CN104987357A - Separation and preparation method of compound with antineoplastic activity - Google Patents

Separation and preparation method of compound with antineoplastic activity Download PDF

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Publication number
CN104987357A
CN104987357A CN201510398649.8A CN201510398649A CN104987357A CN 104987357 A CN104987357 A CN 104987357A CN 201510398649 A CN201510398649 A CN 201510398649A CN 104987357 A CN104987357 A CN 104987357A
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rce
separation
extraction
mouse
ethanol
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贺海波
邹坤
杨小姣
刘呈雄
白彩虹
王倩
汪鋆植
郭志勇
薛艳红
杨进
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China Three Gorges University CTGU
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China Three Gorges University CTGU
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07JSTEROIDS
    • C07J71/00Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
    • C07J71/0005Oxygen-containing hetero ring

Abstract

The invention discloses a separation and preparation method of a compound with antineoplastic activity. The separation and preparation method comprises the steps that reineckia carnea is extracted through solvents, and is filtered and concentrated; concentration products obtained in the first step are dissolved with water, and are extracted with ethyl acetate for a period of time, and ultrasonic extraction is preferably carried out for one to four hours; finally, the ethyl acetate abstract obtained through extraction is concentrated, and is gradiently eluted and separated with chloroform-methyl alcohol, fractions 1-16 are collected, and the obtained fraction 15 is separated and purified through gel chromatography. RCE-4 obtained through separation has weak cytotoxicity on normal cells, and has high cytotoxicity on human hepatoma carcinoma cells H22 and HepG2. A remarkable anti-tumor effect on H22 dutch ascites tumors and solid cancer mice is achieved. Compared with current clinically-used anticarcinogen, the compound has the advantages of being equivalent in effect and small in toxic and side effect.

Description

A kind of method for separating and preparing with active compound for anti tumor
Technical field
The invention belongs to medical art, be specifically related to a kind of method for separating and preparing with active compound for anti tumor.
Background technology
Pink Reineckea Herb (Reineckia carnea (Andr.) Kunth.) belongs to autogenus perennial evergreen herbaceous plant for Liliaceae Convallarieae Pink Reineckea Herb, has another name called that Premna microphylla Turez, Song Shoulan, leaflet are evergreen, Rhizome of Chinese Tupistra, ophiruid seven etc.Its nature and flavor are sweet flat, have moisten the lung and relieve the cough, regulating blood condition, removing toxic substances, kidney tonifying synthetism, the effect of dispelling rheumatism, be mainly used in pulmonary tuberculosis, cough spitting of blood, chronic bronchitis, asthma, rheumatic arthritis, the treatment of the disease such as wound, fracture is controlled in external application, is " sacred grass " by Tujia, among the people look at of seedling man.Comprise according to its main chemical compositions of analysis: the metallic elements such as steroid saponin, flavonoid, triterpenes, fatty acid, alkaloid, ceramide, cyclic alcohol, alkane and Ca, Fe, Cu, Zn, Mn, Mg, Na, K.It is a kind of herbal medicine resource very with the ethnic minority of Development volue.
Current malignant tumour becomes the major disease of serious threat population health.The annual mortality of malignant tumors number of Present Global is about 7,000,000 people, to the year two thousand thirty every year because the number of cancer mortality may reach 1,700 ten thousand.According to World Health Organization's statistics, the current whole world on average just has 1 people to die from cancer in every 8 deaths, and this death toll summation caused than acquired immune deficiency syndrome (AIDS), tuberculosis and malaria is taller.And the annual whole world is made a definite diagnosis more than 1,200 ten thousand people in addition suffers from cancer.Wherein Cancer in China death toll accounts for the whole world 24%, and the trend that becomes to become younger.Operation, radiation and chemotherapy remain the Main Means of oncotherapy clinically, and wherein chemotherapy is one of its important means, and then the expense of its great number and serious toxic side effect, become the bottleneck limiting its clinical application.In recent years, derive from many herbal medicine of ethnic minority, because its antitumor curative effect is definite, toxic side effect is little, in many-sided comprehensive advantages such as clinical application among the people are with a long history, and become the treasure-house for the treatment of tumor promotion lead compound.
Summary of the invention
Inventor is through research extensively and profoundly, from Tujia, the steroidal saponin composition prepared is separated: (1 β in seedling man traditional herbal medicine Pink Reineckea Herb, 3 β, 5 β, 25S)-spirostane-1, 3-glycol 1-[α-L-rhamnosyl-(1 → 2)-β-D-xylopyranoside], i.e. 25 (S)-5 β-spirostan-1 β, 3 β-diol1-O-α-L-rhamnopyranosyl-(1 → 2)-β-D-xylopyranoside (RCE-4), and find that this composition all has good antitumor action to H22 and HepG2 liver cancer cell and to lotus H22 knurl kunming mice after deliberation, by comparing with the positive drug capecitabine of Hepatoma therapy, similar with its antitumous effect, but there are no obvious toxic side effect.Yet there are no the preparative separation of this compound and the relevant report of Hepatoma therapy.
An object of the present invention is to provide one preparative separation steroidal saponin RCE-4 method from Pink Reineckea Herb.The inventive method comprises the extraction of Pink Reineckea Herb sample and the abstraction and purification of RCE-4.
The present invention realizes specifically by following technical scheme:
The invention provides a kind of method that steroidal saponin RCE-4 is prepared in separation, comprise step below:
A) use solvent extraction Pink Reineckea Herb, filter, concentrated;
B) enriched product water dissolution a) step obtained, is extracted with ethyl acetate for some time, preferred ultrasonic extraction 1 ~ 4 hour;
C) b) step is extracted the acetic acid ethyl ester extract obtained to concentrate, gradient elution separation with chloroform-methanol, collect cut 1-16, by the wherein cut 15 of gained through gel chromatography separation purifying.
