CN103610849B - The application of RP-HPLC in preparation treatment carcinoma of prostate medicine - Google Patents
The application of RP-HPLC in preparation treatment carcinoma of prostate medicine Download PDFInfo
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Abstract
<b> the invention discloses the application of RP-HPLC in preparation treatment carcinoma of prostate medicine.RP-HPLC is People's Republic of China's version pharmacopeia in 2010 nomenclature of drug recorded; Tibetan of RP-HPLC system proved recipe; be made up of more than 100 taste medicines such as Margarita, Cinnabaris, Lignum Santali Albi, Lignum Dalbergiae Odoriferae, Lignum Aquilariae Resinatum, Fructus Chebulae, Calculus Bovis, artificial Moschus, Stigma Crocis, function is heat-clearing and toxic substances removing, mediation nourishing.Instant invention overcomes the technology prejudice of above-mentioned application aspect, found by pharmacological evaluation, the ligroin extraction of RP-HPLC is external has good inhibitory action to prostate gland cancer cell, and its effect and cisplatin are equal to or slightly better; The Contained Serum of the multiple extract of RP-HPLC is external also all has significant inhibitory action to carcinoma of prostate.</b>
Description
Technical field
The present invention relates to the application of RP-HPLC in preparation treatment carcinoma of prostate medicine, belong to field of medicaments.
Background technology
Carcinoma of prostate is the modal malignant tumor of male reproductive system, is only second to pulmonary carcinoma, is the second of cancer mortality male.Carcinoma of prostate morbidity increased with the age, and its sickness rate has obvious regional difference, and before China, sickness rate is lower, but due to aged tendency of population, sickness rate increases to some extent in recent years.
RP-HPLC is People's Republic of China's version pharmacopeia in 2010 nomenclature of drug recorded.
Tibetan of RP-HPLC system proved recipe, Cheng Fang is in eightth century of Christian era, and be hide king, the Panchen, Dalai and high official and noble lord's medicine dedicated the earliest, go through the applicating history of more than 1,000 year, nowadays, RP-HPLC records in " Chinese Pharmacopoeia " (2010 editions).RP-HPLC is made up of more than 100 taste medicines such as Margarita, Cinnabaris, Lignum Santali Albi, Lignum Dalbergiae Odoriferae, Lignum Aquilariae Resinatum, Fructus Chebulae, Calculus Bovis, artificial Moschus, Stigma Crocis, and function is heat-clearing and toxic substances removing, mediation nourishing.For " grand, red bar, Baconic " each disease, old gastroenteritis, ulcer, " wooden cloth " is sick, atrophic gastritis, various nosotoxicosis; Syphilis, leprosy, outmoded calentura, anthrax, furuncle pain, yellow fluid reducing, suppurates.Modern pharmacodynamic study result shows, this product has antiinflammatory, suppresses ulcer, promotes the effects such as ulcer healing.Acute toxicity test, long term toxicity test result shows, this product is without overt toxicity.Clinical practice result shows, this product treatment digestive system disease, as aspect display good efficacy such as various acute or chronic gastritis, chronic atrophic gastritis, peptic ulcer, liver cirrhosis, the untoward reaction of chemotherapy metenteron, and having good health-care effect, is one of Tibetan medicine's health care medicine.
In the application for the treatment of field of RP-HPLC, people know the effect of its heat-clearing and toxic substances removing, mediation nourishing, and know it for " grand, red bar, Baconic " each disease, old gastroenteritis, ulcer, " wooden cloth " is sick, atrophic gastritis, various nosotoxicosis; Syphilis, leprosy, outmoded calentura, anthrax, furuncle pain, yellow fluid reducing, the treatment of suppuration etc.The aspects such as the function of RP-HPLC cures mainly, document, patent all do not find that it can be used for treating the research of carcinoma of prostate.
Summary of the invention
The object of the invention is to provide the application of RP-HPLC in preparation treatment carcinoma of prostate medicine.
