CN101380382A - Traditional Chinese medicine extract with antioxidation activity and preparation method and use thereof - Google Patents
Traditional Chinese medicine extract with antioxidation activity and preparation method and use thereof Download PDFInfo
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Abstract
The invention provides a traditional Chinese medicinal extract with antioxidant activity, which is an acetic ester extract of a traditional Chinese medicine that can strengthen muscle and force. A high efficiency liquid chromatography of the acetic ester extract is characterized in mapping that absorption peaks are sequentially arranged at retention times of 5.30 plus or minus 0.05min, 5.80 plus or minus 0.10min and 6.55 plus or minus 0.35min; relative peak areas of the absorption peaks are sequentially 4.43 to 10.48 percent, 32.9 to 56.03 percent and 1.75 to 2.02 percent; the conditions of the high efficiency liquid chromatography are as follows: chromatographic column: Dikma Diamonsil C18 5um 250 multiplied by 4.6mm; sample: solution of absolute ethyl alcohol of 1mg/mL of the extract; sample size: 20muL; flowing phase: water, methanol, acetonitrile and mixture with the volume ratios of 25:70:3:2; flow rate: 0.500ml/min; detection wavelength: 225nm. The extract has stronger antioxidant activity, can protect mesenchymal stem cells and can be used for preparing drugs which can protect the mesenchymal stem cells.
Description
Technical field
The present invention relates to a kind of pharmaceutical product, be specifically related to a kind of extract of Chinese crude drug.
Background technology
The strong power side of strong flesh, be the famous old docter of TCM Deng Tietao professor of China through proved recipe, come from gold dollar four everybody one of Lee east wall BUZHONG YIQI TANG.But differ from former side, and different persons have three: one, and the BUZHONG YIQI TANG medication component of eastern wall is quite light; The 2nd, Dong Yuan stands this side, is the treatment card of " its full and large pulse and having a headache, or thirsty more than, its skin is not appointed wind and cold and the life cold and heat for upward adverse flow of QI and breathing heavily, fever of the body and be tired of " due to " QI of the spleen and stomach is dirty, and it is floating that essence derived from food must not be risen "; The 3rd, the method for the method that eastern wall stood " relieving high fever with drugs of sweet flavor and warm nature "." strong flesh strong power drink " with " BUZHONG YIQI TANG " though flavour of a drug with, component is different, so that the disease that is cured mainly, institute at the pathogenesis and the purport of the prescription of making laws also inequality.The descendant sums up the experience of successive dynasties utilizations BUZHONG YIQI TANG again, has changed the compatibility component and has become " BUZHONG YIQI WAN " (Radix Astragali Preparata 200g, Radix Codonopsis 60g, Radix Glycyrrhizae Preparata 100g, Rhizoma Atractylodis Macrocephalae 60g, Radix Angelicae Sinensis 60g, Rhizoma Cimicifugae 60g, Radix Bupleuri 60g and Pericarpium Citri Reticulatae 60g carry " first one 480 pages of Chinese pharmacopoeia versions in 2000).Pharmacopeia " BUZHONG YIQI WAN ", the Radix Astragali and Radix Glycyrrhizae component are maximum, and all process usefulness, and Radix Codonopsis, the Rhizoma Atractylodis Macrocephalae, Radix Angelicae Sinensis, Rhizoma Cimicifugae, Radix Bupleuri, Pericarpium Citri Reticulatae component equate.Deng Tietao professor controls the experience that atmospheric subsidence should be reused the living arrow Radix Astragali according to Zhang Xichun, and the Radix Astragali is reused, give birth to and need not process, Radix Glycyrrhizae is got its unlikely sweet meaning of keeping too gently with also processing; Pericarpium Citri Reticulatae is got its circulation of qi promoting and the fraud of harmless gas gently in order to do using corrigent.
Professor Deng Tietao treats myasthenia gravis and reuses the Radix Astragali, be subjected to the inspiration of the well-known doctor of Qing Dynasty Wang Qingren errors in Medicine Corrected BUYANG HUANWU TANG, this side gets the meaning of " treating flaccidity syndromes have to treat first YANG MING ", reuses four liang of Radix Astragali (120 gram) and is principal agent, be to rescue taste, main muscle fills lung qi, and main the liter fallen into, the Wei Chonghe of battalion, the QI and blood smoothness reaches the purpose of the strong power of strong flesh, be monarch; Radix Codonopsis, Rhizoma Atractylodis Macrocephalae spleen invigorating, Radix Angelicae Sinensis matter are moistened hot temperature and are gone into blood to join the ginseng stilbene, make the QI and blood interpromoting relation in five elements, coordination of YIN and YANG, muscle is moistened foster, more than 3 medicines appoint the duty of ministerial drug; Radix Bupleuri, Rhizoma Cimicifugae rise sends out the spleen sun, is adjuvant drug altogether.Pericarpium Citri Reticulatae reaches the purpose of regulating qi-flowing for activating stagnancy, happy mechanism of qi simply with using corrigent; Radix Glycyrrhizae and in, coordinating the actions of various ingredients in a prescription is made the usefulness of messenger drug.
The strong power side of strong flesh (being also referred to as the strong power series of strong flesh) comprises the strong power drink of strong flesh, the strong power capsule of strong flesh and the strong three kinds of dosage forms of power oral liquid of strong flesh.
First kind of dosage form, the strong power drink of strong flesh is the prepared slices of Chinese crude drugs, needs to decoct, and bring into use the fifties in last century, and its prescription is: the Radix Astragali 20: Radix Codonopsis 6: the Rhizoma Atractylodis Macrocephalae 5: Radix Angelicae Sinensis 4: Rhizoma Cimicifugae 3: Radix Bupleuri: Pericarpium Citri Reticulatae 1: Radix Glycyrrhizae 1.
Second kind of dosage form, the strong power capsule of strong flesh, beginning in 1988 is used so far in The First Affiliated Hospital of Guangzhou University of Traditional Chinese Med, its prescription is: Radix Astragali 84.0Kg, Radix Bupleuri 12.6Kg, Rhizoma Atractylodis Macrocephalae 21.0Kg, Radix Glycyrrhizae 4.2Kg, Pericarpium Citri Reticulatae 4.2Kg, Radix Angelicae Sinensis 16.8Kg, Radix Codonopsis 25.2Kg, (the strong flesh of professor Deng Tietao is good for the brief introduction of power serial therapy myasthenia gravis for Rhizoma Cimicifugae 12.6Kg and Herba Ipomoeae Cairicae 21.0Kg, be stated from " theoretic discussion clinical practice experimentation Deng Tie great waves fund assistance problem paper compilation " Guangzhou: the Deng Tie of Traditional Chinese Medicine University Of Guangzhou great waves institute, 2006:90).
