CN102349924A - Centella asiatica triterpenic acid single-glucopyranoside composition, its preparation method, its quantitative analysis method and its application - Google Patents
Centella asiatica triterpenic acid single-glucopyranoside composition, its preparation method, its quantitative analysis method and its application Download PDFInfo
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- CN102349924A CN102349924A CN201110234306XA CN201110234306A CN102349924A CN 102349924 A CN102349924 A CN 102349924A CN 201110234306X A CN201110234306X A CN 201110234306XA CN 201110234306 A CN201110234306 A CN 201110234306A CN 102349924 A CN102349924 A CN 102349924A
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- glucoside
- beta
- glucosidase
- herba centellae
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Abstract
The invention relates to a centella asiatica triterpenic acid single-glucopyranoside composition which is composed of ursane madecassic acid single-glucopyranoside, madecassic acid single-glucopyranoside and oleanane chebuloside II, wherein the mass ratio of ursane madecassic acid single-glucopyranoside to madecassic acid single-glucopyranoside to oleanane chebuloside II is 1:0.5-2:0.1-1, and the sum of the mass percentage content of three components is not less than 50%. The composition takes a centella asiatica extract as a substrate, the centella asiatica extract is fermented and hydrolyzed by microbes of beta-glucosidase or microbes capable of generating beta-glucosidase, and extracted by n-butanol or separated and purified by macroporous adsorption resin. The invention also provides a quantitative analysis method which is a HPLC quantitative analysis for three components by adding a proper amount of mobile phase of beta-cyclodextrin. Experimental research of pharmacodynamics proves that the composition has substantial activity for inhibiting tumor cells and fibroblast, the composition can be used for treating tumor and scar hyperplasia.
Description
Technical field
The invention belongs to the field of Chinese medicines, relate to the single glucoside compositions of a kind of asiatic centella triterpenoid acid.In addition, the present invention also provides a kind of biotransformation Herba Centellae extract to prepare the quantitative analysis method and the purposes of asiatic centella triterpenoid single glucoside preparation of compositions method of acid and compositions thereof.
Background technology
Herba Centellae (Centella asiatica (L.) Urb) is claimed Herba sambuci chinensis again, collapses jorum, Herba Christiae Obcordatae, is Umbelliferae Centella plant, and its beginning is stated from Shennong's Herbal, classifies middle article as.It is cold in nature, and bitter in the mouth, suffering have the effect of clearing away heat-damp and promoting diuresis, removing toxic substances and promoting subsidence of swelling, are mainly used in jaundice due to damp-heat, heatstroke diarrhoea, sand Stranguria stranguria with blood, carbuncle sore tumefacting virus, traumatic injury.Herba Centellae is area, a Guangdong among the people Chinese herbal medicine commonly used, utilizes Herba Centellae external and for oral administration curing the disease in existing more than the 2000 year history of China.Modern study shows; Herba Centellae has the effect of anti-inflammation, antidepressant, antitumor, promotion wound healing and treatment hypertrophic cicatrix; Herba Centellae extract has tangible curative effect to fibrotic diseases such as scleroderma and burns scars, can promote wound healing effectively.Thereby Herba Centellae promotes the generation of VEGF to promote the MCP-1 macrophage to generate IL-1 β simultaneously through the expression that increases MCP-1 in the keratinocyte, improves the generation of burn site blood vessel and reaches the purpose of treatment burn.
Herba Centellae is rich in ursane type and two types of pentacyclic triterpene saponins of oleanane type, and wherein main component is asiaticoside (asiaticoside), asiaticoside (madecassoside) and asiaticoside B (asiaticoside B).The structural formula of the pentacyclic triterpene composition that has been separated at present is following:
The A ursane type
1. asiatic acid (asiatic acid): R
1=H, R
2=H
2. Madecassic acid (6 β-hydroxyasiatic acid): R
1=OH, R
2=H
3. asiaticoside (asiaticoside): R
1=H, R
2=Trisaccharide unit
4. asiaticoside (madecassoside): R
1=OH, R
2=Trisaccharide unit
5. Herba Centellae bioside (asiaticodiglycoside): R
1=H,
6. hydroxyl Herba Centellae bioside (centellasaponin B): R
1=OH,
7. asiatic acid list glucoside (asiatic acid-28-O-β-D glucopyranoside): R
1=H, R
2=β-D-glucopyranosyl
8. Madecassic acid list glucoside (centelloside C): R
1=OH, R
2=β-D-glucopyranosyl
The B oleanane type
1.Terminolic?acid：R
1＝OH，R
2＝H
2. asiaticoside B (asiaticoside B): R
1=OH, R
2=Trisaccharide unit
3.scheffoleoside?A：R
1＝H，R
2＝Trisaccharide?unit
4.centellasaponin?B：R
1＝OH，?
