CN1307131C - Antineoplastic extract of Chinese fan palm and its preparation method and uses - Google Patents
Antineoplastic extract of Chinese fan palm and its preparation method and uses Download PDFInfo
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Abstract
The present invention relates to external antineoplastic extract of Chinese fanpalm leaf. The chemical components comprise diosgenin, beta-sitosterol, sitosterol, beta-daucesterol, n-haxacosyl alcohol, etc. In the extraction method, the root, seed and leaf of fresh collected Chinese fanpalm leaf are pulverized, solid phases and liquid phases are lixiviated, filtered and separated with etanol, etanol is recovered by distilling the liquid phases, black extract paste A is obtained, the solid phases are filtered and separated, aqueous extraction and concentration are carried out, extract paste obtained through alcohol extaction and concentrated solution obtained through aqueous extraction are merged, and monomeric compounds are obtained through the extraction of ethyl acetate and petroleum ether and the recrystallization of silica gel columns. Pharmacology verifies that the extract of Chinese fanpalm leaf has inhibiting effect on the growth of seven tumor cell strains.
Description
Technical field
The present invention relates to a kind of method of extracting Chinese medicine effective antitumor composition from plant, is method and the application that raw material extracts antitumor effective constituent with Chinese fan palm (comprising fruit, stem, root, leaf) plant particularly.
Background technology
Malignant tumour is the principal disease of harm humans health and lives.The whole world has the people about 5,000,000 to die from malignant tumour every year approximately according to estimates.Over nearly more than 20 years, because the continuous deterioration of ecotope, the anxiety of people's life and irregular, and transmission of disease, the sickness rate of malignant tumour is in rising trend, and becomes the second largest deadly cause of disease after cardiovascular disorder.Therefore, the annual lot of manpower and material resources that drops in the whole world is used to research and develop the novel drugs of preventing curing cancers.In recent years, the pharmacology expert is except the research Western medicine, also Chinese medicine has been carried out a lot of researchs, the mechanism that malignant tumour forms is also inquired into, the invention of modern techniquies such as combinatorial chemistry, genetically engineered and application have been quickened the malignant tumor medicine development process.Particularly be that lead compound searching new drug has vast potential for future development at present with the active skull cap components.At least assessed the anti-tumor activity of kind of higher plant surplus in the of 40,000 up to now.According to the foreign data introduction; hundreds of kind PTS has been developed in the whole world; the a variety of natural plant extracts that belong to are wherein arranged; in recent years; domestic researchist has carried out extensive and deep research to the antitumous effect of traditional Chinese medicine and traditional herbal medicine; and found large quantities of plant and biologies in succession with antitumous effect, as Pacific yew, Chinese Ramulus et folium taxi cuspidatae, Chinese fan palm, argy wormwood, ginkgo, camplotheca acuminata, glossy ganoderma, Herba Selaginellae Doederleinii, Fructus Cnidii, Herba Lophatheri, Mao Ju, cow-bezoar, Pseudobulbus Cremastrae Seu Pleiones, vervain, Spica Prunellae., Herba Agrimoniae, wartwort, Turkey-galls, Fructus Fici, beet (root), open up tree root, big carry, Chinese juniper skin oak, Williams Elder Twig, propolis, Root of Indigowoad, Bai Yepi, italian-jobi jen, Huang Mao, vanilla etc., people have isolated the activeconstituents that antitumous effect is arranged from these plants.
In above-mentioned these plants, what people studied morely is Ramulus et folium taxi cuspidatae, camplotheca acuminata, glossy ganoderma, ginkgo, but much plant is owing to poor growth, and natural resources shortage is difficult to satisfy the demands.And in south, people begin plant Palmae Chinese fan palm is studied in recent years, find that the material that the Chinese fan palm fruit extracts has the composition that prevents and treat cancer, are a kind of efficient, nontoxic more satisfactory cancer therapy drugs.
Chinese fan palm is plant Palmae Chinese fan palm Livistona chinensis R.Br., another name: flabellum certain herbaceous plants with big flowers, palm-leaf fun wood, Chinese fanpalm seed, Chinese fan palm belongs to aiphyllium, a kind of wild or tame aiphyllium for the Palmae certain herbaceous plants with big flowers.It originates in south China, and there are a plurality of kinds on ground such as Guangxi, Guangdong, Fujian, and the plant height that has reaches 30-50 centimetre, is longer than the Wingdale mountain area, and the plant height that has reaches 10-20 rice, and individual plant is upright.Petiole is about 2 meters, and the hook thorn is arranged, and does and wraps up in brown skin, is used for the townscape tree, and leaf is fan-shaped, about 2 meters of footpath, and drastic crack is to the middle part, and top sliver ribbon is sagging.Plant is stood straight with a canopy of leaves, and blade Rejoice such as silk are graceful foliage plants.The seedling leafage is close, and crown rounding can be done greening in the potted landscape or the hall.The big available shade tree of tree and plant in waterside, garden etc. and locate is the exhibition scape common in the gardens and the seeds of shading.Tender leaf can be made palm-leaf fun, and Lao Ye covers cool canopy, penthouse in other words for braiding; The palm fibre micromicro is made seat cushion, brush, straw or palm-bark rain hat etc.; Handle also can or be made toothpick for braiding, and economic worth is quite high, and its growth cycle is long, cultivation easily, fruit yield height, aboundresources.
According to the record of relevant document, extracting effective components from Chinese fan palm (comprising fruit, root, leaf), to tumour particularly the malignant tumour illness good therapeutic action is arranged, some drug research departments develop Chinese fan palm as anti-cancer agent.
