CN103893242A - Radix trichosanthes triterpene extract as well as preparation method and use thereof - Google Patents
Radix trichosanthes triterpene extract as well as preparation method and use thereof Download PDFInfo
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Abstract
The invention provides a radix trichosanthes triterpene extract. The radix trichosanthes triterpene extract is prepared by use of the following steps: (1) crushing the radix trichosanthes as a raw material, adding a mixed solution of a complex enzyme and water, carrying out enzymolysis and filtering to obtain a filtrate; (2) concentrating the obtained filtrate, extracting the obtained concentrated liquor by using a halogenated hydrocarbon first to separate out a water layer and a halogenated hydrocarbon layer, next, extracting the water layer by using ethyl acetate to separate out the water layer and an ethyl acetate layer, concentrating and drying the halogenated hydrocarbon layer and the ethyl acetate layer, respectively, and then blending to obtain a coarse extract; and (3) dispersing the obtained coarse extract in water or an aqueous solution of ethanol, carrying out resin adsorption separating first and then carrying out hierarchical elution by using the aqueous solution of ethanol, recovering the eluate, and then concentrating and drying the eluate to obtain the radix trichosanthes triterpene extract. The radix trichosanthes triterpene extract can be used for preparing functional food and medicaments for treating the diabetes.
Description
(1) technical field
The invention belongs to medical technical field, relate to medicinal plants Fructus Trichosanthis, be specifically related to radices trichosanthis triterpene extract and preparation method thereof, and in the application of preparing in functional food and the medicine for the treatment of diabetes
(2) background technology
The invention belongs to medical technical field, relate to triterpene extract and preparation method thereof and purposes in Fructus Trichosanthis, the present invention is take radices trichosanthis as raw material, first through enzymolysis auxiliary extraction, after filtering, obtaining extracting solution is concentrated into after certain volume, extract respectively by halogenated hydrocarbons and ethyl acetate, merge halogenated hydrocarbons and ethyl acetate extract, obtain triterpene active site extract through resin absorption purification.Suppress active testing and animal vivo test through vitro enzyme, result shows, triterpene extract of the present invention has obvious 11 11-beta-hydroxysteroid dehydrogenase type 1 types (11 β-HSD1) and suppresses activity and blood sugar decreasing effect, can be used for the medicine of the preparation clinical common diabetes for the treatment of and correlated metabolism diseases.
Diabetes (diabetes mellitus, DM) are to cause insulin secretion biological effect absolute or relative deficiency or insulin to reduce by h and E factor, cause the clinical syndrome of a series of metabolism disorders.Along with raising, the onset diabetes rate of people's living standard worldwide rise year by year, be listed in the third-largest disease of serious threat human health after cardiovascular diseases, tumor, cause the great attention of countries in the world.Treating diabetes oral drugs are mainly divided into 3 classes at present, insulin secretion stimulators, euglycemic agent and alpha-glucosidase inhibitor, but they all have side effect in various degree, as hypoglycemia, body weight increase and cardiovascular disease etc., therefore avoid the antidiabetic thing exploitation of the existing side effect of traditional diabetes medicament gradually to become the focus of domestic and international research.
Glucocorticoid can be induced insulin resistant, and insulin resistant is the key link of type 2 diabetes mellitus morbidity.And 11 11-beta-hydroxysteroid dehydrogenase types (11 β-HSD) the mutual conversion between the C11-ketone metabolite (cortisone, 11-dehydrocorticosterone) of the activated C1-hydroxylating of catalysis glucocorticoid (hydrocortisone, corticosterone) and inactive form in vivo.It is to belong to short-chain dehydrogenase/reductase superfamily (SDR), has two hypotype: 11 β-HSD1 and 11 β-HSD2.11 β-HSD1 is a kind of nicotinamide adenine dinucleotide (reduced form) ［ NADP (H) ］ dependent dehydrogenase/oxidoreductase of low-affinity, wide expression in liver organization and fatty tissue, placenta tissue and cerebral tissue.Its Main Function is to make the corticosterone of non-activity be converted into activated hydrocortisone, thereby improves the glucocorticoid levels of tissue local.