5. cut cloth glycosides II (chebuloside II): R
1=OH, R
2=glucopyranosyl
Also do not see the report that the asiatic centella triterpenoid single glucoside compositions of acid and Study on Preparation, quality standard research and purposes are arranged up to now.
Summary of the invention
The present invention is intended to adopt modern pharmaceutical technology, and a kind of have antitumaous effect and the single glucoside compositions of asiatic centella triterpenoid acid that can suppress the fibroblasts from hypertrophic scars vigor are provided.
In the said composition; Asiatic acid list glucoside, Madecassic acid list glucoside, the mass ratio of cutting cloth glycosides II are 1: 0.5～2: 0.1～1; Three's mass percent sum in compositions is not less than 50%; Preferred asiatic acid list glucoside, Madecassic acid list glucoside, the mass ratio of cutting cloth glycosides II are 1: 0.7～1.5: 0.3～0.8, and three's mass percent sum in compositions is not less than 80%.
Another object of the present invention is to provide a kind of asiatic centella triterpenoid acid single glucoside preparation of compositions method.
The single glucoside preparation of compositions method of above-mentioned asiatic centella triterpenoid acid; With the Herba Centellae extract is raw material; The microorganism that maybe can produce beta-glucosidase with beta-glucosidase comes biotransformation to prepare the single glucoside compositions of asiatic centella triterpenoid acid; Its reaction principle is (1 → 6)-β-D-glucoside bond between beta-glucosidase enzyme hydrolysis ursane type and the acid two of oleanane type asiatic centella triterpenoid and/or 28 last two glucoses of tri-glucose glycosides; Generate Herba Centellae triterpenic acid list glucoside, reaction equation is following:
R is 2 α, 3 β, 23 α-trihydroxy Usu-12-alkene-28-acidic group, or 2 α, 3 β, 6 β; 23 α-tetrahydroxy Usu-12-alkene-28-acidic group, or 2 α, 3 β, 6 β-trihydroxy Usu-12-alkene-28-acidic group, or 2 α, 3 β; 23 α-trihydroxy olive-12-alkene-28-acidic group, or 2 α, 3 β, 6 β, 23 α-tetrahydroxy olive-12-alkene-28-acidic group, or 3 β; 6 β, 23 α-trihydroxy olive-12-alkene-28-acidic group, or 2 α, 3 β, 23 α-trihydroxy olive-13-alkene-28-acidic group.
Compositions of the present invention adopts bioconversion method to extract, and specifically may further comprise the steps:
(1) in the aqueous solution of Herba Centellae extract, adds the beta-glucosidase of Herba Centellae extract weight 0.1～50% or immobilized beta-glucosidase, 20～60 ℃ of hydrolysis 0.5～72 hour; Perhaps add microbiological culture media, sterilization treatment, inoculation can produce the microorganism of β glucosidase, and 20～45 ℃ fermented 2～8 days; The mass concentration of Herba Centellae extract is 0.1～20% in the reaction system;
(2) filtration or centrifugalize obtain containing the fermentation liquid or the enzymolysis solution of the single glucoside compositions of asiatic centella triterpenoid acid;
(3) enzymolysis solution or the zymolysis liquid with the single glucoside compositions of contained asiatic centella triterpenoid acid carries out separation and purification.
Above-mentioned beta-glucosidase derives from plant or microorganism, and ability hydrolysis β-1,2,1,3,1,4 and 1,6 glycosidic bond like emulsin, preferably derive from the beta-glucosidase of main hydrolytic cleavage β-1,6 glycosidic bond of fungus.