The third dosage form, the strong power oral liquid of strong flesh.Prescription is: the Radix Astragali 560 grams, and Radix Codonopsis 168 grams, the Rhizoma Atractylodis Macrocephalae 140 grams, Radix Angelicae Sinensis 113 grams, Rhizoma Cimicifugae 87 grams, Radix Bupleuri 80 grams, Pericarpium Citri Reticulatae 28 grams, Radix Glycyrrhizae 28 grams, potassium sorbate 2 grams, high fructose syrup 50ml makes 1000ml altogether.Said preparation is spent all pharmaceutical factory and Guangzhou Traditional Chinese Medicine College's Chinese medicine and the common development gained of health care developmental research institute by Guangzhou Traditional Chinese Medicine College, be stated from " the strong power drink of strong flesh declaration material " (1994) project 4 " preparation technology and research data thereof " page 2, obtained national Bureau of Drugs Supervision clinical research certification in 2003, batch code is 2003L00874.
Modern Chinese medicine studies show that spleen channel is gone into by the strong power side of strong flesh, and the power of QI invigorating spleen reinforcing is strong.The strong power side of strong flesh commonly used carries out compatibility with other medicines in treatment and mesenchymal stem cells MSCs contents level diseases associated, but the ubiquity treatment cycle is long, the defective of DeGrain.Along with science and technology development, tcm field has been introduced pharmaceutically active ingredient in the modern extracting method extraction, to improve drug effect and to reduce side effect.At present, the extracting method of Chinese medicine mainly contains the chloroform-methanol supersound extraction, ethanol extraction and water extraction.Because chloroform and methanol all are toxic solvents, so the chloroform-methanol sonicated extract mainly carries out fat-soluble chemical composition analysis.Ethanol extraction and water extract are the extracting method of the most frequently used Chinese medicine, but the inventor finds that the antioxidant activity of strong power ethanol extraction of strong flesh and water extract is lower, to the effect of mesenchymal stem cells MSCs unprotect.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of Chinese medicine extract with antioxidant activity.
The technical scheme that the present invention addresses the above problem is:
A kind of Chinese medicine extract with antioxidant activity, this extract is the ethyl acetate extract of the strong power side of strong flesh Chinese medicine, the high performance liquid chromatography of described ethyl acetate extract has following figure spectrum signature: be followed successively by 5.30 ± 0.05min, 5.80 ± 0.10min and there is absworption peak at 6.55 ± 0.35min place in retention time, the relative peak area of described absworption peak is followed successively by 4.43～10.48%, 32.9～56.03% and 1.75～2.02%; Wherein,
Described high-efficient liquid phase chromatogram condition is:
Chromatographic column: Dikma Diamonsil C18 5um 250 * 4.6mm;
The ethanol solution of the described extract of sample: 1mg/mL;
Sample size: 20 μ L;
Mobile phase: volume ratio is 25: 70: 3: 2 water, methanol, acetonitrile and THF mixture;
Flow velocity: 0.500ml/min;
Detect wavelength: 225nm;
The strong power side of described strong flesh Chinese medicine derives from the strong power oral liquid of strong flesh, is made up of following medicinal materials in part by weight: the Radix Astragali 560, Radix Codonopsis 168, the Rhizoma Atractylodis Macrocephalae 140, Radix Angelicae Sinensis 113, Rhizoma Cimicifugae 87, Radix Bupleuri 80, Pericarpium Citri Reticulatae 28 and Radix Glycyrrhizae 28.
Extract of the present invention is dark brown mastic, slightly aromatic odor down at room temperature (25 ℃); Difficulty is dissolved in water, is soluble in organic solvent, and meltage is 20mg/ml in ethanol, and the chloroform meltage is 50mg/ml, and meltage is 30mg/ml in the dimethyl sulfoxine.
Extract of the present invention obtains with the strong power side of the strong flesh of ethyl acetate extraction Chinese medicine, specifically: various pulverizing medicinal materials in the strong power side of strong flesh are become 10～40 purpose fine powders, the ethyl acetate that adds 6～12 times is 80～90 ℃ of following reflux, extract, 8～12 hours, and decompression also heating concentrates and removes ethyl acetate.In order to obtain the extract of better effects if, before ethyl acetate extraction or after extracting, can also use alkali liquor saponification method, enzymatic isolation method or organic solvent extraction defat; Be not damaged in order to protect the strong power side of strong flesh Chinese crude drug effective ingredient as far as possible; we recommend to use the organic solvent method defat; concrete grammar is: with the organic solvent of 6～12 times of the strong power side of strong flesh medicated powder quality 80～90 ℃ of following reflux, extract, 8～12 hours; remove extractive with organic solvent, described organic solvent can be weak polar solvents such as petroleum ether, ether or isopropyl alcohol.
In the said method, preferred 87 ℃ of the temperature of usefulness ethyl acetate extraction, preferred 12 hours of time.
The extract of said method gained can also be used purification by silica gel column chromatography, and concrete grammar is: with silica gel column chromatography on the extract of the present invention, use petroleum ether-ethyl acetate gradient elution 2 times, get the eluent evaporation and concentration and become colourless waxy solid to get final product; The program of described gradient elution is: the consumption of petroleum ether is decremented to 0 from 100% by 1%, and the consumption of ethyl acetate is corresponding to be increased progressively.
Ratio between solid described in the method for preparing extractive of the present invention and the liquid is by weight/volume (usual practice of this area), and promptly solid is a unit of weight, and liquid is volume unit.
The authentication method of extract of the present invention is: dissolve with dehydrated alcohol (analytical pure) extract obtained, be made into 1mg/mL concentration, and with after filter membrane (the 0.45 μ m) filtration, at Yi Shui, methanol, acetonitrile and THF (volume ratio is 25:70:3:2) is mobile phase, wavelength is 225nm, chromatographic column is Dikma Diamonsil C18 5um 250 * 4.6mm, and sample size is 20uL, and flow velocity is to carry out high performance liquid chromatogram HPLC under the condition of 0.500ml/min to detect.
Extract of the present invention has good scavenging action to ABTS free radical and peroxy radical, also can suppress lipid peroxidation, has stronger antioxidant activity.Existing many research points out that the mesenchymal stem cells MSCs damage may be to be caused by oxidative damage; the pharmacological evaluation of extract of the present invention proves that also extract of the present invention has protective effect to mesenchymal stem cells MSCs; therefore, extract of the present invention can be used for preparing the medicine of protecting mesenchymal stem cells MSCs.
Extract of the present invention can add acceptable accessories and be prepared into various regular dosage forms, as tablet, pill, capsule, electuary, oral liquid etc., is good with capsule wherein.Behind purification by silica gel column chromatography, can also be prepared into injection.
Below by pharmacological testing and result thereof the technique effect that the present invention has is described.
One, the comparison of the strong power side of the strong flesh of different solvents Chinese medicine extract
1, the preparation of various extracts
Extract of the present invention (being ethyl acetate extract): press the preparation of embodiment 2 methods.
Ligroin extraction: remove petroleum ether 85 ℃ of following reflux, extract, after 12 hours with the strong power side medicated powder quality 6-12 of strong flesh petroleum ether doubly.
Dehydrated alcohol is carried thing: remove dehydrated alcohol 92 ℃ of following reflux, extract, after 12 hours with the strong power side medicated powder quality 6-12 of strong flesh dehydrated alcohol doubly, promptly.
