CN101804096B - Method for extracting purified total alkaloid of strychnos nux-vomica and application thereof in pharmacy - Google Patents
Method for extracting purified total alkaloid of strychnos nux-vomica and application thereof in pharmacy Download PDFInfo
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Abstract
The invention discloses a method for obtaining purified total alkaloid of strychnos nux-vomica by removing partial strychnine in the total alkaloid of the strychnos nux-vomica, which comprises the following steps: extracting with a certain concentration of ethanol, dissolving the extract with hydrochloric acid ; adjusting the pH value to be alkaline after dissolving; carrying out extracting and pressure reduction on dichloromethane to remove solvent to obtain the total alkaloid of the strychnos nux-vomica; ultrasound dissolving total alkaloid powder of strychnos nux-vomica with ethanol; carrying out pressure reduction and concentration to a certain volume; centrifuging the concentrated solution, taking supernate, refrigerating and standing to remove precipitate; and drying the supernate to obtain the purified total alkaloid of strychnos nux-vomica. The content of strychnine in the total alkaloid of strychnos nux-vomica extracted by the method is remarkably reduced; and in addition, the method has safe extraction process, low cost and is beneficial to industrial scale production. Proved by the evaluation result of a pharmacological test, the effects of analgesia, anti-inflammation, immunity and antitumor activity of the purified total alkaloid of strychnos nux-vomica are remarkably superior to the effects of total alkaloid of strychnos nux-vomica and the toxicity is remarkably reduced.
Description
One, technical field
The present invention relates to a kind of method for extraction and purification of active ingredient of Chinese herbs, specifically relate to remove the method for extraction and purification of the purified total alkaloid of strychnos nux-vomica that obtains behind the part strychnine and the application in pharmacy thereof.
Two, technical background
Semen Strychni has another name called vomiting nut, beginning is stated from " Compendium of Materia Medica rolls up 18 ", it is the dry mature seed of loganiaceae plant Semen Strychni (Strychnounux-vomica L.), effect with removing obstruction in the collateral to relieve pain, mass dissipating and swelling eliminating, be commonly used to treat sore throat, toxic swelling, rheumatic arthralgia, fracture, facial paralysis, diseases such as myasthenia gravis on the tcm clinical practice.Compendium of Material Medica is sayed that it can be controlled in the Chinese patent medicines such as " mass in the abdomen disappears for typhoid fever calentura, throat arthralgia pain " as drug for invigorating blood circulation and eliminating stasis, be everlasting " JIUFEN POWDER ", " dispelling wind Pain-Stopping Pill ", " tiger strong ball ", " Green Dragon ball " and is used.
Total alkaloids is a topmost effective site in the Semen Strychni, and present studies show that: have 16 kinds of alkaloid components in the total alkaloids in Semen Strychni at least, main component is strychnine and strychnine.Strychnine has very strong pharmacologically actives such as antalgic and inflammation relieving as one of main component of Semen Strychni, and be usually used in antitussive clinically, eliminate the phlegm, antibacterial and antitumor, and another main component
StrychnineContent account for total alkaloids pact half, but pharmacologically active is relatively low, is the main toxic component of Semen Strychni.Total alkaloids in Semen Strychni is applied to clinical biggest obstacle and is to have intensive nervus centralis toxicity.
Have document (cross the China etc. of shaking, the research of Semen Strychni pharmacological toxicology is looked back and the safety research prospect, combination of Chinese and Western medicine journal, 2008,6 (6): 645-649) report strychnine and strychnine are given mouse stomach, LD
50Be respectively 3.27mgkg
-1And 233mgkg
-1, lumbar injection is 1.53mgkg
-1And 69mgkg
-1Therefore if can remove part strychnine in the total alkaloids, then can reduce its toxicity, can keep its original pharmacologically active substantially again, improve clinical application safety.It must be noted that, can realize that cost is very high, other alkaloid component loss simultaneously is also big, and is difficult to realize large-scale industrial production, therefore, removes the part strychnine and has more feasibility and application prospect though all remove strychnine.So far do not appear in the newspapers about the method for removing the part strychnine in the total alkaloids in Semen Strychni.