The above-mentioned microorganism that can produce the β glucosidase is selected from Penicillium fungus, aspergillus fungi, fungus Trichoderma, Saccharomyces fungus, bacillus or genus lactubacillus antibacterial; Like Paecilomyces varioti, penicillium expansum, Aspergillus citrimum, aspergillus oryzae, Aspergillus glaucus, aspergillus niger, Li Shi Trichoderma spp., koning trichoderma, Trichoderma viride, saccharomyces cerevisiae, bakery yeast, many viscosity bacillus cereus or lactobacillus; Penicillium funguses such as the Paecilomyces varioti of the main generation of preferred use β-1,6 glucosidase, penicillium expansum, Aspergillus citrimum.
In the said method, described Herba Centellae extract is selected from Herba Centellae total glycosides, Herba Centellae alcohol extract, Herba Centellae water extract or Herba Centellae alcohol water mixed solution extract.
In the said method, the enzymolysis solution of the single glucoside compositions of asiatic centella triterpenoid acid or fermentation liquid are carried out separation and purification specifically can adopt following method:
N-butanol extraction:
(1) with enzymolysis solution or fermentation liquid is concentrated into volume and the Herba Centellae extract part by weight is 1～5L/kg, divide 1～3 extraction with 1～10 times of volume water-saturated n-butanol, merge gained n-butyl alcohol phase, promptly get the butanol extraction liquid of compositions;
(2) with the butanol extraction liquid concentrating under reduced pressure, vacuum drying is pulverized, and promptly gets compositions;
Perhaps, macroporous adsorbent resin method:
(1) with macroporous adsorptive resins absorption on enzymolysis solution or the fermentation liquid, impurity is removed in 3-5 times of bed volume washing, 2-5 times of bed volume 20-80% volumetric concentration ethanol or methanol aqueous solution eluting, alcohol eluen;
(2) with the alcohol eluen concentrating under reduced pressure, vacuum drying is pulverized, and promptly gets compositions.
In the above-mentioned macroporous adsorbent resin method, before macroporous adsorptive resins absorption on enzymolysis solution or the fermentation liquid, a pre-treatment step can be arranged: it is 1-30% that enzymolysis solution or fermentation liquid are evaporated to the solid content weight percentage; Or to be evaporated to the solid content weight percentage be 30-60%, adds 3-4 times of volume 95% ethanol precipitation, filters or the centrifugalize deposition, and supernatant concentration is removed ethanol, and thin up to solid content weight percentage is 1-30%.
Compositions of the present invention proves to have and suppress tumor cell and fibroblastic active function significantly through pharmacodynamic study, can be used for the treatment of tumor and scar hyperplasia.Said composition can be used with pharmaceutically acceptable adjuvant, processes oral formulations such as capsule, tablet, granule, powder and oral liquid; Injections such as liquid drugs injection, powder pin or infusion solutions; Or products such as preparation for external application to skin such as gel, ointment, Emulsion or spirit.
Suppress the experimentation that human hepatoma cell strain is grown with compositions of the present invention, experimental result shows a and shows: when reaching finite concentration, the single glucoside solution of asiatic centella triterpenoid acid has remarkable inhibitory action to the HepG2 cell proliferation.
Suppress the experiment that the fibroblasts from hypertrophic scars strain is grown with compositions of the present invention, experimental result shows: when reaching finite concentration, the single glucoside solution of asiatic centella triterpenoid acid has remarkable inhibitory action to the fibroblasts from hypertrophic scars vigor.
In addition; The present invention also provides the quantitative analysis method of a kind of asiatic centella triterpenoid single glucoside compositions of acid and formulation products thereof; Adopt HPLC to carry out the quantitative analysis of asiatic acid list glucoside, Madecassic acid list glucoside and three kinds of components of Qie Bu glycosides II, mobile phase wherein by beta-schardinger dextrin-, water, methanol or/and acetonitrile form.Mobile phase preferably contains 0.1～1mmol/L beta-schardinger dextrin-, and percent by volume is that 15～50% methanol are or/and the aqueous solution of acetonitrile.
Description of drawings
Fig. 1 is the single glucoside HPLC collection of illustrative plates of embodiment 4 gained asiatic centella triterpenoids acid.
Fig. 2 is for cutting cloth glycosides II reference substance HPLC collection of illustrative plates.
Fig. 3 is a Madecassic acid list glucoside reference substance HPLC collection of illustrative plates.
