CN100406473C - Preparation method and application of Asiatic polysaccharide - Google Patents
Preparation method and application of Asiatic polysaccharide Download PDFInfo
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- CN100406473C CN100406473C CNB2004100668599A CN200410066859A CN100406473C CN 100406473 C CN100406473 C CN 100406473C CN B2004100668599 A CNB2004100668599 A CN B2004100668599A CN 200410066859 A CN200410066859 A CN 200410066859A CN 100406473 C CN100406473 C CN 100406473C
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Abstract
The present invention provides a polysaccharide extracted from herba centellae and an extracting method and the chemical constitution of the polysaccharide. As is proven by pharmacological tests outside the animal body, the polysaccharide has appreciable immunologic enhancement and can be used as the auxiliary medicament for treating immunological function deficiency.
Description
Technical field
The present invention relates to the Chinese herbal medicine extracting polysaccharide, more specifically say so and from Herba Centellae, extract polysaccharide, and the chemical structure characteristic of this polysaccharide and its application, specifically comprise the extracting method, chemical structure of Asiatic polysaccharide HBN and the immunologic enhancement of body.
Background technology
Cancer has become one of human dead principal element at present.But lack still so far that specificity is strong, good effect, the little cancer therapy drug of side effect.Though the antitumor drug kind of using is a lot of now, be the chemicals that adopts the chemical synthesis process preparation mostly, toxic side effect is bigger.In recent years, biotherapy has become the important channel of antineoplaston, and immunopotentiating agent is a class medicine that is used for tumor biotherapy.The polysaccharide that the present invention obtains from Herba Centellae is a kind of immunomodulator.
Herba Centellae is the conventional Chinese medicine material that Chinese Pharmacopoeia records, be recorded in Shennong's Herbal the earliest, classify middle product as, effect with clearing heat and promoting diuresis, detoxify and promote the subsdence of swelling, be applicable to that jaundice due to damp-heat, heatstroke diarrhoea, sand drench blood pouring, carbuncle sore tumefacting virus etc., clinically treatments that are used for wound, tetter etc. more.The present invention extracts from Herba Centellae and obtains still a kind of homogeneous polysaccharide of one, and determines its chemical structure characteristic.Pharmacological testing shows that the polysaccharide of this chemical structure has significant immunologic enhancement.
Summary of the invention
The object of the invention provide a kind of from Herba Centellae separation and Extraction go out to determine the polysaccharide of chemical structure characteristic.
Another object of the present invention provides the extraction and separation method of this polysaccharide.
A further object of the present invention provides the medical usage of this polysaccharide.
The present invention implements like this:
Get dried Herba Centellae herb and pulverize product, add 95% medicinal alcohol, soaked for 2 weeks, immersion finishes, and filters, and residue continues to use alcohol reflux 6 hours with apparatus,Soxhlet's, the elimination mother liquor, and residue dries in the shade.Boiling water extraction 3-5 time of dry product after the degreasing, each extraction time is 5-6 hour, with the extracting liquid filtering that extracts at every turn, the water extract of gained merged, and is concentrated into small volume, to flowing water dialysis 3-4 days, continuation with solution concentration to small volume, with 10%～20% Tricholroacetic Acid deproteinated under 4 ℃ of stirrings, making the solution final pH is 2-3, leaves standstill to place 6-8 hour.Solution precipitates through centrifugal Deproteinization, and clear liquid is neutralized to neutrality with the 1mol/l sodium hydroxide solution, again to flowing water dialysis 3 days.Solution concentration to 1/3～1/5 volume in the bag, add 3-5 doubly to 95% ethanol of sugar soln volume in solution, and spend the night in 4 ℃ of precipitations.Centrifugal collecting precipitation, dehydration obtains Crude polysaccharides JXC1 in 40 ℃ low-temperature vacuum drying case.
