CN104248664A - Method for reducing toxicity of acoitum soogaricum - Google Patents
Method for reducing toxicity of acoitum soogaricum Download PDFInfo
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Abstract
The invention discloses a method for reducing the toxicity of acoitum soogaricum, main effective substance group total alkaloids of the acoitum soogaricum are used as a chemical research object for the establishment of a fingerprint; anti-inflammatory, analgesic, influence on the immune function and other pharmacological effects of the acoitum soogaricum are used as a research object for clarifying the exact pharmacological effects and toxicity parameters; by comparison of chemical constituent and pharmacological effect changes before and after preparation of the acoitum soogaricum, a toxicity-reducing acoitum soogaricum preparation method is identified; the method has the advantages that: the toxicity of the acoitum soogaricum is reduced, the prepared acoitum soogaricum has certain analgesic and anti-inflammatory effects, so that the prepared acoitum soogaricum can reduce the toxicity and preserve the effect, and has a certain medicinal value.
Description
Technical field
The present invention relates to a kind of medicine processing method, specifically a kind of method reducing Aconitum soongaricum toxicity, belongs to Chinese medicine manufacture field.
Background technology
Aconitum soongaricum (
aconitum soongoricum Stapf.) Ranunculaceae, originate in Xinjiang of China Condition in North Xinjiang, expelling wind and cold, reducing swelling and alleviating pain, dredge the meridian passage, in treatment rheumatism, have the advantage of its uniqueness, the value that there is research further and develop.This research emphasis is investigated alkaloid and pharmacological action, toxicity before and after the Aconitum soongaricum process of preparing Chinese medicine and is changed, and determines that the process of preparing Chinese medicine attenuation of Aconitum soongaricum deposits efficacious prescriptions method, and then lays the foundation for Aconitum soongaricum is applied safely and effectively.Due to, Aconitum soongaricum is hypertoxic Chinese crude drug, if just use and will certainly cause potential safety hazard as Chinese medicine without the process of preparing Chinese medicine, but the method that complete set is unfeasible at present determines how can make Aconitum soongaricum attenuation.How to ensure the safe and effective problem that become research of Aconitum soongaricum in clinical practice.
Summary of the invention
In order to solve the problem, the present invention devises a kind of Preparation process method reducing Aconitum soongaricum toxicity, adopt the concocting method process of preparing Chinese medicine Aconitum soongaricum that three kinds different, comprise heating water cooking method, high press-steamed method, add adjuvant cooking method altogether, result shows Aconitum soongaricum three kinds of processed product diester-type alkaloids (aconitines, mesaconitine, hypaconitine) be mostly hydrolyzed into monoester alkaloid (benzoyl aconine), Aconitum soongaricum toxicity is reduced, and there is certain analgesia and antiinflammatory pharmacological action, show that Aconitum soongaricum can deposit effect by attenuation by the process of preparing Chinese medicine, there is certain medicine valuable.
Technical scheme of the present invention is:
The present invention for chemical research object, sets up finger printing with the main active materials total alkaloids of Aconitum soongaricum; The antiinflammatory had with Aconitum soongaricum, analgesia, on pharmacological actions such as the impacts of immunologic function for object of study, specify its definite pharmacological action and toxicity parameter; The change of front and back chemical composition and pharmacological action is concocted by comparing Aconitum soongaricum, determine Aconitum soongaricum and concoct method of attenuating, illustrate its processing excipient, tentatively verify its medical value, and then lay the foundation as the clinical practice of aconitum plants for Aconitum soongaricum.
Reduce a method for Aconitum soongaricum toxicity, described method is determined by following steps:
(1) with the total alkaloids of Aconitum soongaricum for chemical research object, utilize HPLC, Zhunger Basin aconite alkaloid composition is analyzed, and through Method validation, sets up the chemical fingerprint of Aconitum soongaricum and the assay method of the main active such as neoline, songorine;
(2) adopt heating water cooking method, high press-steamed method respectively, add adjuvant cooking method three kinds of methods process of preparing Chinese medicine Aconitum soongaricum altogether;
(3) by the chemical change of chemical fingerprint relative analysis three kinds of processed products before and after concocting;
(4) antiinflammatory had with Aconitum soongaricum, analgesia, on the pharmacological actions such as the impact of immunologic function and toxicity for object of study, set up Aconitum soongaricum pharmacology, acute toxicity test model, pharmacological action that total alkaloids produces and the toxicity change of front and back and different concocting method are concocted in research;
(5) changed in conjunction with corresponding chemical substance by contrast Aconitum soongaricum and processed product pharmacology test result of variations thereof, determine that it concocts method of attenuating;
Due to aconitum plants, often toxicity is cruel, and contained diester-type alkaloids character is unstable, meets water heating easily hydrolysis, generates the monoester alkaloid with mild toxicity, but still have drug action.Therefore often adopt decoction, soak, steam, add the concocting methods such as adjuvant boils altogether, to make in aconitum plants not only poisonous but also effective composition remain in suitable scope, guarantee the safety and effectively of aconitum plants clinical application.
In three kinds of different concocting methods, cooking method processed product toxicity is minimum altogether to add adjuvant, and heating water cooking method processed product and the drug action of high press-steamed method processed product are better than adding adjuvant cooking method processed product altogether, although high press-steamed method processed product have employed " high pressure steaming ", the method that this short time concocts, but its toxicity is only lower than the raw product of Aconitum soongaricum, far above heating water cooking method processed product with add adjuvant altogether cooking method processed product, based on the effective principle of clinical safety, heating water cooking method processed product is all better than other two kinds of processed products in acute toxicity, pharmacodynamics.Therefore, preferably heating decocting in water concocting method deposits the processing procedure of effect as Aconitum soongaricum attenuation.
Wherein, the concrete grammar of described step (4) comprising: antiinflammatory action experiment, analgesic activity experiment, immunoregulation effect experiment, animal acute toxicity test;
Described Aconitum soongaricum antiinflammatory action experiment comprises the following steps:
1, on arthritis (CIA) the synovial cells in rats pathology of II Collagen Type VI induction and the impact of relevant cell factor expression, CIA rat articular swelling rate is detected; Serum SA concentration; IL-2, TNF-alpha levels; Synovial tissue's morphological observation;
2, on the impact of adjuvant-induced arthritis (AA) rat paw Articular swelling, serum cortisol and serum IL-1 β content, Articular swelling is detected; Serum cortisol; IL-1 β.
