Validated High-performance Thin-layer Chromatography Method Research Articles

Mixture Design-driven Statistical Optimization and Method Validation in HPTLC: Targeting Rutin, Quercetin, and Gallic Acid

Rutin (RT), Quercetin (QT), and Gallic Acid (GA) are recognized for their potent antioxidant and anticancer properties, prevalent across numerous plant species. The precise quantification of RT, QT, and GA is pivotal for evaluating the therapeutic potential of plant-based substances. In response, a new, straightforward, cost-effective, and reliable method using High-Performance Thin-Layer Chromatography (HPTLC) has been developed and validated for the quantification of these compounds. The mobile phase optimization employed a mixture design approach, achieving chromatographic separation with a mobile phase mixture of toluene, ethyl acetate, menthol, and formic acid at specific ratios (3.56:3.70:0.94:1.80 v/v/v/v/v/v/v/v). Silica gel 60 F254 HPTLC plates were utilized for the analysis. The retention factors (Rf) observed for RT, GA, and QT were 0.21, 0.58, and 0.74, respectively. This method demonstrated a robust linear relationship for concentrations ranging from 400 to 2000 ng per band, with correlation coefficients (R2) of 0.9921 for RT, 0.9936 for QT, and 0.9912 for GA. The Limits of Detection (LOD) and quantification (LOQ) were established at 100.84, 102.38, and 84.54 ng per band for LOD, and 305.58, 310.25, and 256.18 ng per band for LOQ, respectively, for RT, QT, and GA. This validated HPTLC method developed through a Design of Experiment (DoE) approach was successfully employed for the quantification of GA, QT, and RT from the fruits of Adansonia digitata and leaves of Grewia asiatica ethanolic extracts.

Development of a new validated stability-indicating high-performance thin-layer chromatography method for determination of dosulepin HCl in bulk and marketed formulation with characterization of its degradants by liquid chromatography–Mass spectrometry

Aim: Dosulepin hydrochloride is a tricyclic antidepressant. It acts by inhibition of the reuptake of biogenic amine and increasing available neurotransmitter levels at the synaptic cleft. The objective of this work is to develop a new validated stability-indicating high-performance thin-layer chromatography (HPTLC) method for the determination of dosulepin hydrochloric acid (HCl) in bulk and marketed formulation and characterization of the degradation products by liquid chromatography–mass spectrometry (LC–MS). Materials and Methods: The HPTLC method development of dosulepin HCl was performed on aluminum plates precoated with silica gel 60 F254 using dichloromethane: toulene: methanol: glacial acetic acid (GAA) (4:4:2:0.2 v/v) with densitometric detection at 220 nm. The method validation was done using linearity, precision, accuracy, and robustness parameter. Results: Linearity range for dosulepin HCl was found in between 150 and 900 ng/band correlation coefficient was 0.996. The % relative standard deviation for accuracy and precision was found <2%. The developed and validated HPTLC method is simple, accurate, precise, and specific. Conclusion: This study concludes that the dosulepin HCl undergoes degradation to different extent under different stress conditions. From the peak purity profile studies, it was confirmed that the peak of the degradation product was not interfering with the peak of drugs. Major acidic and oxidation degradation products were isolated and characterized by LC–MS and probable degradation pathway for dosulepin HCl was determined.

Screening of elite germplasms for industrially valuable medicinal crop Stevia rebaudiana for stevioside and rebaudioside A production: An HPTLC-linked chemotaxonomic assessment

