HPTLC fingerprinting and quantification of lignans as markers in sesame oil and its polyherbal formulations

Sesamum (Sesamum indicum) seed and its oil have been in use in Indian traditional medicine, 'Ayurveda' since antiquity. However, there has been no attempt to standardize the polyherbal formulations containing sesamum oil as the main ingredient in terms of its active principle or marker compound. Biologically active lignans in sesamum oil are identified as the marker compound for the oil and its formulations. In this report, a simple, rapid and sensitive HPTLC method is described for the first time to identify and quantify sesamin and sesamolin, the major lignans of the sesamum oil and the method was applied to polyherbal formulations containing the oil for their quantitative estimation. The method was validated in terms of its calibration curve, limits of detection and quantification, precision, accuracy and robustness following standard protocols. The method thus developed was applied to sesamum oil and its commercial herbal formulations to quantify sesamin and sesamolin. The method for fingerprints of the formulations in the form of densitogram following charring of the chromatographic plate was also developed that could be useful for marker-based quality assurance of the polyherbal products containing sesamum oil.     

Comparison of kava (Piper methysticum Forst.) varieties by UV absorbance of acetonic extracts and high-performance thin-layer chromatography

The development of quality standards to regulate the trade of kava (Piper methysticum) in the Pacific necessitates new analytical techniques for routine control of high-quality raw material. In the present study, varieties and parts of the plant were compared for their kavalactone and flavokavin (FK) profiles. Three hundred and eight samples corresponding to 25 noble, 25 two-days and five wichmannii varieties were collected from 203 different plants. Commercial samples were also analysed to test the technique. Overall, 353 samples were prepared (221 roots, 114 stumps, nine basal stems and nine stem peelings). Their acetonic extracts were measured for UV absorbance and analysed by HP-TLC. Two ratios were computed after scanning the plates at 245 nm (K/KL = kavain/total kavalactones) and 366 nm (FK/KL = flavokavins/kavalactones). The results indicate that noble varieties suitable for daily consumption of kava are characterised by high K/KL and low FK/KL. At 400 nm, the mean UV absorbance of acetonic extracts from noble, two-days and wichmannii varieties are 0.69, 0.94 and 1.04, respectively. The significant (+0.757**) correlation between the total FK content and the extracts absorbance at 400 nm indicates that a simple colorimeter can be used to detect poor-quality raw material.     

3种五味子药材HPTLC鉴别与HPLC含量测定

目的 建立五味子HPTLC定性鉴别和HPLC含量测定方法.方法 以五味子甲素、五味子乙素、五味子醇甲、五味子酯甲为对照品,采用HPTLC法带状点样于GF254板上,以石油醚(30～60℃)-甲酸乙酯-甲酸(15:5:1)为展开剂,上行展开8 cm,置于紫外灯254 nm下观察,比较不同来源的五味子活性成分的差异.以五味子醇甲为对照品,采用HPLC法,ZORBAX SB-C<,18>(4.6 mm×250 mm,5 μm),流动相:甲醇-水(65:35);流速:1.0 ml·min<'-1>;柱温:室温;检测波长:254 nm,比较不同来源的五味子醇甲的含量.结果 HPTLC法能够准确快速鉴别不同来源的五味子;3种药材中的五味子醇甲含量有差异.结论 该方法简便、准确、重现性好,可从定性、定量两方面评价五味子药材质量.     

巴戟天寡糖的高效薄层色谱指纹图谱研究

 目的 建立巴戟天寡糖的高效薄层色谱（HPTLC）指纹图谱,并对不同产地巴戟天药材、炮制饮片、混伪品及含巴戟天药材的复方制剂——口服液X中寡糖成分进行分析比较。方法 在湿度40%～55%、温度20～32 ℃条件下,以乙酸乙酯-甲醇-水-冰醋酸为展开剂二次展开,展距13 cm,α-萘酚试液加热至显色,于可见光下检视。将巴戟天寡糖HPTLC指纹图谱导入指纹图谱系统解决方案软件（CHROMAP 1.5）生成共有模式。结果 巴戟天寡糖指纹图谱由11个特征条带（色谱峰）构成,并指认了其中的果糖、蔗糖、1-蔗果三糖、耐斯糖及1F-果呋喃糖基耐斯糖5个峰。结论 通过HPTLC指纹图谱考察,炮制后巴戟天寡糖成分出现质与量的改变;来源于4省的巴戟天药材无明显差异;混伪品中不含巴戟天寡糖不能作为巴戟天替代药材入药;含巴戟天药材的制剂仍可采用该HPTLC法进行制剂质量评价与控制。     

