CN104211633B

CN104211633B - Isoindole compounds and application thereof

Info

Publication number: CN104211633B
Application number: CN201410394697.5A
Authority: CN
Inventors: 刘宏伟; 汪锴; 宝丽; 韩俊杰
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2014-08-12
Filing date: 2014-08-12
Publication date: 2016-06-15
Anticipated expiration: 2034-08-12
Also published as: CN104211633A

Abstract

The present invention discloses isoindole compounds and application thereof. The present invention discloses one group of isoindole compounds. Disclosed by the invention from deposit number be CGMCC? Hericium erinaceus (Bull. Ex Fr.) Pers. (the Hericium of No.9224? erinaceus) compound (wherein having 9 for new isoindole compounds) that L547 isolates has hypoglycemic and antitumor action, has broad application prospects.

Description

Isoindole compounds and application thereof

Technical field

The present invention relates to isoindole compounds and application thereof, belong to pharmacy field.

Background technology

Containing diterpene skeleton and pyranone in Hericium erinaceus (Bull. Ex Fr.) Pers., isoindole compounds, diterpene-kind compound has the activity that promotion nerve growth factor (NGF) is synthesized. There is lower molecular weight NGF inductor active by force and it is considered as treatment nerve degenerative diseases such as the potential drug of Alzheimer ' s disease.

Hericium erinaceus (Bull. Ex Fr.) Pers. has another name called hedgehog hydnum, Hericium erinaceus (Bull. Ex Fr.) Pers., hedgehog hydnum mushroom etc., is a kind of large-scale fungi, belongs to Basidiomycetes, Aphyllophorales, hedgehog fungus section, hedgehog hydnum genus. It is reported, the contained abundant nutritive ingredient of Hericium erinaceus (Bull. Ex Fr.) Pers.: (Yuan Yahong, Yue Tianli, the Wang Yunyang etc. such as protein, fat, Mierocrystalline cellulose, polysaccharide, amino acid. Hericium erinaceus (Bull. Ex Fr.) Pers. nutritive medium Study on extraction. Chinese food journal, 2005,2,65-68. ). Hericium erinaceus (Bull. Ex Fr.) Pers. is applied to gastrointestinal illness treatment clinically, and primary treatment gastritis, duodenal ulcer etc., made hedgehog hydnum syrup, hedgehog hydnum biscuit, hericium erinaceus sheet etc. What at present research was maximum is Hericium erinaceus (Bull. Ex Fr.) Pers. polysaccharide, has physiological function (Chen Ming such as improving immunizing power, antitumor, anti-ageing, reducing blood-fat. The conventional kind of anticancer mushroom medicine and recent studies progress. Edible mushrooms, 1994,40-41. Lu Yaohuan, Li Changli, Zhou Yufen. The experiment of mouse anti-reflecting fatigue effect is studied by Hericium erinaceus (Bull. Ex Fr.) Pers.. Journal of physiology, 1996,98-101. ) in addition Hericium erinaceus (Bull. Ex Fr.) Pers. promote appetite in addition, strengthen barrier of gastric mucosa function, improve lymphocyte transformation rate, promote the effects such as white corpuscle.

Summary of the invention

It is an object of the invention to provide isoindole compounds and application thereof.

The present invention provides the compound shown in formula I, formula I:

In formula I,

Compound shown in formula II, formula II:

In formula II,

Compound shown in formula III, formula III:

In formula III,

Isoindole compounds shown in following compound 1-5,7-10 is arbitrary also belongs to protection scope of the present invention:

(1) compound 1, structural formula is

2) compound 2, structural formula is

(3) compound 3, structural formula is

(4) compound 4, structural formula is

(5) compound 5, structural formula is

(6) compound 7, structural formula is

(7) compound 8, structural formula is

(8) compound 9, structural formula is

(9) compound 10, structural formula is

The described compound 1-compound 5 synthesized by chemical process, compound 7-compound 10 also belongs to protection scope of the present invention.

A kind of method preparing above-mentioned isoindole compounds 1 also belongs to protection scope of the present invention, comprise the steps: that the extract of Hericium erinaceus (Bull. Ex Fr.) Pers. mycelium fermentation thing by preserving number is CGMCCNo.9224 carries out silica gel column chromatography separation, take volume ratio as 1:0, 50:1, 30:1, 20:1, 15:1, 10:1, 4:1, the normal hexane of 2:1: eluent ethyl acetate agent and volume ratio are 1:0, 100:1, 50:1, the methylene dichloride of 30:1: methanol-eluted fractions agent carries out gradient elution successively, each eluent 3 retention volume 1, finally obtain the methylene dichloride that volume ratio is 30:1: the wash-out composition of methanol-eluted fractions agent, by its freeze-drying, it is denoted as HE-E1-19,HE-E1-19 is carried out the separation of ODS reverse phase silica gel post, it is 30% with volumn concentration, the aqueous solution of 50% methyl alcohol carries out wash-out successively as eluent, each eluent 3 retention volume 2, at volumn concentration be the methanol aqueous solution of 50% as eluent, when elution volume is 280-300ml, obtain evaporating points 10, by its freeze-drying, it is denoted as HE-E1-19-10; HE-E1-19-10 is carried out HPLC preparation, the sample solution of 10mg/ml it is formulated as with chromatogram methyl alcohol, applied sample amount is 15ul, with Kromasil10 × 250mmC18 semipreparative column, post temperature is 25 DEG C, is that moving phase carries out wash-out with the sour water containing volumn concentration 26% acetonitrile, flow velocity is 2ml/min, 210nm wavelength detects, and obtains the chromatographic peak that retention time is 26 minutes, to obtain final product;

Described retention volume 1 is 500ml;

Described retention volume 2 is 100ml;

The water of acid described in described HPLC is volumn concentration is the aqueous solution of 0.01% trifluoroacetic acid;

The extract that described preserving number is the Hericium erinaceus (Bull. Ex Fr.) Pers. mycelium fermentation thing of CGMCCNo.9224 is prepared as follows:

(1) by preserving number be CGMCCNo.9224 Hericium erinaceus (Bull. Ex Fr.) Pers. mycelium liquid medium within cultivate, obtain seed culture fluid; Seed culture fluid is inoculated in solid medium and cultivates, obtain fermented product;

Described liquid nutrient medium is made up of solvent and solute, and solvent is water, and solute is glucose, malt extract and yeast extract; The concentration of described glucose in described liquid nutrient medium is 3.0-6.0g/L, and the concentration of described malt extract in described liquid nutrient medium is 8.0-10.0g/L, and the concentration of described yeast extract in described liquid nutrient medium is 2.0-5.0g/L;

The concentration of described glucose in described liquid nutrient medium is specially 4.0g/L, and the concentration of described malt extract in described liquid nutrient medium is specially 10.0g/L, and the concentration of described yeast extract in described liquid nutrient medium is specially 4.0g/L;

Described malt extract is purchased from Beijing Suo Laibao Science and Technology Ltd.;

Described yeast extract is purchased from Beijing Suo Laibao Science and Technology Ltd.;

The condition carrying out in described liquid medium within cultivating is 25-28 DEG C, lucifuge, cultivates 7-10 days, is specially 25 DEG C, lucifuge, cultivates 7 days or 10 days;

Described solid medium is that the arbitrary shown rice in following (1)-(6) and water form according to the ratio of 80-100g:100ml:

(1) rice;

(2) brown rice;

(3) glutinous rice;

(4) seed of Job's tears;

(5) purple rice;

(6) black rice;

Described in described solid medium, the ratio of rice and described water is specially 80g:100ml;

The described condition being inoculated in solid medium by seed culture fluid to carry out cultivating is 25-28 DEG C, lucifuge, cultivates 30-50 days, is specially 25 DEG C, lucifuge, cultivates 30 days or 40 days;

Described in described seed culture fluid and described solid medium, the ratio of rice is 10ml:80-100g;

Described mycelium is inoculated on PDA substratum by the Hericium erinaceus (Bull. Ex Fr.) Pers. that preserving number is CGMCCNo.9224 to carry out preculture to obtain;

The condition of described preculture is 25-28 DEG C, lucifuge, cultivates until mycelium covers with PDA media surface, is specially 25 DEG C, lucifuge, cultivates until mycelium covers with PDA media surface;

(2) fermented product freeze-drying step () obtained, then it is carried out refluxing extraction with aqueous ethanolic solution 95-105 DEG C that volumn concentration is 90-98%, obtain extracting solution, to obtain final product;

After described fermented product freeze-drying, ratio with described aqueous ethanolic solution is specially 1g:3-5ml;

In described aqueous ethanolic solution, the volumn concentration of ethanol is specially 95%;

The temperature of described refluxing extraction is specially 100 DEG C;

The number of times of described extraction is 3-4 time, is specially 3 times;

The time of described refluxing extraction is 50-70min/ time, is specially 60min/ time.