95% ethanol extraction: remove 95 ethanol after 12 hours 95 ℃ of following reflux, extract, with the strong power side medicated powder quality 6-12 of strong flesh 95 ethanol doubly.
Water extract: remove moisture content 100 ℃ of following reflux, extract, after 12 hours with the strong power side medicated powder quality 6-12 of strong flesh distilled water doubly.
2, the mensuration of total phenol content in the various extracts
Measuring method reference " pharmacopeia " (Pharmacopoeia of the People's Republic of China 2005 editions, first one, appendix XB) in 2005: above-mentioned five kinds of extracts are made into 1mg/mL solution with the corresponding solvent dissolving.Each sample thief liquid 200 μ L adds 200 μ L95 ethanol.Place in vitro, add the Folin-Ciocalteu reagent of 500 μ L0.25mol/L, fully mix, leave standstill 3min.Add 1mL 15%Na again
2CO
3Solution fully mixes, and leaves standstill 30min.Mixed liquor is surveyed the A value at the 760nm place, with 15%Na with the filtering with microporous membrane of 0.45 μ M
2CO
3Solution is blank.With the 1,2,3,-thrihydroxy-benzene is reference substance drawing standard working curve, as shown in Figure 1.
3, result
The total phenol content of strong each extract of power of the strong flesh of table 1 (%, in 1,2,3,-thrihydroxy-benzene, n=3, Mean ± SD)
The result is as shown in table 1, and the strong power drink of strong flesh crude drug is with six kinds of different solvent extractions, and is the highest with the total phenol content in the ethyl acetate extract.
Ligroin extraction, ethanol extraction, water extract and extract of the present invention used in the following effect experiment are all by method preparation in the above-mentioned experiment one (being the comparison of the strong power side of the strong flesh Chinese medicine extract of different solvents).
Two, antioxidant activity research
The removing ability of 1 pair of DPPH free radical
DPPH is actually that form with free radical DPPH exists, so, individual single electron is arranged, at 519nm strong absorption is arranged, its ethanol water is very dark bluish violet, after adding is tried thing, can monitor the effect that DPPH is eliminated at the 519nm place, this effect is used usually the suppression ratio of DPPH is represented.Obviously, suppression ratio is big more, and the DPPH free radical scavenging is thorough more, and the non-oxidizability of being tried thing is strong more.(Blois?M.，1958.Antioxidant?determinations?by?the?use?of?astable?free?radical.Nature?181，Letter，1199-1200.)
This paper detects (Brand-Williams, W., Cuvelier, M.E.﹠amp according to the Brand-Williams method; Berset C., 1995.Use of a free radical method to evaluate antioxidant activity.Lebensmittel-Wissenschaft undTechnologies 28,25-30.): will test that each extract is made into the sample liquid that concentration is 5mg/mL in one, dewater and carry the beyond the region of objective existence water and do outside the solvent, all the other all make solvent with DMSO. Sample thief liquid 4,10,20,30,40,50 μ L join in the 1.0mLDPPH test fluid (0.1mmol/L), add 95% ethanol, and making total reaction volume is 1.5mL.Shake well left standstill 30 minutes, surveyed the A value (519nm) of each reactant liquor, was blank with 95 ethanol.Calculate suppression ratio according to following formula: suppression ratio=[1-(A-A sample)/A0] * 100%.Wherein: A0-is the DPPH solution absorbency of application of sample not; DPPH solution absorbency behind the A-application of sample; The absorbance of A sample-sample liquid self.Introducing the A sample in the formula is in order to eliminate the influence of sample liquid intrinsic colour to experimental result.Result's following (table 2)
The strong power of the strong flesh of table 2 is drunk the suppression ratio (%, n=3, Mean ± SD, GSH positive matched group) of each extract to DPPH
| Final concentration μ g/mL | Positive controls GSH | Ligroin extraction | Ethyl acetate extract | The dehydrated alcohol extraction thing | 95% ethanol extraction | Water extract |
| 13.33 | 42.74±0.99 | 35.86±1.79 | 43.33±1.49 | 33.23±0.76 | 33.99±3.36 | 31.03±0.368 |
| 33.33 | 49.43±0.25 | 40.88±1.05 | 58.19±1.15 | 38.28±0.98 | 35.64±0.22 | 33.33±0.57 |
| 66.67 | 58.65±2.64 | 44.87±0.13 | 72.58±2.38 | 42.88±0.19 | 41.08±0.52 | 38.04±0.099 |
| 100 | 64.39±1.08 | 49.95±1.19 | 86.78±9.41 | 46.29±0.58 | 44.74±3.31 | 41.47±0.30 |
| 133.33 | 69.53±0.51 | 58.06±4.27 | 87.098±2.05 | 47.10±0.91 | 47.92±1.14 | 44.80±0.82 |
| 166.67 | 72.93±1.59 | 56.86±0.61 | 90.158±1.98 | 54.01±0.69 | 50.89±1.57 | 47.75±0.63 |
As can be seen from the results: in each extract of the strong power drink of strong flesh, it is the strongest to remove DPPH free radical ability with extract of the present invention.
The removing ability of 2 pairs of ABTS free radicals
The ABTS+ free radical is by ABTS[2,2 '-azino-bis (3-ethylbenzthiazoline-6-sulfpnic acid)] promptly [2,2 '-azino-two-(3-ethyl benzo thiazole phenanthroline-6-sulfonic acid)], the stable aeruginous radical cation that after the active oxygen oxidation, generates, to wherein adding measured matter, if there is antioxidant content in this material, then this material can react with ABTS+ and reaction system is faded.Judge the removing ability [5] of this material according to fading extent to ABTS+.(Re R., Pellegrini N., Proteggente A., Pannala A., et al, 1999.Antioxidant activity applying an improved ABTS radicalcation decolorization assay.Free Radical Biology ﹠amp; Medicine 26,1231-1237.), reflect the oxidation resistance of sample from the angle of removing external free radical.
Mix 7.4mmol/LABTS storing solution and 2.6mmol/L K2S2O8 storing solution are respectively got the 0.55mL equal-volume,, placed 12 hours under the lucifuge condition, form ABTS+ free radical storing solution in room temperature.Add the about 43mL of dehydrated alcohol before using and be diluted to the ABTS+ working solution, require at 30 ℃, the absorbance under the 734nm wavelength is 1.10 ± 0.02.Each extract mother solution of having prepared is diluted to the test fluid 0.3mL of variable concentrations with dehydrated alcohol (water extract distilled water), in corresponding test tube, adds the ABTS of 1.2mL then respectively
+Working solution, after fully mixing, lucifuge was placed 2 hours under the room temperature.With the dehydrated alcohol is blank, measures absorbance under the 734nm wavelength.With the positive contrast of standard substance.End product is got the meansigma methods of 3 groups of parallel laboratory tests.The computing formula of suppression ratio: suppression ratio=(A
0-A)/A
0* 100%, A wherein
0For not adding the ABTS+ solution absorbency of sample; A is sample and the reacted absorbance of ABTS+.The result is as shown in table 3.
