CN106608899B - The preparation method and medical usage of 20 (R)-protopanaxatriols and derivative - Google Patents

Abstract

The present invention relates to a series of preparation process of new ginsenosides such as 20 (R)-protopanaxatriols (PPT) and its derivative and its in vivo and in vitro purposes on hypoglycemic effect, it is related to natural drug purposes field, the ginsenoside being specifically related to are as follows: 25-OH-PPT [20 (R)-dammarane-3 β, 12 β, 20,25-tetrol], 25-OCH₃- PPT [20 (R) -25-methoxyl-dammarane-3 β, 12 β, 20-tetrol], PPT [20 (R)-protopanaxatriol] and PT [20 (S)-panaxatriol].The present invention provides the new methods that sour water solution prepares ginsenoside, and it is separated and has been prepared using the ginsenoside that high performance liquid chromatography (HPLC) method and macroreticular resin-normal pressure forward direction silica gel column chromatography partition method generate hydrolysis, while external hypoglycemic activity test in vivo has been carried out to separating obtained ginsenoside.This two kinds of method for separating and preparing are simple and reliable, and strong operability is high-efficient, and separating obtained ginsenoside has the pharmacological activity of anti-diabetic, can be used for preventing, treating and assisting in the treatment of diabetes, can carry out industrialized production, be conducive to promote and apply greatly, have a good application prospect.

Description

The preparation method and medical usage of 20 (R)-protopanaxatriols and derivative

Technical field

The present invention relates to natural drug purposes fields, are related to the preparation side of 20 (R)-protopanaxatriols (PPT) and derivative Method and medical usage, and in particular to a series of ginsenoside acid degradation compounds and its preparing the application in hypoglycemic drug.

Background technique

With the improvement of living standards, diabetes (diabetes mellitus, DM) have become serious harm human health Public health problem, and China is one of fastest-rising country of diabetes prevalence, is at present the second big country of diabetes.Sugar The main harm of urine disease is the generation that hyperglycemia leads to multisystem, multi viscera complication, and multi viscera complication is that diabetes cause The main reason for dead and disabled.In recent years, post-prandial glucose excursions and in a few days maximum blood glucose fluctuation is that type II diabetes is caused to suffer from An important factor for person's blood vessel endothelium injury.Postprandial hyperglycemia is the independent hazard factor of cardiovascular disease death, and effect is independent In lasting blood glucose level.Level of postprandial blood sugar is kept close to normal range (NR), is control blood glucose fluctuation, prevention cardiovascular and cerebrovascular disease Generation, reduce one of the important channel of mortality of cardio and cerebral vascular disease.The main source of blood glucose is the carbohydrate in food.Carbon water Compound passes through alpha-glucosaccharase enzymatic hydrolysis, and generating monosaccharide can just be absorbed, and α-Portugal of catalyzing hydrolysis carbohydrate Polyglycoside enzyme is distributed mainly in small intestinal mucosa brush border, and it includes have alpha-amylase, limit dextrinase, maltose, invertase With lactase etc..Therefore alpha-glucosidase is the key enzyme for adjusting food source blood glucose, becomes regulation postprandial blood sugar drug Target enzyme is acted on, alpha-glucosidase restrainer (α-glucosidase inhibitor, AGI) is to control suiting the medicine to the illness for postprandial blood sugar Therapeutic agent.Protein tyrosine phosphatase 1B (PTP1B) is a kind of intracellular protein tyrosine phosphorus of wide expression in vivo Sour enzyme plays an important role during adjusting insulin sensitivity and energetic supersession.By inhibiting PTP1B that can increase pancreas islet The activity of element and albumen, while also the determination for hypoglycemic lead compound provides foundation.

Ginsenoside is the main active in ginseng, modern pharmacology research show ginsenoside acid or Resulting ginsengenin is hydrolyzed under conditions of alkalinity with stronger pharmacological activity, traditional hydrolysis mainly use strong acid or General ginsenoside is hydrolyzed in highly basic, gained hydrolysate is separated with simple silica gel column chromatography, to obtain Active saponin member, this method has manufacturing cycle too long, single using solvent, and has toxicity more, greatly limits production The raising of efficiency.The present invention carries out sour water solution general ginsenoside using ultrasound and shaking table for the first time, greatly improves the total soap of ginseng The hydrolysis efficiency of glycosides, makes it easier to industrial amplification production.Gained hydrolysate we be utilized respectively traditional silica gel column chromatography The method combined with macroreticular resin separate, and using macroreticular resin to the enriched character of saponin(e, greatly improves pair The production efficiency of sapogenin reduces energy consumption, while carrying out elution preparation using a variety of flow phase systems, enriches preparation side Method reduces the harm caused by human body of part mobile phase high toxicity, avoids the generation of secondary harm.Efficient liquid is utilized for the first time Phase chromatography establishes 25-OH-PPT, 25-OCH₃The Simultaneous Determination method of-PPT, PPT and PT, guidance is using preparation HPLC to institute Hydrolysate carries out separation preparation, while when HPLC is separated and is prepared using blind point-score and continuous sample introduction method to hydrolysate into Row separation preparation, greatly reduces the loss of time and material, improves production efficiency, is the height of blood sugar reducing active saponin member Quickly preparation provides strong technical support to effect.