One prepares above-mentioned isoindole compounds 2, compound 3, the method of compound 4 and/or compound 5 also belongs to protection scope of the present invention, comprise the steps: that the extract of Hericium erinaceus (Bull. Ex Fr.) Pers. mycelium fermentation thing by preserving number is CGMCCNo.9224 carries out silica gel column chromatography separation, take volume ratio as 1:0, 50:1, 30:1, 20:1, 15:1, 10:1, 4:1, the normal hexane of 2:1: eluent ethyl acetate agent and volume ratio are 1:0, 100:1, 50:1, the methylene dichloride of 30:1: methanol-eluted fractions agent carries out gradient elution successively, each eluent 3 retention volume 1, finally obtain the methylene dichloride that volume ratio is 30:1: the wash-out composition of methanol-eluted fractions agent, by its freeze-drying, it is denoted as HE-E1-19, HE-E1-19 is carried out the separation of ODS reverse phase silica gel post, it is 30% with volumn concentration, 50%, the aqueous solution of 70% methyl alcohol carries out wash-out successively as eluent, each eluent 3 retention volume 2, volumn concentration be 70% methanol aqueous solution as eluent, when elution volume is 60-100ml, obtain evaporating points 12, by its freeze-drying, it is denoted as HE-E1-19-12, HE-E1-19-12 being carried out HPLC preparation, is formulated as the sample solution of 10mg/ml with chromatogram methyl alcohol, applied sample amount is 15ul, with Kromasil10 × 250mmC18 semipreparative column, post temperature is 25 DEG C, is that moving phase carries out wash-out with the sour water containing volumn concentration 22% acetonitrile, flow velocity is that 2ml/min, 210nm wavelength detects, and obtains retention time and is respectively 23, minute 25.2,27.1,29.2 chromatographic peak, namely correspondence obtains compound 2, compound 3, compound 4, compound 5,

Described retention volume 1 is 500ml;

Described retention volume 2 is 100ml;

(1) rice;

(2) brown rice;

(3) glutinous rice;

(4) seed of Job's tears;

(5) purple rice;

(6) black rice;

The temperature of described refluxing extraction is specially 100 DEG C;

The number of times of described extraction is 3-4 time, is specially 3 times;

A kind of method preparing above-mentioned isoindole compounds 7 and/or compound 8 also belongs to protection scope of the present invention, comprise the steps: that the extract of Hericium erinaceus (Bull. Ex Fr.) Pers. mycelium fermentation thing by preserving number is CGMCCNo.9224 carries out silica gel column chromatography separation, take volume ratio as 1:0, 50:1, 30:1, 20:1, 15:1, 10:1, 4:1, the normal hexane of 2:1: eluent ethyl acetate agent and volume ratio are 1:0, 100:1, 50:1, the methylene dichloride of 30:1: methanol-eluted fractions agent carries out gradient elution successively, each eluent 3 retention volume 1, finally obtain the methylene dichloride that volume ratio is 30:1: the wash-out composition of methanol-eluted fractions agent, by its freeze-drying, it is denoted as HE-E1-19, HE-E1-19 is carried out the separation of ODS reverse phase silica gel post, it is 30% with volumn concentration, 50%, the aqueous solution of 70% methyl alcohol carries out wash-out successively as eluent, each eluent 3 retention volume 2, volumn concentration be 70% methanol aqueous solution as eluent, when elution volume is 220-250ml, obtain evaporating points 15, by its freeze-drying, it is denoted as HE-E1-19-15, HE-E1-19-15 is carried out HPLC preparation, the sample solution being formulated as 10mg/ml with chromatogram methyl alcohol, applied sample amount is 15ul, with Kromasil10 × 250mmC18 semipreparative column, post temperature is 25 DEG C, with being that moving phase carries out wash-out containing the sour water of volumn concentration 30% acetonitrile, flow velocity is that 2ml/min, 210nm wavelength detects, obtain retention time and it is respectively 32, the chromatographic peak of 35.5 minutes, namely correspondence obtains compound 7, compound 8,

Described retention volume 1 is 500ml;

Described retention volume 2 is 100ml;

(1) by preserving number be CGMCCNo.9224 Hericium erinaceus (Bull. Ex Fr.) Pers. mycelium liquid medium within cultivate, obtain seed culture fluid;Seed culture fluid is inoculated in solid medium and cultivates, obtain fermented product;

(1) rice;

(2) brown rice;

(3) glutinous rice;

(4) seed of Job's tears;

(5) purple rice;

(6) black rice;

The temperature of described refluxing extraction is specially 100 DEG C;

The number of times of described extraction is 3-4 time, is specially 3 times;

A kind of method preparing above-mentioned isoindole compounds 9 and/or compound 10 also belongs to protection scope of the present invention, comprise the steps: that the extract of Hericium erinaceus (Bull. Ex Fr.) Pers. mycelium fermentation thing by preserving number is CGMCCNo.9224 carries out silica gel column chromatography separation, take volume ratio as 1:0, 50:1, 30:1, 20:1, 15:1, 10:1, 4:1, the normal hexane of 2:1: eluent ethyl acetate agent and volume ratio are 1:0, 100:1, 50:1, the methylene dichloride of 30:1: methanol-eluted fractions agent carries out gradient elution successively, each eluent 3 retention volume 1, finally obtain the methylene dichloride that volume ratio is 30:1: the wash-out composition of methanol-eluted fractions agent, by its freeze-drying, it is denoted as HE-E1-19, HE-E1-19 is carried out the separation of ODS reverse phase silica gel post, it is 30% with volumn concentration, 50%, the aqueous solution of 70% methyl alcohol carries out wash-out successively as eluent, each eluent 3 retention volume 2, volumn concentration be 70% methanol aqueous solution as eluent, when elution volume is 260-280ml, obtain evaporating points 16, by its freeze-drying, it is denoted as HE-E1-19-16,HE-E1-19-16 is carried out HPLC preparation, the sample solution being formulated as 10mg/ml with chromatogram methyl alcohol, applied sample amount is 15ul, with Kromasil10 × 250mmC18 semipreparative column, post temperature is 25 DEG C, with being that moving phase carries out wash-out containing the sour water of volumn concentration 40% acetonitrile, flow velocity is that 2ml/min, 210nm wavelength detects, obtain retention time and it is respectively 26.1, the chromatographic peak of 27.5 minutes, namely correspondence obtains compound 9, compound 10;

Described retention volume 1 is 500ml;

Described retention volume 2 is 100ml;

(1) rice;

(2) brown rice;

(3) glutinous rice;

(4) seed of Job's tears;

(5) purple rice;

(6) black rice;

The temperature of described refluxing extraction is specially 100 DEG C;

The number of times of described extraction is 3-4 time, is specially 3 times;

In above-mentioned arbitrary described method, described silica gel column chromatography be separated in the fill method of pillar and loading method as follows: first fill post with 200g silica gel, by described preserving number be again CGMCCNo.9224 Hericium erinaceus (Bull. Ex Fr.) Pers. mycelium fermentation thing extract dissolve with methanol after with 15g silica gel mixed sample, carry out loading;

Described preserving number is the quality during extract loading of the Hericium erinaceus (Bull. Ex Fr.) Pers. mycelium fermentation thing of CGMCCNo.9224 is 15g;

The specification of described silica gel column chromatography separation center pillar is diameter 5cm, high 40cm;

Described ODS reverse phase silica gel post be separated in the fill method of pillar and loading method as follows: first fill post with 50gODS reverse phase silica gel, then mix sample by after described HE-E1-19 dissolve with methanol with 5gODS reverse phase silica gel, carry out loading;

The quality during loading of described HE-E1-19 is 3.45g;

The diameter of the pillar of described ODS reverse phase silica gel post is 3cm, and height is 30cm.