Fig. 4 is an asiatic acid list glucoside reference substance HPLC collection of illustrative plates.
The specific embodiment
Below in conjunction with specific embodiment, further set forth the present invention.These embodiment are interpreted as only being used to the present invention is described and are not used in restriction protection scope of the present invention.After the content of having read the present invention's record, those skilled in the art can do various changes or modification to the present invention, and these equivalences change and modify and fall into claim of the present invention institute restricted portion equally.
(1) takes by weighing 1kg Herba Centellae water extract, add water to 5kg;
(2) add the aspergillus niger beta-glucosidase of 1g, 40 ℃ of hydrolysis 72 hours;
(3) the isolated by filtration enzymolysis solution is evaporated to 2L, divides 1 extraction with the 6L water-saturated n-butanol, gets butanol extraction liquid; With butanol extraction liquid concentrating under reduced pressure, vacuum drying, promptly get the single glucoside compositions of asiatic centella triterpenoid acid.
Employing contains the 0.1mmol/L beta-schardinger dextrin-; And percent by volume is that the aqueous solution of 15% acetonitrile carries out HPLC mensuration as mobile phase; The quality percentage composition of cutting cloth glycosides II, Madecassic acid list glucoside, asiatic acid list glucoside in the result combinations thing is respectively 14.8%, 23.5%, 26.4%, and three's quality percentage composition sum is 64.7%.
(1) takes by weighing 1kg Herba Centellae alcohol extract, add water to 40Lkg;
(2) add the 50g emulsin, 20 ℃ of hydrolysis 24 hours;
(3) the centrifugalize enzymolysis solution is evaporated to 4L, divides 3 extractions with the 4L water-saturated n-butanol, gets butanol extraction liquid; With butanol extraction liquid concentrating under reduced pressure, vacuum drying, promptly get the single glucoside compositions of asiatic centella triterpenoid acid.
Employing contains the 1.0mmol/L beta-schardinger dextrin-; And percent by volume is that the aqueous solution of 50% acetonitrile carries out HPLC mensuration as mobile phase; The quality percentage composition of cutting cloth glycosides II, Madecassic acid list glucoside, asiatic acid list glucoside in the result combinations thing is respectively 18.2%, 28.1%, 27.4%, and three's quality percentage composition sum is 73.7%.
(1) take by weighing the 1kg asiatic centella total saponins, add water to 1000kg,
(2) add the penicillium expansum beta-glucosidase of 0.5kg, 60 ℃ of hydrolysis 0.5 hour,
(3) isolated by filtration enzymolysis solution, the macroporous resin column absorption that last 10L model is D101, impurity is removed in washing, with 40L 60% volumetric concentration alcoholic solution eluting, gets ethanol elution.With ethanol elution concentrating under reduced pressure, vacuum drying, promptly get the single glucoside compositions of asiatic centella triterpenoid acid.
Employing contains the 0.4mmol/L beta-schardinger dextrin-; And percent by volume is that 30% methanol in water is carried out HPLC mensuration as mobile phase; The quality percentage composition of cutting cloth glycosides II, Madecassic acid list glucoside, asiatic acid list glucoside in the result combinations thing is respectively 22.0%, 31.5%, 29.7%, and three's quality percentage composition sum is 83.2%.
(1) take by weighing 1kg Herba Centellae alcohol water extract, add 10kg glucose, 10kg starch, 10kg soybean cake powder, add water to 1000kg, sterilized 30 minutes for 121 ℃,
(2) insert the Li Shi Trichoderma spp., 20 ℃ fermented 8 days,
(3) isolated by filtration fermentation liquid is evaporated to about 5L, and last 10L model is the absorption of HPD100 type macroporous absorption post; Impurity is removed in washing, with 20L 80% volumetric concentration alcoholic solution eluting, collects eluent; Concentrating under reduced pressure, vacuum drying promptly gets the single glucoside compositions of asiatic centella triterpenoid acid.Employing contains the 0.4mmol/L beta-schardinger dextrin-; And percent by volume is that the aqueous solution of 28% acetonitrile carries out HPLC mensuration as mobile phase; Shown in Figure 1 is the HPLC collection of illustrative plates of present embodiment, according to the contrast of Fig. 2, Fig. 3 and Fig. 4, peak 1 is for cutting cloth glycosides II; Peak 2 is a Madecassic acid list glucoside; Peak 3 is an asiatic acid list glucoside, and the result shows that the quality percentage composition of cutting cloth glycosides II, Madecassic acid list glucoside, asiatic acid list glucoside in the compositions is respectively 21.0%, 32.7%, 27.3%, and three's quality percentage composition sum is 81.0%.
