CN112791108B

CN112791108B - Preparation method of phellinus igniarius active ingredient and application of phellinus igniarius active ingredient in auxiliary hypoglycemic drugs

Info

Publication number: CN112791108B
Application number: CN202110176813.6A
Authority: CN
Inventors: 田雪梅; 张国利; 李喆
Original assignee: Qingdao Agricultural University
Current assignee: Qingdao Agricultural University
Priority date: 2021-02-07
Filing date: 2021-02-07
Publication date: 2022-05-03
Anticipated expiration: 2041-02-07
Also published as: CN112791108A

Abstract

The invention discloses a preparation method of a phellinus igniarius active ingredient, which comprises the following steps of taking a phellinus igniarius strain (A) with the preservation number of CGMCC NO.21068 (A)Sanghuangporus sanghuang) Filtering the fermentation broth, separating to obtain Phellinus linteus fermentation broth, heating and concentrating, soaking in 95% ethanol, centrifuging, collecting supernatant, recovering ethanol with rotary evaporator, collecting ethanol soluble substance, dissolving in water, freezing at-20 deg.C, vacuum freeze drying, and pulverizing to obtain Phellinus linteus fermentation broth ethanol soluble sample; and (3) carrying out fractional extraction on the phellinus igniarius fermentation liquor alcoholic solution sample by using four organic solvents to obtain an extract. The research result provides a certain experimental basis and theoretical basis for the development and utilization of the subsequent phellinus igniarius liquid fermentation active substances and the research and development of related medicines for reducing blood sugar and the like, and has certain theoretical significance and practical application value.

Description

Preparation method of phellinus igniarius active ingredient and application of phellinus igniarius active ingredient in auxiliary hypoglycemic drugs

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a preparation method of a phellinus igniarius active ingredient and application of the phellinus igniarius active ingredient in an auxiliary hypoglycemic drug.

Background

Phellinus igniariusSanghuangporus sanghuangIs a precious large perennial medicinal fungus, usually born in Morus (Morus)Morus L.) On plants, also known as mulberry ear, sangchen and phellinus igniarius, is known as 'forest gold', is a rare Chinese medicinal material, and is used for treating diseases such as dysentery, night sweat, metrorrhagia, bloody stranguria, umbilicus and abdominal pain, rectocele and bloody discharge, leukorrhagia, amenorrhea and the like in the traditional Chinese medicine of China.

With the research of the anticancer mechanism and the medicinal value of phellinus igniarius by experts at home and abroad, phellinus igniarius is widely recognized by people, and scientific research proves that phellinus igniarius polysaccharide can improve human immunity, inhibit the proliferation of tumor cells, remove free radicals, prevent aging, activate pancreas function, reduce blood sugar, contribute to the rehabilitation of diabetics, resist thrombus, stabilize blood pressure and the like. The phellinus igniarius polysaccharides are extracted from phellinus igniarius sporocarp or phellinus igniarius mycelium by alcohol precipitation, but the research on alcohol-soluble components is less,only the study on hypoglycemic activity with the alcohol-soluble fraction of the mycelium of Phellinus igniarius is mentioned in the patent CN111096983A, but Phellinus igniarius is usedPhellinus ignariusWith Phellinus linteus AoshimaS.sanghuangAre respectively the species of different genera and have no comparability. There has been no report on the research on alcohol-soluble active ingredients extracted from fermentation broth after filtering Phellinus linteus mycelium.

Disclosure of Invention

In order to solve the problems, the invention provides a preparation method of a phellinus igniarius active ingredient capable of reducing blood sugar. Different solvent extraction parts of phellinus igniarius fermentation broth are obtained by a systematic solvent method for the first time, and hypoglycemic active parts of the phellinus igniarius fermentation broth are preliminarily screened out through a cell experiment, so that scientific basis is provided for further searching for effective components with hypoglycemic effects of phellinus igniarius.

The invention is realized by the following technical scheme:

a method for preparing a phellinus igniarius active ingredient with a hypoglycemic effect comprises the following steps:

(1) filtering mycelium of Phellinus linteus strain fermentation liquor fermented for 10-12 days, separating to obtain Phellinus linteus fermentation liquor, heating and concentrating, soaking with 95% ethanol for 24 hr, layering, centrifuging at 5000 rpm for 5 min, collecting supernatant, recovering ethanol with rotary evaporator, collecting ethanol soluble substance, dissolving in water, freezing at-20 deg.C, vacuum freeze drying, and pulverizing to obtain Phellinus linteus fermentation liquor ethanol soluble sample;

(2) sequentially extracting the phellinus igniarius fermentation liquor alcoholic solution sample by using four organic solvents of petroleum ether, chloroform, ethyl acetate and n-butanol in a grading manner to obtain a petroleum ether extract, a chloroform extract, an ethyl acetate extract and an n-butanol extract.

Preferably, the alcohol-soluble sample of the phellinus linteus fermentation liquor in the step (2) is extracted by using a mixed solvent of petroleum ether and chloroform in a volume ratio of 1:1, a mixed solvent of chloroform and ethyl acetate in a volume ratio of 2:3, and a mixed solvent of petroleum ether, chloroform, ethyl acetate and n-butanol in a volume ratio of 2:2:3:3, respectively.

Preferably, the volume ratio of the phellinus igniarius fermentation broth alcohol-soluble sample to the petroleum ether, the chloroform, the ethyl acetate and the n-butanol in the step (2) is 1: 1-5.

Preferably, the extraction time of the petroleum ether, the chloroform, the ethyl acetate and the n-butanol in the step (2) is 2 hours respectively.

Preferably, in the step (2), the extraction times of the petroleum ether, the chloroform, the ethyl acetate and the n-butanol are more than 2:2: 6: 8.

preferably, the fermentation process of the phellinus igniarius strain fermentation liquor is as follows: inoculating liquid strain 5-10%; culturing for the first 2-3 days at 27-29 deg.C and 110r/min in dark place under shaking; on the 3 rd to 8 th days of culture, setting the temperature at 25-27 ℃, rotating speed at 130-; on 8-12 days of culture, the temperature is set at 23-25 ℃, the rotation speed is 110-. In different stages of liquid fermentation, different temperatures and rotating speeds are set so as to ensure that the field planting is carried out as soon as possible in the early stage, the field planting is rapidly increased in the middle stage, the aging is slowed down in the later stage, and the accumulation of secondary metabolites is promoted.

Preferably, the preparation method of the liquid strain comprises the following steps:

(1) inoculating slant strain in solid culture medium under aseptic condition, wherein the area of inoculation block is 1 cm²After inoculation, standing and culturing for 5-7 days at 27 ℃ in a dark place;

(2) the flask was filled with 250 mL of a liquid medium in a 500 mL Erlenmeyer flask, and the activated medium was inoculated to 1 cm²Fermenting and activating 10 flat strain blocks at 26-28 deg.C and 150 rpm for 7 d to obtain primary fermentation liquid strain;

(3) the 500 mL triangular flask is filled with 250 mL liquid culture medium, and inoculation is carried out in a sterile operation, wherein the inoculation amount is 10 mL, the temperature is 25-29 ℃, and the constant temperature submerged fermentation culture is carried out at 110-150 rpm for 10 d for later use.