The application in the product preparing prevention and/or treatment diabetes or the product suppressing blood sugar to raise of above-mentioned isoindole compounds, following compound shown in (1) or (2) also belongs to protection scope of the present invention:

The application in preparation prevention and/or the product of Therapeutic cancer or the product of anticancer growth and/or propagation of above-mentioned isoindole compounds, following compound shown in (1) or (2) also belongs to protection scope of the present invention:

In above-mentioned application, described cancer is liver cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, leukemia, cancer of the stomach or mammary cancer;

Described cancer cells is specially liver cancer cell, prostate cancer cell, lung carcinoma cell, colorectal cancer cell, cervical cancer cell, leukemia cell, stomach cancer cell or breast cancer cell, then is specially human liver cancer cell, Human Prostate Cancer Cells, human lung carcinoma cell, Human colorectal cancer cells, human cervical carcinoma cell, human leukemia cell, gastric carcinoma cells or human breast cancer cell;

Described human liver cancer cell is specially human liver cancer cell HepG2, described Human Prostate Cancer Cells is specially Human Prostate Cancer Cells PC3, and described human lung carcinoma cell is specially human lung cancer cell A549, described Human colorectal cancer cells is specially Human colorectal cancer cells HCT-15, described human cervical carcinoma cell is specially human cervical carcinoma cell HeLa, described human leukemia cell is specially Leukemia K562 cell, described gastric carcinoma cells is specially SGC-7901 cells, described human breast cancer cell is specially human breast cancer cell line Bcap-37.

Provided by the invention be CGMCCNo.9224 from deposit number the compound (wherein having 9 for new isoindole compounds) isolated of Hericium erinaceus (Bull. Ex Fr.) Pers. (Hericiumerinaceus) L547 there is hypoglycemic and antitumor action, have broad application prospects.

Accompanying drawing explanation

Fig. 1 is separated 11 compounds obtained in L547.

Fig. 2 is the high-efficient liquid phase chromatogram of L547 fermented product HE-E1.

Fig. 3 is the high-efficient liquid phase chromatogram of L547 fermented product HE-E2.

Fig. 4 is the high-efficient liquid phase chromatogram of L547 fermented product HE-E3.

Fig. 5 is the high-efficient liquid phase chromatogram of L547 fermented product HE-E4.

Fig. 6 is the high-efficient liquid phase chromatogram of L547 fermented product HE-E5.

Fig. 7 is the high-efficient liquid phase chromatogram of L547 fermented product HE-E6.

Embodiment

The experimental technique used in following embodiment if no special instructions, is ordinary method.

Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.

Alpha-glucosidase is purchased from Sigma, and article No. is G5003.

4-oil of mirbane-α-D-glucopyranoside is purchased from Beijing lark prestige Science and Technology Ltd., and article No. is 270305.

U-9889 is purchased from Beijing lark prestige Science and Technology Ltd., and article No. is M02540.

Wistar male rat (SPF level) is purchased from Beijing Wei Tonglihua laboratory animal technology company limited.

High glucose and high fat feed: be made up of basis mouse feed (purchased from Beijing Wei Tonglihua laboratory animal technology company limited), sucrose, refining lard and yolk; The mass percentage of described lard in described high glucose and high fat feed is 18%, the mass percentage of described sucrose in described high glucose and high fat feed is 20%, the mass percentage of described yolk in described high glucose and high fat feed is 3%, and the mass percentage of described basis mouse feed in described high glucose and high fat feed is 59%.

Malt extract is purchased from Beijing Suo Laibao Science and Technology Ltd..

Yeast extract is purchased from Beijing Suo Laibao Science and Technology Ltd..

Human liver cancer cell HepG2 is purchased from ATCC, and catalog number is HB-8065.

Human Prostate Cancer Cells PC3 is purchased from ATCC, and catalog number is CRL-1435.

Human lung cancer cell A549 is purchased from ATCC, and catalog number is CCL-185.

Human colorectal cancer cells HCT-15 is purchased from ATCC, and catalog number is CCL-225.

Human cervical carcinoma cell Hela is purchased from ATCC, and catalog number is CCL-2.

K562 cell is purchased from Zhong Shan medical university Experimental Animal Center.

SGC-7901 cells is purchased from ATCC, and catalog number is SGC-7901.

Human breast cancer cell line Bcap-37 is purchased from ATCC, and catalog number is HCC1428.

The cultivation of embodiment 1, bacterium, the preparation of compound and functional evaluation

Hericium erinaceus (Bull. Ex Fr.) Pers. bacterial strain L547 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 16th, 2014 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCCNo.9224, classification called after Hericium erinaceus (Bull. Ex Fr.) Pers. (Hericiumerinaceus).

One, bacterial strain activation

By Hericium erinaceus (Bull. Ex Fr.) Pers. bacterial strain L547 strain inoculation on the inclined-plane of PDA substratum, carrying out preculture, the condition of preculture is: 25 DEG C, lucifuge, cultivates 7 days (25 DEG C-28 DEG C, lucifuge, cultivates 7-10 days).

Two, seed culture

In the preculture of step one, cover with behind whole inclined-plane until mycelia, aseptically mycelium is transferred in the following liquid nutrient medium of PDA and cultivates, obtain seed culture fluid. The condition cultivated is: 25 DEG C, lucifuge, cultivates 7 days (25 DEG C-28 DEG C, lucifuge, cultivates 7-10 days).

Liquid nutrient medium: glucose 4.0g/L, MaltExtract (malt extract) 10.0g/L, YeastExtract (yeast extract) 4.0g/L, surplus is water.

(in aforesaid liquid substratum, glucose 3.0-6.0g/L, MaltExtract (malt extract) 8.0-10.0g/L, YeastExtract (yeast extract) 2.0-5.0g/L)

Three, fermentation culture

After liquid nutrient medium cultivates 7 days, getting the seed culture fluid that 10ml step 2 obtains is inoculated in the 500ml triangular flask of the following solid medium that 80g (80-100g) rice is housed respectively, repeated inoculation 20 bottles altogether, 25 DEG C, lucifuge, cultivate 40 days (25 DEG C-28 DEG C, lucifuge, cultivate 30-50 days), obtain the fermented product of L547.

Solid medium: be made up of 80g rice and 100ml water.

(in above-mentioned solid medium, the ratio of rice and water is at 80-100g:100ml)

Four, the preparation of extract

L547 fermented product lyophilize step 3 obtained, weighs, and weight is 1800 grams. With the aqueous solution (ratio of the aqueous solution of fermented product and ethanol is 1g:3-5ml) of 6L volumn concentration 95% (90%-98%) ethanol, 100 DEG C of (95 DEG C-105 DEG C) refluxing extraction 3 times (3-4 time), each 1 hour (50-70min), merge ethanol extract, concentrating under reduced pressure drying obtains 19.6 grams of extracts, and extract is denoted as HE-E1.

