CN102349924B - 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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- CN102349924B CN102349924B CN201110234306.XA CN201110234306A CN102349924B CN 102349924 B CN102349924 B CN 102349924B CN 201110234306 A CN201110234306 A CN 201110234306A CN 102349924 B CN102349924 B CN 102349924B
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- glucosidase
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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 asiatic centella triterpenoid acid preparation method of single glucoside compositions and the quantitative analysis method of compositions and purposes.
Background technology
Herba Centellae (Centella asiatica (L.) Urb) claims again Herba sambuci chinensis, collapses jorum, Herba Christiae Obcordatae, is Umbelliferae Centella plant, and its beginning is loaded in Shennong's Herbal, classifies middle product as.It is cold in nature, and bitter in the mouth, pungent has effect of clearing away heat-damp and promoting diuresis, removing toxic substances and promoting subsidence of swelling, is mainly used in jaundice due to damp-heat, heatstroke diarrhoea, sand Stranguria stranguria with blood, carbuncle sore tumefacting virus, traumatic injury.Herba Centellae is the conventional Chinese herbal medicine in In Guangdong Province among the people, 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 obvious curative effect to the fibrotic disease such as scleroderma and burns scar, can effectively promote wound healing.Thereby Herba Centellae promotes the generation of VEGF by increasing the expression of MCP-1 in keratinocyte, promote MCP-1 macrophage to generate IL-1 β simultaneously, improve the generation of burn site blood vessel and reach the object for the treatment of burn.
Herba Centellae is rich in ursane type and oleanane type two class pentacyclic triterpene saponins, and wherein main component is asiaticoside (asiaticoside), asiaticoside (madecassoside) and Asaiticoside B (asiaticoside B).The structural formula of the pentacyclic triterpene composition being separated to is at present as follows:
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. asiaticodiglycoside (asiaticodiglycoside): R
1=H,
6. hydroxyl asiaticodiglycoside (centellasaponin B): R
1=OH,
7. asiatic acid-28-O-BETA-D-glucopyranoside (asiatic acid-28-O-β-D glucopyranoside): R
1=H, R
2=β-D-glucopyranosyl
8. centelloside C (centelloside C): R
1=OH, R
2=β-D-glucopyranosyl
B oleanane type
1.Terminolic acid：R
1＝OH，R
2＝H
2. Asaiticoside 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
Yet there are no up to now the report of the asiatic centella triterpenoid single glucoside compositions of acid and Study on Preparation, quality standard research and purposes.
Summary of the invention
The present invention is intended to adopt modern pharmaceutical technique, and a kind of single glucoside compositions of asiatic centella triterpenoid acid that has antitumaous effect and can suppress fibroblasts from hypertrophic scars vigor is provided.
In said composition, asiatic acid-28-O-BETA-D-glucopyranoside, centelloside C, 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%, preferably asiatic acid-28-O-BETA-D-glucopyranoside, centelloside C, 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 the preparation method of the single glucoside compositions of a kind of asiatic centella triterpenoid acid.
The preparation method of the single glucoside compositions of above-mentioned asiatic centella triterpenoid acid, taking Herba Centellae extract as raw material, carry out biotransformation with the microorganism that beta-glucosidase maybe can produce beta-glucosidase and prepare the single glucoside compositions of asiatic centella triterpenoid acid, its reaction principle is (1 → 6)-β-D-Glucose glycosidic bond between beta-glucosidase enzyme hydrolysis ursane type and the acid two of oleanane type asiatic centella triterpenoid and/or 28 upper two glucoses of tri-glucose glycosides, generate Herba Centellae triterpenic acid list glucoside, reaction equation is as follows:
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 comprises the following steps:
(1) in the aqueous solution of Herba Centellae extract, add the beta-glucosidase of Herba Centellae extract weight 0.1～50% or immobilized beta-glucosidase, 20～60 DEG C are hydrolyzed 0.5～72 hour; Or add microbiological culture media, sterilization treatment, inoculation can produce the microorganism of β glucosidase, and 20～45 DEG C ferment 2～8 days; In reaction system, the mass concentration of Herba Centellae extract is 0.1～20%;
(2) filtration or centrifugalize obtain fermentation liquid or the enzymolysis solution containing the single glucoside compositions of asiatic centella triterpenoid acid;
(3) enzymolysis solution or the zymolysis liquid of the single glucoside compositions of contained asiatic centella triterpenoid acid are carried out to separation and purification.