Described Aconitum soongaricum analgesic activity experiment comprises the following steps:
1, on the impact of mouse hot-plate induced pain reaction, the percentage rate that the threshold of pain is improved after medication is detected, and the action intensity of mapping analysis medicine, effect time started and hold time;
2, the impact of Dichlorodiphenyl Acetate induced mice writhing response, detects the analgesia percentage rate of each medicine.
Described Aconitum soongaricum comprises the following steps immunoregulation effect experiment:
1, to the experimentation of mouse cell Immune Function, fluorescencepositive cell percentage rate and CD4+/CD8+ ratio is detected;
2, to the experimentation of mouse humoral immune function effect, Quantitative Haemolytic OD value and the serum hemolysin OD value of sheep red blood cell (SRBC) (SRBC) is detected;
Described Aconitum soongaricum animal acute toxicity test comprises the following steps: detect LD50; The linear regression equation of dosage (x) and dead quantity (y); Mice toxic reaction symptom; Mice maximum tolerated dose.Verify Aconitum soongaricum and processed product toxicity parameter thereof.
The invention has the advantages that: determine Aconitum soongaricum and concoct method of attenuating, Aconitum soongaricum toxicity is reduced, and there is certain analgesia and antiinflammatory pharmacological action, show that Aconitum soongaricum can deposit effect by attenuation by the process of preparing Chinese medicine, there is certain medicine valuable, ensure safe and effective in clinical practice of Aconitum soongaricum.
Below in conjunction with accompanying drawing, the invention will be further described with enforcement.
Accompanying drawing explanation
Fig. 1 is the chromatogram of diester-type alkaloids reference substance and sample in Radix Aconiti Preparata, Aconitum soongaricum and processed product thereof; In figure, 11-mesaconitine; 12-aconitine; 13-hypaconitine.
Fig. 2 is the chromatogram of monoester alkaloid reference substance and sample in Radix Aconiti Preparata, Aconitum soongaricum and processed product thereof; In figure, 21-benzoyl mesaconitine; 22-benzoyl aconite alkali.
Wherein, A reference substance; B Radix Aconiti; C Aconitum soongaricum; D heating water cooking method processed product; E height press-steamed method processed product; F adds adjuvant cooking method processed product altogether.
Detailed description of the invention
Below the preferred embodiments of the present invention are described, should be appreciated that preferred embodiment described herein is only for instruction and explanation of the present invention, is not intended to limit the present invention.
Except as otherwise noted, the percent adopted in the present invention is percetage by weight.
embodiment 1
1, heating water cooking method concocts Aconitum soongaricum
Get clean Aconitum soongaricum, size is separated, and is soaked in water 7 days to interior without the dry heart, takes out, add water boil 5 hours, get large cut in without the white heart, mouth taste micro-have a numb feeling in the tongue time, take out, dry in the air and cut sheet to sixty percent dry (water content 40%), 70 DEG C of oven drying at low temperatures, to obtain final product.
2, high press-steamed method concocts Aconitum soongaricum
Aconitum soongaricum cleaning, after moistening, fixed temperature is 126 DEG C, and pressure is 0.15 MPa, steams 90 minutes, dries in the air to cut sheet to sixty percent dry (water content 40%), and 70 DEG C of oven drying at low temperatures, to obtain final product.
3, adjuvant cooking method process of preparing Chinese medicine Aconitum soongaricum is altogether added
By Aconitum soongaricum bubble in water, bitterness is removed after changing twice water to 15 day every day, then get 8% Radix Glycyrrhizae and 10% Semen sojae atricolor slurry and Aconitum carmichjaelii Debx. one and reinstate that water boil 5 is little to be tasted without fiber crops without the white heart, mouth up to interior or micro-ly have numb feeling in the tongue degree of being, dry in the air and cut sheet to sixty percent dry (water content 40%), 70 DEG C of oven drying at low temperatures, to obtain final product.
embodiment 2
The research of diester-type alkaloids content assaying method in 1 Aconitum soongaricum and processed product thereof
1.1 chromatographic condition
Adopt X Bridge C18 chromatographic column (250 mm × 4.6 mm, 5 μm), with methanol-water-chloroform (strong aqua ammonia regulates pH for 10.62), (70 ︰ 30 ︰ 2), for mobile phase, flow velocity is 1.0 mL/min, and sample size 10 μ L, determined wavelength is 242 nm.Calculate theoretical plate number by aconitine peak and should be not less than 2000.Result test sample and reference substance chromatographic isolation degree well, are shown in Fig. 1 (A ~ F).
The preparation of 1.2 reference substance solution
Precision takes aconitine, mesaconitine, hypaconitine reference substance in right amount respectively, puts in 10 mL measuring bottles, adds methylene chloride and dissolve and be settled to scale, shake up.Make the mixing reference substance solution of every 1 ml containing aconitine 1.006 mg, mesaconitine 0.108 mg, hypaconitine 0.105 mg.
The preparation of 1.3 need testing solutions
Sample thief powder (crossing No. three sieves after pulverizing) each about 0.5 g, accurately weighed respectively, and put in conical flask, add 10% ammonia solution 4 mL, infiltrate, precision adds ether 20ml, supersound process (power 250 W, frequency 40 kHz; Water temperature is below 25 DEG C) 10 min, let cool, more weighed weight, supply the weight of less loss with ether, placement is spent the night, and filters, and divide 3 washing medicinal residues with 30 mL ether, merging filtrate, puts 40 DEG C of water-baths and volatilizes, and residue dichloromethane dissolves and is transferred in 5 mL measuring bottles, add methylene chloride to scale, shake up, get subsequent filtrate, to obtain final product.
1.2 sample tests
Get Radix Aconiti, Aconitum soongaricum and processed product thereof respectively, obtain need testing solution by legal system below " 1.1.3 " item, sample introduction 10 μ L, measure by above-mentioned chromatographic condition, measurement result is in table 1.
table 1 assay result (
n=3)
Show according to Aconitum soongaricum sample size measurement result, this product is same with the Radix Aconiti of existing legal medical material standard containing diester-type alkaloids (aconitine, mesaconitine and hypaconitine), the content of its mesaconitine is higher than Radix Aconiti medical material about 22 times, the content of mesaconitine is about 2/5 of Radix Aconiti medical material, hypaconitine content is about 1/4 of Radix Aconiti medical material, show that Aconitum soongaricum mesaconitine content is higher, anti-inflammatory and antalgic should be had, on medical values such as immune system impacts.