Through retrieval, we find the disclosed relevant Chinese fan palm of public publication or are main component with the Chinese fan palm as theing contents are as follows that antitumour drug is used:
1, the research of Fushengkang capsule quality standard [author] Huang Ruisong, Ye Yunfeng, Su Qing, Hu Qiuping, Qiu Jizhen, Huang Yongling [mechanism] Guangxi Nationality Medical ﹠ Parmaceutical Research Inst., Guangxi veterinary drug supervision institute, Colleges Of Traditional Chinese Medicine Of Guangxi, [periodical name] herbal medicine .2003,34 (8) .[digests] purpose sets up Fushengkang capsule quality control method.Method adopts tlc that Chinese fanpalm seed, Fructus Camptothecae Acuminatae and curcuma zedoary are carried out qualitative identification, adopts the HPLC method to measure the content of camptothecine.Chromatographic condition is: Dikma Diamonsil C18 post (250mm * 4.6mm, 5 Jing); Moving phase: methanol-water (55: 45); Flow velocity: 0.8mL/min: detect wavelength: 254nm.The thin-layer chromatography clear spot is concentrated as a result, and camptothecine has the good linear relation at 0.0506～0.2530 He, and average recovery rate is 96.90%, RSD-1.74%, (n=6).Conclusion method sensitivity, accurately, favorable reproducibility, specificity be strong, can be used as the capsular quality control standard of Fushengkang.
2, some unifacial leaf xylophyta fresh leaf extracts are removed DPPH free radical activity pre-test [author] Lu Ruili Hu Fenglin [mechanism] Agricultural University Of Anhui that permitted to grow into forest, [periodical name] biology magazine .2003,20 (6) .[digests] research free radical scavenging activity microplate reader method, free radical scavenging activity to the bright leaf of kind of unifacial leaf xylophyta surplus common 30 compares, find not equal genus, 80% methanol extract of the bright leaf of kindred plant does not have very big-difference to the removing activity of DPPH free radical, wherein for the Palmae 7 kind of plant fresh leaf extracts that try, being equivalent to bright leaf concentration is that 2.5mg/ml is when hatching 20min under 37 ℃, on average can reach 40.0% to 0.5mmol/L DPPH free radical scavenging activity, and Agavaceae 5 kind of plant on average only are 7.2%; Bright leaves such as thorn certain herbaceous plants with big flowers, the bamboo palm, muscle head bamboo, Chinese fan palm and palm have stronger free radical scavenging activity, and their free radical scavenging activities when being equivalent to bright leaf concentration and being 2.5mg/ml can reach 83.1%, 79.2%, 64.9%, 60.5% and 51.3% respectively.These seeds have certain potentiality to be exploited.
3, Herba Selaginellae Doederleinii and Chinese fanpalm seed are to the yellow just Qin Yan plum of the influence [author] of protein kinase C activity [periodical name] herbal medicine .1995,26 (8) .[digests] adopt the protein kinase C in the D E 52 column chromatography partial purification rats, studied the influence of Herba Selaginellae Doederleinii and Chinese fanpalm seed alcohol extract to protein kinase C activity.In order to prove the reliability of mensuration system, observed the influence of PXB (known protein kinase c inhibitor) to protein kinase C activity.Show: the Herba Selaginellae Doederleinii alcohol extract has had strong inhibitory effects to protein kinase C, and the Chinese fanpalm seed alcohol extract is 66.6% to the inhibiting rate of protein kinase C, reduces the drug level restraining effect and weakens; PXB has restraining effect to protein kinase C.
4, [author] He Xiaoyu is analyzed in the GC-MS coupling of fatty oil in the Chinese fan palm, Cui Jianguo, yellow initial rise, Zhong Zhenguo, department of chemistry of [mechanism] Guangxi Teachers College, Colleges Of Traditional Chinese Medicine Of Guangxi, [periodical name] chemical industry technology and exploitation .2003,32 (2) .[digests] adopt the GC-MS coupling technique to analyze fatty oil composition in the Chinese fan palm first, isolate 36 peaks altogether, and identify 23 kinds of compounds wherein.This fat main body of oil is Laurate ethyl, ethyl palmitate, linolenic acid, ethyl linoleate, 9-vaccenic acid acetoacetic ester etc.For the development and use of Chinese fan palm resource provide scientific basis.Studies show that the Chinese fan palm seed has the obvious suppression effect to the activity of protein kinase C.The inhibitor on cell proliferation of protein kinase C has restraining effect, and the anticarcinogen that some use clinically is exactly the inhibitor of protein kinase C as Ah mould family etc.China south is among the people with Chinese fan palm seed treatment cancer (as nasopharyngeal carcinoma), it is said suitable effect.Simultaneously, the domestic report that also has the former powder that utilizes the Chinese fan palm seed behind breaking cell wall, to extract to prepare carcinostatic agent.People such as external Pakistani Mala.V once carried out research to the composition of Chinese fan palm seed, therefrom isolate some polysaccharide, amino acid etc., and at present domesticly do not find that also the effective constituent in the seed to Chinese fan palm, root, the leaf separates, therefrom extract compound with physiologically active, and carry out structural characterization, the report of physiologically active test and structure activity study.
5, Chinese patent＜denomination of invention〉be used for the treatment of the Chinese medicine＜applicant of cancer〉Zhang Guangxin＜national provinces and cities〉Anhui (34)＜contact address〉No. 287 Hem Meisheng Transferred, Science and Technology Agency Building ,No. Anhui Province＜claims in Chaohu Road, Hefei City, Anhui Province〉be used for the treatment of the Chinese medicine of cancer, it is characterized in that having following each component: Colchicum autumnale, Chinese fan palm, glossy ganoderma, Vinca, camplotheca acuminata, Cephalotaxus fortunei, crocodile first, Herba Scutellariae Barbatae, black nightshade, Yanhusuo, Chinese blister beetle, Spreading Hedyotis Herb, Rabdosia rubescens, tupelo gum, flea stop, konjaku.＜summary〉be used for the treatment of the Chinese medicine of cancer, it is characterized in that having following each component: Colchicum autumnale, Chinese fan palm, glossy ganoderma, Vinca, camplotheca acuminata, Cephalotaxus fortunei, crocodile first, Herba Scutellariae Barbatae, black nightshade, Yanhusuo, Chinese blister beetle, Spreading Hedyotis Herb, Rabdosia rubescens, tupelo gum, flea stop, konjaku.The present invention effectively anticancer diffusion, kill cancer cells, suppress because the pain that cancer causes alleviates patient's misery.
