Vanillin-sulfuric Acid Reagent Research Articles

TLC-fingerprinting and chemometrics for identification of Curcuma xanthorrhiza from different geographical origins in Indonesia

Abstract. Kartini K, Sabatini SS, Haridsa NM, Jayani NIE, Setiawan F, Hadiyat MA. 2023. TLC-fingerprinting and chemometrics for identification of Curcuma xanthorrhiza from different geographical origins in Indonesia. Biodiversitas 24: 6557-6566. Geographical origin is an important parameter that influences the quality of herbal medicine. The fingerprint of herbal plants is expressed as chemical compounds contributing to said quality. Curcuma xanthorrhiza, popular as java turmeric, is extensively used in Indonesian traditional medicine. This study aimed to validate and develop a Thin Layer Chromatography (TLC) fingerprint of C. xanthorrhiza, followed by chemometric techniques to classify C. xanthorrhiza rhizomes from 15 regions in Indonesia. Under selected TLC conditions (i.e., stationary phase: TLC plate Silica gel 60 GF254, mobile phase: dichloromethane, chloroform, and ethanol (10: 10: 1), detection: vanillin-sulfuric acid reagent), C. xanthorrhiza produced five zones of the compound. Extracts were stable both on the TLC plate and in the extract solution during chromatography and within 60 minutes after derivatization. In terms of precision, the chromatogram meets the requirement of intraday precision. Chemometric analysis with Principal Component Analysis (PCA) and Cluster Analysis (CA) showed that the samples of C. xanthorrhiza from 15 regions in Indonesia were grouped into five clusters based on the similarity of the chemical compounds, namely cluster 1 (Tawangmangu, Bangkalan, Kediri, and Surabaya); cluster 2 (Batu, Sragen, Tulungagung, Pasuruan, Blitar, and Lombok Tengah); cluster 3 (Ngawi); cluster 4 (Gresik); and cluster 5 (Bojonegoro, Banyuwangi, and Palangkaraya). The TLC fingerprinting with chemometrics on C. xanthorrhiza rhizome is useful for quality control based on geographic origin and authenticity identification.

The Development and Application of a HPTLC-Derived Database for the Identification of Phenolics in Honey.

This study reports on the development and validation of a HPTLC-derived database to identify phenolic compounds in honey. Two database sets are developed to contain the profiles of 107 standard compounds. Rich data in the form of Rf values, colour hues (H°) at 254 nm and 366 nm, at 366 nm after derivatising with natural product PEG reagent, and at 366 nm and white light after derivatising with vanillin–sulfuric acid reagent, λ max and λ min values in their fluorescence and λ max values in their UV-Vis spectra as well as λ max values in their fluorescence and UV-Vis spectra after derivatisation are used as filtering parameters to identify potential matches in a honey sample. A spectral overlay system is also developed to confirm these matches. The adopted filtering approach is used to validate the database application using positive and negative controls and also by comparing matches with those identified via HPLC-DAD. Manuka honey is used as the test honey and leptosperine, mandelic acid, kojic acid, lepteridine, gallic acid, epigallocatechin gallate, 2,3,4-trihydroxybenzoic acid, o-anisic acid and methyl syringate are identified in the honey using the HPTLC-derived database.

Separation and detection of apricot leaf triterpenes by high-performance thin-layer chromatography combined with direct bioautography and mass spectrometry

Prunus armeniaca leaf extract was screened for antibacterial compounds by high-performance thin-layer chromatography (HPTLC)-direct bioautography using a Gram-positive Bacillus subtilis bacterium. Six chromatographic zones exhibited characteristic bioactivity. Five of them also appeared after derivatization with vanillin-sulfuric acid reagent and could be characterized with HPTLC-electrospray ionization (ESI)-mass spectrometry (MS), suggesting the presence of triterpenoids and the fatty acids linolenic and palmitic acid. To confirm the identification of triterpenoids an HPTLC method using in situ pre-chromatographic derivatization with iodine was developed to separate the closely related triterpenoids. After development, the iodine could be eliminated from the chromatogram (verified by HPTLC-MS), making it suitable for the B. subtilis assay. Ursolic acid, oleanolic acid, betulinic acid, corosolic acid, and maslinic acid were discovered for the first time as antibacterial components of P. armeniaca leaves. Their presence was proved also by 2D-HPTLC combined with intermediate in situ derivatization by iodine.