Heating water cooking method processed product diester-type alkaloids content is about 1/3191 of Aconitum soongaricum, high press-steamed method processed product diester-type alkaloids content is about 1/3191 of Aconitum soongaricum, add adjuvant altogether cooking method processed product diester-type alkaloids content be about diester-type alkaloids content size sequence in 1/8507, three kinds of processed products of Aconitum soongaricum and be followed successively by high press-steamed method processed product ≈ heating water cooking method processed product > and add adjuvant cooking method processed product altogether.
The research of monoester alkaloid content assay method in 2 Aconitum soongaricum and processed product thereof
2.1 chromatographic condition
Take octadecylsilane chemically bonded silica as filler; With oxolane-acetonitrile (15 ︰ 25) for mobile phase A, with 0.1 mol.L-1 Spirit of Mindererus. (every 1000 ml add glacial acetic acid 0.5 ml), for Mobile phase B, the regulation according to the form below 2 carries out gradient elution; Determined wavelength is 235 nm.Calculate theoretical plate number by benzoyl aconine peak and should be not less than 2000.Result test sample and reference substance chromatographic isolation degree well, are shown in Fig. 2 (A ~ F).
table 2 gradient elution
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0～48 | 15→26 | 85→74 |
| 48～49 | 26→35 | 74→65 |
| 49～58 | 35 | 65 |
| 58～65 | 35→15 | 65→85 |
The preparation of 2.2 reference substance solution
Precision takes Benzoylmesaconine respectively, benzoyl aconine reference substance is appropriate, put in 25 mL measuring bottles, add chloroform-isopropyl alcohol (1 ︰ 1) mixed solution dissolve and be settled to scale, shake up, make the mixing reference substance solution of every 1 ml containing Benzoylmesaconine 0.75mg, benzoyl aconine 1.25 mg.
The preparation of 2.3 need testing solutions
Sample thief powder (crossing No. three sieves after pulverizing) each about 2 g, accurately weighed, put in conical flask, add 10% ammonia solution 5 mL, infiltrate, precision adds ethyl acetate-isopropanol (1 ︰ 1) mixed solution 50 ml, weighed weight, supersound process (power 300 W, frequency 40 kHz; Water temperature is below 25 DEG C) 30 min, let cool, more weighed weight, supply the weight of less loss with isopropyl alcohol-ethyl acetate (1 ︰ 1) mixed solution, shake up, filter.Precision measures subsequent filtrate 45 ml, and less than 40 DEG C decompression and solvent recoveries are to dry, and residue precision adds isopropyl alcohol-chloroform (1 ︰ 1) mixed solution 3 ml and dissolves, and filters, gets subsequent filtrate, to obtain final product.
2.4 sample tests
Get Radix Aconiti Preparata, Aconitum soongaricum and processed product thereof respectively, obtain need testing solution by legal system below " 1.2.3 " item, sample introduction 10 μ L, measure by above-mentioned chromatographic condition, measurement result is in table 3.
table 3 assay result (
n=3)
Show according to Aconitum soongaricum sample size measurement result, this product is same with the Radix Aconiti Preparata of existing legal medical material standard containing monoester alkaloid (benzoyl aconine).In Aconitum soongaricum, the content of benzoyl aconine is about 2/5 of Radix Aconiti Preparata, in Aconitum soongaricum heating water cooking method processed product, the content of benzoyl aconine is similar to Radix Aconiti Preparata, in Aconitum soongaricum height press-steamed method processed product, the content of benzoyl aconine is about 3.4 times of Radix Aconiti Preparata, the content that Aconitum soongaricum adds benzoyl aconine in the common cooking method processed product of adjuvant is about 1/2 of Radix Aconiti Preparata, show that the benzoyl aconine content of heating water cooking method, high press-steamed method processed product in Aconitum soongaricum three kinds of processed products is higher, certain pharmacologically active should be had.
Heating water cooking method processed product monoester alkaloid content is about 2 times of Aconitum soongaricum, high press-steamed method processed product monoester alkaloid content is about 8 times of Aconitum soongaricum, altogether cooking method processed product monoester alkaloid content is close with Aconitum soongaricum to add adjuvant, and in three kinds of processed products, monoester alkaloid content size sequence is followed successively by high press-steamed method processed product > heating water cooking method processed product > and adds the common cooking method processed product of adjuvant.
3 Aconitum soongaricum and the research of processed product acute toxicity testing thereof
The mensuration of 3.1 minimum full lethal doses, the most complete works of unlikely dead amount
Get mice 250, random packet, often organize 4 ~ 6.Each concentration group selection one or two groups of mices (4 ~ 6) of each medicinal liquid are tested.Every mice, all by 0.2 mL/10 g intraperitoneal injection 1 time, is observed the death time that mice occurs in 7 days respectively, finally calculates the minimum full lethal dose of each medicine, the most complete works of unlikely dead amount, for mensuration median lethal dose(LD 50) (LD
50) time reference.
3.2 median lethal dose(LD 50) (LD
50) mensuration
Get mice 200, be divided into 20 groups at random more respectively by after mass segregation, before administration, equal fasting 24 hours, can't help water.Aconitum soongaricum, heating water cooking method, high press-steamed method, altogether cooking method 4 samples are according to the value of minimum full lethal dose and the most complete works of unlikely dead amount to add adjuvant, and geometric ratio establishes 5 dosage groups.Every mice, all by 0.2 mL/10 g administration, observes the poisoning symptom after mouse peritoneal injection respectively, death condition and death time, body weight change situation in 7 days.Median lethal dose(LD 50) (the LD that mouse peritoneal injects each medicine is calculated by improvement Kou Shi (Karber) method
50) and 95% credibility interval.
Result Aconitum soongaricum and processed product toxicity size order thereof are: Aconitum soongaricum > height press-steamed method > heating water cooking method > adds adjuvant cooking method altogether.Aconitum soongaricum LD
50=3.0792 g/kg, 95 % credibility intervals are 2.14 ~ 4.39 g/kg; Heating water cooking method LD
50=26.2191 g/kg, 95 % credibility intervals are 24.77 ~ 27.75 g/kg; High press-steamed method LD
50=10.5873 g/kg, 95 % credibility intervals are 9.95 ~ 11.26 g/kg; Add adjuvant cooking method LD altogether
50=39.8702 g/kg, 95 % credibility intervals are 38.28 ~ 41.52 g/kg.
