CN1194154A - Asiaticoside of self asiatic and hydroxy-asiaticoside and carboxy-asiaticoside water soluble extract and its separating method - Google Patents
Asiaticoside of self asiatic and hydroxy-asiaticoside and carboxy-asiaticoside water soluble extract and its separating method Download PDFInfo
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Abstract
A water-soluble extract of asiaticoside and madecassoside from Centella asiatica(L) Urb. and an isolating method thereof. Centella asiatica is immersed in an alcohol solution in water, to give an extract solution which is, then, treated with a halogenide solvent. The aqueous layer was extracted with a higher. Subsequently, the extract in the alcohol layer was washed several times with an aqueous alkaline solution, then with water, concentrated in vacuo, and crystallized with ethyl acetate. A test using a rat whose liver cells had been damaged showed that the water-soluble extract could restore them up to 40-60 %. In addition, the aqueous mix was found to relieve the fibrosis of liver cell by a bile duct ligation and liver fibrosis test.
Description
The present invention relates to have anti-fibrosis effect, can protect the water solubility extract of hepatocellular asiaticoside (asiaticoside) and asiaticoside (madecassoside) and relate to its separation method from Centella asiatica.
Spontaneous at the samphire Centellaasiatica of African Madagascar and India (L.) Urb, be used for the treatment of local wound (Poizot.A, Dumez.D, CR.Acad.Sci[D] 286,1978) for a long time always.Quantitative (titrated) extract (hereinafter being called " TECA ") of many researcher report Centella asiatica is effective for the local wound of tissue.In fact, TECA is a medicine generally acknowledged by the public and available, and its commodity are called " Madecassol " (asiaticoside: asiatic acid: Madecassic acid=4: 3: 3).Its therapeutic effect is for by improving fibrotic processes, and the protective tissue cell is so that injured tissues returns near original state.But these compositions are only limited to the treatment wound, and disclose it extract and need complicated process in Korean Patent Publication No. 87-1458 number, 87-1573 number and 91-2518 number, thereby its yield and purity are very poor.In addition, the main purpose composition is aglucon (asiatic acid, Madecassic acid and Madasiatic acid) and asiaticoside, and they all are water-fast, thereby, limited its purposes.
Although the TECA that deposits earlier shows superior anti-fibrosis effect, its water-fast physical property forces himself to be used with powder type, and its scope of application is limited in the treatment wound.
Multiple by the present inventor, the further investigation that purpose is to enlarge the pharmaceutical usage of TECA causes finding: handle Centella asiatica with aqueous alcohol, help very much to extract water-soluble and can protect hepatocyte effectively and the Fibrotic composition of anti-hepatocyte.
So purpose of the present invention is for providing from the asiaticoside of Centella asiatica and the water solubility extract of asiaticoside, it shows superior protection hepatocyte and the effect of anti-hepatocyte fibrosis.
Another object of the present invention provides the separation method of described water solubility extract.
According to purpose of the present invention, the method for separating the water solubility extract of asiaticoside and asiaticoside from Centella asiatica is provided, it comprises following process: make the aqueous alcohol freezing precipitation that comprises Centella asiatica; Handle extract in the aqueous alcohol so that leafing with halide solvent; Extract the water-bearing layer with higher alcohol; Use sodium hydroxide, wash the extract in the alcohol layer then with water and concentrate this extract; Make this extract crystallization with ethyl acetate, obtain crystallization and wash crystallization.
According to another object of the present invention, provide from the asiaticoside of Centella asiatica and the water solubility extract of asiaticoside, it has anti-fibrosis effect, can protect hepatocyte.
1. asiaticoside and asiaticoside extract separates
Centella asiatica is immersed in the aqueous alcohol solutions, obtain extract, then, handle this extract with halide solvent.Extract its water layer with alcohol (Cn 〉=4).Then, wash extract in the alcohol layer for several times with aqueous alkali, and vacuum concentration or make its precipitation, solid obtained.
This shows that water miscible solid mainly comprises asiaticoside and the asiaticoside by the following formula I representative: (formula I slightly)
The alcoholic solution that is used for the present invention comprises the alcohol amount of containing and is the methanol of 50-80% or ethanol.The halide solvent that is used for the present invention is selected from dichloromethane, chloroform, dichloroethanes and dichloroethylene.With regard to the extraction in described water-bearing layer, use the alcohol that comprises at least 4 carbon atoms for example to be selected from n-butyl alcohol, sec-butyl alcohol and amylalcohol.Use sodium hydroxide or potassium hydroxide aqueous solution as aqueous slkali.