Simultaneous Quantification of Gymnemic Acid as Gymnemagenin and Charantin as β-Sitosterol Using Validated HPTLC Densitometric Method.

Gymnemic acid and charantin are well-established antidiabetic phytosterols found in Gymnema sylvestre and Momordica charantia, respectively. The fact that these plants are often used together in antidiabetic poly-herbal formulations lured us to develop an HPTLC densitometric method for the simultaneous quantification of their bioactive compounds. Indirect estimation of gymnemic acid as gymnemagenin and charantin as β-sitosterol after hydrolysis has been proposed. Aluminum-backed silica gel 60 F254 plates (20 × 10 cm) were used as stationary phase and toluene-ethyl acetate-methanol-formic acid (60 : 20 : 15 : 5, v/v) as mobile phase. Developed chromatogram was scanned at 550 nm after derivatization with modified vanillin-sulfuric acid reagent. Regression analysis of the calibration data showed an excellent linear relationship between peak area versus concentration of the analytes. Linearity was found to be in the range of 500-2,500 and 100-500 ng/band for gymnemagenin and β-sitosterol, respectively. The suitability of the developed HPTLC method for simultaneous estimation of analytes was established by validating it as per the ICH guidelines. The limits of detection and quantification for gymnemagenin were found to be ≈60 and ≈190 ng/band, and those for β-sitosterol ≈30 and ≈90 ng/band, respectively. The developed method was found to be linear (r(2) = 0.9987 and 0.9943), precise (relative standard deviation <1.5 and <2% for intra- and interday precision) and accurate (mean recovery ranged between 98.43-101.44 and 98.68-100.20%) for gymnemagenin and β-sitosterol, respectively. The proposed method was also found specific and robust for quantification of both the analytes and was successfully applied to herbal drugs and in-house herbal formulation without any interference.

Simultaneous quantification of the anti-inflammatory phytoconstituents betulinic acid, 24β-ethylcholesta-5,22E,25-triene-3β-ol, and lupeol inClerodendrum phlomidis

A new validated high-performance thin-layer chromatographic (HPTLC) method has been developed for the simultaneous determination of anti-inflammatory compounds betulinic acid (BA, 1), 24β-ethylcholesta-5,22E,25-triene-3β-ol (ECTO, 2), and lupeol (LU, 3) in the roots of Clerodendrum phlomidis. Extraction efficiency of marker compounds was studied using normal (cold and hot), ultrasonic, as well as microwave-assisted extraction techniques with various solvents. Well-resolved separation of marker compounds was achieved on silica gel 60F254 plates using the mobile phase consisting of chloroform-methanol (98:2, ½/½). Marker compounds were scanned using the densitometric reflection-absorption mode after post-chromatographic derivatization with vanillin-sulfuric acid reagent. Validation of method was performed as per the International Conference on Harmonization (ICH) guidelines. Report on the occurrence of betulinic acid for the first time in C. phlomidis is of chemotaxonomic importance. In addition, anti-infla...

