CN101406509B - Lonicera confusa extract and preparation method and application thereof - Google Patents
Lonicera confusa extract and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to wild honeysuckle flower extract, which mainly comprises the following compositions: macranthoside A,II b, macranthoside B,IIIb, 3-O-alpha-L-pyran rhamnose (1-2)-alpha-L-pyran arabinose-hederagenin and 3-O-beta-D-glucopyranose (1-4)-beta-D-glucopyranose (1-3)-alpha-L-pyran rhamnose (1-2)-alpha-L-pyran arabinose -oleanolic acid. The invention also relates to a method for preparing the compound and application of the compound in the aspect of cancer.
Description
Technical field
The present invention relates to field of traditional Chinese medicine pharmacy, be specifically related to a kind of Lonicera confusa extract and its production and use, particularly, it is effective active extractive part---the total saponins that from the Chinese medicine Flos Lonicerae, extracts, and determined the preparation method that a kind of technology is simplified, yield is high for preparing this extract, and the application of this extract in preparation prevention and treatment cancer drug.
Background technology
Lonicera (Lonicera) plant mostly is fallen leaves or evergreen upright shrub, brushwood and liana, seldom is the dungarunga shape.Totally 47 kinds of the medicinal woodbines of now having put down in writing have stronger antibacterial action to many pathogenic bacterium, and just there was the record of pair Radix Ophiopogonis medical value in China before more than 1500 years, and its rhizome, leaf, Hua Junke are used as medicine.Flos Lonicerae is the dry flower of caprifoliaceae plant largeflower-like honeysuckle flower Loniceramacranthoides Hand.-Mazz, Flos Lonicerae Lonicera hypoglauca Miq. or Lonicera confusa Lonicera confusa DC. or the flower that band is just opened, one of Zhanjiang genuine medicinal materials.Main effective ingredient is a chlorogenic acid, has heat-clearing and toxic substances removing, and the effect of wind-heat dissipating is used for the treatment of the carbuncle furuncle, sore throat, erysipelas, toxic-heat and blood stasis, anemopyretic cold, epidemic febrile disease.Before 2005 editions pharmacopeia, be used as medicine as Flos Lonicerae, be the principal item of South China " Flos Lonicerae " Chinese crude drug.But because there is apparent in view difference in both chemical constituents, 2005 editions pharmacopeia are recorded Flos Lonicerae and Flos Lonicerae respectively, also substitute (Chen Ya etc., Flos Lonicerae progress, Chinese science and technology information, 2007 years 05 phases: 73-74) during clinical practice no longer mutually.There is its contained saponin component of bibliographical information to have and protects the liver (Shi Jingzhen etc.; several compositions of Lonicera fulvotnetosa Hsu et S. C. Cheng. Ms and largeflower-like honeysuckle flower are to the protective effect of large and small Mus chemical liver injury; CHINA JOURNAL OF CHINESE MATERIA MEDICA; 1999; 24 (6): 363-364), anticancer (Cytotoxic saponins from the root0fDipsacus asper Wall.Arch Pharm Res.2005,28 (9): 1053.6.) activity.Successively separated at present and identified saponin component in the multiple Flos Lonicerae plant, but these separating and extracting process belong to the research method in the laboratory more, the method complexity, the cost height, and isolated saponin component yield is low, be not suitable for suitability for industrialized production, component content is indeterminate in the extract in addition, is unfavorable for being applied to pharmacy and treatment field.
This laboratory is engaged in the separation and Extraction and the research work of Flos Lonicerae medicinal ingredient for many years, the total saponin extracts that extracts from largeflower-like honeysuckle flower is disclosed in the application 200610039294.4 before us, total saponin content is 50%-90% in this extract, lonicera macranthoides hypo-saponin B wherein: Macranthoside A=12～6:1, its preparation method and the purposes that is used to prevent and treat hepatocarcinoma are also disclosed simultaneously, the preparation method and the treatment hepatocarcinoma thereof of monomer component lonicera macranthoides hypo-saponin B are disclosed in our an other application 200710023243.7, breast carcinoma, the purposes of cervical cancer, but the extracting method yield in above-mentioned should the asking is also lower, and the kind of disclosed treatment cancer is limited.
Summary of the invention
This laboratory through great deal of experimental and screening, has been determined a kind of new Lonicera confusa extract and pharmaceutical composition that is prepared into and preparation method, and its anticancer effect has been carried out the checking of system on basis before.This extract component is clear and definite, and anticancer effect is good, helps preparation prevention and treatment cancer drug; This method technical process is simple, and product yield height is suitable for suitability for industrialized production.
The object of the invention is to provide a kind of Lonicera confusa extract, the main component of this Lonicera confusa extract is a lonicera macranthoides hypo-saponin B, Macranthoside A, 3-O-α-L-rhamnopyranosyl (1-2)-α-L-arabopyranose base-helexin and 3-O-β-D-glucopyranosyl (1-4)-β-D-glucopyranosyl (1-3)-α-L-rhamnopyranosyl (1-2)-α-L-arabopyranose base-oleanolic acid, the weight ratio of described 4 kinds of compositions is followed successively by 42～10:9～1:12～1:1, and the gross weight of described 4 kinds of compositions accounts for the 50%-100% of this extract.