More preferably, the solid medium is prepared by: 20 g of bran and 30 g of corn flour are all put into distilled water for 25 min at 100 ℃; filtering with 400 mesh gauze, adding 30 g glucose and 1 g KH into the filtrate₂PO₄、0.5 g MgSO₄·7H₂O, adding 4 g of yeast extract, 3 g of peptone and 20 g of peptone at 100 DEG CAgar and distilled water are added to the volume of 1000 mL, and then plate split charging with the diameter of 9 cm is carried out;

preparing a liquid culture medium: 20 g of bran and 30 g of corn flour are all put into distilled water at 100 ℃ for 25 min. After filtering once with 400 mesh gauze, 30 g glucose and 1 gKH were added to the filtrate₂PO₄、0.5 g MgSO₄·7H₂O, adding 4 g of yeast extract and 3 g of peptone at 100 ℃, and subpackaging in 500 mL triangular flasks after the volume of distilled water is up to 1000 mL.

The phellinus igniarius active ingredient prepared by the preparation method is applied to hypoglycemic drugs. Meanwhile, the compound can also be applied to hypolipidemic drugs.

The active ingredient of the phellinus igniarius is prepared into tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, effervescent tablets, sublingual tablets, capsules, hard capsules, soft capsules, microcapsules, microspheres, granules, pills, dropping pills, powder, paste, oral liquid, suspensions, solutions, aerosols, injections, emulsion injections, freeze-dried powder injections and sustained-release or controlled-release preparations with a medically acceptable carrier.

Advantageous effects

The phellinus igniarius is fermented to extract four groups of effective components of petroleum ether extract, chloroform extract, ethyl acetate extract and n-butyl alcohol extract in fermentation liquor, so that the phellinus igniarius fermentation liquor has an obvious blood sugar reducing effect.

The phellinus igniarius fermentation broth active substance alcohol precipitation sample and alcohol soluble samples PartI and PartII have certain functions of promoting glucose consumption in vivo and reducing blood sugar, and have certain potential blood fat reducing efficacy. The in vivo blood sugar reducing effect of the Part II component is wholly superior to that of the Part I component, and the in vivo test result is basically consistent with the in vitro test result. The research result provides a certain experimental basis and theoretical basis for the development and utilization of the subsequent phellinus igniarius liquid fermentation active substances and the research and development of medicines for reducing blood sugar, blood fat and the like, and has certain theoretical significance and practical application value.

Through the preliminary GC-MS analysis of the 4 kinds of extraction components of the phellinus igniarius fermentation liquor, the fact that the 4 kinds of extraction components contain partial active ingredients with the effects of resisting bacteria, resisting oxidation, resisting cancers, reducing blood sugar and the like is inferred. Through comparative analysis of the components of the 4 kinds of extraction components of the phellinus igniarius alcohol-soluble sample, the potential pharmacological activity of the phellinus igniarius alcohol-soluble sample can be better discovered, and a certain theoretical basis is provided for subsequent research work. Provides a certain theoretical basis for the research of alcohol-soluble components of phellinus igniarius fermentation liquor.

Information on strain preservation

Preservation time: 1 month and 4 days 2021;

the preservation unit: china general microbiological culture Collection center;

the preservation number is: CGMCC NO. 21068;

the address of the depository: the institute of microbiology, national academy of sciences No. 3, Xilu No. 1, Beijing, Chaoyang, Beijing;

and (3) classification and naming:Sanghuangporus sanghuang。

drawings

FIG. 1 Effect of Part I on glucose consumption by IR-HepG2 cells;

FIG. 2 Effect of Part II on glucose consumption by IR-HepG2 cells;

FIG. 3 Effect of EPS on glucose consumption by IR-HepG2 cells;

FIG. 4 Effect of EPS1 on glucose consumption by IR-HepG2 cells 1;

FIG. 5 Effect of EPS1 on glucose consumption by IR-HepG2 cells 2;

FIG. 6 Effect of PartI on IR-HepG2 cell viability;

FIG. 7 Effect of PartI1 on IR-HepG2 cell viability;

FIG. 8 Effect of EPS on IR-HepG2 cell viability;

figure 9 effect of EPS1 on insulin resistant HepG2 cell viability 1;

FIG. 10 Effect of EPS1 on viability of IR-HepG2 cells 2;

FIG. 11 comparison of fasting plasma glucose values after modeling and 4w administration in mice;

FIG. 12 fasting plasma glucose reduction rate after 4w administration;

FIG. 13 blood insulin levels in mice after 4w administration;

FIG. 14 Total Cholesterol levels in mice blood after 4w administration;

FIG. 15 mouse blood triglyceride levels after 4w administration;

FIG. 16 mouse blood low density lipoprotein levels after 4w administration;

FIG. 17 mouse blood high density lipoprotein levels after 4w administration;

FIG. 18 Effect of petroleum ether extraction components on glucose consumption by IR-HepG2 cells;

FIG. 19 Effect of chloroform extraction fractions on glucose consumption by IR-HepG2 cells;

FIG. 20 Effect of ethyl acetate extraction components on glucose consumption by IR-HepG2 cells;

FIG. 21 effect of n-butanol extraction on glucose consumption by IR-HepG2 cells;

FIG. 22 Effect of petroleum ether extract fractions on IR-HepG2 cell viability;

FIG. 23 effect of chloroform extraction components on IR-HepG2 cell viability;

FIG. 24 Effect of ethyl acetate extraction fractions on IR-HepG2 cell viability;

FIG. 25 Effect of n-butanol extraction components on IR-HepG2 cell viability;

FIG. 26 Effect of mixed solvent extract 1 on IR-HepG2 cell viability;

FIG. 27 Effect of mixed solvent extract 2 on IR-HepG2 cell viability;

FIG. 28 Effect of mixed solvent extract 3 on IR-HepG2 cell viability.

Detailed Description

The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.

Example 1

1. Materials and methods

1.1 materials

1.1.1 test samples

Phellinus linteus strain, Shandong province, was preserved in key laboratory of fungi.

As used in this applicationThe strain is obtained by collecting fresh fruiting body from living standing tree of Morus alba L in mountain area of Anshu city, Guizhou province, separating tissue and purifying strain, and is named as Phellinus Linteus (A)Sanghuangporus sanghuang) It has been preserved in China general microbiological culture Collection center (CGMCC) at 1 month and 4 days 2021, with the preservation number of CGMCC NO. 21068.