4 Aconitum soongaricum and the research of processed product analgesic experiment thereof
4.1 radiant heat stimulus method experimentatioies
Get the mice 280 of quality (20.0 ± 2.0) g, male and female half and half.Be divided into 14 groups at random, often organize 20, give the positive group (2 mg/mL) of tramadol hydrochloride, Aconitum soongaricum (0.153 g/mL), heating water cooking method (1.311 g/mL), high press-steamed method (0.529 g/mL) respectively, add adjuvant cooking method (1.994 g/mL) 5 medicinal liquids altogether, gastric infusion (0.2 mL/10 g), matched group is to same volume normal saline.Daily once, continuous 2 W, the pain threshold of 30,60,90,120 min after the administration of mensuration last.
Result compares with matched group, after administration 30 min, heating water cooking method, adds the pain threshold unchanged (P>0.05) of the adjuvant altogether low dose group of cooking method, without analgesic effect; The pain threshold of all the other each administration groups increases (P<0.05 or P<0.01), has analgesic effect.After administration 60,90 min, the pain threshold of each administration group all increases (P<0.05 or P<0.01), and analgesic activity starts onset.After administration 120 min, heating water cooking method, add the pain threshold unchanged (P>0.05) of the adjuvant altogether low dose group of cooking method, analgesic effect weakens; The pain threshold of all the other each administration groups compares with matched group and increases (P<0.05 or P<0.01), still has certain analgesic effect.
After administration 30 min, the pain threshold of the high dose group of Aconitum soongaricum, heating water cooking method, high press-steamed method organizes comparing difference not statistically significant (P>0.05) with positive; After administration 60 min, the pain threshold of the high dose group of Aconitum soongaricum, high press-steamed method organizes comparing difference not statistically significant (P>0.05) with positive; After administration 90,120 min, high, the middle dosage group of Aconitum soongaricum and heating water cooking method, high press-steamed method, the pain threshold adding the high dose group of the common cooking method of adjuvant and the positive organize comparing difference not statistically significant (P>0.05).
4.2 acetic acid cause the research of mouse writhing reaction experiment
Get the mice 140 of quality (20.0 ± 2.0) g, male and female half and half.Be divided into 14 groups at random, often organize 10, give the positive group (2 mg/mL) of tramadol hydrochloride, Aconitum soongaricum (0.153 g/mL), heating water cooking method (1.311 g/mL), high press-steamed method (0.529 g/mL) respectively, add adjuvant cooking method (1.994 g/mL) 5 kinds of medicinal liquids altogether, gastric infusion (0.2 mL/10 g), matched group is to same volume normal saline.Daily 1 time, continuous 2 W, 30 min after last administration, every mouse peritoneal injects 0.6% glacial acetic acid solution 0.1 mL/10 g, and (abdominal part shrinks indent to the writhing response starting to observe in 30 min after injection, and buttocks is raised, caudal vertebra upwarps, stretch hind leg, crawling) number of times and the time, calculate writhing number of times suppression ratio (writhing number of times suppression ratio reaches more than 50% and just thinks there is analgesic activity).
Writhing number of times suppression ratio (%)=(matched group average writhing number of times-administration group average writhing number of times) average writhing number of times × 100% of/matched group.
Result compares with matched group, positive group, Aconitum soongaricum high dose group and middle dosage group, heating water cooking method high dose group and middle dosage group, high press-steamed method high dose group and middle dosage group, add the common cooking method high dose group of adjuvant and all can reduce mouse writhing number of times (P<0.01 or P<0.05), thus generation analgesic activity.
5 Aconitum soongaricum and processed product thereof are to the experimentation of adjuvant-induced arthritis (AA) rat therapeutical effect
Foundation and the grouping administration of 5.1 AA rat models
Get rat 180, male and female half and half, be divided into 15 groups at random, often organize 12; I.e. matched group, model group, the positive group (6.00 mg/kg) of prednisolone acetate, Aconitum soongaricum and processed product thereof (heating water cooking method, high press-steamed method, add adjuvant cooking method altogether) high, medium and low dosage group (dosage is respectively 30.65,15.32,7.66 mg/kg).Except matched group, every rat all causes inflammation in right sufficient sole of the foot intradermal injection 0.1 mL Freund's complete adjuvant (20 mg/mL), and matched group gives the normal saline of same dose.Cause and started administration, each administration group every corresponding test medicine of rat gavage every day, successive administration 23 days the scorching same day.
The mensuration of 5.2 AA rat Secondary cases ankle swelling degree
Before causing inflammation and cause scorching after the 14th, 17,21 day, measure Rat Right metapedes sole of the foot volume respectively, obtain Articular swelling (△ mL=cause scorching after volume-cause scorching before volume), to observe Aconitum soongaricum and processed product thereof to the impact of AA rat secondary inflammation.
After result modeling (before administration), the Articular swelling of model group and each administration group rat is obviously greater than matched group (P<0.01), shows modeling success.After administration the 7th day, compare with model group, Aconitum soongaricum high dose group and high press-steamed method high dose group can reduce the Articular swelling (P<0.05 or P<0.01) of AA rat; After administration the 14th day, with model group ratio, high, the middle dosage group of Aconitum soongaricum high dose group, heating water cooking method and high press-steamed method can reduce the Articular swelling (P<0.05 or P<0.01) of AA rat; After administration the 21st day, compare with model group, positive group and the high, medium and low dosage group of each administration group all can reduce the arthroncus degree (P<0.01) of AA rat.
The mensuration of 5.3 AA rat polyarthritis indexes
Cause scorching latter 14th, 17,21 day and observe rat body arthropathy degree and record, whole body pathological changes is by 5 grades of scoring system in post-therapeutic evaluations.0 point: without red and swollen; 1 point: toe joint is red and swollen; 2 points: toe joint and pedal swelling; 3 points: the sufficient pawl swelling below ankle joint; 4 points: the whole sufficient pawl swelling comprising ankle joint.According to the lesion degree scores accumulated of all the other 3 limbs of not injecting Freund's complete adjuvant, calculate polyarthritis index, namely 3 limb scores are added.