The weight ratio of wherein said 4 kinds of compositions is preferably 27～14:5～1:9～1:1;
The gross weight of described 4 kinds of compositions preferably accounts for the 70%-99.6% of this extract.
The discriminating of main component and assay can adopt conventional assay method in the Lonicera confusa extract of the present invention, but preferred method provided by the present invention:
1. thin layer chromatography is adopted in the discriminating of composition:
With the extract dissolve with methanol, point sample after the equal solvent volatilization is done, places chromatography cylinder on silica gel plate, launch down at 25 degrees centigrade with the developing solvent (lower floor's clear solution of chloroform: methanol: water=7:3:0.5) of newly joining.Silica gel plate after launching is waved spray developer vanillin-concentrated sulphuric acid behind the solvent, in 105 degrees centigrade of heating 1 minute, show same blob with lonicera macranthoides hypo-saponin B, Macranthoside A, 3-O-α-L-rhamnopyranosyl (1-2)-α-L-arabopyranose base-helexin and 3-O-β-D-glucopyranosyl (1-4)-β-D-glucopyranosyl (1-3)-α-L-rhamnopyranosyl (1-2)-α-L-arabopyranose base-oleanolic acid reference substance.
2. the assay of composition adopts high performance liquid chromatography:
High performance liquid chromatograph Aglient 1100 series, quaternary pump, automatic sampler, detector: Alltech ELSD 2000 (Alltech, Deerfield, IL, USA), chromatographic column: Aglient Zorbax SB-C18 post (150 * 4.6mm, 5.0pm), acetonitrile is a chromatographically pure, and phosphoric acid is analytical pure.Chromatographic condition: mobile phase: 0-10min, acetonitrile: 0.5% phosphoric acid solution is kept ratio (29:71), 10.25min, the mobile phase ratio changes to (46:54) by (29:71) is linear, 25-30min, mobile phase is kept ratio (46:54).ELSD manages temperature: 106 ℃.Nitrogen flow rate: 2.6L/min.Accurate respectively lonicera macranthoides hypo-saponin B, Macranthoside A, 3-O-α-L-rhamnopyranosyl (1-2)-α-L-arabopyranose base-helexin and 3-O-β-D-glucopyranosyl (1-4)-β-D-glucopyranosyl (1-3)-α-L-rhamnopyranosyl (1-2)-α-L-arabopyranose base-oleanolic acid reference substance that takes by weighing through the phosphorus pentoxide dried overnight, respectively add methanol and make the solution that every 1ml contains 0.03mg respectively, solution in contrast.Get the about 20mg of this product powder (get in addition simultaneously this product powder measure moisture), accurately claim surely, put in the apparatus,Soxhlet's, add methanol eddy and extracted 4 hours, put cold after, be transferred in the 100ml volumetric flask, methanol constant volume promptly.Each sample detects through high performance liquid chromatogram, and the weight ratio that calculates 4 kinds of compositions is followed successively by 42～10:9～1:12～1:1.
Lonicera confusa extract of the present invention can with one or more pharmaceutically the acceptable adjuvant make pharmaceutically preparation.
Described pharmaceutically acceptable adjuvant is meant the pharmaceutical carrier or the adjuvant of pharmaceutical field routine, for example: water; Diluent, excipient or filler such as starch, dextrin, mannitol, sucrose, lactose, microcrystalline Cellulose etc.; Binding agent such as cellulose derivative, alginate, gelatin and polyvinylpyrrolidone; Wetting agent such as glycerol; Disintegrating agent such as methyl starch sodium, hyprolose, agar, calcium carbonate and sodium bicarbonate; Absorption enhancer such as quaternary ammonium compound; Surfactant such as hexadecanol, Tweens, sodium lauryl sulphate: absorption carrier such as Kaolin and soap clay; Lubricant such as Pulvis Talci, calcium stearate and magnesium, micropowder silica gel and Polyethylene Glycol etc.
Described preparation can be applied to the cancer patient by the mode through intestinal canal administration or parenterai administration.When being used for the preparation through the intestinal canal administration mode, conventional solid preparation such as tablet, capsule, powder, granule etc. be can be made into, liquid preparation such as water or oil-suspending agent made, other liquid preparation such as syrup, mixture, elixir etc.; When being used for the preparation of parenterai administration mode, can be made into solution, powder pin, water or the oiliness suspending agent etc. of injection.The preferred form of the present invention is injectable powder, tablet and capsule.
