CN1252078C - Method for extracting total aglycone of Macrotys and its antitumour action - Google Patents
Method for extracting total aglycone of Macrotys and its antitumour action Download PDFInfo
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- CN1252078C CN1252078C CN 200410008997 CN200410008997A CN1252078C CN 1252078 C CN1252078 C CN 1252078C CN 200410008997 CN200410008997 CN 200410008997 CN 200410008997 A CN200410008997 A CN 200410008997A CN 1252078 C CN1252078 C CN 1252078C
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Abstract
The present invention relates to total glycoside of the aboveground part cimicifuga dahurica, and the application of the total glycoside to the antioxidation injuries in tumor therapy and tumor prevention. The total glycoside is prepared by that a medicinal material of the aboveground part of cimicifuga dahurica is pulverized, sieved, extracted through reflux by heating ethanol with a concentration of 80%, filtered and concentrated by pressure reduction, an adsorbing agent (silica gel or diatomite) is added to the medicinal material to be processed through column chromatography, greases are washed off by using industrial hexane firstly, then, elution is carried out by using ethyl acetate, and obtained ethyl acetate is concentrated by pressure reduction. The present invention has the advantages of simple technology, yield coefficient raising and cost reduction, and is favorable for industrialized production. A pharmacodynamics experiment verifies that the total glycoside can be possibly used for preparing a medicine for the chemical prevention and the treatment of tumors.
Description
Technical field:
The present invention relates to the extraction preparation of the total glycosides of plant rattletop over-ground part and in the chemoprophylaxis of tumour and the application in the treatment.
Background technology:
Rattletop is Ranunculaceae Rattleroot (Cimicifugeae S.I.) plant, comprises rattletop or RHIIZOMA CIMICIFUGAE from Northwest of China (Cimicifuga foetida), rhizoma cimicifugae dahuricae or Xingan's rattletop (Cimicifuga dahurica) and rattleroot (Cimicifuga.racemosa).Xingan's rattletop is the peculiar kind of China, belongs to together not of the same race with rattleroot (claiming European rattletop again).Rattletop family plant is many is used as medicine with underground part.According to record, that the rattletop medicinal material has is clearing heat and detoxicating, the elevate a turnable ladder yang-energy, deliver the function of promoting eruption, cures mainly diseases such as headache due to pathogenic wind-heat, dentalgia, swelling and pain in the throat, uterine prolapse.Xingan's rattletop (C.dahurica) rhizome is as the loaded Chinese Pharmacopoeia of conventional Chinese medicine (2000 editions), and over-ground part is usually as waste.Just begin the over-ground part chemical ingredients is studied nearly one or two years, and the result shows that triterpene and saponin thereof are its main components.Yet the pharmacological research of over-ground part is almost a blank.Total triterpene of rattleroot and homemade rattletop (C.foetida) and saponin thereof can improve castrated rats serum estradiol level, and luteotropic hormone (LH) concentration is reduced, and can suppress the osteoporosis and the climacteric syndrome that cause owing to endocrine regulation, estrogen deficiency.Forulic acid in the rattletop, isoferulic acid etc. have the effect of relieving inflammation and relaxing pain, and coffic acid and derivative thereof have anti-oxidant activity.Take 40% isopropanol extraction thing of rattleroot rhizome simultaneously, can strengthen the estrogenic antagonist in the estrogen-dependent oncotherapy, at the external inhibited proliferation that strengthens tamoxifen to human breast carcinoma (MCF-7) cell.In addition, the rattleroot extract is applicable to that also treatment is optimum and malignant prostate is loose.Contain rattletop, the extract of the coptis and Zanthoxylin can be as the alternative medicine of oral carcinoma.The compound cimigenol 3-O-a-L-arabopyranose (cimigenol 3-O-a-L-arabinopyranoside) and the 23-O-acetyl cimigenol-3-O-a-L-arabopyranose (23-O-acetylshengmanol 3-O-a-L-arabinopyranoside) that extract from the rattleroot rhizome have restraining effect to human oral squamous cell carcinoma (HSC-2), and the half inhibiting rate is respectively 30um and 18um.Extraction is from the 23-of Xingan's rattletop over-ground part oxygen-acetyl cimigenol-3-oxygen-β-D-xyloside (23-O-acetylcimigenol-3-O-β-D-xylopyranoside), 24-oxygen-acetyl cimigenol-3-oxygen-β-D-xyloside (24-O-acetylcimigenol-3-O-β-D-xylopyranoside), (25-O-acetylcimigenol-3-O-β-D-xylopyranoside) has an antitumor action external to 25-oxygen-acetyl cimigenol-3-oxygen-β-D-xyloside, can suppress human hepatocellular carcinoma (HepG2), human hepatocellular carcinoma persister (HepG2-R), people's acute promyelocytic leukemia cell (HL-60), and former being commissioned to train supported the normal mouse liver cell, induce HepG2 cell and HL-60 apoptosis and Cycle Arrest, and the Green Tea Extract oxygenizement.