Result control rats, joint is without redness, agile; Model group rats, slow in one's movements, cause joint, scorching side obvious tumefaction.Modeling the 14th day, non-cause scorching parapodum pawl joint occur swelling, arthritis index is passed in time and increases gradually, within the 21st day, reaches the highest.Administration the 14th day, the swelling degree of each administration group high, middle dosage group rat articular obviously alleviates, all lower than the index (P<0.05 or P<0.01) of model group; Administration the 21st day, the swelling degree of each group rat articular all alleviates, and is starkly lower than the index (P<0.05 or P<0.01) of model group.Prompting Aconitum soongaricum and the secondary affection of processed product to AA rat thereof have certain inhibitory action.
The mensuration of 5.4 AA rat blood serum interleukin-1 ' beta 's, serum cortisol Cort
Above-mentioned each group of rat was put to death after 23 days by drug treatment respectively, and centrifugal 15 min of abdomen arterial blood drawing 5 mL, 3000 r/min, get supernatant-80 DEG C of Refrigerator stores.ELISA method is adopted to detect IL-1 β, Cort level.
Result compares with matched group, and model group AA rat blood serum IL-1 β and Cort level all raises (P<0.01); Compare with model group, positive group, the rat blood serum Cort level of Aconitum soongaricum high, medium and low dosage group and high, the middle dosage group of heating water cooking method and high press-steamed method high dose group all reduces (P<0.01 or P<0.05); Compare with model group, positive group, Aconitum soongaricum and heating water cooking method high, medium and low dosage group and high press-steamed method high dose group, rat blood serum IL-1 β level that adds adjuvant cooking method high dose group altogether all reduce (P<0.01 or P<0.05).
6 Aconitum soongaricum and processed product are to the experimentation of arthritis (CIA) the rat therapeutical effect that II Collagen Type VI is induced
6.1 Emulsion preparations
Get appropriate cattle II Collagen Type VI, be dissolved in 0.01 mol/L acetic acid, stir at 4 DEG C and make it abundant dissolving, place 4 DEG C of refrigerator overnight, be mixed with 0.4% concentration Emulsion.
Foundation and the grouping administration of 6.2 CIA rat models
Get rat 120, male and female half and half, be divided into 15 groups at random, often organize 8; I.e. matched group, model group, the positive group (6.00 mg/kg) of prednisolone acetate, Aconitum soongaricum and processed product thereof (heating water cooking method, high press-steamed method, add adjuvant cooking method altogether) high, medium and low dosage group (dosage is respectively 30.65,15.32,7.66 mg/kg).Equal-volume mixing, emulsifying before modeling by 0.4% N of II collagen emulsion and Freund's complete adjuvant.Except matched group, every rat all causes inflammation in right sufficient sole of the foot intradermal injection 0.1 mL suspension emulsion, and matched group gives the normal saline of same dose.After 7 days, in the identical Emulsion booster injection 1 time of rat root of the tail portion.Continuous gastric infusion after modeling, each administration group every corresponding test medicine of rat gavage every day, successive administration 6 W.
During full 3 W of gastric infusion, adopt afterbody to get blood about 2 ml, full 6 W of administration, each group rat experiment stops front 24 h fast, weighs, with etherization, opens abdominal part, abdomen arterial blood extracting, separation of serum, frozen to be measured in-80 DEG C of refrigerators.
The mensuration of 6.3 CIA rat articular swelling rates
After modeling completes the 1st day, adopt water capacity algoscopy, after measuring the left joints of foot swelling of non-injection, surveyed 1 time every 3 days, until put to death.The data recorded the same day with first immunisation are for base value, and the value later often recorded value is by comparison exactly Joint swelling rate.
Result and matched group, model group, positive group is compared, and the Articular swelling of model group rats is more serious than the Articular swelling of matched group and Aconitum soongaricum and each administration of processed product thereof, without downward trend.Administration is after 42 days, and the left joints of foot swelling rate of Aconitum soongaricum and processed product each administration group CIA rat non-injection side thereof all comparatively model group reduces.
The mensuration of 6.4 CIA rat blood serum interleukin II, serum sialic acid, tumor necrosis factor
Adopt ELISA method to detect the 3rd, 6 W rat blood serum IL-2, TNF-α, SA levels, method is undertaken by test kit read-me, finally reads absorbance with microplate reader 450 nm place, calculates IL-2, TNF-α, SA concentration.
Result compares with matched group, and model group rats Serum SA level, higher than matched group, has significant difference (P<0.01).Administration the 3rd W, compares with model group, positive group, Aconitum soongaricum and high press-steamed method high dose can reduce SA content, has significant difference (P<0.05 or P<0.01), all the other each administration group there was no significant differences.Administration the 6th W, compare with model group, positive group, Aconitum soongaricum and the height of heating water cooking method high, medium and low dosage group and high press-steamed method, middle dosage group all significantly can reduce CIA rat blood serum SA content, have significant difference (P<0.05 or P<0.01).
Compare with matched group, model group rats serum TNF-cc level, higher than matched group, has significant difference (P<0.01).Administration the 3rd W, compare with model group, positive group, Aconitum soongaricum be high, in, dosage group and heating water cooking method high dose group and high, the middle dosage group of high press-steamed method all can reduce TNF Contents, has significant difference (P<0.05 or P<0.01).Administration the 6th W, compare with model group, the high, medium and low dosage group of positive group, Aconitum soongaricum and heating water cooking method and high press-steamed method all significantly can reduce CIA rat blood serum TNF-alpha content, has significant difference (P<0.05 or P<0.01).
Compare with matched group, model group rats serum IL-2 level, higher than matched group, has significant difference (P<0.01).Administration the 3rd W, compares with model group, only has positive group can reduce IL-2 content, has significant difference (P<0.05 or P<0.01), all the other each administration group there was no significant differences.Administration the 6th W, compare with model group, positive group, Aconitum soongaricum high, medium and low dosage group and the height of heating water cooking method high dose group and high press-steamed method, middle dosage group all significantly can reduce CIA rat blood serum IL-2 content, have significant difference (P<0.05 or P<0.01).
6.5 CIA synovial cells in rats morphological observations
Stripping cuts each group of non-injection Ce Zuo ankle synovial tissue of joint, puts into 10% formalin, conventional dehydration, paraffin embedding, and section, HE dyes, the pathological change of optical microphotograph Microscopic observation synovial tissue.
It is as follows that observation through Pathology Deparment of Hospital of TCM, Xinjiang Uygur Autonomous Region director Zhang Yajing learns that CIA respectively organizes synovial cells in rats morphology pathological change result:
Matched group: synovial membrane backing layer is 1 ~ 2 confluent monolayer cells thickness, cell arrangement rule, smooth surface, without cell infiltration, synovium of joint sharpness of border is cell monolayer hypertrophy without exception.