By said process obtain from the extract of Centella asiatica mainly by 4: 6-6: 4 asiaticoside and asiaticoside are formed, found described two kinds of compositions account for this extract 97% or more than.It can water-soluble, pure and mild vegetable oil.Especially, because the dissolubility of described extract in water is very important physical property, so, be called water solublity TECA (being called " WS-TECA " hereinafter) in the present invention.
2. protection hepatocyte test
Use in the hepatocyte of the wistars Mus that known representative poisonous substance carbon tetrachloride and galactosamine cultivate in the early stage and bring out toxicity, toxicity blocking effect and the restitution of check WS-TECA.
A) experimental animal
Make the wistars Mus (male, be that 22 ± 1 ℃ and humidity are 60 ± 5% 150-200g) in temperature, under the condition that per round the clock 12 hours change, freely look for food and drink water.Make its fasting the previous day from testing.
B) liver cell culture of wistars Mus
Separate its hepatocyte by 2 stage collagenase perfusion technology (the Berry-Friend method that makes improvements slightly).Use urethane (1g/lkg body weight) to make the anesthesia of wistar Mus, clean its abdominal part and incision with 70% ethanol.Insert 20 catheter gauges at its portal vein, add the HBBS of 150ml with the flow velocity of 15ml/min by it.At this moment, cut its postcava and emit blood.After it opens breast, 18 catheter gauges are inserted its postcava, it is tied, so that make the digestion juice recirculation that in 95mlHBBS, comprises 0.05% collagen element.Described recirculation was carried out 10 minutes, simultaneously, provide CO
2(5%) and O
2(95%) mixture.When isolating hepatocytes, sort out its liver and place the 60ml HBBS of beaker.With the thin film of a shears strip off encirclement liver, so that hepatocyte is free.Then, it is filtered through the paper of showing on trial.Resulting filtrate centrifugal 2 minutes with 50g.Remove supernatant, then,, obtain hepatocellular suspension by centrifugal culture medium under similarity condition.Make this suspension with 5 * 10
5The concentration of cells/ml is transferred in the culture dish that scribbles collagen.Used culture medium is to comprise 10% fetus sura serum, the bovine serum albumin (component V), 10 of 2.0mg/ml
-6The dexamethasone of M, 10
-7The insulin of M, 5.32 * 10
-2The L-serine, 4.07 * 10 of M
-2M NaHCO
3, the streptomycin of penicillin, 100 μ g/ml of 100IU/ml and 5 μ g/ml Waymouth ' s MB 752/1 culture medium of amphotericin B.Make described cell in constant humidity, air (95%) and CO are provided simultaneously
2In the calorstat of mixture (5%), in 37 ℃ of cultivations.
C) test method
Make liver cell culture after 24 hours, replace former culture medium, made this carbon tetrachloride bring out its toxicity in 1.5 hours that cultivate again with the fresh culture that comprises the 10mM carbon tetrachloride.Made 1.5 hours culture medium of described liver cell culture by replacing with the fresh culture of the galactosamine that comprises 1.5mM, and cultivated its 14 hours, galactosamine also can bring out hepatotoxicity.
By Reitman-Frankel method and improved a little Gerlach method, measure paddy third transaminase (GPT) and sorbito dehy drogenase (SDH) in the described culture medium.
The amount of the product that the described protein of per minute 1mg produces is represented the activity of glutathione-S-transferase, its mensuration process is: the mitochondrion supernatant is blended in and comprises glutathion, 1-chloro-2 after inciting somebody to action, in the test tube of 4-dinitro benzene and kaliumphosphate buffer, and the trap that is determined at 340nm place vertical lifting immediately reaches 5 minutes.
Described back mitochondrion supernatant obtains by following process: cultivate hepatocyte, remove culture medium, the Tris-HCl buffer (pH7.4) that adds the 66mM of 3ml obtains the suspension of cell, makes 15 seconds of described suspension homogenize, and with centrifugal this suspension of 12500g 15 minutes.