Three, summary of the invention
1, goal of the invention
The object of the present invention is to provide a kind of method of removing the purified total alkaloid of strychnos nux-vomica of part strychnine.
Purified total alkaloid of strychnos nux-vomica provided by the present invention is compared with the total alkaloids in Semen Strychni of not removing the part strychnine, pharmacological action at aspects such as analgesia, antiinflammatory, immunity, antitumor significantly improves, and toxicity significantly descends, and is easy to realize suitability for industrialized production.
2, technical scheme
The objective of the invention is to adopt following technical proposal to realize:
A kind of method of extracting purified total alkaloid of strychnos nux-vomica is characterized in that step is as follows:
(1) with the Semen Strychni medical material, is ground into coarse powder, takes by weighing 100-200 gram coarse powder;
(2) with the alcohol reflux of 1000-2000ml, decompression recycling ethanol gets pure extractum;
(3) with the above-mentioned pure extractum of dissolving with hydrochloric acid of 400-1000ml, centrifugal, get supernatant;
(4) add sodium hydroxide, the adjusting pH value is 10-14, and the dichloromethane of this solution with 500-2000ml extracted, and removal of solvent under reduced pressure gets the total alkaloids in Semen Strychni powder;
(5) with above-mentioned total alkaloids in Semen Strychni powder, with 1000-3000ml50-90% ethanol ultrasonic dissolution;
(6) 0.4-0.05 that is evaporated to original volume doubly, centrifugal concentrated solution is got supernatant;
(7) supernatant cold preservation is left standstill the centrifugal precipitation of removing, the dry purified total alkaloid of strychnos nux-vomica that gets of supernatant then.
Concentration of alcohol described in the step (2) is 50-95%, and the reflux number of times is 1-5 time, and each reflux time is 0.5-2h.
Concentration of hydrochloric acid described in the step (3) is 0.1-1mol/L.
Refrigerated storage temperature described in the step (7) is 0-10 ℃, and described drying means is constant pressure and dry or vacuum drying.
The application of described extracting method gained purified total alkaloid of strychnos nux-vomica in the preparation analgesic.
The application of described extracting method gained purified total alkaloid of strychnos nux-vomica in the preparation anti-inflammatory drug.
The application of described extracting method gained purified total alkaloid of strychnos nux-vomica in the preparation antitumor drug.
The application of described extracting method gained purified total alkaloid of strychnos nux-vomica in the preparation immunoregulation medicament.
The purified total alkaloid of strychnos nux-vomica that the present invention extracts can be used to prepare analgesic, anti-inflammatory agent, antineoplastic agent, immunomodulator.But more than use oral administration, lumbar injection and percutaneous administration.
We find that total alkaloids in Semen Strychni and strychnine dissolubility in finite concentration ethanol differs very big, utilize the difference of this dissolution properties can realize keeping total alkaloids in Semen Strychni and remove the part strychnine.Not removing the total alkaloids in Semen Strychni of part strychnine before purified total alkaloid of strychnos nux-vomica that pharmacological evaluation shows acquisition that the present invention extracts and purification compares, pharmacological action at aspects such as analgesia, antiinflammatory, antitumor, immunomodulating significantly strengthens, and toxicity significantly descends.
The content of strychnine measures wherein with the HPLC method that the content of strychnine reaches 44%～52% in the total alkaloids in Semen Strychni before the purification, the content of promptly removing strychnine in the purified total alkaloid of strychnos nux-vomica that obtains behind the part strychnine through the extraction purification is 14%～23%, and the purity of measuring purified total alkaloid of strychnos nux-vomica with acid-dye colorimetry is 75%～90%.
Four, the specific embodiment
Further illustrate content of the present invention below in conjunction with example, but these embodiment do not limit protection scope of the present invention.
