CN101584718B - Antisepalous kiwi fruit leaf extractive as well as preparation method and application thereof - Google Patents
Antisepalous kiwi fruit leaf extractive as well as preparation method and application thereof Download PDFInfo
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- CN101584718B CN101584718B CN2008100375859A CN200810037585A CN101584718B CN 101584718 B CN101584718 B CN 101584718B CN 2008100375859 A CN2008100375859 A CN 2008100375859A CN 200810037585 A CN200810037585 A CN 200810037585A CN 101584718 B CN101584718 B CN 101584718B
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Abstract
The invention relates to the technical field of medicines. The roots of the antisepalous kiwi fruit can be used as a medicament of valvate actinidia root and clinically used for curing malignant tumors, however, no medical record and research report of leaves of the antisepalous kiwi fruit exist. The invention provides a preparation method of antisepalous kiwi fruit leaf extractive, comprises the steps of extraction solution preparation, concentration, sedimentation, edulcoration, decolorization, dryness and pulverization, and has the remarkable advantages of simple and convenient technology, low cost, safety, high efficiency, low organic solvent residues, and the like. The prepared antisepalous kiwi fruit leaf extractive is rich in triterpenoid saponin contents, such as koroso acid, crataegolic acid, ursolic acid, and the like, and is proved by pharmacological experiments to have favorable antitumor function, thereby being applied to the preparation of antitumor medicaments and foods.
Description
Technical field
The present invention relates to medical technical field, be specifically related to being a kind of extract and the method for distilling of feedstock production, and this extract is used to prepare anti-tumor drug and food the calyx kiwi fruit leaf.
Background technology
At present, the malignant tumor that comprises hepatocarcinoma is as commonly encountered diseases, frequently-occurring disease, the serious harm human beings'health.The World Health Organization (WHO) and American Society of Clinical Oncology (ASCO) statistical data shows, death toll accounts for 12.3% of total death toll due to the nineteen ninety whole world tumor, estimates will reach 14.6% in 2015.According to the U.S. state-run cancer research institute (National Cancer Institute) data: the malignant tumor sickness rate was 469.7/10 ten thousand in 1998～2002 years, and the cost that family, country and society pay for this reason is heavy.In the multiple therapy methods of malignant tumor, chemotherapy plays an important role clinically.At present, developing anti-tumor medicaments has become the important directions that anti-cancer agent is researched and developed from Chinese medicine, natural drug.
(Actinidia valvata Dunn) is the Actinidiaceae actinidia to the calyx Fructus actinidiae chinensis; Have another name called tweezer and close Fructus actinidiae chinensis, mainly be distributed in East China such as Zhejiang, Jiangxi, Jiangsu, its root is used as medicine and is the Radix actinidiae valvatae; Clinically being used to treat malignant tumor, is East China antitumor Chinese medicine commonly used.At present, rarely seen seldom about Radix actinidiae valvatae's chemical constituent, Pharmacological action study report [Zhang Yani, Liu Ling, Ling Changquan.Radix actinidiae valvatae's effective site is inquired into inhibitory action and the mechanism of mice transplanted tumor H22.CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2006,36 (11): 918-920; Lu Yin, Chen Shaoyuan, Wu Ping, etc.The analysis of medicinal plants Radix actinidiae valvatae's effective ingredient research (I)-inorganic elements.Journal of Zhejiang university, 2004,30 (2): 185-188], for the calyx kiwi fruit leaf then not being had medicinal record and research report.
At present, aspect the total triterpene saponins extract preparation technology of plant origin, the following process route of many employings:
Medicinal raw material (optionally can carry out appropriateness pulverize) adopts alcohol extraction (ethanol or methanol), and extracting solution is concentrated into not to be had the alcohol flavor and be adjusted to desired concn, and this concentrated solution is after centrifugal; Supernatant is crossed decolorizing resin adsorption-edulcoration decolouring (often selecting models such as HP-20, D101, AB-8, MCI-GEL for use); With alcoholic solution (ethanol or methanol) gradient elution, the eluent segmentation merges, and concentrates and reclaims; Can reach purpose [Lei Jianfei, the Jia Liping of decolouring remove impurity, enrichment saponin component.Macroporous adsorbent resin separation and purification saponin technical study progress.Contemporary Chinese Chinese medicine, 2006,8 (11): 22-24].Certainly owing to the difference of concrete raw material,, need through preferred definite optimum process condition at each processing step.In general, there is following limitation in this type of process route: 1. processing step is loaded down with trivial details, operation inconvenience, poor repeatability; 2. organic solvent such as ethanol, methanol consumption is big, and cost is higher; 3. for containing the more raw materials of pigment such as chlorophyll, decolorizing effect is not ideal enough.
