CN101912446A - Application of forsythia chloroform extracts to preparing medicine for treating diabetes mellitus - Google Patents

Abstract

The invention provides the application of forsythia chloroform extracts to preparing a medicine for treating diabetes mellitus, which relates to Chinese herb extracts. The invention provides the application of the forsythia chloroform extracts to preparing a tyrosine phosphatase SHP2 accelerant and a medicine for preparing diabetes mellitus. The preparation method of the forsythia chloroform extracts comprises the following steps of: soaking dry forsythia with ethanol; combining extracting solutions after being heated and refluxed; decompressing and drying to obtain a total forsythia extract; mixing the total forsythia extract with diatomite or suspending the total forsythia extract with water; extracting by adopting chloroform; decompressing and concentrating; and drying to obtain the forsythia chloroform extracts. The forsythia chloroform extracts can promote the activity of the tyrosine phosphatase SHP2, plays a role of reducing blood sugar concentration and can be widely used for preparing the tyrosine phosphatase SHP2 accelerant and the medicine for preparing diabetes mellitus.

Description

The application of chloroform extract of Fructus Forsythiae in preparation treatment diabetes medicament

Technical field

The present invention relates to a kind of Chinese medicine extract, the particularly application of chloroform extract of Fructus Forsythiae in preparation treatment diabetes medicament.

Background technology

Tyrosine phosphatase SHP2 is an important member in tyrosine phosphatase (tyrosine phosphatase) family, what its discovery made that people recognize for the first time that protein kinase contends with agent and the mechanism of contending with exists (Feng GS, Hui CC, Pawson T:SH2-containing phosphotyrosine phosphatase as a target of protein-tyrosine kinases.Science 1993,259 (5101): 1607-1611).A large amount of subsequently systematic studys show, SHP2 not merely suppresses the signal conduction as the contending with agent of protein kinase signal, also can promote various kinds of cell matter signal (2.Feng GS:Shp2-mediated molecular signaling in control of embryonic stem cell self-renewal and differentiation.Cell Res 2007,17 (1): 37-41.3.Feng GS:Shp2 as a therapeutic target for leptin resistance and obesity.Expert Opin Ther Targets 2006,10 (1): 135-142.4.Mohi MG, Neel BG:The role of Shp2 (PTPN11) in cancer.Curr Opin Genet Dev 2007,17 (1): 23-30.).Aspect metabolic disease, by a series of tissue specificity transgenic animal models, the normal activity of proof SHP2 is the key signal that prevents diabetes, its active reduction is then brought out diabetes (5.Zhang SS, Hao E, Yu J, et al.Coordinated regulation by Shp2 tyrosine phosphatase of signaling events controlling insulin biosynthesis in pancreatic beta-cells.Proc Natl Acad Sci U S A 2009,106 (18): 7531-7536; 6.Princen F, Bard E, Sheikh F, et al.Deletion of Shp2 tyrosine phosphatase in muscle leads to dilated cardiomyopathy, insulin resistance, and premature death.Mol Cell Biol 2009,29 (2): 378-388; 7.Krajewska M, Banares S, Zhang EE, et al.Development of diabesity in mice with neuronal deletion of Shp2 tyrosine phosphatase.Am J Pathol 2008,172 (5): 1312-1324; 8.Zhang EE, Chapeau E, Hagihara K, et al.Neuronal Shp2 tyrosine phosphatase controls energy balance and metabolism.Proc Natl Acad Sci U S A 2004,101 (45): 16064-16069; 9.Bard-Chapeau EA, Yuan J, Droin N, et al.Concerted functions of Gab 1and Shp2 in liver regeneration and hepatoprotection.Mol Cell Biol 2006,26 (12): 4664-4674; 10.Bard-Chapeau EA, Hevener AL, Long S, et al.Deletion of Gab 1in the liver leads to enhanced glucose tolerance and improved hepatic insulin action.Nat Med 2005,11 (5): 567-571.).SHP2 is the crucial modulin of diabetes and obesity, SHP2 is after forebrain is rejected, obesity takes place in mice, body weight is apparently higher than normal mouse, blood insulin and leptin raise, produce type ii diabetes symptoms such as hyperglycemia, insulin, leptin opposing, the typical complication of multiple diabetes then appears in development subsequently.And SHP2 is when forebrain continuous expression high activity, no matter the mice feed how much, is not got fat.In addition, use SHP2 and reject or activated transgenic mice, prove that SHP2 has promotion and safeguards secretion of insulin, promotes the glycometabolic effect of insulin regulation and control hepatocyte at liver, pancreatic tissue specificity.

