Analgesic Effect Of Protein Rich Fraction Of Armadilidium Vulgare Extract

Pains are subjective feelings generated along with existing or potential tissue damage, which are also protective reactions of organism to noxious stimulus. As a concomitant symptom of many diseases, pains frequently cause great anxiety to patients. However the application of analgesics so far is limited due to drug addiction and obvious adverse reaction, so finding high efficiency and low toxicity analgesic drugs would have a profound clinical effect.Armadillidium Vulgare is the whole dried body of Latreille of Armadillidium. It contains reducing sugar and glycogen, and the content of them change due to growth period. The mucopolysaccharide of it contains chondroitin sulfate A and C, haluronic acid, various lipid cholesterols and might contain formic acid. Traditionally, Armadillidium Vulgare was used as analgesic drugs in patients with severe cancer and its lasting effect even is better than Dlantin. The chemical composition exerting analgesic effect are still unknown despite the analgesic effect of the decoction and ethanol extract of this bug have been demonstrated by many studies. In an attempt to characterize the analgesic compounds or fraction in this bug, the protein rich fraction of this bug was prepared and its analgesic effect was evaluated in the current paper. The main methods and results are as follows:(1) The preparation and characterization of chemical composition of protein rich fraction of Armadillidium VulgareIn this party, three methods including enzymatic extraction, acid-alkali extraction and water extraction-ethanol precipitation methods to prepare the protein rich fraction of ArmadiIlidium Vulgare were used and compared. For enzymatic method, each of the three factors including solid-liguid ratio, pH and extracting time was optimumazed by singal factor test, then the extract conditions were optimized by orthogonal test. The results showed that, for enzymatic extraction, the optimum extracting conditions were: solid-liquid ratio1:8(g/mL), extracting time4h, pH2. For acid-alkali extraction, similarly the optimum conditions were:HC1pH4, extracting time4h, NaOH pH9. For water extraction-ethanol precipitation method, the optimum conditions were: solid-liquid ratio1:10(g/mL), extracting time2h, ethanol concentration85%. The extraction rate of crude protein at optimum extraction condition of enzymetic, acid-alkali and water-ethnol precipitation method was50.242%,58.698%and34.224%respectively. Amino acid analysis indicated that18amino acids have been found in the extracts. The extracts were rich in excitatory (aspartic acid and glutamic acid) and inhibitory amino acids (glycine and γ-aminobutyric acid) which are associated with analgesia. The content of excitatory amino aids was4.347%,8.848%,11.43%for acid-alkali extract, enzymatic extract and water extraction-ethnol precipitation extract, respectively. The highest content of inhibitory amino acids was found in acid-alkali extract, followed by enzymatic extract and water extraction-ethnol precipitation extract that are4.951%,3.160%and2.832%. The total sterol content in the extract prepared by enzymatic extraction was84.93mg/g,51.83mg/g in the extract prepared by acid-alkali extraction and38.88mg/g in the extract prepared by water extracton ethnol precipitation. According to these result mentioned above, acid-alkali method was the preferred method to effectively extract the protein from Armadillidium Vulgare. In addition, extract prepared by water extraction-ethanol precipitation method had the lowest value in several indicators and thus was excluded in the follow-up experiment.(2) Analgesic effects of protein rich fraction of Armadillidium VulgareAnalgesic effects of protein rich fraction of Armadillidium Vulgare prepared by enzymatic and acid-alkali method were evaluated in mice using chemical stimulation method and hot plate test. The results in chemical stimulation showed that the extract prepared by acid-alkali extraction at360mg/kg inhibited body writhing by76.86%, only16.59%inhibition was observed for the extract prepared by enzymatic extraction. The results in hot plate test indicated that the pain threshold of extract prepared by acid-alkali extraction at360mg/kg was40.37s, that for extract prepared by enzymatic extraction at360mg/kg was25.75s. Analgesic effect of protein rich fraction of Armadillidium Vulgare made by acid-alkali extraction is better than that by enzymatic method.(3) Preliminary study on the analgesic mechanism of protein rich fraction of Armadillidium VulgareAs the mechanism of analgesic effect of the protein rich fraction of Armadillidium Vulgare is unkonwn, xylene induced mice ear swelling test was carried to investigate the anti-inflammatory effects of the protein rich fraction of Armadillidium Vulgare and Norepinephrine (NE), Dopamine and5-hydroxy tryptamine (5-TH) in mice were determined using HPLC-fluorescence analysis method. The extracts exerted very low anti-inflammation activity in xylene induced mice ear swelling test as the ear swelling inhibition values of5.40%for the extract prepared by enzymatic method at360mg/kg and5.81%for the extract prepared by acid-alkali extraction were observed. The content of NE, DA and5-HT in test group is significantly higher than in blank group. It can be concluded that analgesic effect of the Armadillidium Vulgare is associated with monoamine neurotransmitter.(4) Study on the cytotoxicity and acute toxicity of protein rich fraction of Armadillidium Vulgare.MTT method was separately used to test the toxicity effects of the extracts on HepG2and Hela cells. Results showed that inhibition rate of the protein rich fraction of Armadillidium Vulgare to the HepG2cells was less than16.20%and that to Hela cells was less than24.00%, indicating the protein rich fraction of Armadillidium Vulgare has no obviously effect on these two cell lines. Acute toxicity test on mice showed that protein rich fraction of Armadillidium Vulgare at1.08g/kg had no effect on mice. In addition, there was no mouse died within14days.