Morphological characteristics of fruiting bodies: the basil fruits have many years of growth, do not have a handle cover shape, are usually singly grown, have sour taste when fresh, are suberect in quality, and are woody after being dried; the pileus is shaped like a horseshoe, extends outwards to 5 cm, has a width of 11 cm and a base thickness of 6 cm; the surface of the pileus is yellow brown to grey brown, is smooth by fine villi, and has obvious ring areas and ring ditches; dull edge, bright yellow; the surface of the opening is yellow when fresh, brown after dried and has a slight refraction reaction; the sterile edge is obvious, and the width reaches 3 mm; the orifices are polygonal to circular, and 6-7 orifices per millimeter; the edge of the orifice is thin and the whole edge is thin; the mushroom flesh is yellow, has lighter color than the mushroom tube, is made of hardwood with suppository texture, has obvious ring area, has black lines and has the thickness of 5 cm; the mushroom tube is brown, and the length of the mushroom tube is 4 mm.

Microstructural features: a hypha system II; all diaphragms are united without locks; hyphal tissue darkened in KOH. The germ hyphae in the mushroom meat are colorless to light yellow, thin-walled to slightly thick-walled, multi-branched and frequently separated, and the diameter is 2-3 mu m; the skeleton hyphae in the mushroom meat are golden yellow, thick, have wide or narrow inner cavities, are not branched, are more separated, are nearly regularly arranged and have the diameter of 3-4 mu m. The germ hyphae in the fungus tube are colorless to light yellow, thin wall to thick wall, few branches, more partitions and 2-2.5 μm in diameter; the skeleton hyphae in the fungus tube are golden yellow, thick, have wide or narrow inner cavities, are not branched or separated, are arranged nearly regularly, and have the diameter of 2.5-3.7 mu m; the seta of the fructification layer is common and mostly in the shape of a drum, dark brown, thick wall, sharp tail end and 17-30 multiplied by 7-11 mu m in size; the sub-parenchyma layer is free of cysts and pseudo-cysts; the basilar barrel has 4 small stems, and the base has a simple partition with a size of 6-9X 4-5 μm. Basidiospores are wide oval, yellow, thick-walled, smooth, IKI-, CB-, with a size of (3.4-)3.6-4.6(-4.8) × (2.8-)3-3.5(-3.7) μm, an average length L = 4.03 μm, an average width W = 3.18 μm, and an aspect ratio Q = 1.27 (n = 30/1).

And (3) identifying the strain molecules: extracting DNA from the sporocarp and the mycelium by using a kit, amplifying and sequencing by using ITS5/ITS4 primers, wherein the common rDNA-ITS sequence of the sporocarp and the mycelium is as follows:

CGATGGGTACTGCGGAGGTCATTATCGAGTTTTGAAAGCGAGACCTGCTGCTGGTGCGAAATCGCGCATGTGCACGGTCTTCGCGCTCAAATCCAACTCAAACCCCTGTGCACCTTATATATCGCGAGTCGAAGTTAGTAGCCTGAGGTCTTGTAAGTAATTAGTAGAAGGGCGAAAGCGAGTCTTGCTCGTTAGGTAGCCTTTCGAAAATGAAAGCGAGTGCGTCGGGTGAAGACTTCGGCTTGTCGTTACAAAACACCTTATATTGTCTTTGTGAATGTAATGCTCCTTGTGGGCGAAAATAAATACAACTTTCAACAACGGATCTCTTGGCTCTCGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACCTTGCGCCCCTTGGTATTCCGAGGGGCATGCCTGTTTGAGTGTCATGTTTATCTCAAACCGCTCGTCTTTCTTAATTGAAGGGCTTGAGGTTTGGACTTGGAGGTTTACTGCTGGCGCCTTTCGAGGGGTCGGCTCCTCTTAAATACATTAGCTGGGCTTTGGCTCGCGTTTACGGTGTAATAGTTGATTCCATTCACCAACGAGCGCTTGCCTGACGAGCTTGCTTCTAGCCGTCCGCGTCGTCGGACAAGGAGTCACCTCCTTCTTGACACCTTTGACCTCAAATCAGGTAGGATTACCCGCCGACTTACTAATTAAAGGGGGGGGGGGGGGGAAGAAATTGAA

1.1.2 test cell lines

HepG2 cells.

1.2 methods

1.2.1 preparation of test Medium

Preparing a solid culture medium: 20 g of bran and 30 g of corn flour, all of which are put into distilled water at 100 ℃ for about 25 min. Filtering with 400 mesh gauze, adding 30 g glucose and 1 g KH into the filtrate₂PO₄、0.5 g MgSO₄·7H₂And O, adding 4 g of yeast extract, 3 g of peptone and 20 g of agar at 100 ℃, and subpackaging by using a plate with the diameter of 9 cm after the volume is fixed to 1000 mL by using distilled water.

Preparing a liquid culture medium: 20 g of bran and 30 g of corn flour, all of which are put into distilled water at 100 ℃ for about 25 min. After filtering once with 400 mesh gauze, 30 g glucose and 1 gKH were added to the filtrate₂PO₄、0.5 g MgSO₄·7H₂And O, adding 4 g of yeast extract and 3 g of peptone at 100 ℃, and subpackaging in a 500 mL triangular flask after the constant volume of distilled water is 1000 mL.

1.2.2 plate Strain activation culture

Sterile conditionsInoculating slant strain in solid culture medium, wherein the area of the inoculated block is about 1 cm²And after inoculation, standing and culturing for 5-7 days at 27 ℃ in a dark place.

1.2.3 liquid Strain activated fermentation culture

The flask was filled with 250 mL of a liquid medium in a 500 mL Erlenmeyer flask, and inoculated with about 1 cm of the activated medium²Fermenting and activating the flat strain blocks at 26-28 ℃ for 7 d at 150 rpm to obtain primary fermentation liquid strains.

A 500 mL triangular flask is filled with 250 mL liquid culture medium, and inoculation is carried out in a sterile operation, wherein the inoculation amount is 10 mL, the temperature is 25-29 ℃, the temperature is 110-.

Inoculating liquid strain 5-10%; culturing for the first 2-3 days at 27-29 deg.C and 110r/min in dark place under shaking; on the 3 rd to 8 th days of culture, setting the temperature at 25-27 ℃, rotating speed at 130-; on 8-12 days of culture, the temperature is set at 23-25 ℃, the rotation speed is 110-.

1.2.4 preparation of Phellinus Linteus fermentation broth ethanol-dissolved sample and fermentation broth ethanol-precipitated sample

And (3) separating and collecting fermentation liquor and mycelium of the phellinus igniarius strain which is cultured for 10 days by liquid fermentation. Filtering with 200 mesh gauze. Heating and concentrating the separated phellinus igniarius fermentation liquor to 2300 mL, recording the volume after concentration, and soaking in 9200 mL of 95% ethanol for 24 h. After separation, centrifugation was carried out at 5000 rpm for 5 min. And collecting the precipitate, drying in a 60 ℃ oven, and recording as a phellinus igniarius fermentation liquor alcohol precipitation sample as PartI.