Ginseng is that China's pharmacopeia records kind, is Araliaceae (Araliaceae) Panax (Panax) plant ginseng The drying root and rhizome of (Panax ginseng C.A.Meyer), is traditional blood-enrich Chinese medicine, main product is in China's northeast ground Area, be have won fame both at home and abroad, the rare medicinal herbs that old children all knows.Ginseng is classified as top grade by Shennong's Herbal, there is ginseng " tonifying five zang organs, peace Spirit, determine soul, stop palpitate with fear, remove pathogenic factor, improving eyesight, happily, intelligence development, long term usage makes light of one's life by commiting suicide macrobiosis " record.Ginseng is used as many years The various debilitating conditions of valuable drug therapy are enriched blood, and clinical test it is strong demonstrate ginseng and its effective active composition Ginsenoside has the function of anti-diabetic.Ginsenoside is main active isolated in ginseng, has scholar to people Ginseng root and the extract of panax ginseng fruit have carried out hypoglycemic activity research, are with the ob/ob mouse with fat and hyperglycemic symptoms Model, by the change of blood sugar for measuring the mouse in each group, the results showed that, the blood glucose of panax ginseng fruit and ginseng group mouse is all It significantly reduces.At the same time, the experimental results showed that panax ginseng fruit group the weight of animals is substantially reduced.Show the hypoglycemic mechanism of panax ginseng fruit May be related with anti-obesic action, improve blood lipid metabolism correlation (Kim SH, Park KS.Effect of Panax ginseng extract on lipid metabolism in humans[J].Pharmacol Res.2003,48(5):511-513)。 (Attele AS, Zhou YP, the Xie JT et al.Antidiabetic effects of Panax ginseng such as Attele berry extract the identification of an effective component[J] .Diabetes.2002.51 (6): 1851-1858) by research shows that ginsenoside can significantly improve hyperglycemic patients abdominal cavity Blood-sugar content plays apparent hypoglycemic effect, especially to the significant effect of type-2 diabetes mellitus.Some researches show that different ginsengs Saponin(e can play significant effect to different diabetes, show the hypoglycemic totipotency of ginsenoside, while clinical test Show that they can significantly increase influence of the insulin to metabolism of blood glucose.

Some researches show that for ginseng as traditional Chinese medicine, hypoglycemic effect has obtained extensive research and concern (Vuksana,V,Sievenpipera,J.L.Herbal remedies in the management of diabetes: Lessons learned from the study of ginseng[J]..Nutr.Metab.Cardiovasc.Dis.2005, 15,149-1601).There is scholar by using ob/ob mouse species as model, detecting influence of the ginsenoside Re to mouse blood sugar, The result shows that ginsenoside Re has extremely strong hypoglycemic effect, can by improving mouse to the tolerance of glucose, from And have the function that hypoglycemic (Xie JT, Mehendale SR, Li XM, et al.Anti-diabetic effect of ginsenoside Re in ob/ob mice[J].Biochimicaet Biophysica Acta.2005,1740:319- 325).General ginsenoside, ginsenoside Rb₁、Rg₁、Rg₃, Re and Rh₁There is significant inhibiting effect to alpha-glucosidase, And it is better than 3-10 times of (Song Chunqing of positive control acarbose.Ginsenosides inhibit alpha-glucosidase activity in preparation Application [P] on drug.China: CN200910194673).Han has found metabolin one of of the PPT as ginsenoside, PPT energy Enough dramatically increase PPAR γ transcriptional expression activity, the results showed that PPT is used as PPAR gamma agonist, can enhance insulin and support Anti- generation blood sugar reducing function (Han KL, Jung MH.Ginsenoside 20S-protopanaxatriol (PPT) actvates peroxisome proliferator-activated receptorgamma(PPARgamma)in 3T3-L1adipocytes [J].BiolPharm Bull,2006,29(1):110).(the Ye Yin such as Ye Yin.External insulin and panaxoside Rg₁To 3T3- The influence [D] of L1 fat cell adiponectin mRNA expression.Hangzhou: Zhejiang University, 2005) 3T3-L1 fat cell contained into pancreas Island element and ginsenoside Rg₁Culture environment in co-culture a period of time, insulin active present dose dependent reduce 3T3- L1 fat cell adiponectin mRNA expression, as a result illustrates ginsenoside Rg₁The growth of adipose tissue can be promoted to grow, thus What is connect enhances the consumption of internal glucose, reaches hypoglycemic effect, this is also the another kind of ginseng hypoglycemic mechanism Performance.

Ginsenoside is administered orally to diabetes rat, research shows that ginsenoside Re is dynamic to experiment by research in Cho etc. The effect of object model is significant, and administration animal groups blood glucose value is significantly lower than control group, and one is shown in terms of the tolerance to glucose Fixed positive correlation acts on (Xue JT, Mehendale SR, Li X, et al.Anti-diabetic；effect of ginsenoside Re in ob/ob mice[J].Biochimica et Biophysica Acta.2005,1740(3): 319-325).Mechanism of action Sun Lianqing etc. hypoglycemic to ginsenoside Re carries out the study found that it can be thin by improving pancreas islet B The activity of born of the same parents' neuropile enzyme and hippocampus L-SA play hypoglycemic effect (Sun Lianqing, Liang Xiaochun, etc..The hypoglycemic mechanism of Chinese medicine Recent progress in experimental study [J].Chinese traditional Chinese medicine magazine, 2007,22 (11): 789-791).

Chen Yan etc. carries out the study found that the two has one to alpha-glucosidase ginsenoside Re and ginseng berry extract Fixed inhibiting effect, but under the conditions of same concentrations, ginseng berry extract show higher inhibiting effect, table when 10mg/kg Reveal complete inhibition effect.Later period again studies ginseng pectin, shows centainly to alpha-glucosaccharase enzyme inhibition Dose dependent (Chen Yan.The research [D] of Water extracts from Ginseng hypoglycemic effect.Changchun: Northeast Normal University's doctorate opinion Text, 2010,05).The main active ginsenoside of most of research discovery ginsengs has stronger hypoglycemic work in recent years Property.Wherein ginsenoside Rg₁, ginsenoside Re all show extremely strong hypoglycemic activity, while study discovery protoplast ginseng three Alcohol group (PPT) the protopanoxadiol group (PPD) that compares shows stronger hypoglycemic property.Ginsenoside is as natural, cheap, low The hypoglycemic drug of poison, has had received widespread attention.Purposive structural modification, system are carried out to the lower ginsenoside of activity The standby higher Hydrolizates of activity, improve its pharmacokinetic property, increase the research of its hypoglycemic activity, have wide Wealthy application prospect and development prospect.Inventor has found a kind of noval chemical compound in ginsenoside acid degradation compound, has good Good hypoglycemic work.In addition, ginseng also has a wide range of applications history in South Korea, Japan and some other country, ginseng into Enter the every field such as drug, food, more there is the various products circulation market such as ginseng beverage, biscuit, sugar, tea at present.