6; Chinese patent＜denomination of invention〉pellet for dissolving tumor and strengthening body resistance＜applicant〉Ceng Guangsheng; Ning Weiwei＜national provinces and cities〉Hunan (43)＜contact address〉the medical expert's consultation portion＜contriver of Hengyang City Chinese materia medica association in No. 24 big neighs lane, south of the mountains road in the city of hengyang, hunan province〉Ceng Guangsheng＜claim〉a kind of pellet for dissolving tumor and strengthening body resistance; it is characterized in that preparing in the raw material; according to natural Calculus Bovis 0.5-1g; the precious 20-30g in the end; lichen 10-20g; wheat pseudo-ginseng 10-20g; Chinese fanpalm seed 10-20g; crab first 1-2 only; slug 3-5 bar; Radix Panacis Quinquefolii 5-10g; cun 0.1-0 of unit＜summary〉a kind of pellet for dissolving tumor and strengthening body resistance; with natural Calculus Bovis; end treasure; lichen; the wheat pseudo-ginseng; Chinese fanpalm seed; the crab first; slug; Radix Panacis Quinquefolii; unit is mixed cun by a certain percentage, is processed into powder or gets concentrated solution and produce.Have clearing heat and detoxicating, promoting blood circulation and removing blood stasis, softening and resolving hard mass, the strengthening vital QI to eliminate pathogenic factors effect had both been controlled the knurl knurl, auxiliary again human righteousness, the enhancing body activity is treated both principal and secondary aspect of disease, curative effect is obvious, compares existing cancer therapy drug, has increasing man's lifespan, reduces the special efficacy of mortality ratio.No contraindication, easy administration.
7, Chinese patent＜denomination of invention〉high effect nontoxic natural anti-cancer agent [application (patent) number] 97100103.0＜applicant〉fourth celebrating, fourth record, fourth mirror＜national provinces and cities〉Beijing (11)＜contact address〉404 Room, Cheng Zeyuan the 108th buildings, Beijing, BeiJing, Beijing City＜postcode〉100871＜contriver〉fourth celebrating, fourth record＜summary〉the present invention relates in healthcare products and the field of medicaments preventing cancer, treat an important breakthrough on the cancer.This product is from the natural or artificial former powder that extracts behind breaking cell wall the Palmae Chinese fan palm fruit of training that carries, has rich nutrient substances, have the specific function of preventing cancer, treatment cancer clinically, and can improve body's immunological function effectively, without any side effects.The present invention also is applicable to healthcare products or the Chinese medicine producer that produces all kinds of preparations of the former powder of Chinese fan palm.
Recognize that from our above-mentioned result for retrieval the research medicinal about Chinese fan palm has had some bibliographical informations with utilization.The composition to Chinese fan palm that has is studied, and bright leaf such as prompting Chinese fan palm etc. has stronger free radical scavenging activity and to the restraining effect of protein kinase C; Adding in natural nutrition type healthcare products and composite medicine of having has Chinese fan palm, as anti-cancer, anticancer useful composition.But do not see that the effective constituent of prompting in pair Chinese fan palm plant is arranged carries out in the separating process, what disclose that it has anti-cancer, anticancer physiologically active is that compounds.
Summary of the invention
The objective of the invention is to carry out assay determination, determine the separation method of its extract then, respectively effective constituent is carried out physiologically active test and structure activity study again by the composition of first root, stem, seed, leaf to the Chinese fan palm plant.Adopt compounds effective to be prepared into anti-tumor medicinal preparation at last.
Chinese fan palm plant effective antitumour of the present invention (anticancer) medical compounds comprises following several:
1, diosgenin (diosgenin)
2, β-Gu Zaichun (β-sitosterol)
3, stigmasterone (stigmasterone)
4, β-daucosterol (β-daucosterol)
5, positive hexacosyl alcohol (hexacosyl alcohol)
In addition, we go back mixture and some triterpene compounds that extraction and analysis obtains tricin (tricin), apigenin (apinin), Vitexin (C211120010), unsaturated fatty acids.
More than the molecular formula of several compounds be respectively: diosgenin (diosgenin), molecular formula are C27H4203; (β-sitosterol), molecular formula is C29H500 to β-Gu Zaichun; Stigmasterone (stigmasterone), molecular formula are C29H480; (β-daucosterol), molecular formula is C35H6006 to β-daucosterol; Positive hexacosyl alcohol (hexacosyl alcohol), molecular formula is C26H540).
Above-claimed cpd of the present invention is by infrared spectra, mass spectrum, and proton nmr spectra, carbon spectrum, hydrocarbon relevant spectrum, electronic spectrum is measured structure.
Above-claimed cpd of the present invention is that extract is separated from the root of Chinese fan palm plant, seed, leaf.Concrete extracting method is as follows:
(1) root, seed, the leaf of fresh collection or air dried Chinese fan palm plant is ground into particle below 3 millimeters, with the ethanol lixiviate of pressing 1-3 times of 70-97% of Chinese fan palm plant weight (volumetric concentration) 2-10 hour, filter to isolate solid phase (dregs of a decoction) and liquid phase (Chinese fan palm ethanolic soln).
(2) ethanol is reclaimed in liquid phase (Chinese fan palm ethanolic soln) distillation, obtains the lixiviate cream A of black.
(3) solid phase that filters to isolate (dregs of a decoction) is pressed extraordinarily poach twice of Chinese fan palm plant weight 1-3, and each 0.5-2 hour, filter, waste, reconcentration become moisture concentrated solution B below 50%.
(4) merge alcohol extracting lixiviate cream A and water extracting liquid B, obtain merging intermediates C;
(5) ethyl acetate is added among the intermediates C, and be heated to 50-60 ℃, extract 12 hours three times repeatedly, obtain Chinese fan palm ethyl acetate phase crude extract behind the concentrating under reduced pressure, crude extract purification by silica gel column chromatography crude product then, recycle silicon offset plate chromatography, recrystallization draws monomeric compound, obtains β-Gu Zaichun and n-hexacosane alcohol.
(6) sherwood oil is added among the intermediates C, extract 12 hours three times repeatedly, obtain sherwood oil phase crude extract behind the concentrating under reduced pressure, crude extract purification by silica gel column chromatography crude product then, recycle silicon offset plate chromatography, recrystallization draw the separation of monomeric compound extract phase and obtain compound diosgenin (diosgenin); Stigmasterone (stigmasterone); β-daucosterol (β-daucosterol).