Technical scheme of the present invention is as follows:
The application of RP-HPLC in preparation treatment carcinoma of prostate medicine.
Described RP-HPLC is the pharmaceutical formulations made after preparing active component by its formula medical material.
Described active component is one or more in the ligroin extraction of RP-HPLC formula medical material, ethanol extraction or water extract, wherein:
Described ligroin extraction is prepared as follows: get the fine powder after RP-HPLC porphyrize or its formula medical material, and add the petroleum ether boiling extraction 3 times of envelope-bulk to weight ratio 6 times, each 1 hour, 40 DEG C-50 DEG C reclaim under reduced pressure petroleum ether, to obtain final product;
Described ethanol extraction obtains as follows: get the fine powder after RP-HPLC porphyrize or its formula medical material, adds the ethanol extraction 3 times of envelope-bulk to weight ratio 8 times, each 1.5 hours, 50 DEG C of-60 DEG C of decompression recycling ethanols, dry, to obtain final product; The technological parameter of described recovery ethanol is-0.07 ~-0.09Mpa, and the technological parameter of product drying under reduced pressure is 70 DEG C ,-0.08 ~-0.09Mpa;
Described water extract obtains as follows: get the fine powder after RP-HPLC porphyrize or its formula medical material, adds the soak by water 2 times of envelope-bulk to weight ratio 8 times, each 2 hours, and filter, filtrate evaporate to dryness, to obtain final product.
Described pharmaceutical formulations is concentrated pill, coated tablet, thin membrane coated tablet or capsule preparations.
Described RP-HPLC and chemical drugs or Chinese medicine or natural drug are preparing the application for the treatment of in carcinoma of prostate medicine.
In the present invention, the unit corresponding relation of volume weight is ml/g or l/kg.
Beneficial effect: the technology prejudice that instant invention overcomes RP-HPLC application aspect, is found by pharmacological evaluation, RP-HPLC ligroin extraction is external has good inhibitory action to prostate gland cancer cell, and its effect and cisplatin are equal to or slightly better; And the Contained Serum of the water extract of RP-HPLC is external also all has significant inhibitory action (P<0.05) to carcinoma of prostate.
Detailed description of the invention
Following experimental example and embodiment are used for further illustrating but are not limited to the present invention.
The preparation of each extract of experimental example 1, RP-HPLC
One, sample
RP-HPLC, Qinghai gold is scolded Tibetan medicine Pharmaceutical limited company and is produced, lot number: 20100707
Two, method and step
1, ligroin extraction: get the fine powder 50g after RP-HPLC porphyrize, adds petroleum ether 60 DEG C ~ 90 DEG C boiling extraction 3 times that envelope-bulk to weight ratio is 6 times, each 1 hour.40 DEG C-50 DEG C reclaim under reduced pressure petroleum ether, obtain ligroin extraction 2.71g.
2, ethyl acetate extract: get the medicinal residues after Petroleum ether extraction, waves most petroleum ether, and adding envelope-bulk to weight ratio is that the ethyl acetate of 6 times carries out boiling extraction 3 times, each 1 hour.50 DEG C-60 DEG C reclaim under reduced pressure ethyl acetate, obtain ethyl acetate extract 7.66g.
3, ethanol extraction: get ethyl acetate extract after medicinal residues, wave most ethyl acetate, add the ethanol extraction 3 times that envelope-bulk to weight ratio is 8 times, each 1.5 hours, 50 DEG C of-60 DEG C of decompression recycling ethanols, dry, obtain ethanol extraction 4.68g.
4, water extract: get the medicinal residues after ethanol extraction, adds the soak by water 2 times that envelope-bulk to weight ratio is 8 times.Each 2 hours, filter, filtrate evaporate to dryness, obtained water extract 4.76g.
In the present invention, the unit corresponding relation of volume weight is ml/g or l/kg, and what other was not specified is the upper usual parameter of pharmacy production or method.
