CN100488553C - Extractive of bullbrier genus plants, preparation method and application thereof - Google Patents
Extractive of bullbrier genus plants, preparation method and application thereof Download PDFInfo
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Abstract
An extract of the Xiaobaqia-family plants is prepared through reflux extracting in high-concentration alcohol, concentrating, adding water, sequentially extracting by petroether, ethyl acetate, n-butanol, or other organic solvent while respectively recovering solvent, dispersing the extract in water, adsorbing by macroreticular resin, eluting with water and then with the aqueous solution of alcohol, and recovering solvent. It can be used to prepare the medicine for treating tumor.
Description
Technical field
The present invention relates to a kind of Xiao smilax plant extract and its production and application, specifically, it is Xiao smilax plant extract that contains phenolic glycoside, flavonoid glycoside and its production and application, and the pharmaceutical composition that contains this extract, the present invention especially refers to the Smilax lanceaefolia Roxb. Var.opaca A.DC. medicinal substances extract of former plant for Xiao smilax plant.
Background technology
Rhizoma Smilacis Glabrae is a conventional Chinese medicine, originates to be smilax plant Rhizoma Smilacis Glabrae.Compendium of Material Medica record: " Rhizoma Smilacis Glabrae has red white two kinds, goes into medicinal Bai Zheliang ".Rhizoma Smilacis Glabrae is the red person of Li Shizhen (1518-1593 A.D.) indication, that is to say, the red person in the above-mentioned Rhizoma Smilacis Glabrae is essentially real Rhizoma Smilacis Glabrae.Pharmacological experiments shows: the ethyl acetate extract of Rhizoma Smilacis Glabrae and congener has the certain protection effect to cardiovascular system, and water extract has immunoregulation effect, in addition multiple efficacies such as antibiotic in addition, antitumor of Rhizoma Smilacis Glabrae; And white person is the hargil Poria in fact, habit among the people claims Smilax lanceaefolia Roxb. Var.opaca A.DC., succedaneum at a lot of regional Chang Zuowei Rhizoma Smilacis Glabraes, the Smilax lanceaefolia Roxb. Var.opaca A.DC. source can be divided into Xiao smilax plant Xiao Rhizoma Smilacis Chinensis, short column Xiao Rhizoma Smilacis Chinensis or magnificent Xiao Rhizoma Smilacis Chinensis, or the mixture of three kinds, relative Rhizoma Smilacis Glabrae, chemistry and the pharmacological research carried out at Smilax lanceaefolia Roxb. Var.opaca A.DC. then seem less relatively.
Existing chemical constitution study shows, contains chemical compounds such as saponins, flavone (glycosides), the plain class of phenylpropyl alcohol, sterols in the Rhizoma Smilacis Glabrae, and for the hargil Poria, pair The Chemical Constituents that the liposoluble constituent of Xiao Rhizoma Smilacis Chinensis carries out report is only arranged at present.Because it is less to the basic research of Smilax lanceaefolia Roxb. Var.opaca A.DC., some prescription just indicates the Smilax lanceaefolia Roxb. Var.opaca A.DC. that composition contains Xiao smilax plant, but also only in quality standard, only recorded the general qualitative identification that adopts thin layer chromatography and control medicinal material to contrast to Smilax lanceaefolia Roxb. Var.opaca A.DC., lack quantitative controlling index, cause Smilax lanceaefolia Roxb. Var.opaca A.DC. complex chemical composition in medication and preparation, be difficult for carrying out quality monitoring.
Summary of the invention
The inventor passes through great deal of experimental, obtained the effective site of Xiao smilax plant (Xiao Rhizoma Smilacis Chinensis, short column Xiao Rhizoma Smilacis Chinensis or magnificent Xiao Rhizoma Smilacis Chinensis), this extract is that Xiao smilax plant crude extract makes through making with extra care, wherein effective ingredient is clear and definite, content is higher, the composition of clear and definite Xiao smilax plant (especially Smilax lanceaefolia Roxb. Var.opaca A.DC.) effective site and content, and the purpose of spoke reduction dose greatly have been reached, thereby improved bioavailability, and studied the pharmacological effect of the anti-tumor activity of Xiao smilax plant.
The object of the present invention is to provide a kind of Xiao smilax plant extract, its extract by routine and targetedly process for purification make, this extract component is clear and definite, has tangible antineoplastic activity.
The present invention also aims to provide the preparation method of Xiao smilax plant extract, this method comprises the extraction subtractive process, especially adopt successively the technology such as enrichment of carrying out effective ingredient with organic solvent extraction such as petroleum ether, ethyl acetate, n-butyl alcohol and macroporous adsorbent resin, make that effective ingredient is clear and definite in Xiao smilax plant extract, content is high, impurity is few, more helps its application on anti-tumor agent.