The present invention also provides described Lonicera confusa extract preparation method: the flower and alabastrum of getting Flos Lonicerae, extract with polar solvent, extracting solution reclaims solvent, be cooled to 0-5 degrees centigrade, filter, it is that 0.5-50% NaOH or KOH are hydrolyzed that filtrate adds w/v, use n-butanol extraction, butanol extraction liquid is with n-butyl alcohol saturated pH value〉7 alkaline aqueous solution strips, and the solution pH value after the extraction is adjusted to 7, and reclaim butanol solution and obtain Lonicera confusa extract.
Described Flos Lonicerae mainly is the woodbine under the Flos Lonicerae item in the pharmacopeia, comprises largeflower-like honeysuckle flower (Loniceramacranthoides Hand.-Mazz), Lonicera confusa (Lonicera confusa DC.), Flos Lonicerae (Lonicera hypoglaucaMiq) etc.
Lonicera confusa extract of the present invention can also extract from Lonicera fulvotnetosa Hsu et S. C. Cheng. Ms (Lonicera fulvotomentosa Hsu etSCCheng) and L. similis Hemsl (Lonicera similis) and obtain.
It is 0-100% methanol or in the alcoholic solution one or more that described polar solvent is selected from water, v/v.
The time of described hydrolysis is 3-15 hours.
Pharmaceutical composition of the present invention has anticancer effect, is applied to prepare the medicine of prevention and treatment cancer.
Described cancer comprises cancers such as hepatocarcinoma, pulmonary carcinoma, cervical cancer, ovarian cancer, breast carcinoma, gastric cancer, rectal cancer, colon cancer, ehrlich ascites carcinoma, carcinoma of prostate, osteocarcinoma or leukemia.
To compare composition clearer and more definite for disclosed total saponin extracts in extract among the present invention and the application 200610039294.4, medicine anticancer effect better (seeing Pharmacodynamic test of active extract), the raw material sources of disclosed preparation method are wider among the present invention, the yield of extract is higher, can reach ten times more than of foregoing invention, be more suitable for suitability for industrialized production.
Description of drawings
The HPLC collection of illustrative plates of extract of the present invention.
The specific embodiment
The present invention is further illustrated below by the specific embodiment and Pharmacodynamic test of active extract:
The largeflower-like honeysuckle flower flower and alabastrum, concentration with 8 times of volumes is 60% ethanol, 90 degrees centigrade of extractions, extracting solution reclaims solvent to 1/5th of original volume, be cooled to 0 degree centigrade, filter to get filtrate, filtrate adds 8% KOH, 80 degrees centigrade of hydrolysis 10 hours, cooling back n-butanol extraction, butanol extraction liquid is stripped with the alkaline aqueous solution of the saturated pH value 13 of n-butyl alcohol, it is 7 that butanol solution after stripping is regulated pH value, reclaim butanol solution and obtain Lonicera confusa extract, after testing, by weight, wherein lonicera macranthoides hypo-saponin B (hereinafter to be referred as component a): Macranthoside A (hereinafter to be referred as components b): 3-O-α-L-rhamnopyranosyl (1-2)-α-L-arabopyranose base-helexin (hereinafter to be referred as amount of component b): 3-O-β-D-glucopyranosyl (1-4)-β-D-glucopyranosyl (1-3)-α-L-rhamnopyranosyl (1-2)-α-L-arabopyranose base-oleanolic acid (hereinafter to be referred as component d)=22:3:3:1, and the gross weight of described 4 kinds of compositions accounts for 99.6% of this extract, yield 15%.
Embodiment 2
The Flos Lonicerae flower and alabastrum, water with 10 times of volumes, 60 degrees centigrade of extractions, extracting solution reclaims the sixth of solvent to original volume, be cooled to 2 degrees centigrade, filter to get filtrate, filtrate adds 50% NaOH, 100 degrees centigrade of following hydrolysis 3 hours, cooling back n-butanol extraction, butanol extraction liquid is stripped with 0.1% saturated NaOH aqueous solution of n-butyl alcohol, is 7 with the butanol solution adjusting pH value after stripping, reclaim butanol solution and obtain Lonicera confusa extract, after testing, by weight, component a: components b: amount of component b: component d=42:9:10:1, and the gross weight of described 4 kinds of compositions accounts for 62% of this extract, yield 12%.
Embodiment 3
Weight ratio is Flos Lonicerae flower and alabastrum and the Lonicera fulvotnetosa Hsu et S. C. Cheng. Ms flower and alabastrum of 1:1, concentration with 3 times of volumes is 100% methanol, extract at normal temperatures, extracting solution reclaims solvent to 1/4th of original volume, be cooled to 4 degrees centigrade, filter to get filtrate, filtrate adds 0.5% KOH, 40 degrees centigrade of following hydrolysis 10 hours, n-butanol extraction, butanol extraction liquid is stripped with 0.8% saturated KOH of n-butyl alcohol, and it is 7 that the butanol solution after stripping is regulated pH value, reclaim butanol solution and obtain Lonicera confusa extract, after testing, by weight, composition a: composition b: composition c: ingredient d=14:2:3:1, and the gross weight of described 4 kinds of compositions accounts for 74% of this extract, yield 11%.
