CN102293796B - Active part of lysimachia trientaloides Hemsl., and extraction method and application of active part - Google Patents
Active part of lysimachia trientaloides Hemsl., and extraction method and application of active part Download PDFInfo
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Abstract
The invention relates to an active part of lysimachia trientaloides Hemsl.. The active part of the lysimachia trientaloides Hemsl. may be one or any combination of a petroleum ether part, an ethyl acetate part or an n-butyl alcohol part of the lysimachia trientaloides Hemsl. extracts. The extraction method particularly comprises the following steps of: lixiviating dry lysimachia trientaloides Hemsl. powder with methanol or ethanol aqueous solution; concentrating the immersion liquid to obtain an extractum; dispersing the extractum into distilled water; extracting by using the solvents of petroleum ether, ethyl acetate part and n-butyl alcohol sequentially for three times respectively; and volatilizing the solvents to obtain the petroleum ether part, the ethyl acetate part and the n-butyl alcohol part respectively. The active part of the lysimachia trientaloides Hemsl. has high inhibition activity on alpha-glucosidase and good curative effect on the mesoxyalyurea induced diabetes mice, and can be used for preparing medicines for reducing blood sugar.
Description
Technical field
The invention belongs to technical field of Chinese medicines, be specifically related to extract and extracting method and its application aspect the preparation hypoglycemic medicine of a kind of plant Lysimachia trientaloides Hemsl..
Background technology
Lysimachia trientaloides Hemsl. (
Lysimachia paridiformisFranch. var.
StenophyllaFranch.) another name infantile convulsion umbrella, Rhizoma Arisaematis, public Cortex torricelliae tiliifoliae, broken sunshade, back flower grass, turnsole, umbrella leaf Herba lysimachiae capillipedis, be herb or the root of the narrow leaf Herba lysimachiae Paridiformis of Primulaceae Lysimachia plant, be distributed in the ground such as Guizhou, Hubei, Hunan, Guangdong, Guangxi, Sichuan, Yunnan.Its acrid in the mouth, peppery, bitter, hot in nature.Enter cold warp.Main effect is dispelling wind and removing obstruction in the collateral, promoting blood circulation and stopping pain; Cure mainly rheumatism numbness logical, spasm of the limbs, hemiplegia, infantile convulsion, tumbling down, the diseases such as fracture.
Diabetes are to act on that body causes hypoinsulinism, insulin resistant etc. and a series of metabolism disorder syndromes such as the sugar that causes, protein, fat, power and water Xie Zhi by inherited genetic factors, immunologic function disorder, infected by microbes and toxin thereof, free radical toxin, Nervous and Mental Factors etc. various virulence factors.It is take hyperglycemia as feature, take metabolism disorder as performance, model case polyuria, polydipsia, polyphagia can occur, become thin etc., i.e. " three-many-one-little " symptom, in a single day blood glucose controlled bad meeting and caused complication, the exhaustion pathological changes that causes the positions such as kidney, eye, foot, and can't cure, the mankind's health in serious threat.According to the report of World Health Organization (WHO), nineteen ninety-five the diabetics made a definite diagnosis of the whole world approximately 1.35 hundred million, and IDF reported that global diabetics had reached 2.46 hundred million in 2006 in the recent period, predicted 2025 years diabeticss and will reach 3.88 hundred million.China has become diabetes the second big countries in the time of 2003, diabetics approximately 6,000 ten thousand.Therefore, prevention and treatment diabetes have become the health care problem of China and even whole world concern.Mainly to control fasting glucose as target, medicine mainly concentrates on sulphanylureas and biguanides for a long time in the treatment of diabetes for many years.Recent study is found, postprandial hyperglycemia often first occurs in the sick process of diabetes, then develops into gradually diabetes, and namely the former is the latter's sign in advance.For diabetics, II type patient particularly, considerably beyond hyperglycemia, postprandial hyperglycemia not only very easily brings out various complication to postprandial hyperglycemia, also can greatly improve the mortality rate of diabetes to the harm of body.So reducing post-prandial glycemia is one of important measures of prevent diabetes, complication and reduction mortality rate.That is to say, the control post-prandial glycemia is the control hyperglycemia, prevents and treats the Important Action of diabetes.In the medicine of existing treatment diabetes, sulfonylurea drugs is to reduce blood glucose by the secretion that stimulates insulin; Biguanides is by increasing peripheral tissues, the utilization of glucose to be reduced blood glucose, and both all have good therapeutic effect to reducing the Patients with NIDDM fasting glucose, but very limited to the effect that reduces post-prandial glycemia.