The invention provides a kind of Xiao smilax plant extract, wherein in the gross weight of this extract, it contains phenolic glycoside 10% at least, flavonoid glycoside 10%, described phenolic glycoside comprise 4-carboxyl-phenyl-1-O-glucoside, 4-carboxyl-2,6-dimethoxy-phenyl-1-O-glucoside, 4-hydroxyl-3,5-dimethoxy-phenyl-1-O-glucoside, 4-hydroxyl-3,5-dimethoxy-benzoic acid-glucoside etc., described flavonoid glycoside comprises chimonin, Hesperidin, Deng chromone
Ketone and flavanone glycosides etc.
Record with high performance liquid chromatography wherein that the weight content of Hesperidin is at least 0.5% in the flavonoid glycoside, preferred 1-5%.
Through the pharmacological testing test, the extract that contains mentioned component of the present invention has tangible anti-tumor activity, especially lewis pulmonary carcinoma, hepatocarcinoma H-22 and sarcoma S-180 is all had significant inhibitory effect.
In the preferred embodiment of the present invention, have suppress tumor effect extract its make by following method:
(1) with of the 10-95% concentration alcohol extraction of Xiao smilax plant, obtains extracting solution with at least 2 times of amounts;
(2) extracting solution concentrate concentrated solution, add water and make its suspendible;
(3) static, filter, get filtrate for later use;
(4) get above-mentioned filtrate, last macroporous resin column, negative with water elution to the reaction of eluent sugar, discard water elution liquid;
(5) be no more than 90% pure eluting with concentration, eluent concentrates, promptly.
With the total restatement of extract, contain phenolic glycoside 10% at least in Xiao smilax plant extract, flavonoid glycoside 10%.Wherein phenolic glycoside is preferably 10%-90%, more preferably 40-60%; Flavonoid glycoside is preferably 10%-90%, more preferably 40-60%; Described Xiao smilax plant mainly is meant Xiao smilax plant Xiao Rhizoma Smilacis Chinensis, short column Xiao Rhizoma Smilacis Chinensis or magnificent Xiao Rhizoma Smilacis Chinensis, refers in particular to Smilax lanceaefolia Roxb. Var.opaca A.DC..
Because the effective ingredient that is contained in the extract of the present invention is a mixture, the clear and definite after testing active compound composition that it contained comprises phenolic glycoside and flavonoid glycoside etc., so far there is not document to report that openly Xiao smilax plant (comprises Xiao Rhizoma Smilacis Chinensis, short column Xiao Rhizoma Smilacis Chinensis or magnificent Xiao Rhizoma Smilacis Chinensis) what is at the reasonable composition of effective ingredient aspect the treatment tumor, how many preferred therapeutic amounts is, certainly will cause occurring dose-effect relationship like this is not easy to hold, defective characteristics such as curative effect instability, especially for the Chinese medicine injection dosage form that is fit to the treatment tumor, constituent is definite, content accurately is easier to hold the quality of the pharmaceutical preparations.The applicant finds, definite curative effect chemical compound is the phenolic glycoside and the flavonoid glycoside of (but being not only) treatment effective dose in the extract that preparation method of the present invention obtains, so having carried out a large amount of experiments, the present invention detects, the result confirms, the content of flavonoid glycoside is at least 10% in Xiao smilax plant extract, and Determination of Hesperidin Content wherein accounts at least 0.5% of flavonoid glycoside gross weight, and its therapeutic effect is best.
The present invention also provides a kind of preparation method of Xiao smilax plant extract, comprising: with the 10-95% concentration alcohol of Xiao smilax plant, extract and obtain extracting solution with at least 2 times of amounts; With its concentrate concentrated solution, add water and make its suspendible; Leave standstill, filter, get filtrate for later use; Above-mentioned filtrate is gone up macroporous resin column, and described macroporous adsorbent resin is preferably the adsorbent resin that can remove carbohydrate content, AB-8 for example, and D-101, HP-20 etc., preferred DIALON HP-20, negative with water elution to the reaction of eluent sugar; Be no more than 90% pure eluting promptly with concentration.Also can as required eluent be concentrated, drying under reduced pressure obtains the dry finished product of extract.
Above-mentioned preparation method is wherein extracted the pure preferred alcohol of usefulness.
In a large amount of experiments, the applicant finds, when the determining alcohol that extracts usefulness is 10-75%, can obtain that through the refining of macroporous adsorbent resin tumor cell is had the preliminary target extract of inhibitory action in the described preparation method, and surpass 70% when extracting with pure concentration, especially surpass 75%, comprised also before macroporous resin adsorption preferably that then described low polar solvent is snyder parameter ε with the low polar solvent, the medium polar solvent abstraction impurity removal that are at least 0.5 times of filtrate weight
0＜0.4 solvent, medium polar solvent are snyder parameter 0.4＜ε
0＜0.6 solvent extracts once at least, and reuse high polarity solvent extraction effective ingredient, described high polarity solvent are snyder parameter ε
00.6 solvent, with macroporous resin column on the extract of high polarity solvent.With the solvent of high polarity macroporous resin column on the extract of n-butyl alcohol for example, the extract enrichment that obtains lewis pulmonary carcinoma, hepatocarcinoma H-22 and sarcoma S-180 are all had obvious inhibiting active component, this extract can be called the summation of effective site or effective ingredient.