Stevia rebaudiana Bertoni (Asteraceae) is a South American perennial herb native to the Paraguay region. It is high-value commercial crop in the food industry because of an array of bioactive diterpenoid glycosides, the most important of which are the steviol glycosides, stevioside (Stev) and rebaudioside A (Reb A). Standardized extract from Stevia leaves, which is high in steviol glycosides, can be used as a high-potency sweetener. The current study proposes a high-performance thin layer chromatography (HPTLC) approach for estimating stevioside and rebaudioside that is rapid, verified, and repeatable. A collection of 32 chemotypes of Stevia rebaudiana were collected from 32 districts across nine Indian states. Stevioside and rebaudioside A content ranged between 1.43 - 5.88 % and 0.77 - 1.95 % respectively across 32 chemotypes of S. rebaudiana . Among all the studied samples, SrP1 chemotype was found to possess the highest quantity of stevioside (5.88 %) and rebaudioside A (1.95 %), followed by SrH11 (5.28 % Stev; 1.89 % Reb A) chemotypes obtained from Jalandhar, Punjab and Palampur, Himachal Pradesh respectively. Chemotype SrWb32 (1.43 % Stev; 0.77 % Reb A) collected from Bardhaman, West Bengal exhibited least amount of stevioside and rebaudioside A. The current fast, reproducible, and validated HPTLC method presents an efficacious toolkit to explore the quantitative variation in the stevioside and rebaudioside A contents in natural S. rebaudiana chemotypes. This methodology may be employed to screen high stevioside and rebaudioside A yielding elite genotypes for future propagation as well as commercialization of these highly potent sweeteners to deal with the ever-expanding demand for alternative yet natural sweeteners in food industries. • Thirty two populations or chemotypes of Stevia rebaudiana were analysed to detect steviol glycosides. • Chemotypes of the plant revealed altitudinal variation in steviosides and rebaudisides A content. • Elite chemotypes can further be used for propagation and commercial utilization.

Simultaneous Analysis and Efficient Separation of Anabolic Androgenic Steroids in Dietary Supplement by a Validated HPTLC Method.

Using sports supplements is common among athletes. The presence of anabolic steroids in sports supplements as a hormonal contaminant can increase production efficiency. Since anabolic steroids cause health problems and result in positive doping tests in athletes, it is important to investigate their presence in the supplement preparations consumed by athletes. This paper aims to simultaneously determine ten anabolic steroids by high-performance thin-layer chromatography (HPTLC) method in sports supplements. Chromatographic analysis was conducted on glass silica gel 60F254 plates. The extracts loaded on silica gel plates are subjected to programed multiple development (PMD) to separate anabolic androgenic steroids (AASs). Densitometric scanning is carried out at the wavelength of 245 and 366nm. The method was validated according to the ICH guidelines. Spots at retardation factor (Rf) 0.72 (elution system 1), 0.4 (elution system 1), 0.29 (elution system 2), 0.25 (elution system 2), 0.1 (elution system 1), 0.65 (elution system 2), 0.59 (elution system 1), 0.44 (elution system 1), 0.8 (elution system 3), and 0.82 (elution system 3) values were recognized as 19-nor androstenedione, 19-nortestosterone, methyl testosterone, clostebol, stanozolol, trenbolone enanthate, oxymetholone, oxandrolone, testosterone enanthate, and nandrolone decanoate, respectively. The linear ranges were 25 - 250 μg/mL for oxymetholone, 7 - 50 μg/mL for 19-nor androstenedione, 19-nortestosterone, and oxandrolone, and 3 - 20 μg/mL for methyl testosterone, clostebol, stanozolol, trenbolone enanthate, testosterone enanthate, and nandrolone decanoate. The developed method is validated by acceptable precision (CV < 20%) and good accuracy (94% < R < 114%). The value of limit of detection (LOD) for all derivatives was in the range of 0.02 - 0.16 μg/spot (20-160 μg/g of supplement), while limit of quantitation (LOQ) was found to be in the range of 0.06 - 0.5 μg/spot (60 - 500 μg/g of supplement). Fifty sports supplement samples as real sample were collected and analyzed. None of the samples screened positive using the HPTLC method. In the present study, the fast, cheap, and simple HPTLC method could be used for the multi-residue analysis of ten anabolic androgenic steroids in sports supplements.

Development of a validated high-performance thin-layer chromatography method for quantification of lupeol from different parts of Bauhinia acuminata.