α-glucosidase inhibitor is a class new oral hypoglycemic drug of developing in the seventies later stage, and its mechanism of action is: pass through competitive inhibition
αThe activity of-glucosidase, retardance disaccharidase is hydrolyzed into monosaccharide, delays the absorption of sugar, makes blood glucose steadily and maintains lentamente certain level.
α-glucosidase inhibitor can effectively be controlled the rising of post-prandial glycemia, and the generation of prevent diabetes reduces diabetic complication, reduces mortality rate.
α-glucosidase inhibitor not only has definite curative effect to diabetes, and obesity, chronic viral hepatitis B, acquired immune deficiency syndrome (AIDS) and tumor etc. are also had certain therapeutical effect.
At present, being used for this type of clinical medicine has acarbose, voglibose and miglitol, and the alpha-glucosidase inhibitor in natural product is the focus of Recent study.The research discovery, the major structural types of alpha-glucosidase inhibitor is flavonoid, alkaloids and saponins, also has in addition tea polyphenols.Flavone compound is mainly by polyhydroxy structure performance inhibitory action, and the saccharification meeting of hydroxyl group weakens compound pair
αThe inhibitory action of-glucosidase.Alkaloids is also that polyhydroxylated alkaloid is inhibited.The extract of some Chinese herbal medicine also has good inhibition
αThe effect of-glucosidase, as green tea extract and Radix Et Rhizoma Rhei, Fructus Corni, Radix Paeoniae Rubra, Galla Chinensis water boiling and precipitation with ethanol extract, and the water extract of the full powder of Guangxi Sanguis Draxonis and substep extract, Fructus Schisandrae Chinensis and Rhizoma Polygoni Cuspidati etc.Yet existing this type of said medicine cost is higher, and manufacturer seldom is attended by the intestinal side effect simultaneously.
The plant Lysimachia trientaloides Hemsl. contains abundant chemical composition, but both at home and abroad the report of relevant this phytopharmacology studies and clinical application aspect but seldom, and the people such as rarely seen Qi Liuya propose the Lysimachia trientaloides Hemsl. total flavones and have significant analgesic and anti-inflammatory effects.There is not yet both at home and abroad about Lysimachia trientaloides Hemsl. effective site has inhibitory action, can be used for preparing the report of hypoglycemic medicine alpha-glucosidase.
Summary of the invention
The object of the invention is to provide a kind of Lysimachia trientaloides Hemsl. effective site, and this extract has hypoglycemic activity, can be used for preparing hypoglycemic drug.The present invention also provides the extracting method of this extract.
For achieving the above object, the present invention takes following technical scheme:
A kind of Lysimachia trientaloides Hemsl. effective site, it is selected from a kind of or combination in any in petroleum ether part, ethyl acetate extract or the n-butanol portion of Lysimachia trientaloides Hemsl. ethanol extract.
The extracting method of described Lysimachia trientaloides Hemsl. effective site comprises the steps: to get dry Lysimachia trientaloides Hemsl. powder and carries out lixiviate with methanol or ethanol water, and immersion concentrates to get extractum; Extractum is scattered in distilled water, then extracts with solvent petroleum ether, ethyl acetate and n-butyl alcohol successively, each extracts three times; Namely obtain respectively petroleum ether part, ethyl acetate extract and n-butanol portion after volatilizing solvent.
Preferably, the Lysimachia trientaloides Hemsl. powder of described drying at room temperature soaks 2-4 time with methanol or the ethanol water of 60-80V%, each 3 days, merges each gained immersion and is condensed into extractum.
Above-mentioned Lysimachia trientaloides Hemsl. effective site the preparation hypoglycemic drug and
αThe application of-glucosidase inhibitor class medicine aspect.