Summary of the invention
The applicant is in antitumor Chinese medicine, natural drug screening process; Discovery has stronger antitumor action to the extract of calyx kiwi fruit leaf; Further research shows that this extract is rich in triterpene saponin constituents such as Corosolic acid, Crataegolic acid, ursolic acid, has the bright prospects that are used to prepare antitumor drug and food.
In addition, in process of the present invention, the applicant once attempted adopting resin remove impurity decoloration process path of preparing in the background technology to the extract of calyx kiwi fruit leaf, and the result shows that not only processing step is loaded down with trivial details, and decolorizing effect is also undesirable.For extract of the present invention preparation, good remove impurity discoloration method, for remove impurity, active constituent-enriched, improve the product quality, quality is most important.
The object of the present invention is to provide a kind of being the extract of feedstock production to the calyx kiwi fruit leaf, and method for distilling, and with this extract in preparation antitumor drug and Application in Food.
A kind of method for preparing to the calyx kiwi fruit leaf extractive comprises the steps:
(A) preparation extracting solution
Will be to calyx kiwi fruit leaf medicinal material coarse powder with the ethanol of water or various concentration or methanol aqueous solution as extracting solvent extraction, merge, filter, promptly get extracting solution.
This step prepares the used solvent of extracting solution can be the ethanol or the methanol aqueous solution of water or various concentration, preferred 50%-95% ethanol or methanol aqueous solution, more preferably 70%～95% ethanol or methanol aqueous solution, most preferably 80% ethanol or methanol aqueous solution.Certainly, consider safety factors and production cost factor in possible organic solvent residual, the production, ethanol water is more commonly used.
Extracting mode can select conventional hot reflux extraction, percolation to extract or soaking and extracting, and other pharmaceutically acceptable extracting modes.12 times, 10 times, 10 times be can select like the hot reflux extraction, 1.5 hours, 1 hour, 1 hour, other acceptable extraction solvent loads, extraction time, extraction time also can be selected.Percolation extracts, soaking and extracting can be selected 5-100 times of solvent, selects too much or very few solvent for use, although also can partly or entirely realize the described extract of this patent, can not constitute material alterations to the present invention.
(B) concentrate
Said extracted liquid reclaim under reduced pressure to there not being the alcohol flavor, is promptly got concentrated solution.Concentrated mode can adopt 40-100 ℃ of concentrating under reduced pressure, oven dry to concentrate and other pharmaceutically acceptable concentrated modes.Certainly the too high temperature of scruple possibly impact chemical composition stability property in the extract, and it is more feasible to select for use 50-75 ℃ of temperature to concentrate.
(C) deposition
Above-mentioned concentrated solution is put cold, separated out deposition, obtain deposition.Coprecipitation mode can adopt filtration, centrifugal and other acceptable depositions, divide access method.For example can adopt 5000rpm, 1-30 minute, centrifugal, supernatant is inclined to, can obtain deposition.
(D) remove impurity, decolouring
Above-mentioned deposition is dissolved with appropriate solvent, add depigmenting agent remove impurity decolouring.
The used solvent of this step can be selected ethanol, methanol, acetone etc., but the also aqueous solution of ethanol, methanol, acetone etc.If select ethanol, methanol, the dissolving of acetone equal solvent, preferred alcohol.If select aqueous solutions such as ethanol, methanol, acetone, can select 40-99% concentration, preferred 60-95% concentration most preferably is 80% concentration.
The used depigmenting agent of this step can be selected activated carbon, active hargil or both compositionss for use.If to select the activated carbon consumption for use is the 10%-100% of crude drug weight, preferred 25%-75%, most preferably 30%.If to select the active hargil consumption for use is the 100%-1000% of crude drug weight, preferred 300%-800%, most preferably 600%.If select activated carbon and active hargil compositions for use, proportioning is an activated carbon: the mixture of active hargil=1: 100～100: 1, and consumption is 100%～1000% of a crude drug weight; Optimum ratio is 1: 25～25: 1, and consumption is for being the 100%-500% of crude drug weight; Most preferably proportioning is 1: 10, and consumption is 200% of a crude drug weight.