The strong power of the strong flesh of table 3 is drunk each extract to ABTS
+Suppression ratio (%, n=3, Mean ± SD, the positive matched group of Trolox)
| Concentration μ g/mL | Positive controls Trolox | Ligroin extraction | Ethyl acetate extract | The dehydrated alcohol extraction thing | 95% ethanol extraction | Water extract |
| 0.4152 | 4.24±4.59 | 6.12±0.86 | 5.13±2.32 | 3.51±1.60 | 1.46±0.84 | 3.36±0.14 |
| 2.076 | 31.52±0.81 | 8.66±0.085 | 8.15±1.79 | 5.85±0.46 | 4.95±2.17 | 6.97±1.09 |
| 4.152 | 69.77±0.11 | 9.45±0.499 | 15.39±0.77 | 9.66±2.22 | 9.31±0.82 | 10.61±1.72 |
| 6.228 | 100.67±0.15 | 13.03±0.52 | 23.38±0.55 | 12.68±3.44 | 10.49±1.01 | 15.55±3.02 |
| 8.304 | 100.04±0.20 | 15.12±0.45 | 30.5±0.92 | 14.72±1.20 | 12.48±1.41 | 19.48±3.07 |
| 10.38 | 100.08± 0.056 | 18.21±0.57 | 39.72±2.37 | 20.04±2.88 | 18.58±0.36 | 22.16±2.29 |
| 12.456 | 100.08± 0.056 | 20.23±0.085 | 44.58±1.60 | 23.93±3.00 | 20.82±0.37 | 24.42±0.96 |
| 14.532 | 100.35±0.00 | 21.17±0.17 | 48.91±0.25 | 29.07±2.21 | 25.17±0.26 | 28.79±0.72 |
| 16.608 | 100.12± 0.096 | 26.28±0.085 | 55.79±2.71 | 30.80±1.45 | 30.37±2.83 | 32.36±3.24 |
| 18.684 | 100.08± 0.056 | 26.90±0.59 | 63.84±0.25 | 36.09±0.96 | 34.80±0.49 | 37.10±4.31 |
As can be seen from Table 3, extract of the present invention has good scavenging action to ABTS+.
3 total reducing powers
Total reducing power is usually used in reflecting the antioxidant activity power of sample, and this method is according to the Oyaizu method
[3](Oyaizu, M., 1986.Studies on product of browning reaction prepared from glucose amine.Jpn.J.Nutr.44,307-315.). the strong power of strong flesh is drunk each extract, dewater outside the liquid, all the other all make solvent with DMSO, and concentration is 10mg/mL.Each extract gets 0.05,0.1,0.2,0.3,0.4,0.6,0.8 successively, 1.0mL, join phosphate buffer (0.2mol/L, pH6.6) in, and to make cumulative volume be 3.5mL, adds 2.5mLK again
3Fe (CN)
6Solution (1g/100mL), after fully mixing, in 50 ℃ of water-bath 20min, fully reaction.Then, add 2.5mL trichloroacetic acid solution (10g/100mL) cessation reaction.With the centrifugal 10min of 2500g/min, get supernatant 2.5mL, add 2.5mL distilled water, 2.5mLFeCl successively
3Solution (0.1g/100mL) fully mixes, and picks up counting, and when 90s, surveys the A value in the 700nm place.The A value is big more, and the total relatively reducing power of prompting is strong more.The result is as shown in table 4.
The strong power of the strong flesh of table 4 is drunk the total reducing power (A of each extract
700nm, n=3, mean ± SD, the positive matched group of GSH)
As can be seen from the above results, the strong power of strong flesh is drunk in the various extracts, and is the strongest with total reducing power of extract of the present invention.
4 remove peroxy radical (O
2) ability
Peroxy radical (O
2) be a kind of common free radical that produces in the body, can bring out organism disease and aging.This paper adopts 1,2,3,-thrihydroxy-benzene autoxidation method to measure and removes peroxy radical (O
2) ability [2] (Marklund S, Marklund G, 1974.Involvement of the superoxide anion radical in the autoxidation of pyrogallol and convenient assayfor superoxide dismutase.Eur.J.Biochem 47,469-474). 1,2,3,-thrihydroxy-benzene issues at alkali condition is conigenous oxidation generation peroxy radical (O
2), simultaneously, generate semiquinone.Because semiquinone has absorption at λ 325nm place, therefore, the increase speed (Δ A/ Δ t) of A value that can be by detection λ 325nm place is weighed the removing peroxy radical (O of sample
2) ability.Each extract is made into 1mg/mL, gets 50,100,200,300,400,500 μ L respectively, join in the Tris-HCl buffer (0.05mol/L, pH8.2 contain 1mmol/L EDTA), and to make total reaction volume be 1475 μ L.Add 25 μ L 1,2,3,-thrihydroxy-benzene solution (6mmol/L is dissolved in the 10mmol/L hydrochloric acid solution) again, mix rapidly, pick up counting.Surveying the A value at spectrophotometer 325nm place, is blank with the Tris-HCl buffer.Since 60 seconds readings, after this, every 30 seconds readings once, till 300 seconds.Advance the speed (Δ A/ Δ t) with the A value weighs antioxidant activity, and Δ A/ Δ t value is more little, and it is strong more to show that sample suppresses 1,2,3,-thrihydroxy-benzene autoxidation effect.Suppression ratio calculates according to following formula: (in the formula, when sampling amount is 0 μ L, be matched group.)
The strong power of the strong flesh of table 5 is drunk each extract and is removed peroxy radical O
2Ability (%, n=3, Mean ± SD)
| Concentration μ g/mL | Positive controls GSH | Ligroin extraction | Ethyl acetate extract | The dehydrated alcohol extraction thing | 95% ethanol extraction | Water extract |
| 33.3 | 20.78±0.68 | 3.22±1.64 | 11.25±3.88 | 3.22±2.29 | 3.43±1.29 | 3.70±3.05 |
| 66.7 | 35.62±1.25 | 0.75±1.84 | 27.44±2.20 | 7.18±2.12 | 10.61±1.39 | 4.61±3.56 |
| 133.4 | 50.15±0.15 | 25.40±0.69 | 51.45±2.74 | 18.65±0.45 | 24.54±3.28 | 5.03±3.16 |
| 200.1 | 55.30±0.97 | 33.87±1.09 | 75.35±0.99 | 31.62±1.54 | 37.73±0.76 | 8.36±2.29 |
| 266.8 | 58.96±0.32 | 57.77±2.63 | 90.99±0.45 | 46.52±0.84 | 55.95±1.58 | 9.65±1.64 |
| 333.5 | 62.66±0.23 | 74.28±1.31 | 97.21±0.40 | 65.06±1.54 | 71.81±0.84 | 9.86±2.58 |
As can be seen from the above results, the strong power of strong flesh is drunk in the various extracts, with the removing peroxy radical O of ethyl acetate extract
2Ability is the strongest.