Extract of the present invention is from the plant Lysimachia trientaloides Hemsl., and simple and convenient extraction.Carry out in vitro tests with the positive contrast medicine of acarbose (Acarbose), petroleum ether part (LPFPE), ethyl acetate extract (LPFEA) and the n-butanol portion (LPFBU) of observing Lysimachia trientaloides Hemsl. are right
αThe inhibitory action of-glucosidase.Result of the test shows, above-mentioned Lysimachia trientaloides Hemsl. effective site pair
α-glucosidase all has obvious inhibitory action, when Lysimachia trientaloides Hemsl. is 1500 μ g/mL in mass concentration, each effective site is to the inhibition activity of alpha-glucosidase all higher (suppression ratio is all more than 95%), far above positive control Acarbose (suppression ratio is 57.26%), and the half-inhibition concentration IC of each effective site
50Value is all less than 50 μ g/mL, much smaller than positive control drug Acarbose(IC
50Value is 1103.01 μ g/mL).
Further, carry out in vivo test with the positive contrast medicine of acarbose, the therapeutical effect of the diabetic mice that petroleum ether part (LPFPE), ethyl acetate extract (LPFEA) and the n-butanol portion (LPFBU) of observing Lysimachia trientaloides Hemsl. induced alloxan.shown by diabetic mice experiment in body, ethyl acetate extract is high, middle dosage group (1000, 500 mg/kg) can significantly reduce fasting glucose, n-butanol portion is high, middle dosage group (800, 400mg/kg) has certain reduction post-prandial glycemia effect, in ethyl acetate extract, low dose group (500, 250 mg/kg) and n-butanol portion high, middle dosage group (800, 400 mg/kg) can significantly raise hepatic glycogen and significantly reduce TG in serum, the content of TC, ethyl acetate extract is high, middle dosage group (1000, 500 mg/kg) and n-butanol portion low dose group (200 mg/kg) can significantly reduce MDA content, the content of SOD in rising serum, but without difference.
Description of drawings
Fig. 1 is embodiment 1 gained Lysimachia trientaloides Hemsl. effective site different quality concentration pair
αThe impact of-glucosidase activity;
Fig. 2 is embodiment 2 gained Lysimachia trientaloides Hemsl. effective site different quality concentration pair
αThe impact of-glucosidase activity.
The specific embodiment
The invention will be further described by the following examples, but protection scope of the present invention is not limited to this.
Embodiment 1
A kind of Lysimachia trientaloides Hemsl. effective site, it is selected from a kind of or combination in any in petroleum ether part, ethyl acetate extract or the n-butanol portion of Lysimachia trientaloides Hemsl. ethanol extract; Concrete extracting method is: the Lysimachia trientaloides Hemsl. herb dries in the shade, pulverizes, get the Lysimachia trientaloides Hemsl. powder of 1000 g dryings, methanol aqueous solution (addition of methanol aqueous solution is 3-5 times of Lysimachia trientaloides Hemsl. powder weight) with volume fraction 70%, soak 3 times under room temperature, each 3 days, merge 3 gained immersion and be condensed into extractum; Extractum is scattered in the 1000ml distilled water, then uses successively solvent petroleum ether (3000ml), ethyl acetate (3000ml) and n-butyl alcohol (3000ml) to extract, each extracts three times; Namely obtain respectively solid-state petroleum ether part (LPFPE), ethyl acetate extract (LPFEA) and n-butanol portion (LPFBU) after volatilizing solvent, extraction ratio is respectively 1.8%, 6.2% and 2.4%.
Concrete extraction process is: first use petroleum ether extraction three times, upper strata petroleum ether part (being petroleum ether part after volatilizing) is got in the extract layering, and lower floor is water layer; Water layer is used ethyl acetate extraction three times again, and upper strata ethyl acetate part (being ethyl acetate extract after volatilizing) is got in the extract layering, and lower floor is water layer; Water layer is used n-butanol extraction three times at last, and upper strata n-butyl alcohol part (being n-butanol portion after volatilizing) is got in the extract layering.The purpose of operation is the effective ingredient in extractum is extracted successively by polarity order from small to large like this, because the polarity of solvent petroleum ether, ethyl acetate and n-butyl alcohol increases progressively.