Crude polysaccharides JXC1 is carried in water intaking, adds an amount of dissolved in distilled water, the centrifugal insolubles of removing, and supernatant liquor separates by the DEAE-cellulose chromatography.As elutriant, use 0.1,0.3,0.5 and the sodium chloride solution wash-out of 1.0mol/l with distilled water then, collect respectively and merge each flow point, the component of wherein washing part through concentrate, dialysis and lyophilize obtain secondary polysaccharide fraction JXCH.JXCH passes through the DEAE-cellulose chromatography again, behind the distilled water wash-out, and 0.2mol/l sodium chloride solution wash-out, automatic collection instrument is collected flow point, and sulfuric acid-phynol method detects polysaccharide fraction, collects sugared reacting positive part.Concentrating under reduced pressure, dialysis and lyophilize get polysaccharide fraction, further use the DEAE-cellulose chromatography again, elder generation is with the sodium chloride solution gradient elution of 0-0.05mol/l, use 0.2mol/l sodium chloride solution wash-out again, collect the polysaccharide soln of this component, after pure water dialysis being removed small molecules such as inorganic salt in 1 day, do not see through the liquid concentrating under reduced pressure, lyophilize, the component that obtains is through Sephadex G-200 column chromatography, 0.2mol/L the sodium chloride solution wash-out, the differential detector detects, and collects peak position and partly obtains main sugar component, this component obtains polysaccharide through the concentrating under reduced pressure lyophilize, called after HBN.
Molecular weight through efficient gel column chromatography (HPGPC) analysis revealed HBN is 5.4 * 10
5Da.Specific optical rotation [α]
D=-10.4 ° (c=0.70, water).Each residue mol ratio of HBN is a pectinose: semi-lactosi: rhamnosyl: galacturonic acid: wood sugar=1.00: 1.90: 0.26: 0.30: 0.15.
13In the C NMR spectrum, the signal that is positioned at δ 110.4 and 108.5ppm is the anomeric carbon signal of α-Araf and β-Xylp.The signal that is positioned at the different head region of δ 104.6～104.4ppm is β-Galp signal.The weak signal of δ 102.0 and 100.6ppm is the C1 signal of α-Rhap and α-GalpA.About 76%Araf residue is in end, and therefore main Araf signal is to be produced by t-Araf.
1In the H NMR spectrogram, signal δ 5.2 and 4.4-4.5ppm are corresponding to the anomer hydrogen signal of Araf and β-Galp.The anomer hydrogen signal of α-Rhap, α-GalpA and β-Xylp is positioned at δ 5.1,4.9 and 4.7ppm place.The above results proves that in conjunction with the result of conclusive evidence polysaccharide structures common chemical methods such as methylation reaction and sodium periodate oxidation, part acid hydrolysis and enzyme liberating HBN is an arabogalactan of being made up of rhamnosyl, pectinose, semi-lactosi, galacturonic acid and wood sugar.Wherein rhamnosyl and galacturonic acid residue have constituted the core of HBN polysaccharide, belong to pectin RG-I configuration.The primary structure that connects HBN in the rhamnosyl O-4 position of this core chain, i.e. the wood sugar chain of arabogalactan zone and short chain.In arabogalactan chain zone, 1, the semi-lactosi that 6-connects is occupied an leading position, and promptly with 1, the semi-lactosi that 6-connects forms long-chain, connects other semi-lactosi side chain and pectinose side chain in its O-3 position.Simultaneously also there is part 1 in arabogalactan chain zone, the long-chain that the semi-lactosi that 3-connects constitutes, and connect Arabic sugar chain and gala sugar chain at O-6.
Pharmacological testing:
1. external mice spleen lymphocytes proliferation activity
Get each polysaccharide sample HBN 1-2mg/ml respectively and be dissolved in physiological saline, face the time spent to be diluted to the solution that concentration is 1,10,100 μ l/g.Mouse causes death with dislocation of cervical vertebra, the aseptic spleen of getting, and separating Morr. cell, nylon net filter, distilled water destroys red corpuscle, is adjusted to wait to ooze, give a baby a bath on the third day after its birth time with the RPMI-1640 nutrient solution, numeration, the adjusting cell concn is 5 * 10
6Cell/ml.In 96 well culture plates, every hole adds 100 μ cells, establishes a control group, adds the sample associating ConA or the LPA of different concns again, puts 37 ℃ of 5%CO
2Incubator 48 hours.Cultivate termination and added MTT solution 20ul/ hole in preceding hour, continue to cultivate after 4 hours, add three liquid (dimethyl formamide and 12 alkyl benzene sulphonate (ABS) sodium solutions) 100ul/ hole, after 37 ℃ of placements are spent the night, survey the A570 value in every hole with DG-3022 type enzyme post instrument.The result shows that the proliferation rate to the T cell is respectively 36%, 55% and 76% when concentration is 1,10 and 100 μ g/ml, and the appreciation rate of B cell is reached 42%, 120% and 383% respectively.Be unusual effect.Each HBN that organizes under the concentration does not all show toxicity.