Experimental example 2, RP-HPLC are to the inhibitory action of carcinoma of prostate
One, experimental principle
MTT analytic process with living cells metabolize thing reducing agent 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide, based on MTT tetrazolium bromide.MTT is yellow compound, it is the hydrionic dyestuff of a kind of acceptance, the respiratory chain in living cells mitochondrion can be acted on, under the effect of succinate dehydrogenase and cytochrome C, tetrazolium ring opening, generate blue formazan crystallization, the growing amount of formazan crystallization is only directly proportional to number of viable cells (in dead cell, succinate dehydrogenase disappears, and MTT can not be reduced).The formazan crystallization that reduction generates can be dissolved in the MTT lysate containing the DMF of 50% and the ten dimethyl sodium sulfonates (pH 4.7) of 20%, utilizes microplate reader to measure the optical density OD value at 490 nm places, to reflect number of viable cells.
Two, medicine and reagent
1, animal
The healthy Kunming mouse 90 of SPF level, body weight 30 ± 2g, is provided by the new laboratory animal of Lukang Medical Co., Ltd., Shandong, the animal quality certification number: SCXK Shandong 20130001.
2, experimental cell
Carcinoma of prostate (pc3) cell, is provided by Shandong Prov. Hospital.
3, Experimental agents
Experimental agents: each extract of RP-HPLC prepared by experimental example 1, respectively adds the suspension that normal saline becomes 500ml.
Positive control medicine: cisplatin, (Qilu Pharmaceutical Co., Ltd., lot number: 1010041DC)
Reagent: 1640 cell culture mediums (Hyclone, Sai Mo fly generation that biochemistry goods Beijing company limited); Hyclone (Hyclone, Sai Mo fly generation that biochemistry goods Beijing company limited); Pancreatin cell dissociation buffer (the green skies), penicillin streptomycin mixed liquor is dual anti-, cisplatin for inj (Qilu Pharmaceutical Co., Ltd., lot number: 1010041DC).
4, experimental apparatus
Vertical pressure steam sterilization pan (LDZX-50FBS, Shen, Shanghai is pacified); Double one side clean work station (SW-CJ-1C, Suzhou purifies); CO2 gas incubator (BB16UV/BB5060UV, HERAEUS); Desk centrifuge (H3, Sigma); Microplate reader (Multiskan MK3, Thermo Scientific); Electric heating constant-temperature blowing drying box (101, Shanghai roc is along scientific instrument company limited); Inverted microscope (XDS-1B, Chongqing optical instrument factory); Water-bath constant temperature oscillator (SHZ-82, Medical Instruments factory of Jintan City); Pipettor (Gilson, Eppendorf); Culture bottle (Corning); 96 orifice plates (Costar, USA)
Three, experimental technique
1, the external inhibitory action to prostate gland cancer cell of RP-HPLC is tested
(1) cell recovery and cultivation
From ultra cold storage freezer, get frozen carcinoma of prostate (pc3) cell, rapid input is heated in the aquesterilisa of 37 DEG C in advance, after cryopreserving liquid dissolves, and centrifugal three minutes of 2000r/min, collecting cell.Then to contain 1640 fresh cultures of 10% hyclone at 37 DEG C, 5%CO
2cultivate in incubator.
When Growth of Cells to 80% merges, digest with the pancreatin cell dissociation buffer (containing 0.02%EDTA) of 0.25%, with 1 × 10^
4individual cell per well is inoculated in 96 orifice plates, and reserved three zeroing holes, zeroing hole only adds not containing 1640 culture medium of serum, does not add cell.