Embodiment 2
Replace the methanol aqueous solution of volume fraction 70% with the ethanol water of volume fraction 70%, the other the same as in Example 1, obtain petroleum ether part (LPFPE), ethyl acetate extract (LPFEA) and n-butanol portion (LPFBU), extraction ratio is respectively 1.5%, 5.8% and 2.2%.
Effect test
Below provided respectively the result that petroleum ether part (LPFPE), ethyl acetate extract (LPFEA) and n-butanol portion (LPFBU) that embodiment 1 selects 70% methanol aqueous solution and the embodiment 2 to select 70% ethanol water alcohol extraction to obtain at last carry out the blood sugar lowering test.
The external Inhibiting enzyme activity test of test one, Lysimachia trientaloides Hemsl. effective site
1.1 test method: Microdilution plate method
1.1.1 principle: α-D-Glucose glycosides enzymatic hydrolysis 4-Nitrobenzol-
α-D-pyranglucoside (PNPG) produces nitrophenol (PNP, yellow substance have absorption maximum about 400 nm),
α-glucosidase inhibitor can suppress
αThereby-glucosidase and Binding Capacity reduce the burst size of PNP.Calculate different Lysimachia trientaloides Hemsl. effective sites pair with the changes of contents of PNP in certain hour internal reaction system
α-glucoside enzymeinhibition is active.
Instrument:Multiskan MK3 microplate reader (Thermo Electron); LRH-150 constant incubator (the permanent Science and Technology Ltd. in Shanghai one); DELTA 320 type PH meters (Mettler-Toledo); Electronic balance (Mettler-Toledo); Rotary Evaporators (Heidolph).
Reagent: α-glucosidase (Sigma company, EC 3.2.1.20, from baker ' s yeast, lot number: 105K1313), the 4-Nitrobenzol-
α-
D-pyranglucoside (PNPG, Sigma company, lot number: 026K1516), and phosphate buffer (PH 6.8), (lot number: 16869), other reagent are analytical pure to acarbose for acarbose, Sigma company.
Detection method and date processing
In 112 μ L, pH are 6.8 kaliumphosphate buffer, add 20 μ L concentration 0.2 U/mL's
α-glucosidase, 8 μ L sample solutions (sample solution gets with DMSO dissolving), 37 ℃ of constant temperature 15 min add the substrate PNPG of 20 μ L concentration 2.5 mmol/L, 37 ℃ of isothermal reaction 15 min; The terminator Na that adds again 80 μ L concentration 0.2 mol/L
2CO
3Aqueous solution is surveyed the OD value under 405 nm wavelength.
4 groups are established in experiment altogether, every group of three holes, negative control group (buffer+enzyme liquid+substrate), blank group (buffer), sample sets (sample+buffer+enzyme liquid+substrate), sample matched group (sample+buffer), calculate as follows suppression ratio, and obtain corresponding IC with Origin software
50Value.
Suppression ratio
Utilize respectively said method to measure the IC of embodiment 1 and each Lysimachia trientaloides Hemsl. effective site of embodiment 2 gained (petroleum ether part LPFPE, ethyl acetate extract LPFEA and n-butanol portion LPFBU) and positive controls acarbose sample
50
Experimental result
Table 1 and table 2 are respectively that embodiment 1 and embodiment 2 extract the Lysimachia trientaloides Hemsl. different parts that obtains
α-Glucosidase inhibitor is active, and Fig. 1 is the embodiment 1 Lysimachia trientaloides Hemsl. different parts that extracts
α-Glucosidase inhibitor rate is with the variation diagram of drug level; Fig. 2 is the Lysimachia trientaloides Hemsl. different parts that extracts of embodiment 2
α-Glucosidase inhibitor rate is with the variation diagram of drug level.
As can be seen from Table 1 and Table 2, the IC of Lysimachia trientaloides Hemsl. effective site
50Value is all much smaller than the positive control drug acarbose, and this shows that these several groups of active sites that extract all have well from Lysimachia trientaloides Hemsl.
α-The Glucosidase inhibitor effect.Wherein, the IC of n-butanol portion
50Being worth minimumly, showing that its inhibition is best, is secondly petroleum ether part, and the IC of ethyl acetate extract
50Value is maximum.