Described low polar solvent is for removing the solvent of aliphatic composition, preferred snyder parameter ε
0＜0.4 low polar solvent, it is selected from petroleum ether, ether, acetone and the isopropyl alcohol at least a; The solvent of middle polarity is the solvent that can remove sterol, ter penoids, the medium polar solvent of preferred snyder parameter 0.4＜ε 0＜0.6, and it is chloroform and/or ethyl acetate; Preferred its snyder parameter ε of the solvent of described high polarity
00.6, be mainly n-butyl alcohol.
Eluting is 90% with the concentration of alcohol in the above-mentioned preparation method, what collect is the composition (not comprising the composition that water elution gets off) of 90% following concentration alcohol eluting, but from remove crudely and store essence as far as possible angle weigh, preferred eluting is 60% with the concentration of alcohol, collect 60% and the composition of the pure eluting of concentration below 60%, more preferably collect the eluent of 10%～60% alcohol.
Ethanol (the especially ethanol of higher concentration) extract to Xiao smilax plant in the inventive method adopts successively with petroleum ether, ethyl acetate, acetone and other organic solvent or solvent combination are extracted to few benefit once and are, weeding of grease soluble components farthest, especially 70～95% concentration alcohol extracts, wherein contain some belong to impurity concerning suppressing tumor liposoluble constituent, more preferably adopt the little organic solvent of polarity to be removed, take all factors into consideration the condition and the equipment requirements of industrial production, the organic solvent that can select some to be suitable for, for example petroleum ether, ethyl acetate, acetone, n-butyl alcohol etc. extract respectively; And final evidence, contain in the butanol extraction liquid have plenty of that the present invention wishes to get lewis pulmonary carcinoma, hepatocarcinoma H-22 and sarcoma S-180 etc. are all had obvious inhibiting effective ingredient, its effect is better than the activity of the extract that obtains without above-mentioned solvent extraction.
Extraction in the said method is preferably 3-5 times that are extracted liquid weight with the consumption of each solvent.
In order further to obtain more purified extract, the present invention is dissolved in water after also preferably the above-mentioned butanol extraction liquid that obtains being concentrated, and last macroporous adsorptive resins washes with water earlier, remove carbohydrate content, with 30% ethanol elution, purpose is that the effective ingredient that will be adsorbed in resin column elutes again, and described macroporous adsorbent resin is for removing the resin well known in the art of glucide, AB-8 for example, D-101, HP-20 etc., preferred DIALON HP-20.
Extraction process for purification of the present invention is compared with the extracting method of the disclosed Rhizoma Smilacis Glabrae of prior art, enrichment comprise the effective site of Xiao smilax plant extract of Smilax lanceaefolia Roxb. Var.opaca A.DC. medical material, mostly prior art is the preparation total saponins about the extraction of Rhizoma Smilacis Glabrae, reservation be the composition at n-butanol extraction position.And purpose of the present invention and innovation part are to keep simultaneously phenolic glycoside and flavonoid glycoside, by organic solvent extraction and macroporous resin adsorption, remove saccharide and liposoluble constituent as far as possible, the extract that obtains can directly be prepared into pharmaceutical preparation and be used for the treatment of and suppress tumor, lewis pulmonary carcinoma, hepatocarcinoma H-22 and sarcoma S-180 etc. all had obvious inhibitory action, its suppression ratio is respectively 39.2%, 30.9% and 40%, and its effect and safety have obtained guaranteeing to greatest extent.
The present invention is example with the Smilax lanceaefolia Roxb. Var.opaca A.DC., the content assaying method of the effective site that it obtains through said method be spectrophotography (for example: method uv-spectrophotometric), operating procedure is:
The drafting of standard curve: get Hesperidin reference substance 50mg, put in the 25ml measuring bottle, add dissolve with methanol and be diluted to scale.Precision measures 1,2,3,4,5, and 6ml puts in the 25ml measuring bottle, adds dissolve with methanol and is diluted to scale.According to ultraviolet spectrophotometry (an appendix V of Chinese Pharmacopoeia version in 2005 A), measure absorbance at the 320nm place, be vertical coordinate with the absorbance, concentration is abscissa, the drawing standard curve.
Algoscopy: get this product 10mg, the accurate title, decide, and puts in the 25ml measuring bottle, adds dissolve with methanol and be diluted to scale.Measure absorbance at the 320nm place, read the weight that contains flavonoid glycoside (in Hesperidin) the need testing solution, calculate, promptly get flavonoid glycoside content from standard curve.
The present invention also provides a kind of pharmaceutical composition, and it contains Xiao smilax plant extract and the excipient substance of clinical treatment effective dose, and it is 10-500mg/ day that this clinical drug is recommended effective dose, preferred 30-50mg/ day.