Fructus Forsythiae is the dry fruit of Oleaceae plant Fructus Forsythiae Forsythia suspensa (Thunb.) Vahl.Cold nature, bitter in the mouth.Has heat-clearing and toxic substances removing, the effect of dispersing swelling and dissipating binds.Tradition is used and is mainly used in treatment of diseases such as affection due to external wind and heat, laryngopharynx swelling and pain, carbuncle furuncle, scrofula.Chemical constituent in the Fructus Forsythiae mainly is lignanoids, flavonoid, phenethanol and glycoside thereof and the natural alcohols of C6～C2, terpenoid etc.Pharmacological research shows, that Fructus Forsythiae has is antibiotic, antiviral, antiinflammatory, treatment cardiovascular disease, antiendotoxin, inhibition elastin laminin enzyme activity, suppress pharmacological actions such as cAMP di-phosphate ester enzyme activity, hepatoprotective effect (11. Zhang Haiyans. the progress of Fructus Forsythiae chemical constituent and pharmacologically active. Chinese crude drug, 2000,23 (10): 10).But report is not studied in the application of Fructus Forsythiae aspect blood sugar lowering, therefore, is target spot with SHP2, and the Fructus Forsythiae hypoglycemic medicine of development of new has very significant meaning.

Summary of the invention

The object of the present invention is to provide the application of chloroform extract of Fructus Forsythiae in preparation tyrosine phosphatase SHP2 promoter and preparation treatment diabetes medicament.

Described chloroform extract of Fructus Forsythiae prepares by the following method: through soak with ethanol, merge extractive liquid, after the reflux, drying under reduced pressure get the Fructus Forsythiae total extract with dry Fructus Forsythiae; Again the Fructus Forsythiae total extract is mixed with kieselguhr, or, adopt chloroform extraction Fructus Forsythiae total extract water suspendible, concentrating under reduced pressure, drying promptly gets chloroform extract of Fructus Forsythiae.

Can adopt ethyl acetate, ethanol (n-butyl alcohol) and water extraction successively through the residue Fructus Forsythiae extract behind the chloroform extraction, distinguish concentrating under reduced pressure again, drying can get other Fructus Forsythiae extract.

Described chloroform extract of Fructus Forsythiae can promote the activity of tyrosine phosphatase SHP2, and the effect of performance blood sugar lowering concentration has widely on the medicine of preparation treatment diabetes and uses.

Utilize the reaction of enzyme and substrate, set up the effective SHP2 enzyme activity assay of a maturation system, can high-throughout screening influence the composition of SHP2 enzymatic activity.Utilize the method for GST protein purification, from antibacterial, extract and purification SHP2 albumen.Fluorescence reaction is taken place in SHP2 albumen and its catalytic substrate DiFMUP (8-diXuoro-4-methylumbelliferyl phosphate), utilize the microplate reader reading, establish the use amount of ideal SHP2 albumen and catalytic substrate DiFMUP, set up sensitivity SHP2 enzyme activity assay model efficiently.Utilize described SHP2 enzyme activity assay model, activity to a Chinese medicine separate fraction surplus 2000 is screened, find that described chloroform extract of Fructus Forsythiae has tangible SHP2 enzymatic activity facilitation, and confirm that by zoopery described chloroform extract of Fructus Forsythiae can play the effect of treatment diabetes by this target spot of SHP2.

Description of drawings

Fig. 1 is the facilitations of different Fructus Forsythiae extracts to tyrosine phosphatase SHP2 enzymatic activity.In Fig. 1, abscissa is different Fructus Forsythiae extracts, and concentration is 3.84 μ g/mL, and vertical coordinate is a tyrosine phosphatase SHP2 enzymatic activity (%).

Fig. 2 is the effect of variable concentrations chloroform extract of Fructus Forsythiae to multiple tyrosine phosphatase.In Fig. 2, abscissa is chloroform extract of Fructus Forsythiae concentration (μ g/mL), and vertical coordinate is the promotion rate (%) of chloroform extract of Fructus Forsythiae for different tyrosine phosphatases; Labelling ◆ be SHP2, ■ are SHP1, ▲ be Heptp, ● be VHR.

The specific embodiment

Embodiment 1: the extraction of Fructus Forsythiae extract

Dry Fructus Forsythiae 5kg, 6 times of amount 60% soak with ethanol, reflux 4 times, each 2.5h, merge extractive liquid,, drying under reduced pressure get Fructus Forsythiae total extract 1000g.The Fructus Forsythiae total extract is with the kieselguhr mixing of 3～5 times of amounts, or with total extract water suspendible, the isopyknic chloroform extraction of suspension, concentrating under reduced pressure, drying promptly gets chloroform extract of Fructus Forsythiae 260g.

Extract successively with ethyl acetate, ethanol (or n-butyl alcohol) according to same method again through the residue Fructus Forsythiae extract (aqueous suspension) behind the chloroform extraction, respectively concentrating under reduced pressure, dry each extract.Get Fructus Forsythiae ethyl acetate extract 90g, Fructus Forsythiae ethanol (or n-butyl alcohol) extract 221g and Fructus Forsythiae water extract 400g.