Embodiment 1: the method for extracting purified total alkaloid of strychnos nux-vomica:
(1) takes by weighing Semen Strychni, pulverize, sieve, make coarse powder, take by weighing the 200g coarse powder;
(2) extract with 70% alcohol heating reflux, each 1000mL extracts 1h, extracts altogether 3 times, filters, and merges three times filtrate, and decompression recycling ethanol gets pure extractum;
(3) with 1mol/L hydrochloric acid 400mL ultrasonic dissolution, the centrifugal 10min of 4000rpmin takes out supernatant;
(4) regulate pH to 12 with sodium hydroxide, divide 5 extractions, merge with the 2000mL dichloromethane, vacuum drying is removed dichloromethane, and weight decided in accurate title, obtain the total alkaloids in Semen Strychni powder, measuring wherein with the HPLC method, strychnine content accounts for 52% of total alkaloids in Semen Strychni;
(5) with above-mentioned gained total alkaloids in Semen Strychni powder ultrasonic dissolution in 2000mL 50% ethanol;
(6) put and revolve 0.05 times that steams to original volume on the Rotary Evaporators, have a large amount of white crystals to separate out on the visible flask walls, pour out concentrated solution, 4000rpm, centrifugal 10min gets supernatant;
(7) supernatant is put 4 ℃ of standing over night, 4000rpm, centrifugal 10min, discard precipitation, supernatant is put the vacuum drying oven drying, and precision is weighed, and gets purified total alkaloid of strychnos nux-vomica, measure wherein with the HPLC method that the content of strychnine accounts for 23% of purified total alkaloid of strychnos nux-vomica, the purity that acid-dye colorimetry is measured purified total alkaloid of strychnos nux-vomica is 90%.
Embodiment 2: the method for extracting purified total alkaloid of strychnos nux-vomica:
(1) takes by weighing Semen Strychni, pulverize, sieve, make coarse powder, take by weighing the 200g coarse powder;
(2) extract with 90% alcohol heating reflux, each 2000mL extracts 2h, extracts altogether 2 times, filter, and merging filtrate, decompression recycling ethanol gets pure extractum;
(3) with 0.2mol/L hydrochloric acid 1000mL ultrasonic dissolution, sucking filtration gets filtrate;
(4) regulate pH to 14 with sodium hydroxide, divide 3 extractions, merge with the 1000mL dichloromethane, reclaim under reduced pressure is removed dichloromethane, and weight decided in the precision title, obtains total alkaloids in Semen Strychni, and the HPLC method measures wherein that strychnine content accounts for 46% of total alkaloids in Semen Strychni;
(5) with above-mentioned gained total alkaloids in Semen Strychni powder ultrasonic dissolution in 3000mL 70% ethanol;
(6) vacuum drying takes out concentrated solution to 0.2 times of original volume, and the centrifugal 10min of 4000rpm gets supernatant;
(7) supernatant is put 0 ℃ of standing over night, sucking filtration is got filtrate in the dry pressed powder that gets of vacuum drying oven, precision is weighed, get purified total alkaloid of strychnos nux-vomica, the HPLC method measures wherein that strychnine accounts for 14% of purified total alkaloid of strychnos nux-vomica, and the purity that acid-dye colorimetry is measured purified total alkaloid of strychnos nux-vomica is 84%.
Embodiment 3: the method for extracting purified total alkaloid of strychnos nux-vomica:
(1) takes by weighing Semen Strychni, pulverize, sieve, make coarse powder, take by weighing the 100g coarse powder;
(2) extract with 50% alcohol heating reflux, each 1000mL extracts 1h, extracts altogether 4 times, filter, and merging filtrate, decompression recycling ethanol gets pure extractum;
(3) with 0.5mol/L hydrochloric acid 400mL ultrasonic dissolution, sucking filtration gets filtrate;
(4) regulate pH to 10 with sodium hydroxide, divide 4 extractions, merge with the 500mL dichloromethane, reclaim under reduced pressure is removed dichloromethane, and weight decided in the precision title, obtains total alkaloids in Semen Strychni, and the HPLC method measures wherein that strychnine content accounts for 44% of total alkaloids in Semen Strychni;
(5) with above-mentioned gained total alkaloids in Semen Strychni powder ultrasonic dissolution in 1000mL 75% ethanol;
(6) vacuum drying takes out concentrated solution to 0.1 times of original volume, and the centrifugal 10min of 4000rpm gets supernatant;
(7) supernatant is put 8 ℃ of standing over night, sucking filtration is got filtrate in the dry pressed powder that gets of vacuum drying oven, precision is weighed, get purified total alkaloid of strychnos nux-vomica, the HPLC method measures wherein that strychnine accounts for purified total alkaloid of strychnos nux-vomica 21%, and the purity that acid-dye colorimetry is measured purified total alkaloid of strychnos nux-vomica is 75%.