Model group: the lymphocyte of adjurant arthritis rat epithelial cell number of plies increases, has a large amount of inflammatory cell (mononuclear cell, apocyte, lymphocyte, bite neutrophilic granulocyte) to infiltrate in interstitial; Fusiformis fibrocyte increases; Blood capillary proliferation, the intracavity of expansion is full of erythrocyte.
Positive group: synovial tissue's structure comparatively model group is obviously improved, visible minute quantity lymphocyte and monocyte infiltration, accidental intiltration of acidophilic leukocyte, and blood capillary proliferation and mucoid edema all comparatively model control group alleviate.
The high dose group of Aconitum soongaricum and processed product thereof: synovial membrane region inflammatory cell number comparatively model control group obviously reduces, and mucoid edema alleviates, and blood capillary is also shown in hypertrophy, congested.
The middle dosage group of Aconitum soongaricum and processed product thereof: synovial membrane region inflammatory cell reduces, and edema alleviates, comparatively close to high dose group.
The low dose group of Aconitum soongaricum and processed product thereof: synovial membrane district volume lymphocyte, more monocyte infiltration, companion's blood capillary proliferation, hyperemia and edema, infringement osseous tissue, comparatively positive controls and height, middle dosage group are serious.
The experimentation that 7 Aconitum soongaricum and processed product affect immune function of mice
7.1 humoral immunization experimental sections
7.1.1 the foundation of immune extraordinary model, grouping, administration and serum hemolysin assay method
Kunming mouse 60, weight range 18 ~ 22g, male and female half and half.After adaptability raises one, be divided into 6 groups according to body weight sex stratified random, be respectively: blank group and model (AZP) group, give the distilled water of 0.2ml/10g; Raw product group (1.23g/kg) of Aconitum soongaricum, Aconitum soongaricum adds adjuvant cooking method process of preparing Chinese medicine group (15.95g/kg), Aconitum soongaricum heating water cooking method process of preparing Chinese medicine group (10.49g/kg), Aconitum soongaricum height press-steamed method process of preparing Chinese medicine group (4.24g/kg) altogether.Each group of gastric infusion, continuous 7 days.Administration the 3rd day, every mice equal lumbar injection 30%SRBC sensitization, the 4th day model group and each administration group every mouse peritoneal injection AZP1.5mg/0.2ml, the isopyknic NS of blank group lumbar injection.Last administration is plucked eyeball after 1 hour and is got blood, the centrifugal 10min of 3000r/min, separation of serum.
Hemolysin assay: get serum 1.0 ml after dilution 200 times, 30% SRBC 0.5ml, 10% complement 1.0 ml enters reaction tube, after putting 37 DEG C of water bath heat preservation 30min, move to cessation reaction in ice-water bath, the centrifugal 10min of 2000r/min, gets supernatant 1ml and places 10min, read absorbance (A) in 540nm place.
After result administration terminates, Aconitum soongaricum is given birth to product and three kinds of processed product concentrated solution groups and model group Mouse Weight and is compared with blank group, result not statistically significant.The content of AZP model group serum hemolysin is higher than blank group, and result has statistical significance (P < 0.01).The content of raw product and three kinds of processed product concentrated solution group serum hemolysins is all lower than model group, and wherein raw product group and heating water cooking method process of preparing Chinese medicine group compare with model group, and result has statistical significance (P < 0.01).Experimental result refers to table 4.
the raw product of table 4 Aconitum soongaricum and three kinds of processed products are on the impact of immunity extraordinary mice serum hemolysin content(
, n=10)
| Group | Body weight (g) before test | Body weight (g) after test | Hemolysin content (A) |
| Blank group | 20.0±1.2 | 27.9±2.7 | 0.760±0.211 |
| Model group | 19.6±0.9 | 28.9±1.7 | 0.955±0.213 ** |
| Raw product group | 20.4±1.5 | 28.1±1.7 | 0.698±0.175 ## |
| Add adjuvant cooking method process of preparing Chinese medicine group altogether | 20.0±1.6 | 27.6±1.7 | 0.845±0.138 |
| Heating water cooking method process of preparing Chinese medicine group | 19.8±1.5 | 27.7±2.5 | 0.770±0.147 ## |
| High press-steamed method process of preparing Chinese medicine group | 20.0±1.6 | 28.2±2.0 | 0.814±0.166 |
Note: compare with blank group,
* p< 0.05,
* p< 0.01; Compare with model group,
# p< 0.05,
## p< 0.01
7.1.2 the foundation of immunosuppression model, grouping, administration and method
Kunming mouse 60, weight range 18 ~ 22g, male and female half and half.After adaptability raises one, 6 groups are divided into according to body weight sex stratified random, be respectively: raw product group (1.23g/kg) of blank group, model (CY) group, Aconitum soongaricum, Aconitum soongaricum adds adjuvant cooking method process of preparing Chinese medicine group (15.95g/kg), Aconitum soongaricum heating water cooking method process of preparing Chinese medicine group (10.49g/kg), Aconitum soongaricum height press-steamed method process of preparing Chinese medicine group (4.24g/kg) altogether.Model group and the injection of each administration group mouse peritoneal CY80mg/kg, the isopyknic NS of blank group lumbar injection, for three days on end.Meanwhile, the equal gastric infusion of each administration group, continuous 7 days, the distilled water of CY group, blank group gavage same volume.Administration the 3rd day, every mice equal lumbar injection 30% SRBC sensitization, last administration is plucked eyeball after 1 hour and is got blood, the centrifugal 10min of 3000r/min, separation of serum.Hemolysin assay is the same.
