Drug and Herbal Research Centre, Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia; Faculty of Pharmacy, MAHSA University, Bandar Saujana Putra, Malaysia.
Faculty of Pharmacy, MAHSA University, Bandar Saujana Putra, Malaysia.
J Ethnopharmacol. 2021 Jul 15;275:114120. doi: 10.1016/j.jep.2021.114120. Epub 2021 Apr 20.
Numerous Alphonsea species including Alphonsea elliptica (mempisang) leaves and fruits are indigenously used in inflammatory conditions such as postpartum swelling and rheumatism in southeast Asian countries. In our previous in-vitro findings, A. elliptica methanol extract exhibited platelet-activating factor inhibition, suggesting the presence of phyto-constituents with anti-inflammatory potential.
However, so far there is no literature available on the anti-inflammatory activity of this species. Henceforth, based on the above background and our previous laboratory findings, we hypothesize that phytoconstituents of A. elliptica could possess anti-inflammatory potential against inflammatory mediators including prostaglandin-E (PGE), cyclooxegenase-2 (COX-2) and cytokines (IL-1β and IL-6).
Vacuum and column chromatography techniques were employed for the isolation of phytoconstituents. The structure elucidation was carried out using HRESI-MS, H and C-NMR analysis and compared with the published literature. For cytotoxicity analysis, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed on peripheral blood mononuclear cells. In-vitro anti-inflammatory activities were evaluated against the levels of PGE, COX-2, IL-1β and IL-6 in lipopolysaccharide (LPS)-induced human plasma using enzyme-linked immunosorbent assay and radioimmunoassay.
Unprecedentedly, chromatographic purification of methanolic leaves extract afforded five flavones namely vitexin, isovitexin, orientin, isoorientin, schaftoside with three flavanols; kaempferol, myricetin and rutin from A elliptica. In cell viability analysis, isolates did not present cytotoxicity up to 50 μM. In anti-inflammatory evaluation, orientin and isoorientin exhibited strong (≥70%), while isovitexin and vitexin produced strong to moderate (50-69%) PGE, COX-2, IL-1β and IL-6 inhibition at 25 and 50 μM. Isoorientin, orientin, isovitexin, and vitexin showed significant (p < 0.05) and concentration-dependent PGE inhibition with IC values (μM) of 11.40, 14.71, 17.70 and 20.58 against indomethacin (8.80). Furthermore, isoorientin, orientin, isovitexin, and vitexin produced significant concentration-dependent inhibition with IC values (μM) of COX-2: 7.13, 9.51, 12.81, 16.61; IL-1β 4.80, 6.20, 10.85, 14.51; IL-6: 4.01, 5.90, 11.51 and 14.88 as compared to dexamethasone: 5.29, 2.93, 3.72, respectively (p < 0.05).
Conclusively, isolated phytoconstituents are reported for the first time from the A. elliptica. Moreover, isovitexin, vitexin orientin and isoorientin abrogated LPS-induced inflammatory responses in human plasma at non-cytotoxic concentrations.
许多 Alphonsea 物种，包括 Alphonsea elliptica（mempisang）的叶子和果实，在东南亚国家的产后肿胀和风湿病等炎症情况下被本土使用。在我们之前的体外研究结果中，A. elliptica 甲醇提取物表现出血小板激活因子抑制作用，表明存在具有抗炎潜力的植物成分。
然而，到目前为止，关于该物种的抗炎活性还没有文献报道。因此，基于上述背景和我们之前的实验室研究结果，我们假设 A. elliptica 的植物成分可能具有抗炎潜力，可以对抗包括前列腺素 E（PGE）、环氧化酶-2（COX-2）和细胞因子（IL-1β 和 IL-6）在内的炎症介质。
采用真空和柱层析技术分离植物成分。结构鉴定采用 HRESI-MS、H 和 C-NMR 分析，并与已发表的文献进行比较。对于细胞毒性分析，在人外周血单核细胞上进行 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴盐（3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay）。在体外抗炎活性评估中，采用酶联免疫吸附试验（enzyme-linked immunosorbent assay）和放射免疫测定法（radioimmunoassay），检测脂多糖（lipopolysaccharide，LPS）诱导的人血浆中 PGE、COX-2、IL-1β 和 IL-6 的水平。
出乎意料的是，从 A. elliptica 中分离得到的甲醇叶提取物通过色谱纯化，得到了五种类黄酮，即牡荆素、异牡荆素、Orientin、异 orientin、schaftoside 和三种黄烷醇，即山奈酚、杨梅素和芦丁。在细胞活力分析中，分离物在高达 50 μM 的浓度下没有表现出细胞毒性。在抗炎评价中，Orientin 和 isoorientin 表现出强烈的（≥70%）抑制作用，而 isovitexin 和 vitexin 在 25 和 50 μM 时对 PGE、COX-2、IL-1β 和 IL-6 的抑制作用表现出强至中度（50-69%）。Isoorientin、Orientin、Isovitexin 和 Vitexin 对 PGE 抑制具有显著（p<0.05）和浓度依赖性，IC50 值（μM）分别为 11.40、14.71、17.70 和 20.58，与吲哚美辛（8.80）相比。此外，Isoorientin、Orientin、Isovitexin 和 Vitexin 对 COX-2 产生显著的浓度依赖性抑制，IC50 值（μM）分别为 7.13、9.51、12.81 和 16.61；IL-1β 为 4.80、6.20、10.85 和 14.51；IL-6 为 4.01、5.90、11.51 和 14.88，与地塞米松相比：5.29、2.93、3.72（p<0.05）。
总之，从 A. elliptica 中首次分离出了分离出的植物成分。此外，异牡荆素、牡荆素、Orientin 和 isoorientin 在非细胞毒性浓度下消除了 LPS 诱导的人血浆中的炎症反应。