Five, the separation and purification of compound

(1) crude extract HE-E1 is separated by silica gel column chromatography, first fills post (pillar is diameter 5cm, the glass column of high 40cm) with 200g silica gel, then by 15gHE-E1 dissolve with methanol and 15g silica gel mixed sample, carry out loading. Taking normal hexane: ethyl acetate system (normal hexane: the volume ratio of ethyl acetate is as 1:0,50:1,30:1,20:1,15:1,10:1,4:1,2:1), methylene dichloride: methanol system (methylene dichloride: the volume ratio of methyl alcohol is 1:0,100:1,50:1,30:1,20:1,10:1,5:1) carrying out gradient elution successively, each gradient washes 3 retention volume, each volume 500ml, analyze according to thin-layer chromatography chromatographic behavior, at normal hexane: ethyl acetate (volume ratio is 1:0) system obtains evaporating point 1; At normal hexane: ethyl acetate (volume ratio is 50:1) obtains evaporating points 2,3,4; At normal hexane: ethyl acetate (volume ratio is 30:1) obtains evaporating points 5,6,7; At normal hexane: ethyl acetate (volume ratio is 20:1) obtains evaporating points 8,9,10; At normal hexane: ethyl acetate (1 volume ratio is 10:1) obtains evaporating points 11,12; At normal hexane: ethyl acetate (volume ratio is 4:1) obtains evaporating points 13,14; At normal hexane: ethyl acetate (volume ratio is 2:1) obtains evaporating points 15; At methylene dichloride: methyl alcohol (volume ratio is 1:0) obtains evaporating points 16; At methylene dichloride: methyl alcohol (volume ratio is 100:1) obtains evaporating points 17; At methylene dichloride: methyl alcohol (volume ratio is 50:1) obtains evaporating points 18; At methylene dichloride: methyl alcohol (volume ratio is 30:1) obtains evaporating points 19; At methylene dichloride: methyl alcohol (volume ratio is 20:1) obtains evaporating points 20; At methylene dichloride: methyl alcohol (volume ratio is 10:1,5:1) obtains evaporating points 21; Obtain 21 altogether to evaporate point, after respectively evaporating point lyophilize, called after HE-E1-1 to HE-E1-21 respectively.

(2) HE-E1-12 (1.45g) utilizes LH20 gel filtration chromatography to be separated, before loading, (chromatography column diameter is 1cm to first saturated chromatography column, height is 50cm, the quality of column packing LH20 is 40g), gel is shaken even with the methanol aqueous solution of volumn concentration 50%, upright shaft, allow its natural subsidence, open switch after half an hour, flow out 500ml elutriant; Subsequently with the methanol aqueous solution of 3ml volumn concentration 50% by HE-E1-12 (1.45g) sample dissolution, post in wet method, add eluent (methanol aqueous solution of volumn concentration 50%) wash-out, 15ml collects an elutriant, access and obtain by the monitoring of TLC point plate after elutriant, observation process judges different fractions by irradiating 254nm ultraviolet lamp, obtain 15 and evaporate point, after respectively evaporating point lyophilize, called after HE-E1-12-1 to HE-E1-12-15 respectively. Wherein, HE-E1-12-6 is evaporating point of collecting of 120-150ml wash-out, and HE-E1-12-12 is evaporating point of 260-300ml wash-out collection.

Carrying out HPLC preparation to evaporating point HE-E1-12-6 by eluent of the aqueous solution of volumn concentration 65% methyl alcohol, flow velocity is 2ml/min, collects the chromatographic peak of 24.5 minutes, obtains compound 6; Carrying out HPLC preparation to evaporating point HE-E1-12-12 by eluent of the aqueous solution of volumn concentration 70% methyl alcohol, flow velocity is 2ml/min, collects the chromatographic peak of 32 minutes, obtains compound 11.

All the other conditions of above-mentioned HPLC are as follows:

Taking chromatogram methyl alcohol by the solution of sample preparation as 10mg/ml, applied sample amount is 15ul, and chromatographic column is Kromasil10 × 250mmC18 semipreparative column, and post temperature is 25 DEG C, and 210nm wavelength detects.

(3) HE-E1-19 (3.45g) utilizes ODS reverse phase silica gel post to be separated, (diameter of pillar is 3cm first to fill post with 50gODS reverse phase silica gel, height is 30cm), again HE-E1-19 (3.45g) dissolve with methanol and 5gODS reverse phase silica gel are mixed sample, carry out loading. It is 30% with volumn concentration, 50%, 70%, the aqueous solution of 90% methyl alcohol carries out wash-out successively, each eluent system washes 3 retention volume, and each retention volume is 100ml, according to thin-layer chromatography chromatographic behavior, irradiate 254nm ultraviolet lamp and judge different fractions, be that the methanol aqueous solution of 30% obtains evaporating a point 1-5 at volumn concentration; It is that the methanol aqueous solution system of 50% obtains evaporating a point 6-10 (elution volume wherein evaporating points 10 is 280-300ml) at volumn concentration; It is that the methanol aqueous solution system of 70% obtains evaporating a point 11-16 (elution volume wherein evaporating points 12 is 60-100ml, and the elution volume evaporating points 15 is 220-250ml, and the elution volume evaporating points 16 is 260-280ml) at volumn concentration; It is that the methanol aqueous solution system of 90% obtains evaporating a point 17-20 at volumn concentration; Obtain 20 sons altogether to evaporate point, each is evaporated a point lyophilize, respectively called after HE-E1-19-1 to HE-E1-19-20.

It is that eluent carry out HPLC preparation to HE-E1-19-10 taking the sour water of the volumn concentration 26% acetonitrile aqueous solution of 0.01% trifluoroacetic acid (the sour water herein be) solution as volumn concentration, flow velocity is 2ml/min, collect the chromatographic peak of 26 minutes, obtain compound 1; It is that eluent carry out HPLC preparation to HE-E1-19-12 taking the volumn concentration 22% acedic acid water aqueous solution of 0.01% trifluoroacetic acid (the sour water herein be) solution as volumn concentration, flow velocity is 2ml/min, collect 23 respectively, 25.2, minute 27.1,29.2 chromatographic peak obtains compound 2,3,4,5; It is that eluent carry out HPLC preparation to HE-E1-19-15 taking the volumn concentration 30% acedic acid water aqueous solution of 0.01% trifluoroacetic acid (the sour water herein be) solution as volumn concentration, flow velocity is 2ml/min, the chromatographic peak collected 32,35.5 minutes respectively obtains compound 7,8; It is that eluent carry out HPLC preparation to HE-E1-19-16 taking the volumn concentration 40% acedic acid water aqueous solution of 0.01% trifluoroacetic acid (the sour water herein be) solution as volumn concentration, flow velocity is 2ml/min, the chromatographic peak collected 26.1,27.5 minute obtains compound 9,10.

All the other conditions of above-mentioned HPLC are as follows:

Taking chromatogram methyl alcohol by the solution of sample preparation as 10mg/ml, applied sample amount is that 15ul is each, and chromatographic column is Kromasil10 × 250mmC18 semipreparative column, and post temperature is 25 DEG C, and 210nm wavelength detects

Six, the qualification of compound

By apply various Modern spectroscopy technology as mass spectrum, the structural formula that infrared, nuclear magnetic resonance technique identifies above-claimed cpd 1-11 as shown in Figure 1, wherein compound 1-5,7-10 is new isoindole compounds.

The NMR of each compound confirms data as shown in table 1-table 3.

The NMR data of table 1 compound 2-4

The NMR data of table 2 compound 1,5

The NMR data of table 3 compound 7-10

¹HNMRat500Hz,¹³CNMRat125Hz.

Seven, high performance liquid chromatography detection

Crude extract HE-E1 is carried out high performance liquid chromatography detection.