Steroidal saponin RCE-4 is needle-like crystal, in white.Chemical name is: (1 β, 3 β, 5 β, 25S)-spirostane-1,3-glycol 1-[α-L-rhamnosyl-(1 → 2)-β-D-xylopyranoside], be a kind of spirostanol saponin(e, molecular formula is C38H62O12.It is colourless transparent liquid after dissolving.Its molecular structure is shown below:
In a kind of specific embodiment of the present invention, extract the rhizome of Pink Reineckea Herb by common solvent.Pink Reineckea Herb is preferably clean, dry pulverised form; Extraction solvent can be water, ethanol or its mixture, preferably 60 ~ 95% ethanol, such as 75% ethanol; Adopt refluxing extraction mode, preferably under 40 ~ 70 DEG C such as 65 DEG C of conditions, atmospheric pressure reflux is extracted; Or filter the filter method adopting field of traditional Chinese medicine extraction conventional, concentrate and under 40 ~ 70 DEG C of conditions, can be concentrated by rotary evaporation.
Inventor finds, after the enriched material water dissolution obtain, is only extracted with ethyl acetate extraction for some time, finally just can obtains target product; Adopt other solvents such as sherwood oil or butanol, before immunoassay will can not get this product.Ultrasonic extraction can shorten extraction time, such as 1 ~ 4 hour.
The acetic acid ethyl ester extract above-mentioned extraction obtained is concentrated just can obtain target product with being separated.The present invention adopts chloroform-methanol gradient elution separation, collects cut 1-16, the wherein cut 15 of gained is obtained target product after gel chromatography separation purifying.Here, the concentrated concentration method that this area can be adopted conventional, such as rotary evaporation.The preferred permanent gradient of gradient elution, not only easy and simple to handle, and simple and quick, separate targets product efficiently.
Inventor finds, only has the 15th cut to be separated and obtains target product.Chemical composition in Pink Reineckea Herb by the saponin constituent that obtains after various column chromatography chromatographic separation still containing some pigment compositions; Adopt gel chromatography depigmentation than macroreticular resin absorbing method and active carbon adsorption more effective.
According to the present invention a kind of specific embodiment, wherein step c) separation condition be below one or more:
L) acetic acid ethyl ester extract obtained above is carried out chromatographic separation;
M) chloroform-methanol gradient is permanent gradient 15:1; With
N) gel is dextrane gel, preferred hydroxypropyl dextrane gel.
According to the present invention a kind of specific embodiment, wherein step c) described in chromatographic separation and purification after also comprise a re-crystallization step, purifying obtains purer colorless needle crystals.
According to the present invention a kind of specific embodiment, wherein step b) in be extracted with ethyl acetate before with petroleum ether extraction, resistates is extracted with ethyl acetate again.The productive rate of final product steroidal saponin RCE-4 can be improved like this.
According to a kind of specific embodiment of the present invention, wherein step a) described in solvent be 95% ethanol.
According to a kind of specific embodiment of the present invention, wherein step a) in also comprise the step reclaiming ethanol; Preferably, after filtering extracting solution, under about 55 DEG C of conditions, reclaim ethanol.
According to a kind of more particular embodiment of the present invention,
Raw materials pretreatment: Pink Reineckea Herb rhizome is cleaned and dries, be dried to constant weight in 40 ~ 60 DEG C of constant temperature bellowss;
The extraction of Pink Reineckea Herb chemical composition: 1. refluxing extraction: sample is cut into pieces, with 60 ~ 95% ethanol for solvent, under 40 ~ 70 DEG C of conditions, atmospheric pressure reflux is extracted, and after extracting liquid filtering, under 40 ~ 70 DEG C of conditions, concentrates with Rotary Evaporators; 2. extract: by the medicinal extract of concentrated gained, dissolve, with ethyl acetate ultrasonic extraction 1 ~ 4 hour with distilled water;
The separation and purification of RCE-4 in Pink Reineckea Herb: extraction is obtained ethyl acetate extract chloroform-methanol gradient elution, collects flow point, by wherein first-class part 15 of gained through Sephadex LH-20 wash-out, to obtain final product.
The present invention is separated the RCE-4 for preparing at H 22with on HepG2 human liver cancer cell and lotus H 22knurl kunming mice is tested, shows the therapeutic action with anti-liver cancer.
The present invention also demonstrates from cell and animal level the effect that RCE-4 has good Hepatoma therapy.
Another object of the present invention is to provide the steroidal saponin RCE-4 that a kind of above-mentioned method obtains.
Another object of the present invention is to provide a kind of pharmaceutical composition, and it contains steroidal saponin RCE-4 of the present invention.
According to a kind of specific embodiment of the present invention, wherein said pharmaceutical composition also comprises autophagy inhibitor 3-MA, optionally also containing other cancer therapy drug activeconstituentss and/or carrier, thinner or vehicle.
According to a kind of specific embodiment of the present invention, in wherein said pharmaceutical composition, RCE-4 concentration is between 2 ~ 4 μMs, preferably 4 μMs.
Beneficial effect of the present invention
The invention provides a kind of from Tujia, seedling family traditional herbal medicine---be separated the method preparing RCE-4 Pink Reineckea Herb, RCE-4 is less to the cytotoxicity of normal cell as monkey-kidney cells, Madin-Darby canine kidney(cell line) in research display, but to human liver cancer cell (H 22and HepG2) there is very significant growth-inhibiting effect, and in time-dependent manner and dose-dependently; Lotus H can be suppressed 22ascitic tumor mouse suppresses generation and the survival time of ascites; Can significantly suppress lotus H 22solid tumor mouse tumor volume and knurl weight, the normal blood index of mouse, blood parameters and immunity system and techtology all be there is no to the side effect of significance, although and its tumor killing effect of positive drug capecitabine is slightly better than RCE-4, but it is good at high dosage, strong side effect, therefore, have that toxic side effect is little, the good advantage of curative effect in contrast, be worth Application and Development.
