CN100381434C - Emblic leafflower fruit extract possessing anticancer, antibiotic actions and its manufacturing method of traditional Chinese medicine formulation - Google Patents
Emblic leafflower fruit extract possessing anticancer, antibiotic actions and its manufacturing method of traditional Chinese medicine formulation Download PDFInfo
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- CN100381434C CN100381434C CNB2005100366220A CN200510036622A CN100381434C CN 100381434 C CN100381434 C CN 100381434C CN B2005100366220 A CNB2005100366220 A CN B2005100366220A CN 200510036622 A CN200510036622 A CN 200510036622A CN 100381434 C CN100381434 C CN 100381434C
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Abstract
The present invention discloses a flavonoid compound with the anticancer and antibacterial functions. The flavonoid compound is extracted from emblic leafflower root, bark, leaf or fruit. The extraction method can adopt a boiling method, an alcohol extraction method, a macroporous resin adsorption method and an extraction method. Different traditional Chinese medicine preparations prepared by the flavonoid compound have good anticancer and antibacterial functions.
Description
Technical field
The present invention relates to a kind of Chinese medicine preparation, particularly flavonoid compound Chinese medicine preparation preparation, that have anticancer and anti-microbial effect from the root of phyllanthus emblica, leaf, skin, fruit, to extract.
Technical background
Phyllanthus emblica (phyllanthus emblica L) is Euphorbiaceae (Euphorbiaceae) Leafflower (phyllanthus) defoliation small arbor or shrub, wild a lot of on China Guangxi, Guangdong, the mountain, Yunnan, past is the Chinese Minority Nationalities medicinal herbs most in use, by " Chinese pharmacopoeia is recorded.But the fruit of this plant, root, bark, leaf be hyoscine all.Phyllanthus emblica is hidden sweet, sour, puckery, cool, attaches to the lung and stomach meridians.Its function is clearing heat and cooling blood, food digesting stomach fortifying, the cough-relieving of promoting the production of body fluid with curing mainly; Be used for blood-head blood stasis, maldigestion, abdominal distension, cough, laryngalgia and dry.The main chemical compositions of phyllanthus emblica has vitamins C, multiple amino acids and tannin, comprising the sub-tannin of glucose feeding (glucogallin), throw gallate-based (gallic acid) and throw gallate-based (ellagic acid), corilagin (carilagin), terchebin (terchebin), chebulinic acid (chebulinicacid), glactaric acid (mucic acid), phyllemblic acid (phyllemblic acid) etc.In recent years, people have carried out many-sided research to phyllanthus emblica, and its effective constituent is extracted with organic solvent, finding has some anti-microbial activity compositions in the extract, staphylococcus, Corynebacterium diphtheriae, paratyphosum Bacterium, intestinal bacteria and dysentery bacterium all there is restraining effect, in national study of pharmacy, some countries in Asia as states such as Sri Lanka, Burma, Pakistan, Nepal all have and adopt phyllanthus emblica to treat eye disease, dysentery, gastroenteritis, and what have is used for the treatment of gonorrhoea etc.China is among the people then to be had and utilizes that Phyllanthus emblica L. is antibiotic, the history of promote the production of body fluid cough-relieving and treatment acute and chronic pharyngitis, also finds through modern detecting, contains multiple antioxidant component in the phyllanthus emblica fruit: vitamins C, and tannin, vitamin-E also contains superoxide-dismutase (SOD).Therefore a lot of medical workers have carried out many-sided research to phyllanthus emblica.We also find the open source literature of following relevant phyllanthus emblica through retrieval:
1.[autograph] Tibetan medicine phyllanthus emblica chemical constitution study [author] Zhang Lanzhen [1] Zhao Wenhua [1] Guo Yajian [1] burnish gilding loyalty [2] woods tree [1] Xin Linguang [1] [mechanism] [1] Beijing University Of Chinese Medicine Traditional Chinese Medicine College, [2] Beijing Inst. of Micro-Chemistry, Beijing 100091[prints name] CHINA JOURNAL OF CHINESE MATERIA MEDICA .2003,28 (10) .-940-943[digests] purpose: the chemical ingredients of research Tibetan medicine phyllanthus emblica.Method: various chromatography separation and purification, physics and chemistry and modern spectral method are identified structure.The result: isolation identification goes out 11 compounds, is respectively gallic acid (I), ellagic acid (II); I-O-galloyl-beta-D-glucose (III), 3,6-two-O--galloyl-D-glucose (IV); chebulinic acid (V), Quercetin (VI), chebulinic acid (VII); corilagin (VIII), 3-ethyl gallic acid (3-ethoxy-4,5-dihydroxy-bellzcic acid; IX); isoscrtiniin (X), 1,6-two-O-galloyl-D-glucose (XI).Conclusion: 3-ethyl gallic acid (3-ethoxy-4,5-dihydroxy-benzoic acid) is a new compound, the compound of isostrietiniin for obtaining from phyllanthus emblica first.