Aforementioned pharmaceutical compositions comprises peroral dosage form and injection type.In said medicine, be example with the injection type, as the auxiliary element that is used with effective site, according to different concrete injections, at least a in mannitol, glucose, dextran or the sodium chloride selected in suggestion for use.For example, for lyophilized injectable powder, can adopt conventional freeze-drying, with water is solvent, get extract and add an amount of caffolding agent lyophilizing and form, at least a as auxiliary caffolding agent in mannitol, glucose, the dextran selected in suggestion for use, forms jointly with said extract; Wherein preferred mannitol is as caffolding agent.For can be, then can select at least a in mannitol, glucose, the sodium chloride for use as auxiliary element for the medicine of the infusion preparation form of directly using; Serve as preferred to adopt sodium chloride as auxiliary element especially wherein.During for preparation small-volume injection injection and great transfusion preparation, can add antioxidant, antioxidant can be one or more in ascorbic acid, sodium ascorbate, sodium thiosulfate and the sodium pyrosulfite, also can add metal-chelator, as: EDTA.Oral formulations comprises: tablet (conventional tablet, dispersible tablet, slow release, controlled release tablet, effervescent tablet, oral cavity disintegration tablet), capsule (hard capsule, soft capsule, slow release, controlled release capsule), granule, drop pill, oral liquid, suppository, all can select conventional adjuvant for use.
The present invention also provides the application of pharmaceutical composition in the medicine of preparation treatment tumor of above-mentioned Xiao of containing smilax plant extract.
Xiao smilax plant extract test of pesticide effectiveness result of the present invention following (Xiao smilax plant extract is example with the Smilax lanceaefolia Roxb. Var.opaca A.DC.), represent with X through the Smilax lanceaefolia Roxb. Var.opaca A.DC. Extract that said extracted method of the present invention obtains, conventional without representing with Y except that the pure water extract of the Smilax lanceaefolia Roxb. Var.opaca A.DC. of impurity such as desaccharide, it is about 80% that wherein Y contains carbohydrate content, contain the about 2-5% of phenolic glycoside, the about 2-5% of flavonoid glycoside and on a small quantity other compositions (impurity) as sterol, terpenoid etc.
Inhibitory action to mice H-22 liver cancer growth
Sample: X, brown is thick, does not have obvious abnormal smells from the patient, and is water-soluble.
Laboratory animal: II level KM mice, female, body weight 21～23g is provided by Beijing Vital River Experimental Animals Technology Co., Ltd..
Tumor is former, H-22, the unit preservation of going down to posterity.
Animal feeding is in the IVC system, 22～27 ℃ of temperature, and relative humidity 50～70% freely absorbs normal feedstuff, drinking pure water.Change twice, five one cage of nest weekly.
Reagent and equipment: Cyclophosphamide for injection, Hengrui Medicine Co., Ltd., Jiangsu Prov. produces, lot number 04053121; Balance.
The dosage design: cyclophosphamide repeats original design 100mg/kg; X. because sample size is little, be not LD
50(maximum tolerated dose) is so establish 1g/kg with 5:1,0.2g/kg, 0.04g/kg, four groups of 0.008g/kg.
Method: select vigorous, the no diabrosis of tumor growth, tumor-bearing mice in good health, dislocation is put to death, and tumor is got by the sterile working, weighs 1: 2 adding normal saline by tumor, makes cell suspension, in left axillary fossa subcutaneous vaccination 0.2ml.
72 mices are divided into 6 groups at random, blank group, positive controls, X1g group, X0.2g group, X0.04g group and X0.008g group, 12 every group, male and female half and half.
Administration behind the inoculation 24h.The blank group was irritated stomach continuous 10 days with normal saline 0.4ml/20g body weight every day; The 1g group of X was irritated stomach continuous 10 days with the 0.02g/0.4ml/20g body weight every day; 0.2g group was irritated stomach continuous 10 days with the 0.004g/0.4ml/20g body weight every day; 0.04g group was irritated stomach continuous 10 days with the 0.008g/0.4ml/20g body weight every day; 0.008g group was irritated stomach continuous 10 days with the 0.0016g/0.4ml/20g body weight every day.The cyclophosphamide group in the inoculation 24h after with the administration of 100mg/kg (0.2ml/20g) disposable celiac.
The applicant also obtains conventional extracting method simultaneously, promptly without the pharmacological evaluation of doing according to the method described above except that the pure water extract Y of the Smilax lanceaefolia Roxb. Var.opaca A.DC. of impurity such as desaccharide with same test conditions, with checking without the growth inhibited effect of the extract of remove impurity to mice H-22 hepatocarcinoma, the result shows, waiting under the dosage, contain of the growth inhibited almost not significantly effect of the conventional Smilax lanceaefolia Roxb. Var.opaca A.DC. Extract of composition such as polysaccharide, during correlated experimental data is not provided at and shows to mice H-22 hepatocarcinoma.
Discuss
1. the average tumor of blank group weighs 1.4g, greater than 1g; The heavy 1.1g of tubercle is all greater than 0.4g.Growth of xenografted is good, and test is set up.