Stilbenoids from Vitis labrusca “Isabella” Stems

Studies on the chemical constituents of each part of the Vitis species, especially Vitis vinifera L., have been performed since this species has promising biological activities, including antioxidant [1,2] and anticancer activities [3,4]. Meanwhile, investigations on the chemical composition of Vitis labrusca, to which the American grape belongs, have never been reported. This paper reports for the first time the chemical components of Vitis labrusca “Isabella”. The stems of V. labrusca “Isabella” were collected from Kebun Percobaan Banjarsari, Probolinggo, East Java, Indonesia in April 2008. The dried-powdered stems (3.0 kg) were cold extracted using methanol at room temperature for 62 hours. The methanolic extract (342.14 g) was dissolved in methanol–water (9:1) and re-extracted successively with n-hexane (17.78 g) and ethyl acetate (145.36 g). The ethyl acetate extract was investigated. A portion of the ethyl acetate extract (135.36 g) was fractionated by vacuum liquid chromatography using Merck silica gel 60 (230–400 mesh) with a solvent gradient of n-hexane–ethyl acetate to give 10 major fractions. Fractions 4 and 5 were further purified using several chromatographic techniques, including column chromatography on a glass column using Merck silica gel 60 (70–230 mesh), medium- pressure chromatography using Supelco Versa Pack Silica Cartridge (40 × 75 mm), prep-TLC and centrifugal prep-TLC using Merck silica gel PF254, and also by Sephadex LH-20. TLC analysis was performed on Merck pre-coated silica gel F254 plates (2 mm thickness), and spots were visualized by UV light (254 and 365 nm) and spraying with vanillin sulfuric acid reagent followed by heating. Melting points were measured using a melting point apparatus equipped with a Leica Gallen III microscope and were uncorrected. Five compounds were isolated from the ethyl acetate extract. Based on the spectroscopic analysis and comparison with previously reported data, compounds 1–5 were identified as resveratrol (1) [5,6], e-viniferin (2) [7,8], pallidol (3) [9], ampelopsin A (4) [8,10], and α-viniferin (5) [11]. The 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded on a Bruker Avance 400 spectrometer using acetone-d6 as the solvent. Residual solvent was used as an internal standard. The IR spectra were run on PerkinElmer FT-IR spectrometer recorded in KBr pellet. The UV spectra were taken on a Perkin–Elmer UV-Vis spectrophotometer. Mass and LC-MS spectra were obtained from Kent Mass Spectrometry Services, United Kingdom and Department of Metrology, SIRIM BHD, respectively.

Simultaneous Quantification of Diarylheptanoids in Alnus nepalensis Using a Validated HPTLC Method

Platyphylloside, oregonin and hirsutanonol 5-O-β-D-glucopyranoside are known bioactive metabolites in Alnus nepalensis. In this article, the aforementioned three markers were isolated and simultaneously quantified by the thin-layer chromatography densitometric method. A sensitive, selective and robust qualitative and quantitative densitometric high-performance thin-layer chromatographic method was developed and validated for the determination of above markers in the leaves of A. nepalensis. The separation was performed on silica gel 60F254 high-performance thin-layer chromatography plates using chloroform:methanol:formic acid (75:25:2, v/v) as mobile phase. The quantitation of diarylheptanoids was carried out using the densitometric reflection/absorption mode at 610 nm after post-chromatographic derivatization with vanillin-sulfuric acid reagent. A precise and accurate quantification can be performed in the linear working concentration range of 333-3330 ng/spot with good correlation (r(2) = 0.999). The method was validated for peak purities, precision, robustness, limit of detection and quantitation, etc., as per ICH guidelines. Specificity of quantitation was confirmed using retention factor and spectra correlation of markers in standard and sample tracks.

Antifilarial diarylheptanoids from Alnus nepalensis leaves growing in high altitude areas of Uttarakhand, India

Lymphatic filariasis continues to be a major health problem in tropical and subtropical countries. A macrofilaricidal agent capable of eliminating adult filarial parasites is urgently needed. Platyphyllenone (A), alusenone (B), hirustenone (C) and hirsutanonol (D) are important biologically active diarylheptanoids present in Alnus nepalensis. In the present study, we report the antifilarial activity in diarylheptanoids isolated from the leaves of A. nepalensis. Out of four compounds (A–D) tested in vitro one has shown promising anti-filarial activity both in vitro and in vivo studies. This is the first ever report on antifilarial efficacy of a compound of the plant and warrants further studies around this scaffold.In addition, a sensitive, selective and robust densitometric high-performance thin-layer chromatographic method was developed and validated for the above four biomarker compounds. The separation was performed on silica gel 60F254 high-performance thin layer chromatography plates using chloroform:methanol (9:1, v/v) as mobile phase. The quantitation of marker compounds was carried out using densitometric reflection/absorption mode at 600nm after post-chromatographic derivatization using vanillin–sulfuric acid reagent. The method was validated for peak purity, precision, robustness, limit of detection (LOD) and quantitation (LOQ) etc., as per the International Conference on Harmonization (ICH) guidelines.