Above-mentioned beta-glucosidase derives from plant or microorganism, can be hydrolyzed β-1, and 2,1,3, Isosorbide-5-Nitrae and 1,6 glycosidic bond, as emulsin, preferably derive from main hydrolytic cleavage β-1 of fungus, the beta-glucosidase of 6 glycosidic bonds.
The above-mentioned microorganism that can produce β glucosidase is selected from Penicillium fungus, aspergillus fungi, fungus Trichoderma, Saccharomyces fungus, bacillus or genus lactubacillus antibacterial, as 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, preferably use main β-1 that produces, the Penicillium funguses such as the Paecilomyces varioti of 6 glucosidase, penicillium expansum, Aspergillus citrimum.
In 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 said method, the enzymolysis solution to the single glucoside compositions of asiatic centella triterpenoid acid or fermentation liquid carry out separation and purification and specifically can adopt with the following method:
N-butanol extraction:
(1) by enzymolysis solution or fermentation liquid is concentrated into volume and 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, obtain the butanol extraction liquid of compositions;
(2), by butanol extraction liquid concentrating under reduced pressure, vacuum drying, pulverizes, and obtains compositions;
Or, Flavonoids by Macroporous Adsorption Resin:
(1) by macroporous adsorptive resins absorption on enzymolysis solution or fermentation liquid, impurity is removed in 3-5 times of bed volume washing, and 2-5 times of bed volume 20-80% volumetric concentration ethanol or methanol aqueous solution eluting, obtain alcohol eluen;
(2), by alcohol eluen concentrating under reduced pressure, vacuum drying, pulverizes, and obtains compositions.
In above-mentioned Flavonoids by Macroporous Adsorption Resin, by before macroporous adsorptive resins absorption on enzymolysis solution or fermentation liquid, can there is a pre-treatment step: it is 1-30% that enzymolysis solution or fermentation liquid are evaporated to solid content weight percentage; Or to be evaporated to solid content weight percentage be 30-60%, add 3-4 times of volume 95% ethanol precipitation, to filter or centrifugalize precipitation, supernatant concentration is removed ethanol, and being diluted with water to solid content weight percentage is 1-30%.
Compositions of the present invention proves to have inhibition tumor cell and fibroblastic active function significantly through pharmacodynamic study, can be for the treatment of tumor and scar hyperplasia.Said composition can be used in conjunction with pharmaceutically acceptable adjuvant, makes the oral formulations such as capsule, tablet, granule, powder and oral liquid; The injections such as liquid drugs injection, powder pin or infusion solutions; Or the products such as preparation for external application to skin such as gel, ointment, Emulsion or spirit.
Suppress the experimentation of human hepatoma cell strain growth by compositions of the present invention, the aobvious a of experimental result shows: in the time reaching finite concentration, the single glucoside solution of asiatic centella triterpenoid acid has remarkable inhibitory action to HepG2 cell proliferation.
Suppress the experiment of fibroblasts from hypertrophic scars strain growth by compositions of the present invention, experimental result shows: in the time reaching finite concentration, the single glucoside solution of asiatic centella triterpenoid acid has remarkable inhibitory action to 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-28-O-BETA-D-glucopyranoside, centelloside C and tri-kinds of components of Qie Bu glycosides II, mobile phase wherein by beta-schardinger dextrin-, water, methanol or/and acetonitrile form.Mobile phase is preferably containing 0.1～1mmol/L beta-schardinger dextrin-, and percent by volume is that 15～50% methanol are or/and the aqueous solution of acetonitrile.
Brief description of the drawings
Fig. 1 is the single glucoside HPLC collection of illustrative plates of embodiment 4 gained asiatic centella triterpenoid acid.
Fig. 2 is for cutting cloth glycosides II reference substance HPLC collection of illustrative plates.
Fig. 3 is centelloside C reference substance HPLC collection of illustrative plates.
Fig. 4 is asiatic acid-28-O-BETA-D-glucopyranoside reference substance HPLC collection of illustrative plates.
Detailed description of the invention
Below in conjunction with specific embodiment, further set forth the present invention.These embodiment are interpreted as being only not used in and limiting the scope of the invention for the present invention is described.After having read the content of the present invention's record, those skilled in the art can make various changes or modifications the present invention, and these equivalences change and modification falls into the scope of the claims in the present invention equally.