(E) dry, pulverizing
Above-mentioned deposition is dry, pulverizing promptly get extract.
Drying mode can adopt 50-100 ℃ of normal pressure oven dry, vacuum drying and other acceptable drying modes.Grinding mode can adopt ball mill, impact grinder, fluid energy mill to pulverize and other acceptable grinding modes.As spray drying, Freeze Drying Technique are used for this step, can realize oven dry simultaneously, pulverize, use more convenient.
The present invention also provide with above-mentioned method for preparing make to the calyx kiwi fruit leaf extractive, through adopting cycle chemistry component separating, evaluation, show that it mainly contains triterpenes chemical constituents such as Corosolic acid, ursolic acid, Crataegolic acid.Because raw material employing of the present invention, substitutes soon as raw material the calyx kiwi fruit leaf naturally, and is inexpensive, is easy to get, and helps ecological environmental protection, has conspicuous advantage.Of the present invention have stronger antitumor action to the calyx kiwi fruit leaf extractive through the pharmacological evaluation proof, thereby can be used for preparing anti-tumor drug and food.
It is easy that preparation method of extract of the present invention has technology, with low cost, safe and effective, and the residual remarkable advantage that waits of organic solvent-free can be used as the preparation technology of standard extract fully.In view of in modern society, the rapid rising of malignant tumor sickness rate lacks desirable, efficacious therapy medicine, thereby this extract has broad application prospects, and is containing huge social benefit and economic benefit.
Description of drawings
Fig. 1: the anticancer experiment in vitro result of extract of the present invention.
The specific embodiment
Below in conjunction with accompanying drawing and specific embodiment the present invention is done further elaboration, but enforcement of the present invention is not limited in this.
Embodiment 1 preparation is to the calyx kiwi fruit leaf extractive
To calyx kiwi fruit leaf medicinal material coarse powder 1kg, with 80% ethanol, 12 times, 10 times, 10 times, 1.5 hours, 1 hour, 1 hour; Reflux, extract,, the extracting solution filtered while hot, merge extractive liquid,, decompression and solvent recovery is to there not being the alcohol flavor; It is 1g (crude drug)/ml that distilled water is regulated concentration, 5000rpm, centrifugal 15 minutes; Obtain deposition,, add to 10000ml with an amount of anhydrous alcohol solution.(active carbon: active hargil=1: 10) the 2000g remove impurity is decoloured, and decolouring back solution is utmost point pistac (+), and with its concentrating under reduced pressure, 60 ℃ of vacuum dryings are pulverized, and get extract 23.20g, product appearance faint yellow (+) to add the mixing depigmenting agent.Adopt the UV method to carry out total saponin content and measure, the extract total saponin content is in ursolic acid 74.28%
Embodiment 2 preparations are to the calyx kiwi fruit leaf extractive
To calyx kiwi fruit leaf medicinal material coarse powder 1kg, as extracting solvent, all the other get extract 18.64g with embodiment 1 with 95% ethanol, and decolouring back solution is utmost point pistac (+), product appearance faint yellow (+), and total saponin content is in ursolic acid 70.32%.
Embodiment 3 preparations are to the calyx kiwi fruit leaf extractive
To calyx kiwi fruit leaf medicinal material coarse powder 1kg, to extract as extracting the solvent percolation with 50 times of 80% ethanol, all the other are with embodiment 1; Decolouring back solution is utmost point pistac (+); Get extract 21.54g, product appearance faint yellow (+), total saponin content is in ursolic acid 72.65%.
Embodiment 4 preparations are to the calyx kiwi fruit leaf extractive
To calyx kiwi fruit leaf medicinal material coarse powder 1kg, add activated carbon 300g decolouring, all the other are with embodiment 1, and solution was light green (++) after the result was decoloured, and got extract 27.25g, product appearance yellowish-brown (++), total saponin content is in ursolic acid 49.21%.
Embodiment 5 preparations are to the calyx kiwi fruit leaf extractive
To calyx kiwi fruit leaf medicinal material coarse powder 1kg, add activated carbon 1000g decolouring, all the other are with embodiment 1, and solution water white transparency after the result is decoloured gets extract 7.86g, product appearance white, total saponin content is in ursolic acid 42.21%.