The goal of the study was to determine how much lupeol was present in different Bauhinia acuminata samples. Lupeol was analyzed using a unique (verified) high-performance thin-layer chromatography (HPTLC) method. Separation of various compounds was achieved on silica gel (66 F 254) plates with toluene and methanol (99:1) as the mobile phase, followed by derivatization with Liebermann-Burchard reagent (r2 = 0.996). The proposed approach demonstrated a satisfactory linear relationship in the concentration range of 4-8 μg/mL. The limit of detection and limit of quantification were 63.84 ng/spot and 208.57 ng/spot, respectively. The proposed method was a simple and precise procedure that provided good resolution with other constituents of extracts. The validated HPTLC method described was simple, dependable, and practical and could be useful for standardizing and evaluating the quality of herbal material and formulations containing various Bauhinia species. The leaf, stem, unsaponified matter leaf, and stem petroleum ether extract had good amounts of lupeol (25.75, 38.25, 1.65, and 1.75, respectively). This research will be helpful for the quantitative determination of lupeol from herbal formulations as well as useful for further studies in this area.

Quantification of the bioactive marker resveratrol in Morus alba Linn. fruits by high–performance thin–layer chromatography

The present study focuses on the development and validation of a high-performance thin-layer chromatography (HPTLC)‒densitometry method for the identification and quantification of resveratrol in the 70% methanolic extract of Morus alba fruits. The separation was achieved with the developer hexane‒ethyl acetate‒glacial acetic acid (4:6:0.1, V/V) on TLC aluminum plate pre-coated with silica gel 60 F254. Densitometric scanning was carried out at a wavelength of 302 nm in absorbance mode. The peak corresponding to reference standard resveratrol and that of the extract had the same RF of 0.556. The method was validated by following the International Conference of Harmonization (ICH) guidelines for precision, accuracy and reproducibility. The regression equation showed a polynomial function with a correlation coefficient of 0.994 ± 4.20% (R ± SD). The limit of detection and limit of quantification were 16 and 45 ng/band, respectively. The content of resveratrol was found to be 31.94 μg/100 mg of 70% methanolic extract of M. alba. The validated HPTLC method can be routinely used for the quality control and standardization of the bioactive marker resveratrol and formulations containing M. alba.

Development and validation of stability indicating HPTLC method for estimation of pirfenidone and characterization of degradation product by using mass spectroscopy

Pirfenidone is used as a novel antifibrotic agent approved for mild-to-moderate idiopathic pulmonary fibrosis. An extensive literature search revealed that, method validation by high-performance thin-layer chromatography (HPTLC) and structural determination by tandem mass spectrometry (MS/MS) method was not reported till date. Precoated silica gels plates were used as a stationary phase. Methanol: ethyl acetate: toluene (1:2:7 v/v) was delivered best separation at 315 nm (Rf 0.49±0.03) by densitometry analysis. Degradation analysis was performed as per ICH guidelines Q2 (R1). Isolation of degradation product was done by the HPTLC method and characterized by using MS/MS method. All the validation parameters were found within the range. Moreover, its possible degradation pathway was also proposed. The Proposed developed and validated HPTLC method was found to be more sensitive, simple, precise, accurate, cost-effective and robust. This method could be applied for the analysis of bulk drug and tablet formulation, degradation study. This degradation pathway of the drug will further help to identify the degradation products of Pirfenidone which may be used for the impurity profiling of the drug.

Chemotaxonomy of the ethnic antidote Aristolochia indica for aristolochic acid content: Implications of anti-phospholipase activity and genotoxicity study