The objective of the invention is to extract the preparation total glycosides of Xingan's rattletop over-ground part and on the basis of the influence of studying its cytotoxicity, pair cell morphology, cell cycle, free-radical oxidn and dna damage, provide its practical application in the clinical tumor prophylactic treatment.
Summary of the invention:
Rattletop over-ground part total glycosides in the said Xingan of the present invention extracts from Xingan's rattletop (cimicifugadahurica) over-ground part.Starting material pick up from Inner Mongolia Autonomous Region noise made in coughing or vomiting loudspeaker in September, 1999 and secrete flag Mao Jingba, through being accredited as the over-ground part of Rattleroot plant Xingan rattletop (Cimicifuga dahurica Thurez Maxim).The extracting and preparing technique of said total glycosides is with behind the starting material crushing screening, carries out the chemical ingredients trial test.Result of study shows that Xingan's rattletop over-ground part mainly contains the triterpenoid saponin constituents.The extracting method of total saponins is determined as follows:
After rattletop over-ground part pulverizing medicinal materials sieved, extract each 1 hour 3 times with 80% alcohol heating reflux of 10 times of amounts.Extraction finishes, and filters, and it is to add sorbent material (silica gel or diatomite) at 1.12 o'clock to carry out column chromatography that filtrate decompression is concentrated into the Bomi degree.With the industrial hexane wash away grease, use eluent ethyl acetate then earlier, the ethyl acetate part that obtains gets total glycosides through concentrating under reduced pressure, and yield is 6-7%.
By isolating 30 triterpene compounds of total glycosides proportion in total glycosides
The numbering compound is proportion (%) in total glycosides
1 12beta-Acetoxy-3beta-O-D-xylopyranosyloxy-24,25,26,27-tetranor-9,19-cyclolanost-16,23-lactone 1.0
2 rattletop lactones 1.4
The 3 cimicifugoside C of Xingan 2.3
The 4 cimicifugoside D of Xingan 1.7
The 5 cimicifugoside E of Xingan 1.7
The 6 cimicifugoside F of Xingan 1.7
The 7 cimicifugoside G of Xingan 5.0
The 8 cimicifugoside H of Xingan 1.2
The 9 cimicifugoside I of Xingan 1.6
The 10 cimicifugoside J of Xingan 1.7
11 23-O-acetyl cimigenol xylosides 1.7
12 25-O-acetyl-7,8-two dehydrogenation rattletops 1.2
The alcohol xyloside
13 25-O-acetylcimigenol xylosides 6.6
14 cimicifugoside H-2 1.0
The 15 25-cimigenol xyloside 1.0 that dewaters
16 24-epi-7,8-two dehydrogenation cimigenol wood 1.2
Glucosides
17 cimigenol xylosides 5.8
18 7,8-two dehydrogenation cimigenol xylosides 1.2
19 25-O-methyl cimigenol xylosides 1.7
20 15a-hydroxyl cimicifugoside H-2 1.0
21 7 beta-hydroxy cimigenol xylosides 2.0
22 12 beta-hydroxy cimigenol xylosides 1.0
23 24-O-acetyl-7,8-two dehydrogenation rattletops 1.2
The alcohol xyloside
24 24-O-acetyl cimigenol xylosides 2.0
25 25-O-methyl-24-O-acetyl cimigenol 2.0
Xyloside
The little cimicifugoside A 8.4 of 26 12 β-O-acetyl
The little cimicifugoside B 4.2 of 27 12 β-O-acetyl
28 little cimicifugoside A 1.5
29 little cimicifugoside B 1.8
30 cimigenols 1.7
Rattletop over-ground part total glycosides in Xingan's is made into the concentrated solution of 2.50g/ml with DMSO dissolving, carries out dna damage test, the test of epoxidase 2 protein expressions that cell toxicity test, morphocytology test, cell cycle test, DPPH free radical scavenging test (anti-oxidant), hydroxy radical qiao clearance test, hydroxy radical qiao cause respectively.Cell toxicity test result shows that Xingan's total glycosides of rattletop over-ground part all has tumor-inhibiting action preferably to blood tumor, solid tumor.23-oxygen-acetyl cimigenol-3-oxygen-β-D-the xyloside of the cell toxicant intensity and the application number that patented, 24-oxygen-acetyl cimigenol-3-oxygen-β-D-xyloside, 25-oxygen-acetyl cimigenol-3-oxygen-β-D-xyloside is suitable.IC to the mouse normal liver cell