Summary of the invention

The purpose of the present invention is to provide a series of 20 (R)-protopanaxatriols (PPT) and its derivative to separate preparation side Method.

The present invention additionally provides 20 (R)-protopanaxatriols (PPT) simultaneously and its hypoglycemic activity research of derivative is real It tests.

The structure of 20 (R)-protopanaxatriols (PPT) and its derivative of the invention is as follows:

The present invention provides the preparation methods of described 20 (R)-protopanaxatriols (PPT) and its derivative, including directly from Separation and acid degradation preparation are extracted in former plant.

Select the system repeatedlies dry chromatography such as petroleum ether-ethyl acetate, chloroform-methanol-water or petroleum ether-acetone or HPLC is isolated and purified, and prepares 20 (R)-protopanaxatriols (PPT) and its derivative.

Separation is extracted from former plant: accurately weighed 10.0g notoginseng haulm, with 70% ethyl alcohol extract three times, after will Resulting crude product macroporous resin treatment, (being eluted respectively with 30%, 50%, 70%, 90% methanol-water and pure water), obtains people Join saponin compound.By the corresponding solvent solution-forming of this compound, then prepared by efficient preparation liquid phase or tradition Glass column separates product, merges the collection liquid of each ginsenoside, and solvent is flung in decompression, and vacuum freeze drying is up to 6 kinds The freeze-dried powder of monomeric compound.

Its sour water solution preparation method is: precision weighs 10 parts of 1.0g ginsenosides, with 1.0~6.0 times of amount methanol, surpasses Sound dissolution, it is 1.2~12mol/L acid, 2~20mL, constant temperature (38~60 DEG C) shaking table reaction 0.5 that concentration is separately added into after to be dissolved ~3h.It is neutralized to pH=7 with 1mol/L NaOH, is centrifuged after aqueous precipitation, it is dry, obtain ginsenoside acid degradation crude compound. Gained crude product macroporous resin treatment (is eluted with 30%, 50%, 70%, 90% methanol-water and pure water) respectively, obtains people Join stem-leaf total saponin acid degradation compound.By the corresponding solvent solution-forming of acid degradation compound, then by efficient preparation Liquid phase or tradition prepare glass column and separate to product, merge the collection liquid of each ginsenoside, and solvent, vacuum are flung in decompression It is freeze-dried the freeze-dried powder up to 6 kinds of monomeric compounds.

The present invention and Chinese patent literature CN 101828712A ginsenoside acid degradation object preparation process and its application side Method is compared discovery, and acid hydrolytic reaction condition of the present invention is milder, and reaction process replaces refluxing extraction with shaking table reaction, fits Big industrialized production is closed, there is extremely strong application value realistic.

The present invention is prepared in the method for acid degradation compound, saponin(e used are as follows: general ginsenoside is ginseng stem-leaf total soap Glycosides or Radix Ginseng total saponins or ginseng fruit saponins or ginseng flower total saponine or panaxdiols saponin or American ginseng total soap Glycosides or Quinquefolium saponin or American Ginseng glycol saponins or arasaponin or notoginseng stem and leaf total saponin.

The present invention prepare acid degradation compound method in, acid can be hydrochloric acid or sulfuric acid or glacial acetic acid or oxalic acid or One of citric acid, concentration range most preferably 1.2~12mol/L；Acid degradation is dissolved in 1.0~6.0 times of amount methanol, raw material Amount is the 10%~60% of solvent；Reaction carries out in shaking table.Acid degradation temperature is 38~60 DEG C；Hydrolysis time is 0.5~3h. Alkali can plant for one of alkali metal hydroxide, alkali metal salt, rudimentary sodium alkoxide, and concentration is 0.3~2.0mol/L.

The present invention provides being synchronised the method for detecting PPT and its derivative using efficient liquid chromatography, to utilize efficiently system Standby liquid chromatogram (HPLC) method preparative separation acid hydrolysis products provide guidance.

The chromatographic condition that high performance liquid chromatography of the present invention detects 4 kinds of acid hydrolysis products is 0~30min, 70% A；30~45min, 80%.

The present invention provides the preparation methods of 20 (R)-protopanaxatriols (PPT) and its derivative, efficiently prepare liquid phase color Spectrum (HPLC) method application high-purity ginsenoside standard reference material separation prepares acid hydrolysis products.

The present invention and a kind of Panax Notoginseng saponin R of Chinese patent literature CN 101575357A_l, ginsenoside Rg_l、Re、Rb_lWith The preparation method of Rd is compared, our invention can reach 6 compounds of synchronous preparative separation, and single prepares 6 compounds It is completed in 80min, the production cycle greatly shortens, and compared with previous thin layer separates, obtains product purity height, manufacturing cycle It shortens dramatically, greatly improves production efficiency.

Ginsenoside, high performance preparative liquid chromatography (HPLC) separation, chromatographic condition: with Kromasil C₁₈(4.5mm × 150mm, 5 μm) it is stationary phase, sampling volume 1uL-40mL, 25 DEG C of column temperature, flow velocity 1.0mL/min；Mobile phase: methanol (A) and H₂O (B) is by different volumes than mixing, gradient elution program: 0~30min, 70%A；30~45min, 80%.

It is synchronous to use evaporation photodetector (ELSD), vaporizer temperature 50 C, flow rate of carrier gas 3L/min, monitoring elution curve And guide product is collected.

Collection liquid is concentrated with Rotary Evaporators, after be placed in (4 degrees Celsius) progress crystallization bodies of refrigerator, both each monomer at Point.

The product obtained with this method carries out Purity to it with ultraviolet, infrared and gradient high performance liquid chromatogram, knot Fruit shows that products therefrom purity is all larger than 98%.

Beijing Chuangxin Tongheng Science and Technology Co., Ltd.'s Fuji-C18-10 μ performance liquid chromatographic column；Column temperature: 25 DEG C；Detect wave It is long: 254nm；Mobile phase: methanol-water；Volume flow: 20.0mL/min；Sample volume: 1.0mL (continuous sample introduction method).Choose first Alcohol: water=83:17 eluent gradient, sample introduction concentration are 20mg/mL, and sample injection time interval 13.5min is big as the set skill of handling needles is carried out Amount preparation high-purity ginsenoside standard reference material.Separating degree, peak shape in gained spectrogram, peak height meet the requirements, greatly Preparation efficiency is improved, the consecutive intervals sample introduction of trocar partition method in this way can save the time, improve efficiency, save flowing Phase reduces consumption, reduces the discharge etc. of waste liquid, can be used as the method for largely preparing ginsenoside.