Accompanying drawing explanation
Fig. 1 is Pink Reineckea Herb extraction step schema,
Fig. 2 is Pink Reineckea Herb extraction step figure,
Fig. 3 is the separating step schema of RCE-4 in Pink Reineckea Herb,
Fig. 4 Pink Reineckea Herb ethyl acetate extract separation process figure
Fig. 5 is Pink Reineckea Herb n-butanol portion separation process figure,
Fig. 6 Pink Reineckea Herb n-butanol portion WQ-F series of separate schema,
Fig. 7 RCE-4 on the impact of marc-145 and mdck cell cytotoxic activity,
Fig. 8 RCE-4 on the impact of H22 and HepG2 human liver cancer cell cytotoxic activity,
Fig. 9 RCE-4 on the impact of H22 lotus ascitic tumor mouse survival time,
Figure 10 RCE-4 on the impact of H22 solid tumor mice-transplanted tumor techtology (D:RCE-4 high dose group, E: capecitabine group, HE dye for A: model group, B:RCE-4 low dose group, dosage group in C:RCE-4),
Figure 11 RCE-4 on the impact of H22 solid tumor mouse main organs techtology (E: suprarenal gland, F: spleen, G: stomach, H: duodenum, I: large intestine, J: small intestine, HE dyes for A: brain, B: the heart, C: liver, D: kidney),
Embodiment
Below in conjunction with embodiment, the present invention is described further, and these embodiments are only illustrate of the present invention, in any case can not be interpreted as limitation of the scope of the invention.
Embodiment 1
In Pink Reineckea Herb, one has active compound for anti tumor RCE-4, prepares especially by following methods:
1) Pink Reineckea Herb pre-treatment
Pink Reineckea Herb rhizome is cleaned and is dried, and get 0.005kg and preserve, remaining is dried to constant weight, net weight 4.0kg in 48 DEG C of constant temperature bellowss.
2) Pink Reineckea Herb refluxing extraction
Sample is cut into segment, and with 95% ethanol for solvent, under 65 DEG C of conditions, atmospheric pressure reflux is extracted, after extracting liquid filtering, under 55 DEG C of conditions, concentrate with Rotary Evaporators, volatilize in stink cupboard subsequently to without alcohol taste, obtain dark medicinal extract, weigh to obtain medicinal extract 1.4kg (see Fig. 1).
3) extraction of Pink Reineckea Herb medicinal extract
Add 1 liter of distilled water and dissolve above-mentioned 2) gained medicinal extract, use extraction agent sherwood oil, ethyl acetate, water saturated n-butanol extraction successively.Add 2L extraction agent and the medicinal extract aqueous solution mixes, ultrasonic extraction utilizes siphonage principle by the extraction liquid sucking-off on upper strata, concentrates on a rotary evaporator and reclaim extraction liquid for 2 hours after two phase stratification at every turn, and the solvent after recovery continues on for extraction.Merge the extract of same extraction solvent, in stink cupboard, volatilize solvent, finally each medicinal extract (see Fig. 2).To weigh to obtain each medicinal extract amount: the heavy 130g of sherwood oil part, the heavy 112g of ethyl acetate portion; The heavy 550g of n-butanol fraction.
4) a kind of separation with active compound for anti tumor RCE-4 in Pink Reineckea Herb:
By above-mentioned 3) the ethyl acetate extract medicinal extract (70 grams) that obtains carries out silica gel column chromatography, adopt chloroform-methanol=15:1 gradient elution, be total to obtain 16 components, wherein 5,10,15 principal spots are obvious, and No. 5 and No. 10 components obtain white, needle-shaped crystals WQ-E-4 (β-daucosterol) and WQ-E (RCE-2) through recrystallizing methanol.Then No. 15 components are obtained white, needle-shaped crystals, through being accredited as WQ-E-5 (RCE-4) through SephadexLH-20 (methyl alcohol dress post or chloroform methanol 1:1).No. 16 components are through chloroform-methanol silica gel column chromatography, and cut is once the white, needle-shaped crystals WQ-E-2 of acetone recrystallization; Cut two obtains white, needle-shaped crystals WQ-E-3 through Sephadex LH-20 (chloroform methanol 1:1).(see Fig. 3)
Crystallization WQ-E-5 (RCE-4) after above-mentioned chromatographic separation is purified with ethyl alcohol recrystallization again, obtains purer white, needle-shaped crystals.
Learn a skill be accredited as RCE-4 according to its physico-chemical property, modern POP, nuclear-magnetism is identified, colorless needle crystals, molecular formula: C 38h 62o 12. 1H-NMR(400MHz,C 5D 5N)δ0.85(3H,s,H-18),1.34(3H,s,H-19),1.15(3H,s,H-21),1.08(3H,d,J=6.5Hz,H-27),1.76(3H,d,J=6.1Hz,CH 3-Rha),6.63(1H,s),5.11(1H,d,J=7.3Hz); 13C-NMR(100MHz,C 5D 5N)δ:75.9(C-1),27.3(C-2),67.3(C-3),34.2(C-4),31.8(C-5),26.4(C-6),26.4(C-7),35.6(C-8),42.0(C-9),39.4(C-10),21.4(C-11),40.3(C-12),40.7(C-13),56.4(C-14),32.2(C-15),81.3(C-16),62.9(C-17),16.7(C-18),19.4(C-19),42.5(C-20),14.9(C-21),109.7(C-22),26.3(C-23),26.2(C-24),27.3(C-25),65.1(C-26),16.3(C-27),98.1(xyl,C-1),76.9(xyl,C-2),79.2(xyl,C-3),71.6(xyl,C-4),67.4(xyl,C-5),101.9(rha,C-1),72.0(rha,C-2),72.2(rha,C-3),74.5(rha,C-4),69.9(rha,C-5),18.(rha,C-6)。
The Structural Identification of compound WQ-E-5 is: (1 β, 3 β, 5 β, 25S)-spirostane-1,3-glycol 1-[α-L-rhamnosyl-(1 → 2)-β-D-xylopyranoside].
The Structural Identification of compound WQ-E-3 is: (22S)-cholesterol-1 β, 3 β, 16 β, 22-tetrol-1-O-β-D-rhamnopyranosyl-16-O-β-D-glucopyranoside.NMR data are: 13c-NMR (100MHz, C 5d 5n) δ: 71.9 (C-1), 25.6 (C-2), 66.8 (C-3), 27.9 (C-4), 87.6 (C-5), 31.2 (C-6), 24.1 (C-7), 34.2 (C-8), 43.8 (C-9), 42.8 (C-10), 20.9 (C-11), 39.6 (C-12), 40.3 (C-13), 55.8 (C-14), 26.4 (C-15), 80.9 (C-16), 62.1 (C-17), 15.5 (C-18), 13.3C-19), 42.1 (C-20), 15.0 (C-21), 109.7 (C-22), 25.4 (C-23), 23.8 (C-24), 27.1 (C-25), 64.7 (C-26), 16.1 (C-27), 95.6 (glu, C-1), 74.4 (glu, C-2), 77.1 (glu, C-3), 70.0 (glu, C-4), 76.8 (glu, C-5), 61.1 (glu, C-6).
