Shikhar Verma

Evaluation of Successive Fractions for Optimum Quantification of Bergenin and Gallic Acid in Three Industrially Important Bergenia species by High-Performance Thin-Layer Chromatography

Bergenia (Saxifragaceae) known as paashanbheda in Ayurveda is an evergreen perennial herb with about 30 species known worldwide. It is widely distributed in Central and East Asia. Bergenia ligulata Wall., Bergenia stracheyi Engl., and Bergenia ciliata Sternb. are well known species which are available in high altitude (7000–10,000 ft) of temperate Himalayas from Kashmir to Bhutan and in Khasia hills at 400 ft [1]. It is used for the treatment of pulmonary infection [2], as antioxidant [3, 4], in urolithiasis, anti-inflammatory, for treating boils, menorrhagia, and excessive hemorrhage [5, 6].

Studies on chemotypic variation in Centella asiatica (L.) Urban from Nilgiri range of India

Centella asiatica (L.) Urban (Apiaceae) possesses various healing effects and antioxidant properties. However, there has been very less focus on the investigation of chemotypic variations of C. asiatica found in different geographical zones of the country. In order to conserve C. asiatica, as it is an industrially valuable herb and over-exploitation of this drug from wild is a common practice, different distinct accessions of C. asiatica from Nilgiri range (Deccan zones) of India were compared in relation to the levels of triterpenoid saponins. Physicochemical parameters were also evaluated in all the accessions. The metabolites investigated include madecassoside, asiaticoside, and its sapogenin, asiatic acid by high-performance thin-layer chromatography (HPTLC). CA-45 showed the highest content of asiaticoside, CA-51 showed the highest content of madecassoside, and CA-47 showed the highest content of asiatic acid among other accessions of Nilgiri range. It can be concluded that the geographical conditions (soil type and altitude) of these accessions are comparatively favorable for the production of higher levels of triterpenoid saponins in C. asiatica. The reported data will contribute to the establishment of knowledge about the triterpenoid saponin composition of different chemotypes of C. asiatica found in Nilgiri range of India in comparison to other geographical areas, and lays a foundation for the conservation and commercial cultivation of this plant.

High-performance thin-layer chromatographic analysis for the simultaneous quantification of gallic acid, vanillic acid, protocatechuic acid, and quercetin in the methanolic fraction of Limonia acidissima L. fruits

A high-performance thin-layer chromatography (HPTLC) method for the simultaneous quantitative determination of gallic acid, vanillic acid, protocatechuic acid, and quercetin in methanolic fractions of Limonia acidissima L. fruits was developed for the first time for this species. Methanol was found to be the best for the highest possible recovery of the target analytes. For achieving good separation, a mobile phase of toluene—ethyl acetate—formic acid (5:4:1 v/v) was used. The densitometric determination was carried out at 310 and 254 nm in reflection—absorption mode. The calibration curves were linear in the range of 100–600 ng per spot for gallic acid, vanillic acid, protocatechuic acid, and quercetin. The methanolic fractions of L. acidissima L. fruits showed the presence of gallic acid (0.07%), vanillic acid (0.16%), protocatechuic acid (0.06%), and quercetin (0.14%). The proposed method is simple, precise, speciffic, and accurate. The statistical analysis of the data obtained proves that the method is reproducible and selective and can be used for the routine analysis of the reported phenolic compounds in crude drug and extracts. The simultaneous quantification of these compounds has not been reported yet in L. acidissima L., which may be utilized for the proper standardization of the drug.

A novel method for quantification of lactose in mammalian milk through HPTLC and determination by a mass spectrometric technique

Lactose molecules are disaccharides and the basic core unit of sugars (oligosaccharides), and are enriched in mammalian milk. Lactose plays multiple roles in the health of neonates, by stimulating the growth of selected beneficial bacteria in the gut, and participating in the development and growth of newborns. All mammalians produce lactose with oligosaccharides (LOS) in different concentrations during their lactation period. In this study, we present a novel high-performance thin-layer chromatography (HPTLC) method for the estimation of lactose concentration present in milk of different mammalians, viz. mare (Equus caballus), cow (Bos primigenius indicus), buffalo (Bubalus bubalis), camel (Camelus dromedarius) and donkey (Equus asinus). Separation and quantification were achieved using the ternary mobile phase of n-butanol–glacial acetic acid–water (75:10:15 v/v/v) on precoated silica gel 60F254 aluminium plates; lactose standard in milk samples was also confirmed by mass spectrometric determination, and densitometric determination of HPTLC plates was carried out after derivatization with methanol–sulphuric acid reagent. After HPTLC densitometric scanning, the concentrations of free lactose in milk of the mare, cow, camel, buffalo and donkey were 2.16%, 5.70%, 6.70%, 7.17% and 6.38%, respectively.