Use the erythrocuprein (SOD) in the improved McCord-Frivich method mensuration hepatocyte.The back mitochondrion supernatant and 200 μ l reagent A, 500 μ l deionized waters and the 200 μ l reagent B that add 100 μ l, and in 37 ℃ of placements 30 minutes.After in the supernatant that generates, adding 200 μ l reagent C again,, placed 20 minutes, measure the trap at 550nm place, so that calculate SOD unit in room temperature.
With regard to the activity of GSSG reductase, in the test tube that comprises 600 μ l phosphate buffers, 100 μ 10mM GSSG and 100 μ l 1mM NADPH, add the back mitochondrion supernatant of 200 μ l, and detect the variation of trap in 2 minutes at the 340nm place.
With regard to the mensuration of the total content of glutathion (GSH and GSSG), 700 μ l0.3mM NADPH and 100 μ l 6mM DTNB are added in the back mitochondrial suspension of 200 μ l, then, make it in water-bath, in 30 ℃ of cultivations.After GSH reductase (50 units per ml) initiation reaction that adds 10 μ l, at the 412nm place, measure trap every 15 seconds, surveyed altogether 2 minutes.
(mixture of the 2-vinylpyridine of the back mitochondrial suspension 2 μ l of the per 100 μ l of vigorous stirring was also placed 60 minutes in 25 ℃ for oxidised form, content GSSG) with regard to glutathion, so that produce the derivant of GSH, then, measure its content, so that calculate remaining GSSG.
For detecting the biosynthesis of RNA, [3H]-uridnine is added the radio-labeled uridnine of also measuring in the culture medium that concentration is 1 μ Ci/ml among the RNA that mixes the cultivation cell.For this reason, after removing described culture medium, wash described cell three times and add 10% trichloroacetic acid of 1ml with HBSS, so that precipitating proteins.Add the mixture of the alcohol-ether (3: 1) of 1ml, also this protein is dissolved in the 1N sodium hydroxide of 200 μ l so that remove remaining trichloroacetic acid.Get 100 these protein solutions of μ l and be added among the aquasol (a kind of flicker cocktail) of 3ml, so that measure radioactivity.
D) result
With the amount of 1 μ g/ml, WS-TECA is given and brought out in the hepatocyte of originally cultivating of toxic wistar Mus with carbon tetrachloride up to 300 μ g/ml.Demonstration is from the dose-dependence of 1 μ g/ml up to 100 μ g/ml, and WS-TECA has reduced the GPT that is released in the culture medium and the activity of SDH, shows protection activity to the hepatocyte maximum at 100 μ g/ml.In addition, WS-TECA increases the activity of GSSG reductase and SOD, and they participate in the Detoxication of many enzymes in the liver, in the generation that stops the GST free radical with remove in the free radical of generation and play a role.And WS-TECA has increased because the amount of the GSH that described free radical reduces.As mentioned above, WS-TECA shows the maximum activity (seeing Table 1) of 50-60% at 100 μ g/ml.
When the WS-TECA processing of measuring up to 300 μ g/ml with 1 μ g/ml has caused the wistar Hepar Mus cell of toxic cultivation originally with galactosamine,, produce active maximum inhibition to GPT and SDH at 300 μ g/ml.
With regard to WS-TECA to regard to the synthetic influence of RNA in the hepatocyte of galactosamine infringement, the WS-TECA that adds 300 μ g/ml amount compares with not adding WS-TECA, and in the biosynthesis of RNA, its hepatocyte increases by two times, thereby, make the damage hepatocyte of as many as 40% return to normal condition.