Compound WQ-E is accredited as (25S)-spirostane-1 β, 3 β, 5 β, 26-tetrol-5-O-β-D-Glucose glycosides.Colorless needle crystals, molecular formula: C33H54O10, ESI-MS m/z:633 [M+Na]+.NMR data are: 1h-NMR (500MHz, CD 3oD) δ: 1.1 (3H, s, H-18), 0.80 (3H, s, H-19), 1.16 (3H, d, J=7.6Hz, H-21), 1.06 (3H, d, J=6.5Hz, H-27), 4.95 (1H, d, J=7.8Hz), 13c-NMR (125MHz, CD 3oD) δ: 71.9 (C-1), 25.6 (C-2), 66.8 (C-3), 27.9 (C-4), 87.6 (C-5), 31.2 (C-6), 24.1 (C-7), 34.2 (C-8), 43.8 (C-9), 42.8 (C-10), 20.9 (C-11), 39.6 (C-12), 40.3 (C-13), 55.8 (C-14), 26.4 (C-15), 80.9 (C-16), 62.1 (C-17), 15.5 (C-18), 13.3C-19), 42.1 (C-20), 15.0 (C-21), 109.7 (C-22), 25.4 (C-23), 23.8 (C-24), 27.1 (C-25), 64.7 (C-26), 16.1 (C-27), 95.6 (glu, C-1), 74.4 (glu, C-2), 77.1 (glu, C-3), 70.0 (glu, C-4), 76.8 (glu, C-5), 61.1 (glu, C-6).
Compound WQ-E-4 is accredited as daucosterol, colorless needles (methyl alcohol), mp291 ~ 292 DEG C.Molecular formula: C35H60O6, ESI-MS m/z:599 [M+Na]+, NMR data are: 13c-NMR (100MHz, C 5d 5n) δ: 37.8 (C-1), 30.1 (C-2), 78.6 (C-3), 40.0 (C-4), 141.0 (C-5), 122.2 (C-6), 32.5 (C-7), 32.1 (C-8), 50.8 (C-9), 37.1 (C-10), 21.5 (C-11), 28.8 (C-12), 42.8 (C-13), 56.9 (C-14), 24.6 (C-15), 40.5 (C-16), 56.6 (C-17), 12.2 (C-18), 19.5 (C-19), 36.6 (C-20), 19.6 (C-21), 34.4 (C-22), 26.5 (C-23), 46.3 (C-24), 29.6 (C-25), 20.5 (C-26), 19.7 (C-27), 23.5 (C-28), 12.3 (C-29), 101.1 (glu, C-1), 74.5 (glu, C-2), 78.1 (glu, C-3), 71.5 (glu, C-4), 78.8 (glu, C-5), 62.7 (glu, C-6).
The structure of compound WQ-E-2 can be accredited as: (25S, 5 β)-spirostane-1 β, 3 β, 14 beta-triol-1-O-α-L-rhamnopyranosyl (1 → 2)-β-D-xylopyranoside, white, needle-shaped crystals, molecular formula C38H62O13, positive ion ESI-MS display quasi-molecular ion peak ESI-MS m/z:749.5 [M+Na]+.NMR data are: 13c-NMR (100MHz, C 5d 5n) δ: 76.1 (C-1), 27.1 (C-2), 67.3 (C-3), 34.1 (C-4), 31.8 (C-5), 26.4 (C-6), 20.8 (C-7), 39.6 (C-8), 34.5 (C-9), 39.6 (C-10), 21.4 (C-11), 32.6 (C-12), 45.1 (C-13), 87.3 (C-14), 39.9 (C-15), 82.0 (C-16), 59.7 (C-17), 20.5 (C-18), 19.3 (C-19), 42.5 (C-20), 15.2 (C-21), 110.1 (C-22), 26.5 (C-23), 26.2 (C-24), 27.6 (C-25), 65.1 (C-26), 16.3 (C-27), 98.1 (xyl, C-1), 76.9 (xyl, C-2), 79.2 (xyl, C-3), 71.6 (xyl, C-4), 67.4 (xyl, C-5), 101.9 (rha, C-1), 72.0 (rha, C-2), 72.2 (rha, C-3), 74.5 (rha, C-4), 69.9 (rha, C-5), 18.8 (rha, C-6).
Through nuclear-magnetism qualification, WQ-E-5, WQ-E-4 (β-daucosterol), WQ-E (RCE-2), WQ-E-2 and WQ-E-3 structure are respectively:
Inventor adopts HPLC method to carry out assay to active compound RCE-4 in Pink Reineckea Herb, and compare and have employed 95% ethanol, ethyl acetate, chloroform: obtain extraction yield when Extraction solvent made by methyl alcohol (15:1) mixed solvent, found that employing chloroform: when Extraction solvent made by methyl alcohol (15:1) mixed solvent, the extraction yield of RCE-4 is the highest, up to 0.1229%, the method is accurately easy, favorable reproducibility, can set up as the content assaying method of RCE-4 in Pink Reineckea Herb and for reineckea carnea medicinal materials quality standard and lay the foundation.
Table is separated the compound obtained from Pink Reineckea Herb rhizome
N-butanol fraction (550g), through macroporous adsorbent resin, water: Ethanol System wash-out, remove most of pigment, pass through the means such as reverse C18 silica gel column chromatography (acetonitrile: water gradient elution), silica gel column chromatography (chloroform: methanol elution gradient), the separation and purification of preparative high performance liquid phase again, isolate 17 compounds altogether, be respectively compound WQ-5, WQ-6, WQ-7, WQ-2, WQ-4, WQ-10, WQ-13, WQ-14, WQ-1, WQ-15, WQ-F-9, WQ-F-4, WQ-F-5, WQ-F-6, WQ-F-12, WQ-F-13, WQ-F-15.Concrete separation process is shown in Fig. 5 and Fig. 6.