5 remove the lipid peroxidation ability
Lipid peroxidation also is a common oxidative damage form in the body, and it can influence cell, and human many major diseases are all with it about [8].(Sevanian，A.，Ursini，F.，2000.Lipid?peroxidation?in?membranes?and?lowdensitylipoproteins：similarities?and?differences.Free?Radical?Biol.Med.29，306-311.
The method that this paper adopted is according to document [9] slightly modified (Siddhuraju P., 2006.Antioxidant activity ofpolyphenolic compounds extracted from defatted raw and dry heated Tamarindus indica seed coat.LWT, doi:10.1016/j.1wt.2006.07.010.): get 312.6mg linoleic acid and 78.2mg Tween20 and join in 20mL 30% (v/v) ethanol, fully mix shaking up, get the linoleic acid emulsion.In 1.5mL linoleic acid emulsion, add the sample liquid 0.1mL of various concentration, and transfer to cumulative volume 2.0mL with distilled water.This mixed liquor places open glass, and room temperature reaction took out 0.15mL and places in vitro after 72 hours.Add 3.65mL 75% (V/V) ethanol, 0.1mLNH4SCN (30g/100mL), 0.1mL FeSO4 (0.02mol/L is dissolved among the 3.6%HCl) successively, mix.After 3 minutes, surveying the A value at the 500nm place, is blank (Unico 2100 spectrophotometers, Shanghai) with 75% ethanol.Computing formula:
The suppression ratio of lipid peroxidation=(1-A
Sample/ A
0) * 100%
In the formula, A
SampleA value during=application of sample; A
0=sample size is the A value of 0 μ L.
The strong power of the strong flesh of table 6 is drunk each extract and is removed peroxy radical O
2Ability (Mean ± SD, GSH are matched group for %, n=3)
| Concentration μ g/mL | Matched group GSH | Ligroin extraction | Ethyl acetate extract | The dehydrated alcohol extraction thing | 95% ethanol | Water extract | |
| 8 | 0.30±0.54 | 0.082±1.33 | 0.13±0.63 | 1.36±2.78 | -0.61±1.28 | -0.80±1.06 | |
| 16 | 0.19±1.13 | 1.31±2.43 | 6.38±2.85 | 0.90±0.86 | -0.36±1.39 | -0.71±1.12 | |
| 24 | 0.0032±1.28 | 9.80±3.61 | 14.42±4.79 | 0.73±0.52 | -0.34±1.45 | -0.98±0.71 | |
| 32 | -0.044±0.46 | 12.26±6.58 | 28.0±7.67 | 0.11±0.87 | 0.26±1.33 | -0.90±1.21 | |
| 40 | -0.53±1.048 | 8.65±2.15 | 40.70±2.96 | 0.33±1.46 | -0.61±0.83 | -0.78±0.82 | |
| 60 | 0.11±0.67 | 35.06±2.48 | 61.16±6.84 | 3.38±0.89 | 0.62±1.08 | -0.89±0.99 |
The result is as shown in table 6, and extract of the present invention is to peroxy radical O
2Has good removing ability.
Three, extract of the present invention is to the mesenchymal stem cells MSCs protective effect
1, reagent and laboratory animal
Cell culture and SABC reagent: low sugar DMEM (L-DMEM), Percoll and hyclone (FBS) are GIBCOL company product, and antibody is exempted from anti-CD34, CD44, CD45, biotinylation two anti-(goat-anti rabbit), SABC, DAB staining kit all available from Wuhan doctor's moral company.MTT is a SIGMA company product.10 of cleaning level SD rats, 220-250g, female, provide (Guangdong probatio inspectionem pecuoarem word 2005A010 number) by Guangdong Province's Experimental Animal Center.
2, MSC separates, increases and identifies
Rat marrow is gone out with L-DMEM, and fully mixing changes centrifuge tube over to, and centrifugal 10 minutes of 300g removes supernatant, and D-Hanks is resuspended, centrifugal remove supernatant after, add 4mL D-Hanks mixing.The Percoll stock solution is pressed 0.56:0.44 and is mixed, and gets 4mL and puts into the 10mL centrifuge tube, inhales bone marrow fluid and is slowly adding from liquid level 2cm place subsides tube wall.Centrifugal 30 minutes of horizontal centrifuge 900g, the collecting monocytic cell layer is used the DMEM washed twice.Counting cells then, adjust density, by the inoculation of 1 * 106/cm2 density, 37 ℃, the CO2 incubator of 5% saturated humidity is cultivated, culture fluid is the L-DMEM that contains 10%FBS, changes culture fluid behind the 3d, discards not attached cell, changed liquid in 2-3 days 1 time, digested 2-3 minute with containing 0.25% pancreatin room temperature near the MSC that merges, go down to posterity, reached for the 3rd generation to obtain 1 * 107/cm2 cell by 1 * 104/cm2.Part MSC is seeded in 24 well culture plates that contain cover plate, drips the DMEM culture fluid that contains 10% hyclone, cell is in adherent undifferentiated state after 2 hours, takes out cover plate and is Brdu, CD44 SABC and both bonded SABC double stainings.
3, H
2O
2Damage MSC model passes 3 generation MSC and is divided into matched group, H
2O
2Damage group, extract group of the present invention, ligroin extraction group, dehydrated alcohol extraction thing group, 95% ethanol extraction group and water extract group.Matched group: add the DMEM culture fluid that contains 10% hyclone; H
2O
2Damage group: add the H2O2 damage at the DMEM culture fluid that contains 10% hyclone; Extract group of the present invention, ligroin extraction group, dehydrated alcohol extraction thing group, 95% ethanol extraction group and water extract group: MSC is through H
2O
2Damage after 3 hours, change the culture fluid of the ethyl acetate extract, ligroin extraction, dehydrated alcohol extraction thing, 95% ethanol extraction and the water extract that contain variable concentrations respectively.Above-mentioned 7 groups of mtt assay detections respectively after acting on 24 hours.
Contain the preparation of the culture fluid of various extracts: described 4 extracts are taken by weighing 30mg respectively, ethyl acetate extract, ligroin extraction, dehydrated alcohol extraction thing, 95% ethanol extraction are dissolved in respectively in the 1ml dimethyl sulfoxide, water extract is dissolved in the 1ml distilled water, obtains each 30mg/ml of different solvents extract test liquid.
4, mtt assay survey cytoactive mtt assay principle is that dehydrogenase can be reduced into tetrazolium salts water-fast blue product in the living cells, and is deposited in the cell, and dead cell does not have this function.Therefore, how many MTT absorbance values has reacted living cells quantity.The preparation single cell suspension, counting, the adjustment cell concentration is 1 * 105/mL, is inoculated in 96 orifice plates, every hole 200 μ L.Every hole adds MTT (5mg/mL) 20 μ L, hatches 4 hours, and abandons supernatant for MSC37 ℃.Every hole adds dimethyl sulfoxide 150 μ L, and jolting 10 minutes is in 490nm wavelength photometry absorption value (A).