Ethnopharmacological relevanceAristolochia indica L. (Aristolochiaceae) is a common medicinal plant described in many traditional medicine as well as in Ayurveda used against snakebites. Besides, the plant has also been reported traditionally against fever, rheumatic arthritis, madness, liver ailments, dyspepsia, oedema, leishmaniasis, leprosy, dysmenorrhoea, sexual diseases etc. The plant is known to contain its major bioactive constituent aristolochic acid (AA) known for its anti-snake venom, abortifacient, antimicrobial and antioxidant properties. Materials and methodsThis present work describes a validated, fast and reproducible high performance thin layer chromatography (HPTLC) method to estimate AA from the roots of 20 chemotypes of A. indica procured from 20 diverse geographical locations from the state of West Bengal, India. Further, an evidence-based approach was adopted to investigate the reported anti-venom activity of the aqueous extracts of the A. indica roots by assessing its phospholipase A2 (PLA2) inhibitory properties since PLA2 is a major component of many snake-venoms. Finally, the cytotoxicity and genotoxicity of the aqueous root extract of the Purulia (AI 1) chemotype were assessed at various concentrations using Allium cepa root meristematic cells. ResultsThe highest amount of AA (7643.67 μg/g) was determined in the roots of A. indica chemotype collected from Purulia district followed by the chemotypes collected from Murshidabad, Jalpaiguri and Birbhum districts (7398.34, 7345.09 and 6809.97 μg/g respectively). This study not only determines AA in the plants to select pharmacologically elite chemotypes of A. indica, but it also identifies high AA producing A. indica for further domestication and propagation of the plants for pharmacological and industrial applications. The method was validated via analyzing inter-day and intra-day precision, repeatability, reproducibility, instrumental precision, limit of detection (LOD) and limit of quantification (LOQ) and specificity. Chemotypes with high AA content exhibited superior anti-PLA2 activity by selectively inhibiting human-group PLA2. Moreover, A. indica root extract significantly inhibited mitosis in Allium cepa root tips as a potent clastogen. ConclusionsThe present quick, reproducible and validated HPTLC method provides an easy tool to determine AA in natural A. indica plant populations as well as in food and dietary supplements, a potential antivenin at one hand and a possible cause of aristolochic acid nephropathy (AAN) at another. Besides, the cytotoxic and mitotoxic properties of the root extracts should be used with caution especially for oral administration.

Failure Mode Critical Effect Analysis and Design of Experiment-Based Robust Chromatographic Method for Simultaneous Estimation Lornoxicam and Eperisone Hydrochloride.

Failure mode critical effect analysis and design of experiment-based high performance thin layer chromatography (HPTLC) method has been developed for simultaneous estimation of lornoxicam (LOC) and eperisone hydrochloride (EPR). Failure modes were identified on the basis of prior knowledge and experimental data with the help of Ishikawa diagram for the development of method. The criticality of failure mode was assessed by giving risk priority number and criticality rank on the basis of preliminary experimental trials. The identified critical failure modes were analyzed for their effect by design of experiment (DoE)-based Plackett-Burman screening design. From 11 critical factors, the volume of methanol and modifier in mobile phase composition were found as critical failure modes. Critical failure mode was further analyzed by DoE based on central composite design for study of their relationship with resolution of both drugs. Quadratic model suggested by design was further used for failure mode risk control and navigation of design space for a resolution of both drugs more than 1.5. Failure mode risk control strategy was set for robust HPTLC method for simultaneous estimation of both drugs in laboratory mixture. Developed and validated HPTLC method was applied for assay of LOC and EPR in their laboratory mixture and assay values were found in good agreement with a spiked amount of drugs.

HPTLC method for the simultaneous determination of six bioactive terpenoids in Putranjiva roxburghii Wall.

High-throughput and sensitive determination of triterpenoids is challenging by high-performance liquid chromatographic methods due to their non-chromophoric nature, lipophilic properties, and sequential run. The present study demonstrates the development of a validated high-performance thin-layer chromatography (HPTLC) method for the simultaneous determination of bioactive terpenoids in the bark of Putranjiva roxburghii with uncertainty measurement to fulfill the technical requirement of ISO 17025:2017. Optimum separation of friedelin (1), methylputranjate (2), putrone (3), roxburghonic acid (4), putranjivadione (5), and roxburghonol (6) was achieved on silica gel 60 F254 plates using n-hexane–ethyl acetate (90:10, v/v). The sensitive estimation of targeted terpenoids was achieved by screening 15 different post-chromatographic derivatization reagents. The method was validated as per the ICH guidelines. Additionally, an uncertainty measurement for reliable and accurate determination was established following EURACHEM/CITAC guide CG-4 statistical procedure. The expanded uncertainty of terpenoids (1–6) measurement in P. roxburghii bark was in the range of 0.44–2.17%, which confirmed that at a confidence level of 95%, the unknown true value was within ± 5% range of the measured value. The compound methylputranjate showed the minimum uncertainty (0.4424), while putranjivadione showed the maximum uncertainty (2.1722). This is the first validated HPTLC method with new derivatization reagent providing the simultaneous, sensitive, and high-throughput analysis of six bioactive triterpenes in the bark of P. roxburghii with defined traceability and accuracy profile.