Comparative example 1
Adopt the method identical with above-described embodiment 1, difference is only the 3rd) in the extraction process of step Pink Reineckea Herb medicinal extract, add 1 liter of distilled water and dissolve above-mentioned 2) after gained medicinal extract, use extraction agent sherwood oil and water saturated n-butanol extraction respectively.As a result, final product steroidal saponin RCE-4 is not obtained in extraction liquid.
Pink Reineckea Herb complex chemical composition, think that its main pharmacodynamics composition is steroidal saponin at present, people find under study for action, and the aglycon of steroidal saponin is different, or sugar chain is different, and its pharmacologically active exists larger difference.Inventor has carried out anti tumor activity in vitro research to being separated the compound R CE-4 obtained from Pink Reineckea Herb, find that it all has obvious restraining effect to tumour cells such as Caski, SH-SY5Y, HepG2, A549, wherein have best cytotoxicity to Caski cell (human cervical carcinoma cell), IC50 value is 3.71 μMs.
Experimental example 1
Below in conjunction with concrete experimental example, illustrate the RCE-4 anti-liver cancer application on a cellular level that the embodiment of the present invention 1 obtains.
RCE-4 is to normal cell monkey-kidney cells (marc-145), Madin-Darby canine kidney(cell line) (MDCK) and human liver cancer cell H 22with the impact of the cytotoxic activity of HepG2.
Test method
1) cell cultures
Marc-145 and mdck cell DMEM-F12 culture medium culturing, in CO 25%CO in incubator 2, cultivate under 37 DEG C and saturated humidity.When cell monolayer grows to 80%, use contains the tryptic digestion of 0.25% of EDTA, goes down to posterity.
H 22cultivate, in CO with HepG2 human liver cancer cell RPMI-1640 nutrient solution 2cultivate under 5%CO2 in incubator, 37 DEG C and saturated humidity.When cell monolayer grows to 80%, with the tryptic digestion of 0.25%, go down to posterity.
2) 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide bromine salt (MTT) colorimetry test
Collect the normal cell of logarithmic phase and tumour cell, adjustment concentration of cell suspension with 1 × 10 5cell concn kind 96 orifice plate, every hole adds 100 μ L, and surrounding PBS seals, in 5%CO 2hatch 12h for 37 DEG C, the dilution of the storing solution substratum of steroidal saponin RCE-4 is mixed with 6 concentration gradients (50,25,12.5,6.25,3.13,1.62 μMs), every hole adds the medicine 100 μ L of concentration gradient, system becomes 200 μ l, each concentration arranges 6 multiple holes, and arranges blank (celliferous nutrient solution+MTT+ dimethyl sulfoxide (DMSO) (DMSO)), positive drug hole (celliferous nutrient solution+25 μ g/ml mitomycin+MTT+DMSO) and zeroing hole (nutrient solution+MTT+DMSO).5%CO 2, cultivate 48 hours, then add MTT (5mg/mL) 20 μ L for 37 DEG C, continue cultivation and stop after 4 hours cultivating, take out 96 orifice plates, carefully remove liquid in hole, every hole adds 150 μ L DMSO, and concussion shakes up 10 minutes, and crystallisate is fully dissolved.Survey its OD value under microplate reader 490nm wavelength, and calculate inhibiting rate.
Calculation formula: inhibiting rate=(1-medicine hole OD/ blank well OD) × 100%.
3) statistical study
All experimental result mean ± standard deviations represent, adopt SPSS 13.0 statistical software to process experimental data, analytical procedure adopts variance analysis, with P ﹤ 0.05 for significant difference.
RCE-4 is on the impact of normal cell marc-145 and mdck cell cytotoxic activity
Tested by Fig. 7 MTT and show, RCE-4 shows less cytotoxicity, its IC to marc-145 and mdck cell 50be respectively 26.29 and 19.49 μMs.Experimental result shows, RCE-4 has more weak cytotoxicity to normal cell.
RCE-4 is to H 22with the impact of HepG2 human liver cancer cell cytotoxic activity
From Fig. 8 test-results, RCE-4 is to human liver cancer cell H 22restraining effect clearly is all had, its effect IC of 24 hours with HepG2 50be respectively 3.721 μMs, 12.034 μMs.
Experimental example 2
Below in conjunction with specific embodiment, illustrate the anti-liver cancer application of RCE-4 in animal level of the embodiment of the present invention 1.
RCE-4 is to lotus H 22the impact of the anti-tumor activity of knurl mouse.
RCE-4 is to lotus H 22the antitumor test of ascitic tumor mouse
Test method
1) experiment material
Female KM small white mouse (18 ~ 22g), purchased from SanXia University's Experimental Animal Center, animal productiong credit number: SCXK (Hubei Province) 2011-0012.H22 tumor cell line, purchased from Wuhan University's China typical culture collection center, to be gone down to posterity preservation with utilizing key lab of Hubei Province by SanXia University's Natural products research.
2) foundation of cell cultures and animal model
H 22cell cultures is in containing 10% newborn calf serum, 10 5in the RPMI-1640 substratum of U/L penicillin, 100mg/L Streptomycin sulphate, be placed in 37 DEG C, CO 2volume fraction is cultivate under the saturated humidity condition of 5%, treats H 22cell attachment grows, and cell in vegetative period of taking the logarithm, prepares single cell suspension, adjustment density to 5 × 10 6individual/mL, abdominal injection 0.2mL/ are only.By inoculation well-grown H after 7 days 22mouse, cervical dislocation is put to death, and after skin of abdomen sterilization, strip off skin of abdomen, extracts milky dense thick ascites with asepsis injector, put into sterile chamber, put in ice cube and preserve.Separately take a morsel ascites, is placed in the test tube being added with heparin, and for observation of cell form and cell counting, tumor cell number is more than 97% can to use.Ascites stroke-physiological saline solution 1:4 dilution, makes tumor cell number be 5 × 10 6individual/mL.Tumor cell suspension should be oyster white translucent, if there is bloody ascites to use, and every mouse web portion injection 0.2mL ascites.
3) grouping and administration
Female KM small white mouse (18 ~ 22g) 30, mouse peritoneal inoculation H 22be divided into group at random after tumour cell 24h, often organize 6.Be respectively model group, RCE-4 group (50,100,200mg/kg), blank group gavage gives 0.5% carboxymethylcellulose sodium solution of respective volume, every day 1 time, continuous 2 weeks.