Embodiment 4: the toxicity research of purified total alkaloid of strychnos nux-vomica---and the acute toxicity testing to mice carries out evaluating drug effect to embodiment 1 gained purified total alkaloid of strychnos nux-vomica:
1. experiment material
1.1 sample: take by weighing total alkaloids in Semen Strychni, purified total alkaloid of strychnos nux-vomica is an amount of,, be mixed with a series of concentration with containing 20% alcoholic acid PBS (pH=7.4) dissolving.
1.2 reagent: ethanol.
1.3 animal: the ICR mice, body weight 18～22g, ♀ ♂ half and half is provided by Yangzhou University comparative medicine center.
2. method and result
Trial test: at first find out 0% and 100% estimate lethal dose (Dn, Dm).At first use a series of medicinal liquids of 2 times of dilutions, respectively try 4 mices with 0.2mL/10g dosage, and then cause death between the group, insert 3～5 grades of dosage by the geometric ratio grade again 4/4 and 0/4, dosage successively decreases with 0.14mL, 0.1mL, 0.07mL, 0.05mL, 0.035mL by every 10g, respectively tries 4 mices again.If certain group mortality rate is 4/4, its last group is 2/4 or 3/4, is Dm with this group dosage then; If last group is 0/4 or 1/4, then 1.5 times with this group dosage are Dm.Find out Dn with method.
Grouping: packet count is 4～9 groups, guarantees minimum dose group 100% animals survived.
Formal experiment: the medicine that gives respectively to organize variable concentrations by the 0.2mL/10g dosage.Add up every group of mortality rate and ask the median lethal dose(LD 50) of calculating every kind of medicine.
Result: the LD of mouse peritoneal injection purified total alkaloid of strychnos nux-vomica
50Be 12.303mgkg
-1, LD
5095% fiducial limit scope be: 12.303 ± 2.098mgkg
-1The LD of mouse peritoneal injection total alkaloids in Semen Strychni
50Be 6.400mgkg
-1, LD
5095% fiducial limit scope be: 6.400 ± 0.726mgkg
-1As seen the toxicity of purified total alkaloid of strychnos nux-vomica has descended about 1.9 times, and toxicity significantly reduces.
Embodiment 5: the analgesic activity of purified total alkaloid of strychnos nux-vomica---the mouse writhing experiment
Embodiment 1 gained purified total alkaloid of strychnos nux-vomica is carried out evaluating drug effect:
1. experiment material
1.1 sample: take by weighing total alkaloids in Semen Strychni, purified total alkaloid of strychnos nux-vomica is an amount of, with containing 20% alcoholic acid PBS (pH=7.4) dissolving, concentration is 1mgmL
-1Aspirin.
1.2 reagent: ethanol, glacial acetic acid.
1.3 animal: the ICR mice, body weight 18～22g, ♀ ♂ half and half is provided by Yangzhou University comparative medicine center.
2. method and result
Experiment mice is divided into 6 groups at random, 10 every group.Irritate stomach for every treated animal and give purified total alkaloid of strychnos nux-vomica solution, total alkaloids in Semen Strychni solution, aspirin solution, the blank group is given equivalent PBS (containing 20% ethanol), respectively organize mice lumbar injection 0.6% acetum (0.2mL/10g) respectively after the administration behind the 30min, observe the number of times that writhing response appears in the interior mice of 25min, record, and the analgesia percentage rate of calculating medicine.The results are shown in Table 1.