Embodiment 1
(1) take 1kg Herba Centellae water extract, add water to 5kg;
(2) add the aspergillus niger beta-glucosidase of 1g, 40 DEG C of hydrolysis 72 hours;
(3) isolated by filtration enzymolysis solution, is evaporated to 2L, divides 1 extraction with 6L water-saturated n-butanol, obtains butanol extraction liquid; By butanol extraction liquid concentrating under reduced pressure, vacuum drying, obtain the single glucoside compositions of asiatic centella triterpenoid acid.
Adopt containing 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, centelloside C, asiatic acid-28-O-BETA-D-glucopyranoside in result compositions is respectively 14.8%, 23.5%, 26.4%, and three's quality percentage composition sum is 64.7%.
Embodiment 2
(1) take 1kg Herba Centellae alcohol extract, add water to 40Lkg;
(2) add 50g emulsin, 20 DEG C are hydrolyzed 24 hours;
(3) centrifugalize enzymolysis solution, is evaporated to 4L, divides 3 extractions with 4L water-saturated n-butanol, obtains butanol extraction liquid; By butanol extraction liquid concentrating under reduced pressure, vacuum drying, obtain the single glucoside compositions of asiatic centella triterpenoid acid.
Adopt containing 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, centelloside C, asiatic acid-28-O-BETA-D-glucopyranoside in result compositions is respectively 18.2%, 28.1%, 27.4%, and three's quality percentage composition sum is 73.7%.
Embodiment 3
(1) take 1kg asiatic centella total saponins, add water to 1000kg,
(2) add the penicillium expansum beta-glucosidase of 0.5kg, 60 DEG C of hydrolysis 0.5 hour,
(3) isolated by filtration enzymolysis solution, the macroporous resin column absorption that upper 10L model is D101, impurity is removed in washing, with 40L 60% volumetric concentration alcoholic solution eluting, obtains ethanol elution.By ethanol elution concentrating under reduced pressure, vacuum drying, obtain the single glucoside compositions of asiatic centella triterpenoid acid.
Adopt containing 0.4mmol/L beta-schardinger dextrin-, and percent by volume is that the aqueous solution of 30% methanol carries out HPLC mensuration as mobile phase, the quality percentage composition of cutting cloth glycosides II, centelloside C, asiatic acid-28-O-BETA-D-glucopyranoside in result compositions is respectively 22.0%, 31.5%, 29.7%, and three's quality percentage composition sum is 83.2%.
Embodiment 4
(1) take 1kg Herba Centellae alcohol water extract, add 10kg glucose, 10kg starch, 10kg soybean cake powder, add water to 1000kg, 121 DEG C of sterilizings 30 minutes,
(2) access Li Shi Trichoderma spp., 20 DEG C ferment 8 days,
(3) isolated by filtration fermentation liquid, be evaporated to about 5L, upper 10L model is the absorption of HPD100 type macroporous absorption post, impurity is removed in washing, with 20L 80% volumetric concentration alcoholic solution eluting, collect eluent, concentrating under reduced pressure, vacuum drying, obtains the single glucoside compositions of asiatic centella triterpenoid acid.Adopt containing 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, Figure 1 shows that the HPLC collection of illustrative plates of the 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 centelloside C, peak 3 is asiatic acid-28-O-BETA-D-glucopyranoside, result shows that the quality percentage composition of cutting cloth glycosides II, centelloside C, asiatic acid-28-O-BETA-D-glucopyranoside in compositions is respectively 21.0%, 32.7%, 27.3%, and three's quality percentage composition sum is 81.0%.
Embodiment 5
(1) take 1kg asiatic centella total saponins, add 1kg glucose, 1kg starch, 0.5kg soybean cake powder, add water to 50kg, 121 DEG C of sterilizings 30 minutes;
(2) access bakery yeast, 28 DEG C ferment 5 days;
(3) centrifugalize fermentation liquid, carries out pretreatment: be evaporated to about 3L, add 10L95% ethanol precipitation, filter or centrifugalize, supernatant concentration is removed ethanol, is diluted with water to 10L;
(4) pretreatment after completing upper 10L model be HPD700 type macroporous absorption post absorption, impurity is removed in washing, with 50L 20% volumetric concentration alcoholic solution eluting, collects eluent, concentrating under reduced pressure, vacuum drying, obtain the single glucoside compositions of asiatic centella triterpenoid acid.