Beijing Chuangxin Tongheng Science and Technology Co., Ltd.'s Fuji-C18-10 μ performance liquid chromatographic column；Column temperature: 25 DEG C；Mobile phase: Methanol-water.Choose methanol: water=81:19 eluent gradient weighs sample 0.5g, is dissolved in methanol and is configured to saturated solution, sample introduction 2.5mL is measured, largely prepares high-purity ginsenoside standard reference material as blind point-score is carried out.The advantage of blind point-score is when needs When carrying out the separation preparation of large dosage of substance and being limited sensorless detection PM signals by experiment condition, just with blind point-score It can achieve purpose.Because its detection for not needing detector can separate and prepare required substance, have Wide application range.

The present invention provides the traditional colours of 20 (R)-protopanaxatriols (PPT) and its derivative to compose Preparation Method, macropore tree Rouge and the separation preparation of normal pressure forward direction silica gel column chromatography.

The present invention improves extracting method on the basis of early-stage study, got rid of Sephadex LH-20 and ODS purification process repeatedly, improved extracting method is simple process, easy, and cost substantially reduces, and yield is high.

Preparation method of the invention includes: ginseng acid hydrolysis products, and extracting solution first uses macroporous resin column chromatography, and chromatographic column is used Ethanol elution carries out abstraction impurity removal after eluent concentration again, and remaining aqueous fraction is separated through silica gel column chromatography after removal of impurities, Recrystallization to get.

Wherein before macroporous resin column chromatography ethanol elution, preferably first cleaned with water and 20%-30% ethanol rinse, this hair Percentage in bright is percent by volume.

The preferred 50%-90% of concentration of alcohol for extraction, macroporous resin column chromatography preferably collect 60%-70% when eluting The flow point of ethyl alcohol.

It extracts preferred with the amount of ethyl alcohol: medicinal material: ethanol water 1:10-1:12w/v.

Abstraction impurity removal solvent ethyl acetate.

When silica gel column chromatography separates, preferably with, petroleum ether-ethyl acetate (1:5,1:4,1:3,1:2,1:1,2:1, 3:1,5:1) chloroform-methanol (20:1,15:1,10:1,10:2,10:2.5,10:3,10:4,10:5.5,10:6,10:7) petroleum The mixed solvents such as ether-acetone (5:1,4:1,3:1,2:1,1:1,1:2) are eluted.

The preferred D101 type of macroporous resin column chromatography or HPD-100 type.

The aqueous solution of the preferred methanol of recrystallization solvent, ethyl alcohol or n-butanol or methanol, ethyl alcohol or n-butanol.Most preferably The aqueous solution of methanol.It is preferred that methanol: water 1:1-10:1v/v.

The present invention additionally provides the hypoglycemic activity research experiment of PPT and its derivative simultaneously.

Ginsenoside acid degradation compound blood sugar decreasing effect is illustrated by test result below:

External hypoglycemic enzyme experiment:

To the inhibiting effect of alpha-glucosidase.

Its principle is to pass through the yellow green pair of detection PNPG and alpha-glucosaccharase enzyme reaction generation using PNPG as reaction substrate The absorbance (OD) of nitrophenol (PNP) solution carrys out judgement sample inhibitory activity.Experimental method is by 30 μ L alpha-glucosidases Solution, 20 μ L inhibitor are added in test tube, and 150 μ L PNPG, 800 μ l phosphate-buffered salts are added in 37 DEG C of warm bath 5min.Sealing, puts After entering 37 DEG C of warm bath 30min, 2mLNa is added₂CO₃Terminate liquid stops reaction.With microplate reader at 405nm measured value.DMSO makees empty White control, acarbose make positive control.It is calculated using following formula, as a result as shown in Fig. 1, table 1.

Inhibiting rate=(blank group OD- test sample OD)/blank group OD × 100%

Statistical procedures: with SPSS10.0 software IC for statistical analysis₅₀。

1 the compounds of this invention alpha-glucosidase activity of table studies (x scholar S, n=2)

Alpha-glucosidase activity studies have shown that 20 (R)-protopanaxatriols (PPT) etc. series ginsenoside to α-grape Glycosidase has extremely strong inhibitory activity, wherein 25-OCH₃- PPT, 25-OH-PPT and PPT are to alpha-glucosaccharase enzyme inhibition activity It is better than positive control drug acarbose (acarbose).25-OH-PPT is to alpha-glucosaccharase enzyme inhibition in several derivatives Most strong, inhibiting rate reaches 2.5 ± 0.15, while also compared with triol saponins Rg₁It is strong very much.

To the inhibiting effect of alpha-amylase activity

Ill vitro test method: the measurement of alpha-amylase inhibition is according to Japan and the measurement examination of light Co., Ltd. alpha-amylase Agent cassette method carries out.200mM phosphate buffer (PH=7.0) 0.5ml is added i.e. in the matrix liquid of 0.5mL, adds after keeping the temperature 5min Enter commercially available alpha-amylase solution 0.01ml, be allowed to react 5min, pure water 2.5mL and iodine solution 0.5mL is then added, sufficiently stirs It mixes, is measured at 660um, calculate the activity of 5min amylase.The series such as the PPT of various concentration are added in this measurement system The test fluid of ginsenoside observes result.The enzymatic activity of no added control group is 100%.

2 the compounds of this invention alpha-glucosidase activity of table studies (x scholar S, n=2)

Alpha-amylase activity is studies have shown that the series ginsenoside such as 20 (R)-protopanaxatriols (PPT) has alpha-amylase There is extremely strong inhibitory activity, wherein 25-OCH₃- PPT and 25-OH-PPT is better than positive control to alpha-glucosaccharase enzyme inhibition activity Medicine acarbose (acarbose).25-OH-PPT is most strong to alpha-glucosaccharase enzyme inhibition in several derivatives, inhibiting rate Reach 4.33 ± 0.21, while also compared with triol saponins Rg₁It is strong very much.