Research shows to play an important role in the generation of 11 β-HSD1 to insulin sensitivity and diabetes, development.11 β-the HSD1 expressing in islet cells is lowered the expression of insulin receptor protein in cell and is suppressed beta Cell of islet by activation glucocorticoid and causes insulin resistant, reduces insulin secretion and rising blood glucose, promotes the generation of type 2 diabetes mellitus.Pharmaceutical research shows that optionally 11 beta-HSD 1 inhibitors can reduce concentration of cortisol in liver and fatty tissue, and local enhancement insulin sensitivity reaches the effect that reduces blood glucose, fat-reducing.
In Chinese herbal medicine resource, screen in recent years 11 beta-HSD 1 inhibitors and cause gradually relevant concern.TCM Treatment of Diabetes (diabete) has long history.A lot of Chinese medicine is truly had curative effect by clinical proof, and Fructus Trichosanthis is one of them.Medicine food dual purpose plant Fructus Trichosanthis (Trichosanthes kirilowii Maxim) is the perennial prehensile liana of Cucurbitaceae snake gourd, and conventional Chinese medicine Fructus Trichosanthis, Semen Trichosanthis, Pericarpium Trichosanthis and Radix Trichosanthis are dry mature fruit, seed, peel and the dry root of this plant.In successive dynasties multi-section Chinese medicine ancient books and records, all there is Fructus Trichosanthis to treat the record of quench one's thirst (diabetes), as " Bencao Tujing ", Compendium of Material Medica, amplification on Canon of Materia Medica " Yaoxing Fu ", " book on Chinese herbal medicine covers bamboo fish trap ", Jing-Yue Complete Works etc.The clinical Chinese patent medicine that is used for the treatment of diabetes has the Gualou Qumai Pill coming from " Medical Treasures of the Golden Chamber " now, and Fructus Trichosanthis is also principal agent.Since the seventies in last century, Chinese scholars has been done certain research work to the aspect such as chemical composition, pharmacological action of snake gourd.Cucurbitane type and oleanane type triterpene, flavone, albumen and organic acid isoreactivity compound from seed and rhizome, are isolated.Less to Fructus Trichosanthis hypoglycemic activity effective ingredient correlational study, and mainly with water extract [Ma Yan, Hebei United University's Master's thesis; 2011] or agglutinin class glycoprotein components [Hiroshi Hikino, planta med., 1989; 55 (4), 349-350; Li Qiong etc., Changchun University of Traditional Chinese Medicine's journal, 2012,28 (1), 9-11] be main study subject, Fructus Trichosanthis triterpene blood sugar lowering and the selective inhibition activity of 11 β-HSD1 enzyme is had no to report, and the present invention mainly pays close attention to preparation and the hypoglycemic activity thereof of Fructus Trichosanthis triterpene.
(3) summary of the invention
The object of this invention is to provide a kind of radices trichosanthis triterpene extract and preparation method thereof.
Another object of the present invention is to provide the medicinal usage of described triterpene extract, especially 11 β-HSD1 enzyme inhibition activity and hypoglycemic activity.Extract of the present invention can be for the preparation of the medicine of the clinical common diabetes for the treatment of and correlated metabolism diseases.
The present invention is achieved by the following technical solutions:
A kind of radices trichosanthis triterpene extract, described radices trichosanthis triterpene extract is prepared as follows:
(1) after raw material radices trichosanthis is pulverized, add the mixed liquor of compound enzyme and water, under the condition that is 4.5～6.5 at pH, in 25～65 ℃ of enzymolysis 10～120min, filter to get filtrate; Described compound enzyme is obtained by mixing with mass ratio 0.2～5:1 by cellulase and papain; The specific activity of described cellulase is 4000U/g, and the specific activity of described papain is 5000U/g; The quality consumption of described compound enzyme is 0.2%～10% of radices trichosanthis quality;
(2) gained filtrate in step (1) is concentrated into 1/5～1/3 of original volume and obtains concentrated solution, gained concentrated solution first extracts with halogenated hydrocarbons, isolate water layer and halo-hydrocarbon layer, after halo-hydrocarbon layer concentrate drying, obtain extract A, water intaking layer is extracted with ethyl acetate, and isolates water layer and ethyl acetate layer, after ethyl acetate layer contracting is dry, obtains extract B, extract A and extract B are merged, obtain crude extract; Described halogenated hydrocarbons is dichloromethane or chloroform;
(3) gained crude extract in step (2) is scattered in the aqueous solution of 0wt%～20wt% ethanol, and separate through resin absorption, then first use the aqueous solution eluting of 20wt%～30wt% ethanol, use again the aqueous solution eluting of 75wt%～90wt% ethanol, the ethanol water elution liquid that reclaims 75wt%～90wt%, obtains radices trichosanthis triterpene extract after concentrate drying; Described resin is selected from AB-8, D101 or H103.