50Value illustrates that all greater than other tumor cell line rattletop over-ground part total glycosides in Xingan's is lower to Normocellular toxicity when suppressing tumour cell.Morphocytology test and cell cycle test-results show that rattletop over-ground part total glycosides in Xingan's mainly works by inducing apoptosis of tumour cell and G0/G1 cell-cycle arrest.Xingan's total glycosides of rattletop over-ground part has the effect of good removing free radical, and can effectively suppress the damage that hydroxy radical qiao causes DNA.Therefore, the Xingan's total glycosides of rattletop over-ground part among the present invention is expected as clinical tumor prevention and medicine.
The invention effect:
(HepG2, SF-268 MCF-7) all have good lethal effect to the total glycosides of Xingan's rattletop over-ground part that the present invention relates to, and faint relatively to normal mouse liver cell damaging action to blood tumor (HL-60) and solid tumor external; Morphocytology and cell cycle evidence, Xingan's total glycosides of rattletop over-ground part mainly are to block by cell death inducing and G0/G1 to work, and cyclooxygenase-2 is one of mechanism of cell death inducing and G0/G1 retardance; Xingan's total glycosides of rattletop over-ground part has good removing free radical and dna damage provide protection, can prevent free-radical oxidn to damage of biomacromolecule and the canceration that may cause thereof.The cytotoxicity of the total glycosides of Xingan's rattletop over-ground part is suitable with the action intensity of three monomeric compounds that therefrom extract; but remove free radical and dna damage provide protection and be better than three monomeric compounds; and compare with three monomeric compounds; the extraction process of the total glycosides of rattletop is simple; yield improves (reaching 6-7% approximately); cost significantly reduces (reducing more than 10 times than the monomeric compound cost), more helps its practical application in preparation tumor prevention and medicine.
Below listed embodiment help those skilled in the art better to understand the present invention, but do not limit the present invention in any way.
Cell toxicity test:
1, material: human hepatoma cell strain (HepG2), human breast cancer cell strain (MCF-7), human glioma cell's strain (SF-268), people's acute promyelocytic leukemia cell strain (HL-60), all enough in U.S. ATCC (AmericanType Culture Collection).Mouse liver cell separates from Kunming mouse (available from Traditional Chinese Medicine University Of Guangzhou's Experimental Animal Center, animal conformity certification number: Guangdong Province State Scientific and Technological Commission laboratory animal detects institute's conformity certification) liver, through former be commissioned to train to support obtain.Substratum RPMI1640, DMEM, foetal calf serum are all available from Gibico company.Tetrazole bromine MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) be AMRESCO company product, by the packing of Shanghai biotechnology company limited, lot number: 0481B50.
2, mouse liver cell former be commissioned to train foster: sterilization exposes liver behind the mouse anesthesia, portal catheterization, use heparin, preceding filling liquid, collagenase perfusion successively, take off liver and clean twice with no calcium magnesium Hanks liquid, be transferred to then in the clean culture dish liver cell is shaken off in the collagenase solution, it is centrifugal to sieve, wash 3 times with the DMEM substratum, use DMEM 10% foetal calf serum resuspended then, the trypan blue dyeing counting, living cell rate is more than 80%.