With Polyethylene Glycol 120g, lactose 30g mixing, add said extracted thing 300g mixing more earlier, add the hypromellose slurry again, granulate, drying, granulate incapsulates shell, promptly gets capsule.
Embodiment 4
The Lonicera fulvotnetosa Hsu et S. C. Cheng. Ms flower and alabastrum, concentration with 20 times of volumes is methanol, extract down at 10 degrees centigrade, extracting solution reclaims solvent to 1/10th of original volume, be cooled to 5 degrees centigrade, filter to get filtrate, filtrate adds 3% KOH, hydrolysis at normal temperatures 15 hours, n-butanol extraction, butanol extraction liquid is stripped with the saturated 1%NaOH of n-butyl alcohol, and it is 7 that the butanol solution after stripping is regulated pH value, reclaim butanol solution and obtain Lonicera confusa extract, after testing, by weight, composition a: composition b: composition c: ingredient d=27:5:12:1, and the gross weight of described 4 kinds of compositions accounts for 67.2% of this extract, yield 9%.
Extract is granulated, drying, granulate incapsulates shell, promptly gets capsule.
Embodiment 5
The Lonicera confusa flower and alabastrum, concentration with 25 times of volumes is 100% methanol, 0 degree centigrade of extraction, extracting solution reclaims solvent to 1/20th of original volume, filter to get filtrate, filtrate adds 10% KOH, hydrolysis at normal temperatures 14 hours, n-butanol extraction, butanol extraction liquid is stripped with the aqueous solution of the saturated pH value 14 of n-butyl alcohol, it is 7 that butanol solution after stripping is regulated pH value, reclaims butanol solution and obtains Lonicera confusa extract, after testing, by weight, composition a: composition b: composition c: ingredient d=34:5:1:1, and the gross weight of described 4 kinds of compositions accounts for 66.2% of this extract, yield 14%.
With Polyethylene Glycol 120g, lactose 30g mixing, add said extracted thing 300g mixing more earlier, add the hypromellose slurry again, granulate, drying, granulate adds the magnesium stearate mixing, tabletting, coating promptly gets slow releasing tablet.
Embodiment 6
The L. similis Hemsl flower and alabastrum, water with 18 times of volumes, 100 degrees centigrade of extractions, extracting solution reclaims solvent to 1/10th of original volume, filter to get filtrate, filtrate adds 1% KOH, at 60 degrees centigrade of following hydrolysis 11 hours, n-butanol extraction, butanol extraction liquid is stripped with 0.1% saturated NaOH of n-butyl alcohol, it is 7 that butanol solution after stripping is regulated pH value, reclaims butanol solution and obtains Lonicera confusa extract, after testing, by weight, composition a: composition b: composition c: ingredient d=10:1:2:1, and the gross weight of described 4 kinds of compositions accounts for 80.3% of this extract, yield 10%.
Extract adds the injection water makes dissolving in right amount, boils, and puts coldly, filters, and filtrate is with mannitol, and it is an amount of to add water for injection, transfers pH, and lyophilizing promptly gets lyophilized injectable powder.
Above embodiment is only for the present invention is further illustrated, and scope of the present invention is not subjected to the limitation of illustrated embodiment.
Pharmacodynamic test of active extract
Adopt the antitumor drug experimental technique (People's Health Publisher publishes for " pharmacological experimental methodology " third edition, chief editors such as Xu Shuyun) of this area classics to select for use in vitro tests and In vivo assay Cells that the active anticancer of Lonicera confusa extract of the present invention is verified.
One, cell in vitro test
1, is subjected to the reagent thing: the Lonicera confusa extract (extract I) of the embodiment of the invention 1 preparation; The Lonicera confusa extract (extract II) of embodiment 2 preparation faces with preceding and is made into respective concentration with normal saline.
2, subject cell: MDA-MB-231 (human breast carcinoma), Hela (cervical cancer), HepG2 (hepatocarcinoma), IA-9 (ovarian cancer), U-87-MG (human brain astrocytoblast tumor), CAKI-1 (people's clear cell carcinoma of kidney cutaneous metastatic cell), PC-3 (human prostata cancer), KB (human oral cavity epithelial cancer), KB-VIN (nasopharyngeal carcinoma), A-549 (pulmonary carcinoma), SK-MEL-2 (skin carcinoma), HCT-15 (people ties rectal adenocarcinoma) cell.
3, method and result: test method is international mtt assay: the tumor cell that experimentize that will be in exponential phase is by quantitatively being inoculated in 96 well culture plates, cultivate and add dose after 24 hours, cell trembles at 37 ℃, 5～, continue under the C02 condition to cultivate after 48 hours, the MTT liquid 20M.1 that adds 5mg/ml, continue again to cultivate 4 hours, add DMSO dissolving back and measure the OD value at the 570nm place with microplate reader rapidly, calculate inhibitory rate of cell growth by following formula, experimental result sees Table 1 (table 1.1 and table 1.2).