Collecting supernatant, recovering ethanol with rotary evaporator, collecting ethanol soluble substance, dissolving in water, freezing at-20 deg.C, vacuum freeze drying, and pulverizing to obtain Phellinus linteus fermentation broth ethanol soluble sample labeled as Part II.

1.2.5 preparation of crude polysaccharide EPS and EPS1 fractions from Phellinus linteus fermentation broth

2.5 g of phellinus igniarius fermentation liquid alcohol precipitation PartI powder is dissolved by adding 250 mL of distilled water, then dissolved by ultrasonic oscillation at 50 ℃ for 3 h, dissolved for 30 min by magnetic stirring, and centrifuged for 10 min at 5000 rpm. And (3) continuously dissolving the filter residue twice by using 100 mL of distilled water, centrifuging, combining 3 times of centrifuged supernatant, concentrating, and freeze-drying to obtain crude polysaccharide (EPS) of the phellinus igniarius fermentation liquor.

Taking 1.0G EPS sample, dissolving with 10 mL distilled water, centrifuging at 8000 rpm for 10 min to remove insoluble substances, separating and purifying the supernatant by Sephadex G-100, collecting the main peak, and freeze drying to obtain Phellinus linteus fermentation broth polysaccharide (EPS 1).

1.2.6 reagent preparation

2 mg/mL metformin hydrochloride solution: metformin hydrochloride 0.01 g, 5 mL complete medium, can be dissolved at 37 ℃. Filter sterilized and stored at 4 ℃ to obtain solution A. When used, 200. mu.L of solution A was taken, and 1800. mu.L of solvent was adjusted to 200. mu.g/mL.

Part i and Part ii and EPS1 solutions: 100 mg of sample was taken accurately and dissolved in 5 mL of complete medium. Carrying out ultrasonic treatment at 37 ℃ and 100W for 40 min, carrying out filtration sterilization after centrifugation at 5000 rpm for 10 min to obtain a sample solution of 20000 mu g/mL, and diluting the final reaction concentration to 625 mu g/mL, 1250 mu g/mL, 2500 mu g/mL, 5000 mu g/mL and 10000 mu g/mL when the sample solution is added into a cell culture plate hole, wherein the final reaction concentration is respectively marked as S1-S5, and the sample solution is stored for later use at 4 ℃.

1.2.7 detection of the Effect of the glucose oxidase method on glucose consumption

Preparing a 96-well cell culture plate, setting a blank zero-adjustment group (KB), a normal group (CK), a model group (IR) and a positive control group (MET), and setting a phellinus igniarius fermentation liquor alcohol precipitation sample and an alcohol soluble sample: part I (S1-S5) and Part II (S1-S5), EPS and EPS1 components obtained by separating and purifying phellinus igniarius fermentation liquor by alcohol precipitation, and a positive control group is 100 mu g/mL metformin hydrochloride (MET).

1.2.8 MTT method for detecting influence on cell activity

1.3 Effect of different Components on glucose consumption by IR-HepG2 cells

The results of observing the effect of the PartI component on the glucose consumption of the IR-HepG2 cells are compared with the IR (the IR is an insulin resistant group which is a model group, the cell state is close to the cells in a diabetic state, the glucose consumption is abnormally low, and if the glucose consumption of each administered concentration group is higher than that of the model group, the PartI component has the effect of promoting the glucose consumption and has the potential hypoglycemic effect), and each concentration group of the PartI components S1-S5 has a certain effect of promoting the glucose consumption of the IR-HepG2 cells, but the effect is not obvious.

Compared with IR, Part II has extremely obvious promoting effect on the glucose consumption of IR-HepG2 cells in the S1 concentration group, the glucose consumption is improved by 49.4%, the glucose consumption of IR-HepG2 cells in both the S2 and S3 concentration groups is obviously promoted, the glucose consumption is respectively improved by 39.0% and 32.9%, and the S4 group also has certain promoting effect on the glucose consumption, but the effect is not obvious. The analysis of the test results of the PartI component shows that the PartII component has better effect of promoting the glucose consumption of IR-HepG2 cells than the PartI component, and has good blood sugar reducing effect within the concentration range of S1-S3.

The EPS sample has a very obvious effect of promoting the glucose consumption of IR-HepG2 cells at the S2 concentration group, the glucose consumption is improved by 25.0%, the glucose consumption of IR-HepG2 cells at the S5 concentration group is obviously promoted, the glucose consumption is improved by 6%, and the effects of the rest concentration groups are not obvious.

Compared with the IR group, the EPS1 sample has very obvious promotion effect on the glucose consumption of the IR-HepG2 cells in the concentration range of S1-S4, the glucose consumption is respectively improved by 60.0%, 57.6%, 100.1% and 57.8%, the EPS1 sample also has obvious promotion effect on the glucose consumption of the IR-HepG2 cells in the concentration range of S5, and the glucose consumption is improved by 39.3%. Experiments were therefore carried out to continue to design lower concentration gradients C1-C4 to evaluate their effect on glucose consumption by IR-HepG2 cells at low concentrations.

The influence of the EPS1 sample C1-C4 concentration group on the glucose consumption of IR-HepG2 cells is shown in FIG. 5, the C3 and C4 concentration groups have the effect of remarkably promoting the glucose consumption of the IR-HepG2 cells, the glucose consumption is respectively improved by 12.8% and 17.2%, and the effect is equivalent to that of the MET group. The two lowest concentration groups of C1 and C2 had some effect but no significant effect.

As can be seen from fig. 6, compared with the CK group (it is ensured that the degree group of each administration group used does not significantly inhibit the cell activity), the OD value represents the cell activity, and the higher the OD value, the higher the cell activity, and the lower the OD value, the lower the activity. Each group of the PartI component has no obvious influence on the cell activity and has no obvious inhibition effect on the cell activity of an insulin resistant group.

As can be seen from FIG. 7, the Part II fraction showed no inhibitory effect on cell activity in the low concentration S1-S3 groups and the middle concentration S4-S5 groups showed significant inhibitory effect on cell activity, compared to the CK group.

As can be seen from FIG. 8, the EPS fraction had no significant effect of inhibiting the cellular activity at S1-S5, as compared to the CK fraction.

As can be seen from fig. 9, the EPS1 fraction had no significant inhibitory effect on cell viability at both the S1-S5 concentration groups compared to the CK group.

Compared with the CK group, the EPS1 component C3-C4 concentration group has certain inhibition effect on cell viability, and the rest concentration groups have no obvious inhibition effect on cell viability.

2. Establishment of in vivo type II diabetes animal model

Insulin resistance, which is caused by the decrease in the ability of the body to promote glucose consumption and decrease blood glucose levels due to excessive insulin secretion, is a major cause of type ii diabetes. By combining the relevant research practices of the predecessors, the model animal finally selected in the research is Kunming mouse.

An in vivo type II diabetes mouse model is established by high-sugar high-fat feed combined with streptozotocin injection induction. The test can provide a test and theoretical basis for evaluating the blood sugar reducing efficacy of a phellinus igniarius fermentation liquid extraction sample in vivo at the level of a subsequent in vivo type II diabetes animal model.