3. tissue culture: HepG2, MCF-7, SF-268 and HL-60 cell all add conventional cultivation of 10% foetal calf serum with the RPMI1640 substratum and go down to posterity.Inoculating cell is to 96 orifice plates, and inoculum density is the 10000-20000/ hole.Mouse liver cell is former is commissioned to train to support and adds 10% foetal calf serum with the DMEM substratum, and inoculum density is 8000/hole.The 5%CO2 incubator is cultivated after 24 hours for 37 ℃, dosing routinely, and control group adds isometric DMSO.Continuation was cultivated 48 hours in 5%CO2 and 37 ℃ of incubators.Add MTT, final concentration is 50ug/ml.After 37 ℃ of 5%CO2 incubators are hatched 4 hours, measure absorbance on the Bio-RAD Model 680 Micro Plate Reader.Calculate inhibiting rate.
4. result: Xingan's total glycosides of rattletop over-ground part can suppress HepG2, MCF-7, SF-268, the growth (Fig. 1) of HL-60 and mouse normal liver cell, IC
50See Table 1.The total glycosides of Xingan's rattletop over-ground part is to HepG2 as shown in Table 1, MCF-7, SF-268, the IC of HL-60
50Close, illustrate that it all has tumor-inhibiting action preferably to blood tumor, solid tumor.Rattletop over-ground part total glycosides in Xingan's is to the IC of mouse normal liver cell
50Big other tumor cell line of son, illustrate the total glycosides of Xingan's rattletop over-ground part in the inhibition tumour cell to Normocellular toxicity a little less than.
The total glycosides in table 1. rattletop ground is to the IC of five cell strains
50(μ g/ml) value
| HepG2 | MCF-7 | SF-268 | HL-60 | |
| 20 | 72 | 46 | 19 | 95 |
Morphocytology test (apoptosis):
1, method: add 400ul acridine orange/bromine second pyridine (AO/EB) solution (100ug/mlAO solution, 100ug/ml EB solution is prepared with PBS) in the 35*10mm culture dish, be mixed at 1: 1, under 40*10 fluorescence inverted microscope, observe immediately.AO all can dye to viable cell and dead cell, and EB is only to losing the cell dyeing of film conformability, and viable cell still presents uniform green.It is green that viable apoptotic cell is, because chromatin condensation and karyorhexis have viridian spot in its nucleus.Apoptosis cell in late period can be dyed orange by EB, but different with dead cell, the nuclear cohesion and the usually cracking of apoptosis cell in late period.Non-viable non-apoptotic cell is dyed orange, but possesses the nuclear morphology of viable cell sample, does not have chromatin condensation.
2, result: observe of the influence of rattletop over-ground part total glycosides in Xingan's to HepG2 and HL-60 morphocytology, discovery all can cause the morphological change of apoptosis at 25 μ g/ml and 50 μ g/ml to two cell strains, and the positive drug harringtonine also can cause similar apoptosis morphological change (Fig. 2-7).
The cell cycle test:
1, material: RNase A, USB company product, 81.4units/mg, lot number: 110266-007; Propidium iodide (PI, propidium iodide), sigma company product, lot number: 042k3655.
2, method: ordinary method is cultivated HepG2 and HL-60 cell, and collecting cell after the administration is washed twice back with PBS and spent the night with 70% alcohol fixation.The centrifugal ethanol that goes of 1000rpm/10min, wash twice with the PBS that contains 1% foetal calf serum, it is resuspended to contain the PBS of 1% foetal calf serum with 2ml again, adds RNase enzyme (final concentration 0.5mg/ml) digestion 1 hour, add PI (2.5ug/ml) dyeing then, place 20min on ice after the upflowing cell instrument detect.
3, result:
Rattletop over-ground part total glycosides in Xingan's acts on after 24 hours at 50 μ g/ml, can make HepG2 stop at the G0/G1 phase.At 25 μ g/ml, made the HL-60 cell stop at G0/G1 phase (table 2) and (Fig. 8-11) in 12 hours.
