 times with ethyl acetate to obtain the effective part of Selaginella chinensis for lowering blood fat, that is, the ethyl acetate part, and the volume ratio of acetone to water is acetone:water=(6- 8): (2-4).

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