Table 1 different dosing dosage to Dichlorodiphenyl Acetate cause mouse writhing reaction influence (X ± S, n=10)
Annotate: compare with the blank group
△P＜0.05
The result of table 1 shows: what positive controls can reduce mice turns round the body number of times, with the blank group significant difference is arranged relatively; The effective dose of purified total alkaloid of strychnos nux-vomica is 12mgkg
-1But middle dosage group and low dose group do not have effect.And total alkaloids in Semen Strychni is at 12mgkg
-1Relatively do not have significant difference with the blank group, the analgesic effect of prompting purified total alkaloid of strychnos nux-vomica is better than total alkaloids in Semen Strychni.
Embodiment 6: the analgesic activity of purified total alkaloid of strychnos nux-vomica---the experiment of mice hot plate
Embodiment 1 gained purified total alkaloid of strychnos nux-vomica is carried out evaluating drug effect:
1. experiment material
1.1 sample: take by weighing total alkaloids in Semen Strychni, purified total alkaloid of strychnos nux-vomica is an amount of, with containing 20% alcoholic acid PBS (pH=7.4) dissolving, concentration is 1mgmL
-1YUNNAN BAIYAO DING.
1.2 reagent: ethanol.
1.3 instrument: HH-8 type digital display thermostat water bath, state China Electrical Appliances Co., Ltd.
1.4 animal: the ICR mice, body weight 18～22g, ♀ is provided by Yangzhou University comparative medicine center.
2. method and result
The rosette of a diameter 10cm is put into 55 ± 0.5 ℃ of water-baths, on disk, place the glass cylinder that 15cm is high, during mensuration mice is placed on rapidly on the disk, picks up counting, lick metapedes or jump out of rosette until animal from touching rosette.Still reactionless as surpassing 30s, then immediately mice is taken out in case the animal of burning, and count the response time (s).Get 60 mices, be divided into 6 groups (blank group, positive controls, each 10 of high, medium and low three the dosage groups of purified total alkaloid of strychnos nux-vomica, total alkaloids in Semen Strychni groups) at random, 24h before the experiment, to the mouse web portion depilation, depilation area 2 * 2cm
2, standby.Before the administration, measure the basic reaction time (s) of respectively organizing mice, smear purified total alkaloid of strychnos nux-vomica solution, total alkaloids in Semen Strychni solution, YUNNAN BAIYAO DING in every treated animal abdominal part, the blank group is smeared equivalent PBS (containing 20% ethanol), administration is 3 times altogether, each 10min at interval, last administration 15min, 45min, 75min measure its response time once more, the results are shown in Table 2.
The influence that table 2. different dosing dosage reacts the mice thermostimulation (X ± S, n=10)
Annotate: with comparison before the administration
1)P＜0.05,
2)P＜0.01,
3)P＜0.001
The result of table 2 shows: positive controls can significantly improve the mice pain threshold, with the blank group significant difference is arranged relatively; The effective dose of purified total alkaloid of strychnos nux-vomica is 12mgkg
-1But middle dosage group and low dose group do not have effect.And total alkaloids in Semen Strychni is at 12mgkg
-1Relatively do not have significant difference with the blank group, the analgesic effect of prompting purified total alkaloid of strychnos nux-vomica is better than total alkaloids in Semen Strychni.
Embodiment 7: the antiinflammatory action of purified total alkaloid of strychnos nux-vomica---the mice ear experiment
Embodiment 1 gained purified total alkaloid of strychnos nux-vomica is carried out evaluating drug effect:
1. experiment material
1.1 sample: take by weighing total alkaloids in Semen Strychni, purified total alkaloid of strychnos nux-vomica is an amount of, with containing 20% alcoholic acid PBS (pH=7.4) dissolving, concentration is 1mgmL
-1YUNNAN BAIYAO DING.
1.2 reagent: ethanol, dimethylbenzene.
1.3 instrument: electronic scale, card punch.
1.4 animal: the ICR mice, body weight 18～22g, ♀ ♂ half and half is provided by Yangzhou University comparative medicine center.