Adopt containing 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, centelloside C, asiatic acid-28-O-BETA-D-glucopyranoside in result compositions is respectively 22.2%, 33.8%, 29.5%, and three's quality percentage composition sum is 85.5%.
Embodiment 6
(1) take 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, 121 DEG C of sterilizings 15 minutes;
(2) access lactobacillus, 45 DEG C ferment 2 days;
(3) centrifugalize fermentation liquid, is evaporated to about 1L, divides 2 extractions with 2L water-saturated n-butanol, obtains butanol extraction liquid; By butanol extraction liquid concentrating under reduced pressure, vacuum drying, obtain the single glucoside compositions of asiatic centella triterpenoid acid.
Adopt containing 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, centelloside C, asiatic acid-28-O-BETA-D-glucopyranoside in result compositions 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 triterpenoid acid
Take asiatic centella triterpenoid acid single glucoside 1kg, starch 0.3kg, microcrystalline Cellulose 0.2kg that embodiment 4 prepares, mix, with 10% starch slurry wet granulation, dry, add magnesium stearate 8g, always mixed, tabletting, obtains the single glucoside sheet of asiatic centella triterpenoid acid.
The preparation of the single glucoside aqueous injection of embodiment 8 asiatic centella triterpenoid acid
Take the single glucoside sterling 1kg of asiatic centella triterpenoid acid that embodiment 1 prepares, water for injection adds to 10L, and stirring and dissolving is filtered, sterilizing, and subpackage, obtains the single glucoside aqueous injection of asiatic centella triterpenoid acid.
The preparation of the single glucoside ointment of embodiment 9 asiatic centella triterpenoid acid
Get stearic acid 15kg, vaseline 10kg, glyceryl monostearate 3kg, heat and stir, be oil phase; Separately get asiatic centella triterpenoid acid single glucoside 1kg, sodium hydroxide 0.5kg, glycerol 10kg, ethyl hydroxybenzoate 0.1kg, distilled water 60.4kg that embodiment 5 prepares, stirring and dissolving, is water.Water is heated to 50 DEG C, and oil phase is slowly poured in water, and limit bevelling stirs, and stirs, and obtains the single glucoside ointment of asiatic centella triterpenoid acid.
The preparation of the single glucoside gel of embodiment 10 asiatic centella triterpenoid acid
Get Carbomer-940 1kg, add distilled water immersion, stir and make it be swelled into even pasty state, then add respectively ethyl hydroxybenzoate 25g, tween 80 0.5kg, drips triethanolamine and regulates pH to 6.0～8.0 (for subsequent use as blank substrate); Separately get the single glucoside 1kg of asiatic centella triterpenoid acid that embodiment 6 prepares, add PEG-600 2.5kg, glycerol 2.5kg, stir and make to add in blank substrate after its dissolving, add water to 50L, stir, to obtain final product.
The single glucoside compositions of embodiment 11 asiatic centella triterpenoid 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 embodiment 3 method preparations, purity is greater than 80%); MEM culture medium (Gibco company product, the U.S.); Calf serum (Hangzhou Ilex purpurea Hassk.[I.chinensis Sims bio-engineering corporation product); MTT (Amresco company product, the U.S.); All the other not special dated reagent are import or domestic analytical pure.
1.2 key instrument
Spectra Max 190 microplate reader (Molec-ular device, the U.S.); Cell culture incubator (SHELLAB, the U.S.).
2 experimental techniques
2.1 cell culture
MEM culture medium culturing containing 10% calf serum for HepG2 cell.Cell is inoculated in culture bottle, is placed in 37 DEG C, 5%CO
2, in the cell culture incubator of saturated humidity, cultivating, 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 96 porocyte culture plates and cultivate 24h, add the single glucoside of variable concentrations asiatic centella triterpenoid acid and process 24h, discard culture medium, every hole add 100 μ l MTT (D-hank ' S liquid dissolve, concentration is 1g/L), 37 DEG C are continued to hatch after 4h, suck culture fluid, every hole adds DMSO 100 μ l, and thing to be crystallized fully dissolves, and uses microplate reader in 570nm place photometry density after 1h.Result represents with suppression ratio, and computing formula is as follows: suppression ratio=(the average D570 of the average D570-administration of matched group group)/(the average D570 of the blank group of the average D570-of matched group) × 100%
3 results
3.1 centelloside C inhibition tumor cell propagation
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, Growth of Cells is suppressed, and cellular morphology changes, and as projection disappears, cell space shrinkage becomes circle etc.With matched group comparison, low concentration asiatic centella triterpenoid acid single glucoside (20 μ mol/L) group does not have a significant effect to the growth of HepG2 cell; In the time that dosage reaches 40 μ mol/L, the single glucoside of asiatic centella triterpenoid acid has remarkable inhibitory action to HepG2 cell proliferation, and suppression ratio reaches 81%, and the Ic50 value of the single glucoside of asiatic centella triterpenoid acid on HepG2 cell strain is 26 μ mol/L thus.