Described in recommendation step of the present invention (1), compound enzyme and the mixed liquor of water and the mass ratio of radices trichosanthis are 5～20:1, preferably 11:1.
The preparation method of radices trichosanthis triterpene extract of the present invention, in described step (1), preferably described enzymolysis pH value is 5, and hydrolysis temperature is 50 ℃, and enzymolysis time is 60min; Preferred described compound enzyme cellulase and papain mass ratio are 1:1; The quality consumption of preferred described compound enzyme is 5% of radices trichosanthis quality.
The preparation method of radices trichosanthis triterpene extract of the present invention, in described step (2), the volumetric usage of preferred described halogenated hydrocarbons is 0.5～2 times of described concentrated solution volume; The volumetric usage of preferred described ethyl acetate is also 0.5～2 times of described concentrated solution volume; Preferred described halogenated hydrocarbons is dichloromethane.
The preparation method of radices trichosanthis triterpene extract of the present invention, in described step (3), the aqueous solution of preferred described 0wt%～20wt% ethanol and the liquid-solid mass ratio of crude extract are 2～3:1,0wt% represents wherein not contain ethanol here, only has water, represents with 0wt%; The quality consumption of preferred described resin is 1/5～1/3 of raw material radices trichosanthis quality, particularly preferably 1/4.
In the preparation method step (3) of radices trichosanthis triterpene extract of the present invention, the volumetric usage of the aqueous solution of preferred described 20wt%～30wt% ethanol is counted 0.2～1L/kg with the quality of radices trichosanthis; The volumetric usage of the aqueous solution of preferred described 75wt%～90wt% ethanol is counted 0.3～2L/kg with the quality of radices trichosanthis.
Radices trichosanthis triterpene extract of the present invention has effect to treatment diabetes, and radices trichosanthis triterpene extract demonstrates the application prospect in medicine and the functional food of preparation treatment diabetes.
Extractive content measuring method of the present invention: adopt ultraviolet spectrophotometry to measure triterpenes components content in extract: precision measures ring Fructus Trichosanthis glycol reference substance 28mg, with acetone standardize solution in 10mL volumetric flask.Precision measures ring Fructus Trichosanthis glycol acetone soln 0.05,0.1,0.15,0.2,0.25, and 0.3mL, is placed in respectively 6 10mL volumetric flasks.Accurately add respectively 5% vanillin-glacial acetic acid solution 0.4mL, then add 1.4mL perchloric acid, be placed in 60 ℃ of water-bath 20min, be cooled to room temperature with cold water, acetone standardize solution, shakes up, and places 40min.Measure absorbance at 552nm place, draw Fructus Trichosanthis triterpene uv absorption standard curve.Precision takes extract 10mg of the present invention, is dissolved in 10mL volumetric flask with acetone, accurately adds respectively 5% vanillin-glacial acetic acid solution 0.5mL, then adds 1.6mL perchloric acid, is placed in 60 ℃ of water-bath 20min, is cooled to room temperature with cold water, and acetone standardize solution, shakes up.Detect absorbance, calculate related concentrations and content by above standard curve
Radices trichosanthis triterpene extract biological activity of the present invention and purposes analytical method: in the test of external 11 β-HSD1 enzyme inhibition activity and animal body, checking is investigated in blood sugar decreasing effect test, specifically sees embodiment 3.Result shows that the Fructus Trichosanthis triterpene extract making by the inventive method has obvious 11 β-HSD1 enzyme inhibition activity and blood sugar decreasing effect, can be used for functional food and the medicine of preparation treatment diabetes and correlated metabolism diseases thereof.Also can be by adding the medicine of pharmaceutic adjuvant through conventional method preparation treatment diabetes.