Embodiment 5
(1) takes by weighing the 1kg asiatic centella total saponins, add 1kg glucose, 1kg starch, 0.5kg soybean cake powder, add water to 50kg, sterilized 30 minutes for 121 ℃;
(2) insert bakery yeast, 28 ℃ fermented 5 days;
(3) centrifugalize fermentation liquid, carry out pretreatment: be evaporated to about 3L, add the 10L95% ethanol precipitation, filter or centrifugalize, supernatant concentration is removed ethanol, and thin up is to 10L;
(4) going up the 10L model after pretreatment is accomplished is the absorption of HPD700 type macroporous absorption post, and impurity is removed in washing, with 50L 20% volumetric concentration alcoholic solution eluting, collects eluent, and concentrating under reduced pressure, vacuum drying promptly get the single glucoside compositions of asiatic centella triterpenoid acid.
Employing contains the 0.5mmol/L beta-schardinger dextrin-; And percent by volume is that the aqueous solution of 10% methanol and 20% acetonitrile carries out HPLC mensuration as mobile phase; The quality percentage composition of cutting cloth glycosides II, Madecassic acid list glucoside, asiatic acid list glucoside in the result combinations thing is respectively 22.2%, 33.8%, 29.5%, and three's quality percentage composition sum is 85.5%.
(1) takes by weighing the 1kg asiatic centella total saponins, add 50g glucose, 50g yeast extract, Carnis Bovis seu Bubali cream 50g, soybean cake powder 100g, add water to 5kg, sterilized 15 minutes for 121 ℃;
(2) insert lactobacillus, 45 ℃ fermented 2 days;
(3) centrifugalize fermentation liquid is evaporated to about 1L, divides 2 extractions with the 2L water-saturated n-butanol, gets butanol extraction liquid; With butanol extraction liquid concentrating under reduced pressure, vacuum drying, promptly get the single glucoside compositions of asiatic centella triterpenoid acid.
Employing contains the 0.4mmol/L beta-schardinger dextrin-; And percent by volume is that the aqueous solution of 28% acetonitrile carries out HPLC mensuration as mobile phase; The quality percentage composition of cutting cloth glycosides II, Madecassic acid list glucoside, asiatic acid list glucoside in the result combinations thing is respectively 21.6%, 31.1%, 30.1%, and three's quality percentage composition sum is 82.7%.
The preparation of the single glucoside tablet of embodiment 7 asiatic centella triterpenoids acid
Take by weighing the single glucoside 1kg of asiatic centella triterpenoid acid, starch 0.3kg, microcrystalline Cellulose 0.2kg that embodiment 4 prepares, mixing, with 10% starch slurry wet granulation, drying adds magnesium stearate 8g, total mixing, tabletting promptly gets the single glucoside sheet of asiatic centella triterpenoid acid.
The preparation of the single glucoside aqueous injection of embodiment 8 asiatic centella triterpenoids acid
Take by weighing the pure article 1kg of the asiatic centella triterpenoid single glucoside of acid that embodiment 1 prepares, water for injection adds to 10L, and stirring and dissolving is filtered, sterilization, and packing promptly gets the single glucoside aqueous injection of asiatic centella triterpenoid acid.
The preparation of the single glucoside ointment of embodiment 9 asiatic centella triterpenoids acid
Get stearic acid 15kg, vaseline 10kg, glyceryl monostearate 3kg, heat and stir, be oil phase; Other gets the single glucoside 1kg of asiatic centella triterpenoid acid, sodium hydroxide 0.5kg, glycerol 10kg, ethyl hydroxybenzoate 0.1kg, distilled water 60.4kg that embodiment 5 prepares, and stirring and dissolving is water.Water is heated to 50 ℃, slowly pours oil phase into aqueous phase, and the limit bevelling stirs, and stirs, and promptly gets the single glucoside ointment of asiatic centella triterpenoid acid.