(2) dosing
After the cell in 96 orifice plates covers with monolayer, discard culture medium, zeroing group, blank group add 100 μ l not containing 1640 culture medium of serum, and positive controls adds 10 μ g/ml cisplatin 100 μ l.Other every holes of administration group add 1640 culture medium 100 μ l of the screening of medicaments containing gradient dilution concentration respectively, and dosing one is arranged.Then cell is at 37 DEG C, 5%CO
2continue in incubator to cultivate 24h, added drug level is the former concentration of RP-HPLC after conversion.
(3) measure
Mtt assay measures cell survival rate: add 5mg/mlMTT solution 20 μ l lucifuge in incubator in every hole and react 4 h.Then add 100 μ l DMSO microplate reader and measure OD value in 492nm, calculate suppression ratio: inhibitory rate of cell growth=(matched group absorbance values-experimental group absorbance values)/matched group absorbance values × 100%.
2, the external inhibitory action to prostate gland cancer cell of RP-HPLC Contained Serum is tested
(1) cell recovery and cultivation
From ultra cold storage freezer, get frozen carcinoma of prostate (pc3) cell, rapid input is heated in the aquesterilisa of 37 DEG C in advance, after cryopreserving liquid dissolves, and centrifugal three minutes of 2000r/min, collecting cell.Then to contain 1640 fresh cultures of 10% hyclone at 37 DEG C, 5%CO
2cultivate in incubator.
(2) preparation of Contained Serum
Kunming mouse 90, is divided into 6 groups at random, blank group, cisplatin group, RP-HPLC ligroin extraction group, RP-HPLC ethyl acetate extract group, RP-HPLC ethanol extraction group, RP-HPLC water extract group, often organizes 15.The administration concentration of RP-HPLC each extract group is equivalent to the former medicine/ml of 10mg RP-HPLC, and cisplatin group administration concentration is 10 μ g/ml.Each group of continuous gavage of mice 3 days, every day every, 0.5ml, was equivalent to 100 times of the maximum recommended doses of human body.Wherein blank group gives normal saline.After last day gavage 1 hour, 10% chloral hydrate anesthesia, abdominal aortic blood, 3000 turns/min was centrifugal, gets upper serum ,-80 DEG C of preservations.With the mixing of group serum, 0.22 μm of filtering with microporous membrane is degerming in for subsequent use.
(3) mtt assay measures Contained Serum to the suppression situation of different growth of tumour cell
By CO
2carcinoma of prostate (pc3) cell cultivated in incubator, digests with the pancreatin cell dissociation buffer (containing 0.02%EDTA) of 0.25%, with 1 × 10^
4individual cell per well is inoculated in 96 orifice plates, and reserved three zeroing holes, zeroing hole only adds not containing 1640 culture medium of serum, does not add cell.After the cell in 96 orifice plates covers with monolayer, discard culture medium, zeroing group, blank group add 100 μ l not containing 1640 culture medium of serum, and every hole adds 1640 culture medium 100 μ l of Contained Serum respectively, and the medicine of each concentration adds 6 holes.Then cell is at 37 DEG C, 5%CO
2continue in incubator to cultivate 24h.Add 5mg/mlMTT solution 20 μ l lucifuge in incubator and react 4 h.Then add 100 μ l DMSO microplate reader and measure OD value in 492nm, calculate suppression ratio: inhibitory rate of cell growth=(matched group absorbance values-experimental group absorbance values)/matched group absorbance values × 100%.
Four, experimental result
All data are all with (X ± SD) display, and adopt GraphPad Prism 5 software analysis process data, utilize between t survey each group and blank group whether there is significant difference, P<0.05 is significant difference.
1, the external inhibitory action to prostate gland cancer cell of RP-HPLC is tested
Carry out IC50 calculating to various medicine, IC50 value is verified lower than the medicine of 250 μ g/ml.All data after checking represent with X ± SD, and the significance of group difference is compared in t inspection, and P<0.05 is significant difference.
IC50 value lower than the medicine of 250 μ g/ml is: RP-HPLC ligroin extraction, and IC50 value is 68.74 μ g/ml.Other drug IC50 value is bigger than normal or do not have inhibitory action to cancerous cell, no longer does demonstration test.