Growth inhibition ratio (%)=(1-dosing hole average A value/blank hole average A value) * 100%.
Table 1.1 cell in vitro poison experimental result
Table 1.2 cell in vitro poison experimental result
The result shows, handles each tumor cell with the Lonicera confusa extract of variable concentrations, and the growth of tumor cell all is suppressed, and is concentration and time dependence.Show that Lonicera confusa extract of the present invention has stronger inhibitory action to tumor cell.Under the simultaneously same concentration, Lonicera confusa extract of the present invention is to HepG
2Be better than disclosed total saponin extracts in the application 200610039294.4 with the A549 cytotoxicity.
Two, anti-tumor in vivo test
1, to the effect of rat liver cancer (H22) cell inhibiting
1.1 be subjected to the reagent thing: 5-fluorouracil, the general Pharmaceutical product in the rising sun East Sea, Shanghai; The Lonicera confusa extract (extract I) of the embodiment of the invention 1 preparation; The Lonicera confusa extract (extract II) of embodiment 2 preparations; Face with preceding and be made into respective concentration with normal saline.
1.2 animal subject: Male Kunming strain mice, body weight (20 ± 2g), 20 ± 3 ℃ of laboratory temperatures, relative humidity: 75%.
1.3 method and result: strip the hepatocarcinoma H22 cancer piece that went down to posterity 10 days under the aseptic condition, make homogenate, with normal saline 1:3 dilution, it is 2 * 10 that counting is adjusted cell concentration
6L
-1, every 0.2ml, it is subcutaneous to be inoculated in the right armpit of mice, is divided into 8 groups at random after 24 hours, i.e. negative control group (normal saline); Extract I 80mg/kg group, 40mg/kg group, 20mg/kg; Extract I 80mg/kg group, 40mg/kg group, 20mg/kg; Positive controls (5-fluorouracil) is all irritated stomach respective concentration medicine or normal saline for every group, once a day, the execution of weighing after ten days, tumor mass is weighed and is calculated inhibition rate of tumor growth by following formula, the results are shown in Table 2.
Inhibition rate of tumor growth suppression ratio (%)=(the 1-treatment. it is heavy to organize the average tumor of average tumor weight/matched group) * 100%.
2, to the inhibitory action of mice lung cancer (Lewis)
2.1 be subjected to the reagent thing: 5-fluorouracil, the general Pharmaceutical product in the rising sun East Sea, Shanghai; The Lonicera confusa extract (extract I) of the embodiment of the invention 1 preparation; The Lonicera confusa extract (extract II) of embodiment 2 preparation faces with preceding and is made into respective concentration with normal saline.
2.2 animal subject: C
57The BL/6 male mice, and body weight (20 ± 2g), 20 ± 3 ℃ of laboratory temperatures, relative humidity: 75%.2.3 method and result: strip the hepatocarcinoma Lewis cancer piece that went down to posterity 10 days under the aseptic condition, make homogenate, with normal saline 1:3 dilution, it is 1 * 10 that counting is adjusted cell concentration
7L
-1, every 0.2ml, it is subcutaneous to be inoculated in the right armpit of mice, is divided into 8 groups at random after 24 hours, i.e. negative control group (normal saline); Extract I 80mg/kg group, 40mg/kg group, 20mg/kg; Extract I 80mg/kg group, 40mg/kg group, 20mg/kg; Positive controls (5-fluorouracil) is all irritated stomach respective concentration medicine or normal saline for every group, once a day, the execution of weighing after ten days, tumor mass is weighed and is calculated inhibition rate of tumor growth by following formula, the results are shown in Table 2.
Inhibition rate of tumor growth suppression ratio (%)=(the 1-treatment. it is heavy to organize the average tumor of average tumor weight/matched group) * 100%.
3, to the inhibitory action of mouse breast cancer (MA737)
3.1 be subjected to the reagent thing: 5-fluorouracil, the general Pharmaceutical product in the rising sun East Sea, Shanghai; The Lonicera confusa extract (extract I) of the embodiment of the invention 1 preparation; The Lonicera confusa extract (extract II) of embodiment 2 preparation faces with preceding and is made into respective concentration with normal saline.
3.2 animal subject: Kunming kind female mice, body weight (20 ± 2g), 20 ± 3 ℃ of laboratory temperatures, relative humidity: 75%.
3.3 method and result: according to a conventional method under aseptic condition the subcutaneous injection of every Mus right fore armpit growth 7d contain 1 * 10
7Individual thin/the cell suspension 0.1ml of ml, be divided into 8 groups then at random, i.e. negative control group (normal saline); Extract I 80mg/kg group, 40mg/kg group, 20mg/kg; Extract I 80mg/kg group, 40mg/kg group, 20mg/kg; Positive controls (5-fluorouracil).Tumour transplatation begins to irritate stomach next day.Negative control group is irritated the stomach normal saline.Put to death whole mices on the 9th, separate the tumor piece, weighing tumor mass weight is calculated inhibition rate of tumor growth by following formula, the results are shown in Table 2.