After result administration terminates, Aconitum soongaricum gives birth to product and three kinds of processed product concentrated solution groups and model group Mice Body weight average lower than blank group, and result has statistical significance (P < 0.01).The content of CY model group serum hemolysin is lower than blank group, and result has statistical significance (P < 0.01).The content of raw product and three kinds of processed product concentrated solution group serum hemolysins is all higher than model group, and wherein raw product group and heating water cooking method process of preparing Chinese medicine group compare with model group, and result has statistical significance (P < 0.01).Experimental result refers to table 5.
the raw product of table 5 Aconitum soongaricum and three kinds of processed products are on the impact of immunosuppressed mice serum hemolysis cellulose content(
, n=10)
| Group | Body weight (g) before test | Body weight (g) after test | Hemolysin content (A) |
| Blank group | 20.4±1.6 | 29.8±2.9 | 0.842±0.344 |
| Model group | 20.4±1.0 | 23.8±2.9 ** | 0.247±0.085 ** |
| Raw product group | 19.9±1.6 | 24.4±2.4 ** | 0.311±0.047 ## |
| Add adjuvant cooking method process of preparing Chinese medicine group altogether | 20.2±1.9 | 24.0±2.2 ** | 0.260± 0.038 |
| Heating water cooking method process of preparing Chinese medicine group | 20.6±1.3 | 24.1±2.1 ** | 0.298±0.021 ## |
| High press-steamed method process of preparing Chinese medicine group | 20.1±1.4 | 24.4±2.7 ** | 0.270±0.043 |
Note: compare with blank group,
* p< 0.05,
* p< 0.01; Compare with model group,
# p< 0.05,
## p< 0.01
7.2 cell immunization experiments parts
7.2.1 the foundation of immune extraordinary model, grouping, administration and T cell Subsets method
Kunming mouse 60, weight range 18 ~ 22g, male and female half and half.After adaptability raises one, be divided into 6 groups according to body weight sex stratified random, be respectively: blank group and model (AZP) group, give the distilled water of 0.2ml/10g; Raw product group (1.23g/kg) of Aconitum soongaricum, Aconitum soongaricum adds adjuvant cooking method process of preparing Chinese medicine group (15.95g/kg), Aconitum soongaricum heating water cooking method process of preparing Chinese medicine group (10.49g/kg), Aconitum soongaricum height press-steamed method process of preparing Chinese medicine group (4.24g/kg) altogether.Each group of gastric infusion, continuous 14 days.Administration the 4th day and the 11st day, model group and each administration group every mouse peritoneal injection AZP1.5mg/0.2ml, the isopyknic NS of Normal group lumbar injection.Eyeball blood sampling is plucked in last administration after 1 hour, use the anticoagulant of EDTA-2K+ pipe.
T cell Subsets: get 120ul anticoagulation in streaming sample feeding pipe, adds FITC anti-mouse CD4 1ul and PE anti-mouse CD8 2.5ul, room temperature lucifuge 15min; Add erythrocyte cracked liquid 500ul, mix gently, room temperature lucifuge 10min; Add 1mlPBS mixing, the centrifugal 5min of 2000r/min, abandons supernatant; Add 500ul PBS resuspended, use flow cytomery, analyze the percentage composition of CD4+ and CD8+.
After result administration terminates, Aconitum soongaricum is given birth to product and three kinds of processed product concentrated solution groups and model group Mouse Weight and is compared with blank group, result not statistically significant.AZP model group CD4+ cell and CD8+ cell proportion are all higher than blank group, and CD4+/CD8+ ratio is lower than blank group, and wherein CD4+ cell and CD8+ cell proportion result have statistical significance (P < 0.05).Raw product and three kinds of processed product concentrated solution group CD4+ cells, CD8+ cell proportion and CD4+/CD8+ ratios compare with model group, result not statistically significant.Experimental result refers to table 6.
the raw product of table 6 Aconitum soongaricum and three kinds of processed products are on the impact of the extraordinary mouse T cell subgroup of immunity(
, n=10)
| Group | CD4 +（%） | CD8 +（%） | CD4 +%/ CD8 +% |
| Blank group | 34.44±7.03 | 17.59±5.04 | 1.99±0.50 |
| Model group | 42.28±5.94 * | 23.56±3.83 * | 1.82±0.30 |
| Raw product group | 35.74±6.39 | 24.70±8.07 | 1.67±0.54 |
| Add adjuvant cooking method process of preparing Chinese medicine group altogether | 33.09±10.41 | 19.09±4.74 | 1.76±0.56 |
| Heating water cooking method process of preparing Chinese medicine group | 39.93±9.02 | 21.03±2.92 | 1.90±0.35 |
| High press-steamed method process of preparing Chinese medicine group | 37.10±8.55 | 20.64±4.93 | 1.82±0.26 |
Note: compare with blank group,
* p< 0.05,
* p< 0.01
7.2.2 the foundation of immunosuppression model, grouping, administration and T cell Subsets method
Kunming mouse 60, weight range 18 ~ 22g, male and female half and half.After adaptability raises one, 6 groups are divided into according to body weight sex stratified random, be respectively: raw product group (1.23g/kg) of blank group, model (CY) group, Aconitum soongaricum, Aconitum soongaricum adds adjuvant cooking method process of preparing Chinese medicine group (15.95g/kg), Aconitum soongaricum heating water cooking method process of preparing Chinese medicine group (10.49g/kg), Aconitum soongaricum height press-steamed method process of preparing Chinese medicine group (4.24g/kg) altogether.Model group and the injection of each administration group mouse peritoneal CY80mg/kg, the isopyknic NS of blank group lumbar injection, for three days on end.Meanwhile, the equal gastric infusion of each administration group, continuous 14 days, the distilled water of CY group, blank group gavage same volume.Eyeball blood sampling is plucked in last administration after 1 hour, use the anticoagulant of EDTA-2K+ pipe.T cell Subsets is the same.
After result administration terminates, Aconitum soongaricum gives birth to product and three kinds of processed product concentrated solution groups and model group Mice Body weight average lower than blank group, and result has statistical significance (P < 0.01).CY model group CD4+ cell and CD8+ cell proportion are all lower than blank group, and result has statistical significance (P < 0.01).The CD4+ cell proportion of raw product group, heating water cooking method process of preparing Chinese medicine group and high press-steamed method process of preparing Chinese medicine group is higher than model group, and result has statistical significance (P < 0.05); The CD8+ ratio of raw product group and high press-steamed method process of preparing Chinese medicine group is higher than model group, and result has statistical significance (P < 0.05).Cooking method process of preparing Chinese medicine group CD4+/CD8+ ratio is lower than model group altogether to add adjuvant, and result has statistical significance (P < 0.05).Experimental result refers to table 7.