The inhibitory action (suppression ratio: %) of the single glucoside compositions of table 1 hydroxyl asiatic centella triterpenoid acid to 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 triterpenoid 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 embodiment 3 method preparations, 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); Microplate reader (Finland Wellscan MK3 type); Inverted microscope (German MPS30 type).
2 methods
2.1 medicine preparations
The single glucoside solution of asiatic centella triterpenoid acid: the single glucoside of asiatic centella triterpenoid acid adds a small amount of culture fluid, 0.45 μ m membrane filtration degerming after fully dissolving, with culture fluid be diluted to 0.125,0.25,0.50,1.00,2.00mg/mL.
2.2 fibroblasts from hypertrophic scars are cultivated
Aseptic condition menisectomy cuts the about 0.5-1mm of cicatrix skin corium piece of tissue, is inoculated in the culture bottle containing DMEM culture fluid, and the 4-7 generation in exponential phase is chosen in experiment.
2.3 fibroblasts from hypertrophic scars morphological observations
Digestion single-layer culturing cell, with l × 10, every hole
4individual being inoculated in 96 well culture plates, stays 1 hole only to add culture fluid as blank well.Sucking-off stock solution after cell attachment, sets up matched group and experimental group, and each sample standard deviation is established 4 multiple holes.Matched group changes liquid, the culture fluid 100L of every Kong Jiahan serum, and experimental group adds the each 100 μ L of drug solution containing variable concentrations, moves in calorstat and continues to cultivate, and observes and makes a video recording respectively at 24h, 48h, 72h under inverted microscope.
2.4MTT colorimetry observation fibroblasts from hypertrophic scars vigor
After the cell of above 96 orifice plates was made a video recording respectively at 3 fixing time periods, every hole 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 hole, every hole adds the dimethyl sulfoxide (DMSO) of 150 μ L, and vibration 5min fully dissolves crystallization.With blank well zeroing, in microplate reader, detect the light absorption value that wavelength is 490nm (A value), calculate the suppression ratio of drug effect after 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
Under mirror, long and narrow, the karyon of visible profile is the cell that fusiformis, trend reach unanimity, and is more and is arranged in parallel and has certain radian.There are circle or oval core in cell central authorities, and the visible protruding 2-3 of part cell projection different in size, is fibroblast.Along with the increase of incubation time, cell quantity increases, and intercellular substance narrows, and is streak distribution.
3.1.2 the single glucoside experimental group of asiatic centella triterpenoid acid
In the time that drug level is 0.50mg/mL, after 12h, cell just shortens, and occurs a little de-wall, floating cell.At 24h, 48h, in these 3 time periods of 72h, along with the increase of incubation time, attached cell quantity further reduces, and it is large that intercellular substance becomes.The cell being shaped in culture fluid after 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 respectively the suppression ratio to fibroblasts from hypertrophic scars, in table 2 according to the single glucoside of asiatic centella triterpenoid acid.As can be seen here, the single glucoside of asiatic centella triterpenoid acid has remarkable inhibitory action to fibroblasts from hypertrophic scars vigor.
The inhibitory action (suppression ratio: %) of the single glucoside of table 2 asiatic centella triterpenoid acid to fibroblasts from hypertrophic scars vigor
。
Claims (11)
1. the single glucoside compositions of asiatic centella triterpenoid acid, it is characterized in that, said composition is mainly made up of the cloth glycosides II of cutting of asiatic acid-28-O-BETA-D-glucopyranoside, centelloside C and the oleanane type of ursane type, asiatic acid-28-O-BETA-D-glucopyranoside, centelloside C, 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%.
