| Background:Pain, being one of the most common symptoms in clinic, is a kind of unpleasant emotional response or pathological process. Inflammatory pain is associated with inflammation, characterized by pain. This disorder is commonly encountered in clinical practice and can be caused by many diseases, such as osteoarthritis, rheumatoid arthritis, and periodontitis. Non-steroidal anti-inflammatory drug, hormone and CNS analgesics are commonly used in the treatment of nociception. However, their clinical applications have been limited owning to their side effects. Therefore, there is a pressing need for more effectively anti-nociceptive agents with less adverse event.Armadillidium vulgare Latreille, known as "Shufu" in ancient China, is a small arthropod belonging to the subphylum Crustacea. It is a terrestrial isopod animal with the innate ability to curl into a ball shape when disturbed. It lives and reproduces best under humid conditions, and feeds on decayed plants or mosses. A. vulgare (AV) is a medicinal animal that is widespread in China, where it has been used to treat various kinds of diseases for centuries, such as toothache, malaria, dysuria and skin diseases. The extract of AV is a folk medicine commonly prescribed for the treatment of injuries, wound pain, skin warts, stomatitis, and cancer-related pain. The ancient Chinese medical books report that the aqueous extract and powdered raw materials of AV were mainly orally administered. In modern China, research studies have focused on the antinociceptive effect of AV in clinical practice. It has been recommended as a potential therapeutic agent for the treatment of moderate to severe painful conditions because its analgesic activity is associated with fewer side effects than those of conventional analgesics.However, to the best of our knowledge, the bioactive properties including the potential analgesic effect of AV have not been investigated, to date. Therefore, we sought to evaluate the antinociceptive and anti-inflammatory activity of the aqueous extract of AV. In addition, we analyzed the bioactive constituents of AV to further elucidate possible underlying mechanisms and determine its potential clinical usefulness. Finally, the quality standard (draft) was set up according to CHP (2010), in order to utilize and develop medicinal animal resources.Experimental methods:1. Screening of the effective parts of AVWe determined the effective parts of AV by studying the analgesic effect of those extracts obtained by different polar solvent (petroleum ether, Ethyl acetate,50% ethyl alcohol, absolute ethyl alcohol and water). Acetic acid-induced writhing tests were performed to investigate the effects of those extracts at the same doses (0.6 g/kg), using indomethacin as the reference and the number of writhes was counted over 20 min.2. The anti-nociceptive effect of AVFirstly, we study the acute toxicity of the AV extract at the dosages of 2.0,2.7, 3.7,5.0 g/kg to measure the LD50. The mice were allowed food and water ad libitum and observed for toxic symptoms, and mortality for 14 days following the treatment.The acetic acid-induced writhing test, formalin test and hot plate test were carried out to study the analgesic effect of AV at 3 doses (0.4 g/kg,0.6 g/kg, and 0.8 g/kg).2.1. Acetic acid-induced writhing testThis experiment was performed according to the procedure described above, using indomethacin (10 mg/kg) as the reference.2.2. Formalin testThe mice were treated with AV by gavage. After 1 h, formalin solution (30 μL) was injected into the subplantar surface of the right hind paw. The mice were placed in a transparent observation chamber and the time spent licking and biting the injected paw was recorded during the first (0-5 min) and second (15-30 min) phases following the formalin injection. Indomethacin and tramadol were used as standards.2.3. Hot plate testFollowing treatments with AV, tramadol, or vehicle, the mice were placed on a hot plate apparatus maintained at 50±0.5 ℃. The latency to the nociceptive behavior was recorded before and 0.5,1,1.5, and 2 h after the respective treatment.3. Anti-inflammatory effect of AVThe anti-inflammatory actions of AV were evaluated by phlogogen-induced paw edema test, acetic acid-induced increase in capillary permeability test. The content of histamine in plasma was determined by ELISA to assess the ability of AV inhibiting the inflammatory exudation.3.1. Carrageenan testThe mice were treated with AV, Indo, or vehicle. After 60 min, a 1% carrageenan solution (50 μL) was injected into the subplantar tissue of the left hind paw. The paw volume (up to the ankle joint) was measured before and 1,2,3,4,5, and 6 h after the injection using a plethysmometer.3.2. Acetic acid-induced increase in capillary permeabilityMice were treated with AV, Indo, or vehicle. The tail of each animal was injected with 0.5%　Evans blue followed by 0.6%　acetic acid (10 mL/kg, i.p.) 60 min after the treatments. After 20 min, the mice were euthanized by cervical dislocation followed by the injection of 5 mL of normal saline into the peritoneal cavity. After massaging and exposing the viscera, the peritoneal fluids were collected. The absorbance of the supernatant was measured using a spectrophotometer.3.3. Dextran and compound 48/80 induced edema testHind paw edema was induced by a subplantar injection of phlogogen 60 min after pretreatment with the vehicle, AV, or cyproheptadine (Cyp). The volume of the injected paw was measured using a plethysmometer before and after the injection.3.4. Determination of histamine in plasmaThe test was carried out according to the procedure descrived in compound 48/80 induce paw edema test. The mice were treated with AV, dexamethasone, saline 1 h before the injection. After 30 min, the blood was collected and the content of histamien was determined by ELISA.4. Carrageenan induced peritonitis testThe mice were orally pre-treated with AV (0.8 g/kg,0.6 g/kg), vehicle, Indo 1 h before carrageenan injection. After 4 h, mice were killed and the cells and peritoneal fluids were collected for cellular conounting and cytokine quantification in the peritoneal lavage (TNF-a, IL-1β, IL-6, and IL-10), respectively.5. Study on the material base of AVThe aqueous extract of AV was investigated by physicochemical identification and TLC methods to explore the existence of alkaloid, flavone, saponin, steroid, amino acid and saccharide.We employed HPLC to identified the presence of bioactive compound in AV, using