2. immunity test in the mouse body
ICR (♀) mouse body weight 20 ± 2g, control group physiological saline 0.2ml/ mouse, qd. * 4d administration group abdominal injection dosage respectively is 0.1,1.0 and the HBN of 10mg/kg, administration 4 days.The 3rd day sensitization after the mouse administration: with physiological saline washing three times, by 5% dilution proportion SRBC hematocrit, every mouse peritoneal is injected 0.2ml with bright sheep red blood cell.Dissected in the 5th day and measure every immune parameter.The result shows polysaccharide HBN not influence of spleen antibody forming cell secretory antibody to mouse under the dosage of 0.1-10mg/kg; Under the dosage of 0.1-1mg/kg, HBN does not have obvious influence to mice serum IgG; Effect to mice serum IgG inhibition appears when heavy dose of (10mg/kg); Under 1.0mg/kg dosage, it has obvious promoter action (P＜0.01) to T, bone-marrow-derived lymphocyte proliferative response.
According to the inside and outside result of above-mentioned pharmacological testing, show that HBN can be used as immunoregulation druge and uses.
Description of drawings
Fig. 1 is an Asiatic polysaccharide separation and purification synoptic diagram.
Embodiment
Embodiment:
Dried Herba Centellae herb is pulverized product after 95% medicinal alcohol soaked for 2 weeks, filters, and the apparatus,Soxhlet's alcohol reflux is used in the residue continuation, the elimination mother liquor, and residue dries in the shade.Dry product after the degreasing (6 kilograms) is used boiling water extraction, adds 30 liters in water at every turn, and extraction time is 5-6 hour, and is not obvious until the sugar reaction with the sugared content of phenol sulfuric acid process Detection and Extraction liquid, extracts altogether 4 times.The water that at every turn extracts the filtration gained is proposed the mother liquor merging, be concentrated into small volume, use glass dialyzing paper flowing water dialysis 3 days, continue solution concentration to small volume, with 15% Tricholroacetic Acid stirring deproteinated under 4 ℃.Solution is through the centrifugal precipitation of going, and clear liquid is neutralized to neutrality with the 1mol/l sodium hydroxide solution, again to flowing water dialysis 3 days.Solution concentration is to proper volume (about 1/3-1/5) in the bag, and 4 times of volumes to sugar soln of ethanol of adding 95% spend the night in 4 ℃ of precipitations.Centrifugal collecting precipitation washs after three times the solid (JXCl, productive rate 2.1%) that dehydration in the low-temperature vacuum drying case at 40 ℃ obtains the 360g taupe successively through ethanol and anhydrous propanone.
Get 6g water and carry Crude polysaccharides JXC1, add an amount of dissolved in distilled water, centrifugal, supernatant liquor carries out initial gross separation with DEAE-Mierocrystalline cellulose (chlorine type) column chromatography.At first with distilled water as elutriant, use 0.1,0.3,0.5 and the NaCl eluant solution of 1.0mol/l then, collect each stream part respectively, wherein from the washing elution fraction, obtain polysaccharide fraction JXCH.
JXCH uses DEAE-Mierocrystalline cellulose (acetic acid type) column chromatography again, behind the distilled water wash-out, and 0.2mol/l NaCl wash-out, automatic collection instrument is collected flow point, and sugar component is collected in the sulfuric acid-phynol method colour developing.This polysaccharide fraction of concentrating under reduced pressure, dialysis and lyophilize, further use DEAE-Mierocrystalline cellulose (acetic acid type) column chromatography again, elder generation is with the sodium acetate solution gradient elution of 0-0.05mol/l, use 0.2mol/l sodium chloride solution wash-out again, collect the polysaccharide soln of this component, behind the small molecules such as removing inorganic salt of dialysing, concentrating under reduced pressure, lyophilize, the component that obtains obtains the main sugar component of wash-out through Sephadex G-200 column chromatography, and this component obtains polysaccharide HBN (400mg) through the concentrating under reduced pressure lyophilize.