2.[autograph] ethnic drug phyllanthus emblica influence [author] Li Ping that D-galactosamine is caused the chmice acute liver injury thanks to Jin Xianlin and opens pharmacology teaching and research room of cloud [mechanism] Colleges Of Traditional Chinese Medicine Of Guangxi; [periodical name] Chinese national folk medical magazine .2003 (3) .-161-164[digest] method: this experiment utilizes D-galactosamine (D-Gal-N) disposable celiac injection (ip) to bring out small white mouse acute liver damage model; by measuring serum glutamic pyruvic transminase (ALT); glutamic-oxal(o)acetic transaminase (AST); alkaline phosphatase (ALP); superoxide-dismutase (SOD); mda (MDA) and liver glycogen; the liver coefficient is observed the provide protection of phyllanthus emblica water extracting alcohol hypostasis to liver injury.The result: each dosage composition of phyllanthus emblica water extracting alcohol hypostasis all reduces Serum ALT, AST, ALP, MDA content and liver coefficient, improves activity of SOD in serum and promotes liver glycogen synthetic, and can improve the liver organization pathology damage, and its effect is dose-dependently.Conclusion: phyllanthus emblica water extracting alcohol hypostasis has to a certain degree Green Tea Extract and lipoid peroxidization resistant, and the acute injury due to the D-Gal-N is had significant protective effect.
3.[autograph] the pharmacology teaching and research rooms of [mechanism] Colleges Of Traditional Chinese Medicine Of Guangxi such as acute toxicology and pharmacodynamic study [author] Li Ping Xie Bin of ethnic drug phyllanthus emblica, [periodical name] Chinese materia medica academic periodical .2002,20 (6) .[digests] purpose: the effect of observation phyllanthus emblica anti-liver injury.And disclose its mechanism of action, method: the one, observe the acute toxicity effect of phyllanthus emblica to mouse.The 2nd, adopt tetracol phenixin (CC14) disposable celiac injection (ip) to cause the chmice acute liver injury model.Detect serum glutamic pyruvic transminase (ALT), glutamic-oxal(o)acetic transaminase (AST), alkaline phosphatase (ALP), the liver glycogen level is calculated the liver coefficient, and makes pathology and observe.The result: the LD50 of (1) phyllanthus emblica aqueous extract is 35.16 ± 2.5g/kg.(2) each dosage group of phyllanthus emblica aqueous extract all can reduce the acute liver damage mice serum ALT due to the CC14, AST, the activity of ALP and liver coefficient, and can increase liver glycogen content, improve the liver organization pathology damage, its effect is dose-dependently, conclusion: the phyllanthus emblica aqueous extract has prophylactic effect to the acute liver damage due to the CC14; The oral safety of this medicine, toxicity is minimum.
4.[autograph] chemical ingredients of phyllanthus emblica and the Chinese forest-science academy of application [author] Hou Kaiwei [mechanism] the resource insect institute in the national folk traditional medicine, [periodical name] Chinese national folk medical magazine .2002 (6) .-345-348[digest] in the chemical ingredients of carrying out phyllanthus emblica, the Dietotherapy health function, in the time of aspect such as pharmacology research, collected pertinent data both domestic and external, chemical ingredients from the sweet son of section, pharmacology, clinical application and should be used as the comparatively summary of system in the national folk traditional medicine at home and abroad is for the exploitation of phyllanthus emblica compound pharmaceutical preparation provides scientific basis.
This agrostology overview [author] Zhou Tao [1] Qiu Dewen [2] [mechanism] [1] Guiyang College of Traditional Chinese Medicine of [autograph] ethnic drug phyllanthus emblica, [periodical name] journal .2002 of Guiyang College of Traditional Chinese Medicine, 24 (3) .-3-5[digests] title source, phytomorph, ecotope and the national administration form of phyllanthus emblica are summarized.Phyllanthus emblica is as an important traditional national medicine, and bentsaological research will have been established the communication and the alternative theoretical basis of Chinese materia medica and ethnic drug deep layer along with carrying out that its modern pharmacology is studied to its resource of development and utilization.