Protein tyrosine phosphatase 1B (PTP1B) activity experiment.

PNPP (p-nitrophenyl phosphate) is used to carry out phosphatase activity measurement as substrate.Principle are as follows: protein junket Propylhomoserin phosphatase -1B (PTP1B), is tyrosine phosphatase widely distributed in vivo, mainly by insulin receptor substrate - The dephosphorylation of 1 (IRS-1) and Insulin receptor substrate-2 (IRS-2), the final conduction for adjusting insulin receptor signal. I.e. insulin is by conjunction with insulin receptor, leading to insulin receptor and intracellular protein phosphorylation step by step, thus by signal It is passed to and generates physiological effect into the cell.And PTP1B is hindered the progress of this process, is caused by catalysis dephosphorylation The phenomenon that insulin resistance.Insulin resistance is the basic reason of type-2 diabetes mellitus, so that body is sensitive to own insulin Property reduce, illustrate PTP1B and type-2 diabetes mellitus have close relationship.

Compound inhibits the experiment of PTP1B enzymatic activity to carry out in 96 orifice plates, and it is molten containing enzyme buffer that experimental group sequentially adds 83 μ L Liquid (0.4 μ L enzyme), 10 μ L compound solutions and 4 μ L substrate solutions, positive controls sequentially add 83 μ L containing enzyme buffer solution (0.4 μ L enzyme), 10 μ L sodium vanadate solutions and 4 μ L substrate solutions.Blank control group sequentially adds 93 μ L (0.4 μ containing enzyme buffer solution L enzyme) and 4 μ L substrate solutions.83 μ L are added containing enzyme buffer solution (0.4 μ L enzyme), 10 μ L DMSO solutions and 4 μ L in reagent controls group Substrate solution, in constant incubator 37 DEG C reaction 30 minutes after, with 5 μ L NaOH terminate react.Product is measured by microplate reader The absorption intensity at 405nm wavelength, and sodium vanadate is the positive control drug of PTP1B activity inhibitor.

3 the compounds of this invention PTP1B enzyme activity research research of table

PTP1B enzyme activity research shows that the serial ginsenoside such as 20 (R)-protopanaxatriols (PPT) has pole to PTP1B Strong inhibitory activity, activity is nearly all better than positive control drug sodium vanadate, while Re is ground as Ginsenosides hypoglycemic effect Study carefully a kind of most ingredients, it is weaker than positive control medicine sodium vanadate to the inhibiting effect of PTP1B, while being also weaker than our 20 (R)-protopanaxatriol (PPT) series derivates.

Vitro enzyme activity experiment the result shows that, two kinds of enzymes of the compounds of this invention have inhibiting effect, show stronger Inhibiting rate, the compound equally has stronger inhibitory activity compared with the control group.

Hypoglycemic enzyme experiment in vivo

Experimental example 1 causes the research of hyperglycemia mouse hypoglycemic effect to STZ by taking 25-OH-PPT (T19) as an example.

4 week old C57/BL male mices are selected, STZ is injected intraperitoneally after a week with 50mg/kg body weight dose in adaptable fed Sodium citrate solution totally 3 times, every minor tick 2 days), induce its blood glucose rise.The drop of mouse fasting 16h rearward end extracting vein blood one is surveyed Its blood glucose value selects mouse of the blood glucose value between 10~14 as ideal model group, constructs the type-2 diabetes mellitus mouse of STZ induction Model, under the conditions of normal feeding, (positive is right by the T19 and dimethyldiguanide tablet that give various dose respectively using stomach-filling mode According to), administration time is surrounding, tests its weight and postprandial plasma glucose level weekly.After drug withdrawal, sugar tolerance experiment is carried out；Detect blood Middle triglycerides and the analysis of cholesterol level and organ index.

Weight detects weekly:

STZ group mouse weight continues to increase, and is apparently higher than blank control group.Give melbine and the Mice Body of T19 It overweights and third week starts to be substantially reduced.

Fig. 2

Blood sugar monitoring weekly

According to Fig. 3 data as it can be seen that STZ group mouse blood sugar is persistently higher than blank control group, also, melbine and three kinds of agent The T19 of amount can be substantially reduced mouse blood sugar, and middle dosage is that blood still drops in the either hypoglycemic ability of the T19 of 20mg/kg The stability of sugar, better than positive drug melbine.

Sugar tolerance test is injected intraperitoneally

After four weeks is administered, sugar tolerance experiment show dose is that the T19 of 20mg/kg can significantly increase the sugar tolerance of mouse, Such as Fig. 4, Fig. 5.

Organ index

Organ index (specific gravity of internal organ and weight) simultaneously has no difference, such as Fig. 6.

Lipid determination

Triglycerides (TG) and cholesterol (CHO) measure lipids in serum assay in blood, select TG and CHO content Carry out representative measurement.The results show that T19 can be substantially reduced triglycerides and cholesterol level in blood, and effect is better than sun Property medicine, such as Fig. 7, Fig. 8.

In conclusion T19 hypoglycemic effect under the conditions of giving in short term is obvious.It tests and finds for sugar tolerance, low dosage T19 Improvement (20mg/kg) resistance to body sugar under the conditions of giving for a long time is obviously even better than commercially available melbine.For body The influence of interior internal organs has no difference, and the effect of T19 is also superior to positive drug melbine in lipid determination.

In conclusion the series ginsenoside acid degradation compound such as T19 has good hypoglycemic effect, can be developed into very Good clinical application.

The hypoglycemic work for the diabetic mice that 2 ginsengenin 25-OH-PPT (T19) of experimental example induces four oxygen crash smack one's lips With:

Animal system Kunming mouse, weight 20-22g, female.