(7) monomeric compound with (5) and (6) step passes through infrared spectra, mass spectrum, and proton nmr spectra, carbon spectrum, hydrocarbon relevant spectrum, electronic spectrum is measured structure.
(8) respectively above-claimed cpd is carried out antitumor test.Found that: the effect of antitumour activity is arranged as follows by size:
Diosgenin compound＞steroid compound＞fatty alkanoic acid compounds＞triterpene compound.The reason of antitumour activity may to contain basic spiral shell steroid structure relevant with diosgenin, or relevant with the group of steroid compound, fatty alkanoic acid compounds.
Other unsaturated fatty acids can adopt carbon spectrum and gas chromatography-mass spectrum, proton nmr spectra to analyze.
Other steroid compound also can adopt thin-layer chromatography, hydrogen spectrum, carbon spectrum etc. to analyze.
Embodiment
Below be embodiments of the invention:
Root, seed, the leaf of the Chinese fan palm plant of fresh collection is ground into particle below 3 millimeters, with the ethanol lixiviate of pressing Chinese fan palm plant weight 2 times 70% (volumetric concentration) 7 hours, filter to isolate the dregs of a decoction and Chinese fan palm ethanolic soln, ethanol is reclaimed in the distillation of Chinese fan palm ethanolic soln, obtain the lixiviate cream A of black; The solid phase that filters to isolate (dregs of a decoction) is pressed extraordinarily poach twice of Chinese fan palm plant weight 1, and each 0.5-2 hour, filter, waste, reconcentration become moisture concentrated solution B below 50%; Merge alcohol extracting lixiviate cream A and water extracting liquid B, obtain merging intermediates C;
Ethyl acetate is added among the intermediates C, and be heated to 50-60 ℃, extract 12 hours three times repeatedly, obtain Chinese fan palm ethyl acetate phase crude extract behind the concentrating under reduced pressure, crude extract purification by silica gel column chromatography crude product then, recycle silicon offset plate chromatography, recrystallization draws monomeric compound, identify through spectrum and nuclear magnetic resonance spectrum, obtain β-Gu Zaichun; Perhaps identify, determine that molecular formula is C26H540 and determine that its structure is positive 26 carbon alcohol through carbon spectrum and hydrogen spectrum structure.
Sherwood oil is added among the intermediates C, extract 12 hours three times repeatedly, obtain sherwood oil phase crude extract behind the concentrating under reduced pressure, crude extract purification by silica gel column chromatography crude product then, recycle silicon offset plate chromatography, recrystallization draw the monomeric compound extract phase to be separated, and monitors at thin-layer chromatography, measure through hydrogen spectrum and carbon spectrum, obtain the compound diosgenin; Stigmasterone; β-daucosterol.
Root, seed, the leaf of the Chinese fan palm plant of fresh collection is ground into particle below 3 millimeters, with the ethanol lixiviate of pressing Chinese fan palm plant weight 2 times 80% (volumetric concentration) 6 hours, filter to isolate the dregs of a decoction and Chinese fan palm ethanolic soln, ethanol is reclaimed in the distillation of Chinese fan palm ethanolic soln, obtain the lixiviate cream A of black; The solid phase that filters to isolate (dregs of a decoction) is pressed extraordinarily poach twice of Chinese fan palm plant weight 2, and each 0.5-2 hour, filter, waste, reconcentration become moisture concentrated solution B below 50%; Merge alcohol extracting lixiviate cream A and water extracting liquid B, obtain merging intermediates C;
Ethyl acetate is added among the intermediates C, and be heated to 50-60 ℃, extract 12 hours three times repeatedly, obtain Chinese fan palm ethyl acetate phase crude extract behind the concentrating under reduced pressure, crude extract purification by silica gel column chromatography crude product then, recycle silicon offset plate chromatography, recrystallization draws monomeric compound, identify through spectrum and nuclear magnetic resonance spectrum, obtain β-Gu Zaichun; Perhaps identify, determine that molecular formula is C26H540 and determine that its structure is positive 26 carbon alcohol through carbon spectrum and hydrogen spectrum structure.
Sherwood oil is added among the intermediates C, extract 12 hours three times repeatedly, obtain sherwood oil phase crude extract behind the concentrating under reduced pressure, crude extract purification by silica gel column chromatography crude product then, recycle silicon offset plate chromatography, recrystallization draw the monomeric compound extract phase to be separated, and monitors at thin-layer chromatography, measure through hydrogen spectrum and carbon spectrum, obtain the compound diosgenin; Stigmasterone; β-daucosterol.
Root, seed, the leaf of the Chinese fan palm plant of fresh collection is ground into particle below 3 millimeters, with the ethanol lixiviate of pressing Chinese fan palm plant weight 2 times 95% (volumetric concentration) 4 hours, filter to isolate the dregs of a decoction and Chinese fan palm ethanolic soln, ethanol is reclaimed in the distillation of Chinese fan palm ethanolic soln, obtain the lixiviate cream A of black; The solid phase that filters to isolate (dregs of a decoction) is pressed extraordinarily poach twice of Chinese fan palm plant weight 2, and each 0.5-2 hour, filter, waste, reconcentration become moisture concentrated solution B below 50%; Merge alcohol extracting lixiviate cream A and water extracting liquid B, obtain merging intermediates C;
Ethyl acetate is added among the intermediates C, and be heated to 50-60 ℃, extract 12 hours three times repeatedly, obtain Chinese fan palm ethyl acetate phase crude extract behind the concentrating under reduced pressure, crude extract purification by silica gel column chromatography crude product then, recycle silicon offset plate chromatography, recrystallization draws monomeric compound, identify through spectrum and nuclear magnetic resonance spectrum, obtain β-Gu Zaichun; Perhaps identify, determine that molecular formula is C26H540 and determine that its structure is positive 26 carbon alcohol through carbon spectrum and hydrogen spectrum structure.