Table 1
WS-TECA is to by CCl
4The effect of the hepatotoxicity of bringing out
Protective effect (%)
*Condition GPT SDH GST GSH GSSG SOD blank
*100 100 100 100 100 100 positive controls
* *0 0 0 0 0 0WS-TECA 1μg/ml 10.1 3.0 2.8 6.6 13.9 15.8WS-TECA 10μg/ml 26.9 14.9 5.4 12.7 55.8 31.6WS-TECA 50μg/ml 35.2 34.3 36.8 26.1 60.0 36.8WS-TECA 100μg/ml 64.5 55.8 49.2 57.0 63.9 57.9WS-TECA 300μg/ml 53.6 41.7 38.0 45.5 55.8 47.4
* protect (%)=(positive control-sample)/(positive control-blank)
* blank: untreated hepatocyte
* * positive control: CCl
4The hepatocyte of handling
Table 2
WS-TECA is to by CCl
4The effect of the hepatotoxicity of bringing out
Protective effect (%)
*Condition GPT SDH RNA synthesizes (cpm) blank
*100 100 615 ± 48 positive controls
* *00 125 ± 11WS-TECA, 1 μ g/ml, 10.8 12.2 109 ± 32WS-TECA, 10 μ g/ml, 18.5 19.1 204 ± 29WS-TECA, 100 μ g/ml, 30.8 32.7 234 ± 14WS-TECA, 300 μ g/ml 41.5 43.9 267 ± 58
* protect (%)=(positive control-sample)/(positive control-blank)
* blank: untreated hepatocyte
* * positive control: CCl
4The hepatocyte of handling
E) anti-fibrosis effect
Handle provocative test animal liver fibrosis by bile duct ligation technology or by carbon tetrachloride.Give the WS-TECA that obtains in the present invention with experimental animal,, thereby measure the biosynthesis of the principal element collagen protein that causes fibrotic processes in the tissue and accumulate so that estimate serum procollagen N end group peptide (PIIIP) and hydroxyproline quantitatively.
Process of the test and WS-TECA fibrosis result are as follows.
Described bile duct ligating methods carries out in the following manner, promptly cuts the abdominal part of male SD mouse (200-220g) and its bile duct binode is pricked to reach for 4 weeks, so that bring out the hepatocyte fibrosis.Once-a-day, peritoneal administration.Use the anti-PIIIP antibody of rabbit, use the PIIIP in the ELISA method quantitative assay serum.Use staining to finish the quantitative process of collagen protein in the described hepatic tissue, wherein, when making described hepatic tissue acid hydrolysis, the hydroxyproline that discharges is handled with toluene-sodium-sulfonchloramide and ER solution (to dimethylamino benzaldehyde, perchloric acid, isopropyl alcohol).
With regard to anti-fibrosis effect in the described ligation bile duct test, WS-TECA can reduce as the amount of the hydroxyproline of hepatic fibrosis index (seeing Table 3).In addition, use WS-TECA can suppress to be used as the PIIIP serum-concentration (seeing Table 4) of collagen protein biosynthesis index.Obviously, WS-TECA is effective to alleviating hepatic fibrosis.
Following hepatic fibrosis test for bringing out by carbon tetrachloride.
By body weight, weekly twice, altogether around, peritoneum is given the CCl with male SD mouse 480mg
4Simultaneously, once a day, around being total to, peritoneum is given and the described trial drug that is dissolved in the normal saline.With PIIIP in the method quantitative assay serum identical and the collagen protein in the hepatic tissue with bile duct ligation test.
Described CCl
4Handle producing the fibrosis fall out effect more light, and, use CCl not using under the condition of medicine than described bile duct ligation
4It is the twice of matched group that the hepatic tissue of handling makes the collagen protein precipitation capacity.
WS-TECA suppresses the concentration (seeing Table 6) that collagen protein precipitates (seeing Table 5) and reduces PIIIP in the serum in the hepatic tissue, its show dose dependence.
These data show, because WS-TECA increases the synthetic of collagen protein, can be used for alleviating hepatic fibrosis and because of hindering the precipitation of the hepatic tissue collagen protein that collagen protein synthesis causes.
Table 3
WS-TECA is to total hydroxyproline blank 32.11 ± 6.07 ligation bile ducts 162.2 ± 127WS-TECA administration in the sedimentary inhibitory action group of hydroxyproline in the hepatic tissue hepatic tissue
1.5mg/kg 111.2±60.6
5.0mg/kg 96.4±51.7
Table 4
WS-TECA is to reduction effect group PIIIP serum-concentration blank 6.1 ± 2.9 ligation bile ducts 37.4 ± 12.9WS-TECA administration of PIIIP concentration in the serum
1.5mg/kg 25.5±6.4
Table 5
WS-TECA is to CCl
4Total hydroxyproline blank 1.67 ± 0.26CCl in the sedimentary inhibitory action group of the collagen protein hepatic tissue in the processing Mus
4-processing 3.02 ± 0.78WS-TECA administration
0.5mg/kg 3.29±1.34
1.5mg/kg 2.39±0，89
5.0mg/kg 2.18±1.03
Table 6
WS-TECA is for CCl
4Handle reduction effect group PIIIP serum-concentration blank 7.4 ± 2.9 ligation bile ducts 47.2 ± 7.8WS-TECA administration of PIIIP concentration in the Mus serum
0.5mg/kg 32.8±9.2
1.5mg/kg 26.1±3.5
5.0mg/kg 27.6±6.3
The present invention may be better understood by the following example, and they just explain the present invention, and it are not limited.