High-efficient liquid phase chemistry fingerprint map analyzing condition is as follows:

Use Agilent1200 high performance liquid chromatograph, quaternary gradient pump, DAD detector, Agilent chromatographic working station. Chromatographic column YMCC8 analytical column (4.6mm × 150mm, 5 μm), moving phase is the aqueous solution of the trifluoroacetic acid of acetonitrile-volumn concentration 0.04%, gradient elution (volumn concentration of elution program and each composition is as follows) is carried out, flow velocity 1.00mL/min when temperature room temperature (20～30 DEG C). Being dissolved by crude extract HE-E1 acetonitrile, be made into the solution of 10mg/mL, sample size 10 μ L, ultraviolet detection wavelength is 210nm.

Gradient elution step is as follows:

The color atlas obtaining extract HE-E1 according to above-mentioned elution requirement is as shown in Figure 2.

In Fig. 2, the peak that 1-11 indicates and the compound 1-11 one_to_one corresponding that step 5 obtains.

Eight, the function of extract

(1) hypoglycemic activity

1, external alpha-glucosidase (α-Glucosidase) inhibit activities test

The preparation of test sample solution: final concentration is respectively the crude extract HE-E1 of 200 μ g/ml, 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, final concentration is each compound 1-11 of 25.0 μMs, 12.5 μMs, 6.3 μMs, 3.1 μMs, after crude extract HE-E1 and each compound being dissolved in a small amount of DMSO during preparation, again with distilled water diluting to respective concentration, control the final volumn concentration < 1% of DMSO;

The preparation of positive control sample solution: final concentration is respectively the acarbose solution of 800 μMs, 400 μMs, 200 μMs, 100 μMs, after acarbose being dissolved in a small amount of DMSO during preparation, again with distilled water diluting to respective concentration, control the final volumn concentration < 0.1% of DMSO.

Sample sets: each above sample solution (test sample solution and positive control sample solution) getting 25 μ L different concns, add alpha-glucosaccharase enzyme aqueous solution and 175 μ L phosphate buffer soln (50mM that 25 μ L concentration are 0.2U/mL respectively, pH7.0), obtain mixing solutions. Mixing solutions is added, after room temperature places 10min, the 4-oil of mirbane-α-D-glucopyranoside aqueous solution that 25 μ L concentration are 2.5mM, 37 DEG C of constant temperature hatch 30min, the centrifugal 5min of 5000rpm, gets in supernatant 150 μ L to 96 orifice plate, measures light absorption value at wavelength 405nm place.

Sample controls group: use the phosphate buffered saline buffer (50mM, pH7.0) of 25 μ L to replace alpha-glucosaccharase enzyme aqueous solution to carry out above-mentioned experiment.

Experiment arranges blank group and blank group simultaneously, and method to set up is as follows:

Blank group: use the phosphate buffered saline buffer (50mM, pH7.0) of 25 μ L to replace sample solution;

Blank group: use the phosphate buffered saline buffer (50mM, pH7.0) of 25 μ L to replace sample solution, the phosphate buffered saline buffer (50mM, pH7.0) of 25 μ L replaces alpha-glucosaccharase enzyme aqueous solution simultaneously;

Each group above-mentioned all repeats three times, results averaged. Use following formulae discovery sample to the inhibiting rate of alpha-glucosidase:

Inhibiting rate (%)=[1-(sample sets light absorption value-sample controls group light absorption value)/(blank group light absorption value-blank group light absorption value)] × 100%

To experimental data statistical study, it may also be useful to IC₅₀The IC of each test sample of computed in software₅₀Value result is as shown in table 4, and table 4 shows, the IC of HE-E1₅₀It is 90.96 μMs, the IC of compound 1-11₅₀At 5-150 μM, the particularly IC of compound 6,7,11₅₀At about 10 μMs, far away higher than positive drug acarbose.

The alpha-glucosaccharase enzyme inhibition activity detected result of table 4 crude extract HE-E1 and each compound

2, to the effect of diabetes rat

1) getting wistar male rat 75, adaptability raises one week: room temperature 18-25 DEG C, humidity 50-60%, in 12/12 hour light and shade cycle, freely ingests, drinks water, gives standard rat feed.

2) following group is arranged

Blank group: 5 wistar male rats, gives basis mouse feed.

Model group: 70 wistar male rats, give high glucose and high fat feed.

After blank group and model group rats are all fed 4 weeks, fasting 8 hours, got rat tail vein blood and measures fasting plasma glucose in the 29th day, and intravenous injection U-9889 45mg/kg body weight, feeding after intravenous injection 72 hours, fasting 8 hours, gets rat tail vein blood and surveys fasting plasma glucose.

Result shows, and after injection U-9889, the fasting blood sugar of model group rats is 18.21 ± 0.86mmol/L, is significantly higher than the 4.83 ± 0.92mmol/L (P < 0.01) of blank group rat. And the situation of model group rats is not good enough, occurs that the diabetic symptom such as many drinks, many urine, many foods is bright, these results suggest that type ii diabetes rat model modeling success.

3) administration experiment

Administration experiment grouping is as shown in table 5, wherein, test group, for model group rats (giving high glucose and high fat forage feed 70 wistar male rats of 1 week) is divided into 14 groups (model control group, positive drug group, HE-E1, compound 1-compounds 11) at random, often organizes 5; Blank group is 5 normal wistar male rats (giving 5 wistar male rats of basis mouse feed).

Table 5 administration experiment grouping and process

Note: compound 1-compound 11, HE-E1 and acarbose are all dissolved in distilled water.

Each group rat is with feeding corresponding feed under condition, and gastric infusion, once a day, successive administration one week, the tail vein then getting each group of rat measures fasting plasma glucose. Result is as shown in table 6, result shows, the extract HE-E1 of embodiment 1 has the blood sugar decreasing effect suitable with positive drug (acarbose), and other 10 compounds except compound 5 have the effect of significantly control type II diabetes rat model blood glucose value, relatively positive drug (acarbose) is stronger for its action effect.

Rat fasting blood-glucose value respectively organized by table 6

Note: compared with model control group, * * P < 0.01, * P < 0.05; Compared with blank group,^##P<0.01。

(2) antitumor action

Test tumour cell: human liver cancer cell HepG2, the Human Prostate Cancer Cells PC3 DMEM nutrient solution of the foetal calf serum containing volumn concentration 10% is cultivated, and human lung cancer cell A549, Human colorectal cancer cells HCT-15, human cervical carcinoma cell Hela, Leukemia K562 cell, SGC-7901 cells, the human breast cancer cell line Bcap-37 RPMI1640 nutrient solution of the foetal calf serum containing volumn concentration 10% are cultivated. The culture condition of above-mentioned cell is 37 DEG C, 5%CO₂Cellar culture in incubator, goes down to posterity every other day.

Sample solution: test sample is the crude extract HE-E1 that obtains of step 5 and be therefrom separated 11 compounds (compound 1-compound 11) obtained. Accurately take each sample, it is mixed with 50mM solution (after being dissolved in a small amount of DMSO during preparation, then with distilled water diluting to respective concentration with DMSO, control DMSO final volumn concentration < 1%), 2 times of gradient dilutions are carried out, totally 8 concentration, for active testing with DMEM substratum.

Positive control solution: 5-Fluorouracil (5-FU) DMSO is mixed with 2mM solution (after being dissolved in a small amount of DMSO during preparation, again with distilled water diluting to respective concentration, control DMSO final volumn concentration < 1%), 2 times of gradient dilutions are carried out, totally 8 concentration with DMEM culture medium solution.

Cell inhibitory effect activity test method (mtt assay) is adopted to test the anti-tumour cell proliferative activity of sample, its principle is, in viable cell plastosome desaturase can metabolism reduction yellow bromination 3-(4,5-dimethylthiazole)-2,5-phenylbenzene tetrazole is the first (formazan) being insoluble to water of bluish voilet, and the amount of first measures its absorbancy by microplate reader and tries to achieve. Owing to the amount of first is directly proportional to viable count, so the number of viable cell can be calculated according to absorbancy, thus obtain the ability of Drug inhibition or killing tumor cell.