Rattletop over-ground part total glycosides in table 2. Xingan is to HepG2 and the influence of HL-60 cell cycle
| Cell strain | Sample | Dosage (μ g/ml) | Time (hr.) | G0/G1％ | S％ | G2/M％ |
| HepG2 | Contrast | - | 24 | 60.07±3.26 | 27.54±1.89 | 12.38±2.57 |
| The total glycosides of Xingan's rattletop | 50 | 24 | 72.93±2.69 | 20.07±3.54 | 7.00±2.93 | |
| HL-60 | Contrast | - | 12 | 53.3±2.36 | 41.22±2.30 | 5.47±1.53 |
| The total glycosides of Xingan's rattletop over-ground part | 25 | 12 | 75.39±3.12 | 21.41±2.89 | 3.20±0.45 |
Embodiment 4
Hexichol is for the anti-oxidant test of bitter taste diazanyl (DPPH) free radical scavenging:
1, materials and methods: DPPH (1, be a kind of more stable free radical 1-Diphenyl-2-picrylhydrazyl), 1 free electron is arranged on its N, therefore maximum absorption honeybee is arranged at the 517nm place, its methanol solution is purple.Add after the antioxidant, DPPH catches 1 electronics and free electron pairing, and the absorption at the 517nm place disappears, and purple takes off.Therefore by measure add antioxidant after, DPPH the decline of 517nm place absorption value as can be seen antioxidant to the scavenging(action) of DPPH.
Add the total glycosides of Xingan's rattletop over-ground part to DPPH (concentration is 2.4mg/ml MeOH), react after 1.5 hours, measure its absorbancy on UV, the detection wavelength is 515nm.
Blank: sample+methyl alcohol
Contrast: dimethyl sulfoxide (DMSO) (DMSO)+DPPH
Sample: sample+DPPH
Free radical scavenging activity: SR=(1-A
1/ A
0) * 100%
A
1: the absorbancy of sample
A
0: the absorbancy of contrast
2, result: Xingan's total glycosides of rattletop over-ground part can make absorbance reduce, and the effect of good removing free radical is arranged, and be dose-dependence (Figure 12).
Embodiment 5
Hydroxy radical qiao is removed and the dna damage protection test
1, material TOPCOUNT luminescence assays instrument, PH determinator, BMG 96 orifice plates, phenanthroline (3500uM), copper sulfate (500Um), vitamins C (3500uM), hydrogen peroxide (3%), DNA, Sigma product, acetic acid, other reagent of sodium-acetate are commercially available analytical pure
2, method Cu
2+-xitix-H
2O
2Effect can produce hydroxy radical qiao, and the latter and phenanthroline (phen) effect produces chemoluminescence.In this system, add DNA, hydroxy radical qiao simultaneously can with the DNA effect, cause the dna structure damage, and follow this process to have one greater than making the characteristic luminescence of time spent luminous intensity separately with phen at 445-450nm.Therefore, detect to add the luminous intensity before and after the DNA respectively, can reflect indirectly that medicine is to the removing ability of hydroxy radical qiao and to the degree of protection of dna damage.Get one of the light tight plate of BMG 96 hole black, place precooling on the ice cube.The Cu that adds precooling respectively
2+-phen 10 μ l/ holes, Vc 10 μ l/ holes, DNA (10ug/ml) 10 μ l/ holes (not adding when surveying the hydroxy radical qiao clearance test), H
2O
2The 20ul/ hole, (blank well does not add H
2O
2), wait to sieve sample 10 μ l/ holes (control wells adds the equivalent solvent), mend to 100ul with acetate buffer solution (0.1M, PH 5.5) in each hole.Fully mixing is gone up Topcount immediately and is measured luminous intensity values.And calculating inhibiting rate.Inhibiting rate (%)=(contrast-sample)/(contrast-blank) * 100%.Salvianolic acid B is as positive control.
3, result: with the luminous intensity is ordinate zou, is X-coordinate with time, and record sample luminescence kinetics curve the results are shown in Figure 13-16.Adding has obvious rising before the peak value that as seen reacts behind the DNA does not add.Total glycosides of Xingan's rattletop over-ground part and positive drug salvianolic acid B can both make luminosity curve generation considerable change, show as the remarkable reduction of peak of luminous intensity and area under curve.Show that total glycosides of Xingan's rattletop over-ground part and positive drug salvianolic acid B have tangible scavenging(action) to hydroxy radical qiao and can effectively prevent the dna damage (table 3, Figure 13-16) that hydroxy radical qiao causes.