Beneficial effect of the present invention is:
Because radices trichosanthis quality is hard, and contain a large amount of glycoprotein compositions, affect the extraction effect of Fructus Trichosanthis triterpene, contrast experiment shows with water extraction or with triterpene in alcohol extraction radices trichosanthis, the response rate is less than 3%, and the product separation difficulty obtaining, due to the easy saponification of protide composition, and protein surface, to active substance Adsorption Effect, causes the radices trichosanthis response rate sharply to decline, Simultaneous purification weak effect in extraction separation process.And method of the present invention adopts protease method to be first hydrolyzed glycoprotein composition in radices trichosanthis, and the utilization of cellulase is conducive to triterpenes components stripping, and the triterpene response rate reaches more than 7%, obtains product triterpene purity and exceedes 70%.Obviously be better than traditional extraction process
On the other hand the hypoglycemic research of radices trichosanthis forefathers are published an article and mainly pay close attention to glycoprotein composition in radices trichosanthis, and without clear and definite hypoglycemic activity Mechanism Study.The present invention thinks that by test Fructus Trichosanthis blood-sugar decreasing active is triterpene, and it is the hypoglycemic main cause of Fructus Trichosanthis that its triterpenes components has 11 β-HSD1 enzyme inhibition activity.
(4) specific embodiment
Below by embodiment, the present invention is further described in detail, but protection scope of the present invention is not limited to this.
The preparation of triterpene extract in embodiment 1 radices trichosanthis
After 20 kilograms of pulverizing of Fructus Trichosanthis dry rhizome, and compound enzyme 1 kilogram (cellulase: papain/1:1, mass ratio) evenly mixes, add 220 premium on currency, in 50 degree, enzymolysis 60 minutes under the condition that pH is 5, after filtering, filtrate is concentrated into 65 liters of left and right, first use 50 liters of dichloromethane extractions, with 50 liters of ethyl acetate extractions, reclaim respectively the solvent of dichloromethane layer and ethyl acetate layer again, obtain two parts of extracts, merge two extracts, 451 grams of dry crude extracts.Crude extract is scattered in 1.35 premium on currency, and adopt 5 kilograms of AB-8 resin absorptioies to separate, then first with 10 liters of eluting of aqueous solution of 20wt% ethanol, then use 30 liters of eluting of aqueous solution of 80wt% ethanol, reclaim the ethanol water elution liquid of 80wt%, concentrate drying obtains 138 grams of triterpene extracts.Through determined by ultraviolet spectrophotometry, triterpene content 74.2% in extract.
The preparation of triterpene extract in embodiment 2 radices trichosanthises
After 1 kilogram of pulverizing of Fructus Trichosanthis dry rhizome, and compound enzyme 0.1 kilogram (cellulase: protease/1:1, mass ratio) evenly mixes, add 10 premium on currency, in 55 degree, enzymolysis 50 minutes under the condition that pH is 4.5, after filtering, filtrate is concentrated into 2 liters of left and right, first use 1.5 liters of dichloromethane extractions, with 1 liter of ethyl acetate extraction, reclaim respectively the solvent of dichloromethane layer and ethyl acetate layer again, obtain two parts of extracts, merge two extracts, 23.1 grams of dry crude extracts.Crude extract is scattered in 60 ml waters, and adopt 250 grams of AB-8 resin absorptioies to separate, then first with 0.5 liter of eluting of aqueous solution of 20wt% ethanol, then use 1.5 liters of eluting of aqueous solution of 80wt% ethanol, reclaim the ethanol water elution liquid of 80wt%, concentrate drying obtains 7.5 grams of triterpene extracts.Through determined by ultraviolet spectrophotometry, triterpene content 72.3% in extract.
The preparation of triterpene extract in embodiment 3 radices trichosanthises
After 4.3 kilograms of pulverizing of Fructus Trichosanthis dry rhizome, and compound enzyme 0.2 kilogram (cellulase: papain/1:1, mass ratio) evenly mixes, add 50 premium on currency, in 60 degree, enzymolysis 60 minutes under the condition that pH is 5.5, after filtering, filtrate is concentrated into 16 liters of left and right, first use 12 liters of dichloromethane extractions, with 12 liters of ethyl acetate extractions, reclaim respectively the solvent of dichloromethane layer and ethyl acetate layer again, obtain two parts of extracts, merge two extracts, 113 grams of dry crude extracts.Crude extract is scattered in the aqueous solution of 330 milliliters of 10wt% ethanol, and adopt 1.3 kilograms of AB-8 resin absorptioies to separate, then first 2.3 liters of eluting of aqueous solution with 30wt% ethanol, use again 7.2 liters of eluting of aqueous solution of 87wt% ethanol, reclaim the ethanol water elution liquid of 87wt%, concentrate drying obtains 34.1 grams of triterpene extracts.Through determined by ultraviolet spectrophotometry, triterpene content 73.8% in extract.