Above-mentioned various test tumour cell (the human liver cancer cell HepG2 taken the logarithm vegetative period, Human Prostate Cancer Cells PC3, human lung cancer cell A549, Human colorectal cancer cells HCT-15, human cervical carcinoma cell Hela, K562 cell, SGC-7901 cells, human breast cancer cell line Bcap-37), respectively with trysinization, make every milliliter containing 1 × 10⁵The single cell suspension of individual cell, is inoculated in 96 orifice plates (every hole 200 μ L), and often group establishes 3 parallel holes. At 37 DEG C, after 24 hours, add the sample solution of the above-mentioned different concns of 2ul, as administration group, cultivate 48 hours. Establish blank group (2ulDMSO substitutes above-mentioned sample solution) and positive controls (the 5-Fluorouracil solution of 2ul different concns substitutes above-mentioned sample solution) simultaneously. Then in each group, add the RPMI-1640 solution (MTT final concentration is 5mg/L) of 20 μ L containing MTT, cultivate 4 hours again, add 150 μ LDMSO after moving out of 150 μ L nutrient solutions and dissolve first, measure its optical density at 540nm place, calculate inhibiting rate (%).

The calculation formula of inhibiting rate (%): (1-administration group OD value/blank group OD value) × 100%.

Calculate IC₅₀Value: IC₅₀=[CL (IH-50)+CH (50-IL)]/(IH-IL)

CL: lower concentration values; CH: high concentration value; IH: the inhibiting rate under high density; IL: the inhibiting rate under lower concentration

Result is as shown in table 7.

The anti-tumor activity of table 7 crude extract HE-E1 and each compound

Note: 5-FU is as antineoplastic positive control.

Table 7 shows, HE-E1 and 11 compound is all inhibited to above-mentioned tumour cell, can be used as the treatment of antineoplastic agent (i.e. antitumor drug) for tumour, it is possible to as cell inhibitory effect low molecule bioprobe for explore biological phenomena essence Life Science Experiment study in.

The cultivation of embodiment 2, bacterium, the analysis of extract

One, bacterial strain activation and seed culture are with embodiment 1.

Two, fermentation culture

Liquid nutrient medium cultivate 10 days (25 DEG C-28 DEG C, lucifuge, cultivate 7-10 days) after, get the seed culture fluid that 10 milliliters of embodiment 1 step 2 obtain and it is inoculated in 500 milliliters of triangular flasks of the following solid medium that 80 grams of brown rice are housed respectively, altogether repeated inoculation 10 bottles, 25 DEG C, lucifuge, cultivate 30 days (25 DEG C-28 DEG C, lucifuge, cultivate 30-50 days), obtain the fermented product of L547.

Solid medium: be made up of 80g brown rice and 100 ml waters.

(in above-mentioned solid medium, the ratio of brown rice and water is at 80-100g:100ml)

Three, the preparation of extract

(1) preparation of the fermented product of L547

By cultivating the fermented product lyophilize of the L547 obtained, weighing, weight is 960 grams. By the aqueous solution (ratio of the aqueous solution of fermented product and ethanol is 1g:3-5ml) 100 DEG C of (95 DEG C-105 DEG C) refluxing extraction 3 times (3-4 time) of 5 liters of volumn concentrations 95% (90%-98%) ethanol, each 1 hour (50-70min), merge ethanol extract, concentrating under reduced pressure drying obtains 10.2 grams of extracts, and extract is denoted as HE-E2.

(2) high performance liquid chromatography detection

HE-E2 is carried out high performance liquid chromatography detection, and analysis condition is with step 7 in embodiment 1, and result is as shown in Figure 3.

In Fig. 3, the compound 1-11 one_to_one corresponding that obtains of step 5 in the peak that 1-11 indicates and embodiment 1.

Fig. 3 shows, has 11 compounds being separated in embodiment 1 and obtaining in HE-E2.

The cultivation of embodiment 3, bacterium, the preparation of extract

One, bacterial strain activation and seed culture are with embodiment 1.

Two, fermentation culture

Liquid nutrient medium cultivate 10 days (25 DEG C-28 DEG C, lucifuge, cultivate 7-10 days) after, get the seed culture fluid that 10 milliliters of embodiment 1 step 2 obtain and it is inoculated in 500 milliliters of triangular flasks of the following solid medium that 80 grams of glutinous rice are housed respectively, altogether repeated inoculation 10 bottles, 25 DEG C, lucifuge, cultivate 30 days (25 DEG C-28 DEG C, lucifuge, cultivate 30-50 days), obtain the fermented product of L547.

Solid medium: be made up of 80g glutinous rice and 100 ml waters.

(in above-mentioned solid medium, the ratio of glutinous rice and water is at 80-100g:100ml)

Three, the preparation of extract

(1) the fermented product preparation of L547

By cultivating the fermented product lyophilize of the L547 obtained, weighing, weight is 920 grams. By the aqueous solution (ratio of the aqueous solution of fermented product and ethanol is 1g:3-5ml) 100 DEG C of (95 DEG C-105 DEG C) refluxing extraction 3 times (3-4 time) of 5 liters of volumn concentrations 95% (90%-98%) ethanol, each 1 hour (50-70min), merge ethanol extract, concentrating under reduced pressure drying obtains 9.8 grams of extracts, and extract is denoted as HE-E3.

(2) high performance liquid chromatography detection

HE-E3 is carried out high performance liquid chromatography detection. Analysis condition is with step 7 in embodiment 1, and result is as shown in Figure 4.

In Fig. 4, the compound 1-11 one_to_one corresponding that obtains of step 5 in the peak that 1-11 indicates and embodiment 1.

Fig. 4 shows, has 11 compounds being separated in embodiment 1 and obtaining in HE-E3.

The cultivation of embodiment 4, bacterium, the preparation of extract

One, bacterial strain activation and seed culture are with embodiment 1.

Two, fermentation culture

Liquid nutrient medium cultivate 10 days (25 DEG C-28 DEG C, lucifuge, cultivate 7-10 days) after, get the seed culture fluid that 10 milliliters of embodiment 1 step 2 obtain and it is inoculated in 500 milliliters of triangular flasks of the following solid medium that 80 grams of seeds of Job's tears are housed respectively, altogether repeated inoculation 10 bottles, 25 DEG C, lucifuge, cultivate 30 days (25 DEG C-28 DEG C, lucifuge, cultivate 30-50 days), obtain the fermented product of L547.

Solid medium: be made up of the 80g seed of Job's tears and 100 ml waters.

(in above-mentioned solid medium, the ratio of the seed of Job's tears and water is at 80-100g:100ml)

Three, the preparation of extract

(1) the fermented product preparation of L547

By cultivating the fermented product lyophilize of the L547 obtained, weighing, weight is 990 grams. By the aqueous solution (ratio of the aqueous solution of fermented product and ethanol is 1g:3-5ml) 100 DEG C of (95 DEG C-105 DEG C) refluxing extraction 3 times (3-4 time) of 5 liters of volumn concentrations 95% (90%-98%) ethanol, each 1 hour (50-70min), merge ethanol extract, concentrating under reduced pressure drying obtains 9.4 grams of extracts, and extract is denoted as HE-E4.

(2) high performance liquid chromatography detection

HE-E4 is carried out high performance liquid chromatography detection, and analysis condition is with step 7 in embodiment 1, and result is as shown in Figure 5.

In Fig. 5, the compound 1-11 one_to_one corresponding that obtains of step 5 in the peak that 1-11 indicates and embodiment 1.

Fig. 5 shows, has 11 compounds being separated in embodiment 1 and obtaining in HE-E4.

The cultivation of embodiment 5, bacterium, the preparation of extract

One, bacterial strain activation and seed culture are with embodiment 1.