Purification of vetiver alcohols and esters for quantitative high-performance thin-layer chromatography determination in Haitian vetiver essential oils and vetiver acetates

A simple, fast, and efficient High-Performance Thin-Layer Chromatography (HPTLC) method was developed for the simultaneous quantitative determination of alcohols and acetates in Haitian vetiver essential oils (Chryzopogon zizanioides) and its acetylated form. Analytes were separated by using a mixture of n-hexane–chloroform–ethyl acetate (8:6:0.5, v/v/v) as mobile phase under 47% humidity. Quantification was achieved by densitometric evaluation of the analytes in absorbance mode under visible light (λ=530nm) after staining with a vanillin–sulfuric acid reagent. Reference mixtures of alcohols and acetates were obtained by fractionation of Haitian vetiver oil or vetiver acetates, followed by purification of the fractions of interest by means of Over-Pressured Layer Chromatography (OPLC). The chemical composition of each reference fraction was determined by using GC–MS and GC×GC–MS, and their overall purity was determined by GC/FID and HPTLC. The TLC method provided compact spots for alcohols (Rf2=0.18±0.01 and Rf1=0.28±0.01) and acetates (Rf3=0.65±0.01). Calibration plots showed good linear correlation with r2=0.9995±0.0001 and r2=0.9995±0.0001 for alcohols and r2=0.9996±0.0001 for acetates in a 40–200ngspot−1 concentration range with respect to peak areas. The method was validated for precision and accuracy. Limit of detection (LOD) and quantification (LOQ) were determined. Method specificity was confirmed using retention factor (Rf) and GC–MS control of the standards reference mixtures.

Development and validation of hptlc densitometric method for identification and quantification of geraniol in palmarosa oil

India is the main producer of palmarosa oil obtained from rosha grass (Cymbopogan martini var. motia) of family Graminae. The essential oil obtained from the grass is rich in geraniol content. The oil is commercially obtained by hydro-steam distillation of rosha grass. Oil of palmarosa and its separated fraction geraniol are widely used in perfumery industry. The palmarosa oil is valued due to geraniol and largely used as base for fine perfumery. In this article, a simple, rapid, cost-effective and accurate method using high-performance thin-layer chromatographic (HPTLC) method has been developed for separation, identification and quantification of geraniol in palmarosa oil. Separation and quantification of palmarosa are achieved by HPTLC using mobile phase of toluene-ethylacetate (92.5:7.5) followed by separation on precoated silica gel 60 F254 aluminium plates and densitometric determination carried out after derivatization with vanillin-sulfuric acid reagent at λmax, 400 nm, in absorption-reflectance m...

Simultaneous quantification of diterpenoids in Premna integrifolia using a validated HPTLC method

A sensitive, selective and robust densitometric high-performance thin layer chromatographic method was developed and validated for the determination of diterpenoids in the root bark of Premna integrifolia. Diterpenoids 1β,3α,8β-trihydroxy-pimara-15-ene (A), 6α,11,12,16-tetrahydroxy-7-oxo-abieta-8,11,13-triene (B) and 2α,19-dihydroxy-pimara-7,15-diene (C) were used as chemical markers for the standardization of P. integrifolia plant extracts. The separation was performed on silica gel 60F(254) high-performance thin layer chromatography plates using hexane/acetone/ethylacetate (60:20:20 v/v) as mobile phase. The quantitation of diterpenoids was carried out using densitometric reflection/absorption mode at 475 nm after post-chromatographic derivatization using vanillin-sulfuric acid reagent. A precise and accurate quantification can be performed for compounds A, B and C in the linear working concentration range of 1-10 μg/spot with good correlations (r(2) =0.9985, 0.9996 and 0.9992, respectively). The method was validated for peak purity, precision, robustness, limit of detection (LOD) and quantitation (LOQ) etc., as per the International Conference on Harmonization (ICH) guidelines. Specificity of quantitation was confirmed using retention factor (R(f)) and spectra correlation of markers in standard and sample tracks. The method reported here is simple and reproducible which may be applied for quantitative analysis of above diterpenoids in the root bark of P. integrifolia.

