| Genus Zanthoxylum is the most widely distributed genus of Rutaceae. It comprises of 250 species growing in the tropical and subtropical zones of Asia, Africa, America and Oceania. There are 39 species and 14 varieties in China. As a folk medicine, Z. armatum is widely used for the prevention of stomachache and toothache. Z. avicennae can promote blood circulation and relieve pain.Pain is a complex physiological and psychological activity. It is one of the most common clinical symptoms. Analgesic drugs are usually used in our daily life. Many lignans and alkaloids from Genus Zanthoxylum have been reported to exhibit analgesic and anti-inflammatory activities. As a result, the studies to discover new compounds with Analgesic and anti-inflammatory effects from Genus Zanthoxylumhas aroused researchers’ extensive concern.1. Phytochemical studiesThe compounds have been isolated by CC, PTLC, recrystallization, and identified by UV, IR, MS,1H-NMR,13C-NMR, DEPT,2D-NMR, CD.Stem and root of Z. armatum were collected in Nanning, Guangxi Province. Phytochemical investigation on the ethyl acetate fraction of ethanol extract from the plant, led to the isolation of forty-eight compounds. Among them, forty-one compounds were identified, including twenty furofuran lignans, four triterpenoids, two steroids, four isoquinoline alkaloids, two furoquinoline alkaloids,4 aromatic compouds, two coumarins, a fatty acid and a alkane. armatumin (Ⅰ-5) and armatunine (Ⅰ-39) are new compounds. Thirteen compounds were isolated for the first time from Z. armatum, including norchelerythrine (Ⅰ-7), isodecaline (Ⅰ-8), lupeol (Ⅰ-13), pinoresinol-di-3,3-dimethylally lether (Ⅰ-19), n-nornitidine (Ⅰ-20), methylxanthoxylol (Ⅰ-22),5’-demethoxyepiexcelsin (Ⅰ-23), benzoic acid (Ⅰ-24), demethoxyaschantin (Ⅰ-25), epiaschantin (Ⅰ-29), piperitol (Ⅰ-30), pinoresinol-3,3-dimethylallylether (Ⅰ-31). and 6-acetonyldihydrochelerythrine(I-41).Ⅰ-23 andⅠ-29 were isolated for the first time from Rutaceae.From the ethyl acetate fraction of the stem and root of Z. avicennae collected in Nanning of Guangxi, sixteen compounds were isolated, and ten compounds were identified, including coumarins (avicennin,Ⅱ-1; Luvangetin,Ⅱ-8; Xanthoxyletin,Ⅱ-10), a lignan (pinoresinol-di-3,3-dimethylally lether,Ⅱ-9), a steroids (β-sitosterol,Ⅱ-2), triterpenoids (α-amyrin,Ⅱ-5;β-amyrin,Ⅱ-6; lupol,Ⅱ-7), alkaloid (norchelery-thrine,Ⅱ-3), a aromatic compound (diisobutyl phthalate,Ⅱ-4). Among these, compundsⅡ-2～Ⅱ-10 were isolated for the first time from Z. avicennae.2. HPLC for identification of the main components of ethyl acetate from Z. armatumThe solvent system was composed of A (acetonitrile) and B (water). An elution program performed as follows:0-20 min, linear gradient from 30% A to 50% A; 20-35 min,50% A; 35-50 min, linear gradient from 50% A to 90% A,50min-57 min, 90% A. The flow rate was 1 ml/min. The detection was conducted using the wavelength of 233 nm. The main chromatographic peaks reached the baseline separation. Eight main peaks were identified as eudesmin, horsfieldin, (-)-fargesin, kobusin, sesamin, L-asarinin, planispine A and pinoresinol-di-3,3-dimethylallyl.3. Pharmacological Studies PartⅠTests in vivoThe classical animal models were used in the research of pharmacological activities, including the acetic acid-induced writhing test, formalin test, hot plate test, and xylene-induced ear edema test. Z. armatum, as a folk analgesic medicine, was utilized for several hundred years. Z. armatum exhibit good effects in clinical. So we choose it for evaluating the effects and explaining the mechanism of analgesic and anti-inflammatory. the activity guided method are adopted. These were tested for activities in the animal models by turns, including the ethanol extract, some soluble fractions, ethyl acetate fraction in different doses, four subfractions of ethyl acetate fraction and some compounds. Main results were obtained as follows:1. Ethanol extract showed the analgesic activity in three pain model. Especially in the acetic acid-induced writhing test and the second phase of formalin test, ethanol extract at all test doses displayed significantly analgesic activity.2. The opioid receptor antagonist naloxone hydrochloride obviously antagonized analgesic effect of ethanol extract on the first phase of formalin test, also partly on the second phase. 