5, result
By table 7 as seen, H
2O
2Damage group cell light absorption value obviously reduces, and relatively has significant difference (P＜0.01) with matched group, and H is described
2O
2Damage MSC model modeling success; With the increase of ethyl acetate extract concentration, the cell light absorption value increases gradually, with matched group and and H
2O
2The damage group compares, and there was a significant difference (P＜0.01).Ethyl acetate extract relies on mode to H with concentration
2O
2Damage MSC has repair.Experimental result shows that ligroin extraction, ethanol extraction and water extract are to H
2O
2Damage MSC repair does not have influence.Result of study shows that extract of the present invention is to H
2O
2Damage MSC has protective effect.
Table 7
| Ultimate density μ g/mL | Positive controls GSH | Ligroin extraction | Ethyl acetate extract | The dehydrated alcohol extraction thing | 95% ethanol extraction | Water extract |
| 13.33 | 42.74±0.99 | 35.86±1.79 | 43.33±1.49 | 33.23±0.76 | 33.99±3.36 | 31.03±0.368 |
| 33.33 | 49.43±0.25 | 40.88±1.05 | 58.19±1.15 | 38.28±0.98 | 35.64±0.22 | 33.33±0.57 |
| 66.67 | 58.65±2.64 | 44.87±0.13 | 72.58±2.38 | 42.88±0.19 | 41.08±0.52 | 38.04±0.099 |
| 100 | 64.39±1.08 | 49.95±1.19 | 86.78±9.41 | 46.29±0.58 | 44.74±3.31 | 41.47±0.30 |
| 133.33 | 69.53±0.51 | 58.06±4.27 | 87.098±2.05 | 47.10±0.91 | 47.92±1.14 | 44.80±0.82 |
| 166.67 | 72.93±1.59 | 56.86±0.61 | 90.158±1.98 | 54.01±0.69 | 50.89±1.57 | 47.75±0.63 |
The modern medicine result of study shows that glutathion is the strong antioxidant of (GSH) effect, so the present invention selects positive contrast of GSH and extract effect of the present invention comparison for use, and the result shows that extract of the present invention is stronger than the GSH effect.Above results suggest extract of the present invention has the novel potentiality to mesenchymal stem cells MSCs protective effect medicine of further research and development, and may obtain than the better effect of traditional antioxidants.
Description of drawings
Fig. 1 is to be the standard working curve that reference substance is drawn with the 1,2,3,-thrihydroxy-benzene.
Fig. 2 is the extract obtained gas chromatography-mass spectrum figure of embodiment 1
Fig. 3 is the high performance liquid chromatogram collection of illustrative plates of embodiment 2 described extracts.
Fig. 4 is the gas chromatography-mass spectrum figure of embodiment 2 described extracts.
Fig. 5 is the high performance liquid chromatogram collection of illustrative plates of embodiment 3 described extracts.
Fig. 6 is the high performance liquid chromatogram collection of illustrative plates of embodiment 4 described extracts.
The specific embodiment
Example 1
One, extract preparation of the present invention:
1, pulverizes
Adopt universal mill that the strong power of strong flesh is pulverized, crossing 20 mesh sieves is the strong power fine powder of required strong flesh.
2, the extraction of effective site
Get the strong power fine powder of the strong flesh of 40g and be heated to 90 ℃ of continuation reflux, extract, 12 hours with 250ml ethyl acetate (analytical pure), concentrating under reduced pressure reclaims ethyl acetate then, obtains coffee-like paste extract.
Two, extract obtained evaluation
The strong flesh of resulting coffee-like paste is good for the power extract through silica gel column chromatography, 1% gradient petroleum ether-ethyl acetate eluting 2 times, get colourless waxy solid, (gas chromatography-mass spectrum method) demarcates then by the following method: extract (the solidifying shape) 18.3mg that gets strong flesh ethyl acetate is dissolved in 330mg oxolane (THF, chromatographically pure, Di Ma company) in, and, carry out GC/MS in following condition and detect with after filter membrane (the 0.45 μ m) filtration:
GC conditions: (30m * 0.25mmi.d.), carrier gas is a helium, and split ratio is 1/10, and injector temperature is 280 ℃ to separate employing DB-5MS quartz capillary column; Heating schedule is: initial temperature is 60 ℃, rises to 280 ℃ with 8 ℃/min, keeps 20min;
Mass spectrum condition: EI source, 250 ℃ of ion source temperatures, bombardment electron energy 70eV, electron multiplier voltage 1.7kV, mass scanning scope 28～400amu.Sample size 1 μ L;
Instrument: Agilent 5973N GC/MSD.
The result as shown in Figure 6, No. 1 peak (14.8min) and No. 2 peaks (16.8min) are the atypia peak because signal too a little less than; No. 3 peaks (18.65 ± 0.05min) structure exist two kinds may:
Or
The structure the unknown at No. 4 peaks (19.58min), No. 5 peaks (21.39min) and No. 6 peaks (27.49min); No. 7 peaks (34.05 ± 0.05min) is steroid compound, has four kinds of different possible structures:
One, extract preparation of the present invention:
1, pulverizes
Adopt universal mill that the strong power of strong flesh is pulverized, crossing 20 mesh sieves is the strong power fine powder of required strong flesh.
2, defat
Get the strong power fine powder of the strong flesh of 40g, in the filter cylinder of packing into, put into apparatus,Soxhlet's then, add 250ml petroleum ether (analytical pure), be heated to 50 ℃, reflux, extract, 12 hours is removed the Petroleum ether extraction position.
3, the extraction of effective site
To be heated to 90 ℃ with 250ml ethyl acetate (analytical pure) through the residue after the Petroleum ether extraction remove impurity and continue reflux, extract, 12 hours, concentrating under reduced pressure reclaims ethyl acetate then, obtains coffee-like paste extract 0.4g.
Two, extract obtained evaluation
1, efficient liquid phase chromatographic analysis: be made into 1mg/mL concentration with extract obtained with dehydrated alcohol (analytical pure) dissolving, and, carry out high performance liquid chromatogram HPLC and detect with after filter membrane (the 0.45 μ m) filtration.Elution requirement is a water: methanol: acetonitrile: THF=25:70:3:2.Detecting used instrument is high performance liquid chromatogram instrument DIONEX summit P680 (single pump), detector UVD-170, wavelength 250nm; Chromatographic column Dikma Diamonsil C18 5um 250 * 4.6mm; Sample size 20uL; Flow velocity 0.500mL/min.Testing result as shown in Figure 2, extract obtained distinctive main absworption peak is: No. 1 peak retention time Ret.Time is 5.307min, and relative peak area Rel.Area is 5.99%; No. 2 peak retention time Ret.Time is 5.898min, and relative peak area Rel.Area is 48.52%; No. 3 peak retention time Ret.Time is 6.270min, and relative peak area Rel.Area is 2.02%.
2, chemical analysis analysis: the strong flesh of resulting coffee-like paste is good for the power extract through silica gel column chromatography, 1% gradient petroleum ether-ethyl acetate eluting 2 times, get colourless waxy solid, use the gas chromatography-mass spectrum method to demarcate (concrete grammar is with example 1) then, the result is shown in Fig. 3 and table 8.