Post-ischemic administration of nimodipine following focal cerebral ischemic-reperfusion injury in rats alleviated excitotoxicity, neurobehavioural alterations and partially the bioenergetics

The present study focuses on the temporal calcium significance in middle cerebral artery occluded (2 h ischemia)-reperfused (70 h reperfusion) rats treated with nimodipine (NM) through concurrent measurements of excitotoxicity, bioenergetics and neurobehavioural paradigms. Further, the suitable therapeutic time window of calcium channel antagonism in stroke was also ascertained. NM (5 mg/kg, i.p.) was administered at pre (30 min before the induction of ischemia), during (1 h following occlusion of MCA) and post-ischemic (3 h after begin of reperfusion) states. The magnitude of neuroprotection in terms of excitotoxicity (glutamate, glutamine synthetase, Na⁺K⁺ATPase), bioenergetics (ATP, NAD⁺) and neurobehavioural paradigms (neurological score and open field exploratory behaviour) were measured and compared to ensure the therapeutic time-window of NM in stroke. Middle cerebral artery occlusion-reperfusion (MCAO/R) was found to elevate glutamate, glutamine synthetase levels and deplete Na⁺K⁺ATPase activity in the vehicle treated group (IR group). Significant decrease in bioenergetics such as ATP and NAD⁺ levels was also observed. Further, IR group demonstrated grievous oxidative stress (increase in lipid peroxidation, protein carbonyl content, nitrite/nitrate levels and decrease in superoxide dismutase and glutathione levels) along with anxiogenic behaviour, neurological deficits and neuronal damage and decreased nuclear to cytoplasm ratio in CA1 hippocampal region. Post-ischemic NM administration reversed the excitotoxicity, neurobehavioural and histopathological alterations significantly, but it restored bioenergetics level in MCAO/R rats only partially.These findings were further confirmed with the combination treatment (CT) of post-ischemic NM and pre-ischemic memantine (MN) administration, since MN showed protective effect in the pre-ischemic administration (Babu and Ramanathan, 2009). The failure of NM to forefend the neurodegeneration on pre- and during-ischemic administration suggests that the initial phase damages in ischemic-reperfusion (IR) might be mediated through other mechanism(s) such as glutamergic overstimulation or reverse operation of glutamate transporters. From the present study, it is concluded that calcium plays a crucial role in post-ischemic status and the suitable therapeutic time window of calcium antagonism is the post-ischemic state.     

The hydroalcoholic extract of Cassia alata (Linn.) leaves and its major compound rhein exhibits antiallergic activity via mast cell stabilization and lipoxygenase inhibition

Ethnopharmacological relevance

Leaves of Cassia alata (family: Caesalpiniaceae) are ethnomedically claimed as anti-asthmatic. In the current study we aimed to investigate the anti-allergic activities of hydro-methanolic extract of Cassia alata (Linn.) and its constituents rhein and kaempferol on triple antigen/sheep serum-induced mast-cell degranulation in rats.

Materials and methods

Antiallergic activity of hydroalcoholic extract of Cassia alata along with its two components rhein and kaempferol was evaluated using in vivo mast cell stabilization assay. Inhibitory effect on lipoxygenase (LOX) enzyme was also evaluated in vitro. Further chemical standardization of Cassia alata extract was done using rhein and kaempferol by HPTLC-densitometric method.

Results

The hydroalcoholic extract of Cassia alata significantly inhibited mast cell degranulation at 200 mg/kg dose. Both chemical constituents rhein and kaempferol also showed potent (>76%) inhibition of mast-cell degranulation at 5 mg/kg. Extract and rhein inhibited LOX enzyme with IC₅₀ values of 90.2 and 3.9 μg/mL, respectively, whereas kaempferol was inactive.

Conclusion

Our results suggest that Cassia alata exhibit anti-allergic activity through mast cell stabilization and LOX inhibition. Thus, Cassia alata or its active constituents could be potential alternative treatment for allergic diseases.     