Embodiment 1
32 liter of 70% ethanol is added among the exsiccant Centella asiatica of 4.7kg, then, make its freezing precipitation 48 hours.The ethanol extract that obtains is placed bottle, add 17 liters of dichloromethane, and stirred 1 hour.After complete layering, get the upper strata, add the dichloromethane of 5 liters of ethanol and 7 liters, and stirred 1 hour.Deng once more fully after the layering, get the upper strata, use 17 liters of n-butanol extractions.Divide several to wash the n-butanol layer extract with the 0.1N sodium hydroxide, then, wash with water several times.Vacuum concentration n-butanol layer to ten/one (volume) makes the concentrate that obtains mix with 5 liters of ethyl acetate, so that crystallization.The crystallization of sucking filtration gained is so that the solid that obtains being left.Then, wash with a spot of ethyl acetate, in 50 ℃, vacuum drying 24 hours obtains the 120g yellow solid.
Embodiment 2
35 liter 70% ethanol is added among the Centella asiatica of 5kg, then, it was placed 48 hours in 10 ℃.
The gained ethanol extraction is placed bottle, press embodiment 1 same procedure and handle, obtain the asiaticoside of 125g and the mixture of asiaticoside (4: 6).
Embodiment 3
Obtain the mixture of asiaticoside and asiaticoside (6: 4) by embodiment 1 same procedure.
Embodiment 4
Use mobile phase (n-butyl alcohol: ethanol: ammonia: water=60: 40: 5: 10) silica gel (70-230 order) is packed into diameter 6cm, in the pillar of length 26cm.Then, the WS-TECA with 1g is dissolved in the least possible mobile phase and application of sample.Mobile phase is flowed down with constant speed and collect, obtain pure asiaticoside of 400mg and asiaticoside respectively with the 25ml bottle.
Experimental example
5gWS-TECA is dissolved in the 100ml methanol, then, added 3ml 5N sodium hydroxide and reflux 12 hours.Make the solution and the conventional TECA of generation on immobile phase, use mobile phase (n-butyl alcohol: ethanol: ammonia: water=60: 40: 5: 10) launch, and with the colour developing of the mixture of anhydrous acetic acid and sulphuric acid (9: 1), so that identify the hydrolyzate of the WS-TECA that comprises Madecassic acid and asiatic acid.
The HPLC analysis demonstration asiaticoside of its hydrolyzate and asiaticoside exist with 5: 5 ratio.
In many countries, annual chronic hepatitis for example viral hepatitis and the alcoholic hepatitis of often taking place, mortality rate is very high.Described hepatopathy causes hepatocellular damage constantly, thereby causes hepatic fibrosis.So far, attempt using colchichine and UDCA to treat described hepatopathy always, yet, about its effect many different viewpoints are arranged.So an urgent demand exploitation can reduce hepatocyte injury and Fibrotic new therapeutic agent.As above-mentioned, the asiaticoside that expection is extracted from Centella asiatica and the mixture of asiaticoside can reduce the serosity and because the mortality rate that this disease causes of chronic hepatopathy.
The present invention is illustrative, and used term is in order to illustrate rather than to be limited.
According to the above description, can be to the many changes of the present invention's row.So, in the scope of these claims, implement the present invention with diverse ways, be understandable.
Claims (4)
1. separate the method for the water solubility extract of asiaticoside and asiaticoside from Centella asiatica, it comprises the steps:
Make the aqueous alcohol solutions freezing precipitation that comprises Centella asiatica;
Handle extract in this aqueous alcohol solutions with halide solvent, so that layering;
Extract its water layer with higher alcohol;
Use sodium hydroxide, wash its alcohol layer extract then with water, and concentrate this extract;
With this extract of ethyl acetate crystallization, obtain crystallization; With
Wash crystallization.