2. compositions claimed in claim 1, is characterized in that, asiatic acid-28-O-BETA-D-glucopyranoside, centelloside C, 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%.
3. the preparation method of compositions claimed in claim 1, is characterized in that, comprises the following steps:
(1) in the aqueous solution of Herba Centellae extract, add the beta-glucosidase of Herba Centellae extract weight 0.1～50% or immobilized beta-glucosidase, 20～60 DEG C are hydrolyzed 0.5～72 hour; Or add microbiological culture media, sterilization treatment, inoculation can produce the microorganism of β glucosidase, and 20～45 DEG C ferment 2～8 days; In reaction system, the mass concentration of Herba Centellae extract is 0.1～20%; Described Herba Centellae extract is asiatic centella total saponins;
(2) filtration or centrifugalize obtain fermentation liquid or the enzymolysis solution containing the single glucoside compositions of asiatic centella triterpenoid acid;
(3) enzymolysis solution or the zymolysis liquid of the single glucoside compositions of contained asiatic centella triterpenoid acid are carried out to separation and purification, comprise the following steps:
A, enzymolysis solution is concentrated into volume and Herba Centellae extract part by weight is 1～5L/kg, divides 1～3 extraction with 1～10 times of volume water-saturated n-butanol, merge gained n-butyl alcohol phase, obtain the butanol extraction liquid of compositions; Or by macroporous adsorptive resins absorption on 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, obtain alcohol eluen;
B, by butanol extraction liquid or alcohol eluen concentrating under reduced pressure, vacuum drying, obtain compositions.
4. the preparation method of compositions claimed in claim 3, is characterized in that, enzymolysis solution has a pre-treatment step before upper macroporous absorption post absorption:
Being evaporated to solid content weight percentage is 1-30%;
Or being evaporated to solid content weight percentage is 30-60%, add 3-4 times of volume 95% ethanol precipitation, to filter or centrifugalize precipitation, supernatant concentration is removed ethanol, and being diluted with water to solid content weight percentage is 1-30%.
5. the preparation method of compositions claimed in claim 3, is characterized in that, described beta-glucosidase is main hydrolytic cleavage β-1, the beta-glucosidase of 6 glycosidic bonds.
6. the preparation method of compositions claimed in claim 3, is characterized in that, the described microorganism that can produce β glucosidase is selected from Penicillium fungus, aspergillus fungi, fungus Trichoderma, Saccharomyces fungus, bacillus or genus lactubacillus antibacterial.
7. the preparation method of compositions claimed in claim 3, it is characterized in that, the described microorganism that can produce 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.
Described in claim 1 compositions in the application of preparing in treating acne medicine or liver cancer diseases medicine.
9. described in claim 1, compositions coordinates oral formulations, injection or the preparation for external application to skin made with pharmaceutically acceptable adjuvant.
10. the HPLC quantitative analysis method of compositions described in claim 1, is characterized in that, HPLC mobile phase is made up of at least one in beta-schardinger dextrin-, water and methanol and acetonitrile.
The HPLC quantitative analysis method of compositions described in 11. claim 1, it is characterized in that, HPLC mobile phase is the aqueous solution containing 0.1～1mmol/L beta-schardinger dextrin-, and to contain percent by volume be 15%～50% methanol or acetonitrile, or to contain percent by volume be 15%～50% methanol and acetonitrile.
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| 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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| CN109879926B (en) * | 2019-02-27 | 2021-07-23 | 江西省药品检验检测研究院 | Triterpene glycoside compounds in Glechomae herba and extraction and separation method thereof |
| CN109824752A (en) * | 2019-04-02 | 2019-05-31 | 吉林大学 | A kind of triterpenoid and purposes of anti-listeria |
| CN111286810B (en) * | 2019-08-14 | 2022-04-15 | 上海交通大学医学院附属第九人民医院 | Asiaticoside biocompatible degradable electrospinning fiber and preparation method and application thereof |
| CN111544357B (en) * | 2020-06-05 | 2022-12-13 | 上海全丽生物科技有限公司 | Centella fermented raw stock and preparation method and application thereof |
| CN113509415B (en) * | 2021-07-08 | 2022-10-28 | 江苏瑞霆生物科技有限公司 | Preparation method of centella asiatica fermentation filtrate, product and application thereof |
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