The preparation of the single glucoside gel of embodiment 10 asiatic centella triterpenoids acid
Get carbomer-940 1kg, add distilled water immersion, stirring makes it be swelled into even pasty state, adds ethyl hydroxybenzoate 25g more respectively, and tween 80 0.5kg drips triethanolamine and regulates pH to 6.0～8.0 (subsequent use as blank substrate); Other gets the single glucoside 1kg of asiatic centella triterpenoid acid that embodiment 6 prepares, and adds PEG-600 2.5kg, glycerol 2.5kg, stirs its dissolving back is added in the blank substrate, adds water to 50L, stirs, and promptly gets.
The single glucoside compositions of embodiment 11 asiatic centella triterpenoids acid suppresses the experimentation of human hepatoma cell strain growth
1 material and instrument
1.1 experiment material
HepG2 (human hepatoma cell strain) is provided by Fudan University; The single glucoside of asiatic centella triterpenoid acid (press the preparation of embodiment 3 methods, purity is greater than 80%); MEM culture medium (Gibco Company products, the U.S.); Calf serum (Hangzhou Ilex purpurea Hassk.[I.chinensis Sims bio-engineering corporation product); MTT (Amresco Company products, the U.S.); All the other not special dated reagent are import or homemade analytical pure.
1.2 key instrument
Spectra Max 190 ELIASAs (Molec-ular device, the U.S.); Cell culture incubator (SHELLAB, the U.S.).
2 experimental techniques
2.1 cell culture
The HepG2 cell is with the MEM culture medium culturing that contains 10% calf serum.Cell inoculation in culture bottle, is placed 37 ℃, 5%CO
2, cultivating in the cell culture incubator of saturated humidity, 2～3d goes down to posterity 1 time.
2.2MTT method is surveyed cell survival rate
4 * 10
4Ml
-1Cell 100 μ l are inoculated in the 96 porocyte culture plates and cultivate 24h, add the single glucoside of variable concentrations asiatic centella triterpenoid acid and handle 24h, discard culture medium; Every hole adds 100 μ l MTT (D-hank ' S liquid dissolving, concentration is 1g/L), 37 ℃ continue to hatch 4h after; Culture fluid is removed in suction; Every hole adds DMSO 100 μ l, and thing to be crystallized fully dissolves, behind the 1h with ELIASA in 570nm place photometry density.The result representes that with suppression ratio computing formula is following: suppression ratio=(the average D570 of the average D570-administration of matched group group)/(the average D570 of the average D570-blank control group of matched group) * 100%
3 results
3.1 Madecassic acid list glucoside suppresses tumor cell proliferation
The HepG2 cell is respectively 10,20 through final concentration, and after the single glucoside solution-treated of asiatic centella triterpenoid acid of 30,40 μ mol/L, the cell growth is suppressed, and cellular morphology changes, and disappears like projection, and the cell space shrinkage becomes circle etc.Compare with matched group, the low concentration asiatic centella triterpenoid single glucoside of acid (20 μ mol/L) group does not obviously influence the growth of HepG2 cell; When dosage reached 40 μ mol/L, the single glucoside of asiatic centella triterpenoid acid had remarkable inhibitory action to the HepG2 cell proliferation, and suppression ratio reaches 81%, and the Ic50 value of the asiatic centella triterpenoid single glucoside of acid on the HepG2 cell strain is 26 μ mol/L thus.
The single glucoside compositions of table 1 hydroxyl asiatic centella triterpenoid acid is to the inhibitory action (suppression ratio: %) of human hepatoma cell strain (HepG2) and cervical cancer cell strain (Hela)
| Dosage (μ g/ml) | 10 | 20 | 40 | 80 | 100 | IC 50 |
| Hela | -7.17±4.45 | 1.33±8.41 | 13.07±19.35 | 32.26±2.46 | 33.15±5.15 | 142 |
| HepG2 | -5.58±9.28 | -4.21±12.49 | 35.89±11.46 | 70.13±1.37 | 71.85±0.59 | 58 |
The single glucoside compositions of embodiment 12 asiatic centella triterpenoids acid suppresses the experimentation of fibroblasts from hypertrophic scars strain growth
1 material and instrument
1.1 material
The have drawn from scar tissue of keloid patient skin of fibroblasts from hypertrophic scars; The single glucoside of asiatic centella triterpenoid acid (press the preparation of embodiment 3 methods, purity is greater than 80%); DMEM culture medium (Hyclone company); Tetramethyl azo azoles salt (MTT) (Sigma company).