Embodiment 4 radices trichosanthis triterpene extract anti-diabetic activity researchs
Get 60 of healthy adult rats, female, raise with cobalt-60 irradiation normal feedstuff, Animal House temperature is controlled at 22 ℃ of left and right, and adaptability was fed after 7 days, and rat body weight maintains 200 ± 10g, by the method for lumbar injection alloxan, set up Alloxan-diabetes rat model.Rat be can't help after water 16h in fasting, the alloxan solution of lumbar injection 2%, and dosage is 200mg/kg body weight, causes diabetes model.After injection 7d, get blood, select blood sugar concentration >=11mmol/L to be defined as diabetes rat model.According to experiment demand, rat model is divided into Normal group by table of random number, (radices trichosanthis obtains product for 3 hours with liquid-solid ratio 8L/g reflux, extract, to radices trichosanthis water extract group in hot water, main component is saccharide and glycoprotein composition), radices trichosanthis triterpene extract (embodiment 1 obtains) group, model control group and rosiglitazone group, 10 every group.Once a day, successive administration 28 days, carries out following 3 experiments.
1. the impact of extract of Trichosanthes on Alloxan-diabetes rat fasting blood-glucose
Before administration, when administration 14 days, administration 28 days, adopt glucose assays kit measurement rat fasting blood-glucose.The mensuration of fasting glucose is mainly that fasting glucose is high for weighing basic blood sugar level, illustrates that this rat blood sugar is high, is one of symptom of diabetes.Said determination result shows: radices trichosanthis water extract and radices trichosanthis triterpene extract all have the effect of the rat blood sugar of reduction, and triterpene extract effect is better compared with water extract, suitable with rosiglitazone group, specifically in table 1.
The variation of table 1 rat blood sugar value (mmol/L,
)
Note: a and model control group comparison, P < 0.05.Before b and administration, compare P < 0.05.
2. the impact of extract of Trichosanthes on Alloxan-diabetes rat glycated serum protein
Glycated serum protein can reflect 2～3 weeks in the past interior average blood sugar levels of body, is proportionate with blood sugar level, more can reflect the variation of blood sugar level, is that diabetes patient blood sugar controls very suitable good index.Extract of Trichosanthes is as shown in table 2 on the impact of Alloxan-diabetes rat and normal rat serum glycated serum protein.Result shows: radices trichosanthis triterpene extract has the effect that reduces diabetes rat glycated serum protein content.
The mensuration (mmol/L) of table 2 glycated serum protein
Note: a and model control group comparison, P < 0.05.B and Normal group comparison, P < 0.05.
3., separate the compound obtaining 11 β-HSD1 is suppressed to active
Enzyme inhibition rate detects and carries out in 96 orifice plates, every hole solution volume 100 μ L, containing 30mM, the HEPES buffer of pH7.4,1mM EDTA, substrate mixture cortisone/NADPH(200nM/200 μ M) and the inhibitor of serial dilution.Add 10 μ L(2 μ g) from colibacillary 11 β-HSD1, and the hepatomicrosome component of rat (2.5 μ are g) to start reaction, then mix, 37 ℃ of jolting plates 150 minutes, by 10 μ L enoxolone cessation reactions.Detect cortisol levels in prepared product with microplate reader, calculate enzyme inhibitor rate.
The radices trichosanthis triterpene extract that embodiment 1 is obtained carries out further separation and purification and obtains compound 1～8, the method of described separation and purification is: radices trichosanthis triterpene extract 13.2g, separate with silica gel column chromatography, methylene chloride-methanol (15～7:1, V:V) gradient elution, separates and obtains position I (2.1g), position II (3.3g) and position III (1.5g).Position I is adopted to silica gel column chromatography, carries out eluting with petroleum ether-ethyl acetate (2:1, V:V), obtain respectively compound 1 (312mg), 2(154mg) and 3(43mg); Position II is adopted to silica gel column chromatography, with methylene chloride-methanol (20～10:1, V:V) gradient elution, separation obtains compound 4 (42mg), position III is adopted to silica gel column chromatography, with ethyl acetate-methanol-water (4～2:1:0.1, V:V:V) gradient elution, respectively compound 5 (35mg), 6 (42mg), 7 (12mg) and 8 (23mg).
