The present paper reports a validated high-performance thin-layer chromatography (HPTLC)‒densitometric method for the simultaneous quantification of phenolic (ferulic acid and caffeic acid) and terpenoid (β-sitosterol and lupeol) markers in Convolvulus pluricaulis Choisy. According to Ayurveda, it is commonly known as ‘Shankhpushpi’ due to its ‘Conch’ or ‘Shankh’-shape flower. The plant species, viz., Clitoria ternatea L., Evolvulus alsinoides (L.) L., and Tephrosia purpurea (L.) Pers., also having similar flowers are reported as its adulterants/substitutes. This creates a problem in its quality and efficacy in the commercial drug market of India. Therefore, a HPTLCmethod was performed on a pre-coated silica gel 60 F254 plate with the aforesaid markers. The solvent system toluene–ethyl acetate–formic acid (8.5:1.5:0.1) was determined to be the best system for the simultaneous separation of caffeic acid, ferulic acid, β-sitosterol, and lupeol at RF values of 0.14, 0.29, 0.48, and 0.63, respectively. A densitometric scanning profile of all the samples at 580 nm showed peaks for all the four markers of varying heights in the samples, except the absence of caffeic acid in Tephrosia purpurea. The developed method was standardized and validated for the quantification of active principal-based quality-control markers in terms of precision, accuracy, linearity, recovery, and repeatability. It will help to maintain batch-to-batch consistency and identification of adulterants/substitutes in raw materials during production of drug in the pharmaceutical units.
Authors:Sayyada Khatoon, Harsh Singh, and Anil Goel
Dendrophthoe falcata (Linn. f.) Etting. is a stem parasite commonly known as ‘Vanda’ in the Ayurvedic system of medicine. HPTLC has been used to detect the chemotypes of D. falcata growing on Mangifera indica Linn., Melia azedarach Linn., Wrightia tinctoria R. Br., and Callistemon lanceolatus DC. Phenolic compounds were selected as markers for this study. HPTLC results revealed qualitative and quantitative variations in the phenolic compounds in the leaves. Caffeic acid, (+)-epicatechin, and kaempferol were present in all four samples although the quantities varied from sample to sample. In contrast, ellagic acid was present only in the sample growing on Mangifera indica and gallic acid was absent only from the sample growing on the tree Melia azedarach. This variation in the phenolic compounds in these samples might be because of interaction with the host trees.
Authors:Sayyada Khatoon, Harsh Singh, Kripal Singh, and Anil Goel
(Linn. f.) Etting. is a stem parasite commonly known as ‘
’ in the Ayurvedic system of medicine. A TLC method has been established for simultaneous identification and quantification of phenolic compounds in different parts of this plant. Chromatography was performed on silica gel with tolueneethyl acetate-formic acid 6:4:1 (
) as mobile phase, and characteristic bands of (+)-catechin, ellagic acid, quercetin, and ferulic acid were observed at
0.35, 0.41, 0.63, and 0.66, respectively. The method was validated for precision, repeatability, and accuracy. The method enables reliable quantification with good resolution and separation from other constituents of the plant extract. The accuracy of the method was checked by measuring recovery of all four phenolic compounds at three different concentrations and by matching the spectra of reference markers with those of the corresponding bands in the extract at three different positions, i.e. the start, apex, and end positions of the band. The presence and quantity of these phenolic compounds varies from part to part of this parasitic plant. For example, quercetin was present only in the leaves, ferulic acid in the stem, and ellagic acid and (+)-catechin in stem and root. More (+)-catechin and ellagic acid were found in the stem than in the root.