Sherwood oil is added among the intermediates C, extract 12 hours three times repeatedly, obtain sherwood oil phase crude extract behind the concentrating under reduced pressure, crude extract purification by silica gel column chromatography crude product then, recycle silicon offset plate chromatography, recrystallization draw the monomeric compound extract phase to be separated, and monitors at thin-layer chromatography, measure through hydrogen spectrum and carbon spectrum, obtain the compound diosgenin; Stigmasterone; β-daucosterol.
Root, seed, the leaf of the Chinese fan palm plant of fresh collection is ground into particle below 3 millimeters, with the ethanol lixiviate of pressing Chinese fan palm plant weight 2 times 97% (volumetric concentration) 3 hours, filter to isolate the dregs of a decoction and Chinese fan palm ethanolic soln, ethanol is reclaimed in the distillation of Chinese fan palm ethanolic soln, obtain the lixiviate cream A of black; The solid phase that filters to isolate (dregs of a decoction) is pressed extraordinarily poach twice of Chinese fan palm plant weight 3, and each 0.5-2 hour, filter, waste, reconcentration become moisture concentrated solution B below 50%; Merge alcohol extracting lixiviate cream A and water extracting liquid B, obtain merging intermediates C;
Ethyl acetate is added among the intermediates C, and be heated to 50-60 ℃, extract 12 hours three times repeatedly, obtain Chinese fan palm ethyl acetate phase crude extract behind the concentrating under reduced pressure, crude extract purification by silica gel column chromatography crude product then, recycle silicon offset plate chromatography, recrystallization draws monomeric compound, identify through spectrum and nuclear magnetic resonance spectrum, obtain β-Gu Zaichun; Perhaps identify, determine that molecular formula is C26H540 and determine that its structure is positive 26 carbon alcohol through carbon spectrum and hydrogen spectrum structure.
Sherwood oil is added among the intermediates C, extract 12 hours three times repeatedly, obtain sherwood oil phase crude extract behind the concentrating under reduced pressure, crude extract purification by silica gel column chromatography crude product then, recycle silicon offset plate chromatography, recrystallization draw the monomeric compound extract phase to be separated, and monitors at thin-layer chromatography, measure through hydrogen spectrum and carbon spectrum, obtain the compound diosgenin; Stigmasterone; β-daucosterol.。
It below is the anticancer experiment in vitro (testing crew: Zhong Zhenguo, Zhang Fengfen etc.) that carries out according to the Chinese fan palm plant extract compound that the embodiment of the invention is made
Summary purpose: with the restraining effect of cell in vitro culture method research Chinese fan palm extract to the growth of 7 class tumor cell lines.Method: adopt mtt assay, cell colony forming method, growth curve method to measure the inhibition situation of Chinese fan palm extract to leukemia cell line L1210 and P388D1, cervical cancer cell strain Hela, human stomach cancer cell line SGC7901, melanoma cell strain B16, neural tumor cell strain NG108-15, human hepatoma cell strain Hele7404 growth.The result: the Chinese fan palm extract has restraining effect to the growth of 7 class tumor cell lines.Drug level is lower than (0.5 μ g/ml), and is not obvious to the effect of tumour cell.Along with drug level is increased to 5.0 μ g/ml, the growth of tumor cell line is subjected to obvious inhibition.Conclusion: the Chinese fan palm extract has an antitumor action external.
Materials and methods
1 material
1.1 Chinese fan palm extract Chinese fan palm repeatedly after the lixiviate, obtains the concentrating part (hereinafter to be referred as the Chinese fan palm extract) of extract with ethyl acetate, soup faces with preceding standby with DMSO dissolving back sterile filtration.
1.2 tumor cell line leukemia cell line L1210 and P388D1, cervical cancer cell strain Hela, human stomach cancer cell line SGC7901, melanoma cell strain B16, neural tumor cell strain NG108-15 etc. are all available from Shanghai cell biological institute cell bank, human hepatoma cell strain Hele7404 is provided by pharmacology teaching and research room of Guangxi Medical University.
1.3 reagent MTT (tetramethyl-tetrazolium bromide) is a German Sigma company product, lot number: 020609; FBS (embryo's bovine serum) is a U.S. Hyclone company product, lot number: 0405; RPMI1640 and DMEM substratum are U.S. GIBCO company product, lot number: 1120708 and 0311; Trypsin (trypsinase) provides lot number by Tianjin Hao ocean biological products limited liability company: 200503; DMSO (dimethyl sulfoxide (DMSO)) provides lot number by Tianjin remittance English chemical reagent company limited: 20030526.
1.4 laboratory apparatus CO
2Incubator (ThermO Forma), the U.S. produces.Inverted microscope (Olympus), Japan produces.Microplate reader (Biocell), Australia's land productivity produces.
2 experimental techniques
2.1 mtt assay reference report
[3], every hole adds the cell suspension (except that NG108-15 cell usefulness contains the DMEM substratum of 10%FBS) that 200 μ l (containing 5000/ml tumour cell) contain the RPMI1640 substratum of 10%FBS in 96 well culture plates (NUNC), puts 37 ℃ of 10%CO
2After cultivating 24h in the incubator, experimental group adds the nutrient solution of the Chinese fan palm extract of ultimate density 0.5ug/ml, 1.0ug/ml, 5.0ug/ml respectively, and control group then adds the nutrient solution of isoconcentration equal-volume solvent, and repeat 4 times in every group 4 hole.Put 37 ℃, 10%CO
2Incubator was cultivated after 5 days, abandoning supernatant, the serum-free medium that adds the freshly prepared 0.2mg/ml of the containing MTT in 200 μ l/ holes, 37 ℃ are continued to cultivate 4h. and abandon supernatant liquor, add 200 μ l DMSO, behind the vibration mixing, be 550nm with the wavelength on microplate reader, reference wavelength is that 450nm measures the OD value. calculation result.
Be calculated as follows the inhibiting rate of medicine to growth of tumour cell.