4) RCE-4 is to lotus H 22the impact of ascitic tumor Bearing Mice Life Prolongation rate
Often organize 6 mouse, calculate from inoculation day, observe mouse hair color, mobility, measure the abdominal circumference of each test group mouse and weigh; Record the survival time of each group of mouse, draw survival curve.With increase in life span, [increase in life span (%)=[(administration group the average survival time number of days-model group the average survival time number of days)/model group the average survival time number of days] represents ascitic type tumor efficiency.
5) statistical study
All experimental result mean ± standard deviations represent, all data analyses are carried out on SPSS13.0 statistical package, carry out the comparison of many group differences with one-way analysis of variance, compare the difference of mean between two groups with Dunnett-t inspection; P<0.05 thinks that difference has significant difference.
Test-results
1) RCE-4 is to lotus H 22the impact of ascitic tumor mouse generalized case
3 groups to RCE-4 test mice during administration and in the observation period without changes such as hair color obfuscation, depilations, and stool and urine is normal.
2) RCE-4 is to H 22the impact of lotus ascitic tumor Mouse Weight
From table 1, the weight of model group all obviously increases, and compares have significant difference (P < 0.01) with normal group; After RCE-4 (50,100,200mg/kg) treatment, its weight is not made significant difference, compares there was no significant difference (P > 0.05) with model.
Table 1 RCE-4 is to H 22the impact of lotus ascitic tumor Mouse Weight
Compare with the normal group of same time point #p < 0.05, ##p < 0.01; * P < 0.05, * * P < 0.01 is compared with the model group of same time point.
3) RCE-4 is to H 22the impact of lotus ascitic tumor mouse abdominal circumference
From table 2, the abdominal circumference of model group all obviously increases, and compares have significant difference (P < 0.01) with normal group; After RCE-4 (50,100,200mg/kg) treatment, all have good reducing effect to abdominal circumference, wherein RCE-4100 and 200mg/kg dosage group compares with model group and has significant difference (P < 0.05).
Table 2 RCE-4 is on the impact of H22 lotus ascitic tumor mouse abdominal circumference
Compare with the normal group of same time point #p < 0.05, ##p < 0.01; * P < 0.05, * * P < 0.01 is compared with the model group of same time point.
4) RCE-4 is to H 22the impact of lotus ascitic tumor mouse survival time
Model group during administration the 5th day, not dead one of middle dosage component, other groups have no dead; Dead 1 of model group during administration the 6th day, other groups have no dead; During administration the 7th day, each group is showed no death; Model group group during administration the 8th day, each dead 1 of middle dosage group, other groups have no dead; During administration the 9th day, RCE-4 high dose group, model group, in, low dose group dead 1,2,1,3 respectively; During administration the 10th day, in RCE-4 high dose group, model group, RCE-4, low dose group dead 2,1,3,3 respectively; During administration 11 days, RCE-4 high dose group dead 3 (Fig. 9).
Conclusion (of pressure testing)
By RCE-4 to H 22lotus ascitic tumor mouse experiment is known, and RCE-4 can suppress lotus H 22ascitic tumor mouse suppresses generation and the survival time of ascites.
Experimental example 3
RCE-4 is to lotus H 22the antitumor test of solid tumor mouse
Test method
1) experiment material
Female KM small white mouse (18 ~ 22g), purchased from SanXia University's Experimental Animal Center, animal productiong credit number: SCXK (Hubei Province) 2011-0012.H 22tumor cell line, purchased from Wuhan University's China typical culture collection center, to be gone down to posterity preservation with utilizing key lab of Hubei Province by SanXia University's Natural products research.
2) foundation of cell cultures and animal model
H 22cell cultures is in containing 10% newborn calf serum, 10 5in the RPMI-1640 substratum of U/L penicillin, 100mg/L Streptomycin sulphate, be placed in 37 DEG C, CO 2volume fraction is cultivate under the saturated humidity condition of 5%, treats H 22cell attachment grows, and cell in vegetative period of taking the logarithm, prepares single cell suspension, adjustment density to 5 × 10 6individual/mL, abdominal injection 0.2mL/ are only.By inoculation well-grown H after 7 days 22mouse, cervical dislocation is put to death, and after skin of abdomen sterilization, strip off skin of abdomen, extracts milky dense thick ascites with asepsis injector, put into sterile chamber, put in ice cube and preserve.Separately take a morsel ascites, is placed in the test tube being added with heparin, and for observation of cell form and cell counting, tumor cell number is more than 97% can to use.Ascites stroke-physiological saline solution 1:4 dilution, makes tumor cell number be 5 × 10 6individual/mL.Tumor cell suspension should be oyster white translucent, if there is bloody ascites to use, every mouse is in right side rear dorsal sc injection 0.2mL ascites, and obvious skin mound appears in injection site.After tumour grows, every day measures gross tumor volume, and after 7 days, all mouse all occur being greater than 100mm 3subcutaneous nodule, Transplanted tumor model is successfully established.
3) experiment grouping
The mouse that after inoculation, solid tumor is successful by about 7 days is divided into 5 groups at random: model group, RCE-4 (50,100,200mg/kg) group and positive drug capecitabine group (755mg/kg), often organize gavage and give corresponding medicine, every day 1 time, successive administration 4 weeks, separately sets 6 mouse of non-replication entity knurl as normal group.
4) RCE-4 is to H 22the impact of lotus solid tumor mouse generalized case
Every day observation experiment mouse dietary amount, amount of drinking water, observe every day such as the mental status of mouse, motility, reaction, tumor growth situation, stool form etc.
5) RCE-4 is to H 22the impact of lotus solid tumor Mouse Weight
Measure 2 Mouse Weights weekly.
6) RCE-4 is to H 22the impact of solid tumor mouse tumor volume
After grouping, the 2nd day (d1) starts length (L) and the width (W) of measuring each group of transplanted tumor in nude mice, within every 3 days, measures 1 time, gross tumor volume (V)=(L × W 2)/2, using observation time (d) as transverse axis, the average (mm of knurl volume 3) be longitudinal axis drafting growth of xenografted curve.