A validated HPTLC method for the quantitative determination of duloxetine hydrochloride and 1-naphthol in bulk and pharmaceutical formulation

Duloxetine hydrochloride is an anti-depressant drug, while 1-naphthol is one of its hepatotoxic impurities. So, there was an urgent need to do simultaneous analysis for both the drug and its toxic impurity. Therefore, a validated high-performance thin-layer chromatography (HPTLC) method was established for the simultaneous separation and quantification of duloxetine hydrochloride and 1-naphthol in their binary mixture and pharmaceutical formulation. The utilized developing system consisted of a mixture of chloroform–ethyl acetate–benzene–33% ammonia solution (6:3:1:0.1, v/v). Densitometric scanning was used to detect the separated peaks at 233 nm using an ultraviolet detector. The validation of the estimated method was performed according to the International Conference on Harmonization guidelines. The results indicated that it can be used for the quality control analysis of the proposed mixture. The developed method showed advantages over the previously published TLC methods of having higher sensitivity and allowing the determination of duloxetine hydrochloride with and in the presence of its toxic impurity 1-naphthol.

Quantitative determination of canagliflozin in human plasma samples using a validated HPTLC method and its application to a pharmacokinetic study in rats.

Canagliflozin (CNZ) is the first sodium-glucose co-transporter-2 inhibitor approved for treatment of type 2 diabetes mellitus. In the proposed work, a sensitive, rapid and validated high-performance thin-layer chromatography (HPTLC) method was established for the estimation of CNZ in human plasma for the first time. HPTLC analysis of CNZ and internal standard (sildenafil) was performed on glass coated silica gel 60 F254 HPTLC plates using a binary mixture of chloroform-methanol 9:1 (%, v/v) as the mobile phase. Densitometric detection was done at 295 nm. Retardation factor values were obtained as 0.22 and 0.52 for the CNZ and the IS, respectively. The linearity range of CNZ was obtained as 200-3,200 ng/ml. A simple protein precipitation method was used for the extraction of analyte from plasma using methanol. The proposed HPTLC technique was validated for linearity, accuracy, precision and robustness. The proposed HPTLC technique was successfully utilized for the assessment of pharmacokinetic profile of CNZ in rats after oral administration. After oral administration, the peak plasma concentration of CNZ was obtained as 1458.01 ng/ml in 2 h. The proposed HPTLC method could be applied to the study of the pharmacokinetic profile of pharmaceutical formulations containing CNZ.

A validated HPTLC method for quantitative analysis of morin in Maclura cochinchinensis heartwood

ObjectiveThe study was conducted to develop and validate a high performance thin-layer chromatography (HPTLC)-densitometric method for the quantitative analysis of morin in Maclura cochinchinensis heartwood collected from different locations in Thailand. MethodsHPTLC analysis was performed on an aluminium sheet of silica gel 60 F254 using toluene: ethyl acetate: formic acid (36:12:7, volume percent) as a mobile phase. The densitometric scanning was performed at the wavelength 410 nm. HPTLC method was validated according to ICH guideline. ResultsThe proposed HPTLC method showed acceptable validation parameters. The content of morin in M. cochinchinensis heartwood collected from eight different provinces in Thailand were in the ranges of 1.53%−2.73%. ConclusionThe simple and sensitive HPTLC method was successfully developed and validated for determination of morin in M. cochinchinensis heartwood. The proposed HPTLC method was found to be simple, fast and inexpensive, and can be used for the routine quality control of raw materials.

Response surface methodology-based optimization of ultrasound-assisted extraction of β-sitosterol and lupeol from astragalus atropilosus (roots) and validation by HPTLC method