Table 1, different extract are to the suppression ratio (n=6, ± SD) of prostate gland cancer cell
* compare with blank group, P<0.05
Adopt GraphPad Prism 5 software analysis process data, utilize between t survey each group and blank group and whether there is significant difference, result shows, cisplatin, RP-HPLC ligroin extraction and blank group have significant difference, significant difference is not had between other each group and blank groups, illustrate that RP-HPLC ligroin extraction has good inhibitory action to prostate gland cancer cell, its effect and cisplatin are equal to or slightly better.
2, the external inhibitory action to prostate gland cancer cell of RP-HPLC Contained Serum is tested.The results are shown in Table 2.
Table 2, RP-HPLC Contained Serum to the suppression ratio of carcinoma of prostate (n=15,
± SD, unit: %)
Note: compare with blank group, * P<0.05
As can be seen from table 2 result, the Contained Serum of RP-HPLC water extract has significant inhibitory action (P<0.05) to carcinoma of prostate, and ethanol extraction has suppression trend, but does not have statistical significance.
Following embodiment all can realize the effect described in above-mentioned experimental example.
Embodiment 1
RP-HPLC, 1g/ ball, 1 ball on the 1st, took every 3-7 days 1 gram (1 ball); Grinding (medicine) being taken before meal at rear dawn is used.
Embodiment 2
RP-HPLC concentrated pill, 0.2g/ ball, 1 ball on the 1st, took 1 ball, prepares as follows every 3-7 days:
A, ligroin extraction: get the fine powder after RP-HPLC porphyrize or its formula medical material, add the petroleum ether boiling extraction 3 times that envelope-bulk to weight ratio is 6 times, each 1 hour, 40 DEG C-50 DEG C reclaim under reduced pressure petroleum ether, to obtain final product.
B, ethanol extraction: get the fine powder after RP-HPLC porphyrize or its formula medical material, add the ethanol extraction 3 times that envelope-bulk to weight ratio is 8 times, each 1.5 hours, 50 DEG C of-60 DEG C of decompression recycling ethanols, dry, to obtain final product.
C, water extract: get the fine powder after RP-HPLC porphyrize or its formula medical material, add the soak by water 2 times that envelope-bulk to weight ratio is 8 times, each 2 hours, filter, filtrate evaporate to dryness, to obtain final product.
After above-mentioned ligroin extraction, ethanol extraction and water extract being pulverized, mix homogeneously, becomes 0.2g/ ball with water pill, to obtain final product, and every ball is equivalent to RP-HPLC former preparation 2 ball.
The unit corresponding relation of volume weight is ml/g or l/kg.
Embodiment 3
RP-HPLC coated tablet, 0.5g/ sheet, 1 day 1, took 1 every 3-7 days, containing ligroin extraction, ethanol extraction and water extract three kinds of active components, wherein the preparation method of ligroin extraction, ethanol extraction and water extract is with embodiment 2, after finally three kinds of active components being pulverized, mix homogeneously, adds the adjuvant such as starch, Pulvis Talci and makes plain sheet and sugar coating, obtain, every sheet is equivalent to RP-HPLC former preparation 2 ball.
Embodiment 4
RP-HPLC thin membrane coated tablet, 0.5g/ sheet, 1 day 1, took 1 every 3-7 days, containing ligroin extraction, ethanol extraction and water extract three kinds of active components, wherein the preparation method of ligroin extraction, ethanol extraction and water extract is with embodiment 2, after finally three kinds of active components being pulverized, mix homogeneously, adds the adjuvant such as starch, Pulvis Talci and makes plain sheet and film coating, obtain, every sheet is equivalent to RP-HPLC former preparation 2 ball.