7) RCE-4 is to H 22the impact of solid tumor mouse tumor weight and tumour inhibiting rate
After drug withdrawal, 24h is by mouse, and mouse is plucked eyeball and gets blood, and then de-cervical approach is put to death, and completely strips out tumor tissue, and observe each group of knurl block situation and infiltrate with or without surrounding tissue, routine is cut open the belly and observed with or without ascites and other Organ relative weight, claims tumor weight, calculates tumour inhibiting rate.Tumour inhibiting rate=[1-(treatment group knurl weight/control group knurl weight)] × 100%.
8) RCE-4 is to H 22the impact of solid tumor mouse tumor techtology
After getting knurl, 10% neutral formalin is liquid-solid fixed, paraffin embedding, 4 μm of serial section, and HE dyes, and under light microscopic, pathologic examination respectively organizes the morphological change of mice-transplanted tumor tissue.
9) RCE-4 is to H 22the impact of solid tumor mouse blood index
After drug withdrawal, 24h is by mouse, and mouse is plucked eyeball and gets blood, with Blood cell analyzer detection blood rbc, white corpuscle and classification thereof, thrombocyte, the determination of tube method clotting time.
10) RCE-4 is to H 22the impact of solid tumor mouse blood biochemical indicator
Every mouse mouse residue whole blood is with 3500r/min, centrifugal 10min, get serum, detect gpt (ALT), glutamic-oxal(o)acetic transaminase (AST), total bilirubin (TBIL), total protein (TP), serum albumin (ALB), alkaline phosphatase (ALP), total cholesterol (CHOL), blood urea nitrogen (BUN), creatinine (CREA), blood sugar (Glu) content in serum with automatic clinical chemistry analyzer.
11) RCE-4 is to H 22the impact of solid tumor mouse blood amynologic index
Every mouse mouse residue whole blood is with 3500r/min, and centrifugal 10min, gets serum, measures the level of IgA, IgM in blood with Immunity transmission turbidity.
11) RCE-4 is to H 22the morphologic impact of solid tumor mouse tissue
Core, liver, spleen, lung, kidney, brain, suprarenal gland, thymus gland, stomach, large intestine, small intestine, ovary, internal organs 10% formaldehyde such as uterus fixes, conventional paraffin embedding, section, HE dyeing, light microscopy checking, all the other experimental rats do same inspection in drug withdrawal after 2 weeks.
12) statistical study
All experimental result mean ± standard deviations represent, all data analyses are carried out on SPSS13.0 statistical package, carry out the comparison of many group differences with one-way analysis of variance, compare the difference of mean between two groups with Dunnett-t inspection; P<0.05 thinks that difference has significant difference.
Test-results
1) RCE-4 is to H 22the impact of lotus solid tumor mouse generalized case
H 22within 3rd day, rise after cell inoculation, there is the transplanted tumor of soya bean size in KM right side of mice back leg back, 25 mouse 20 one-tenth knurls, mean tumour volume size there was no significant difference (P > 0.05) between each group before medication.After mouse becomes knurl and administration, model group, the food-intake of RCE-4 (50,100,200mg/kg) and positive drug capecitabine (755mg/kg), drinking-water, stool and urine are all normal, the mental status is good, movable normal, reaction is quick, and skin color is as usual, and transplanted tumor tissue has no ulceration.
2) RCE-4 is on the impact of H22 lotus solid tumor Mouse Weight
From table 3, at treatments period, the weight of model group all obviously increases, and compares have significant difference (P < 0.05) with normal group; After RCE-4 (50,100,200mg/kg) and the treatment of capecitabine group, from the 11st day, there is reduction in various degree in the body weight of each administration group, compares have significant difference (P < 0.05 & P < 0.01) with model.
Table 3 RCE-4 is to H 22the impact of solid tumor Mice Body quality
Continued 3
Compare with the normal group of same time point #p < 0.05, ##p < 0.01; * P < 0.05, * * P < 0.01 is compared with the model group of same time point.
3) RCE-4 is to H 22the impact of solid tumor mouse tumor volume
Model group mouse inoculation H 22after hepatoma cell strain, knurl bulk-growth is rapid, after RCE-4 (50,100,200mg/kg) and capecitabine treatment, although gross tumor volume continues to increase, the speed of growth obviously slowed down, from after treatment the 15th day, compare with model group group, there is significant difference (P < 0.05 or P < 0.01), and along with the prolongation for the treatment of time, its curative effect further obviously (see table 4).
Table 4 RCE-4 is to H 22the impact of solid tumor mouse tumor volume
Continued 4
* P < 0.05, * * P < 0.01 is compared with the model group of same time point.
4) RCE-4 is to H 22the impact of solid tumor mouse tumor weight and tumour inhibiting rate
From table 5 and, model group knurl volume and the heavy obviously increase of knurl, after RCE-4 (50,100,200mg/kg) and capecitabine treatment, its knurl is heavy obviously to be reduced, and compares have significant difference (P < 0.01) with model group; Tumour inhibiting rate increases gradually with the increase of dosage, compares have significant difference (P < 0.01) with model group.
Table 5 RCE-4 is to H 22the impact of solid tumor mouse tumor weight and tumour inhibiting rate
* P < 0.05, * * P < 0.01 is compared with model group.
6) RCE-4 is to H 22the impact of solid tumor mouse blood index
As shown in Table 6, model group mouse blood index as: WBC, RBC, HGB, PLT, MON%, NEU, BASO, EOS and EOS% etc. are badly damaged, and compare have significant difference (P < 0.01) with normal group; After RCE-4 (50,100,200mg/kg) treatment, hematological indices has improvement to a certain degree, and capecitabine is not obvious to this improvements, some index deterioration in various degree in addition on the contrary.
Table 6 RCE-4 is to H 22the impact of solid tumor mouse blood index
Continued 6
Continued 6
Continued 6
Continued 6
Continued 6
Compare with normal group #p < 0.05, ##p < 0.01; * P < 0.05, * * P < 0.01 is compared with the model group of same time point.7) RCE-4 is to H 22the impact of solid tumor mouse blood biochemical indicator
As shown in Table 7, there is abnormal rising in model group mouse blood biochemical indicator, compares have significant difference (P < 0.01) with normal group; After RCE-4 (50,100,200mg/kg) treatment, hematological indices has improvement to a certain degree, and capecitabine has deterioration side effect to blood parameters.