Physico-chemical property is measured: according to the ordinary method of polysaccharide, HPGPC method determining molecular weight is 5.4 * 10
5Da, specific optical rotation [α]
D=-10.4 ° (c 0.70, water).
Determination of chemical structure: according to the ordinary method of polysaccharide, comprise spectrographic technique (
13C NMR, IR) and chemical process (all-hydrolytic, methylation reaction, sodium periodate oxidation-Smith degraded, part acid hydrolysis and enzymolysis) prove that HBN is an arabogalactan of being made up of rhamnosyl, pectinose, semi-lactosi, galacturonic acid and wood sugar.Rhamnosyl and galacturonic acid have constituted the core of HBN polysaccharide among the HBN, belong to the RG-I configuration.The primary structure that connects HBN in the rhamnosyl O-4 position of this core chain, i.e. the wood sugar chain of arabogalactan zone and short chain.In arabogalactan chain zone, 1, the semi-lactosi that 6-connects is occupied an leading position, and promptly with 1, the semi-lactosi that 6-connects forms long-chain, connects other semi-lactosi side chain and pectinose side chain in its O-3 position.Simultaneously also there is part 1 in arabogalactan chain zone, the long-chain that the semi-lactosi that 3-connects constitutes, and connect Arabic sugar chain and gala sugar chain at O-6.
Pharmacological testing: get above-mentioned HBN and carry out the vivo and vitro immunity test according to the pharmacological testing method of mentioning in the test respectively.The result shows in the proliferation test to T, bone-marrow-derived lymphocyte that concentration is that the in vitro tests of 1,10,100 μ g/ml can reach significantly and highly significant.When dosage reached 1mg/kg in the body, (ConA, LPS) inducing mouse splenic T, bone-marrow-derived lymphocyte proliferative response had obvious facilitation to HBN to mitogen.
Claims (3)
1. polysaccharide HBN who is extracted by Herba Centellae, its structure is that molecular weight is 5.4 * 10
5Da, specific optical rotation [α]
D=-10.4 ° (c=0.70, water); Each residue mol ratio of HBN is a pectinose: semi-lactosi: rhamnosyl: galacturonic acid: wood sugar=1.00: 1.90: 0.26: 0.30: 0.15;
13In the C NMR spectrum, the signal that is positioned at δ 110.4 and 108.5ppm is the anomeric carbon signal of α-Araf and β-Xylp; The signal that is positioned at the different head region of δ 104.6～104.4ppm is β-Galp signal; The weak signal of δ 102.0 and 100.6ppm is the C1 signal of α-Rhap and α-GalpA; About 76%Araf residue is in end, and therefore main Araf signal is to be produced by t-Araf;
1In the H NMR spectrogram, signal δ 5.2 and 4.4-4.5ppm are corresponding to the anomer hydrogen signal of Araf and β-Galp; The anomer hydrogen signal of α-Rhap, α-GalpA and β-Xylp is positioned at δ 5.1,4.9 and 4.7ppm place; The above results is measured the result that the polysaccharide structures common chemical is reacted in conjunction with methylation reaction and sodium periodate oxidation, part acid hydrolysis and enzyme liberating, proves that HBN is an arabogalactan of being made up of rhamnosyl, pectinose, semi-lactosi, galacturonic acid and wood sugar; Wherein rhamnosyl and galacturonic acid residue have constituted the core of HBN polysaccharide, belong to pectin RG-I configuration; The primary structure that connects HBN in the rhamnosyl O-4 position of this core chain, i.e. the wood sugar chain of arabogalactan zone and short chain; In arabogalactan chain zone, 1, the semi-lactosi that 6-connects is occupied an leading position, and promptly with 1, the semi-lactosi that 6-connects forms long-chain, connects other semi-lactosi side chain and pectinose side chain in its O-3 position; Simultaneously also there is part 1 in arabogalactan chain zone, the long-chain that the semi-lactosi that 3-connects constitutes, and connect Arabic sugar chain and gala sugar chain at O-6.