Embodiment 6 preparations are to the calyx kiwi fruit leaf extractive
To calyx kiwi fruit leaf medicinal material coarse powder 1kg, as extracting solvent, all the other are with embodiment 1 with 60% ethanol, and decolouring back solution is pistac (++), gets extract 19.53g, product appearance faint yellow (++), and total saponin content is in ursolic acid 58.52%.
Embodiment 7 preparations are to the calyx kiwi fruit leaf extractive
To calyx kiwi fruit leaf medicinal material coarse powder 1kg, mix depigmenting agent consumption 1000g, all the other are with embodiment 1, and decolouring back solution is pistac (++), gets extract 33.23g, the product appearance yellowish-brown (+++), total saponin content is in ursolic acid 47.87%.
Embodiment 8 preparations are to the calyx kiwi fruit leaf extractive
To calyx kiwi fruit leaf medicinal material coarse powder 1kg, (active carbon: active hargil=1: 5), all the other are with embodiment 1 to mix depigmenting agent consumption 2000g; Decolouring back solution is utmost point pistac (+); Get extract 16.74g, product appearance faint yellow (++), total saponin content is in ursolic acid 64.85%.
Embodiment 9 preparations are to the calyx kiwi fruit leaf extractive
To calyx kiwi fruit leaf medicinal material coarse powder 1kg, (active carbon: active hargil=1: 1), all the other are with embodiment 1 to mix depigmenting agent consumption 1000g; Decolouring back solution is pistac (++); Get extract 13.73g, product appearance faint yellow (++), total saponin content is in ursolic acid 48.98%.
Embodiment 10 preparations are to the calyx kiwi fruit leaf extractive
To calyx kiwi fruit leaf medicinal material coarse powder 1kg, active hargil consumption 5000g, all the other are with embodiment 1, decolouring back solution is bottle green (++ ++), extract 37.74g, product appearance yellowish-brown (++ ++), total saponin content is in ursolic acid 39.46%.
Embodiment 11 preparations are to the calyx kiwi fruit leaf extractive
To calyx kiwi fruit leaf medicinal material coarse powder 1kg, and the mixing depigmenting agent (active carbon: 6000g active hargil=1: 25), decolouring back solution is pistac (+), gets extract 17.25g, product appearance faint yellow (+), total saponin content is in ursolic acid 52.64%.
The pharmacological evaluation of 12 pairs of calyx kiwi fruit leaf extractives of embodiment
1. anticancer experiment in vitro (MTT test)
Take the logarithm K562, the HL60 tumor cell (being introduced by Chinese Academy of Sciences's Shanghai cell research) of trophophase are made into 7 * 10 respectively with RPMI 1640 culture medium that contain 10% calf serum
4, 1.05 * 10
5The cell suspension of individual/ml is seeded in respectively in 96 well culture plates, and 90 μ l are inoculated in every hole; Add the PBS solution 10 μ l that contain variable concentrations extract (embodiment 1 preparation) respectively; Make final concentration be respectively 50,100,200,300,400,500 μ g/ml, the blank group adds 10 μ l RPMI, 1640 culture medium, establishes 5 parallel holes for every group; 37 ℃, 5%CO
2Cultivate 3d; Every hole adds 5mg/ml MTT normal saline solution 10 μ l, and 37 ℃ are continued to cultivate 4h; The centrifugal 10min of 96 orifice plate 800g, supernatant discarded, every hole adds DMSO 150 μ l, and the vibration mixing is measured OD value with the 492nm wavelength on ELIASA.Be calculated as follows the suppression ratio of medicine to growth of tumour cell:
Growth of tumour cell suppression ratio (%)=(1-experimental group OD/ matched group OD value) * 100
With concentration to the growth of tumour cell suppression ratio map amount effect relation curve (as shown in Figure 1), use the logarithm probit method, calculating the growth of tumour cell suppression ratio with linear regression equation is 50% drug level, is IC50 half casualty-producing concentrations.Extract is to the IC of K562, HL60 tumor cell line
50Be respectively 217.31 μ g/ml and 207.61 μ g/ml.Show that extract has stronger cytotoxicity.