Development of chromatographic and free radical scavenging activity fingerprints by thin‐layer chromatography for selected Salvia species

Plant-derived free radical scavengers have become the subject of intensive scientific interest. Recently, the concept of coupling chromatographic fingerprints with biological fingerprinting analysis has gained much attention for the quality control of plant extracts. However, identification of free radical scavenging activity of each single compound in a complex mixture is a difficult task. Thin-layer chromatography with post-chromatographic derivatisation with the methanol solution of DPPH can be a valuable tool in such analyses. Development of chromatographic and free radical scavenging fingerprints of nineteen Salvia species grown and cultivated in Poland. Chromatography was performed on the silica gel layers with use of two eluents, one for the resolution of the less polar compounds, and the other one for the resolution of the medium and highly polar ones. The plates were sprayed with the vanillin-sulfuric acid reagent to produce chemical fingerprints, and with DPPH solution to generate free radical scavenging fingerprints. With four Salvia species, it was revealed that their strong free radical scavenging properties are not only due to the presence of polar flavonoids and phenolic acids, but also due to the presence of several free radical scavengers in the less polar fraction. Because of the similarities in both the chromatographic and the free radical scavenging fingerprints, S. triloba can be introduced as a possible equivalent of the pharmacopoeial species, S. officinalis. Fingerprints developed in the experiments proved useful for the analysis of complex extracts of the different Salvia species.

CHIEF CONSTITUENTS AND BIOLOGICAL ACTIVITIES DETERMINATION OF STEPHANIA VENOSA

CHIEF CONSTITUENTS AND BIOLOGICAL ACTIVITIES DETERMINATION OF STEPHANIA VENOSA

COLORIMETRIC DETERMINATION OF CRUDE POWDERED MYRRH, PURIFIED MYRRH EXTRACT, OILY FRACTION, AND ITS DIFFERENT PHARMACEUTICAL DOSAGE FORMS

ABSTRACT Myrrh is an effective anti-microbial agent that is considered an excellent external remedy for mouth, throat and skin infections and it is also useful in systematic treatment of glandular fever and brucellosis. The purified myrrh extract has been formulated as soft gelatin capsules, suppositories and emulsion known commercially as Mirazid. Recently, Mirazid has been proved to be a safe drug of potential effects as antibilharzial and fasciolicidal agent. In this study, a simple, precise and accurate, colorimetric method for determination of crude powdered myrrh, petroleum ether extract (oily fraction) and purified myrrh extract, in soft gelatin capsules, emulsion and in suppositories, is put forward. The procedure involves reaction with vanillin-sulfuric acid reagent to form a violet colored reaction product with maximum absorption at 518 nm. Optimum conditions for color formation are reported and the color has been found to be stable for six hours. Beer's law is obeyed in a concentration range 40–200 µg/mL, with correlation coefficient of 0.9994. The mean percentage recoveries have been found to be 101.31±0.54 for soft gelatin capsule, 100.18±0.39 for suppositories and 100.14±0.41 for emulsion. No interference has been observed either from capsule shell, suppository base or emulsifying agent. In comparison to a another colorimetric method based on the use of blue tetrazolium salt, the proposed method is more selective, in addition of being simple, of good accuracy, and high precision for determination of the major bioactive components of the oily fraction rather than resin extract. The procedure has been validated for use in quality control applications.