Influence to Alloxan-diabetes mouse blood sugar

Healthy mice 60 are taken, takes 8 to be only used as blank control at random, i.e., normal group, four oxygen are injected intraperitoneally in remaining mouse The successful mouse of modeling after 6 days, is randomly divided into four groups (every group 8): model control group, ginsengenin by pyrimidine 20mg/kg Tri- dosage treatment groups of 25-OH-PPT (T19) 5mg/kg, 10mg/kg, 20mg/kg.Gastric infusion, every the sky are distinguished after grouping Noon, 8:30-9:30 was administered once, and successive administration 14 days.Respectively at the 0th day, the 7th day, the 14th day survey empty stomach 12h blood glucose.

Influence to Alloxan-diabetes glucose tolerance in mice

Healthy mice 60 are taken, takes 8 to be only used as blank control at random, i.e., normal group, four oxygen are injected intraperitoneally in remaining mouse The successful mouse of modeling after 6 days, is randomly divided into four groups (every group 8): model control group, ginseng soap former times by pyrimidine 20mg/kg Tri- dosage treatment groups of 25-OH-PPT (T19) 5mg/kg, 10mg/kg, 20mg/kg.Gastric infusion, every the sky are distinguished after grouping Noon, 8:30-9:30 was administered once, and successive administration 14 days.14th day, empty stomach mouse glucose 2g/kg was given in stomach-filling, and docking takes blood 0min, 30min, 60min, 120min blood glucose are measured respectively.Influence to Alloxan-diabetes mouse weight

Healthy mice 60 are taken, takes 8 to be only used as blank control at random, i.e., normal group, four oxygen are injected intraperitoneally in remaining mouse The successful mouse of modeling after 6 days, is randomly divided into four groups (every group 8): model control group, ginsenoside by pyrimidine 20mg/kg Tri- dosage treatment groups of 25-OH-PPT (T19) 5mg/kg, 10mg/kg, 20mg/kg.Gastric infusion, every the sky are distinguished after grouping Noon, 8:30-9:30 was administered once, and successive administration 14 days.Mouse weight is recorded before daily administration.

Influence to Alloxan-diabetes mouse food ration

Healthy mice 60 are taken, takes 8 to be only used as blank control at random, i.e., normal group, four oxygen are injected intraperitoneally in remaining mouse The successful mouse of modeling after 6 days, is randomly divided into four groups (every group 8): model control group, ginsenoside by pyrimidine 20mg/kg Tri- dosage treatment groups of 25-OH-PPT (T19) 5mg/kg, 10mg/kg, 20mg/kg.Gastric infusion, every the sky are distinguished after grouping Noon, 8:30-9:30 was administered once, and successive administration 14 days.Every morning claims mouse grain, calculates food ration.

Influence to Alloxan-diabetes mouse amount of drinking water

Healthy mice 60 are taken, takes 8 to be only used as blank control at random, i.e., normal group, four oxygen are injected intraperitoneally in remaining mouse The successful mouse of modeling after 6 days, is randomly divided into four groups (every group 8): model control group, ginsenoside by pyrimidine 20mg/kg Tri- dosage treatment groups of 25-OH-PPT (T19) 5mg/kg, 10mg/kg, 20mg/kg.Gastric infusion, every the sky are distinguished after grouping Noon, 8:30-9:30 was administered once, and successive administration 14 days.Every morning records amount of drinking water.

Experimental result

Influence to Alloxan-diabetes mouse fasting blood glucose level

Respectively in the 0th day, the 7th day, the 14th day measurement mouse blood sugar.0th day, ginsenoside 25-OH-PPT (T19) treatment The blood glucose level of group 5mg/kg, 10mg/kg, 20mg/kg and model group is all apparently higher than normal group.14th day, 10mg/kg group was small Mouse blood glucose (6.58mmol/L) divides compared with model group (10.40mmol/L) with 20mg/kg group mouse blood sugar (6.50mmol/L) Not Jiang Di by 36.7% and 37.5%, the blood glucose of 25mg/kg group mouse has restored normal level.(see attached drawing 9)

Influence to Alloxan-diabetes mouse oral sugar tolerance

It was treated by 14 days, the sugar tolerance of 10mg/kg group mouse is obviously improved compared with model group, and AUC reduces by 34.47% (P<0.05).When 120min, the blood glucose of 10mg/kg group mouse has restored normal level.However, the AUC of three dosage groups is still bright It is aobvious to be higher than normal group.(see attached 10)

Influence to Alloxan-diabetes mouse weight

Ginsenoside 25-OH-PPT (T19) does not influence Alloxan-diabetes mouse weight significantly.

Influence to Alloxan-diabetes mouse food ration

Compared with model group, after ginsenoside 25-OH-PPT (T19) treatment, 5mg/kg group is taken the photograph with 20mg/kg group mouse Appetite significantly reduces (P < 0.01).(see attached drawing 11)

Influence to Alloxan-diabetes mouse amount of drinking water

By 5mg/kg, the treatment of tri- dosage ginsenoside 25-OH-PPT (T19) of 10mg/kg, 20mg/kg, diabetes The amount of drinking water of mouse significantly reduces compared with model group.(see attached drawing 12)

It discusses

Ginsenoside 25-OH-PPT (T19) can reduce Alloxan-diabetes mouse blood sugar, improve Mouse oral sugar Amount reduces diabetic mice food ration and amount of drinking water, however, ginsenoside 25-OH-PPT (T19) does not have diabetic mice weight There is apparent influence.To sum up, ginsenoside 25-OH-PPT (T19) has certain function that is hypoglycemic and improving glucose tolerance Energy.

The beneficial effects of the present invention are: the present invention is resourceful, and reaction condition is mild, low in cost, quality controllable, week Phase is short, high income, and production extraction process is simple and easy, is suitble to industrialized production, can be made into various Food and hygienical foods, nontoxic Side effect can take for a long time or eat, easy to spread.

Detailed description of the invention

Fig. 1 is the present invention 5 kinds of ginsenosides lower to different quality concentration to alpha-glucosaccharase enzyme inhibition activity.

Fig. 2 is weekly Avoirdupois monitoring.

Fig. 3 is weekly blood sugar monitoring.

Fig. 4 is intraperitoneal injection sugar tolerance test.

Fig. 5 is intraperitoneal injection sugar tolerance test.

Fig. 6 is organ index.

Fig. 7 is Triglycerides in Serum assay.

