利用体外和计算机模拟方法研究 Leucas cephalotes 的超临界提取物和分离出来的齐墩果酸的抗登革热活性。
Anti-dengue activity of super critical extract and isolated oleanolic acid of Leucas cephalotes using in vitro and in silico approach.
机构信息
Department of Genetics, Maharshi Dayanand University, Rohtak, Haryana, 124001, India.
Department of Microbiology, All India Institute of Medical Sciences, Delhi, 110029, India.
出版信息
BMC Complement Med Ther. 2021 Sep 8;21(1):227. doi: 10.1186/s12906-021-03402-2.
BACKGROUNDS
Leucas cephalotes is a common ethnomedicinal plant widely used by traditional healers for the treatment of Malaria and other types of fever. Oleanolic acid and its derivatives have been reported for various types of pharmacological activities, such as anti-inflammatory, antioxidant, anticancer, hepatoprotective, anti-HIV and anti-HCV activity.
METHODS
L.cephalotes plant extracts were prepared by supercritical fluid extraction (SFE) method and oleanolic acid was isolated by preparatory thin-layer chromatography. The compound was identified and characterize by using ultraviolet-visible spectroscopy (UV-VIS), Fourier transform infra-Red spectroscopy (FT-IR) and high-performance thin-layer chromatography (HPTLC). The structure of the compound was elucidated by proton nuclear magnetic resonance (HNMR) and carbon nuclear magnetic resonance (CNMR) and the purity checked by differential scanning calorimetry (DSC). The MTT assay was used to determine the toxicity of plant extract and oleanolic acid using a microplate reader at 595 nm. The anti-dengue activity of plant extract and oleanolic acid was tested in vitro and in silico using real-time RT-PCR.
RESULTS
The optimum yield of the extract was obtained at 40 °C temperature and 15Mpa pressure. The maximum non-toxic dose (MNTD) of plant extract and oleanolic acid were found as 46.87 μg/ml and 93.75 μg/ml, respectively in C6/36 cell lines. UV spectrophotometer curve of the isolated compound was overlapped with standard oleanolic acid at 232 nm. Superimposed FT-IR structure of the isolated compound was indicated the same spectra at 3433, 2939, 2871, 1690, 1500,1463, 1387, 1250, 1209, 1137 and 656 position as per marker compound. HPTLC analysis showed the retention factor of L. cephalotes extract was 0.19 + 0.06 as similar to the standard oleanolic acid chromatogram. The NMR structure of the isolated compound was identified as similar to the marker oleanolic acid structure. DSC analysis revealed the purity of isolated oleanolic acid was 98.27% with a melting point of 311.16 °C. Real-time RT PCR results revealed that L. cephalotes supercritical extract and isolated oleanolic acid showed 100 and 99.17% inhibition against the dengue - 2 virus when treated with MNTD value of plant extract (46.87 μg/ml) and the test compound (93.75 μg/ml), respectively. The molecular study demonstrated the binding energy of oleanolic acid with NS1and NS5 (non-structural protein) were - 9.42 & -8.32Kcal/mol, respectively.
CONCLUSIONS
The SFE extract L. cephalotes and its active compound, oleanolic acid inhibiting the activity of dengue-2 serotype in the in vitro and in silico assays. Thus, the L.cephalotes plant could be an excellent source for drug design for the treatment of dengue infection.
背景
Leucas cephalotes 是一种常见的民间药用植物，传统治疗者广泛用于治疗疟疾和其他类型的发热。齐墩果酸及其衍生物已报道具有多种类型的药理活性，如抗炎、抗氧化、抗癌、保肝、抗 HIV 和抗 HCV 活性。
方法
采用超临界流体萃取（SFE）法制备 L.cephalotes 植物提取物，用制备薄层层析法分离齐墩果酸。采用紫外可见光谱（UV-VIS）、傅里叶变换红外光谱（FT-IR）和高效薄层色谱（HPTLC）对化合物进行鉴定和表征。通过质子核磁共振（HNMR）和碳核磁共振（CNMR）阐明了化合物的结构，并通过差示扫描量热法（DSC）检查了其纯度。采用 MTT 法在 595nm 处用微孔板读数器测定植物提取物和齐墩果酸的毒性。采用实时 RT-PCR 在体外和体内检测植物提取物和齐墩果酸的抗登革热活性。
结果
在 40°C 温度和 15Mpa 压力下，提取物的最佳产率最高。在 C6/36 细胞系中，植物提取物和齐墩果酸的最大无毒剂量（MNTD）分别为 46.87μg/ml 和 93.75μg/ml。分离化合物的紫外分光光度计曲线在 232nm 处与标准齐墩果酸重叠。分离化合物的叠加 FT-IR 结构表明，在 3433、2939、2871、1690、1500、1463、1387、1250、1209、1137 和 656 位与标记化合物的相同光谱。HPTLC 分析表明，L.cephalotes 提取物的保留因子为 0.19+0.06，与标准齐墩果酸色谱相似。分离化合物的 NMR 结构与标记齐墩果酸结构相似。DSC 分析表明，分离出的齐墩果酸纯度为 98.27%，熔点为 311.16°C。实时 RT-PCR 结果表明，当用植物提取物（46.87μg/ml）和测试化合物（93.75μg/ml）的 MNTD 值处理时，L.cephalotes 超临界提取物和分离出的齐墩果酸对登革热-2 型病毒的抑制率分别为 100%和 99.17%。分子研究表明，齐墩果酸与 NS1 和 NS5（非结构蛋白）的结合能分别为-9.42 和-8.32Kcal/mol。
结论
SFE 提取物 L.cephalotes 及其活性化合物齐墩果酸在体外和体内抑制登革热-2 型血清型的活性。因此，L.cephalotes 植物可能是治疗登革热感染的药物设计的极好来源。
Zotero 插件