Research [author] Sun Xiaofang [1] Du Guiyou [mechanism] [1] Xiyuan Hospital, Chinese Medicine Academy of China such as [2] of anti-human immunodeficiency virus medicine in [autograph] Egyptian medicinal plant, [2] China Academy Of Traditional Chinese Medicine Traditional Chinese Medicine Research Institute, [periodical name] CHINA JOURNAL OF CHINESE MATERIA MEDICA .2002,27 (9) .[digests] to carrying inhibition reverse transcription (RT) enzyme of carrying thing on the back from the natural phant of 97 kinds of medicinal plants of Egypt and methanol extract thereof and water and suppressing HIV-1 inductive cell pathology effect (CPE) and carry out in-vitro evaluation.The methanol extract of phyllanthus emblica Phyllanthus emblica L. fruit demonstrates the RT effect of very strong inhibition HIV-1, and its 50% inhibition concentration (IC50) value is 9 μ gmL^-1.To methanol extract by biological activity fractional separation (bioactivity-guided fractionation) the being inhibited property active substance Putranjivain Λ (P Λ) that leads. its IC50 value is 3.9 μ m.The aqueous extract of crotons Crotontiglium seed and methanol extract can suppress the CPE of infectivity and HIV-1 inductive MT-4 cell significantly, their IC50 value is respectively 0.025 μ gmL^-1 and 2.0 μ gmL^-1, and all is lower than generation Cytotoxic concentration (selectivity index is respectively 34.4 and 50.0).By biological activity guiding fractional separation, from methanol extract, separate obtaining 8 kinds of phorbol esters, they are suppressed the activity of the CPE of HIV-1 inductive MT-4 cell and estimate.Finding that 12-O-is the inhibitor (the IC100 value is 0.48ngmL^-1) of HIV-1 inductive CPE from four (alkane) acyl phorbol-13-acetic ester (TPA), is again the activator of PKC.Prove in addition, 12-O-acetyl phorbol-13-decylate (APD) has the effect (the IC100 value is 7.6ngmL^-1) of anti-HIV-1, but the activation that does not have PKC, prove in addition, 12-O-acetyl phorbol-13-decylate (APD) has the effect (the IC100 value is 7.6ngmL^-1) of anti-HIV-1, but does not have the activation of PKC.
Basic institute in modern study progress [author] Zhou Tao [1] Liao Bo [2] [mechanism] [1] Kweiyang of [autograph] ethnic drug phyllanthus emblica, [2] Xingyi City medicine inspecting institute, [periodical name] journal .2002 of Guiyang College of Traditional Chinese Medicine, 24 (2) .-50-52[digests] the important simply traditional national medicine of phyllanthus emblica conduct, along with modern chemistry, the further expansion of pharmaceutical research, not only explained its national administration form, and make phyllanthus emblica in help, anti-ageing, the function of aspect such as antitumor obtains understanding, has created condition for more reasonably developing it.
Pharmacological research [author] Li Changling [mechanism] the Fujian An Mole pharmacy Industrial Co., Ltd. of [autograph] phyllanthus emblica, [periodical name] pharmacy progress .2001,25 (4) .-210-213[digests] the herbal medicine phyllanthus emblica having using value aspect new drug and the protective foods exploitation.The pharmacological research of phyllanthus emblica shows, various biological activity such as its tool anti-microbial infection, anti-oxidant, antitumor, reducing blood-fat and blood sugar, step-down, help, and do not have obvious toxic-side effects.
Guangzhou Chenliji Pharmaceutical Factory of the Peng Fuquan of herbal textual [author] Zhouning County [1] [2] [mechanism] [1] Guangzhou of myrobalan, Fructus Terminaliae Billericae and phyllanthus emblica medicine technical secondary school [2] in the Tibetan medicine in [autograph], [periodical name] herbal medicine .2001,32 (4) .-355-357[digests] purpose: tentatively inquire into the myrobalan, Fructus Terminaliae Billericae and the phyllanthus emblica relation in Chinese medicine and Tibetan medicine.Method: herbal textual and applicating history are reviewed, result: myrobalan, Fructus Terminaliae Billericae and phyllanthus emblica all are the external medicines in the Chinese medicine, be to follow Buddhist sutra to import China into, on using, relatively have and accept or reject and lay particular stress on conclusion with Tibetan medicine: meet the tendency with the modern scientific research achievement to Chinese materia medica and ethnic drug carry out each other in-depth communication with exchange to enrich traditional Chinese medicine and pharmacy.
The crude drug of [autograph] phyllanthus emblica is identified the herbal medicine .2000 of institute for drug control, [author] Luo Gan bright [1] Xu Jiwen [2] [mechanism] [1] Foshan, Guangdong Province [2] Fushan City, Guangdong Province pharmacist industry main office [periodical name], 31 (6) .-461-463[digests] tissue and the microscopic character of powder of phyllanthus emblica are identified, the research of correcting and having replenished former document, differentiating for the medicinal material of phyllanthus emblica provides foundation.