Growth of tumour cell inhibiting rate %=(the average OD value of the 1-experimental group/average OD value of contrast) * 100%
The attached cell in vegetative period 2.2 colony forming method is taken the logarithm (Hela, SGC7901, B16) is made into 500/ml cell suspension with the RPMI1640 nutrient solution of 10%FBS and NG108-15 with the DMEM nutrient solution of 10%FBS, be inoculated in the 35mm culture dish (NUNC), 4 is one group, each 2ml; It is the cell suspension 200ul of the Chinese fan palm extract of 0.5ug/ml, 1.0ug/ml, 5.0ug/ml that experimental group adds final concentration.The cell suspension 200ul. that control group adds isoconcentration equal-volume solvent puts in 37 ℃ of incubators of 5%CO2 and leaves standstill 7d; Discard nutrient solution, contain the above cell colony of 50 cells with Rui Shi-Ji's nurse Sa dyeing back counting.To suspension cell (L1210 and P388D
1) be made into 1000/ml cell suspension with the RPMI1640 nutrient solution of 10%FBS, put 37 ℃ of pre-temperature; Get 35mm culture dish (NUNC), 4 is one group, and experimental group adds the Chinese fan palm extract that concentration is 0.5ug/ml, 1.0ug/ml, 5.0ug/ml respectively, and control group then adds isopyknic solvent; Get 1 part of 5% agar solution that melted, join in the 9 parts 37 ℃ fresh RPMI1640 nutrient solutions that contain 15%FBS, shake up and add in the plate, each 1ml puts room temperature it is solidified; The cell suspension of getting pre-temperature adds the ratio mixing of 5% agar 0.6ml in every 9.4ml, is added in the plate of shop fixtures layer agar, and each 1ml puts 37 ℃, 5%CO
2Cultivate 7d in the incubator; Contain 50 cell colonies more than the cell at the microscopically counting.
Calculate the medicine cell cluster by following formula and form the inhibition percentage
The tumour cell colony forms and suppresses percentage %=(1-experimental group colony number of cell/cellular control unit colony number) * 100%
2.3 in the RPMI1640 substratum that contains 10%FBS, NG108-15 then with the DMEM substratum that contains 10%FBS, is made into 10 * 10 respectively to the growth curve method with L1210, P388D1, Hela, SGC7901, B16, Hele7404 cell inoculation
3The cell suspension of individual/ml adds in 96 well culture plates then, every hole 200 μ l, it is the Chinese fan palm extract of 0.5ug/ml, 1.0ug/ml, 5.0ug/ml that experimental group adds ultimate density, control group adds isoconcentration equal-volume solvent. and every kind of every concentration of cell is established three holes, repeats 4 times, puts 10%CO
2Cultivate in 37 ℃ of incubators, by 0d, 1d, 2d, 3d, 4d, 5d, 6d, the 7d 40 μ l that respectively take a sample, add 0.4% trypan blue liquid, 10 μ l, the living cell counting number then.Every hole counting 4 times is got average, adds up 3 hole averages again, plots figure according to the logarithmic value and the time effect of cell concn value, is cell growth curve.
2.4 statistical procedures is with the analysis of SPSS 10.0 statistical softwares, the mean between two groups relatively adopts the t check.
3. experimental result
3.1 mtt assay is measured the inhibition of different concns Chinese fan palm extract to the tumor cell line growth.The results are shown in Table 1.
The influence (%) that table 1 different concns Chinese fan palm extract suppresses tumour cell
| Group | Extract concentrations (μ g/ml) | ||
| 0.5 | 1.0 | 5.0 | |
| Hela B16 SGC7901 P388D1 L1210 Hele7404 NG108-15 | -0.96±3.93 4.49±1.28 ▲ -2.33±1.95 ▲ 28.66±7.40 ▲▲ -0.26±2.42 2.43±6.84 -4.45±7.30 ▲ | 11.66±2.10 ▲▲▲▲ 22.32±1.99 ▲ 11.96±2.60 ▲ 36.78±6.15 ▲▲▲ 16.60±3.82 ▲▲ 21.81±1.63 ▲ 23.63±3.36 ▲ | 38.24±3.78 ▲▲▲▲ 52.22±6.34 ▲ 56.89±2.11 ▲▲▲▲ 94.89±0.93 ▲▲▲▲ 32.66±7.71 ▲▲▲▲ 45.25±8.26 ▲▲▲▲ 53.00±8.13 ▲▲▲▲ |
Annotate: compare with control group
▲P＜0.05,
▲ ▲P＜0.01,
▲ ▲ ▲P＜0.005,
▲ ▲ ▲ ▲P＜0.001
The result shows that the Chinese fan palm extract has restraining effect to 7 class tumor cell lines, and relevant with concentration.When extract concentrations lower (0.5 μ g/ml), medicine is not obvious to the tumour cell effect.Along with drug level raises, medicine kills and wounds more and more obvious to the inhibition of tumour cell.Its Chinese traditional medicine dialogue blood disease cell line P388D1 is comparatively responsive.
3.2 colony forming method is observed different concns Chinese fan palm extract to the tumour cell restraining effect.The results are shown in Table 2.
The influence (%) that table 2 different concns Chinese fan palm extract suppresses tumour cell
| Group | Extract concentrations (μ g/ml) | ||
| 0.5 | 1.0 | 5.0 | |
| Hela B16 SGC7901 P388D1 L1210 Hele7404 NG108-15 | 1.74±1.63 ▲ 1.37±0.98 ▲ 3.13±2.29 ▲ 25.34±6.18 ▲ 2.15±5.01 0.85±2.13 -0.47±1.03 ▲ | 13.11±2.10 ▲▲▲▲ 26.17±3.21 ▲ 27.01±3.93 ▲ 41.33±4.03 ▲ 21.53±3.08 ▲ 34.92±7.04 ▲ 21.95±3.22 ▲▲▲ | 43.90±4.09 ▲▲▲ 63.18±5.35 ▲ 45.79±4.86 ▲ 89.08±10.04 ▲▲▲ 46.25±6.13 ▲▲▲▲ 64.31±8.13 ▲▲▲▲ 43.90±1.34 ▲ |
Annotate: compare with control group
▲P＜0.05,
▲ ▲P＜0.01,
▲ ▲ ▲P＜0.005,
▲ ▲ ▲ ▲P＜0.001
The result of colony forming method shows that the Chinese fan palm extract is formed with restraining effect to the colony of 7 class tumor cell lines.Its result is more consistent with mtt assay.