Table 7 RCE-4 is to H 22the impact of solid tumor mouse blood biochemical indicator
Continued 7
Compare with normal group #p < 0.05, ##p < 0.01; * P < 0.05, * * P < 0.01 is compared with the model group of same time point.
8) RCE-4 is to H 22the impact of solid tumor mice-transplanted tumor techtology
As shown in Figure 10, blank group tumor cell core increases, and core is in two or multiple, and size, form differ, and heteromorphism is obvious, nuclear hyperchromatism, and kernel is clear, and chromatin increases, thickening of nuclear membrane; RCE-4 treatment group has more oncocyte shrinkage, and kytoplasm is intensive, and the necrotic zone of the red dye of homogeneous, appears in nuclear chromatin limit collection, visible cell apoptosis, and with the increase of RCE-4 dosage, the necrotic zone of tumor tissue increases gradually, and apoptotic cell increases gradually.
9) RCE-4 is to H 22the impact of solid tumor mouse main organs techtology
Histopathology detected result shows, RCE-4 is to H 22solid tumor mouse brain, the heart, liver, spleen, lung, kidney, suprarenal gland, stomach, small intestine, ovary, uterus naked eyes and microscopy all change without pathologic, show that RCE-4 is without significantly effect (Figure 11).
Experiment conclusion
By RCE-4 to H 22lotus solid tumor mouse experiment is known, and RCE-4 can suppress lotus H 22solid tumor mouse tumor volume and knurl weight, the normal blood index of mouse, blood parameters and techtology all be there is no to the side effect of significance, although and positive drug capecitabine tumor killing effect is slightly better than RCE-4, it is good at high dosage, strong side effect.Therefore, in contrast, RCE-4 of the present invention has good advantage.
In sum, RCE-4 has more weak cytotoxicity to normal cell, and to human liver cancer cell H 22with HepG2, there is stronger cytotoxicity; To H 22lotus ascitic tumor and solid tumor mouse all have significant antitumor action, effectively can extend the survival time of mouse, reduce the abdominal circumference of lotus ascitic tumor mouse and knurl volume, the knurl weight of solid tumor mouse, and liquid normal blood index, blood parameters and techtology all be there is no to the side effect of significance.Experimental result shows, no matter RCE-4 is the effect that cell levels or animal level all show certain anti-liver cancer, compared with the current anticarcinogen used clinically, has effect suitable, but the advantage that toxic side effect is little.
Above-described embodiment and experimental example illustrate invention has been the present invention.Any those skilled in the art may utilize the technology contents of above-mentioned announcement to be changed to the Equivalent embodiments of equal change.Everyly do not depart from the present invention program's content, according to technical spirit of the present invention to any simple modification made for any of the above embodiments or equivalent variations, all drop in protection scope of the present invention.

Claims (10)

1. a method of steroidal saponin RCE-4 is prepared in separation, comprises step below:
A) use solvent extraction Pink Reineckea Herb, filter, concentrated;
B) enriched product water dissolution a) step obtained, is extracted with ethyl acetate for some time, preferred ultrasonic extraction 1 ~ 4 hour;
C) b) step is extracted the acetic acid ethyl ester extract obtained to concentrate, gradient elution separation with chloroform-methanol, collect cut 1-16, by the wherein cut 15 of gained through gel chromatography separation purifying.
2. method according to claim 1, wherein step a) in extraction conditions be below one or more:
I) Extraction solvent is water, ethanol or its mixture, preferably 60 ~ 95% ethanol, such as 75% ethanol;
Ii) Pink Reineckea Herb is clean, dry pulverised form;
Iii) extracting mode is that atmospheric pressure reflux is extracted under 40 ~ 70 DEG C such as 65 DEG C of conditions; With
Iv) under 40 ~ 70 DEG C of conditions, concentrated by rotary evaporation.
3. method according to claim 1 and 2, wherein step c) in separation condition be below one or more:
L) acetic acid ethyl ester extract that above-mentioned b) step obtains is carried out chromatographic separation;
M) chloroform-methanol gradient is permanent gradient 15:1; With
N) gel is dextrane gel, preferred hydroxypropyl dextrane gel.
4. the method according to any one of claim 1-3, uses petroleum ether extraction before being wherein extracted with ethyl acetate in b).
5. method according to claim 4, wherein step c) described in chromatographic separation and purification after also comprise a re-crystallization step, obtain white, needle-shaped crystals.
6. the method according to any one of claim 1-5, wherein step a) described in solvent be 95% ethanol.
7. method according to claim 6, wherein step a) in also comprise and reclaim the step of ethanol; Preferably, after filtering extracting solution, under about 55 DEG C of conditions, reclaim ethanol.
8. the steroidal saponin RCE-4 that the method according to any one of claim 1-7 obtains.
9. a pharmaceutical composition, it contains steroidal saponin RCE-4 according to claim 8.
10. pharmaceutical composition according to claim 9, wherein also comprises autophagy inhibitor 3-MA, optionally also containing other cancer therapy drug activeconstituentss and/or carrier, thinner or vehicle;
More preferably, in wherein said pharmaceutical composition, RCE-4 concentration is between 2 ~ 4 μMs, preferably 4 μMs.
CN201510398649.8A 2015-07-08 2015-07-08 Separation and preparation method of compound with antineoplastic activity Pending CN104987357A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111700963A (en) * 2020-08-10 2020-09-25 云南中医药大学 Application of pink reineckea herb extract

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
NA HAN, ET AL.: "Steroidal Glycosides from Reineckia carnea herba and Their Antitussive Activity", 《PLANTA MED》 *
ZHONG-QUAN ZHANG, ET AL.: "Two New Spirostanol Saponins from Reineckia carnea", 《HELVETICA CHIMICA ACTA》 *
王桂萍等: "保护性自噬对吉祥草皂苷RCE-4诱导的人宫颈癌Ca Ski细胞凋亡的抑制作用", 《中药药理与临床》 *

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111700963A (en) * 2020-08-10 2020-09-25 云南中医药大学 Application of pink reineckea herb extract

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