The CHINA JOURNAL OF CHINESE MATERIA MEDICA .1997 of plant research institute of Northwest Normal University of Beijing consonance medical university Institute of Medical Plants [2] of national study of pharmacy [author] Xia Quan [1] Kong Jie [2] [mechanism] [1] Chinese Academy of Medical Sciences plant research department [periodical name] of [autograph] conventional medicament phyllanthus emblica, 22 (9) .-515-518, the 525[digest] systematic study the national pharmacy of phyllanthus emblica, introduced the biological characteristics of phyllanthus emblica, the regional distribution general layout, chemical constitution and the pharmacological research achievement of being carried out in recent years, and introduced 17 countries and the nationality tradition to phyllanthus emblica in its medical practice and used.Point out by cross-cultural comparison, as a kind of important conventional medicament, phyllanthus emblica with protect the liver, anticancer, be good for the stomach, antimutagenic, the anti-ageing 35 kinds of functions of waiting for a long time are relevant, are a kind of medicinal plants with wide DEVELOPMENT PROSPECT.
Recognize from above-mentioned patent documentation, at present to the research of phyllanthus emblica except its anti-microbial activity composition, the pharmaceuticals researcher finds also that phyllanthus emblica has and protects the liver, anticancer, be good for the stomach antimutagenic, effects such as anti-ageing, anti-immune deficiency, and the oral safety of phyllanthus emblica, toxicity is little.But present open source literature does not see that to the chemical ingredients of Fructus Phyllanthi extract and antibiotic and anticancer mechanism detailed report is arranged.
Summary of the invention
The objective of the invention is from phyllanthus emblica and root, leaf, skin or fruit, to isolate activeconstituents, and the starting material of and cancer therapy drug antibiotic as preparation with these activeconstituentss, thereby utilize the Chinese herbal medicine resource of China better, enrich the field of Chinese herbal treatment disease.
The inventor is by studying the extract of phyllanthus emblica root, bark, leaf or fruit, and discovery has antibiotic and composition antitumous effect is a flavonoid compound.The inventor's experimental result also shows, the Chinese medicine preparation of using the flavonoid compound that extracts from phyllanthus emblica and root thereof, leaf, skin to make is used on the in vitro tests and the bacterial infection disease person, and the result shows good antibacterial effect; Anticancer experimental study in vivo and in vitro shows that its flavonoid compound has good anticancer effect.
Antibiotic and the anticancer effective component that the present invention extracts, its chemical identification and spectral response curve are as follows:
(1) complex reaction of metallic salt reagent
1. hydrochloric acid--magnesium powder reaction: add the small amount of magnesium powder in the sample ethanolic soln, drip 1～2 concentrated hydrochloric acid again, heat 3min in water-bath, solution colour becomes redness.
2. AlCl
3Color reaction: the ethanolic soln of sample dripped on filter paper, form spot, on spot, spray 1%AlCl again
3Ethanolic soln is put to examine under the ultraviolet lamp and is shown the spot displaing yellow.
(2) alkaline reagents color reaction
Normal temperature drips the NaOH aqueous solution down in sample liquid, shake up, and solution colour becomes orange red.
(3) ultra-violet absorption spectrum feature
Sample liquid is measured absorption peak with methyl alcohol or dissolve with ethanol at ultraviolet section wavelength.The result shows, maximum absorption band occurs at 265nm and 336nm.
Above presentation of results, the chemical identification result of the effective constituent that the inventor extracts from phyllanthus emblica and root thereof, leaf, skin and the feature of determination of uv absorption characteristic conforms flavonoid compound.
It below is the example of preparation phyllanthus emblica flavonoid compound.These examples just are used for the present invention is illustrated, rather than to the restriction of extracting method of the present invention:
Raw material: comprise the mixture of root, bark, leaf, fruit or the above plant position of phyllanthus emblica, hereinafter to be referred as medicinal material; Can be used in product or bright product, but recommend to use dry product,, guarantee the consistence of material quality so that store.
Processing before extracting:, make the above meal of 60 orders earlier with the phyllanthus emblica raw material pulverizing.
Extracting method one
Medicinal material coarse powder, boiling 3-4 time merges water cooking liquid, filters, and filtrate is used macroporous resin adsorption.With 30-95% or 95% above aqueous ethanolic solution wash-out, elutriant reclaims solvent, promptly gets Fructus Phyllanthi total flavones behind the washing post.
Extracting method two
Medicinal material coarse powder, 95% alcohol reflux 2-3 time merges alcohol extract, filters, and is condensed into dried cream, and last silicagel column with ethyl acetate-sherwood oil gradient elution, is collected the stream part that contains flavonoid compound.Reclaim solvent, promptly get Fructus Phyllanthi total flavones.