3.3 growth curve method different concns Chinese fan palm extract is to the influence process of tumor cell line growth.The results are shown in figure below: 1,2,3,4,5,6,7.
Description of drawings
Fig. 1 is the influence of Chinese fan palm extract to the L1210 cell growth curve;
As seen from the figure, after the Chinese fan palm extract acts on tumor cell line L1210, relatively find, can find out when concentration is 1.0 μ g/ml that medicine has than the obvious suppression lethal effect tumour cell by medicine group and control group.When being 5.0 μ g/ml, drug level shows stronger lethal effect.
Fig. 2 is the influence of Chinese fan palm extract to the HELE7404 cell growth curve;
As seen from the figure, after the Chinese fan palm extract acts on tumor cell line HELE7404, relatively find, can find out when concentration is 1.0 μ g/ml that medicine has than the obvious suppression lethal effect tumour cell by medicine group and control group.When being 5.0 μ g/ml, drug level shows stronger inhibition lethal effect.
Fig. 3 Chinese fan palm extract to the Hela cell growth curve influence;
As seen from the figure, after the Chinese fan palm extract acts on tumor cell line Hela, relatively find, can find out when concentration is 1.0 μ g/ml that medicine has than the obvious suppression lethal effect tumour cell by medicine group and control group.When being 5.0 μ g/ml, drug level shows stronger inhibition lethal effect.
Fig. 4 Chinese fan palm extract is to the influence of P388D1 cell growth curve.
As seen from the figure, after the Chinese fan palm extract acts on tumor cell line P388D1, relatively find, can find out when concentration is 1.0 μ g/ml that medicine has than the obvious suppression lethal effect tumour cell by medicine group and control group.When drug level was 5.0 μ g/ml, cell cultures to the showed pair cell in 3 days stronger inhibition lethal effect, to cell mass mortality later in the 4th day.
Fig. 5 Chinese fan palm extract is to the influence of B16 cell growth curve
As seen from the figure, after the Chinese fan palm extract acts on tumor cell line B16, relatively find, can find out when concentration is 1.0 μ g/ml that medicine has than the obvious suppression effect tumour cell by medicine group and control group.When drug level was 5.0 μ g/ml, cell cultures to the showed pair cell in 3 days stronger inhibition lethal effect, and cell engendered death later on by the 4th day.
Fig. 6 Chinese fan palm extract influences the NG108-15 cell growth curve
As seen from the figure, after the Chinese fan palm extract acts on tumor cell line NG108-15, relatively find, can find out when concentration is 1.0 μ g/ml that medicine has than the obvious suppression effect tumour cell by medicine group and control group.When drug level was 5.0 μ g/ml, cell cultures to the showed pair cell in 2 days stronger restraining effect, and cell engendered death later on by the 4th day.
Fig. 7 Chinese fan palm extract is to the influence of SGC7901 cell growth curve
As seen from the figure, after the Chinese fan palm extract acts on tumor cell line SGC7901, relatively find, can find out when concentration is 1.0 μ g/ml that medicine has restraining effect to tumour cell by medicine group and control group.When being 5.0 μ g/ml, drug level shows stronger inhibition lethal effect.
The result shows from above seven map analysis Chinese fan palm extracts the growth of tumour cell process is had the inhibition lethal effect.
4, discuss
This experiment is by adopting three kinds of different experimental techniques, and research Chinese fan palm extract is at extracorporeal anti-tumor function. The result of mtt assay and colony forming method shows that growth all has inhibitory action to extract to 7 class tumor cell lines, and its inhibiting rate is along with drug concentration increases and corresponding increasing. The result of the shown result of cell growth curve method result and mtt assay and colony forming method matches. Prompting Chinese fan palm extract has an antitumor cell effect external.
Mtt assay, colony forming method, growth curve method and Hoechst 33258 fluorescence colours are the common methods of observing anti tumor activity in vitro, and especially mtt assay is fast and convenient, the external Primary Screening Test of Chang Zuowei antineoplastic[4], this research adopts the use in conjunction of several experimental techniques to increase the reliability of experimental result.
Malignant tumour be there is no very good medicine at present. In recent years, from natural plants, seek new antineoplastic and become one of main development direction of new drug research. The compound that Chinese fan palm root, stem, leaf, fruit are extracted all has antitumor action. Studies show that protein kinase C (PKC) is the protein kinase that a kind of Ca+ and phosphatidylserine (PS) activate, and plays an important role in the transmission of cell transmembrane signal. Show that on evidence protein kinase C is relevant with cell proliferation, many protein kinase C inhibitor on cell proliferation have inhibitory action, and some anticarcinogens itself are exactly the inhibitor of protein kinase C. Chinese fan palm extract that there are some researches show 100 μ g/ml is 66.6% to the inhibiting rate of protein kinase activity, reduces the concentration inhibitory action and weakens, and prompting Chinese fan palm extract active anticancer may be relevant with the inhibition to protein kinase C.
Recognize that from above-mentioned result of the test the Chinese fan palm extract produces effect to treatment leukaemia, cervical carcinoma, people's cancer of the stomach, melanoma, neural tumor, people's liver cancer.
Concrete case 1:
Lee xx, the man, 55 years old, feel upper right abdomen pain, find lobus dexter liver cancer 15 days through CT examination, without other malaise symptoms, sign. With this medicine after two weeks, check CT found that tumour dwindles the existing medication treatment that still continues.
Concrete case 2:
Xx, the woman, 12 years old, come medically with weak, heating, bone marrow smear sees that early myelocyte accounts for 70%, blood platelet 32 * 109/l, red is Lymphatic System, all obviously suppressed, examines to be " Acute Non-lymphocytic Leukema: ", is in hospital take blood transfusion and symptomatic treatment as main. Take upon somebody's introduction this Chinese medicine medicine series, antisecosis after two months, weak, heating disappears, check blood picture, blood platelet 83 * 109/l, early, myelocyte accounts for 13%, reached partial rcsponse.