Objective: To optimize the ultrasonication method for efficient extraction of P-sitosterol and lupeol from the roots of Astragalus atropilosus using Box-Behnken design of response surface methodology (RSM), and its validation by high performance thin layer chromatography (HPTLC) method. Methods: Ultrasonication method was used to extract β-sitosterol and lupeol from Astragalus atropilosus (roots). RSM was used to optimize the different extraction parameters viz. liquid to solid ratio (10-14 mL/g), temperature (60-80 °C) and time (40-60 min) to maximize the yield of β-sitosterol and lupeol. The quantitative estimation of β-sitosterol and lupeol was done in chloroform extract of Astragalus atropilosus by validated HPTLC method on 10 cm × 20 cm glass-backed silica gel 60F254 plate using hexane and ethyl acetate (8:2, v/v) as mobile phase. Results: A quadratic polynomial model was found to be most appropriate with regard to R1 (yield of total extraction; R2/% CV = 0.994 8/0.28), R2 (β-sitosterol yield; R2/% CV = 0.992 3/0.39) and R3 (lupeol yield; R2/% CV = 0.994 2/0.97). The values of adjusted R2/predicted R2/signal to noise ratio for R1, R2, and R 3 were 0.978 2/0.955 1/48.77, 0.990 4/0.911 0/31.33, and 0.992 7/0.940 1/36.08, respectively, indicating a high degree of correlation and adequate signal. The linear correlation plot between the predicted and experimental values for R1, R2, and R3 showed high values of R2 ranging from 0.990 5-0.997 3. β-sitosterol and lupeol in chloroform extract of Astragalus atropilosus were detected at Rf values of 0.22 and 0.34, respectively, at X max = 518 nm. The optimized ultrasonic extraction produced 8.462% w/w of Rl, 0.451% w/w of R2 and 0.172% w/w of R3 at 13.5 mL/g liquid to solid ratio, 78 C of temperature and 60 min of time. Conclusions: The experimental findings of RSM optimized extraction and HPTLC analysis can be further applied for the efficient extraction of β-sitosterol and lupeol in other species of Astragalus.

Development of high-performance thin layer chromatography method for identification of phenolic compounds and quantification of rosmarinic acid content in some species of the Lamiaceae family.

ABSTRACTAim:Representatives of Nepetoideae Burnett subfamily are promising species of the Lamiaceae Martinov family because of accumulating such valuable groups of secondary metabolites as terpenoids and polyphenols. A high-performance thin layer chromatography (HPTLC) fingerprint method for the qualitative determination of phenolic compounds and for the quantification of rosmarinic acid (RA) content in methanol extracts of five species of this subfamily was developed for the first time.Materials and Methods:Dried aerial parts of Dracocephalum moldavica L., Ocimum americanum L., Lophanthus anisatus (Nutt.) Benth., Monarda fistulosa L., and Satureja hortensis L. collected in flowering period were macerated with methanol. The HPTLC analysis was conducted using the CAMAG analytical system (Muttenz, Switzerland). The comparative analysis of RA contents was performed by HPTLC densitometric detection at λ = 366nm.Results:Identification of polyphenols in the investigated herbs was performed by comparison of a color and Rf of the chromatographic zones with six reference standards: rutin, apigenin, luteolin, caffeic acid, chlorogenic acid, and RA. HPTLC method was also validated for the quantification of RA in the extracts of investigated herbs. RA contents decreased in such a sequence: D. moldavica (24.83 ± 0.78mg/g) > M. fistulosa (20.32 ± 0.64mg/g) > O. americanum (19.59 ± 0.61mg/g) > S. hortensis (18.77 ± 0.52mg/g) > L. anisatus (12.61 ± 0.43mg/g).Conclusion:Obtained data can facilitate the differentiation of investigated species using the chromatographic fingerprints of their phenolic compounds. Developed and validated HPTLC method provides an approach to estimate RA content as a common marker of investigated herbs.

Pharmacognostical specification and validated high-performance thin-layer chromatographic method for the estimation of quercetin in Phoenix sylvestris root