3. Evaluation of in vivo blood sugar lowering function of active ingredients in phellinus igniarius fermentation liquor

A II-type diabetes mouse model established by combining high-sugar high-fat feed and STZ intraperitoneal injection induction is utilized, two samples of gastric perfusion phellinus igniarius fermentation liquor PartI and PartII are orally taken, and the blood sugar reducing effect of the sample to be tested is comprehensively evaluated by detecting the condition of fasting blood sugar reduction and indexes such as insulin, total cholesterol, triglyceride, low-density lipoprotein, high-density lipoprotein and the like in blood.

3.1 materials

3.1.1 test samples

The method comprises the steps of carrying out alcohol precipitation on a Phellinus linteus fermentation liquor PartI sample, carrying out alcohol extraction on a Phellinus linteus fermentation liquor Part II sample, and providing the Phellinus linteus fermentation liquor by using a fungi key laboratory in Shandong province.

3.1.2 test animals

KM mice, male, 5-6 weeks old, purchased from dawn rich city ltd, Qingdao, Shandong province.

3.2 methods

3.2.1 preparation of test reagents

Preparation of a test sample: PartI and PartII are respectively provided with 3 dose groups of 100 mg/kg, 200 mg/kg and 400 mg/kg, which are respectively marked as PartI-1, PartI-2, PartI-3, PartII-1, PartII-2 and PartII-3, the mass of the PartI and the PartII-3 is weighed according to the weight of the mouse, the PartI and the PartII are dissolved in 0.9 percent of sodium chloride solution, and the filtration sterilization is carried out. Storing at 4 ℃ for later use.

Preparing metformin hydrochloride: the dosage of the metformin hydrochloride is 200 mg/kg, a certain amount of the medicament is weighed according to the weight of a mouse, the medicament is dissolved in a corresponding volume of 0.9 percent physiological saline solution, and bacteria are removed by a filter membrane with the aperture of 0.22 mu m. Treating at 4 deg.C for use.

EPS1 (secondary test) solution: accurately weighing 40 mg of sample, dissolving in 4 mL of complete culture medium, carrying out ultrasonic treatment at 37 ℃ for 40 min and 5000 rpm, centrifuging for 10 min, and then carrying out filtration sterilization to obtain 5000 mug/mL of sample solution. When added to the wells of the cell culture plate, the final reaction concentration was diluted to 4. mu.g/mL, 20. mu.g/mL, 100. mu.g/mL, 500. mu.g/mL, respectively labeled C1-C4, and stored at 4 ℃ until use.

3.2.2 type II diabetic mice gavage feeding

The results of early in vitro blood sugar reducing tests show that the PartI and Part II components of the phellinus igniarius fermentation broth have a certain effect of promoting the consumption of glucose, and the blood sugar reducing effects of the PartI and Part II components are further verified by in vivo type II diabetes mouse model tests. The early-stage modeling group DM1 is divided into 8 groups, namely a model group DM, a positive drug group MET, and a sample group Part-1, Part-2, Part-3, Part II-1, Part II-2 and Part II-3. Marking each group of mice on the back by using marker pens with different colors, re-marking every 1d, adding mouse grains and water, cleaning excrement in a mouse cage, and keeping the growth environment of the mice clean. And (3) performing intragastric feeding on different dose groups of mouse physiological saline, metformin hydrochloride, PartI and PartII according to different intragastric doses corresponding to the body weight of the mouse. Gavage was performed 8 am each day. The gavage is performed for 1 time every day, and the gavage volume is not more than 0.8 mL.

3.2.3 detection of fasting plasma glucose amplitude decrease in mice

After the fasting state of the mice for 12 hours at 8 o 'clock, the mice begin to take blood from the tail vein at 8 o' clock of the next day, and the fasting blood sugar of the mice is detected by using a glucometer and test paper and recorded. During which time the status of the mice was observed.

3.2.4 mice after gastric lavage 4w blood sampling and detection content

After gavage 4w, the fasting plasma glucose value of the mice was measured. When the fasting blood sugar value is lower than 11 mmol/L, the blood sugar value shows that the medicine has good blood sugar reduction effect.

Blood is taken from orbital venous plexus of mice to detect content changes of insulin, total cholesterol, triglyceride, high density lipoprotein and low density lipoprotein.

When the orbital venous plexus of the mouse is bled, the transparent box is firstly inverted, the water-absorbing cloth immersed in ether is placed in the box, and then the mouse is placed in the inverted transparent box, and the operations are all carried out in an enamel plate. After about 15 s, the unconscious mice were removed. Dipping a proper amount of heparin sodium in a capillary tube, and drying by spinning. The operation of blood sampling of orbital venous plexus of the mouse is carried out, and the blood volume of 0.4 mL to 0.5 mL can be obtained. The removed blood was placed on an ice box, labeled, transferred to 4 ℃, 1000 rpm, and centrifuged for 10 min. After that, the supernatant was transferred to a 1.5 mL centrifuge tube, and the 1.5 mL centrifuge tube was labeled with the label. Can be stored at-20 deg.C for 1 month.

3.2.5 detection of blood insulin values in mice

TABLE 1 dilution of standards

Table 2 insulin assay protocol

3.3 results and analysis

3.3.1 detection of fasting blood glucose levels in mice

As can be seen from the test results of fig. 11: after 4w administration, fasting blood glucose values were reduced to different extents in each group of the test compared to those after modeling. Wherein, the fasting blood glucose of the model group (DM group) is reduced from 17.4 mmol/L to 11.1 mmol/L, and is still higher than the standard of 11.0 mmol/L although the fasting blood glucose is reduced to a certain extent. The reason for the decrease of blood sugar in DM group is presumed to be that the feeding of high-sugar and high-fat feed is stopped after the successful modeling, and the feeding of common rat is replaced by the feeding of common rat. The fasting blood sugar of the positive control group (MET group) and all 6 drug groups is reduced to a level higher than that of the DM group from 15-20 mmol/L to about 7.0-8.0 mmol/L, so that the normal level is achieved, and the blood sugar reducing effect is good.

According to the decrease of fasting blood glucose of the mice after modeling and 4w administration, the result of calculating the reduction rate of fasting blood glucose of each test mouse is shown in figure 12. Wherein the blood sugar reduction rate of DM group is 36.2 percent at least, the blood sugar reduction rate of MET group is 59.3 percent, the blood sugar reduction rates of PartI-3 and PartII-3 groups are 59.5 percent and 62.5 percent respectively, the effect of promoting the blood sugar reduction is equivalent to that of MET group, and the blood sugar reduction in vivo effect is good.

The blood glucose reduction rate of each group of PartII was slightly higher than that of each group of PartI when compared between two sample groups at the same dose. The blood glucose reduction rates for each dose group of the two samples tested Part i and Part ii exhibited some degree of dose dependence. Comprehensive analysis test results show that both the phellinus igniarius fermentation broth PartI and the Phellinus igniarius fermentation broth PartII have good effects of promoting blood sugar reduction in vivo, wherein the PartII is superior to the PartI on the whole.