2. the dosage that uses cyclophosphamide to do positive control drug usually is the 50mg/kg body weight, lumbar injection, and concentration is the 0.2ml/20g body weight.To the suppression ratio of H-22 generally 50～60%.
3.X1g no matter group is that tumour inhibiting rate or t check show that all this medicine has certain curative effect to H-22.The X0.2g group has poisoning manifestations, drug withdrawal subsequently the back animal that is administered four times.X0.04g group and X0.008g organize equal administration 10 times, and wherein X0.04g group tumour inhibiting rate is 30%, compare (t check) with the blank group, and P=0.09 has significant difference.
Growth inhibited effect to Mice Bearing Lewis Lung Cancer
Sample: X, brown is thick, does not have obvious abnormal smells from the patient, and is water-soluble.
Laboratory animal: II level C57 mice, male and female half and half, body weight 18～23g grinds animal housing of institute by Chinese medical courses in general institute blood credit number scxk (Tianjin) 2004-0001 is provided; Animal feeding is in the IVC system, 20～22 ℃ of temperature, and relative humidity 40～58% freely absorbs the normal feedstuff drinking pure water.Change twice, five one cage of nest weekly.Tumor is former: Lewis lung cancer, tumor institute of Chinese medical courses in general institute test center provides.
Reagent and equipment
1. Cyclophosphamide for injection, Hengrui Medicine Co., Ltd., Jiangsu Prov. produces, lot number 04053121.
2. electronic balance, the T500 type, d=0.1g, the two outstanding test instrunment in Changshu, two outstanding brother (group) the company Changzhou of U.S. factory produces.
The dosage design
1. cyclophosphamide is by traditional dosage 50mg/kg.
Since X to mice H-22 inhibition test in, 0.04g/kg body weight group is effective, 0.008mg/kg body weight group is invalid, 1g/kg and 0.2g/kg poisoning manifestations all occurs for two groups, so establish 0.08g/kg, 0.04g/kg, 0.02g/kg, three dosage groups.
Method
Select vigorous, the no diabrosis of tumor growth, tumor-bearing mice in good health, dislocation is put to death, and tumor is got by the sterile working, by the heavy 1:2 adding of tumor normal saline, makes cell suspension, in left axillary fossa subcutaneous vaccination 0.2ml.
60 C57 mices are divided into 5 groups at random, blank group, positive controls, X0.08g/kg group, X0.04g/kg group and X0.02g/kg group, 12 every group, male and female half and half.
Administration behind the inoculation 24h.The blank group was irritated stomach continuous 8 days with normal saline 0.4ml/20g body weight every day; The X0.08g/kg group was irritated stomach continuous 8 days with the X0.016g/0.4ml/20g body weight every day; X0.04g/kg organizes every day with F0.008g/0.4ml/20.Body weight was irritated stomach continuous 8 days; The X0.02g/kg group was irritated stomach continuous 8 days with the F0.004g/0.4ml/20g body weight every day.The cyclophosphamide group in the inoculation 24h after with the administration of 50mg/kg (0.2ml/20g) disposable celiac.
Discuss
1. the average tumor of blank group weighs 1.76g, greater than 1g; The heavy 1.22g of tubercle is all greater than 0.4g.Growth of xenografted is good, the success of transplanted tumor model.
2. to do the dosage of positive control drug be the 50mg/kg body weight to cyclophosphamide, lumbar injection, and concentration is the 0.2ml/20g body weight.Suppression ratio to Lewis lung cancer is 62.5%, with result of the test was close in the past.
3. all show after 8 days that about X0.08,0.04, the administration of three dosage groups of 0.02g/kg Lewis lung cancer is had the good restraining effect, with the blank group significant difference is arranged more all, and tumour inhibiting rate is all greater than 30%.
Growth inhibited effect to mice S-180
Sample: X, brown is thick, does not have obvious abnormal smells from the patient, and is water-soluble.
Laboratory animal: II level C57 mice, male and female half and half, body weight 18～23g, grinding animal housing of institute by Chinese medical courses in general institute blood provides, credit number scxk (Tianjin) 2004-0001, and animal feeding is in the IVC system, 20～22 ℃ of temperature, relative humidity 40～58% freely absorbs the normal feedstuff drinking pure water.Change twice, five one cage of nest weekly.Tumor is former: Lewis lung cancer, tumor institute of Chinese medical courses in general institute test center provides.
Reagent and equipment:
1. Cyclophosphamide for injection, Hengrui Medicine Co., Ltd., Jiangsu Prov. produces, lot number 04053121.
2. electronic balance, the T500 type, d=0.1g, the two outstanding test instrunment in Changshu, two outstanding brother (group) the company Changzhou of U.S. factory produces.
The dosage design:
1. cyclophosphamide is by I traditional dosage 50mg/kg.
2. establish 0.2g/kg, 0.04g/kg, three dosage groups of 0.008g/kg.