Fig. 1 and Fig. 2 show, Lysimachia trientaloides Hemsl. effective site of the present invention is when concentration is 1500 μ g/mL pair
α-glucoside enzymeinhibition activity is higher (suppression ratio is all more than 95%) all, all far above positive controls acarbose Acarbose (suppression ratio 57.26%), shows that Lysimachia trientaloides Hemsl. effective site has preferably
α-Glucosidase inhibitor is active.The petroleum ether part LPFPE of Lysimachia trientaloides Hemsl., ethyl acetate extract LPFEA and n-butanol portion LPFBU pair
α-glucoside enzymeinhibition activity all first presents dose dependent, but when suppression ratio acquires a certain degree, then increase its mass concentration, right
α-glucoside enzymeinhibition activity no longer increases.
Concrete, Fig. 1 shows, in the concentration range of experiment, Lysimachia trientaloides Hemsl. petroleum ether part LPFPE and n-butanol portion LPFBU are respectively when concentration is 62.5 μ g/mL and 46.88 μ g/mL, and be right
αThe suppression ratio of-glucosidase reaches respectively 93.10% and 89.42%, then increases the almost no longer increase of its suppression ratio of concentration, and ethyl acetate extract LPFEA is 93.75 μ g/mL pairs in mass concentration
α-Glucosidase inhibitor rate reaches 97.58%, and increasing its suppression ratio of concentration has downward trend.
Generally speaking, above-mentioned external Inhibiting enzyme activity test confirms, several groups of Lysimachia trientaloides Hemsl. effective sites that the present invention provides pair
α-Glucosidase all has very strong inhibitory action, and wherein the n-butanol portion effect with Lysimachia trientaloides Hemsl. is the most obvious, is good
α-Glucosidase inhibitor can be for the preparation of hypoglycemic drug.
Test two: blood sugar lowering test in the glycosuria sick body of Lysimachia trientaloides Hemsl. effective site
2.1 test material:
Main agents and instrument:Alloxan (ALX, Alfa Aesar company, lot number: 10122281); Acarbose (acarbose, Sigma company, lot number: 091010); T-CHOL TC, triglyceride TG (North Sea, Shanghai Bioisystech Co., Ltd); Superoxide dismutase SOD, malonaldehyde MDA (Nanjing build up bio-engineering research institute); UV 2000 type ultraviolet-uisible spectrophotometers (but You Ni Shanghai Instr Ltd.); Multiskan MK3 microplate reader (Thermo Electron); LRH-150 constant incubator (the permanent Science and Technology Ltd. in Shanghai one).
Experimental technique
120 4～6 week age, body weight 20 ± 2 g male and healthy kunming mices, be divided at random 12 groups, 10 every group.Mice is first fasting 12 h all, and then except Normal group, all the other organize equal tail vein injection 80 mg/kg alloxan (ALX).Fasting 12h again after 72 h then gets blood from mouse orbit, surveys blood sugar level.Select blood sugar level the mice of 11.1 mmol/more than L, be divided at random diabetic model group, and the high, medium and low dosage group of Lysimachia trientaloides Hemsl. petroleum ether part (LPFPE) (500,250,125mg/kg); The high, medium and low dosage group of ethyl acetate extract (LPFEA) (1000,500,250mg/kg); The high, medium and low dosage group of n-butanol portion (LPFBU) (800,400,200mg/kg); Acarbose positive controls (75 mg/kg) and Normal group.Except diabetic model group, Normal group is with 0.5% Carboxymethyl cellulose sodium aqueous solution (CMC-Na) gavage, and other test group is mixed with respectively the respective concentration gastric infusion with 0.5%CMC-Na, and 1 times/day, successive administration 7 days.2 h eye sockets are got the hematometry level of postprandial blood sugar after the last administration, and in fasting on that night 12h, anesthetized mice is plucked eyeball and got blood, and separation of serum is measured fasting glucose, malonaldehyde MDA, superoxide dismutase SOD, triglyceride TG, T-CHOL TC.Put to death animal, get liver and weigh, and measure hepatic glycogen content.In addition, observe the data such as animal feed, drinking-water, urine amount every day, and observe the situations such as fur, activity.
Embodiment 1 selects the result of the test of petroleum ether part (LPFPE), ethyl acetate extract (LPFEA) and n-butanol portion (LPFBU) that 70% methanol aqueous solution alcohol extraction obtains at last, sees Table 3 to 6.