HPTLC measured values of primula saponins in extracts ofPrimulae radix. A comparison after derivatization between handspraying, dipping and autospraying

The primula saponins were measured after derivatization with the DESAGA densitometer CD 60. The critical procedure of post-chromatographic derivatization with vanillin-sulfuric acid reagent basically defines the quality of quantification. Uniform colouring of the spots is decisive for standardization. In this paper we judge the pros and cons of the diverse derivatizing systems as e. g. handspraying, dipping and autospraying with the new equipment ChromaJet DS 20. We consider the autospraying device ChromaJet DS 20 the appropriate method to recieve better and more precise results constantly and to economize the ecological harmful spray solutions as much as possible. This means in our study only 1.8 ml of spray solution were used.

Thin-layer chromatography of gallic acid, methyl gallate, pyrogallol, phloroglucinol, catechol, resorcinol, hydroquinone, catechin, epicatechin, cinnamic acid, p-coumaric acid, ferulic acid and tannic acid

Six solvent systems of varying suitability are reported for the thin-layer chromatographic separation of simple phenolics and related compounds such as gallic acid, methyl gallate, pyrogallol, phloroglucinol, catechol, resorcinol, hydroquinone, catechin, epicatechin, cinnamic acid, p-coumaric acid, ferulic acid and tannic acid. The solvent system chloroform-ethyl acetate-acetic acid (50:50:1) facilitated the separation of all the compounds except pyrogallol and ferulic acid; resorcinol and hydroquinone, which were in turn resolved in benzene-dioxane-acetic acid (85:15:1). Detection was carried out using iodine vapour, ferric chloride reagent, ferric chloride-ferricyanide reagent and vanillin-sulfuric acid reagent.

The Development and Use of a Rapid Analytical Technique for Estimation of Free and Potentially Volatile Monoterpene Flavorants of Grapes

A technique has been developed for the rapid assay of monoterpene flavorants of grape juices. Steam distillation of juice at neutrality yielded free aroma compounds of the grape while, at low pH, monoterpenes derived from the polyols and glycosidically bound forms were collected. Reaction of these distillates with a vanillinsulfuric acid reagent gave a color, the intensity of which was proportional to the concentration of monoterpenes in the two fractions. Thus analytical figures for free volatile terpenes (FVT) and potential volatile terpenes (PVT) of the grape were obtained. The presence of sulfur dioxide in juice interfered with the determination, necessitating treatment with H₂O₂ before color development. Other non-monoterpenoid juice constituents did not interfere, except <i>trans</i>-hex-2-en-1-ol, which would have contributed to FVT figures. Experiments with different categories of reference monoterpenes, including polyols and glycoside derivatives, demonstrated that the technique gave quantitative recovery of FVT while yields of PVT were 55 to 80 percent. Solutions containing mixtures of different monoterpene categories on assay gave satisfactory recoveries of FVT and PVT. In a preliminary survey, the assay was applied to samples of mature grapes of several varieties and figures for FVT and PVT correlated with the recognized aroma characteristics of the cultivars examined. The value of the assay was demonstrated in observing the increase in concentration of monoterpene flavor compounds in developing Muscat of Alexandria grapes.

Colorimetric Determination of Cyclamate

A colorimetric method for the determination of cyclamate in foods, based on the reaction of cyclohexanol with vanillin-sulfuric acid reagent, was established. After separation of cyclamate from other components by dialysis pretreatment, cyclamate was converted to cyclohexyl nitrite with nitrous acid under cooling in an ice bath and then to cyclohexanol with sulfamic acid. Cyclohexanol was extracted with ether. After the addition of water to the extract, the ether was evaporated by vigorous stirring using a mixer at room temperature. Vanillin-sulfuric acid reagent was added little by little to the remaining water layer containing cyclohexanol under cooling in an ice bath and warmed for 20 minutes at 70°to form a greenish product. The colored solution was measured spectrophotometrically at 655 mμ. The recovery of sodium cyclamate added to some foods was 93.2-98.8%.