1.2 instrument
CO
2Cell culture incubator (German Heraeus); ELIASA (Finland Wellscan MK3 type); Inverted microscope (German MPS30 type).
2 methods
2.1 medicine preparation
The single glucoside solution of asiatic centella triterpenoid acid: the single glucoside of asiatic centella triterpenoid acid adds a small amount of culture fluid, fully dissolving back 0.45 μ m membrane filtration degerming, with culture fluid be diluted to 0.125,0.25,0.50,1.00,2.00mg/mL.
2.2 fibroblasts from hypertrophic scars is cultivated
Aseptic condition operation down cuts the about 0.5-1mm of cicatrix skin corium piece of tissue, is inoculated in the culture bottle that contains the DMEM culture fluid, and the 4-7 generation that is in exponential phase is chosen in experiment.
2.3 fibroblasts from hypertrophic scars morphological observation
The digestion single-layer culturing cell is with every hole l * 10
4Individual being inoculated in 96 well culture plates stays 1 hole only to add culture fluid as blank well.Treat sucking-off stock solution behind the cell attachment, set up matched group and experimental group, each sample standard deviation is established 4 multiple holes.Matched group changes liquid, and every hole adds the culture fluid 100L that contains serum, and experimental group adds each the 100 μ L of drug solution contain variable concentrations, moves into and continues in the calorstat to cultivate, and under inverted microscope, observes and makes a video recording respectively at 24h, 48h, 72h.
2.4MTT colorimetry observation fibroblasts from hypertrophic scars vigor
More than the cell of 96 orifice plates respectively at fixed 3 time periods shooting after, every hole promptly adds MTF20 μ L (blank well does not add), puts back to calorstat and continues to cultivate 4.5h.Take out culture plate, absorb the liquid in the hole, every hole adds the dimethyl sulfoxide (DMSO) of 150 μ L, and vibration 5min fully dissolves crystallization.With the blank well zeroing, on ELIASA, detecting wavelength is the light absorption value (A value) of 490nm, calculates the suppression ratio of drug effect behind the fibroblast vigor.
Suppression ratio=(blank group A
490Value-trial drug group A
490Value)/blank group A
490Value * 100%
3 results
3.1 fibroblasts from hypertrophic scars morphological change
3.1.1 blank group
More the cell that the down visible profile of mirror is long and narrow, karyon is fusiformis, trend reaches unanimity is and is arranged in parallel and certain radian is arranged.There are circle or oval nuclear in cell central authorities, and visible protruding 2-3 the projection different in size of part cell is fibroblast.Along with the increase of incubation time, cell quantity increases, and intercellular substance narrows down, and is streak distribution.
3.1.2 the single glucoside experimental group of asiatic centella triterpenoid acid
When drug level was 0.50mg/mL, cell just shortened behind the 12h, a little occurred and took off wall, buoyant cell.In this 3 time periods, along with the increase of incubation time, attached cell quantity further reduces at 24h, 48h, 72h, and it is big that intercellular substance becomes.The cell that is shaped in the culture fluid behind the 72h is rare.
3.2MTT method is measured the variation of fibroblasts from hypertrophic scars vigor
To fibroblasts from hypertrophic scars vitality test result, calculate suppression ratio according to the single glucoside of asiatic centella triterpenoid acid respectively, see table 2 fibroblasts from hypertrophic scars.This shows that the single glucoside of asiatic centella triterpenoid acid has remarkable inhibitory action to the fibroblasts from hypertrophic scars vigor.
The single glucoside of table 2 asiatic centella triterpenoid acid is to the inhibitory action (suppression ratio: %) of fibroblasts from hypertrophic scars vigor
。
Claims (13)
1. the single glucoside compositions of asiatic centella triterpenoid acid is characterized in that, said composition mainly is made up of the cloth glycosides II that cuts of asiatic acid list glucoside, Madecassic acid list glucoside and the oleanane type of ursane type.
2. the described compositions of claim 1 is characterized in that, asiatic acid list glucoside, Madecassic acid list glucoside, the mass ratio of cutting cloth glycosides II are 1: 0.5～2: 0.1～1, and three's mass percent sum in compositions is not less than 50%.