Inhibition rate of tumor growth suppression ratio (%)=(it is heavy that the average tumor of average tumor weight/matched group is organized in the 1-treatment) * 100%.
4, to the inhibitory action of mouse cervical cancer (U14)
4.1 be subjected to the reagent thing: 5-fluorouracil, the general Pharmaceutical product in the rising sun East Sea, Shanghai; Extract I; Extract II is faced with preceding and is made into respective concentration with normal saline.
4.2 animal subject: Kunming kind female mice, body weight (20 ± 2g), 20 ± 3 ℃ of laboratory temperatures, relative humidity: 75%.
4.3 method and result: according to a conventional method under aseptic condition the subcutaneous injection of every Mus right fore armpit growth 7d contain 3 * 10
6～4 * 10
6The cell suspension 0.2ml of individual cell/ml is divided into 8 groups then at random, i.e. negative control group (normal saline); Extract I 80mg/kg group, 40mg/kg group, 20mg/kg; Extract I 80mg/kg group, 40mg/kg group, 20mg/kg; Positive controls (5-fluorouracil).Tumour transplatation begins to irritate stomach next day.Negative control group is irritated the stomach normal saline.Put to death whole mices on the 16th, separate the tumor piece, weighing tumor mass weight is calculated inhibition rate of tumor growth.The results are shown in Table 2
Inhibition rate of tumor growth suppression ratio (%)=(it is heavy that the average tumor of average tumor weight/matched group is organized in the 1-treatment) * 100%.
5, to the inhibitory action of nude mice gastric cancer (people SGC7901)
5.1 be subjected to the reagent thing: 5-fluorouracil, the general Pharmaceutical product in the rising sun East Sea, Shanghai; Extract I; Extract II is faced with preceding and is made into respective concentration with normal saline.
5.2 animal subject: nude mice male and female half and half, body weight (20 ± 2g), 20 ± 3 ℃ of laboratory temperatures, relative humidity: 75%.
5.3 method and result: the subcutaneous injection of every Mus left side groin contains 1 * 10 under aseptic condition according to a conventional method
7The cell suspension 0.2ml of individual cell/ml is divided into 8 groups then at random, i.e. negative control group (normal saline); Extract I 80mg/kg group, 40mg/kg group, 20mg/kg; Extract I 80mg/kg group, 40mg/kg group, 20mg/kg; Positive controls (5-fluorouracil).Tumour transplatation begins to irritate stomach next day.Negative control group is irritated the stomach normal saline.Put to death whole mices on the 12nd, separate the tumor piece, weighing tumor mass weight is calculated inhibition rate of tumor growth by following formula, the results are shown in Table 2.
Inhibition rate of tumor growth suppression ratio (%)=(it is heavy that the average tumor of average tumor weight/matched group is organized in the 1-treatment) * 100%.
6, to the inhibitory action of murine sarcoma (SRS-82).
6.1 be subjected to the reagent thing: 5-fluorouracil, the general Pharmaceutical product in the rising sun East Sea, Shanghai; Extract I; Extract II is faced with preceding and is made into respective concentration with normal saline.
6.2 animal subject: Male Kunming strain mice, body weight (20 ± 2g), 20 ± 3 ℃ of laboratory temperatures, relative humidity: 75%.
6.3 method and result: the 8th dying young behind the SRS-82 sarcoma cell inoculation kunming mice of cultivating, extract the about 5ml of ascites in the ascites tumor mouse peritoneal, being diluted to cell number with normal saline is 1 * 10
7/ ml, every mice left side groin subcutaneous injection 0.2ml (2. * 10
6/ only).Be divided into 8 groups then at random, i.e. negative control group (normal saline); Extract I 80mg/kg group, 40mg/kg group, 20mg/kg; Extract I 80mg/kg group, 40mg/kg group, 20mg/kg; Positive controls (5-fluorouracil).Intraperitoneal injection, all beginning of the 2nd day behind inoculated tumour administrations, 1/ day, totally 10 days.In postvaccinal the 12nd day, disconnected neck was put to death and is respectively organized mice, peels off tumor mass, takes by weighing tumor weight, and calculates tumour inhibiting rate as follows, sees Table 2.
Inhibition rate of tumor growth (%)=(it is heavy that the average tumor of average tumor weight/matched group is organized in the 1-treatment) * 100%.
Table 2 couple H
22The influence of tumor-bearing mice and Lewis tumor-bearing mice tumour inhibiting rate
The result shows that Lonicera confusa extract of the present invention can significantly suppress H22, Lewis, MA737, U14, people SGC7901, SRS-82 tumor-bearing mice tumor heavily, is concentration dependent.Show that Lonicera confusa extract of the present invention has the tumor growth of inhibition and anticancer effect.