Secondary metabolite credentials of Evolvulus alsinoides by high performance thin layer chromatography (HPTLC)

Plants and plant-based products are the bases of many modern pharmaceuticals that are current in use today for various diseases.The aim of the study was to investigate the biochemical constituents and high performance thin layer chromatography(HPTLC) finger printing of the ethanolic extract of Evolvulus alsinoides.Phytochemical screening was done by standard procedures and HPTLC method was also established to analyze alkaloids,flavonoids and phenolic compounds from the ethanolic extract of Evolvulus alsinoides.Preliminary phytochemical screening showed that ethanol extracted more secondary metabolites than other solvents.HPTLC fingerprinting analysis showed the presence of various alkaloids,flavonoids and phenols(quercetin) in the ethanolic extract.It can be concluded that Evolvulus alsinoides may serve as a source of potent antioxidants that may be used in the prevention of various diseases such as cancer,diabetes and cardiovascular diseases due to the presence of phenolic compounds.HPTLC finger print of Evolvulus alsinoides may be useful in the differentiation of the species from adulterants and act as a biochemical marker for this medicinally important plant in the pharmaceutical industry and plant systematic studies.     

巴戟天寡糖在提取过程中化学稳定性的研究简

 目的 考察巴戟天寡糖在不同溶媒中的化学稳定性。方法 以水、乙醇、不同pH值水溶液为介质提取巴戟天寡糖, 利用高效薄层色谱法检测其变化规律, 探讨不同提取介质对其化学稳定性的影响。结果 巴戟天寡糖在以不同提取时间的水溶液为介质进行提取时, 0.5 h后即开始水解, 巴戟天寡糖含量逐渐下降, 果糖与蔗糖含量逐渐增高, 并产生新的未知成分;巴戟天寡糖在以不同体积分数的乙醇溶液为介质进行提取时, 仅在乙醇溶液体积分数为10%~30%内发生少量水解, 并产生少量未知成分;巴戟天寡糖在以不同pH值水溶液为介质进行提取时, 在pH 2和3时基本被水解, pH 4和5时大部分被水解, 并产生较多新的未知成分, pH 为6~10时巴戟天寡糖趋于稳定。结论 巴戟天寡糖在水中不稳定, 在乙醇溶液中相对稳定, 在不同pH值水溶液中变化差异较大。     

HPTLC fingerprint profile analysis of coffee polyphenols during different roast trials

Coffee beans are a rich source of bioactive phytochemicals such as chlorogenic acids (CGA). During the roasting process, the CGA content changes dramatically. An effective HPTLC method was developed which allows a fast characterization of the eight main CGA present in green and roasted coffee beans and their distribution during roasting trials. The method was optimized with regard to three caffeoylquinic acids (CQA), two feruloylquinic acids (FQA) and three dicaffeoylquinic acids (di-CQA).The best separation for a fingerprint consisting of eight phenolic compounds as markers was achieved on silica gel 60 F₂₅₄ HPTLC plates with a solvent mixture of diethyl ether, formic acid, acetic acid, water, acetophenone and heptane (30:3:9:50:30:10) (v/v/v/v/v/v). Staining with natural product reagent A enabled visualization and quantitative evaluation. Principal component analysis can help to visualize the evolution of the chlorogenic acids throughout the process also with the aim of controlling the roasting degree.     

人子宫肌瘤鞘糖脂的分离纯化及其薄层层析图谱

本文将手术切除的人子宫肌瘤及瘤旁正常肌组织纯化分离的神经节苷脂和中性鞘糖脂进行高效薄层层析,扫描结果发现子宫肌瘤较同一病人正常子宫肌的GD_3的%明显减少,GM_1、多唾液酸神经节苷脂有增高的趋势,而肌瘤的GM_3则有降低的趋向。子宫肌瘤及瘤旁组织的中性鞘糖脂薄层层析图谱相近似,均以红细胞糖苷脂(GbOse_4Cer)及三己糖苷神经酰胺(GbOse_3Cer)为主要成分,乳糖苷及半乳糖苷神经酰胺(Lac-Cer、Gal-Cer)二者的%都少。鉴于子宫肌瘤是激素依赖性肿瘤,这些鞘糖脂组分的改变在肿瘤发生过程中起什么作用,以及它们是否与女性激素作用有关,有待进一步研究。