Method:
Select vigorous, the no diabrosis of tumor growth, tumor-bearing mice in good health, dislocation is put to death, and tumor is got by the sterile working, by the heavy 1:2 adding of tumor normal saline, makes cell suspension, in left axillary fossa subcutaneous vaccination 0.2ml.
60 C57 mices are divided into 5 groups at random, blank group, positive controls, X0.08g/kg group, X0.04g/kg group and X0.02g/kg group, 12 every group, male and female half and half.
Administration behind the inoculation 24h.The blank group was irritated stomach continuous 8 days with normal saline 0.4ml/20g body weight every day; The administration group was irritated stomach continuous 8 days.The cyclophosphamide group in the inoculation 24h after with the administration of 100mg/kg (0.2ml/20g) disposable celiac.
Discuss
1. the average tumor of blank group weighs 1.76g, greater than 1g; The heavy 1.22g of tubercle is all greater than 0.4g.Growth of xenografted is good, the success of transplanted tumor model.
2. to do the dosage of positive control drug be the 100mg/kg body weight to cyclophosphamide, lumbar injection, and concentration is the 0.2ml/20g body weight.Suppression ratio to Lewis lung cancer is 92.9%, with result of the test was close in the past.
3. data extract of the present invention as can be seen has the activity that suppresses tumor from each table, and significantly reduces with its side effect of cyclophosphamide comparison, has developable prospect.
All show after 8 days that about 0.2,0.04 two dosage group administration the S-180 sarcoma is had the good restraining effect, with the blank group utmost point significant difference is arranged more all, and tumour inhibiting rate is all greater than 30%, especially the tumour inhibiting rate of X0.2 group is greater than 40%.
The extract that relates in the pharmacological evaluation of the present invention makes according to the method for embodiment 1.
Description of drawings
Fig. 1: the hydrogen-nuclear magnetic resonance map of isolating Hesperidin in the extract of the present invention;
Fig. 2: the carbon-nuclear magnetic resonance map of isolating Hesperidin in the extract of the present invention.
The specific embodiment
Following examples are in order to illustrate in greater detail the present invention, are not that the present invention is construed as limiting.
Embodiment 1 Xiao smilax plant extract
Get the dry rhizome of short column Xiao Rhizoma Smilacis Chinensis, section, measure 95% alcohol reflux 3 times with 8 times, each 2 hours, merge extractive liquid,, being evaporated to does not have the alcohol flavor, add water and make suspendible in right amount, respectively extract 5 times with petroleum ether, ethyl acetate, n-butyl alcohol successively, reclaim solvent respectively, n-butanol portion (extraction ratio be about primary dose 1%); The extractum of n-butanol portion is dissolved in water, and last macroporous resin column (DIALON HP-20) is with the water flushing, colourless to eluent with 60% ethanol elution more earlier, gets final product (that is the X in the pharmaceutical research).
After testing, phenolic glycoside is about 50% in this extract, and flavonoid glycoside is about 50%; Wherein use high effective liquid chromatography for measuring, through to the calculating of collection of illustrative plates absworption peak, obtain that the weight content of Hesperidin is about 2% in the flavonoid glycoside.
Through the separation and the purification of chemical compound, and, confirmed that above-mentioned phenolic glycoside comprises 4-carboxyl-phenyl-1-O-glucoside by methods such as physical and chemical reaction and Spectrum Analysis, 4-carboxyl-2,6-dimethoxy-phenyl-1-O-glucoside, 4-hydroxyl-3,5-dimethoxy-phenyl-1-0-glucoside, 4-hydroxyl-3,5-dimethoxy-benzoic acid-glucoside, described flavonoid glycoside comprises chimonin, Hesperidin, Deng chromone
Ketone and flavanone glycosides.
Separation and purification process are as follows:
Get the dry rhizome decoction pieces of Xiao smilax plant (for example short column Xiao Rhizoma Smilacis Chinensis), measure 95% alcohol reflux 3 times with 8 times, each 2 hours, merge extractive liquid,, being evaporated to does not have the alcohol flavor, adds water and makes suspendible in right amount, respectively extracts 5 times with petroleum ether, ethyl acetate, n-butyl alcohol successively, reclaim solvent, get petroleum ether part, ethyl acetate extract and n-butanol portion respectively.
N-butanol portion is distinguished water, 10%, 30%, 60% ethanol gradient elution through macroporous adsorbent resin Diaion HP-20 column chromatography.Be divided into four parts by eluting order.