Extracting method three
Medicinal material coarse powder, boiling 3-4 time merges water cooking liquid, micro-filtration (filter opening 0.2 μ m-0.45 μ m), 95% ethanol is used in macroporous adsorptive resins absorption on the filtrate, perhaps than the little solvent of ethanol polarity such as the mixture of ethyl acetate, mixed solvent such as ethyl acetate and sherwood oil, wash-out.Elutriant reclaims solvent, promptly gets Fructus Phyllanthi total flavones.
Extracting method four
Medicinal material coarse powder, 95% alcohol reflux 2-3 time, merge alcohol extract, filter, be condensed into thick paste or in cream, use propyl carbinol, perhaps than the little solvent of propyl carbinol polarity such as the mixture of ethyl acetate, sherwood oil, mixed solvent such as ethyl acetate and propyl carbinol, dissolving is filtered, filtrate is reclaimed solvent, mainly contains flavonoid compound in the extract.
Extracting method five
The aqueous extract of extracting method one gained, be concentrated to 1: 1 (medicinal material weight: liquor capacity), use propyl carbinol, perhaps than the little solvent of propyl carbinol polarity such as the mixture of ethyl acetate, sherwood oil, mixed solvent such as ethyl acetate and propyl carbinol, extraction.Collect organic phase, reclaim solvent, mainly contain flavonoid compound in the extract.
The method of the invention is extracted the phyllanthus emblica flavonoid compound that obtains, and on producing, can prepare different preparations (or formulation) as required: oral Preparation, as oral liquid, tablet, capsule, lozenge; Percutaneous drug administration preparation is as tincture, liniment, emplastrum; Injecting and administering preparations is as injection; The spray delivery preparation is as nasal spray, throat spraying agent.
Extracting method adopts common pharmaceutical technology and method, no matter, be principle all, promptly obtain flavonoid compound in the extract to contain antibiotic active substance in the extract with what method preparation, other may contain a spot of alkaloid, saponins, but few in chromatography process residue.
Below be preparation example anticancer, antibiotic Chinese medicine preparation:
In anticancer, antibiotic Chinese medicine preparation of the present invention, contain phyllanthus emblica and root, leaf, skin or fruit extract dry-matter 0.00001-100%, auxiliary material 0-99.99999%.Can prepare different formulations as required: oral Preparation, as oral liquid, tablet, capsule, lozenge; Percutaneous drug administration preparation is as tincture, liniment, emplastrum; Injecting and administering preparations is as injection; The spray delivery preparation is as nasal spray, throat spraying agent.
Embodiment
It below is example of the invention process.These examples only are used to illustrate enforcement of the present invention, are not limitation of the invention.
Extracting method embodiment one
Medicinal material coarse powder adds 5 times of decoctings and boils 3-4 time, merges water cooking liquid, filters, and filtrate is used macroporous resin adsorption.With 75% aqueous ethanolic solution wash-out, elutriant recovery solvent promptly gets Fructus Phyllanthi total flavones behind the washing post.
Extracting method embodiment two
Medicinal material coarse powder, 85% alcohol reflux 2-3 time merges alcohol extract, filters, and is condensed into dried cream, and last silicagel column with ethyl acetate-sherwood oil gradient elution, is collected the stream part that contains flavonoid compound.Reclaim solvent, promptly get Fructus Phyllanthi total flavones.
Extracting method embodiment three
Medicinal material coarse powder adds 3 times of decoctings and boils 3-4 time, merges water cooking liquid, micro-filtration (filter opening 0.2 μ m-0.45 μ m), 95% ethanol is used in macroporous adsorptive resins absorption on the filtrate, perhaps than the little solvent of ethanol polarity such as the mixture of ethyl acetate, mixed solvent such as ethyl acetate and sherwood oil, wash-out.Elutriant reclaims solvent, promptly gets Fructus Phyllanthi total flavones.
Extracting method embodiment four
Medicinal material coarse powder, 55% alcohol reflux 2-3 time, merge alcohol extract, filter, be condensed into thick paste or dried cream, use propyl carbinol, perhaps than the little solvent of propyl carbinol polarity such as the mixture of ethyl acetate, sherwood oil, mixed solvent such as ethyl acetate and propyl carbinol, dissolving is filtered, filtrate is reclaimed solvent, mainly contains flavonoid compound in the extract.