3.3.2 detection of blood insulin values in mice

The change of the blood insulin level of the mice after 4w administration is shown in figure 13, compared with the DM group, the blood insulin level of the mice of each administration group is increased, the difference of the MET group is extremely obvious, each concentration group of 2 samples has certain effect of promoting the increase of insulin, shows more obvious trend of increasing along with the increase of concentration, has certain dose dependence, but the whole effect is not obvious.

3.3.3 detection of Total Cholesterol in blood of mice

As can be seen from the test results in fig. 14: change in blood total cholesterol levels in mice after 4w administration. Compared with DM group, the blood total cholesterol level of mice of each administration group is reduced, the total cholesterol of MET group, PartI-3 group and PartII 3 concentration group is reduced remarkably, and the other administration groups have certain effect of promoting the total cholesterol reduction but have insignificant effect. Both test samples showed a tendency to decrease in the total cholesterol level in blood with increasing concentration, with dose dependence.

3.3.4 measurement of blood triglycerides in mice

As can be seen from fig. 15: compared with DM group, the mice in each administration group have reduced blood triglyceride level and reduced amplitude. The triglyceride reduction of the Part II-3 concentration group is more obvious, and the other sample concentration administration groups also have certain effect of promoting the triglyceride reduction but are not obvious. The 2 samples tested all exhibited a dose-dependent profile with increasing blood triglyceride levels with increasing concentration.

3.3.5 detection of Low Density lipoprotein in mouse blood

As can be seen in fig. 16: change in blood low density lipoprotein levels in mice after 4w administration. Compared with the DM group, the mice in each administration group show extremely remarkable reduction of the blood low-density lipoprotein level, and show the characteristic of dose dependence with larger reduction amplitude along with the increase of the concentration.

3.3.6 detection of high-Density lipoprotein in mouse blood

As can be seen from fig. 17: change in blood high density lipoprotein levels in mice after 4w administration. Compared with the DM group, the mouse blood high-density lipoprotein level of each administration group is increased to a certain degree, the high-density lipoprotein of the PartI-3 group and the Part II-3 group is increased obviously, and the effect of promoting the increase of the mouse blood high-density lipoprotein level of the PartI total concentration group and the Part II-3 group is better than that of the MET group.

Type ii diabetes is a metabolic syndrome of the body due to insufficient insulin secretion or decreased insulin sensitivity. Decreased insulin secretion leads to decreased blood glucose metabolism and increased blood glucose levels in the body. The onset of type II diabetes is often accompanied by a series of symptoms such as hypertension and hyperlipidemia. Triglycerides, total cholesterol, low density lipoproteins and high density lipoproteins are intimately involved in the development of hypertension and hyperlipidemia. The increase of triglyceride, total cholesterol and low density lipoprotein can lead to the increase of lipid content in the organism, the decrease of high density lipoprotein can also lead to the increase of lipid content, the lipid content in the organism can be abnormally increased in the past, the formation of hypertension and hyperlipaemia diseases is caused, and the traditional Chinese medicine composition also has a certain inducing effect on cardiovascular and cerebrovascular diseases.

The insulin content of the body of the type II diabetes is low, the consumption effect on blood sugar is low, under the action of 2 test samples, the consumption on the blood sugar is enhanced due to the increase of the insulin content, and the blood sugar content in the body can be reduced. The increase of triglyceride, total cholesterol and low density lipoprotein content and the decrease of high density lipoprotein content can cause the abnormal increase of lipid content in the organism, under the action of 2 test samples, the index of abnormal increase and decrease in the II type diabetes mellitus machine is reduced and increased to a certain extent, and the hypertension and hyperlipidemia degree are reduced by reducing the lipid content, so that the II type diabetes mellitus has a certain adjuvant therapy effect.

The influence of the active substances of the phellinus igniarius fermentation liquor on various physiological indexes of blood of in vivo type II diabetic mice is detected to discover that: after the oral gavage of 4w in a diabetes model mouse, the blood sugar is obviously reduced, the indexes of insulin, triglyceride, total cholesterol, low-density lipoprotein and the like related to the blood sugar reducing activity are also reduced, the high-density lipoprotein is increased, and the obvious dose dependence characteristic is shown. Although the insulin content is increased to some extent after administration, the difference is not significant, and the decrease in blood glucose may be caused by other routes, for example, acceleration of the decomposition of blood glucose by increasing the activity of α -glucosidase or acceleration of the synthesis of hepatic glycogen.

The test result shows that: the active substances PartI and PartII of the phellinus igniarius fermentation liquor have the effects of promoting the glucose consumption in a certain body and reducing blood sugar, and have certain potential blood fat reducing efficacy. The in vivo blood sugar reducing effect of the Part II component is wholly superior to that of the Part I component, and the in vivo test result is basically consistent with the in vitro test result. The research result provides a certain experimental basis and theoretical basis for the development and utilization of the subsequent phellinus igniarius liquid fermentation active substances and the research and development of related medicines for reducing blood sugar, blood fat and the like, and has certain theoretical significance and practical application value.

4. The crude extract is extracted and purified by different solvents and activity tracking is carried out by using cell tests

4.1 preparation of ethanol-soluble petroleum ether, chloroform, ethyl acetate and n-butanol extract components of Phellinus linteus fermentation broth

The phellinus igniarius fermentation liquor alcohol-soluble sample is extracted by grades by using four organic solvents of petroleum ether, chloroform, ethyl acetate and n-butanol in sequence according to the polarity from small to large. V (Phellinus linteus fermentation liquor alcohol soluble) according to the extraction volume ratio: v (petroleum ether): v (chloroform): v (ethyl acetate): v (n-butanol) = 1: 2:2:3:3, ratio of extraction times, V (petroleum ether): v (chloroform): v (ethyl acetate): v (n-butanol) = 2:2: 6: 8; extraction was carried out for 2h each time. Preparing a 50 mL or 100 mL rotary steaming bottle, binding a preservative film on a bottle mouth by using a rubber band, marking on a bottle body, weighing the original bottle on an electronic balance, and marking. Sequentially extracting, combining and collecting several extraction liquids of each stage of extraction phase, sequentially recovering petroleum ether at 40 ℃, chloroform at 55 ℃, ethyl acetate at 70 ℃ and n-butanol at 80 ℃ by using a rotary evaporator, collecting the extract, adding water, storing at-20 ℃, freeze-drying in a vacuum freeze-drying machine, recording the total weight, and finally subtracting the original bottle weight from the net weight of the extract. And (4) obtaining the extraction rate of each extract by using the net weight ratio of each extract to the volume of each extracted liquid. Finally, the extraction rates of the extraction components are respectively 0.2%, 0.54%, 4.12% and 5.18%.