Two, fermentation culture

Liquid nutrient medium cultivate 10 days (25 DEG C-28 DEG C, lucifuge, cultivate 7-10 days) after, get the seed culture fluid that 10 milliliters of embodiment 1 step 2 obtain and it is inoculated in 500 milliliters of triangular flasks of the following solid medium that 80 grams of purple rice are housed respectively, altogether repeated inoculation 10 bottles, 25 DEG C, lucifuge, cultivate 30 days (25 DEG C-28 DEG C, lucifuge, cultivate 30-50 days), obtain the fermented product of L547.

Solid medium: be made up of the purple rice of 80g and 100 ml waters.

(in above-mentioned solid medium, the ratio of purple rice and water is at 80-100g:100ml)

Three, the preparation of extract

(1) preparation of the fermented product of L547

By cultivating the fermented product lyophilize of the L547 obtained, weighing, weight is 1010 grams. By the aqueous solution (ratio of the aqueous solution of fermented product and ethanol is 1g:3-5ml) 100 DEG C of (95 DEG C-105 DEG C) refluxing extraction 3 times (3-4 time) of 5 liters of volumn concentrations 95% (90%-98%) ethanol, each 1 hour (50-70min), merge ethanol extract, concentrating under reduced pressure drying obtains 9.6 grams of extracts, and extract is denoted as HE-E5.

(2) high performance liquid chromatography detection

HE-E5 is carried out high performance liquid chromatography detection, and analysis condition is with step 7 in embodiment 1, and result is as shown in Figure 6.

In Fig. 6, the compound 1-11 one_to_one corresponding that obtains of step 5 in the peak that 1-11 indicates and embodiment 1.

Fig. 6 shows, has 11 compounds being separated in embodiment 1 and obtaining in HE-E5.

The cultivation of embodiment 6, bacterium, the preparation of extract

One, bacterial strain activation and seed culture are with embodiment 1.

Two, fermentation culture

Liquid nutrient medium cultivate 10 days (25 DEG C-28 DEG C, lucifuge, cultivate 7-10 days) after, get the seed culture fluid that 10 milliliters of embodiment 1 step 2 obtain and it is inoculated in 500 milliliters of triangular flasks of the following solid medium that 80 Ke Hei meter are housed respectively, altogether repeated inoculation 10 bottles, 25 DEG C, lucifuge, cultivate 30 days (25 DEG C-28 DEG C, lucifuge, cultivate 30-50 days), obtain the fermented product of L547.

Solid medium: be made up of the black rice of 80g and 100 ml waters.

(in above-mentioned solid medium, the ratio of Hei meter and water is at 80-100g:100ml)

Three, the preparation of extract

(1) preparation of the fermented product of L547

By cultivating the fermented product lyophilize of the L547 obtained, weighing, weight is 890 grams. By the aqueous solution (ratio of the aqueous solution of fermented product and ethanol is 1g:3-5ml) 100 DEG C of (95 DEG C-105 DEG C) refluxing extraction 3 times (3-4 time) of 5 liters of volumn concentrations 95% (90%-98%) ethanol, each 1 hour (50-70min), merge ethanol extract, concentrating under reduced pressure drying obtains 9.1 grams of extracts, and extract is denoted as HE-E6.

(2) high performance liquid chromatography detection

HE-E6 is carried out high performance liquid chromatography detection, and analysis condition is with step 7 in embodiment 1, and result is as shown in Figure 7.

In Fig. 7, the compound 1-11 one_to_one corresponding that obtains of step 5 in the peak that 1-11 indicates and embodiment 1.

Fig. 7 shows, has 11 compounds being separated in embodiment 1 and obtaining in HE-E6.

The evaluated biological activity of embodiment 7, embodiment 1-6 extract HE-E1 to HE-E6

One, to the effect of diabetes rat

Specific experiment method is with reference to (one) hypoglycemic activity of step 8 in embodiment 1.Experimental result is as shown in table 8, and HE-E1 to HE-E66 kind extract all has good alpha-glucosaccharase enzyme inhibition activity, is better than positive control acarbose. Wherein that the medication of diabetes rat is as shown in table 9, experimental result is as shown in table 10, and table 10 shows, the effect in this experiment of 6 kinds of extracts is suitable with acarbose.

The alpha-glucosaccharase enzyme inhibition activity detected result of 6 extracts (HE-E1 to HE-E6) in table 8 embodiment 1-6

	IC₅₀μM		IC₅₀μM
				HE-E1	90.96	HE-E2	100.32
HE-E3	95.93	HE-E4	95.64
				HE-E5	97.80	HE-E6	91.20
Acarbose	751.68

Table 9 administration experiment grouping and process

The hypoglycemic detected result of table 10 embodiment 1-6 extract (HE-E1 to HE-E6)

	Fasting plasma glucose mmol/L		Fasting plasma glucose mmol/L
				Blank group	4.76±0.73	Model control group	21.08±1.52^##
HE-E1	11.25±1.56**	HE-E2	12.63±1.36**
				HE-E3	16.25±2.82**	HE-E4	12.12±1.03**
HE-E5	11.79±2.26**	HE-E6	10.20±1.86**
				Positive drug group	10.68±0.89

Two, antitumor action

Specific experiment method is with reference to (two) antitumor action of embodiment 1 step 8.

Result is as shown in table 11.

The anti-tumor activity result of 6 extracts (HE-E1 to HE-E6) in table 11 embodiment 1-6

Table 11 shows, 6 kinds of extracts have anti-tumor activity to a certain degree, suitable with 5-FU. And 6 kinds of extract difference are little, this conclusion conclusion similar to the finger printing of Fig. 2-Fig. 7 is also coincide, and illustrates that the basic substance of 6 kinds of extracts is consistent.

Claims

1. the arbitrary shown isoindole compounds of following compound 1-5,7-10:

(1) compound 1, structural formula is

(2) compound 2, structural formula is

(3) compound 3, structural formula is

(4) compound 4, structural formula is

(5) compound 5, structural formula is

(6) compound 7, structural formula is

(7) compound 8, structural formula is

(8) compound 9, structural formula is

(9) compound 10, structural formula is

2. prepare the method for isoindole compounds 1 in claim 1 for one kind, comprise the steps: that the extract of Hericium erinaceus (Bull. Ex Fr.) Pers. mycelium fermentation thing by preserving number is CGMCCNo.9224 carries out silica gel column chromatography separation, take volume ratio as 1:0, 50:1, 30:1, 20:1, 15:1, 10:1, 4:1, the normal hexane of 2:1: eluent ethyl acetate agent and volume ratio are 1:0, 100:1, 50:1, the methylene dichloride of 30:1: methanol-eluted fractions agent carries out gradient elution successively, each eluent 3 retention volume 1, finally obtain the methylene dichloride that volume ratio is 30:1: the wash-out composition of methanol-eluted fractions agent, by its freeze-drying, it is denoted as HE-E1-19, HE-E1-19 is carried out the separation of ODS reverse phase silica gel post, it is 30% with volumn concentration, the aqueous solution of 50% methyl alcohol carries out wash-out successively as eluent, each eluent 3 retention volume 2, at volumn concentration be the methanol aqueous solution of 50% as eluent, when elution volume is 280-300ml, obtain evaporating points 10, by its freeze-drying, it is denoted as HE-E1-19-10, HE-E1-19-10 is carried out HPLC preparation, the sample solution of 10mg/ml it is formulated as with chromatogram methyl alcohol, applied sample amount is 15ul, with Kromasil10 × 250mmC18 semipreparative column, post temperature is 25 DEG C, is that moving phase carries out wash-out with the sour water containing volumn concentration 26% acetonitrile, flow velocity is 2ml/min, 210nm wavelength detects, and obtains the chromatographic peak that retention time is 26 minutes, to obtain final product,

Described retention volume 1 is 500ml;

Described retention volume 2 is 100ml;

(1) rice;

(2) brown rice;

(3) glutinous rice;

(4) seed of Job's tears;

(5) purple rice;

(6) black rice;

(2) fermented product freeze-drying step () obtained, then it is carried out refluxing extraction with aqueous ethanolic solution 95-105 DEG C that volumn concentration is 90-98%, obtain extracting solution, to obtain final product.