Extracting method embodiment five
The aqueous extract of extracting method one gained, be concentrated to 1: 1 (medicinal material weight: liquor capacity), use propyl carbinol, perhaps than the little solvent of propyl carbinol polarity such as the mixture of ethyl acetate, sherwood oil, mixed solvent such as ethyl acetate and propyl carbinol, extraction.Collect organic phase, reclaim solvent, mainly contain flavonoid compound in the extract.
Preparation embodiment one
Get above extracting method one gained phyllanthus emblica flavonoid compound 10 grams, add an amount of water dissolution, add 60 gram powdered sugar, dissolving, sterilization, packing makes oral liquid 100ml.Every 100ml oral liquid is equivalent to 100g phyllanthus emblica raw medicinal herbs.
Preparation embodiment two
Get above extracting method two gained phyllanthus emblica flavonoid compounds,, make the tincture solution that every ml contains crude drug 10g, for the trauma wounds sterilization with an amount of 30% dissolve with ethanol.
Preparation embodiment three
Get above extracting method three gained phyllanthus emblica flavonoid compounds 10 grams, add the pure water dissolving, millipore filtration divides the freeze-drying bottle of packing into, lyophilize, injection.Every bottle is equivalent to 10g phyllanthus emblica raw medicinal herbs.
Preparation embodiment four
Get above extracting method four gained phyllanthus emblica flavonoid compounds,, add 1% azone and make permeate agent, make the skin liniment solution that every ml contains crude drug 5g with an amount of 30% dissolve with ethanol.
Preparation embodiment five
Get above extracting method five gained phyllanthus emblica flavonoid compounds, add appropriate amount of starch and granulate, be pressed into tablet, the heavy 0.3g of substrate, or dressing, every is equivalent to 4g phyllanthus emblica raw medicinal herbs.
Preparation embodiment six
Preparation embodiment five gained particles, the zero capsule of packing into, every capsule are adorned 0.6 gram, and every capsules is equivalent to 8g phyllanthus emblica raw medicinal herbs.
Preparation embodiment seven
Extracting method one gained phyllanthus emblica flavonoid compound 100 grams add 1400 gram powdered sugar, make particle, are distributed into the electuary of every bag heavy 15 gram.Every bag is equivalent to 10g phyllanthus emblica raw medicinal herbs.
Preparation embodiment eight
Extracting method one gained phyllanthus emblica flavonoid compound 1 gram is ground into the above fine powder of 200 orders, packs in the spray bottle, can prepare nasal spray or throat spraying agent.
Concrete pharmacology of phyllanthus emblica flavonoid compound of the present invention and pharmacodynamics clinical trial.
One, antitumous effect test
1 experiment material
State provides 1.1 the Fructus Phyllanthi total flavones clock shakes, and faces with preceding standby with sterile filtration behind the dmso solution.
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) provide 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
2.1MTT method 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 Fructus Phyllanthi total flavones 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 lDMSO, 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 used 10%FRS
RPMI1640 nutrient solution and NG108-15 be made into 500/ml cell suspension 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 Fructus Phyllanthi total flavones 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 5%CO
237 ℃ of incubators in leave 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, experimental group adds the Fructus Phyllanthi total flavones that concentration is 0.5ug/ml, 1.0ug/ml, 5.0ug/ml respectively, 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, add then in 96 well culture plates, every hole 200 μ l, it is the Fructus Phyllanthi total flavones 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. and repeat 4 times, put 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 results
3.1MTT method is measured the inhibition of different concns Fructus Phyllanthi total flavones to the tumor cell line growth.The results are shown in Table 1.
The influence (%) that table 1 different concns Fructus Phyllanthi total flavones suppresses tumour cell
Annotate: compare with control group
1)P＜0.05,
2)P＜0.01,
3)P＜0.005,
4)P＜0.001
The result shows that Fructus Phyllanthi total flavones has restraining effect to 7 class tumor cell lines, and relevant with concentration.When Fructus Phyllanthi total flavones concentration 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 the different concns Fructus Phyllanthi total flavones to the tumour cell restraining effect.The results are shown in Table 2.
The influence (%) that table 2 different concns Fructus Phyllanthi total flavones suppresses tumour cell
Annotate: compare with control group
1)P＜0.05,
2)P＜0.01,
3)P＜0.005,
4)P＜0.001
The result of colony forming method shows that Fructus Phyllanthi total flavones 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 Fructus Phyllanthi total flavones is to the influence process of tumor cell line growth.The results are shown in Figure of description.
Description of drawings
Figure of description is to the influence process of growth curve method different concns Fructus Phyllanthi total flavones to the tumor cell line growth.Wherein:
Fig. 1 has showed different concns Fructus Phyllanthi total flavones effect L1210 cell growth curve situation.
Fig. 2 different concns Fructus Phyllanthi total flavones effect HELE740 cell growth curve situation.