2. the preparation method of the polysaccharide HBN that is extracted by Herba Centellae as claimed in claim 1 is made up of following steps:
After a, dried Herba Centellae herb crushed material soaked for 2 weeks with 95% medicinal alcohol, filtration, residue apparatus,Soxhlet's extraction using alcohol, the elimination mother liquor, residue dries in the shade;
B, dry product boiling water extraction after above-mentioned degreasing 3-5 time, extracting liquid filtering, united extraction liquid is evaporated to small volume, with flowing water was dialysed 3-4 days, continuation is arrived small volume with solution concentration, stir deproteinated with 10%～20% Tricholroacetic Acid at 4 ℃, solution centrifugal goes precipitation, supernatant liquor is neutralized to neutrality with 1mol/l NaOH solution, uses flowing water dialysis 3 days again, and solution concentration is to the 1/3-1/5 volume in the bag, add 3～5 times to 95% ethanol of sugar soln volume, in being set to 4 ℃ refrigerator, place and spend the night, centrifugal collecting precipitation, in 40 ℃ low-temperature vacuum drying case, dewater Crude polysaccharides JXCl;
C, adding dissolved in distilled water Crude polysaccharides, the centrifugal insolubles of removing, supernatant liquor separates by the DEAE-cellulose chromatography, with distilled water as elutriant, then with containing 0.1,0.3 with every liter successively, 0.5 and 1 molar sodium chloride solution wash-out, collect respectively and merge each flow point, wherein wash the part component through concentrate, dialysis and lyophilize get secondary polysaccharide component JXCH;
D, above-mentioned JXCH are by the DEAE-cellulose chromatography, behind the distilled water wash-out, with 0.2 mol/liter sodium chloride solution wash-out, automatic collection instrument is collected flow point, sulfuric acid-phynol method detects the polysaccharide component, collect sugared reacting positive part concentrating under reduced pressure, dialysis and this polysaccharide component of lyophilize, use the DEAE-fibre column chromatography earlier with 0-0.05 mol/liter sodium chloride solution gradient elution again, use 0.2 mol/liter NaCl eluant solution again, collect this component polysaccharide soln, after the inorganic salt small molecules is removed in dialysis, concentrating under reduced pressure, lyophilize gets the polysaccharide component;
E, above-mentioned polysaccharide component are through S ephadex G 200 column chromatographies 0.2 mol/liter sodium chloride solution wash-out, and the polysaccharide soln of collecting this component concentrates, and lyophilize gets polysaccharide HBN.
3. the application of polysaccharide HBN in the preparation immunoregulation druge of extracting as claimed in claim 1 by Herba Centellae.
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| CN110317279B (en) * | 2019-08-19 | 2021-07-27 | 烟台新时代健康产业日化有限公司 | Herba Centellae polysaccharide, application of herba Centellae polysaccharide as plant source antiseptic, and application of herba Centellae polysaccharide in cosmetics |
| CN113456796B (en) * | 2020-03-31 | 2024-04-12 | 无限极（中国）有限公司 | Anti-aging composition and preparation method and application thereof |
| CN112656707A (en) * | 2020-12-29 | 2021-04-16 | 中华全国供销合作总社南京野生植物综合利用研究所 | Extraction method of centella asiatica polysaccharide extract, centella asiatica polysaccharide extract and application thereof |
| CN113527532B (en) * | 2021-08-20 | 2022-08-05 | 山东农业大学 | Long-chain algal polysaccharide and separation and purification method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1194154A (en) * | 1997-03-24 | 1998-09-30 | 东国制药株式会社 | Asiaticoside of self asiatic and hydroxy-asiaticoside and carboxy-asiaticoside water soluble extract and its separating method |
| JP2000080044A (en) * | 1998-09-02 | 2000-03-21 | Ranka Aayurubeedick Haabu Yakuhin Kk | Aldose reductase inhibitor, new saponin and new sapogenol |
| JP2000109498A (en) * | 1998-10-05 | 2000-04-18 | Kiyoteru Tobinaga | Iron citrate absorption promoter |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1194154A (en) * | 1997-03-24 | 1998-09-30 | 东国制药株式会社 | Asiaticoside of self asiatic and hydroxy-asiaticoside and carboxy-asiaticoside water soluble extract and its separating method |
| JP2000080044A (en) * | 1998-09-02 | 2000-03-21 | Ranka Aayurubeedick Haabu Yakuhin Kk | Aldose reductase inhibitor, new saponin and new sapogenol |
| JP2000109498A (en) * | 1998-10-05 | 2000-04-18 | Kiyoteru Tobinaga | Iron citrate absorption promoter |
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