Rattletop over-ground part total glycosides in table 3. Xingan is to the provide protection of hydroxy radical qiao removing and dna damage thereof
| Sample | Dosage (μ g/ml) | Hydroxy radical qiao inhibiting rate (%) | Dna damage inhibiting rate (%) |
| Salvianolic acid B | 4 | 49.15±2.31 | 48.94±3.02 |
| The total glycosides of Xingan's rattletop over-ground part | 25 | 50.63±1.54 | 47.25±1.76 |
Embodiment 6
The expression that oxygenase-2 is gone back in rattletop total glycosides inhibition on the ground:
1, material: the anti-cyclooxygenase-2 antibody of rabbit polyclonal, anti-rabbit igg antibody, all available from Santa CruzBiotechnology, Inc. (Santa Cruz, CA). proteinase inhibitor (Protease inhibitor cocktailtablets), protein standard (BenchMark
TMPre-stand Protein ladder) available from Invitrogen. western hybridization detection reagent (ECL) available from Amersham Pharmacia Biotech. nitrocellulose filter available from Schleicher ﹠amp; Schuell (Keene, NH)., other reagent are commercially available analytical pure.
2, method: when HepG2 cell 70% converges, add the medicine to be measured of different concns, compare with solvent.37 ℃ of cultivations of 5%CO2 incubator with ice-cold phosphoric acid buffer (PBS) washed cell twice, are used the lysate lysing cell after 4.5 hours then, choose big DNA, collect lysate and measure protein content with the bradford method.The 10%SDS polyacrylamide gel electrophoresis is transferred to nitrocellulose with albumen behind the albumen applied sample amount 25ug. electrophoresis and spreads.Nitrocellulose filter is resisted with one and two anti-hatching successively, add the ECL detection reagent, on Bio-Radchemi Doc, detect chemiluminescence intensity.
3, result: the total glycosides of rattletop over-ground part is 25,50, and the 100/ml effect all can suppress the expression of cyclooxygenase-2 after 4.5 hours, and protein band obviously weakens compared with the control, expresses descend (Figure 17).
Description of drawings:
The cytotoxicity of Fig. 1-total glycosides of rattletop over-ground part
Fig. 2-HepG2 normal control
Fig. 3-HepG2-harringtonine-20 μ M-6hr positive control
Fig. 4-HepG2-rattletop over-ground part total glycosides-50 μ g-24hr
Fig. 5-HL-60-normal control
Fig. 6-HL-60-harringtonine-20 μ M-6hr
Fig. 7-rattletop over-ground part total glycosides-25 μ the g/ml-12hr of HL-60-Xingan
Fig. 8-HepG2-normal control
Fig. 9-rattletop over-ground part total glycosides-50 μ the g/ml-24hr of HepG2-Xingan
Figure 10-HL-60-normal control
Figure 11-rattletop over-ground part total glycosides-25 μ the g/ml-12hr of HL-60-Xingan
Figure 12-total glycosides of Xingan's rattletop over-ground part is to the removing and the antioxygenation of DPPH free radical
Figure 13-salvianolic acid is to the scavenging(action) of hydroxy radical qiao
Figure 14-total glycosides of Xingan's rattletop over-ground part is to the scavenging(action) of hydroxy radical qiao
The provide protection of the dna damage that Figure 15-salvianolic acid causes hydroxy radical qiao
The provide protection of the dna damage that Figure 16-total glycosides of Xingan's rattletop over-ground part causes hydroxy radical qiao
The restraining effect that Figure 17-total glycosides of Xingan's rattletop over-ground part is expressed cyclooxygenase-2
Claims (1)
1, the application of the total glycosides of rattletop in preparation anti-tumor medicine and removing free radical chemoprevention of cancer medicine of extracting by Xingan's rattletop over-ground part.
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| CN100443495C (en) * | 2006-06-07 | 2008-12-17 | 肖培根 | Triterpene saponin compound of cycro jackfruit alkyl, and effect for anti tumor |
| CN102512491A (en) * | 2012-01-01 | 2012-06-27 | 山东大学威海分校 | Preparation method for meehania urticaefolia preparation and anti-oxidation application thereof |
| CN102614208B (en) * | 2012-02-25 | 2013-09-18 | 中国科学院昆明植物研究所 | Application of compound (20R,24R)-24,25-16,23-23,27-triepoxy-12-acetoxyl-9,19-cyclolanostanol-3-O-beta-D xylopyranoside in pharmacy |
| CN105497169A (en) * | 2016-01-06 | 2016-04-20 | 南京海源中药饮片有限公司 | Method for extracting phenolic acid effective constituents of rhizome cimicifugae |
| CN110946852A (en) * | 2018-09-27 | 2020-04-03 | 东莞广州中医药大学中医药数理工程研究院 | Application of salvianolic acid B in preparation of gamma-ray radiation resistant medicines |
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