3. In activity screening for different soluble fractions, ethyl acetate fraction showed the strongest analgesic activity (P＜0.001), while other soluble fractions also exhibited significant activity (P＜0.05).4. In three animal models, ethyl acetate fraction of all test doses (800,400,200, 100mg/kg), showed analgesic and anti-inflammatory activities obviously.5. Screening for subfractions of ethyl acetate fraction, analgesic and anti-inflammatory activities decreased gradually with the polarity increasing. In the same dose, ethyl acetate fraction showed more marked analgesic activity than all subfractions of ethyl acetate fraction.6. In acetic acid-induced writhing test, the norchelerythrine in doses of 20mg/kg and 40mg/kg significantly decreased writhing times, (-)-fargesin in dose of 80mg/mg and (-)-yangambin in 30mg/mg significantly inhibited xylene-induced ear swelling.Based on the above results, some conclusions were drawn as follows:1. The mechanisms of the analgesic effect of Z. armatum might be involved in the periphery inflammatory effect, and at least in part, the opiod system.2. The analgesic activity of Z. armatum might be mutual synergymay of various components.3. Considering related research reports, alkaloids and lignans might be responsible for analgesic activity, while lignan for its anti-inflammatory activity.4. Sesamin and norchelerythrine as analgesic ingredients, (-)-fargesin and (-)-yangambin as anti-inflammatory ingredients were obtained.PartⅡTests in vitroDuring the progress of inflammation, macrophages actively participate in inflam-matory responses by releasing the pro-inflammatory cytokines TNF-a and IL-6, as well as other inflammatory factors, such as NO and PGE2. TNF-a and IL-6 are critical cytokines involved in inflammation. On the other hand, the massive production of NO and PGE2 played an important physiological role in inflammation. In this paper, we established inflammatory models by LPS-induced RAW264.7 cells, and studied the mechanisms of the anti-inflammatory effect of Z. armatum. Some results were obtained as follows:1. Sesamin, (-)-fargesin, L-asarinin, pinoresinol-di-3,3-dimethylally lether, norchelerythrine and magnoflorine Show NO-suppressing activity respectively.2. Both L-asarinin and magnoflorine can inhibit the expression of IL-6 in LPS-treated RAW264.7cells in the concentration of 100μM respectively.3. Sesamin suppressed production of TNF-αin LPS-induced RAW264.7 cells4. Sesamin. (-)-fargesin, L-asarinin, pinoresinol-di-3,3-dimethylally lether, norchelerythrine and magnoflorine can block the secretion of PGE2 from LPS-stimulated RAW264.7 cells respectively.The mechanisms of the anti-inflammatory effect of Z. armatum are involved in inhibiting the expression of pro-inammatory cytokines (TNF-α、IL-6), and the overproduction of inflammatory factors.In the future, we will enrichment and prepare the main compounds isolated from the plant previous, especially lignans and alkaloids, continue to screen active ingredients, and study on the mechanisms of anti-inflammatory effect of main active ingredients.The studies on chemical constituents of Z. armatum and Z. avicennae, and pharmacology of Z. armatum were expected to complement the phytotaxonomy, to enrich the database of natural products, and to afford important academic basis for the exploitation and utilization of natural resources with this genus. |
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