7, to the influence of mice ehrlich carcinoma (ECA)
7.1 be subjected to the reagent thing: 5-fluorouracil, the general Pharmaceutical product in the rising sun East Sea, Shanghai; This extract I; Extract II is faced with preceding and is made into respective concentration with normal saline.
7.2 animal subject: Male Kunming strain mice, body weight (20 ± 2g), 20 ± 3 ℃ of laboratory temperatures, relative humidity: 75%.
7.3 method and result: every Mus abdominal cavity inoculation cancerous cell suspension 0.2ml/ only inoculates back 24 hours random packet under aseptic condition according to a conventional method, is divided into 8 groups then at random, i.e. negative control group (normal saline); Extract I 80mg/kg group, 40mg/kg group, 20mg/kg; Extract I 80mg/kg group, 40mg/kg group, 20mg/kg; Positive controls (5-fluorouracil).Tumour transplatation begins to irritate stomach next day.Negative control group was irritated the stomach normal saline 9.Observe the mice survival condition after the drug withdrawal, record death toll and survival natural law calculate the increase in life span % of each treated animal, and organize and ask and comparison the results are shown in Table 3:
Increase in life span (ILS)=(administration group The average survival time natural law/matched group The average survival time natural law-1) * 100%
The influence of table 3 pair ehrlich ascites carcinoma ECA tumor-bearing mice time-to-live
The result shows, the Lonicera confusa extract energy significant prolongation ehrlich ascites carcinoma ECA tumor-bearing mice time-to-live of the present invention, be dose dependent, and the result has significant difference.Show that Lonicera confusa extract of the present invention has the tumor growth of inhibition and anticancer effect.
8, to the inhibitory action of mouse ascites tumor (S180)
8.1 be subjected to the reagent thing: 5-fluorouracil, the general Pharmaceutical product in the rising sun East Sea, Shanghai; Extract I; Extract II is faced with preceding and is made into respective concentration with normal saline.
8.2 animal subject: Male Kunming strain mice, body weight (20 ± 2g), 20 ± 3 ℃ of laboratory temperatures, relative humidity: 75%.
8.3 method and result: behind the S180 cell inoculation kunming mice of cultivating the 8th day, extract the about 7ml of ascites in S180 ascites tumor mouse peritoneal, being diluted to cell number with normal saline is 1 * 10
7/ ml, every mouse peritoneal injection 0.2m] (2.0 * 10
6/ only).Be divided into 8 groups then at random, i.e. negative control group (normal saline); Extract I 80mg/kg group, 40mg/kg group, 20mg/kg; Extract I 80mg/kg group, 40mg/kg group, 20mg/kg; Positive controls (5-fluorouracil).Beginning administration in the 2nd day behind inoculated tumour, other gastric infusion 0.2ml, totally 10 days.Write down dead mice quantity every day, and calculate mice prolonged survival period ratio as follows.See Table 4.
Mice prolonged survival period rate=(this group mice mean survival time/negative control group mice mean survival time-1) * 100%.
The influence of table 4 pair sarcoma S180 tumor-bearing mice time-to-live
The result shows, the Lonicera confusa extract energy significant prolongation sarcoma S180 tumor-bearing mice time-to-live of the present invention, be dose dependent, and the result has significant difference.Show that Lonicera confusa extract of the present invention has the tumor growth of inhibition and anticancer effect.
9, to the inhibitory action of mouse leukemia (L1210)
9.1 be subjected to the reagent thing: 5-fluorouracil, the general Pharmaceutical product in the rising sun East Sea, Shanghai; Extract I; Extract II is faced with preceding and is made into respective concentration with normal saline.
9.2 animal subject: the L615 mice, body weight (20 ± 2g), 20 ± 3 ℃ of laboratory temperatures, relative humidity: 75%.
9.3 method and result: behind the L1210 cell inoculation L615 mice of cultivating the 8th day, extract the about 7ml of ascites in L1210 leukemia mouse abdominal cavity, being diluted to cell number with normal saline is 1 * 10
7/ ml, every mouse subcutaneous injection 0.2m] (4 * 10
7/ only).Be divided into 8 groups then at random, i.e. negative control group (normal saline); Extract I 80mg/kg group, 5mg/kg group, 2.5mg/kg; Extract I 80mg/kg group, 40mg/kg group, 20mg/kg; Positive controls (5-fluorouracil).Beginning administration in the 2nd day behind inoculated tumour, difference gastric infusion 0.2ml, totally 10 days.Write down dead mice quantity every day, and calculate mice prolonged survival period ratio as follows.See Table 5.
Mice prolonged survival period rate: (this group mice mean survival time/negative control group mice mean survival time-1) * 100%
The influence of table 5 pair leukemia L12100 tumor-bearing mice time-to-live
The result shows, the Lonicera confusa extract energy significant prolongation leukemia L12100 tumor-bearing mice time-to-live of the present invention, be dose dependent, and the result has significant difference.Show that Lonicera confusa extract of the present invention has the tumor growth of inhibition and anticancer effect.