2. method and result
Get 60 mices, be divided into 6 groups (blank group, positive controls, each 10 of high, medium and low three the dosage groups of purified total alkaloid of strychnos nux-vomica, total alkaloids in Semen Strychni groups) at random.Mouse right ear is smeared 20 μ L dimethylbenzene, and left ear is not coated with in contrast, behind the 30min, each is organized about mice two ears and all smears and be subjected to the reagent thing accordingly, blank group contains 20% alcoholic acid PBS, and the administration group gives purified total alkaloid of strychnos nux-vomica, and administration is 3 times altogether, each 80 μ L, each 10min at interval behind the last administration 40min puts to death the dislocation of mice cervical region two ears about cutting along auricle, with the card punch of diameter 6mm about two ear same area lay round auricle, weigh.
Ear swelling degree=auris dextra weight-left ear is heavy
Press down swollen rate=(blank group average ear swelling degree-average ear swelling degree of administration group)/average ear swelling degree of blank group
The results are shown in Table 3.
Table 3 different dosing dosage is to antiphlogistic influence
Annotate: compare with the blank group
△P＜0.05,
△ △P＜0.01,
△ △ △P＜0.001, YUNNAN BAIYAO group administration volume is 240 μ L
Table 3 is the result show, positive controls can obviously suppress the ear swelling of mice, compares significant difference (P＜0.001) with the blank group, shows the modeling success.The total alkaloids in Semen Strychni group does not relatively have significant difference with blank group, and purified total alkaloid of strychnos nux-vomica high dose group and middle dosage group and blank group are relatively, and difference also has significance (P＜0.05), the low dose group zero difference.
Embodiment 8: the immunization of purified total alkaloid of strychnos nux-vomica---the experiment of mice delayed hypersensitivity
Embodiment 1 gained purified total alkaloid of strychnos nux-vomica is carried out evaluating drug effect:
1. experiment material
1.1 sample: take by weighing total alkaloids in Semen Strychni, purified total alkaloid of strychnos nux-vomica is an amount of, with containing 20% alcoholic acid PBS (pH=7.4) dissolving, concentration is 1mgmL
-1Dexamethasone.
1.2 reagent: ethanol, dinitrochlorobenzene, acetone.
1.3 animal: the ICR mice, body weight 18～22g, ♀ ♂ half and half is provided by Yangzhou University comparative medicine center.
2. method and result
Laboratory animal is handled: get the ICR mice, be divided into 7 groups at random, every group 10, be two kinds of administering modes of percutaneous and lumbar injection, the administration of percutaneous mode be divided into dosage group, purified total alkaloid of strychnos nux-vomica high dose group in blank group, purified total alkaloid of strychnos nux-vomica low dose group, the purification strychnine total alkaloids, 24h scrapes unhairing, the about 2 * 2cm of area with the blade after lubricated before positive group of intraperitoneal injection, every group of mouse web portion administration of percutaneous
2Draw medicinal liquid with pipettor and evenly be applied to mouse web portion for every group of percutaneous dosing during administration, blank group is coated with and contains 20% alcoholic acid PBS in contrast, and three groups of dosage of the basic, normal, high dosage of purified total alkaloid of strychnos nux-vomica are respectively 3mgmL
-1, 6mgmL
-1, 12mgmL
-1, total alkaloids in Semen Strychni group dosage is 12mgmL
-1Every group of lumbar injection gives dexamethasone, and dosage is 12mgmL
-1Every group of every day be administered once successive administration 10 days.
Sensitization: after the administration in first day, every group of mice used the DNCB solution 50 μ L sensitization of microsyringe back subcutaneous injection 3%, and after administration in the 4th day, same operation is strengthened once again.
The generation and the mensuration of DTH reaction: administration the 10th day, 1%DNCB solution 20 μ L are applied to mouse right ear (two sides) attack, attack back 24h, mice is put to death in the cervical vertebra dislocation, cut left and right sides auricular concha, take off auricle at identical position, scales/electronic balance weighing with the card punch of diameter 6mm.Getting mouse thymus simultaneously and spleen is weighed, is the swelling degree with the difference of left and right sides auricle weight, and heavy and thymus recast is spleen index and thymus index with the spleen of every 10g mice respectively, observes difference that each is organized.The results are shown in Table 4,5.