Chemotaxonomic Studies on Centella asiatica (L.) Urb. from Varied Phytogeographical Conditions of India for Its Industrial Prospection

Centella asiatica (L.) Urb. (Apiaceae) with high commercial value is an important herb in traditional Indian medicine. In the present study, comparative chemoprofiling was performed on various samples of C. asiatica (CA) collected from different phytogeographical zones of India, using high-performance thin-layer chromatography. Three biologically active phytochemicals, viz. asiaticosides, madeccasosides and asiatic acid, were investigated in 35 samples of CA. Variation in concentrations of asiaticoside, madeccasoside and asiatic acid was observed as 0.03–0.66%, 0.007–2.688% and 0.006–0.44%, respectively. The mobile phase which was used for asiaticoside and madecassoside was butanol:ethyl acetate:water, and for asiatic acid, it was toluene:ethyl acetate:formic acid. Among all samples, significant variation was observed in asiaticoside and madeccasoside from Northern Himalayas, while very small variation was found in asiatic acid from Deccan region. The results of the present study are of industrial importance in selection of phytogeographical regions of India for collection and to get maximum contents of active targeted phytochemicals.

High-performance thin-layer chromatographic analysis for the simultaneous quantification of lupeol and ursolic acid in the methanolic fraction of four different species of Bauhinia

A high-performance thin-layer chromatography (HPTLC) method for the simultaneous quantitative determination of lupeol and ursolic acid in the methanolic fraction of four different species of Bauhinia leaves was developed for the first time. For achieving good separation, a mobile phase of toluene—ethyl acetate—formic acid (8:2:0.1, v/v) was used. The densitometric determination was carried out at 550 nm and 520 nm in reflection—absorption mode for lupeol and ursolic acid, respectively, which were linear in the range of 100–600 ng per band. During the analysis, lupeol (0.15%) and ursolic acid (0.11%) were found to be the highest in the leaves of B. acuminata. The proposed method is simple, precise, specific, and accurate. The statistical analysis of the obtained data proves that the method is reproducible and selective and can be used for the routine analysis of the reported terpenoids in crude drug and extracts. The simultaneous quantification of these compounds has not yet been reported in the leaves of the studied Bauhinia species which may be utilized for the proper standardization of these species.

High-Performance Thin-Layer Chromatographic Analysis for the Simultaneous Quantification of Four Phenolics in Flowers and Flower Buds of Bauhinia purpurea L., Bauhinia variegata L., and Bauhinia acuminata L.

A high-performance thin-layer chromatographic (HPTLC) method for the simultaneous quantitative determination of caffeic acid, vanillic acid, syringic acid and kaempferol in flowers and buds of three different Bauhinia species was developed for the first time in the case of these species. Methanol was found to be the best for the highest possible recovery of target analytes. For achieving good separation, a mobile phase of toluene—ethyl acetate—formic acid (5:4:1, v/v) was used. The densitometric determination was carried out at 350 nm in reflection—absorption mode. The calibration curves were linear in the range of 100–700 ng per spot for caffeic acid, vanillic acid, syringic acid, and kaempferol. The methanolic fractions of Bauhinia variegata L. flowers (BVFM) showed the highest amount of caffeic acid (0.08%), B. variegata L. buds (BVBM) and Bauhinia purpurea L. flowers (BPFM) showed the highest amount of kaempferol (1.53%), Bauhinia acuminata L. flowers showed the highest amount of vanillic acid (0.40%), and B. acuminata L. buds showed the highest amount of syringic acid (0.08%). The proposed method is simple, precise, specific, accurate, less time-consuming, and cost-effective. The statistical analysis of data obtained proves that the method is reproducible and selective and can be used for routine analysis of reported phenolic compounds in crude drug and extracts. The simultaneous quantification of these phenolic compounds has not yet been reported in the case of these species which may be utilized for the proper standardization of the drug.