3. prepare isoindole compounds 2 in claim 1 for one kind, compound 3, the method of compound 4 and/or compound 5, comprise the steps: that the extract of Hericium erinaceus (Bull. Ex Fr.) Pers. mycelium fermentation thing by preserving number is CGMCCNo.9224 carries out silica gel column chromatography separation, take volume ratio as 1:0, 50:1, 30:1, 20:1, 15:1, 10:1, 4:1, the normal hexane of 2:1: eluent ethyl acetate agent and volume ratio are 1:0, 100:1, 50:1, the methylene dichloride of 30:1: methanol-eluted fractions agent carries out gradient elution successively, each eluent 3 retention volume 1, finally obtain the methylene dichloride that volume ratio is 30:1: the wash-out composition of methanol-eluted fractions agent, by its freeze-drying, it is denoted as HE-E1-19, HE-E1-19 is carried out the separation of ODS reverse phase silica gel post, it is 30% with volumn concentration, 50%, the aqueous solution of 70% methyl alcohol carries out wash-out successively as eluent, each eluent 3 retention volume 2, volumn concentration be 70% methanol aqueous solution as eluent, when elution volume is 60-100ml, obtain evaporating points 12, by its freeze-drying, it is denoted as HE-E1-19-12, HE-E1-19-12 being carried out HPLC preparation, is formulated as the sample solution of 10mg/ml with chromatogram methyl alcohol, applied sample amount is 15ul, with Kromasil10 × 250mmC18 semipreparative column, post temperature is 25 DEG C, is that moving phase carries out wash-out with the sour water containing volumn concentration 22% acetonitrile, flow velocity is that 2ml/min, 210nm wavelength detects, and obtains retention time and is respectively 23, minute 25.2,27.1,29.2 chromatographic peak, namely correspondence obtains compound 2, compound 3, compound 4, compound 5,

Described retention volume 1 is 500ml;

Described retention volume 2 is 100ml;

Described liquid nutrient medium is made up of solvent and solute, and solvent is water, and solute is glucose, malt extract and yeast extract;The concentration of described glucose in described liquid nutrient medium is 3.0-6.0g/L, and the concentration of described malt extract in described liquid nutrient medium is 8.0-10.0g/L, and the concentration of described yeast extract in described liquid nutrient medium is 2.0-5.0g/L;

(1) rice;

(2) brown rice;

(3) glutinous rice;

(4) seed of Job's tears;

(5) purple rice;

(6) black rice;

4. prepare the method for isoindole compounds 7 and/or compound 8 in claim 1 for one kind, comprise the steps: that the extract of Hericium erinaceus (Bull. Ex Fr.) Pers. mycelium fermentation thing by preserving number is CGMCCNo.9224 carries out silica gel column chromatography separation, take volume ratio as 1:0, 50:1, 30:1, 20:1, 15:1, 10:1, 4:1, the normal hexane of 2:1: eluent ethyl acetate agent and volume ratio are 1:0, 100:1, 50:1, the methylene dichloride of 30:1: methanol-eluted fractions agent carries out gradient elution successively, each eluent 3 retention volume 1, finally obtain the methylene dichloride that volume ratio is 30:1: the wash-out composition of methanol-eluted fractions agent, by its freeze-drying, it is denoted as HE-E1-19, HE-E1-19 is carried out the separation of ODS reverse phase silica gel post, it is 30% with volumn concentration, 50%, the aqueous solution of 70% methyl alcohol carries out wash-out successively as eluent, each eluent 3 retention volume 2, volumn concentration be 70% methanol aqueous solution as eluent, when elution volume is 220-250ml, obtain evaporating points 15, by its freeze-drying, it is denoted as HE-E1-19-15, HE-E1-19-15 is carried out HPLC preparation, the sample solution being formulated as 10mg/ml with chromatogram methyl alcohol, applied sample amount is 15ul, with Kromasil10 × 250mmC18 semipreparative column, post temperature is 25 DEG C, with being that moving phase carries out wash-out containing the sour water of volumn concentration 30% acetonitrile, flow velocity is that 2ml/min, 210nm wavelength detects, obtain retention time and it is respectively 32, the chromatographic peak of 35.5 minutes, namely correspondence obtains compound 7, compound 8,

Described retention volume 1 is 500ml;

Described retention volume 2 is 100ml;

(1) rice;

(2) brown rice;

(3) glutinous rice;

(4) seed of Job's tears;

(5) purple rice;

(6) black rice;

5. prepare the method for isoindole compounds 9 and/or compound 10 in claim 1 for one kind, comprise the steps: that the extract of Hericium erinaceus (Bull. Ex Fr.) Pers. mycelium fermentation thing by preserving number is CGMCCNo.9224 carries out silica gel column chromatography separation, take volume ratio as 1:0, 50:1, 30:1, 20:1, 15:1, 10:1, 4:1, the normal hexane of 2:1: eluent ethyl acetate agent and volume ratio are 1:0, 100:1, 50:1, the methylene dichloride of 30:1: methanol-eluted fractions agent carries out gradient elution successively, each eluent 3 retention volume 1, finally obtain the methylene dichloride that volume ratio is 30:1: the wash-out composition of methanol-eluted fractions agent, by its freeze-drying, it is denoted as HE-E1-19, HE-E1-19 is carried out the separation of ODS reverse phase silica gel post, it is 30% with volumn concentration, 50%, the aqueous solution of 70% methyl alcohol carries out wash-out successively as eluent, each eluent 3 retention volume 2, volumn concentration be 70% methanol aqueous solution as eluent, when elution volume is 260-280ml, obtain evaporating points 16, by its freeze-drying, it is denoted as HE-E1-19-16, HE-E1-19-16 is carried out HPLC preparation, the sample solution being formulated as 10mg/ml with chromatogram methyl alcohol, applied sample amount is 15ul, with Kromasil10 × 250mmC18 semipreparative column, post temperature is 25 DEG C, with being that moving phase carries out wash-out containing the sour water of volumn concentration 40% acetonitrile, flow velocity is that 2ml/min, 210nm wavelength detects, obtain retention time and it is respectively 26.1, the chromatographic peak of 27.5 minutes, namely correspondence obtains compound 9, compound 10,

Described retention volume 1 is 500ml;

Described retention volume 2 is 100ml;

(1) rice;

(2) brown rice;

(3) glutinous rice;

(4) seed of Job's tears;

(5) purple rice;

(6) black rice;

6. according to the arbitrary described method of claim 2-5, it is characterized in that: described silica gel column chromatography be separated in the fill method of pillar and loading method as follows: first fill post with 200g silica gel, by described preserving number be again CGMCCNo.9224 Hericium erinaceus (Bull. Ex Fr.) Pers. mycelium fermentation thing extract dissolve with methanol after with 15g silica gel mixed sample, carry out loading;

The quality during loading of described HE-E1-19 is 3.45g;

7. the application that isoindole compounds according to claim 1 prevents in preparation and/or treats in the product of diabetes or the product of suppression blood sugar rising.

8. the application of isoindole compounds according to claim 1 in the product of preparation prevention and/or the product of Therapeutic cancer or anticancer growth and/or propagation.

9. application according to claim 8, it is characterised in that: described cancer is liver cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, leukemia, cancer of the stomach or mammary cancer;

Described cancer cells is liver cancer cell, prostate cancer cell, lung carcinoma cell, colorectal cancer cell, cervical cancer cell, leukemia cell, stomach cancer cell or breast cancer cell.

10. application according to claim 9, it is characterised in that: described cancer cells is human liver cancer cell, Human Prostate Cancer Cells, human lung carcinoma cell, Human colorectal cancer cells, human cervical carcinoma cell, human leukemia cell, gastric carcinoma cells or human breast cancer cell.