2, anti-tumor in vivo experiment
Rat liver cancer cell line H22 (being introduced by Chinese Academy of Sciences's Shanghai cell research) recovery cultivations of going down to posterity, collection logarithmic (log) phase cell is adjusted to 1 * 10 with normal saline
6Individual/ml; Be inoculated in the mouse peritoneal with every 0.5ml; Mouse ascites is extracted in one week back, and according to being diluted to cell suspension at 1: 4, it is subcutaneous only to be inoculated in mice (Kunming mouse is provided by Fudan University's Experimental Animal Center) right fore armpit with 0.2ml/ with normal saline.Behind inoculation 24h, the mice completely random is dispensed to blank group, extract low dose group, middle dose groups, high dose group (extract is embodiment 1 preparation), 10 every group.Adopt the gastric infusion method, successive administration 14 days; Adopt the cervical vertebra dislocation method to put to death mice, peel off the tumor piece and weigh, calculate inhibition rate of tumor growth according to following formula:
Inhibition rate of tumor growth (the %)=average tumor of (it is heavy that average tumor is organized in the average tumor weight-treatment of matched group)/matched group heavy * 100
The result shows that giving the basic, normal, high dosage of extract all can suppress growth of tumor (table 1), shows that extract has stronger antitumous effect.
Table 1 pair calyx Radix Actinidiae Chinensis, stem are to the inhibitory action (n=10) of mice-transplanted tumor
Annotate: compare with matched group
*P＜0.01
Claims (3)
1. method for preparing to the calyx kiwi fruit leaf extractive, form by following steps:
To calyx kiwi fruit leaf medicinal material coarse powder 1kg, with 80% ethanol, 12 times, 10 times, 10 times, 1.5 hours, 1 hour, 1 hour; Reflux, extract,, the extracting solution filtered while hot, merge extractive liquid,, decompression and solvent recovery is to there not being the alcohol flavor; It is 1g crude drug/ml that distilled water is regulated concentration, 5000rpm, centrifugal 15 minutes; Obtain deposition,, add to 10000ml with an amount of anhydrous alcohol solution; Add active carbon: the mixing depigmenting agent 2000g remove impurity decolouring of active hargil=1: 10, decolouring back solution is utmost point pistac, with its concentrating under reduced pressure, 60 ℃ of vacuum dryings are pulverized, extract 23.20g, product appearance is faint yellow.
A method for preparing as claimed in claim 1 make to the calyx kiwi fruit leaf extractive.
One kind as claimed in claim 2 to the calyx kiwi fruit leaf extractive in preparation antitumor drug and Application in Food.
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| CN102212093B (en) * | 2010-04-01 | 2013-11-06 | 中国人民解放军第二军医大学 | Flavonoid glycoside compounds, method for preparing same and application |
| CN101897738B (en) * | 2010-05-20 | 2012-05-02 | 长春瑞德医药科技有限公司 | Application of actinidia kolomicta leaf flavonoids in preparing medicines for preventing and treating vascular dementia |
| CN101891792B (en) * | 2010-06-29 | 2014-04-16 | 河南科技大学 | Triterpene compounds in salvia miltiorrhiza flower and extraction method and application thereof |
| CN102846680B (en) * | 2011-07-01 | 2014-06-11 | 中国人民解放军第二军医大学 | Actinidia valvata root extract, preparation method and application thereof |
| CN102617656B (en) * | 2012-02-02 | 2014-04-23 | 中国人民解放军第二军医大学 | Method for separating and preparing high purity flavonoid glycoside compounds from actinidia valvata dunn leaves |
| CN105687319A (en) * | 2016-02-23 | 2016-06-22 | 中国人民解放军第二军医大学 | Anti-osteoporosis hops extract, method for preparing same and application of anti-osteoporosis hops extract |
| CN105943571B (en) * | 2016-07-13 | 2020-06-02 | 秦小波 | Preparation method and application of kiwifruit waste anti-tumor extract |
| CN107114644A (en) * | 2017-05-27 | 2017-09-01 | 周安琪 | Kiwi fruit leaf health beverages and preparation method thereof |
| CN110496144A (en) * | 2019-08-14 | 2019-11-26 | 右江民族医学院 | A kind of Kiwi extract preparation method and its application for preventing and treating cardiovascular and cerebrovascular disease |
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