Then compound 1～8 has been carried out to the preliminary inhibition screening active ingredients to 11 beta-hydroxysteroid dehydrogenases, compound 5 has stronger inhibitory action to 11 β-HSD1, suitable with enoxolone, and main component be the radices trichosanthis water extract of glycoprotein compound to 11 β-HSD1 enzyme inhibition a little less than, specifically in table 3.
It is as follows that table 3 separates calabash alkyl-type triterpenoids 1～8 active testing result obtaining
Note: labelling * represents positive control; In table, the concentration of compound 1～8 is 10 μ M.
The structural formula that separates the compound 1～8 obtaining is as follows:
The carbon spectrum data that separate the compound 1～8 obtaining are as follows:
Compound 1 (Hemslecin A):
13c-NMR (C
5d
5n, 125MHz) δ: 216.1 (C-22), 214.2 (C-11), 171.1 (MeCO), 143.4 (C-5), 120.2 (C-6), 82.6 (C-25), 82.5 (C-3), 81.2 (C-20), 72.1 (C-16), 71.5 (C-2), 60.1 (C-17), 52.1 (C-14), 50.2 (C-12), 49.8 (C-9), 49.6 (C-13), 47.1 (C-15), 44.2 (C-8), 43.8 (C-4), 36.4 (C-24), 35.7 (C-1), 35.4 (C-10), 33.3 (C-23), 27.1 (C-27), 26.9 (C-26), 26.5 (C-21), 26.5 (C-29), 25.2 (C-7), 23.4 (MeCO), 23.2 (C-28), 21.4 (C-30), 21.4 (C-18), 20.2 (C-19).
Compound 2 (23,24-dihydrocucurbitacin B):
13c-NMR (CD
3oD, 125MHz) δ: 214.6 (C-22), 213.6 (C-3), 212.9 (C-11), 171.1 (MeCO), 141.2 (C-5), 121.1 (C-6), 82.1 (C-25), 79.6 (C-20), 72.4 (C-16), 71.7 (C-2), 58.5 (C-17), 51.3 (C-13), 51.1 (C-14), 49.4 (C-12), 49.1 (C-9), 49.1 (C-4), 46.2 (C-7), 43.1 (C-15), 36.7 (C-15), 35.4 (C-24), 34.4 (C-10), 31.4 (C-23), 30.1 (C-21), 27.0 (C-26), 26.5 (C-27), 25.2 (C-28), 24.6 (C-1), 23.1 (C-29), 21.9 (MeCO), 20.8 (C-19), 20.5 (C-18), 19.5 (C-30).
Compound 3 (3-epicucurbitacin B):
13c-NMR (CDCl
3, 125MHz) δ: 214.6 (C-22), 213.2 (C-3), 211.7 (C-11), 171.1 (MeCO), 140.6 (C-5), 122.7 (C-6), 82.1 (C-25), 80.2 (C-16), 79.6 (C-20), 71.7 (C-2), 58.5 (C-17), 51.3 (C-13), 49.5 (C-12), 49.2 (C-14), 48.6 (C-9), 46.3 (C-7), 43.0 (C-8), 41.6 (C-4), 37.1 (C-15), 35.4 (C-24), 33.0 (C-10), 31.4 (C-23), 28.4 (C-21), 27.0 (C-29), 26.6 (C-28), 25.2 (C-27), 25.1 (C-26), 24.4 (C-1), 23.1 (MeCO), 20.7 (C-19), 19.7 (C-18), 19.1 (C-30).
Compound 4 (Scandenogenin D):
13c-NMR (CD
3oD, 125MHz) δ: 216.5 (C-11), 143.5 (C-25), 142.1 (C-5), 130.9 (C-24), 120.6 (C-6), 82.5 (C-3), 74.1 (C-20), 72.5 (C-16), 72.4 (C-23), 72.2 (C-2), 65.8 (C-26), 59.0 (C-27), 57.1 (C-17), 50.8 (C-13), 50.8 (C-14), 50.1 (C-22), 49.9 (C-9), 47.6 (C-12), 44.6 (C-8), 44.0 (C-4), 42.6 (C-15), 35.4 (C-10), 30.4 (C-21), 26.1 (C-30), 25.5 (C-7), 22.9 (C-29), 22.2 (C-28), 21.3 (C-19), 20.8 (C-18).