The measurement of Fig. 8 serum cholesterol levels.

Influence of the Fig. 9 to Alloxan-diabetes mouse fasting blood glucose level.

Influence of the Figure 10 to Alloxan-diabetes mouse oral sugar tolerance.

Influence of the Figure 11 to Alloxan-diabetes mouse weight.

Influence of the Figure 12 to Alloxan-diabetes mouse amount of drinking water.

Figure 13 is the analytic type high-efficient liquid phase chromatogram of 6 product purity detections contained by the present invention.

The preparative high performance liquid chromatography figure of the commercially available general ginsenoside acid hydrolysis products of Figure 14.

Figure 15 Figure 16 is R the and S isomers (T19) that the separation of continuous sample introduction method prepares ginsengenin 25-OH-PPT.

Specific embodiment

The present invention is further described by following case study on implementation, is not limit the invention in any way, without departing substantially from this Under the premise of the technical solution of invention, those of ordinary skill in the art made for the present invention any change easy to accomplish Or changes and fall within scope of the presently claimed invention.

Embodiment 1

Precision weighs 1.0g ginsenoside, and with 1.0~6.0 times of amount methanol, ultrasonic dissolution is separately added into after to be dissolved Concentration is 1.2mol/L HCl2~20mL, and constant temperature (38~60 DEG C) shaking table reacts 0.5~3h.PH is neutralized to 1mol/L NaOH =7, it is centrifuged after aqueous precipitation, it is dry, obtain ginsenoside acid degradation crude compound.By gained crude product macroporous resin treatment, Obtain ginseng stem and leave general saponin acid degradation compound.Ginseng stem and leave general saponin acid hydrolysis products 100mg is taken, 20ml first is fully dissolved in In alcohol, by the solution of above-mentioned hydrolysate be applied to preparative reversed-phase high performance liquid chromatography, with Kromasil C18 (4.5mm × 150mm, 5 μm) it is stationary phase, sampling volume 3mL, 25 DEG C of column temperature, flow velocity 1.0mL/min；Mobile phase: methanol (A) and water (B) are pressed Different volumes are than mixing, gradient elution program: 0~30min, 70%A；30~63min, 80%；ELSD vaporizer temperature 50 C, Flow rate of carrier gas 3L/min collects ginsenoside 25-OCH respectively₃The eluting peak of-PPT, 25-OH-PPT, PT and PPT.Merge each one Join the collection liquid of saponin(e, solvent is flung in decompression, vacuum freeze drying up to 4 kinds of monomeric compounds freeze-dried powder.

Embodiment 2

Precision weighs 1.0g ginsenoside, and with 1.0~6.0 times of amount methanol, ultrasonic dissolution is separately added into after to be dissolved Concentration is each 2~20mL of 1.2mol/L HCl, and constant temperature (38~60 DEG C) shaking table reacts 0.5~3h.It is neutralized to 1mol/L NaOH PH=7 is centrifuged after aqueous precipitation, dry, obtains ginsenoside acid degradation crude compound.At gained crude product macroreticular resin Reason, obtains ginseng stem and leave general saponin acid degradation compound.Ginseng stem and leave general saponin acid hydrolysis products 100mg is taken, is fully dissolved in In 20ml methanol, the solution of above-mentioned hydrolysate is applied to preparative reversed-phase high performance liquid chromatography, with Kromasil C18 (4.5mm × 150mm, 5 μm) be stationary phase, sampling volume 3mL, 25 DEG C of column temperature, flow velocity 1.0mL/min；Mobile phase: acetonitrile (A) With water (B) by different volumes ratio mixing, gradient elution program: 0~30min, 70%A；30~63min, 80%；ELSD vaporizer Temperature 50 C, flow rate of carrier gas 3L/min collect ginsenoside 25-OCH respectively₃The elution of-PPT, 25-OH-PPT, PPT and PT Peak.Merge the collection liquid of each ginsenoside, solvent is flung in decompression, vacuum freeze drying up to 4 kinds of monomeric compounds freeze-dried powder.

Embodiment 3

Precision weighs 1.0g ginsenoside, and with 1.0~6.0 times of amount methanol, ultrasonic dissolution is separately added into after to be dissolved Concentration is each 2~20mL of 1.2mol/L HCl, and constant temperature (38~60 DEG C) shaking table reacts 0.5~3h.It is neutralized to 1mol/L NaOH PH=7 is centrifuged after aqueous precipitation, dry, obtains ginsenoside acid degradation crude compound.At gained crude product macroreticular resin Reason, obtains ginseng stem and leave general saponin acid degradation compound.Ginseng stem and leave general saponin acid degradation compound sample 0.4g is weighed, is configured to The solution that concentration is 20mg/mL carries out trocar preparation, and mobile phase is methanol: water=83:17, sample volume 1mL, between sample injection time Every 13.5min.Ginsenoside 25-OCH is collected respectively₃The eluting peak of-PPT, 25-OH-PPT, PPT and PT.Merge each ginseng soap Solvent is flung in the collection liquid of glycosides, decompression, vacuum freeze drying up to 4 kinds of monomeric compounds freeze-dried powder.

Embodiment 4

Precision weighs 1.0g ginsenoside, and with 1.0~6.0 times of amount methanol, ultrasonic dissolution is separately added into after to be dissolved Concentration is each 2~20mL of 1.2mol/L HCl, and constant temperature (38~60 DEG C) shaking table reacts 0.5~3h.It is neutralized to 1mol/L NaOH PH=7 is centrifuged after aqueous precipitation, dry, obtains ginsenoside acid degradation crude compound.At gained crude product macroreticular resin Reason, obtains ginseng stem and leave general saponin acid degradation compound.Using blind point-score in methanol: under water=81:19 eluent gradient, weighing people Join stem-leaf total saponin acid degradation compound sample 0.5g, is dissolved in methanol and is configured to saturated solution, sample volume 2.5mL is received made Standby fraction is detected using analytic type high performance liquid chromatograph to mobile phase is received.Identify 25-OCH₃-PPT、25-OH- Each fraction of PPT, PPT and PT.Merge the collection liquid of each ginsenoside, solvent is flung in decompression, and vacuum freeze drying is up to 4 kinds of monomers The freeze-dried powder of compound.