The present study was aimed to assess the scientific appraisal of Phoenix sylvestris in the course of pharmacognostical properties and phytochemical parameters, as this has not been done yet. Pharmacognostic study mainly covered the macroscopic and microscopic features of the roots including powder microscopy and revealed the presence of pitted and spiral xylem vessels, lignified xylem fiber, and brown-colored stone cells. Physicochemical constants such as ash value, extractive value, and fluorescence analysis were assessed in the preliminary physicochemical screening. Qualitative analysis revealed the existence of certain chemical constituents such as flavonoids, tannins, steroids, and phenolic compounds. The crude extract of the root was subjected to high-performance thin-layer chromatography (HPTLC) for the separation of components. HPTLC analysis was carried out on the methanolic extract of the root by using toluene—ethyl acetate—formic acid acid (5:4:1, v/v) as the mobile phase at 254-nm detection to quantify the amount of quercetin. The calibration range was 50–250 ng per band (r2 = 0.994). The method was accurate in triplicate results at diff erent standard addition levels with average recovery of 99.40% for quercetin. Limits of detection and quantification were calculated as 20.94 ng per band and 43.90 ng per band, respectively. The quantity of quercetin in the methanolic extract was found to be as 3.13 mg g−1. The developed and validated HPTLC method can be used as a tool for standardization of roots in diff erent formulations using quercetin as the marker.

Effect of different extraction techniques on total phenolic and flavonoid contents, and antioxidant activity of betelvine and quantification of its phenolic constituents by validated HPTLC method.

The objectiveof the study is to analyze the effect of different extracting methods on the polyphenolic content and antioxidant activities in Piper betle leaves. In the present research, P. betle leaf extract was prepared by sonication, Soxhlet and maceration methods using acetone (100%, v/v). The efficiency of the extraction methods was estimated by quantifying the total phenolic content (TPC) by the Folin-Ciocalteu method and total flavonoid content (TFC) by AlCl3 colorometric methods, and antioxidant power of the various extracts was determined by DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical scavenging assay. Thin-layer chromatography (TLC) bioautography was carried out to identify antioxidants, and their amount was determined by the newly developed high-performance thin-layer chromatography (HPTLC) method. DPPH free radical scavenging capacity of the different extracts from strongest to weakest was as follows: ascorbic acid (4.27µg/mL) > sonication (5.35µg/mL) >, maceration (5.53µg/mL)>, soxhlet extraction (5.83µg/mL). Same trend was also observed for the ABTS radical scavenging capacity. Similarly, findings of this study also showed that sonication extract possessed highest phenolic and flavonoid contents followed by maceration and Soxhlet extraction. In addition, important bioactive phenolic constituents which contribute largely towards antioxidant potential such as eugenol and eugenol acetate were quantified using HPTLC (high-performance thin-layer chromatography) method. The average percent recovery of eugenol and eugenol acetate was found to be 97.28% and 98.04%, respectively. The LOD (limit of detection) and LOQ (limit of quantification) for eugenol were 5 and 15ng/spot, whereas that of eugenol acetate were 10 and 30ng/spot. The HPTLC densitometric determination also supported the results of antioxidant assays by revealing the presence of higher amount of identified antioxidants in sonication followed by maceration and Soxhlet extraction. The developed HPTLC chromatogram profile may be used as a reference for the standardization of P. betle leaf extracts.

Evaluation of diosgenin content in Costus speciosus germplasm collected from Eastern Ghats of India and identification of elite chemotypes

Background: Costus speciosus known as insulin plant for its anti-diabetic potential. It has commercial significance due to the presence of industrially viable metabolite diosgenin. Objective: The identification of elite chemotypes of C. speciosus (Rhizome) through high-performance thin-layer chromatography (HPTLC) from the Eastern Ghats (India). Materials and Methods:A validated HPTLC method for the quantification of diosgenin was developed in accordance with the International Conference on Harmonization Guidelines. Results: In total, 11 populations of species were collected from their natural habitat with all the Global Positioning System (GPS) coordinates. The method was developed on HPTLC pre-coated silica gel 60 F254plates under a binary solvent system of n-hexane and ethyl acetate (7:2 v/v). The linearity was established at concentration range of 0.1–0.9 μg/spot having regression equation, 0.010× + 0.002. The limit of detection and limit of quantification were 0.907 and 2.751 with a regression coefficient of 0.999. The diosgenin content varies significantly (P

Quantification of the secondary metabolites by HPTLC, analgesic and antipyretic activity evaluation of Ficus racemosa L. leaves