Compound 5 (Khekadaengoside C):
13c-NMR (C
5d
5n, 125MHz) δ: 121.2 (C-1), 147.1 (C-2), 197.3 (C-3), 49.9 (C-4), 137.3 (C-5), 121.0 (C-6), 24.2 (C-7), 42.1 (C-8), 51.2 (C-9), 35.9 (C-10), 214.2 (C-11), 49.9 (C-12), 49.4 (C-13), 49.0 (C-14), 46.8 (C-15), 70.7 (C-16), 59.4 (C-17), 20.5 (C-18), 18.6 (C-19), 80.3 (C-20), 25.8 (C-21), 215.1 (C-22), 32.5 (C-23), 36.1 (C-24), 145.8 (C-25), 110.6 (C-26), 23.0 (C-27), 20.7 (C-28), 27.8 (C-29), 21.1 (C-30), 100.9 (C-1'), 74.7 (C-2'), 78.7 (C-3'), 71.0 (C-4'), 78.9 (C-5'), 62.3 (C-6').
Compound 6 (Khekadaengoside D):
13c-NMR (C
5d
5n, 125MHz) δ: 120.9 (C-1), 147.1 (C-2), 197.3 (C-3), 49.8 (C-4), 137.3 (C-5), 121.4 (C-6), 24.2 (C-7), 42.1 (C-8), 51.4 (C-9), 35.8 (C-10), 214.8 (C-11), 50.3 (C-12), 49.4 (C-13), 48.7 (C-14), 45.9 (C-15), 71.7 (C-16), 57.2 (C-17), 20.5 (C-18), 18.4 (C-19), 76.6 (C-20), 25.0 (C-21), 81.9 (C-22), 126.3 (C-23), 142.0 (C-24), 70.2 (C-25), 31.0 (C-26), 31.1 (C-27), 20.6 (C-28), 27.9 (C-29), 21.1 (C-30), 101.0 (C-1'), 74.7 (C-2'), 78.7 (C-3'), 71.0 (C-4'), 78.9 (C-5'), 62.3 (C-6').
Compound 7 (bryoamaride):
13c-NMR (C
5d
5n, 125MHz) δ: 121.2 (C-1), 147.1 (C-2), 197.3 (C-3), 49.8 (C-4), 137.3 (C-5), 121.1 (C-6), 24.2 (C-7), 42.1 (C-8), 51.3 (C-9), 35.8 (C-10), 214.3 (C-11), 49.9 (C-12), 49.7 (C-13), 48.9 (C-14), 46.8 (C-15), 70.7 (C-16), 59.2 (C-17), 20.5 (C-18), 18.6 (C-19), 80.4 (C-20), 25.8 (C-21), 216.4 (C-22), 33.0 (C-23), 38.7 (C-24), 69.4 (C-25), 30.1 (C-26), 30.3 (C-27), 20.6 (C-28), 27.9 (C-29), 21.1 (C-30), 101.0 (C-1'), 74.7 (C-2'), 78.7 (C-3'), 71.0 (C-4'), 78.9 (C-5'), 62.2 (C-6').
Compound 8 (25-O-acetyl-bryoamaride):
13c-NMR (C
5d
5n, 125MHz) δ: 121.2 (C-1), 146.8 (C-2), 197.8 (C-3), 49.6 (C-4), 137.0 (C-5), 121.1 (C-6), 24.1 (C-7), 42.0 (C-8), 51.0 (C-9), 35.8 (C-10), 214.2 (C-11), 49.8 (C-12), 49.4 (C-13), 48.7 (C-14), 46.6 (C-15), 70.7 (C-16), 59.3 (C-17), 20.4 (C-18), 18.4 (C-19), 80.3 (C-20), 25.8 (C-21), 215.4 (C-22), 32.4 (C-23), 35.5 (C-24), 81.9 (C-25), 26.1 (C-26), 26.2 (C-27), 20.5 (C-28), 27.7 (C-29), 20.9 (C-30), 100.6 (C-1'), 74.4 (C-2'), 78.3 (C-3'), 70.5 (C-4'), 78.6 (C-5'), 62.2 (C-6').