As can be seen from Table 3, diabetic model group mice post-prandial glycemia obviously raises, and relatively there were significant differences (p＜0.001) with Normal group.After gavage treatment in 7 days, each Lysimachia trientaloides Hemsl. effective site administration group and model group compare, there was no significant difference.
As can be seen from Table 4, before administration, each test group mouse blood sugar is compared difference significantly (p＜0.001) with Normal group.The gavage treatment was compared with diabetic model group after 7 days, and LPFEA is high, middle dosage (1000,500 mg/kg) is organized and the mice fasting glucose of acarbose positive controls significantly reduces (p＜0.001, p＜0.05); Compare with Normal group, significant difference (p＜0.05, p＜0.001) is arranged.
As can be seen from Table 5, gavage is after 7 days, except LPFBU low dosage (200 mg/kg) group, the hepatic glycogen content of other Lysimachia trientaloides Hemsl. effective site dosage group is all high than diabetic model group, and all has significant difference (p＜0.05, p＜0.01, p＜0.001); Compare dosage group (250mg/kg) and LPFEA low dose group (250mg/kg) in LPFPE, there was no significant difference with Normal group.The TG level of each administration group is lower than diabetic model group, and all has significant difference (p＜0.05, p＜0.01, p＜0.001); And compare difference that there are no significant with Normal group.Compare with model group, (1000,500,250mg/kg), TC level middle dosage (800,400 mg/kg) high with LPFBU obviously reduces the high, medium and low dosage group of LPFEA, has significant difference (p＜0.05, p＜0.001); And compare there was no significant difference with Normal group.
By as seen from Table 6, in LPFPE, low dose group (250,125mg/kg), LPFEA is high, middle dosage group (1000,500mg/kg), the MDA content in LPFBU low dose group (200 mg/kg) and the little serum of acarbose positive controls is lower than diabetic model group, and has significant difference (p＜0.05, p＜0.01, p＜0.001).In LPFPE high dose group (500mg/kg), LPFEA low dose group (250 mg/kg), LPFBU, low dose group (400,200mg/kg) and the SOD level in acarbose positive controls mice serum higher than diabetic model group, but do not have significant difference.In each administration group mice serum, the SOD level is compared there was no significant difference with model group, and with normal group, significant difference (p＜0.01, p＜0.001) is arranged more all.
Embodiment 2 selects the result of the test of petroleum ether part (LPFPE), ethyl acetate extract (LPFEA) and n-butanol portion (LPFBU) that 70% ethanol water alcohol extraction obtains at last, sees Table 7 to table 10.
Claims (2)
1. Lysimachia trientaloides Hemsl. effective site is in the application of preparation aspect hypoglycemic drug, and described Lysimachia trientaloides Hemsl. effective site is selected from a kind of or combination in any in petroleum ether part, ethyl acetate extract or the n-butanol portion of Lysimachia trientaloides Hemsl. ethanol extract; Described Lysimachia trientaloides Hemsl. effective site is extracted through following step and obtained: get dry Lysimachia trientaloides Hemsl. powder and carry out lixiviate with methanol aqueous solution, immersion concentrates to get extractum; Extractum is scattered in distilled water, then extracts with solvent petroleum ether, ethyl acetate and n-butyl alcohol successively, each extracts three times; Namely obtain respectively petroleum ether part, ethyl acetate extract and n-butanol portion after volatilizing solvent.
2. Lysimachia trientaloides Hemsl. effective site is in preparation
αThe application of-glucosidase inhibitor class medicine aspect, described Lysimachia trientaloides Hemsl. effective site are selected from a kind of or combination in any in petroleum ether part, ethyl acetate extract or the n-butanol portion of Lysimachia trientaloides Hemsl. ethanol extract; Described Lysimachia trientaloides Hemsl. effective site is extracted through following step and obtained: get dry Lysimachia trientaloides Hemsl. powder and carry out lixiviate with methanol aqueous solution, immersion concentrates to get extractum; Extractum is scattered in distilled water, then extracts with solvent petroleum ether, ethyl acetate and n-butyl alcohol successively, each extracts three times; Namely obtain respectively petroleum ether part, ethyl acetate extract and n-butanol portion after volatilizing solvent.
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