There have been no reports of simultaneous quantification of kaempferol and quercetin in Ficus racemosa L. Objective of the present work included to perform extraction using petroleum ether (PEE) and ethanol (EE), to perform preliminary phytochemical test, quantitative estimation of phytochemicals and simultaneous estimation of kaempferol and quercetin in Ficus racemosa L. using high performance thin layer chromatography (HPTLC) method and to assess the analgesic and antipyretic activity of the extracts. PEE and EE obtained from cold maceration technique. Extracts were subjected to qualitative test and estimated quantitatively. Extracts were applied on silica gel G 60 F254 plate; solvent-Toluene: Chloroform: Acetone 4.5:4.5:1 (v/v) and scanned at 254 nm. The extracts were also subjected to analgesic activity (hot plate method and tail immersion method) and antipyretic (Brewer’s yeast induced pyrexia model and Lipopolysaccharide induced pyrexia mode) activity. Findings of preliminary phytochemical test and quantitative estimation of phytochemicals confirms the presence of flavonoids in both the extracts (PEE and EE). The amount of flavonoids in PEE and EE were found to be 24.58 ± 0.60 mg/100 g and 21.01 ± 0.58 mg/100 g respectively. HPTLC system resulted well resolved bands for quercetin (Rf 0.38) and kaempferol (Rf 0.55). The validated HPTLC method was found suitable for kaempferol and quercetin quantification in Ficus racemosa L. Hot plate reaction time response was improved in a dose-dependent manner in a group of rats treated with PEE suggesting its analgesic potential. In view of analgesic potential by tail immersion method, PEE (400 mg/kg b.w.) showed no significant difference in tail flick reaction time as shown by standard treated animal (Aspirin 100 mg/kg b.w.). The findings of antipyretic activity suggested that PEE (400 mg/kg b.w.) have no significant difference in treating pyrexia when compared with Paracetamol (100 mg/kg). PEE (400 mg/kg b.w.) was found to be most potent. It may be due to the presence of comparatively high amount of flavonoids in PEE confirmed by the preliminary phytochemical test, quantitative estimation of phytochemicals and HPTLC quantification the PEE was found to be more effective when compared to EE.

Bioprofiling for the quality control of Egyptian propolis using an integrated NIR-HPTLC-image analysis strategy

Quality control of propolis being a complex mixture of compounds that are very difficult to analyze and standardize is certainly challenging. Shown on the example of 35 Egyptian propolis samples, a strategy for an improved quality control was demonstrated in which efficacy-directed fingerprint analysis of high-performance thin-layer chromatography (HPTLC) fingerprints were evaluated in combination with Near Infra-Red spectrometry (NIRS) to realize precise discrimination and high quality assessment of chemical and effective consistency of propolis samples that are collected from various geographical locations in Egypt. The fingerprints obtained after derivatization and fluorescence detection (FLD) at λ 366 nm were analyzed by using multivariate data analysis and data were used to identify nine marker ingredients of the different propolis samples. These markers were then quantified by a new validated HPTLC method and an attempt to classify the samples by using a targeted approach was implemented. All investigated propolis samples were initially divided into two types in the untargeted and targeted HPTLC image multivariate analysis. The antimicrobial activities of propolis samples were then evaluated and the results showed that there was significant variation in the biological activities of the different samples. The quantitative data of marker compounds as well as the samples bioactivity results were then subjected to partial least squares regression (PLS-R) analysis which revealed that chrysin, galangin‑5‑O‑methylether, pinostrobin, and islapinin were the main bioactive markers. Based on the efficacy-associated marker ingredients, pretreated spectral patterns of NIRS were selected as a complementary evaluation technique directed to the comprehensive efficacy-directed discrimination of the different Egyptian propolis samples which could successfully classify the propolis samples tested into three different types. Hierarchical cluster analysis and orthogonal projection to latent structures discriminant analysis (OPLS-DA) were applied to the quantitative results from the efficacy-associated markers only. From this systematic method, the 35 samples were subsequently divided into three types; orange, green and blue, displaying a more intricate division than OPLS-DA which is based only on the untargeted HPTLC analysis or the full NIR spectral data. This study initiated the research on the Egyptian propolis and confirmed the existence of three different types of Egyptian propolis, the blue, green and orange types. The study also emphasizes how it is critical to implement the fingerprint- efficacy relationship analysis in finding out the main health-relevant biomarkers for the quality assessment especially for samples displaying delicate differences in their chemical composition and bioactivity.