Table 8
Three, the preparation of extract injection of the present invention (Injectio)
This product is the aseptic aqueous solution that the strong power of the strong flesh of Chinese medicine is made through effective component extracting.2ml is equivalent to the strong power effective ingredient 10mg of strong flesh.
1, refining
Through silica gel column chromatography, 1% gradient petroleum ether-ethyl acetate eluting 2 times gets colourless waxy solid, is extract highly finished product of the present invention with the strong power extract of the strong flesh of resulting coffee-like paste.
2, prescription
Extract highly finished product 500mg of the present invention, 1,2 propylene glycol 20ml, tween 80 2ml.Make injection 100ml.
3, method for making
Get extract highly finished product of the present invention (colourless waxy solid) 500mg, add 1,2 propylene glycol 20ml dissolving, jolting is dissolved the refining effective ingredient of the strong power of strong flesh fully, adds the injection water again to 100ml.Then, add activated carbon 0.5% (V/V), fully stir, filter, filter with G3 remelting glass funnel at last, embedding, 100 ℃ of circulation steam sterilization 30min, promptly.
Four, the using method of extract injection of the present invention
Intramuscular injection and acupoint injection therapy, 4ml first, later on each 2ml, 1-2 time on the one.
One, extract preparation of the present invention:
1, pulverizes
Adopt universal mill that the strong power medicine of strong flesh is pulverized, crossing 40 mesh sieves is the strong power fine powder of required strong flesh.
2, defat
Get the strong power fine powder of the strong flesh of 60g, in the filter cylinder of packing into, put into apparatus,Soxhlet's then, add 500ml petroleum ether (analytical pure), be heated to 50 ℃, reflux, extract, 10 hours is removed the Petroleum ether extraction position.
3, the extraction of effective site
To be heated to 70 ℃ with 500ml ethyl acetate (analytical pure) through the residue after the Petroleum ether extraction remove impurity and continue reflux, extract, 10 hours, concentrating under reduced pressure reclaims ethyl acetate then, obtains the strong power extract 0.6g of the strong flesh of coffee-like paste.
Two, extract obtained evaluation
1, efficient liquid phase chromatographic analysis: be made into 1mg/mL concentration with extract obtained with dehydrated alcohol (analytical pure) dissolving, and, carry out high performance liquid chromatogram HPLC and detect with after filter membrane (the 0.45 μ m) filtration.Elution requirement is a water: methanol: acetonitrile: THF=25:70:3:2.Detecting used instrument is high performance liquid chromatogram instrument DIONEX summit P680 (single pump), detector UVD-170, wavelength 275nm; Chromatographic column Dikma Diamonsil C18 5um 250 * 4.6mm; Sample size 20uL; Flow velocity 0.500mL/min.Testing result as shown in Figure 3, extract obtained distinctive main absworption peak is: No. 1 peak retention time Ret.Time is 5.266min, and relative peak area Rel.Area is 10.48%; No. 2 peak retention time Ret.Time is 5.746min, and relative peak area Rel.Area is 32.9%; No. 3 peak retention time Ret.Time is 6.591min, and relative peak area Rel.Area is 1.75%.
2, chemical analysis is analyzed: the strong flesh of resulting coffee-like paste is good for the power extract through silica gel column chromatography, 1% gradient petroleum ether-ethyl acetate eluting 2 times, get colourless waxy solid, use the gas chromatography-mass spectrum method to demarcate (concrete grammar is with example 1) then, contain 14 carbon carboxylic acids 5.5% in the described extract, 16 carbon carboxylic acids 10.36%, 18 carbon carboxylic acids 14.33%, 16 carbon carboxylate methyl esters 16.80%, 16 carbon carboxylic acid, ethyl esters 10.8%, 18 carbon carboxylic acid, ethyl esters 7.00%, Linolenic Acid-olefinic carboxylic acid methyl ester 14.00%, polyphenol 8.11%, 3-cholesterol 6.64%, 3,5 two cholestene 3.01%.
Three, the preparation of extract capsule of the present invention (Capsulae)
The strong flesh of coffee-like paste of writing out a prescription is good for power extract 250mg, calcium carbonate 45mg, starch 5mg.The heavy 300mg of each capsule.
Method for making is sieved calcium carbonate and starch mixing, mixes with the extractum of an amount of ethanol dilution again, crosses 120 mesh sieves, and is in 60-70 ℃ of oven dry 2h, encapsulated.
Four, the using method of extract capsule of the present invention
Usual amounts every day 1 time, each 1-2 grain, 4 weeks were a course of treatment, can obey two courses of treatment again, discretionary reduction can be made in a dose.
Example 4
One, extract preparation of the present invention:
1, pulverizes
Adopt universal mill that the strong power of strong flesh is pulverized, crossing 10 mesh sieves is the strong power fine powder of required strong flesh.
2, defat
Get the strong power fine powder of the strong flesh of 60g, in the filter cylinder of packing into, put into apparatus,Soxhlet's then, add 600ml petroleum ether (analytical pure), be heated to 70 ℃, reflux, extract, 8 hours is removed the Petroleum ether extraction position.
3, the extraction of effective site
To be heated to 80 ℃ with 600ml ethyl acetate (analytical pure) through the residue after the Petroleum ether extraction remove impurity and continue reflux, extract, 8 hours, concentrating under reduced pressure reclaims ethyl acetate then, obtains the strong power extract 0.6g of the strong flesh of coffee-like paste.
Two, extract obtained evaluation
1, efficient liquid phase chromatographic analysis: be made into 1mg/mL concentration with extract obtained with dehydrated alcohol (analytical pure) dissolving, and, carry out high performance liquid chromatogram HPLC and detect with after filter membrane (the 0.45 μ m) filtration.Elution requirement is a water: methanol: acetonitrile: THF=25:70:3:2.Detecting used instrument is high performance liquid chromatogram instrument DIONEX summit P680 (single pump), detector UVD-170, wavelength 300nm; Chromatographic column Dikma Diamonsil C18 5um 250 * 4.6mm; Sample size 20uL; Flow velocity 0.500mL/min.Testing result as shown in Figure 4, extract obtained distinctive main absworption peak is: No. 1 peak retention time Ret.Time is 5.304min, and relative peak area Rel.Area is 4.43%; No. 2 peak retention time Ret.Time is 5.887min, and relative peak area Rel.Area is 56.03%; No. 3 peak retention time Ret.Time is 6.235min, and relative peak area Rel.Area is 1.80%.
2, chemical analysis is analyzed: the strong flesh of resulting coffee-like paste is good for the power extract through silica gel column chromatography, 1% gradient petroleum ether-ethyl acetate eluting 1 time, get colourless waxy solid, use the gas chromatography-mass spectrum method to demarcate (concrete grammar is with example 1) then, contain 14 carbon carboxylic acids 2.5% in the described extract, 16 carbon carboxylic acids 13.36%, 18 carbon carboxylic acids 7.33%, 16 carbon carboxylate methyl esters 15.60%, 16 carbon carboxylic acid, ethyl esters 9.36%, 18 carbon carboxylic acid, ethyl esters 6.50%, Linolenic Acid-olefinic carboxylic acid methyl ester 13.00%, polyphenol 7.53%, 3-cholesterol 3.64%, 3,5 two cholestene 2.4%.