Authors:Sayyada Khatoon, Manjoosha Srivastava, A. Rawat, and Shanta Mehrotra
The roots of
, known as ‘Bala’ in the Ayurvedic system of medicine, are used to treat a variety of ailments, including pulmonary tuberculosis, rheumatism, hematuria, urinary and heart diseases. The roots have recently been used to cure
disease and as a food supplement for fat loss. During a survey of Indian herbal drug markets it was observed that the roots and whole plants of other species of
S. acuta, S. cordata
, were being sold under the same vernacular name. In this context, HPTLC markers and a method for quantitative estimation of ephedrine, one of the therapeutically active constituents of this genus, have been developed. The study concluded that common and distinguishing bands were observed for all four
species in UV light at
= 366 nm. The roots of
had a similar HPTLC profile but the whole plant of
had characteristic blue bands at
0.60 and 0.81 and a red band at
0.87. Similarly, the whole plant of
can easily be differentiated on the basis of blue bands at
0.15 and 0.67; the roots can be differentiated by blue bands at
0.84 and 0.90. Although no characteristic bands were observed for
whole plant, the roots of this plant could also be identified by the presence of yellow and blue bands at
0.18 and 0.79, respectively. Amount of ephedrine varied in the four species studied. The maximum amount (0.112%) was found in
whole plant and the minimum amount (0.005%) in the roots of
Buch.-Ham., commonly known as ‘Kaphal’ in indigenous systems of medicine, is used for treatment of a variety of ailments. Three samples were collected from the Nainital, Ranikhet, and Shimla regions of India for qualitative and quantitative standardization by HPTLC on silica gel plates. The profiles obtained by use of two mobile phases revealed the presence of almost similar components. Amethod was established for identification of the biomarkers gallic acid, lupeol, oleanolic acid, and stigmasterol in extracts of the plant, because of the therapeutic importance of these compounds. The characteristic band of gallic acid occurred at
0.56 when toluene-ethyl acetate-formic acid 5:5:1 was used as mobile phase. Bands of oleanolic acid, stigmasterol, and lupeol occurred at
0.38, 0.49, and 0.62, respectively, when toluene-ethyl acetate 8:2 was used. These biomarkers were also estimated quantitatively. The amounts of the biomarkers varies from sample to sample - stigmasterol from 0.2229–0.4489%, oleanolic acid from 0.0079–0.0384%, lupeol from 0.0233–0.0783%, and gallic acid from 0.0795–0.0918%. The amounts were highest in the Nainital sample and lowest in the Shimla sample.
species are widely used as important timber-yielding plants and are rich source of tannins and dyes. The stem bark of these species (waste from the timber industry) is used in Ayurveda as a cardiac tonic and diuretic and is also of ethnobotanical importance. It can be exploited as a source of important polyphenols, for example ellagic and gallic acids, which are potent antioxidants. In this context, a TLC method has been established for quantification of ellagic and gallic acids in seven
(Roxb.) Wight & Arn,
W. & A. Prodr.), and TLC markers of the species have been identified. The study concluded that common and distinguishing bands were observed for all seven
species in UV light at 254 and 366 nm and after derivatization with anisaldehyde sulfuric acid reagent.
have a similar TLC profile under UV light at 254 nm and
has two characteristic yellow bands at
0.22 and 0.39 under UV light at 366 nm.
could be identified by blue bands at
0.57 and 0.66 under UV light at 366 nm and by a purple band at
0.37 after derivatization.
can easily be differentiated by a fluorescent blue band at
0.39 under UV light at 366 nm and a blue band at the same
T. bellirica, T. manii
furnished faint blue, fluorescent blue, and greenish blue bands, respectively, at
0.66 under UV light at 366 nm. Only a single faint blue band is visible for
0.57 under UV light at 366 nm. The amounts of ellagic and gallic acids vary from species to species. The maximum concentrations of ellagic and gallic acids, 2.69% and 1.04%, respectively, were found in
Authors:Veena Dixit, Saba Irshad, Harsh Singh, Priyanka Agnihotri, Tariq Husain, and Sayyada Khatoon
This study was designed to develop a simple, sensitive, selective, and precise high-performance thin-layer chromatography (HPTLC) fingerprint and quantitative estimation method for the analysis of three phenolic compounds from nine Leucas species. The developed HPTLC method was validated according to the International Conference on Harmonization guidelines. The method permits reliable quantification of one important phenolic acid (gallic acid) and two hydroxycinnamic acids (caffeic and ferulic acids) in 50% hydroethanolic extracts on silica gel with toluene—ethyl acetate—formic acid 8:2:1 (v/v) as the mobile phase. The system showed good resolution and separation of caffeic, ferulic, and gallic acids (at Rf values 0.48, 0.60, and 0.30, respectively) from the other constituents of the extract. Densitometric scanning was done at 300 nm in absorbance mode. All the results obtained by the developed method were statistically compared for validation parameters, for accuracy and good precision. Caffeic, ferulic, and gallic acids were estimated in all the aforesaid nine Leucas species, but the quantity varied from species to species. Further, these phenolics were reported from the aforesaid Leucas species for the first time, except for Leucas lanata and Leucas urticifolia. However, Leucas biflora, Leucas decemdentata, and Leucas stricta were documented for their chemical constituents for the first time. The proposed HPTLC method can be used for routine quality testing of Leucas species.
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.