Analgesic And Anti-inflammatory Effects And Toxicity Of Wuling Jianzhou Pellet

Background&Objective:Scapulohumeral periarthritis, which shows a predilection to labourers around50-year-old, is one of the aseptic inflammations that occur in parenchyma around shoulder joint. Clinical manifestation of scapulohumeral periarthritis includes shoulder joint pain, activity limitation and so on. Deterioration, relapse, muscular dystrophy or dyskinesia may happen in patients if timely treatments are not received. The disease will cause great inconvenience in daily work once the "inflammatory exudate�'adhesion formation�'grievous pain�'activity limitation�'muscular dystrophy" vicious circle is established. Scapulohumeral periarthritis development is devided into three stages including acute, chronic and stiff stages. Acute stage is the best time to treat scapulohumeral periarthritis because in this peroid adhesion formation hasn’t appeared. If timely and sufficient drug, appropriate exercise are used to intervene, development of the disease will be reversed and "pain remission�'exudate absorption�'functional recovery�'tissuse recovery" virtuous circle can be built.Western and Chinese medicine are commonly used to treat patients with scapulohumeral periarthritis in earlier stage. Western medicine refers chiefly to NSAIDs, like aspirin and indometacin. Chinese medicine, expecially compound Chinese traditional medicine, is the optimal option for scapulohumeral periarthritis patients. Wuling Jianzhou water extracts, which is composed of many kinds of Chinese medicine including Angelica, Radix Clematidis, G.macrophylla and so on, is one of the effective empirical formulas that have used in treating scapulohumeral periarthritis. But Wuling Jianzhou water extracts is sharp tasted, time wasting to prepare and inconveniet when going outside. Therefore, the research is focus on the screening of a better extracting method to prepare Wuling Jianzhhou pellet, which is time saving, more convenient and effective.Methods:1. Formalin test:NIH mice were devided into control group(0.5%CMC-Na), each tehnology low (3.8g/kg) and high (11.4g/kg) dose groups and positive control group (0.025g/kg indometacin) according to body weight,10in each Wuling Jianzhou group and20in control and positive group respectively, male and female was fifty-fifty, intragastric administration for3days,1hour after the last administration, animals were hypodermic injected with25ul of1%formalin and pain reaction in0-5min (phase I) and20-30min (phase II) after injection were recorded and graded.2. Cotton ball granuloma test in rat:SD rats were devided into control group(0.5%CMC-Na), each tehnology low (2.25g/kg) and high (7.64g/kg) dose groups and positive control group (0.04mg/kg prednisone acetate) according to body weight,10in each Wuling Jianzhou group and20in control and positive group respectively. Animals were anaesthetized by aether, then cotton ball around50mg were buried into subcutane at inguen. Intragastric administration was carried out after operation and last for10days (once a day).24hours after the last administration, rats were executed and granuloma were taken out, dried in oven at the temperature of50℃, then weight of each granuloma was recorded.3. Writhing test: NIH mice were divided into5groups according to body weight, including control group, Wuling Jianzhou pellet low, middle, sub-high and high dose groups, positive control group,10animals in each group, male and female was fifty-fifty, animals of control group were administrated with0.5%CMC-Na, low, middle, sub-high and high dose groups with1.9,3.8,7.6,15.2g/kg Wuling Jianzhou pellet respectively, positive control group with0.015mg/kg indometacin, for3days. After the last administration, intraperitoneal injection was carried out with0.6% acetic acid at the dose of0.01ml/g of body weight. The number of writhing was counted in15min after injection.4. Ear swelling test:Animals were grouped and administrated as writhing test above.30min after the last administration, right ear of each mouse was smeared with0.025ml of xylene. Mice were excecuted after30min and both the right and left ears were perforated. Round ear tissue of diameter6mm were weighed and ear swelling rates were caculated.5. Acute toxicity trial:Dosees in formal experiment were caculated into6levels (216.8.184.3.156.6.133.1、113.2.96.2g/kg)according to the principle of1:0.85between Dm and Dn, at the same time control group is required. There were12mice in each group, in half respectively male and female, administrate for40ml/kg of volume1time only, then obseration was last for14days. Status and death of the mice were recorded to compute LD50and LD5of Wuling Jianzhou pellet.6. Long term toxicity trial:SD rats were devided into4group including control group, low, middle and high dose goup randomly on the base of body weight,24in each group, male and female was fifty-fifty. Volume of10ml/kg aministration