Concrete case 3:
Grandson xx, woman, 26 years old, Acute Non-lymphocytic Leukema:. The patient is admitted to hospital with " it is weak to become thin, and faints suddenly ", looks into RBC1.6 * 1012/l, HB60g/l, PLT19 * 109/l. Wear through hospital's bone and to make a definite diagnosis: acute non-lymphatic leukemia. Take are through taking the treatment of this medicine, and hemochrome rose to 111g/l in ten days. Serve on half a year, routine blood test transfers to normally, and bone marrow smear is alleviated.
Concrete case 4:
The xx of Soviet Union, man, 3 years old, because of anaemia, hemorrhage, heating, in hospital's bone marrow examination, former ten childrens drench 79%, be diagnosed as acute lymphoblastic type leukaemia, delay more than the moon, after two months because the head of a family refuses chemotherapy, infant is on the point of dying, 39 ℃-40 ℃ of high fevers are not moved back, and bleeding gums are more than, and bulbar conjunctiva is hemorrhage, the skin purpura, extremely anaemia has several enlarged lymph nodes large such as broad bean, blood count: hemochrome 24g/l under the jaw, blood platelet 36 * 109/l, former ten childrens drench 93%, and newspaper is critically ill, through taking the treatment of this medicine, after heat in five days retreats to normal level, and bone marrow smear showed basic alleviation in 35 days.
Concrete case 5:
King xx, the woman, 50, go to see a doctor because of hypogastralgia, colporrhagia, be thought of as cervical carcinoma by gynecologial examination. The pathology biopsy, assay: uterine neck 3,9 points, the low differentiation of uterine neck 12 points-uterine neck squamous cell carcinoma. Make a definite diagnosis cervical carcinoma, hypogastralgia, hemorrhage, feed is few. Through using this medicine after 1 month, pain obviously alleviates, stopped bleeding, feed increase. Cervical carcinoma is clearly better. 1 first quarter moon that continues to take medicine, lower abdomen pendant pain disappears, and without hemorrhage, has no adverse reaction. Again through 3 months take medicine, the state of an illness without significant change, have no adverse reaction. Through hospital's gynecologial examination and ultrasonic examination: cure cervical carcinoma.
Concrete case 6:
Woods xx, the man, 58 years old, the treatment of performing a surgical operation because getting a cancer of the stomach, afterwards recurrence in a year finds that liver has metastatic lesion. Behind this medicine one wheat harvesting period, check CT discovery focus is obviously dwindled. Existing continual cure still.
Claims (3)
1, the extracorporeal anti-tumor extract of a kind of Chinese fan palm is characterized in that: its chemical ingredients is diosgenin, β-Gu Zaichun, stigmasterone, β-daucosterol and positive hexacosyl alcohol, and its chemical structure is as follows:
Diosgenin
β-Gu Zaichun
Stigmasterone
β-daucosterol
Positive hexacosyl alcohol
2, the extracorporeal anti-tumor method for preparing extractive of a kind of Chinese fan palm as claimed in claim 1 is characterized in that:
(1) root, seed, the leaf of the Chinese fan palm plant of fresh collection is ground into particle below 3 millimeters,, filters to isolate solid phase and liquid phase with by the ethanol lixiviate of 1-3 times of 70-97 volume of Chinese fan palm plant weight % 2-10 hour;
(2) ethanol is reclaimed in the liquid phase distillation, obtains the lixiviate cream A of black;
(3) solid phase that filters to isolate is by Chinese fan palm plant weight 1-3 poach twice extraordinarily, and each 0.5-2 hour, filter, waste, reconcentration become the following concentrated solution B of moisture 50 volume %;
(4) merge alcohol extracting lixiviate cream A and water extracting liquid B, obtain merging intermediates C;
(5) ethyl acetate is added among the intermediates C, and be heated to 50-60 ℃, extract 12 hours three times repeatedly, obtain Chinese fan palm ethyl acetate phase crude extract behind the concentrating under reduced pressure, crude extract purification by silica gel column chromatography crude product then, recycle silicon offset plate chromatography, recrystallization draws monomeric compound, obtains β-Gu Zaichun and n-hexacosane alcohol;
(6) sherwood oil is added among the intermediates C, extract 12 hours three times repeatedly, obtain sherwood oil phase crude extract behind the concentrating under reduced pressure, crude extract purification by silica gel column chromatography crude product then, recycle silicon offset plate chromatography, recrystallization draw the separation of monomeric compound extract phase and obtain compound diosgenin (diosgenin); Stigmasterone (stigmasterone); β-daucosterol (β-daucosterol);
(7) monomeric compound with (5) and (6) step passes through infrared spectra, mass spectrum, and proton nmr spectra, carbon spectrum, hydrocarbon relevant spectrum, electronic spectrum is measured structure.
3, the purposes of the extracorporeal anti-tumor extract of Chinese fan palm as claimed in claim 1, the application that it is characterized in that being used for preparing treatment leukemia, cervical cancer, people's cancer of the stomach, melanoma, neural tumor or people's liver cancer medicine.
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| CN101637550B (en) * | 2009-07-27 | 2011-12-28 | 林新华 | Chinese fanpalm seed polysaccharide extract and preparation method and purpose thereof |
| CN101735885B (en) * | 2009-12-14 | 2012-07-04 | 山东省中医药研究院 | Method for extracting shelled cedar seed oil and daucosterol from shelled cedar seed |
| CN102807771B (en) * | 2012-08-08 | 2014-06-04 | 江门市宝发纺织服饰制造有限公司 | Vegetable dye and preparation method thereof |
| CN102838648A (en) * | 2012-08-29 | 2012-12-26 | 陕西上格之路生物科学有限公司 | Steroid enol compound as well as preparation method thereof and application thereof |
| CN102940773A (en) * | 2012-12-04 | 2013-02-27 | 邓鑫 | Chinese medicinal preparation for inhibiting liver cancer metastasis and preparation method thereof |
| CN104861024B (en) * | 2015-04-03 | 2017-06-13 | 上海交通大学 | The method that Sitosterolum linoleate is prepared from black nightshade plant |
| CN107854590B (en) * | 2016-09-22 | 2021-11-26 | 西南大学 | Sweet potato saponin extract and application thereof |
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