LH-20 gel column in the first with the water-methanol gradient elution, obtains 4-carboxyl-2 respectively in the water elution flow point, 6-dimethoxy-phenyl-1-O-glucoside (180mg) and 4-hydroxyl-3,5-dimethoxy-phenyl-1-O-glucoside (250mg); Obtain 4-hydroxyl-3 in the 10% methanol-eluted fractions flow point, 5-dimethoxy-benzoic acid-glucoside (350mg), 4-carboxyl-phenyl-1-O-glucoside (220mg); Obtain obtaining 3,5,7 in a chromogen ketoside (120mg, structure is undetermined), the methanol-eluted fractions flow point 4 '-tetrahydroxy-flavanone-6-C-rhamnose (1-4) glucoside (300mg) in the 30% methanol-eluted fractions flow point;
LH-20 gel column on the second portion with the water-methanol gradient elution, obtains obtaining one in chimonin (320mg), the methanol-eluted fractions flow point in 90% methanol-eluted fractions flow point
Ketoside, promptly 1,3,6, the 7-tetrahydroxy
Ketone-3-O-glucoside (200mg);
LH-20 gel column on the third part, with the water-methanol gradient elution, obtain Hesperidin (300mg) in 50% methanol-eluted fractions flow point, Hesperidin is dissolved in dimethyl sulfoxine (DMSO) solution, (model of instrument is INOVA-500 through the hydrogen of nuclear magnetic resonance, NMR spectrum and carbon analysis of spectrum
1H-NMR and INOVA-500
13C-NMR, spectrogram see Fig. 1 and Fig. 2), and with the contrast of the nuclear magnetic resonance map of Hesperidin standard substance, prove that the composition of mensuration is Hesperidin.
The first position enrichment composition all belongs to phenolic glycoside and flavonoid glycoside, and calculating its content is to account for 50% of extract gross weight; The one-tenth of second and third part enrichment belongs to flavonoid glycoside, calculates its content and accounts for extract gross weight 50%.
Get the dry rhizome (10 kilograms) of Xiao Rhizoma Smilacis Chinensis, section is measured 90% alcohol reflux 4 times, each 2 hours with 8 times, merge extractive liquid,, being evaporated to does not have the alcohol flavor, adds water and makes suspendible in right amount, use ethyl acetate, 1 times of amount of n-butyl alcohol successively, each extracts 3 times, reclaims solvent respectively.Get n-butanol portion, the extractum of n-butanol portion is dissolved in water, and last macroporous resin column (DIALON HP-20) is with the water flushing, colourless to eluent with 60% ethanol elution more earlier, gets final product (that is the X in the pharmaceutical research).
After testing, phenolic glycoside content is about 45% in this extract, and flavonoid glycoside content is about 55%.Wherein use high effective liquid chromatography for measuring, obtain that the weight content of Hesperidin is about 3% in the flavonoid glycoside.
Embodiment 3 Xiao smilax plant extracts
Get the dry rhizome of magnificent Xiao Rhizoma Smilacis Chinensis, section, with 8 times of amount 40% alcohol reflux 3 times, each 2 hours, merge extractive liquid,, being evaporated to does not have alcohol and distinguishes the flavor of, and adds water and makes suspendible in right amount, leaves standstill 24 hours; Filter, macroporous resin column on the filtrate (DIALON HP-20), with the water flushing, colourless with 60% ethanol elution more earlier to eluent, reclaim solvent and get final product.
After testing, phenolic glycoside content is about 50% in this extract, and flavonoid glycoside content is about 50%.
Wherein use high effective liquid chromatography for measuring, obtain that the weight content of Hesperidin is about 3.5% in the flavonoid glycoside.
The preparation of embodiment 4 Xiao smilax plant extract freeze-dried powders
Take by weighing the 310g dextran, be dissolved in 4500 milliliters of waters for injection, take by weighing extract 350g again, be dissolved in the above-mentioned solution by the inventive method preparation.Sodium hydroxide solution with 20% is regulated pH to 7.0 ± 0.2.The 0.1% needle-use activated carbon heated and stirred 15 minutes that adds amount of preparation, sucking filtration, filtrate replenishing adds water for injection to 5000 milliliter.Fine straining, intermediate inspection, fill, lyophilizing, gland, finished product inspection, packing.
The preparation of embodiment 5 infusion solutionses
Get the extract 50g that makes as stated above, add the water for injection of 834.7g sodium chloride and 90L, stirring and dissolving, it is an amount of to add 0.1mol/L NaOH, transfer pH to be about 7.0, replenish water for injection, add the needle-use activated carbon of liquid measure 0.15% again to capacity, heated and stirred 15 minutes, the sucking filtration de-carbon, measure content, pH value qualified after, fine straining packing again, (105 ℃) pressure sterilizing, promptly.
The preparation of embodiment 6 little liquid drugs injections
Get the extract 300g that makes as stated above, add the water for injection of 80.0g sodium chloride and 10L, stirring and dissolving, it is an amount of to add 0.1mol/L NaOH, transfers pH to be about 7.0, adds 10g antioxidant sodium thiosulfate, replenish water for injection to capacity, the needle-use activated carbon that adds liquid measure 0.1% again stirred the sucking filtration de-carbon 15 minutes, measure content, pH value qualified after, fine straining packing again, (105 ℃) pressure sterilizing, promptly.
The preparation of embodiment 7 oral liquids
Get the extract 250g that makes as stated above, glucose 50g adds water 1L, and stirring and dissolving adds 10g antioxidant sodium thiosulfate, and supplementing water is to 10L, and mixing filters, packing, and pressure sterilizing, promptly.