Claims (6)
1. Lonicera confusa extract, the main component that it is characterized in that described extract is a lonicera macranthoides hypo-saponin B, Macranthoside A, 3-O-α-L-rhamnopyranosyl (1-2)-α-L-arabopyranose base-helexin and 3-O-β-D-glucopyranosyl (1-4)-β-D-glucopyranosyl (1-3)-α-L-rhamnopyranosyl (1-2)-α-L-arabopyranose base-oleanolic acid, weight ratio is followed successively by 27～14: 5～1: 9～1: 1, and the gross weight of described 4 kinds of compositions accounts for the 70%-99.6% of this extract, Flos Lonicerae is selected from largeflower-like honeysuckle flower, Lonicera confusa, Flos Lonicerae, in Lonicera fulvotnetosa Hsu et S. C. Cheng. Ms or the L. similis Hemsl one or more, its preparation method is a flower and alabastrum of getting Flos Lonicerae, extract with polar solvent, extracting solution reclaims solvent, be cooled to 0-5 degree centigrade, filter, filtrate adds NaOH or the KOH that w/v is 0.5-50% and is hydrolyzed, the time of hydrolysis is 3-15 hour, use n-butanol extraction, butanol extraction liquid is stripped with the alkaline aqueous solution of saturated pH value＞7 of n-butyl alcohol, solution pH value after the extraction is adjusted to 7, reclaim butanol solution and obtain Lonicera confusa extract, described polar solvent is selected from water, v/v is the methanol of 0-100% or in the alcoholic solution one or more.
2. pharmaceutical preparation, it is characterized in that Lonicera confusa extract as claimed in claim 1 and one or more pharmaceutically the acceptable adjuvant make.
3. preparation as claimed in claim 2 is characterized in that it being by using through the mode of intestinal canal administration or parenterai administration.
4. preparation as claimed in claim 3 is characterized in that being tablet, capsule, powder, granule, spray, syrup, mixture, injection.
5. the purposes of Lonicera confusa extract as claimed in claim 1 is characterized in that the application in the medicine of preparation prevention and treatment cancer.
6. purposes as claimed in claim 5, described cancer are hepatocarcinoma, pulmonary carcinoma, cervical cancer, ovarian cancer, breast carcinoma, gastric cancer, rectal cancer, colon cancer, carcinoma of prostate, osteocarcinoma or leukemia.
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| TWI410431B (en) * | 2010-12-22 | 2013-10-01 | Ind Tech Res Inst | Use of oleanolic acid derivatives in the manufacture of a medicament to prevent or treat hepatitis c |
| CN102199184B (en) * | 2011-04-07 | 2013-03-06 | 江西本草天工科技有限责任公司 | Hederasaponin derivative, preparation method of salts thereof and application thereof to resisting tumor |
| CN102424699B (en) * | 2011-09-23 | 2016-02-17 | 江苏省中国科学院植物研究所 | A kind of new largeflower-like honeysuckle flower saponin(e and its production and use |
| CN102924418B (en) * | 2011-11-03 | 2014-04-02 | 成都中医药大学 | Method for separating and purifying effective ingredients from fine felt hairy honeysuckle |
| CN103183616B (en) * | 2012-12-06 | 2014-12-03 | 长沙理工大学 | Method for preparing chlorogenic acid from leaves of lonicera hypoglauca miq |
| CN103860622B (en) * | 2012-12-07 | 2016-05-25 | 廉吉芳 | A kind of method of extracting saponins compound from honeysuckle |
| CN103690549A (en) * | 2012-12-24 | 2014-04-02 | 江西本草天工科技有限责任公司 | Preparation of alpha-hederin solid dispersion and its cyclodextrin inclusion compound |
| CN104523700A (en) * | 2013-09-28 | 2015-04-22 | 杨小林 | Application of hederagenin to preparation of drug for resisting to MGC-803 tumors of gastric cancer |
| CN104523699B (en) * | 2013-09-28 | 2019-09-13 | 杨小林 | Hederagenin is preparing the application in ovarian cancer resistance HO-8910PM tumour medicine |
| CN105053174B (en) * | 2015-09-17 | 2018-08-17 | 邯郸学院 | A kind of Preparation method and use of plant source composite preservative |
| CN108078922B (en) * | 2017-02-23 | 2020-08-11 | 佑嘉（上海）医药科技有限公司 | Lonicera macranthoides saponin B self-microemulsion preparation and application thereof |
| CN107602717B (en) * | 2017-09-29 | 2020-05-26 | 贵州中科健生物医药有限公司 | Preparation method and application of lonicera confusa polysaccharide |
| CN114259510A (en) * | 2021-12-24 | 2022-04-01 | 亿利耐雀生物科技有限公司 | Total saponins of flos Lonicerae, and its extraction method and application |
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