Table 4 purified total alkaloid of strychnos nux-vomica to dinitrochlorobenzene induce the ear swelling degree of tardy paraphilia reaction mice influence (X ± S, n=10)
Annotate: compare with the blank group
△P＜0.05,
△ △P＜0.01,
△ △ △P＜0.001
Experimental results show that, the purified total alkaloid of strychnos nux-vomica high dose group of percutaneous dosing can obviously suppress DNCB induced mice ear swelling degree, compare with the blank group, significant difference (p＜0.01), but middle dosage and low dosage purified total alkaloid of strychnos nux-vomica group compare zero difference with blank group.The dexamethasone of intraperitoneal injection and blank comparing difference be (p＜0.001) significantly, and the purified total alkaloid of strychnos nux-vomica that prompting reaches doses can obviously alleviate the ear swelling degree of mice, and the pair cell immunity has inhibitory action.
Table 5 purified total alkaloid of strychnos nux-vomica to mouse immune organ weight's influence (X ± S, n=10)
Annotate: with blank group separately relatively:
△ △P＜0.01
The experiment proved that, with blank group comparison separately, the purification Strychnos alkaloid of high, medium and low three the dosage groups of percutaneous dosing and Strychnos alkaloid group do not have obvious influence (p＞0.05) to the ponderal index of immune organ, and lumbar injection group dexamethasone has the effect (p＜0.01) that suppresses immune organ extremely significantly.The administration of prompting short-term, purified total alkaloid of strychnos nux-vomica has immunosuppressive action, but the weight of immune organ is not had influence.
Embodiment 9: the antitumor action of purified total alkaloid of strychnos nux-vomica---and the influence to the tumor cell of In vitro culture is carried out evaluating drug effect to embodiment 1 gained purified total alkaloid of strychnos nux-vomica:
1 experiment material
1.1 sample: take by weighing total alkaloids in Semen Strychni, purified total alkaloid of strychnos nux-vomica is an amount of, with dimethyl sulfoxide (DMSO) hydrotropy, the DMSO ultimate density is no more than 1.0%.0.22 μ m microporous filter membrane (GVMP 01230, Millipore, and USA) filtration sterilization, 4 ℃ of preservations are stand-by; Cisplatin.
1.2 reagent: DMEM culture medium (GIBCO, the U.S.); Newborn calf serum (Hangzhou Ilex purpurea Hassk.[I.chinensis Sims) (56 ℃ of water-bath deactivation 0.5h); Trypsin AMRESCO, the U.S.); MTT (AMRESCO, the U.S.); Penicillin, streptomycin (Lukang Medical Co., Ltd., Shandong)
1.3 instrument: CO
2Cell culture incubator (FORMA SCIENTIFIC, the U.S.); Enzyme mark spectrophotometric is read plate instrument (Bio-RAD, Model 680, the U.S.)
1.4 cell: human hepatoma cell strain SMMC-7721, HepG2, human stomach cancer cell line MGC-803 are available from Nanjing KaiJi Biology Science Development Co., Ltd; Human lung carcinoma cell line A549 is so kind as to give by doctor Zhang Xu of preclinical medicine institute of Nanjing University of Traditional Chinese Medicine; Human colon cancer cell strain LoVo is so kind as to give by professor Cai Yunqing of Nanjing Medical University; People's adenocarcinoma of stomach cell strain SGC-7901 is so kind as to give by Nanjing drum tower doctor Li Rutian of hospital; Human oophoroma cell line A2780 is provided by Jiangsu TCM Hospital.