was carried out1time each day. Behavious of the animals were observed each day and body weight and food intake were recorded and doses of Wuling Jian were adjusted every week according to body wight. After3months,12animals of each group(male: femal was1:1) were anaesthetized by pentobarbital randomly according to body weight. After24hours of the last administrion, blood was collected for haematology testand blood biochemical examination. Autopsy was carried out and organs including heart, liver, spleen, lung, kidneys and adrenal glands of both side, thymus gland, brain, testicle and epididymis of both side were weighed and organ index were caculated. After1month of drug withdrawal, the same process was carried out as above.Results:1. Formalin test: comparing to control group, score of phase Ⅰ but not phase Ⅱ pain reaction of mice in water extract high dose, technology B and technology C group were obviously lower; Cotton ball granuloma test:Wuling Jianzhou pellet inhibited the formation of granuloma in varying degrees but only the inhibiton of technology B low dose, technology C low and high dose group were significant.2. Writhing test:Wuling Jianzhou pellet significantly reduced the number of mice’s writhing at the dose levels of3.8、7.6、15.2g/kg; Ear swelling test:3.8-7.6g/kg of Wuling Jianzhou pellet showed obviously inhibiting effect on ear swelling induced by xylene and inhibiting rate reached to25.0%and38.7%respectively3. Acute toxicity trail:LD50and LD5of Wuling Jianzhou pellet to Kunming mice were148.4g/kg and110.7g/kg respectively which were caculated by Bliss method. Animals were observed to have less activity and week response before death. Abnormal change of the organs was not found when observed with an unaided eye after autopsy.4. Long term toxicity trail:After3months’ administration:General conditions: animals behaved without abnormality; Body weight: Wuling Jianzhou pellet can reduce the body weight of the male rats but not femle obviously; Food intake:Wuling Jianzhou tablet had effect, mainly inhibiting effect on food intake of male rats. Haematology:male rats in middle dose group had more neutrophils while male rats in middle and high dose groups had less hemoglobin comparing with control group. Wuling Jianzhou pellet had little effect on the haematology of the female rats. Blood biochemical:Middle and high dose of Wuling Jianzhou pellet decreased triglyceride of the male rats obviously and high dose of that can also increased potassium ion concentration in female rats markly. Autopsy:abnormal change of the organs was not found when observed with an unaided eye.Organ index:male rats in middle and high dose groups had an obvious rise in liver, kidney, testicle, epididymis index and female rats in middle and high dose goups also had a significant rise in liver index.1month after drug withdrawal:General conditions:animals behaved without abnormality; Body weight:both male and female rats had no significance in body weight compared to control group respectively. Food intake:aministration of Wuling Jianzhou pellet still had significance in food intake of male and female rats but difference can not be regarded as the result of the drug after analysis. Haematology:red cells in low dose male group, hemoglobin and hematocrit of middle dose male group have signifiance in numbers when compared with control group but no dose dependence can be found after analysis. All the indicatiors of haematology show no significance in female rats. Blood biochemical:male rats of low dose group had obviously lower chloridion ion concentration and higher usea nitrogen compared to that in control group while female rats in low dose group showed markly higher in albumin and total bilirubin levels. But valures of the indicators are still in normal range so difference can not be regarded as the effect of Wuling Jianzhou pellet. Autopsy:abnormal change of the organs was not found when observed with an unaided eye. Organ index:comparing to control groups, both the male and female rats’organ index had no significance.We can conclude that ralative weights of organ have resumed after1month of drug withdrawal.Conclusions:1Wuling Jianzhou pellet prepared by technology C exhibits the most effective in analgesia and anti-inflammation among4methods.2Wuling Jianzhou pellet produced by technology C shows obviously analgesic effect ranging from3.8-15.2g/kg and significantly anti-inflammatory effect at doses of3.8g/kg and15.2g/kg.3LD50=148.4g/kg,95%confidence interval is (138.1-159.5)g/kg;LD5=110.7g/kg,95%confidence interval is (97-126.3)g/kg.4Administration of4g/kg (as much as clinical relevant dose) for3months and after1month’s of drug withdrawal, Wuling Jianzhou pelelt shows no effect on body weight, food intake, organ index, haematologies and blood biochemical of both male and female rats. Therefore, we can draw a conclusion that Wuling Jianzhou pellet exhibits no toxicity under clinical relevant dose.