2. according to the process of claim 1 wherein, described aqueous alcohol is ethanol or methanol solution, and described halide solvent is selected from dichloromethane, chloroform and dichloroethanes, and described higher alcohol is selected from n-butyl alcohol, sec-butyl alcohol and amylalcohol.
3. from the asiaticoside of Centella asiatica and the water solubility extract of asiaticoside, it can be protected hepatocyte and have anti-fibrosis effect.
4. according to the water solubility extract of claim 3, wherein, the ratio of described asiaticoside and asiaticoside is 4: 6-6: 4.
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| CN101407536B (en) * | 2008-11-27 | 2011-05-18 | 浙江大学 | Process for preparing high-purity asiaticoside by solvent crystallization |
| CN102532244A (en) * | 2011-12-31 | 2012-07-04 | 广西大学 | Method for preparing high-purity asiaticosid |
| CN102973581A (en) * | 2011-09-07 | 2013-03-20 | 杭州赛利药物研究所有限公司 | Use of madecassoside of centella asiatica in preparing medicaments for treating liver fibrosis |
| CN102973580A (en) * | 2011-09-07 | 2013-03-20 | 杭州赛利药物研究所有限公司 | Use of asiatic acid of centella asiatica in preparing medicaments for treating liver fibrosis |
| CN102973579A (en) * | 2011-09-07 | 2013-03-20 | 杭州赛利药物研究所有限公司 | Use of madecassic acid of centella asiatica in preparing medicaments for treating liver fibrosis |
| US8449924B2 (en) | 2007-08-29 | 2013-05-28 | Development Center For Biotechnology | Process for the preparation of plant extracts for treating skin disorders and enhancing healing of wounds |
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| CN100406473C (en) * | 2004-09-29 | 2008-07-30 | 中国科学院上海药物研究所 | Preparation method and application of Asiatic polysaccharide |
| EP1925310A1 (en) | 2006-11-24 | 2008-05-28 | Development Center For Biotechnology | Plant extracts from genera Plectranthus and Centella for treating skin disorders and enhancing healing of wounds for diabetics patients |
| US8449924B2 (en) | 2007-08-29 | 2013-05-28 | Development Center For Biotechnology | Process for the preparation of plant extracts for treating skin disorders and enhancing healing of wounds |
| CN101323637B (en) * | 2008-07-29 | 2010-12-22 | 卢照凯 | Asiaticoside and preparation thereof |
| CN101407536B (en) * | 2008-11-27 | 2011-05-18 | 浙江大学 | Process for preparing high-purity asiaticoside by solvent crystallization |
| CN102973581A (en) * | 2011-09-07 | 2013-03-20 | 杭州赛利药物研究所有限公司 | Use of madecassoside of centella asiatica in preparing medicaments for treating liver fibrosis |
| CN102973580A (en) * | 2011-09-07 | 2013-03-20 | 杭州赛利药物研究所有限公司 | Use of asiatic acid of centella asiatica in preparing medicaments for treating liver fibrosis |
| CN102973579A (en) * | 2011-09-07 | 2013-03-20 | 杭州赛利药物研究所有限公司 | Use of madecassic acid of centella asiatica in preparing medicaments for treating liver fibrosis |
| CN102532244A (en) * | 2011-12-31 | 2012-07-04 | 广西大学 | Method for preparing high-purity asiaticosid |
| CN103796631A (en) * | 2012-06-29 | 2014-05-14 | 生物光谱公司 | Compositions for whitening skin comprising madecassoside |
| US10758584B2 (en) | 2016-07-17 | 2020-09-01 | Oneness Biotech Co. LTD | Topical formulation for promoting wound healing |
| CN108398493A (en) * | 2017-02-08 | 2018-08-14 | 桂林三金药业股份有限公司 | A kind of quality determining method of centella medicinal material and its extract and preparation |
| CN108398493B (en) * | 2017-02-08 | 2021-05-28 | 桂林三金药业股份有限公司 | Quality detection method for centella asiatica and its extract and preparation |
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Correction item: Denomination of Invention Correct: Water soluble extract of Centella asiatica and asiaticoside from Centella asiatica and separation method thereof False: Water soluble extract of Centella asiatica, asiaticoside and hydroxyl asiaticoside and asiaticoside from Centella asiatica and separation method thereof Number: 39 Page: 12 Volume: 14 |
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