3. the described compositions of claim 2 is characterized in that, asiatic acid list glucoside, Madecassic acid list glucoside, the mass ratio of cutting cloth glycosides II are 1: 0.7～1.5: 0.3～0.8, and three's mass percent sum in compositions is not less than 80%.
4. the described preparation of compositions method of claim 1 is characterized in that, may further comprise the steps:
(1) in the aqueous solution of Herba Centellae extract, adds the beta-glucosidase of Herba Centellae extract weight 0.1～50% or immobilized beta-glucosidase, 20～60 ℃ of hydrolysis 0.5～72 hour; Perhaps add microbiological culture media, sterilization treatment, inoculation can produce the microorganism of β glucosidase, and 20～45 ℃ fermented 2～8 days; The mass concentration of Herba Centellae extract is 0.1～20% in the reaction system;
(2) filtration or centrifugalize obtain containing the fermentation liquid or the enzymolysis solution of the single glucoside compositions of asiatic centella triterpenoid acid;
(3) enzymolysis solution or the zymolysis liquid with the single glucoside compositions of contained asiatic centella triterpenoid acid carries out separation and purification, may further comprise the steps:
A, enzymolysis solution is concentrated into volume and the Herba Centellae extract part by weight is 1～5L/kg, divides 1～3 extraction, merge gained n-butyl alcohol phase, promptly get the butanol extraction liquid of compositions with 1～10 times of volume water-saturated n-butanol; Or with macroporous adsorptive resins absorption on the enzymolysis solution, impurity is removed in 3～5 times of bed volumes washings, 2～5 times of bed volume 20～80% volumetric concentration ethanol or methanol aqueous solution eluting, alcohol eluen;
B, with butanol extraction liquid or alcohol eluen concentrating under reduced pressure, vacuum drying, promptly get compositions.
5. the described preparation of compositions method of claim 4 is characterized in that, described Herba Centellae extract is selected from Herba Centellae total glycosides, Herba Centellae alcohol extract, Herba Centellae water extract or Herba Centellae alcohol water mixed solution extract.
6. the described preparation of compositions method of claim 4 is characterized in that, enzymolysis solution has a pre-treatment step before last macroporous absorption post absorption:
Being evaporated to the solid content weight percentage is 1-30%;
Or being evaporated to the solid content weight percentage is 30-60%, adds 3-4 times of volume 95% ethanol precipitation, filters or the centrifugalize deposition, and supernatant concentration is removed ethanol, and thin up to solid content weight percentage is 1-30%.
7. the described preparation of compositions method of claim 4 is characterized in that, described beta-glucosidase is the beta-glucosidase of main hydrolytic cleavage β-1,6 glycosidic bond.
8. the described preparation of compositions method of claim 4 is characterized in that, the described microorganism that can produce the β glucosidase is selected from Penicillium fungus, aspergillus fungi, fungus Trichoderma, Saccharomyces fungus, bacillus or genus lactubacillus antibacterial.
9. the described preparation of compositions method of claim 4; It is characterized in that the described microorganism that can produce the β glucosidase is selected from Paecilomyces varioti, penicillium expansum, Aspergillus citrimum, aspergillus oryzae, Aspergillus glaucus, aspergillus niger, Li Shi Trichoderma spp., koning trichoderma, Trichoderma viride, beer yeast, bakery yeast, many viscosity bacillus cereus or lactobacillus.
10. the application of the said compositions of claim 1 in preparation treating acne medicine or tumor disease therapeutic medicine.
11. the said compositions of claim 1 cooperates oral formulations, injection or the preparation for external application to skin of processing with pharmaceutically acceptable adjuvant.
12. the HPLC quantitative analysis method of the said compositions of claim 1 is characterized in that, HPLC mobile phase is by at least a composition the in beta-schardinger dextrin-, water and methanol and the acetonitrile.
13. the HPLC quantitative analysis method of the said compositions of claim 12; It is characterized in that; HPLC mobile phase is the aqueous solution that contains 0.1～1mmol/L beta-schardinger dextrin-, and to contain percent by volume be 15%～50% methanol or acetonitrile, and perhaps containing percent by volume is 15%～50% methanol and acetonitrile.
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