Fig. 3 different concns Fructus Phyllanthi total flavones effect Hela cell growth curve situation.
Fig. 4 different concns Fructus Phyllanthi total flavones effect P388D1 cell growth curve situation.
Fig. 5 different concns Fructus Phyllanthi total flavones effect B16 cell growth curve situation.
Fig. 6 different concns Fructus Phyllanthi total flavones effect NG108-15 cell growth curve situation.
Fig. 7 different concns Fructus Phyllanthi total flavones effect SGC7901 cell growth curve situation.
Show that from the result of Fig. 1-Fig. 7 Fructus Phyllanthi total flavones has inhibitory action to the growth of tumour cell process. 7 accompanying drawings all Can see that during drug concentration lower (0.5 μ g/ml), compare with control group, medicine is not obvious to the effect of tumour cell. Along with drug concentration raises, the effect performance of inhibition tumor cell is comparatively obvious. From figure, also can know control group DMSO Follow concentration enrichment that tumour cell is also had certain inhibitory action. Fructus Phyllanthi total flavones is with to leukemia cell line P388D1 comparatively Responsive. The result is consistent with front two kinds of methods.
4 conclusions
In in vitro test, Fructus Phyllanthi total flavones has obvious inhibitory action to the growth of tumour cell.
Although just provided the situation of emblic root extract general flavone among the figure, extracted from emblic root, leaf, skin or fruit The chemical composition of general flavone be more or less the same, the verification experimental verification data are omitted.
Two, antibacterial action test
1. the preparation of extract: emblic and root thereof, leaf, skin dry, and break into meal, respectively water, 95% ethanol, oil Ether, n-butanol, ethyl acetate extraction reclaim solvent, make extract separately. In addition, the water extraction liquid (medicine that is concentrated to 1: 1 Material weight: liquor capacity), use petroleum ether extraction, collect organic phase, reclaim solvent, get petroleum ether extract; 95% ethanol The extract acetic acid ethyl dissolution filters, and collects filtrate, reclaims solvent, gets the ethyl acetate extract.
2. method: solid medium serial dilution.
3. result: see the following form.
4. result: each extract when low concentration to Escherichia coli, staphylococcus aureus, Candida albicans, typhoid fever sramana Bacterium, Klebsiella Pneumoniae, alpha streptococcus and beta hemolytic streptococcus namely show inhibitory action.
To above example one gained oral liquid, entrust pharmacology teaching and research room of Colleges Of Traditional Chinese Medicine Of Guangxi to test, the result shows that acute toxicity and long term toxicity reaction do not appear in tested small white mouse, can not survey LD50。
Six, clinical effectiveness
Because the present invention not yet obtains the clinical official written reply of new drug, can not enter clinical research, therefore only face with it 2 routine volunteers The bed application experiment.
Example one Huang * *, the woman, the Nanning people, 43 years old, the hand knife wound reached muscle layer deeply, nursed improper and inflammation, change Purulence. With above-mentioned preparation example two gained tinctures, put on the skin once every 4 hours, inflammation and pus disappear after three days, and wound is healed gradually Close.
Example two not * *, the man, the Nanning people, 37 years old, unhygienic diet caused bacillary dysentery diarrhoea. Give above-mentioned preparation example one The gained oral liquid is oral, every day three times, and each 50ml, taking suffer from diarrhoea behind the secondary stops, and continuously takes one, all fully recovers.
Example three Huangs * *, man, Hengxian County peasant, 52 years old, lung cancer patient. Because financial difficulties are interrupted hospital's normal therapeutic, Change into and took preparation example six gained capsules 30 days, during the state of an illness do not have malignant development.
Example four Dong * *, man, Gongcheng Guangxi peasant, 60 years old, Chronic bronchitis pulmonary infection patient. Due to illness condition is sent out repeatedly Do, add financial difficulties and interrupt hospital's normal therapeutic, change into and took preparation example five gained tablets 10 days, the state of an illness has inclination Turn to, asthma disappears, and continues to take 3 all rear pulmonary emphysema and is clearly better.
Claims (7)
1. the production method of the Chinese medicine preparation of the Fructus Phyllanthi extract with anticancer, anti-microbial effect, it is characterized in that: earlier with phyllanthus emblica meal boiling 3-4 time, merge water cooking liquid, filter, filtrate use macroporous resin adsorption, behind the washing post with the aqueous ethanolic solution wash-out more than 30-95% or 95%, elutriant reclaims solvent, obtain Fructus Phyllanthi extract, add pharmaceutical excipient then, extract is made Chinese medicine preparation.
