Studies On The Bioactive Constituents Of Analgesia And Anti-inflammation From Toddalia Asiatica And Its Mechanisms

Toddalia asiatica Lam. belongs to Toddalia genus of Rutaceae family, and is mainly distributed around the south of Qinling Mountains in China. The property and flavor of this plant is spicy, bitter and warm, with dampness elimination, wind dispelling, stasis bleeding, swelling detoxification effects. It is mainly used to treat bruises, rheumatism, lumbago, dysmenorrhea and snake bites and other diseases with significant effects, and it has been used for the treatment of rheumatoid arthritis with long folk medicinal history. The main chemical constituents from Toddalia asiatica are coumarins and alkaloids. Reports on the active constituents from this plant with analgesic and anti-inflammatory effects and their mechanisms are rare. This paper intends to further study the active extracts and compounds from Toddalia asiatica and to investigate their analgesic and anti-inflammatory mechanisms.I. Studies on the active extracts from Toddalia asiatica with analgesic and anti-inflammatory activities, and immunomodulatory effects.Hot reflux extraction method was used to yield the total extract of Toddalia asiatica, and the alkaloid extract and ethyl acetate extract (subdivided into E I, EII, EIII parts) were isolated in turn, taking them as subjects, the experimental contents and results were as follows:1. Immunomodulatory effects of extracts:the impacts of alkaloid extract and ethyl acetate extract on ConA and LPS-induced T, B lymphocyte proliferation in mice were studied with MTT assay, while effects on its T lymphocyte subsets were tested using flow cytometry, results showed that they could regulate the body’s immune function by changing the ratio and number or T-lymphocytes subpopulations (CD4+and CD8+).2. Studies on the analgesic and anti-inflammatory activities in vivo:the acetic acid-induced writhing, hot plate and xylene-induced ear edema in mice were performed and we found that the alkaloid extract, ethyl acetate extract and E I part showed better analgesic and anti-inflammatory activity, which could significantly inhibit the writhing response and ear edema in mice, and obviously decrease the pain threshold values. And their analgesic mechanisms were studied preliminarily by the effects of naloxone and reserpine in hot plate, results showed that the analgesia of alkaloid extract was antagonized by reserpine, while the analgesia of ethyl acetate extract and E I part was antagonized by naloxone.3. Study on anti-RA activity:the CIA model induced by type II collagen in mice was established to test effects of each extract on the body weight, joint swelling, spleen index, pathological changes and scores of joint, cytokine levels in serum of CIA mice, and results found that alkaloid extract and E I part of ethyl acetate showed significant improvement of arthritis, which could alleviate the hind paw and ankle joint swelling, and inhibit the excessive hyperplasia of articular cartilage and synovial membrane by alleviating the spleen enlargement and regulating levels of TNF-a, IL-17, IL-6and IL-10in serum.II. Studies on the anti-inflammatory activity of Toddalia asiatica in vitro and their anti-inflammatory mechanisms.Toddalia asiatica extracts and several compounds were taken as experimental subjects, and the contents and results were as follows:1. Effects on the proliferation LPS-induced RAW264.7macrophages:MTT assay was used to study whether they had a cytotoxic effect on this model, we found that total extract, E I extract and alkaloid extract from Toddalia asiatica and pimpinellin, toddaculin, isopimpinellin, oxychelerythrine in high concentrations showed certain cytotoxicity, while omphalocarpin, dihydrochelerythrine, skimmianine and y-fagarine had no significant cytotoxicity.2. Effects on NO level in RAW264.7macrophages:their impacts on the level of NO in cell culture supernatant were detected using Griess method, and results found that except for pimpinellin, toddaculin, isopimpinellin and oxychelerythrine, the rest all had a certain role of NO inhibition.3. Effects on levels of TNF-α, IL-6, COX-2and iNOS in RAW264.7macrophages:the levels of inflammatory mediators in cell culture supernatant were detected by ELISA, and we found that total extract, E I extract and alkaloid extract from Toddalia asiatica and omphalocarpin, skimmianine may reduce the levels of TNF-a, IL-6, COX-2and iNOS obviously.4. Effects of skimmianine and omphalocarpin on the COX-2, iNOS expression and NF-κB pathway:Western Blotting method was used, and we found that they both could significantly inhibit COX-2, iNOS expression and transcriptional activity of NF-κB (inhibition of the phosphorylation of cytosolic protein IκB-α and the translocation of NF-κB p65protein to the nucleus), which indicated that they could down-regulate levels of various inflammatory mediators such as NO, TNF-a, IL-6, COX-2and iNOS by inhibiting the transcriptional activity of NF-κB in RAW264.7macrophages.5. Effects on the proliferation of RA FLS and the levels of MMP-13, IL-1β released by RA FLS:CCK-8assay was used to study their effects on the proliferation of RA FLS, flow cytometry was used to detect the effects on the cell cycle, and ELISA method was used to detect their effects on the levels of MMP-13and IL-1β, results showed that the total extract, E I extract and alkaloid extract from Toddalia asiatica and omphalocarpin, skimmianine all could inhibit the proliferation of RA FLS by making a large number of cells stay in the G1phase of DNA, and obviously down-regulate levels of MMP-13and IL-1β released by RA FLS.Ⅲ. Studies on major compounds with analgesia and anti-inflammation.Four main compounds including pimpinellin, isopimpinellin, omphalocarpin and skimmianine were taken as experimental subjects, and the contents and results were as follows:1. Studies on analgesic and anti-inflammatory activities in vivo:the acetic acid writhing, hot plate and xylene-induced ear edema in mice were performed and we found that skimmianine and omphalocarpin had good activity, which could significantly inhibit the writhing response and ear edema of mice, and obviously decrease the pain threshold values. And their analgesic mechanisms were studied preliminarily by the effects of naloxone and reserpine in hot plate, results showed that the analgesia of them was antagonized by reserpine, but was unaffected by naloxone.2. Study on anti-RA activity:the CIA model induced by type II collagen in mice was established to test effects of each compound on the body weight, joint swelling, spleen index, pathological changes and scores of joint, and cytokine levels in serum of CIA mice, results found that skimmianine showed significant alleviation of arthritic symptoms in CIA mice, which could improve the hind paw and ankle joint swelling, and inhibit the excessive hyperplasia of articular cartilage and synovial membrane by alleviating the spleen enlargement and regulating levels of TNF-α, IL-1β, IL-6and IL-10in serum.In this paper we further confirmed the bioactive constituents of analgesia and anti-inflammation from Toddalia asiatica and its mechanisms, and screened the analgesic and anti-inflammatory activities of major compounds isolated from Toddalia asiatica for the first time. The T, B lymphocyte proliferation model in vitro was established to study its immunomodulatory effects, and the analgesic mechanisms were studied preliminarily by the effects of naloxone and reserpine for the first time. Meanwhile, we deeply investigated its anti-inflammatory mechanism by establishing models of LPS-induced the proliferation of RAW264.7macrophages in mice and TNF-a induced the proliferation of RA FLS in rats for the first time. Results demonstrate that Toddalia asiatica can probably inhibit the transcriptional levels of NF-κB in various cells associated with RA, thereby regulating levels of inflammatory mediators released by these cells and inhibiting the activity and expression of enzymes associated with inflammation, thus affecting the proliferation and activation of immune cells such as macrophages participating in RA, and synovial cells with characteristic lesions in RA synovial tissues, to improve the inflammatory and immune response in RA. In short, the national drug Toddalia asiatica has good therapeutic effects on RA.