Claims (10)
1. a radices trichosanthis triterpene extract, the radices trichosanthis triterpene extract described in it is characterized in that is prepared as follows:
(1) after raw material radices trichosanthis is pulverized, add the mixed liquor of compound enzyme and water, under the condition that is 4.5～6.5 at pH, in 25～65 ℃ of enzymolysis 10～120min, filter to get filtrate; Described compound enzyme is obtained by mixing with mass ratio 0.2～5:1 by cellulase and papain; The specific activity of described cellulase is 4000U/g, and the specific activity of described papain is 5000U/g; The quality consumption of described compound enzyme is 0.2%～10% of radices trichosanthis quality;
(2) gained filtrate in step (1) is concentrated into 1/5～1/3 of original volume and obtains concentrated solution, gained concentrated solution first extracts with halogenated hydrocarbons, isolate water layer and halo-hydrocarbon layer, after halo-hydrocarbon layer concentrate drying, obtain extract A, water intaking layer is extracted with ethyl acetate, and isolates water layer and ethyl acetate layer, after ethyl acetate layer contracting is dry, obtains extract B, extract A and extract B are merged, obtain crude extract; Described halogenated hydrocarbons is dichloromethane or chloroform;
(3) gained crude extract in step (2) is scattered in the aqueous solution of 0wt%～20wt% ethanol, and separate through resin absorption, then first use the aqueous solution eluting of 20wt%～30wt% ethanol, use again the aqueous solution eluting of 75wt%～90wt% ethanol, the ethanol water elution liquid that reclaims 75wt%～90wt%, obtains radices trichosanthis triterpene extract after concentrate drying; Described resin is selected from AB-8, D101 or H103.
2. radices trichosanthis triterpene extract as claimed in claim 1, is characterized in that compound enzyme described in step (1) and the mixed liquor of water and the mass ratio of radices trichosanthis are 5～20:1.
3. radices trichosanthis triterpene extract as claimed in claim 1, is characterized in that, in described step (1), described enzymolysis pH value is 5, and hydrolysis temperature is 50 ℃, and enzymolysis time is 60min; Described compound enzyme cellulase and papain mass ratio are 1:1; The quality consumption of described compound enzyme is 5% of radices trichosanthis quality.
4. radices trichosanthis triterpene extract as claimed in claim 2, is characterized in that compound enzyme described in step (1) and the mixed liquor of water and the mass ratio of radices trichosanthis are 11:1.
5. radices trichosanthis triterpene extract as claimed in claim 1, the volumetric usage that it is characterized in that halogenated hydrocarbons described in step (2) is 0.5～2 times of described concentrated solution volume.
6. radices trichosanthis triterpene extract as claimed in claim 1, the volumetric usage that it is characterized in that ethyl acetate described in step (2) is 0.5～2 times of described concentrated solution volume.
7. radices trichosanthis triterpene extract as claimed in claim 1, is characterized in that the aqueous solution of the 0wt%～20wt% ethanol described in step (3) and described crude extract mass ratio are 2～3:1.
8. radices trichosanthis triterpene extract as claimed in claim 1, the quality consumption that it is characterized in that resin described in step (3) is 1/5～1/3 of described radices trichosanthis quality.
9. radices trichosanthis triterpene extract as claimed in claim 1, is characterized in that, in step (3), the volumetric usage of the aqueous solution of described 20wt%～30wt% ethanol is counted 0.2～1L/kg with the quality of radices trichosanthis; The volumetric usage of the aqueous solution of described 75wt%～90wt% ethanol is counted 0.3～2L/kg with the quality of radices trichosanthis.
10. the application of radices trichosanthis triterpene extract as claimed in claim 1 in functional food and the medicine of preparation treatment diabetes.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101983637A (en) * | 2010-11-10 | 2011-03-09 | 河南科技学院 | Radix trichosanthis saponin and application of radix trichosanthis saponin in preparing medicine for treating ischemic cerebrovascular diseases |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101983637A (en) * | 2010-11-10 | 2011-03-09 | 河南科技学院 | Radix trichosanthis saponin and application of radix trichosanthis saponin in preparing medicine for treating ischemic cerebrovascular diseases |
Non-Patent Citations (3)
| Title |
|---|
| 李晓芳等: "天花粉降血糖活性成分的分离和活性观察", 《中成药》 * |
| 李琼等: "天花粉降糖作用有效部位的研究", 《长春中医药大学学报》 * |
| 陈胜发等: "栝楼属植物化学成分的研究进展", 《中成药》 * |
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