4.2 detection of the Effect of the glucose oxidase method on glucose consumption

A96-well cell culture plate is prepared, and a blank zero-adjustment group (KB), a normal group (CK), a model group (IR), a positive control group (MET) and a phellinus igniarius fermentation liquor alcohol-soluble group with different dosages of 4 extraction components C1-C5 are arranged, wherein the positive control group is 100 mu g/mL metformin hydrochloride (MET).

Petroleum ether extract, chloroform extract, ethyl acetate extract, n-butanol extract solution: accurately weighing 40 mg of sample, dissolving in 4 mL of complete culture medium, carrying out ultrasonic treatment at 37 ℃ for 40 min and 5000 rpm, centrifuging for 10 min, and then carrying out filtration sterilization to obtain 5000 mug/mL of sample solution. When added to the wells of the cell culture plate, the final reaction concentrations were diluted to 4. mu.g/mL, 20. mu.g/mL, 100. mu.g/mL, 500. mu.g/mL were labeled C1-C4, respectively, and stored at 4 ℃ until use.

As shown in fig. 18, the petroleum ether extract fraction showed a very significant effect of promoting the glucose consumption in the C2-C4 concentration range, which was increased by 30.2%, 33.5%, 42.9%, respectively, as compared to IR. And compared with the MET group, the effect of promoting the glucose consumption is relatively superior to that of the MET group, and the glucose reducing agent has good blood sugar reducing effect.

As seen from FIG. 19, compared with IR, the chloroform extraction components have very significant promoting effects on glucose consumption of IR-HepG2 cells in the concentration range of C1-C3, the glucose consumption is respectively improved by 61.0%, 63.1% and 46.0%, wherein the promoting effects of the C1-C2 in the low-concentration group on the glucose consumption are equivalent to those of the MET group, and the low-concentration group has good blood sugar reducing effects and great development and utilization potentials.

As seen from fig. 20, compared with IR, the ethyl acetate extract component has a very significant effect of promoting glucose consumption of IR-HepG2 cells at C3 concentration, and the glucose consumption is increased by 37.4%, and has a significant effect of promoting glucose consumption of IR-HepG2 cells at C1 and C2 concentration, and the glucose consumption is increased by 18.7% and 19.9%, respectively. The C4 concentration group also showed some glucose consumption-promoting effect, but was not significant.

As seen from fig. 21, the n-butanol extraction component showed very significant effects in promoting glucose consumption in the C1-C4 concentration range, which was increased by 19.2%, 26.2%, 26.4% and 34.3%, respectively, as compared to the IR group. However, the effects of promoting glucose consumption were all lower than those of the MET group.

2.2 Effect of different Components on the viability of IR-HepG2 cells

As can be seen from fig. 22, the concentration groups of the petroleum ether extract fraction had no significant inhibitory effect on cell activity compared to the CK group.

As can be seen from FIG. 23, the chloroform-extracted fraction had no effect of inhibiting cell activity at C1-C4 as compared with the CK fraction.

As can be seen from fig. 24, the ethyl acetate-extracted fraction had no significant inhibitory effect on cell viability at each concentration group compared to the CK group.

As can be seen from FIG. 25, none of the petroleum ether extract fractions had the effect of inhibiting cell activity at the C1-C4 concentration group as compared with the CK group.

5 ingredient identification

5.1 materials

5.1.1 test samples

The method comprises the steps of extracting a petroleum ether extraction component, a chloroform extraction component, an ethyl acetate extraction component and an n-butanol extraction component from an alcohol extract of phellinus igniarius fermentation liquor.

5.2 methods

5.2.1 treatment of the test sample

5.2.2 conditions of analysis

5.3 results and analysis

5.3.1 detection of Petroleum Ether extracted Components

TABLE 3 detection of chemical composition of petroleum ether extract fraction

As can be seen from the test results of table 3: 33 components are detected in alcohol-soluble petroleum ether extraction components of phellinus igniarius fermentation liquor, the similarity reaches more than 85 percent, 7 components exist, and most of the 33 components are lipid substances. Among them, 7-methyl-Z-tetradecene-1-ol acetate, ethyl isocholate, 1, 2-benzenedicarboxylic acid, bis (2-xylyl) ester and the like have antibacterial activity, and ethyl isocholate has antioxidant activity.

5.3.1 chloroform extraction component detection

TABLE 4 chloroform extraction component chemical composition test results

The results in table 4 show: the method comprises the following steps of detecting 12 components in an alcohol soluble sample Part II chloroform extraction component of phellinus igniarius fermentation liquor, wherein 6 components with the similarity of more than 85 percent exist, and the detected components contain active substances such as phenols, fatty substances, alkaloids and the like.

Wherein, heptacosane and the like have antibacterial activity, 5-hydroxymethyl furfural, [1,2-a ] pyrazine-1, 4-dione, hexahydro-3- (2-methylpropyl) -pyrrole and the like have antioxidant activity and anticancer activity, and 5-hydroxymethyl furfural has hypoglycemic activity.

5.3.3 Ethyl acetate extract component detection

TABLE 5 detection of the chemical composition of the ethyl acetate extract fraction

3 components are detected in the alcohol-soluble ethyl acetate extraction component of the phellinus igniarius fermentation liquor, and only 1 component with the similarity of more than 85 percent is detected. Among them, 2' -methylenebis [ 6- (1, 1-dimethylethyl) -4-methylphenol and diisooctyl phthalate are reported to have antioxidant activity in the literature.

5.3.4 detection of constituents extracted with n-butanol

TABLE 6 detection results of chemical components of n-butanol extract

Table 6 the test results show: and (3) carrying out the co-separation detection on 6 components in the n-butanol extraction component of the phellinus igniarius fermentation liquor alcoholic solution sample, wherein the similarity of the phellinus igniarius fermentation liquor is 85%. Among them, 2-methyl, (S) -1-butanol, 2-hydroxybutyric acid propionate, 2-ethyl-1-hexanol and the like have been reported to have antibacterial activity, and 2-methylbutyric acid propyl ester has antioxidant activity.

6 conclusion and discussion

The research results on the structure and the function of related components are combined with the GC-MS analysis result obtained by the research, and the fact that part of the 4 extraction components contains active components with the effects of resisting bacteria, resisting oxidation, resisting cancer, reducing blood sugar and the like is inferred. Through comparative analysis of the components of the 4 kinds of extraction components of the phellinus igniarius alcohol-soluble sample, the potential pharmacological activity of the phellinus igniarius alcohol-soluble sample can be better discovered, and a certain theoretical basis is provided for subsequent research work.

Example 2

In this example, the preparation method of the phellinus linteus fermentation broth alcoholic solution sample is the same as that in example 1, and the extraction agents are different: and extracting the alcohol-soluble sample of the phellinus igniarius fermentation liquor by using a mixed solvent of petroleum ether and chloroform in a volume ratio of 1:1, a mixed solvent of chloroform and ethyl acetate in a volume ratio of 2:3 and a mixed solvent of petroleum ether, chloroform, ethyl acetate and n-butyl alcohol in a volume ratio of 2:2:3:3 respectively to obtain a mixed solvent extract 1 of 1:1, a mixed solvent extract 2 of 2:3, and a mixed solvent extract 3 of 2:2:3:3 in sequence.