Embodiment 2: Fructus Forsythiae extract is to the facilitation of SHP2

1, the activity rating method of SHP2 facilitation

Utilize the reaction of enzyme and substrate, set up an effective enzyme activity assay system of maturation, can high-throughout screening influence the composition of SHP2 enzymatic activity.Utilize the method for GST protein purification, from antibacterial, extract and purification SHP2 albumen.Fluorescence reaction is taken place in SHP2 albumen and its catalytic substrate DiFMUP (8-diXuoro-4-methylumbelliferyl phosphate), utilize the microplate reader reading, establish the use amount of ideal SHP2 albumen and catalytic substrate DiFMUP, set up sensitivity SHP2 activity analysis efficiently model.Utilize 96 hole ELISA Plate, different fractions or natural micromolecule are added treatment S HP2 activity analysis system, screening promotes the active positive micromolecule of SHP2.The positive micromolecule that screening is obtained is handled the activity analysis system of other tyrosine phosphatases VHR, Heptp, SHP1, and whether analyze them is the active specificity promoter of SHP2.

2, activity rating result

(3.84 μ g/ml) carries out the evaluation of SHP2 enzymatic activity facilitation to the different Fructus Forsythiae extracts among the embodiment 1, the result shows, chloroform extract of Fructus Forsythiae has good SHP2 enzymatic activity facilitation, the Fructus Forsythiae ethyl acetate extract, Fructus Forsythiae ethanol (n-butyl alcohol) extract and Fructus Forsythiae water extract promote that ability is more weak or do not have facilitation (referring to Fig. 1) for the SHP2 enzymatic activity.

Subsequently, chloroform extract of Fructus Forsythiae with variable concentrations, other three member VHR, the Heptp of treatment S HP2 and tyrosine phosphatase enzyme family, SHP1 observe the facilitation of chloroform extract of Fructus Forsythiae to each proteinase activity, estimate its facilitation to the SHP2 enzymatic activity and whether have specificity.The result shows: chloroform extract of Fructus Forsythiae increases with fraction concentration the facilitation of SHP2 enzymatic activity, show good concentration dependent, its other 3 member VHR, Heptp to the tyrosine phosphatase enzyme family, the enzymatic activity of SHP1 do not have obvious facilitation.Show that chloroform extract of Fructus Forsythiae has specificity (referring to Fig. 2) for the facilitation of SHP2.

Embodiment 3: the blood sugar reducing function of chloroform extract of Fructus Forsythiae

Adopt the inductive diabetic mice model of streptozotocin (STZ) to implement.Mice selects for use Kunming white mice male and female to buy during half and half, 4 age in week, in one week of the feeding adaptation of freely drinking water, begins to utilize the streptozotocin modeling.Mice is divided into 2 groups, diabetes-induced group, injection citrate buffer solution group.Before the injection each mice is weighed, mice should first fasting when injecting streptozotocin liquid 6 hours.The diabetes-induced group is according to the ratio lumbar injection STZ-citrate buffer solution (STZ is dissolved in the citrate buffer solution of 0.1mol/L pH4.5) of mice body weight 40mg/kg; The citrate buffer solution of matched group injection equivalent; Injection is 5 days continuously.Subsequently, mice places under the home and raises, and surveys blood glucose value (cut tail and get blood) in per 7 days, and in continuous 3 weeks, the mice of blood glucose value more than 16.7mmol/L is the diabetic mice model under the non-fasted conditions.Press the balanced random packet of blood glucose value, every group of 20 mices, every group of mouse blood sugar average basically identical.With the positive contrast medicine of metformin hydrochloride, the negative contrast of the solvent of chloroform extract of Fructus Forsythiae, chloroform extract of Fructus Forsythiae is established 100mg/kg, 400mg/kg, 3 dosage of 800mg/kg.Other gets 1 group of mice, and intraperitoneal injection of saline is irritated stomach according to the amount of 0.02ml/g to mice according to the mice body weight as the normal control group, once a day, and continuous two weeks.In at 10 o'clock in the morning every day mice is carried out fasting, fasting is irritated stomach to mice after 6 hours again.At the 7th day and the 14th day, fasting 3h respectively surveyed blood glucose once.

The result is as shown in table 1, and the chloroform extract of Fructus Forsythiae of 3 various dose all shows the effect of tangible blood sugar lowering.

Table 1 forsythia chloroform layer extract is to the effect of mouse blood sugar

A. compare the p value of t-check with the solvent model group.*p＜0.05，**p＜0.01。

Claims (2)

1. the application of chloroform extract of Fructus Forsythiae in preparation tyrosine phosphatase SHP2 promoter.

2. the application of chloroform extract of Fructus Forsythiae in preparation treatment diabetes medicament.

Images