The preparation of embodiment 8 dispersible tablets
Get the extract 25g that makes as stated above, microcrystalline Cellulose 35g, starch 10g, carboxymethyl starch 5g is that binding agent is granulated with 5%PVP solution, tabletting, promptly.
The preparation of embodiment 9 drop pill
Get PEG-6000 50-100g, heating and melting adds the extract 25g that makes as stated above, drips into drop pill, promptly.
The preparation of embodiment 10 drop pill
Get PEG-4000 60-100g, heating and melting adds the extract 25g that makes as stated above, drips into drop pill, promptly.
Get the extract 250g that makes as stated above, starch 100g is that binding agent is granulated with 20% ethanol, and is encapsulated, promptly.
The preparation of embodiment 12 granules
Get the extract 250g that makes as stated above, glucose 100g, dextrin 200g is that binding agent is granulated with rare gelatinized corn starch, packing, promptly.
The preparation of embodiment 13 tablets
Get the extract 25g that makes as stated above, add starch 50g, mixing is a binding agent with the starch slurry, granulate, and oven dry, granulate, tabletting, promptly.
The preparation of embodiment 14 tablets
Get the extract 25g that makes as stated above, add starch 40g, dextrin 10g, mixing is a binding agent with the starch slurry, granulates, oven dry, granulate, tabletting, promptly.
Embodiment 15 preparation of soft capsule
Get the extract 250g that the method by embodiment 1 makes, add vegetable oil 550g, Cera Flava 50g, the heating mixing in the capsule material of packing into, is made soft capsule promptly.
Claims (9)
1, a kind of Xiao smilax plant extract with the total restatement of this extract, wherein contains phenolic glycoside 10%, flavonoid glycoside 10% at least; This Xiao smilax plant extract is made by following method:
(1) Xiao smilax plant is extracted and obtains extracting solution with the 10-95% concentration alcohol of at least 2 times of amounts;
(2) extracting solution concentrate concentrated solution, add water and make its suspendible;
(3) leave standstill, filter, get filtrate for later use;
(4) get above-mentioned filtrate, last macroporous resin column, negative with water elution to the reaction of eluent sugar, discard water elution liquid;
(5) be no more than 90% pure eluting with concentration, eluent concentrates, and promptly obtains Xiao smilax plant extract.
2, Xiao smilax plant extract as claimed in claim 1, wherein the phenolic glycoside that contains in this extract is that 10%-90%, flavonoid glycoside are 10%-90%.
3, the preparation method of the described Xiao smilax of claim 1 plant extract, comprising:
(1) Xiao smilax plant is extracted and obtains extracting solution with the 10-95% concentration alcohol of at least 2 times of amounts;
(2) extracting solution concentrate concentrated solution, add water and make its suspendible;
(3) leave standstill, filter, get filtrate for later use;
(4) get above-mentioned filtrate, last macroporous resin column, negative with water elution to the reaction of eluent sugar, discard water elution liquid;
(5) be no more than 90% pure eluting with concentration, promptly.
4, the described preparation method of claim 3, the determining alcohol that wherein extracts usefulness in the step (1) is 70-95%.
5, claim 3 or 4 described preparation methoies comprise also afterwards successively that in step (3) described low polar solvent is snyder parameter ε with the low polar solvent, the medium polar solvent abstraction impurity removal that are at least 0.5 times of filtrate weight
0＜0.4 solvent, medium polar solvent are snyder parameter 0.4＜ε
0＜0.6 solvent extracts once at least, and reuse high polarity solvent extraction effective ingredient, described high polarity solvent are snyder parameter ε
00.6 solvent, with macroporous resin column on the extract of high polarity solvent.
6, the described preparation method of claim 5, wherein snyder parameter ε
0＜0.4 low polar solvent is selected from petroleum ether, ether, acetone and the isopropyl alcohol at least a, snyder parameter 0.4＜ε
0＜0.6 medium polar solvent is chloroform and/or ethyl acetate, snyder parameter ε
00.6 high polarity solvent is n-butyl alcohol.
7, the described preparation method of claim 3, wherein eluting is 60% with the concentration of alcohol.
8, a kind of pharmaceutical composition is characterized in that containing the claim 1 for the treatment of effective dose described Xiao smilax plant extract and pharmacy acceptable auxiliary.
9, the application of the described Xiao smilax of claim 1 plant extract in preparation treatment anti-tumor drug.
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| Title |
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| 白土苓挥发性成分的GC-MS分析. 张思巨，潘炯光，吉力，刘丽.中国中药杂志，第24卷第12期. 1999 |
| 白土苓挥发性成分的GC-MS分析. 张思巨，潘炯光，吉力，刘丽.中国中药杂志，第24卷第12期. 1999 * |
| 肖菝葜化学成分的研究. 于江泳，张思巨，刘丽.中国药学杂志，第40卷第1期. 2005 |
| 肖菝葜化学成分的研究. 于江泳，张思巨，刘丽.中国药学杂志，第40卷第1期. 2005 * |
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