Three, the preparation of extract tablet of the present invention (Tablet)
Prescription: the strong flesh of coffee-like paste is good for power extract 400mg, calcium carbonate 90mg, starch 10mg.Every heavy 500mg.
Method for making: with calcium carbonate and starch mixing, sieve, mix with the extractum of an amount of ethanol dilution again, cross 90 mesh sieves, in 60-70 ℃ of oven dry, tabletting.
Four, the using method of extract tablet of the present invention
Effect has the MSC of promotion growth and promotes the MSC effect with purposes this product, is mainly used in treatment osteopathia, aplastic anemia and sacred disease, bone marrow protection behind the chemotherapy of tumors.
Usage and consumption usual amounts every day 1 time, each 1-2 sheet, 4 weeks were a course of treatment, can obey two courses of treatment again, discretionary reduction can be made in a dose.
Example 5
Extract preparation of the present invention:
1, pulverizes
Adopt universal mill that the strong power of strong flesh is pulverized, crossing 10 mesh sieves is the strong power fine powder of required strong flesh.
2, the extraction of effective site
Get the strong power fine powder of the strong flesh of 60g, 720ml ethyl acetate (analytical pure) is heated to 80 ℃ and continued reflux, extract, 8 hours, and concentrating under reduced pressure reclaims ethyl acetate then.
3, defat
The extract of step 2 gained is packed in the filter cylinder, put into apparatus,Soxhlet's then, add 720ml petroleum ether (analytical pure), be heated to 70 ℃, reflux, extract, 12 hours is removed the Petroleum ether extraction position, obtains the strong power extract of the strong flesh of coffee-like paste.
Example 6
Extract preparation of the present invention:
1, pulverizes
Adopt universal mill that the strong power of strong flesh is pulverized, crossing 10 mesh sieves is the strong power fine powder of required strong flesh.
2, the extraction of effective site
Get the strong power fine powder of the strong flesh of 60g, 720ml ethyl acetate (analytical pure) is heated to 80 ℃ and continued reflux, extract, 8 hours, and concentrating under reduced pressure reclaims ethyl acetate then, obtains the strong power extract of the strong flesh of coffee-like paste.
3, defat
The extract of step 2 gained is packed in the filter cylinder, put into apparatus,Soxhlet's then, add 720ml ether (analytical pure), be heated to 70 ℃, reflux, extract, 8 hours is removed the ether extraction position.
Example 7
Extract preparation of the present invention:
1, pulverizes
Adopt universal mill that the strong power of strong flesh is pulverized, crossing 20 mesh sieves is the strong power fine powder of required strong flesh.
2, defat
Get the strong power fine powder of the strong flesh of 40g, in the filter cylinder of packing into, put into apparatus,Soxhlet's then, add 250ml isopropyl alcohol (analytical pure), be heated to 90 ℃, reflux, extract, 12 hours is removed the isopropanol extraction position.
3, the extraction of effective site
To be heated to 87 ℃ with 250ml ethyl acetate (analytical pure) through the residue after the Petroleum ether extraction remove impurity and continue reflux, extract, 12 hours, concentrating under reduced pressure reclaims ethyl acetate then, obtains coffee-like paste extract.
Claims (7)
1, a kind of Chinese medicine extract with antioxidant activity, this extract is the ethyl acetate extract of the strong power side of strong flesh Chinese medicine, the high performance liquid chromatography of described ethyl acetate extract has following figure spectrum signature: be followed successively by 5.30 ± 0.05min, 5.80 ± 0.10min and there is absworption peak at 6.55 ± 0.35min place in retention time, the relative peak area of described absworption peak is followed successively by 4.43～10.48%, 32.9～56.03% and 1.75～2.02%; Wherein,
Described high-efficient liquid phase chromatogram condition is:
Chromatographic column: Dikma Diamonsil C18 5um 250 * 4.6mm;
The ethanol solution of the described extract of sample: 1mg/mL;
Sample size: 20 μ L;
Mobile phase: volume ratio is 25: 70: 3: 2 water, methanol, acetonitrile and THF mixture;
Flow velocity: 0.500ml/min;
Detect wavelength: 225nm;
The strong power side of described strong flesh Chinese medicine is made up of following medicinal materials in part by weight: the Radix Astragali 560, Radix Codonopsis 168, the Rhizoma Atractylodis Macrocephalae 140, Radix Angelicae Sinensis 113, Rhizoma Cimicifugae 87, Radix Bupleuri 80, Pericarpium Citri Reticulatae 28 and Radix Glycyrrhizae 28.
2, the described preparation method of extract of claim 1, this method may further comprise the steps:
(1) pulverizes: various pulverizing medicinal materials in the strong power side of strong flesh are become 10～40 purpose fine powders;
(2) extract: remaining medicinal residues add 6～12 times ethyl acetate 80～90 ℃ of following reflux, extract, 8～12 hours, and decompression and heating concentrate removes ethyl acetate and get final product.
3, method as claimed in claim 2 is characterized in that the temperature with ethyl acetate extraction is 87 ℃, and the time is 12 hours.
4, as claim 2 or 3 described preparation method of extract, it is characterized in that this method also comprises a defatting step that carries out after the preceding or step (2) in step (2), this step is: 80～90 ℃ of following reflux, extract, 8～12 hours, discard extractive with organic solvent with 6～12 times organic solvents; Wherein said organic solvent is petroleum ether, ether or isopropyl alcohol.
5, method as claimed in claim 4 is characterized in that this method is further comprising the steps of: with the extract obtained silica gel column chromatography of going up, use petroleum ether-ethyl acetate gradient elution 2 times, get the eluent evaporation and concentration and become colourless waxy solid to get final product; The program of described gradient elution is: the consumption of petroleum ether is decremented to 0 from 100% by 1%, and the consumption of ethyl acetate is corresponding to be increased progressively.
6, the application of the described extract of claim 1 in preparation protection mesenchymal stem cells MSCs medicine.
7, a kind of medicine of protecting mesenchymal stem cells MSCs, this medicine is made up of described extract of claim 1 and acceptable accessories.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102133290A (en) * | 2011-03-16 | 2011-07-27 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Application of invigorator tea pill in electromagnetic radiation protection |
| CN113736731A (en) * | 2021-11-05 | 2021-12-03 | 保信亚太生物科技（深圳）有限公司 | Serum-free medium of adipose tissue-derived mesenchymal stem cells and preparation method and application thereof |
-
2008
- 2008-10-16 CN CNA2008101991985A patent/CN101380382A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102133290A (en) * | 2011-03-16 | 2011-07-27 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Application of invigorator tea pill in electromagnetic radiation protection |
| CN113736731A (en) * | 2021-11-05 | 2021-12-03 | 保信亚太生物科技（深圳）有限公司 | Serum-free medium of adipose tissue-derived mesenchymal stem cells and preparation method and application thereof |
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