2. the production method of the Chinese medicine preparation of the Fructus Phyllanthi extract with anticancer, anti-microbial effect is characterized in that: phyllanthus emblica meal, alcohol reflux 2-3 time, merge alcohol extract, filter, be condensed into dried cream, last silicagel column, with ethyl acetate-sherwood oil gradient elution, collection contains stream part of flavonoid compound, reclaims solvent, obtains Fructus Phyllanthi extract, add pharmaceutical excipient then, extract is made Chinese medicine preparation.
3. the production method of the Chinese medicine preparation of the Fructus Phyllanthi extract with anticancer, anti-microbial effect, it is characterized in that: the phyllanthus emblica meal, boiling 3-4 time merges water cooking liquid, 0.2 μ m-0.45 μ m filter opening micro-filtration, macroporous adsorptive resins absorption on the filtrate is with the mixture of ethanol, ethyl acetate or ethyl acetate and sherwood oil, wash-out, elutriant reclaims solvent, obtain Fructus Phyllanthi extract, add pharmaceutical excipient then, extract is made Chinese medicine preparation.
4. the production method of the Chinese medicine preparation of the Fructus Phyllanthi extract with anticancer, anti-microbial effect is characterized in that: phyllanthus emblica meal, alcohol reflux 2-3 time, merge alcohol extract, filter, be condensed into thick paste or dried cream, with the mixture of propyl carbinol, ethyl acetate, sherwood oil or ethyl acetate and propyl carbinol, dissolving, filter, filtrate is reclaimed solvent, obtains Fructus Phyllanthi extract, add pharmaceutical excipient then, extract is made Chinese medicine preparation.
5. the production method of the Chinese medicine preparation of the Fructus Phyllanthi extract with anticancer, anti-microbial effect, it is characterized in that: with the phyllanthus emblica meal, boiling 3-4 time merges the water cooking liquid aqueous extract, be concentrated to 50% of original solution volume, with the mixture of propyl carbinol, ethyl acetate, sherwood oil or ethyl acetate and propyl carbinol, organic phase is collected in extraction, reclaim solvent, obtain Fructus Phyllanthi extract, add pharmaceutical excipient then, extract is made Chinese medicine preparation.
6. as the Chinese medicine preparation of the production method preparation of the Chinese medicine preparation of each described Fructus Phyllanthi extract with anticancer, anti-microbial effect of claim 1-5, it is characterized in that: described Chinese medicine preparation formulation is oral liquid, tablet, capsule, tincture, liniment, emplastrum, injection, nasal spray or throat spraying agent.
7. as the Chinese medicine preparation of the production method preparation of the Chinese medicine preparation of each described Fructus Phyllanthi extract with anticancer, anti-microbial effect of claim 1-5, it is characterized in that: the chemical identification and the spectral response curve of flavonoid compound is as follows in the Fructus Phyllanthi extract:
(1) complex reaction of metallic salt reagent:
1. hydrochloric acid-magnesium powder reaction: add the small amount of magnesium powder in the sample ethanolic soln, drip 1～2 concentrated hydrochloric acid again, heat 3min in water-bath, solution colour becomes redness;
2. AlCl
3Color reaction: the ethanolic soln of sample dripped on filter paper, form spot, on spot, spray 1%AlCl again
3Ethanolic soln is put to examine under the ultraviolet lamp and is shown the spot displaing yellow;
(2) alkaline reagents color reaction:
Normal temperature drips the NaOH aqueous solution down in sample liquid, shake up, and it is orange red that solution colour becomes;
(3) ultra-violet absorption spectrum feature:
Sample liquid is measured absorption peak with methyl alcohol or dissolve with ethanol at ultraviolet section wavelength, and the result shows, maximum absorption band occurs at 265nm and 336nm.
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| CN101032322B (en) * | 2007-03-30 | 2011-06-08 | 华南理工大学 | Phyllanthus emblica extract and its preparation and application |
| CN102836200B (en) * | 2012-09-06 | 2013-12-25 | 广东松山湖臻德生物医药科技有限公司 | Application of oil orange to preparation of anti-H1N1 flu medicament |
| CN107441148A (en) * | 2017-08-17 | 2017-12-08 | 吉林省中医药科学院（吉林省中医药科学院第临床医院） | A kind of serum grass extractive of general flavone and preparation method thereof and antitumor activity application |
| CN108030092A (en) * | 2017-12-22 | 2018-05-15 | 福建康凌谷生物科技有限公司 | A kind of emblic cotyledon cream and preparation method thereof |
| CN111772227B (en) * | 2020-08-10 | 2022-05-03 | 四川中烟工业有限责任公司 | Phyllanthus emblica targeted refined substance, preparation method and application thereof in cigarettes |
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