Embodiment 5
RP-HPLC capsule, 0.5g/ grain, 1 day 1, took 1 every 3-7 days, containing ligroin extraction, ethanol extraction and water extract three kinds of active components, wherein the preparation method of ligroin extraction, ethanol extraction and water extract is with embodiment 2, after finally three kinds of active components being pulverized, mix homogeneously, adds the adjuvant such as starch, Pulvis Talci and makes capsule, obtain, every is equivalent to RP-HPLC former preparation 2 ball.
Embodiment 6
RP-HPLC capsule, 0.5g/ grain, 1 day 1, took 1 every 3-7 days, containing ligroin extraction, water extract two kinds of active components, the preparation method of two kinds of active components is with embodiment 2, after finally two kinds of active components being pulverized, mix homogeneously, adds the adjuvant such as starch, Pulvis Talci and makes capsule, obtain, every is equivalent to RP-HPLC former preparation 2 ball.
Embodiment 7
RP-HPLC concentrated pill, 0.5g/ ball, 1 ball on the 1st, took 1 ball every 3-7 days, containing a kind of active component of ligroin extraction, the preparation method of active component with embodiment 2, after finally this active component being pulverized, mix homogeneously, with water pill and get final product, every ball is equivalent to RP-HPLC former preparation 2 ball.
Embodiment 8
RP-HPLC capsule, 0.5g/ grain, 1 day 1, took 1 every 3-7 days, a kind of active component of aqueous extract, the preparation method of this active component is with embodiment 2, after finally this active component being pulverized, mix homogeneously, adds the adjuvant such as starch, Pulvis Talci and makes capsule, obtain, every is equivalent to RP-HPLC former preparation 2 ball.
Claims (3)
1. the application of RP-HPLC in preparation treatment carcinoma of prostate medicine, it is characterized in that, described RP-HPLC is the pharmaceutical formulations made after preparing active component by its formula medical material; Described active component is one or more in the ligroin extraction of RP-HPLC formula medical material, ethanol extraction or water extract, wherein:
Described ligroin extraction is prepared as follows: get the fine powder after RP-HPLC porphyrize or its formula medical material, and add the petroleum ether boiling extraction 3 times that envelope-bulk to weight ratio is 6 times, each 1 hour, 40 DEG C-50 DEG C reclaim under reduced pressure petroleum ether, to obtain final product;
Described ethanol extraction obtains as follows: get the fine powder after RP-HPLC porphyrize or its formula medical material, adds the ethanol extraction 3 times that envelope-bulk to weight ratio is 8 times, each 1.5 hours, 50 DEG C of-60 DEG C of decompression recycling ethanols, dry, to obtain final product; The technological parameter of described recovery ethanol is-0.07 ~-0.09Mpa, and the technological parameter of product drying under reduced pressure is 70 DEG C ,-0.08 ~-0.09Mpa;
Described water extract obtains as follows: get the fine powder after RP-HPLC porphyrize or its formula medical material, and add the soak by water 2 times that envelope-bulk to weight ratio is 8 times, each 2 hours, filter, filtrate evaporate to dryness, to obtain final product;
The unit corresponding relation of described volume weight is ml/g or l/kg.
2. the application of RP-HPLC according to claim 1 in preparation treatment carcinoma of prostate medicine, it is characterized in that, described pharmaceutical formulations is concentrated pill, coated tablet, thin membrane coated tablet or capsule preparations.
3. the application of RP-HPLC according to claim 1 and 2 in preparation treatment carcinoma of prostate medicine, is characterized in that, described RP-HPLC and chemical drugs or Chinese medicine or natural drug are preparing the application for the treatment of in carcinoma of prostate medicine.
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Effective date of registration: 20171227 Address after: 810003, No. two, No. 22, Xining Road, Qinghai, Qinghai Province Patentee after: JINHE TIBETAN MEDICINE CO., LTD. Address before: The 250101 Ji'nan Road, Shandong province hi tech Development Zone, No. 322 room 501-506 Patentee before: Shandong Jin He drug development research company limited |