Embodiment 5

Precision weighs 1.0g ginsenoside, and with 1.0~6.0 times of amount methanol, ultrasonic dissolution is separately added into after to be dissolved Concentration is each 2~20mL of 1.2mol/L HCl, and constant temperature (38~60 DEG C) shaking table reacts 0.5~3h.It is neutralized to 1mol/L NaOH PH=7 is centrifuged after aqueous precipitation, dry, obtains ginsenoside acid degradation crude compound.At gained crude product macroreticular resin Reason, obtains ginseng stem and leave general saponin acid degradation compound.By a small amount of acetone solution of acid degradation crude compound, proper silica gel is added Stir evenly, fling to solvent, using normal-phase chromatography silica gel dry column-packing, by eluent system petroleum ether-ethyl acetate (1:5,1: 4,1:3,1:2,1:1,2:1,3:1,5:1) elution, merge the collection liquid of each ginsenoside, solvent is flung in decompression, and vacuum refrigeration is dry The dry freeze-dried powder up to 4 kinds of monomeric compounds.

Embodiment 6

Precision weighs 10 parts of 1.0g ginsenosides, and with 1.0~6.0 times of amount methanol, ultrasonic dissolution is backward respectively after to be dissolved It is 12mol/L HC1,1mL, constant temperature (38~60 DEG C) refluxing extraction 2h that concentration is added dropwise in each test tube.Use 1mol/L NaOH is neutralized to pH=7, is centrifuged after aqueous precipitation, dry, obtains ginsenoside acid degradation crude compound.By gained crude product with greatly Hole resin treatment obtains ginseng stem and leave general saponin acid degradation compound.The a small amount of acetone solution of product is dropped into acid, proper silica gel is added Stir evenly, fling to solvent, using normal-phase chromatography silica gel dry column-packing, by eluent system chloroform-methanol (20:1,15:1, 10:1,10:2,10:2.5,10:3,10:4,10:5.5,10:6,10:7) elution, merge the collection liquid of each ginsenoside, depressurizes Fling to solvent, vacuum freeze drying up to 4 kinds of monomeric compounds freeze-dried powder.

Embodiment 7

Precision weighs 10 parts of 1.0g ginsenosides, and with 1.0~6.0 times of amount methanol, ultrasonic dissolution is backward respectively after to be dissolved It is 12mol/L sulfuric acid, 1mL, constant temperature (38 DEG C) refluxing extraction 2h that concentration is added dropwise in each test tube.With in 1mol/L NaOH With to pH=7, it is centrifuged after aqueous precipitation, it is dry, obtain ginsenoside acid degradation crude compound.By gained crude product macroreticular resin Processing, obtains ginseng stem and leave general saponin acid degradation compound.By a small amount of acetone solution of acid degradation product, proper silica gel stirring is added Uniformly, fling to solvent, using normal-phase chromatography silica gel dry column-packing, by eluent system petroleum ether-acetone (5:1,4:1,3:1,2: 1,1:1,1:2) elution, merge the collection liquid of each ginsenoside, solvent is flung in decompression, and vacuum freeze drying is up to 4 kinds of singulations Close the freeze-dried powder of object.

Embodiment 8

By the ginsenoside acid degradation compound 50mg of above-mentioned preparation, with the edible shuttle sodium carboxymethylcellulose pyce of 10mL 0.3% Dissolution is added in 500mL milk and food is made.

Embodiment 9

By the ginsenoside acid degradation compound 50mg of above-mentioned preparation, 5mg isomaltose is added, solution is made, be distributed into guarantor Health food.

Embodiment 10

By the ginsenoside acid degradation compound 50g of above-mentioned preparation, starch 100g dextrin 50g, made with appropriate 50% ethyl alcohol Wetting agent is made softwood, pelletizes according to a conventional method, and quality magnesium stearate is added and mixes, and is made 0.2g/ piece through tablet press machine, and every 50mg containing dammar-20(22)-ene-3BETA,12BETA,25-triol.

Embodiment 11

By the ginsenoside acid degradation compound 50mg of above-mentioned preparation, dosage forms are made with auxiliary material appropriate and excipient Drug.

Claims (2)

1. The preparation method of 1.20 (R)-protopanaxatriols and its derivative, which is characterized in that specific step is as follows for preparation:

   (1) general ginsenoside is placed in container, and organic solvent is added, and is stirred, dissolution；

   (2) acid adding, heating, shaking table reaction；

   (3) end of reaction after reaction solution alkali neutralization, is centrifuged off organic solvent, dry, obtains acid degradation crude compound；

   20 (the R)-protopanaxatriols and its derivative have the following structure:

   Solvent described in step (1) is water or methanol or ethyl alcohol or propyl alcohol or butanol, material quantity be solvent 10%~ 60%；

   Acid described in step (2) acid hydrolytic reaction is one in hydrochloric acid or sulfuric acid or glacial acetic acid or oxalic acid or citric acid Kind, concentration range is 1.2~12mol/L, and temperature is 38~60 DEG C, and the reaction time is 0.5~3h, the preparation of sour water solution；

   General ginsenoside described in step (1), be ginseng stem and leave general saponin or Radix Ginseng total saponins or ginseng fruit saponins, Or ginseng flower total saponine or panaxdiols saponin or American ginseng total saponins or Quinquefolium saponin or American Ginseng two Alcohol saponins or arasaponin or notoginseng stem and leaf total saponin；

   Alkali in the step (3) is one of alkali metal hydroxide or alkali metal salt or rudimentary sodium alkoxide, concentration range For 1mol/L.
2. 2. the preparation method of 20 (R)-protopanaxatriol as described in claim 1 and its derivative, which is characterized in that from people Ginseng, gen-seng haulms, panax ginseng fruit, flower of Panax ginseng, ginseng, American Ginseng, stem and leaves of American ginseng, American Ginseng flower, Fructus Panacis Quinquefolii, American Ginseng Root, notoginseng haulm, Radix Notoginseng fruit, Roots of Panax Notoginseng, is prepared in sanchi flower Radix Notoginseng.