standards as references, in order to contribute to its pharmacological activities. Besides, the residues after being roasted in muffle furnace were also analysed by HPLC to study the presence of inorganic substances.6. Study on quanlity control of Armadillidium vulgare LatreilleThe quality control research was carried out according to CHP (2010), with the aim of controlling the quality of those medicinal animals, especially to develop a method for determination of the content of amino sugar.7. Statistical analysisThe results are presented as the mean ± standard error of the mean (SEM). The data were analyzed using a one-way analysis of variance (ANOVA) followed by Fisher’s least significant difference (LSD) post hoc test (SPSS 13.0 software). P-values<0.05 were considered significant.Experimental results1. Screening of the effective parts of AVOur study showed that the administration of aqueous extract of AV and Indo significantly reduced the number of acetic acid-induced wrhthes (P<0.05) but not the constituents extracted by other organic solvents (P>0.05)2. The anti-nociceptive effect of AVAV did not elicit any observable acute toxic effects at the doses of 2.0,2.7,3.7, and 5.0 g/kg body weight (BW) during the 14-day testing period, indicating the LD50 value of AV exceeded 5.0 g/kg via gavage in mice.The animals treated with AV showed postive results in acetic acid-induced writhes test and formalin test (second phase), as well as the references (P<0.05). However, Compared to the control vehicle-treated group, the AV-treated mice did not show an increase in the latency time to the thermal stimulus (P>0.05) and only the positive control group exhibited reduced nociception (P<0.05).3. Anti-inflammatory effect of AV3.1. Carrageenan testAV significantly reduced the edema volume throughout the test period (1-6h) compared to that of the vehicle control group (P<0.05). AV at all doses exhibited positive result in the first hour and Indo-treated mice show inhibitory effect during the 2-5 h period (P<0.05).3.2. Acetic acid-induced increase in capillary permeabilityThe high-dose AV and Indo significantly reduced the absorbance of Evans blue compared to that of the vehicle (P<0.05).3.3. Dextran and compound 48/80 induced edema testAV and reference significantly reduced the phlogogen-induced paw edema in these tests (P<0.01).3.4. Determination of histamine in plasmaThe animals treated with AV and dexamethasone show lower level of histamine in plasma, compared with the control group (P<0.05).4. Carrageenan induced peritonitis testPeritonistis induction with carrageenan caused increase in the leukocyte counts, MPO, and cytokines (TNF-a, IL-1β, IL-6, IL-10) compared to the sham group. AV pretreatment lead to a reduction significantly in the leukocyte migration, MPO and other indicators when compared to the control group. Mice treated with reference show positive results in concentration detection of cell counts, MPO, TNF-a, IL-1β IL-10 but not IL-6.5. Study on the material base of AVFirstly, qualitative tests of amino acid, saccharides and steroids showed positive results. Secondly, glucosamine, taurine and amino sugar have been identified using HPLC-ELSD, UPLC-MS/MS in aqueous extract of AV. Thirdly, we also found some other amino acids in AV, such as arginine, valine, cysteine, isoleucine, tyrosine, phenylalanine, tryptophan. Finally, the presence of inorganic substances in the residues of AV was affirmed based on the HPLC spectrum.6. Study on quantity control of Armadillidium vulgare LatreilleWe set up a content determination method of total amino sugar in this traditional medicine and made a systematic study on methodology on the method, which is simple, stable and repeatable and can be used to control the quality of these traditiional medicine. Besides, we drawed up the quanlity standard (draft) of this medicinal animals by studying character, identification, examination, conten of extraction and quantitative determination of 5 batches of those animal materials.Conclusions:1. Screening of the effective parts of AVWe found out that the aqueous extract of AV is the effective fraction, indicating the bioactive components in AV are soluble in water but not ethanol. The preparation process is belowed:powdered material was extracted twice by maceration in distilled water (1:20, w/v) with occasional stirring at room temperature (25 ± 2℃) for 8 h. After filtration and centrifugation, the supernatant was concentrated. The residue (AV) was redissolved in distilled water.2. The anti-nociceptive effect of AVThe absence of lethal effects at 5.0 g/kg indicated that AV has low toxicity profile, confirming the safety of all doses of AV used in our tests.Analgesic tests confirmed the anticociceptive activities of AV and the mechanism of the analgesic action of AV was more likely to be related to its peripheral anti-inflammatory effect.3. Anti-inflammatory effect of AVOur research revealed that AV can releave inflammatory pain, verifing the conclusion described above. Furthermore, inhibition of mast cell activities is the most important mechanism responsible for the pharmacological function of AV, providing evidences for the antihistamine action and inhibition of degranulation of mastocyte.4. Carrageenan induced peritonitis testThose tests confirmed the anti-inflammatory actions of AV, which can be explained by the reduction of leukocyte migration, MPO, TNF-a, IL-1β, IL-6, and IL-10.5. Study on the material base of AVGlucosamine, taurine and other amino acids are the bioacitve substances responsible for the pharmocological effect of AV. There are many other amino acid existed in these medicinal animals, some of which contributed to the anti-inflammatory effect of AV. Besides, there are remarkably high conent of inorganic substances in AV, which is the key point for the effective dose at a high level.6. Study on quanlity control of Armadillidium vulgare LatreilleThe quanlity standard of these medicinal insects is important and significant in exploitating and utilizing the animal medicines, providing the evidencdes for their further application in clinical practices.In addition, based on the TCM theory and what we found out in our pharmacological research and bioactive substances analysis, we discuss the potential medical application of Armadillidium vulgare in the treatment of some rheumatic diseases, establishing the theoretical basis for their further study and clinical applications. |
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