the raw product of table 7 Aconitum soongaricum and three kinds of processed products are on the impact of immunosuppressed mice T cell subgroup(
, n=10)
| Group | CD4 +（%） | CD8 +（%） | CD4 +%/ CD8 +% |
| Blank group | 46.90±4.69 | 23.48±4.09 | 2.04±0.37 |
| Model group | 22.26±8.71 ** | 13.03±6.03 ** | 1.80±0.44 |
| Raw product group | 35.70±10.30 # | 19.56±5.61 # | 2.02±1.03 |
| Add adjuvant cooking method process of preparing Chinese medicine group altogether | 19.69±9.84 | 14.59±5.85 | 1.33±0.19 # |
| Heating water cooking method process of preparing Chinese medicine group | 34.37±12.11 # | 20.86±9.69 | 1.82±0.66 |
| High press-steamed method process of preparing Chinese medicine group | 33.23±10.28 # | 17.46±4.84 # | 1.97±0.69 |
Note: compare with blank group,
* p< 0.05,
* p< 0.01; Compare with model group,
# p< 0.05,
## p< 0.01
8 Aconitum soongaricum concoct the dependency relation analysis of front and back chemical composition and antiinflammatory pharmacological action change
8.1 analytical methods and result
Adopt SPSS19.0 statistical software rank correlation statistical analysis technique, give birth to the high, medium and low dosage group of product and three kinds of processed products to Aconitum soongaricum respectively and analyze the dependency between AA rat, CIA rat antiinflammatory pharmacodynamics index, data are in Table 8-11.Result shows various dose, diester-type alkaloids (aconitine, mesaconitine, hypaconitine), height correlation between monoester alkaloid (benzoyl aconine) and rat antiinflammatory pharmacodynamics index in the raw product of Aconitum soongaricum, show that the raw product of Zhunger Basin crow have certain antiinflammatory action, there is certain dose-effect relationship (P ﹤ 0.05) between its curative effect and dosage, the diester-type alkaloids (aconitine, mesaconitine, hypaconitine) simultaneously contained by it and monoester alkaloid (benzoyl aconine) are its curative effect compositions.
the raw product of table 8 Aconitum soongaricum and AA rat pharmacodynamics index correlation coefficient
the raw product of table 9 Aconitum soongaricum and CIA rat pharmacodynamics index correlation coefficient
table 10 Aconitum soongaricum three kinds of processed products and AA rat pharmacodynamics index correlation coefficient
table 11 Aconitum soongaricum three kinds of processed products and CIA rat pharmacodynamics index correlation coefficient
9 conclusions
Experimental result shows that different processed product diester-type alkaloids (aconitine, mesaconitine, hypaconitine) of Aconitum soongaricum is hydrolyzed into monoester alkaloid (benzoyl aconine) mostly, Aconitum soongaricum toxicity is reduced, and processed product has the analgesia similar with prednisolone acetate with tramadol and antiinflammatory pharmacological action, show that Aconitum soongaricum can deposit effect by attenuation by the process of preparing Chinese medicine, there is certain medical value.
In three kinds of different concocting methods, cooking method processed product toxicity is minimum altogether to add adjuvant, and heating water cooking method processed product and the drug action of high press-steamed method processed product are better than adding adjuvant cooking method processed product altogether, although high press-steamed method processed product have employed " high pressure steaming ", the method that this short time concocts, but its toxicity is only lower than Aconitum soongaricum, far above heating water cooking method processed product with add adjuvant altogether cooking method processed product, based on the effective principle of clinical safety, heating water cooking method processed product is all better than other two kinds of processed products in acute toxicity, pharmacodynamics.Therefore, preferred pharmacopeia concocting method concocts as Aconitum soongaricum the processing procedure that attenuation deposits effect.
Last it is noted that the foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, although with reference to previous embodiment to invention has been detailed description, for a person skilled in the art, it still can be modified to the technical scheme described in foregoing embodiments, or carries out equivalent replacement to wherein portion of techniques feature.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (2)
1. reduce a method for Aconitum soongaricum toxicity, it is characterized in that: described method is determined by following steps:
(1) with the total alkaloids of Aconitum soongaricum for chemical research object, utilize HPLC, Zhunger Basin aconite alkaloid composition analyzed, and through Method validation, set up the assay method of the main active such as chemical fingerprint and aconitine of Aconitum soongaricum;
(2) adopt heating water cooking method, high press-steamed method respectively, add adjuvant cooking method three kinds of methods process of preparing Chinese medicine Aconitum soongaricum altogether;
(3) by the chemical change of total alkaloids before and after concocting of chemical fingerprint relative analysis three kinds of processed products;
(4) antiinflammatory had with Aconitum soongaricum, analgesia, on the pharmacological actions such as the impact of immunologic function and acute toxicity for object of study, set up Aconitum soongaricum pharmacology, acute toxicity test model, research is concocted front and back and pharmacological action that different concocting method produces and toxicity and is changed;
(5) changed in conjunction with corresponding chemical substance by contrast Aconitum soongaricum and processed product total alkaloids pharmacology test result of variations thereof, determine to concoct method of attenuating.
2. method according to claim 1, is characterized in that: the concrete grammar of described step (4) comprising: antiinflammatory action experiment, analgesic activity experiment, immunoregulation effect experiment, animal acute toxicity test;
Described Aconitum soongaricum antiinflammatory action experiment comprises the following steps:
1), on the rats with arthritis Synovial histopathology of II Collagen Type VI induction and the impact of relevant cell factor expression, CIA rat articular swelling rate is detected; Serum SA concentration; IL-2, TNF-alpha levels; Synovial tissue's morphological observation;
2) impact, on adjuvant arthritis rats foot sole of the foot Articular swelling, serum cortisol and serum IL-1 β content, detects Articular swelling; Serum cortisol; IL-1 β;
Described Aconitum soongaricum analgesic activity experiment comprises the following steps:
1) impact, on the reaction of mouse hot-plate induced pain, detects the percentage rate that the threshold of pain is improved after medication, and the action intensity of mapping analysis medicine, effect time started and hold time;
2), the impact of Dichlorodiphenyl Acetate induced mice writhing response, detect the analgesia percentage rate of each medicine;
Described Aconitum soongaricum comprises the following steps immunoregulation effect experiment:
1) experimentation, to mouse cell Immune Function, detects fluorescencepositive cell percentage rate and CD4+/CD8+ ratio;
2) experimentation, to mouse humoral immune function effect, detects Quantitative Haemolytic OD value and the serum hemolysin OD value of sheep red blood cell (SRBC);
Described Aconitum soongaricum animal acute toxicity test comprises the following steps: detect LD
50; The linear regression equation of dosage and dead quantity; Mice toxic reaction symptom; Mice maximum tolerated dose.
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