2 methods and result
Get the various cancerous cell that are in exponential phase, insert (cell density 6 * 10 in 96 orifice plates
4Cells/ml), every hole 80 μ l.Merge to 60-70% dosing processing at cell attachment and growth, every kind of medicine is established 5 Concentraton gradient, and (the medicine final concentration: total alkaloids in Semen Strychni, purified total alkaloid of strychnos nux-vomica are 200,100,50,25,12.5ugmL
-1Cisplatin is 20,10,5,2.5,1.25ugmL
-1) every kind of concentration establishes four multiple holes.Behind 72h, every hole adds MTT (5mgmL
-1) 20 μ l, behind the mixing of plane, place incubator to continue to hatch 4h, discard the supernatant in each hole then, add 150 μ l DMSO again, planar oscillation mixing 10min, after treating that purple crystal in its each hole dissolves fully, place enzyme mark spectrophotometric to read light absorption value (A, the λ in each hole of the mensuration plate instrument under
490nm).Calculate cell proliferation inhibition rate IR:IR (%)=(1-administration group absorbance/blank group absorbance) * 100% by following formula.The experiment triplicate, the IC of drug effect behind various cell 72h
50Value sees Table 6.
The IC of table 6 drug effect behind various cell 72h
50(ugmL
-1)
Claims (8)
1. method of extracting purified total alkaloid of strychnos nux-vomica is characterized in that step is as follows:
(1) with the Semen Strychni medical material, is ground into coarse powder, takes by weighing 100-200 gram coarse powder;
(2) with the alcohol reflux of 1000-2000ml 50-95%, decompression recycling ethanol gets pure extractum;
(3) with the above-mentioned pure extractum of dissolving with hydrochloric acid of 400-1000ml, centrifugal, get supernatant;
(4) add sodium hydroxide, the adjusting pH value is 10-14, and the dichloromethane of this solution with 500-2000ml extracted, and removal of solvent under reduced pressure gets the total alkaloids in Semen Strychni powder;
(5) with above-mentioned total alkaloids in Semen Strychni powder, with 1000-3000ml50-90% ethanol ultrasonic dissolution;
(6) 0.4-0.05 that is evaporated to original volume doubly, centrifugal concentrated solution is got supernatant;
(7) supernatant cold preservation is left standstill the centrifugal precipitation of removing, the dry purified total alkaloid of strychnos nux-vomica that gets of supernatant then.
2. the method for extraction purified total alkaloid of strychnos nux-vomica according to claim 1 is characterized in that the concentration of alcohol described in the step (2) is 50-95%, and the reflux number of times is 1-5 time, and each reflux time is 0.5-2h.
3. the method for extraction purified total alkaloid of strychnos nux-vomica according to claim 1 is characterized in that the concentration of hydrochloric acid described in the step (3) is 0.1-1mol/L.
4. the method for extraction purified total alkaloid of strychnos nux-vomica according to claim 2 is characterized in that the refrigerated storage temperature described in the step (7) is 0-10 ℃, and described drying means is constant pressure and dry or vacuum drying.
5. as the application of claim 1 extracting method gained purified total alkaloid of strychnos nux-vomica in the preparation analgesic.
6. as the application of claim 1 extracting method gained purified total alkaloid of strychnos nux-vomica in the preparation anti-inflammatory drug.
7. as the application of claim 1 extracting method gained purified total alkaloid of strychnos nux-vomica in the preparation antitumor drug.
8. as the application of claim 1 extracting method gained purified total alkaloid of strychnos nux-vomica in the preparation immunoregulation medicament.
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| CN103159774B (en) * | 2011-12-09 | 2015-07-08 | 北京因科瑞斯医药科技有限公司 | Extraction, separation and purification method for strychnos total alkaloid |
| CN103908493B (en) * | 2014-03-20 | 2018-04-10 | 浙江工业大学 | The preparation method and its pharmaceutical applications of a kind of semen strychni total alkaloids |
| CN105384747A (en) * | 2015-11-24 | 2016-03-09 | 正源堂（天津）生物科技有限公司 | Brucine extraction method |
| CN105524073A (en) * | 2015-12-09 | 2016-04-27 | 正源堂（天津）生物科技有限公司 | Method for extracting brucine N-oxide from nux vomica |
| CN112641827A (en) * | 2020-11-30 | 2021-04-13 | 山东中医药大学 | Preparation method of compatible traditional Chinese medicine for preventing and treating arthralgia syndrome |
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