The above extracts were tested according to the method of example 1 and the results are shown in FIGS. 26-28: none of the three mixed solvent extracts had a significant effect on the activity of insulin resistant HepG2 cells.

On the basis of the research of example 1, the extractant for extracting the active ingredients of the phellinus linteus fermentation broth is already optimized, and the experiment of this example was performed in order to further improve the extraction efficiency. In the test, a plurality of solvents and a plurality of proportions of the mixed solvent are selected, and in order to save space in the specification, only the mixed solvent with a good effect and the proportions thereof are listed. Through the test, the ethanol-soluble sample of the phellinus igniarius fermentation liquid is extracted by using a mixed solvent with the volume ratio of petroleum ether to chloroform being 1:1, a mixed solvent with the volume ratio of chloroform to ethyl acetate being 2:3 and a mixed solvent with the volume ratio of petroleum ether, chloroform, ethyl acetate and n-butyl alcohol being 2:2:3:3 respectively, and compared with a single petroleum ether, chloroform, ethyl acetate and n-butyl alcohol extracting agent, the phellinus igniarius fermentation liquid has higher extraction efficiency and better effect of the obtained active ingredients. The effect of the obtained component is not good when the ethyl acetate is used alone for extraction, but when the ethyl acetate, the petroleum ether, the chloroform and the n-butyl alcohol are compounded and used according to a specific proportion, the extraction efficiency can be improved, and the active component with better effect can be obtained.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Sequence listing

<110> Qingdao agricultural university

<120> preparation method of phellinus igniarius active ingredient and application of phellinus igniarius active ingredient in auxiliary hypoglycemic drugs or foods

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 764

<212> DNA

<213> Phellinus linteus (Sanghuangpolus sanghuang)

<400> 1

cgatgggtac tgcggaggtc attatcgagt tttgaaagcg agacctgctg ctggtgcgaa 60

atcgcgcatg tgcacggtct tcgcgctcaa atccaactca aacccctgtg caccttatat 120

atcgcgagtc gaagttagta gcctgaggtc ttgtaagtaa ttagtagaag ggcgaaagcg 180

agtcttgctc gttaggtagc ctttcgaaaa tgaaagcgag tgcgtcgggt gaagacttcg 240

gcttgtcgtt acaaaacacc ttatattgtc tttgtgaatg taatgctcct tgtgggcgaa 300

aataaataca actttcaaca acggatctct tggctctcgc atcgatgaag aacgcagcga 360

aatgcgataa gtaatgtgaa ttgcagaatt cagtgaatca tcgaatcttt gaacgcacct 420

tgcgcccctt ggtattccga ggggcatgcc tgtttgagtg tcatgtttat ctcaaaccgc 480

tcgtctttct taattgaagg gcttgaggtt tggacttgga ggtttactgc tggcgccttt 540

cgaggggtcg gctcctctta aatacattag ctgggctttg gctcgcgttt acggtgtaat 600

agttgattcc attcaccaac gagcgcttgc ctgacgagct tgcttctagc cgtccgcgtc 660

gtcggacaag gagtcacctc cttcttgaca cctttgacct caaatcaggt aggattaccc 720

gccgacttac taattaaagg gggggggggg gggaagaaat tgaa 764

Claims

1. A preparation method of a phellinus igniarius active ingredient with a hypoglycemic effect is characterized by comprising the following steps:

(2) sequentially carrying out fractional extraction on the phellinus igniarius fermentation broth alcoholic solution sample by using four organic solvents of petroleum ether, chloroform, ethyl acetate and n-butanol to obtain a petroleum ether extract, a chloroform extract, an ethyl acetate extract and an n-butanol extract;

wherein the Phellinus linteus strainSanghuangporus sanghuangThe preservation number is CGMCC NO. 21068.

2. A preparation method of a phellinus igniarius active ingredient with a hypoglycemic effect is characterized by comprising the following steps:

(2) respectively extracting the phellinus igniarius fermentation liquor alcohol-soluble sample by using a mixed solvent of petroleum ether and chloroform in a volume ratio of 1:1, a mixed solvent of chloroform and ethyl acetate in a volume ratio of 2:3 and a mixed solvent of petroleum ether, chloroform, ethyl acetate and n-butyl alcohol in a volume ratio of 2:2:3:3 to obtain a mixed solvent extract 1 of 1:1, a mixed solvent extract 2 of 2:3, and a mixed solvent extract 3 of 2:2:3:3 in sequence;

3. The preparation method according to claim 1, wherein the volume ratio of the alcoholic solution sample of the phellinus linteus fermentation broth to the petroleum ether, chloroform, ethyl acetate and n-butanol in the step (2) is 1: 1-5.

4. The method according to claim 1, wherein the extraction time of the petroleum ether, chloroform, ethyl acetate and n-butanol in the step (2) is 2 hours.

5. The preparation method according to claim 1, wherein the number of extraction times of the petroleum ether, chloroform, ethyl acetate and n-butanol in the step (2) is 2:2: 6: 8.

6. the method according to claim 1, wherein the fermentation process of the phellinus igniarius strain fermentation broth comprises: inoculating liquid strain 5-10%; culturing for the first 2-3 days at 27-29 deg.C and 110r/min in dark place under shaking; on the 3 rd to 8 th days of culture, setting the temperature at 25-27 ℃, rotating speed at 130-; on 8-12 days of culture, the temperature is set at 23-25 ℃, the rotation speed is 110-.

7. The method according to claim 6, wherein the liquid spawn is prepared by:

8. The method according to claim 7, wherein the solid medium is prepared by: 20 g of bran and 30 g of corn flour, and putting all the components into distilled water at 100 ℃ for 25 min; filtering with 400 mesh gauze, adding 30 g glucose and 1 g KH into the filtrate₂PO₄、0.5 g MgSO₄·7H₂O, adding 4 g of yeast extract, 3 g of peptone and 20 g of agar at 100 ℃, and subpackaging a flat plate with the diameter of 9 cm after the constant volume of 1000 mL of distilled water is obtained;

preparing a liquid culture medium: 20 g of bran and 30 g of corn flour are all put into distilled water for 25 min at 100 ℃, after the distilled water is filtered by 400 meshes of gauze, 30 g of glucose and 1 gKH are added into the filtrate₂PO₄、0.5 g MgSO₄·7H₂And O, adding 4 g of yeast extract and 3 g of peptone at 100 ℃, and subpackaging in a 500 mL triangular flask after the constant volume of distilled water is 1000 mL.

9. Use of Phellinus linteus active ingredient prepared by the preparation method according to any one of claims 1 to 8 in the preparation of hypoglycemic drugs.

10. Use of Phellinus linteus active ingredient prepared by the preparation method according to any one of claims 1 to 8 in preparing hypolipidemic drugs.