HPTLC

Riluzole is used in the treatment for amyotrophic lateral sclerosis (ALS). The present work describes simple, precise and accurate HPTLC method for the determination of riluzole in bulk and its dosage form. Quantification of riluzole was carried out with Aluminum packed silica gel 60 F 254 precoated TLC plates as a stationary phase using a mixture of mobile phase consists of hexane: ethyl acetate in the ratio 1:1 v/v and in the absorbance/ reflectance mode at 222 nm using a CAMAG TLC scanner 3 with winCATS software version 1.4.3. The Rf value of riluzole was found to be 0.34 (±0.02). The proposed method has permitted the quantification of riluzole over the linearity range of 200-1000 ng/spot and its percentage recovery was found to 99.17%. The intraday and interday precisions were found to be 1.26% and 1.40%, respectively. The limit of detection and the limit of quantification were found to be 18 ng/spot and 54 ng/spot respectively. The Coefficient of determination (r 2 ) was 0.9992...

Berberis aristata DC and Nigella sativa L. are officially listed in various Indian Pharmacopoeia and AYUSH official documents. Prescribed for different ailments for proven medicinal activities, they thus became part of polyherbal medications. With reverse pharmacology and scientific validation, more than 30 patents are filed on different formulations of B. aristata and granted. Nigella sativa L. has been broadly studied for its therapeutic potential and wide range of activities against cardiovascular, diabetic, cancer, and life style disorders. Thus, this study is aimed at standardizing B. aristata and N. sativa and their antineoplasia activity in 7, 12-dimethylbenz[a]anthracene (DMBA)-induced mouse models. Molecular docking was done using the Schrodinger program Maestro 9.0. Herbal extracts and essential oil (B. aristata and N. sativa) were standardized and quantified using high-performance thin-layer chromatography (HPTLC) (CAMAG) and gas chromatography-mass spectrometry (GCMS) (Agilent 2010GC System) with validated methods. DMBA was administered orally once a week (1mg/ 200 µL) to each animal except the normal control. Hematology, histopathology, and immunoassays were performed, and data were analyzed and depicted with GraphPad and SPSS. In molecular docking, thymoquinone showed the highest docking score (9.519, 9.211, and 9.042, respectively) in the active site pockets of IL6 (PDB ID: 4CNI and 5FCU), TNF (PDB ID: 2AZ5), and VEGF (PDB ID: 4KZN). Out of all four target sites, thymoquinone and berberine showed good binding affinity with IL6 (PDB ID: 4CNI) compared to αand β-pinenes. HPTLC analysis of the hydroalcoholic extract showed the presence of berberine both qualitatively and quantitatively (5.4% berberine), and thymoquinone detected 0.17% in the N. sativa extract. GCMS for essential oil showed 26 compounds including ±pinene. Leukocytes and erythrocytes of N. sativa and B. aristata were analyzed, and significant improvements were recorded (P < 0.05) and graphically presented. Mean survival time was calculated by the Kaplan Meier method (119 days). Immunoassay analyses were conducted, namely, TNF-α and VEGF, and interpreted and marked.

Acronychia pedunculata (L.) (Ankenda) under family RUTACEAE is widely used in Traditional medicine system. The present study was aimed at the development of quality control parameters for identification and authentication of A. pedunculata. Hence, standardization is important in maintaining the purity, quality, safety and efficacy of Ayurveda formulations. Phytochemical, physico-chemical parameters and HPTLC fingerprints of methanol extracts of leaves and stem barks were determined as tools for quality standards. Preliminary phytochemical analysis revealed the presence of carbohydrates, reducing sugars, tannins, saponins, alkaloids, flavonoids, anthranol glycosides, phenols, terpenoids and proteins in both leaf and stem bark extracts. Under physico-chemical parameters; total ash, acid insoluble ash, water soluble ash, loss on drying and extractability in methanol for A. pedunculata leaves were 40.38% w/w, 12.64% w/w, 34.42% w/w, 21.80% w/w and 17.84% w/w respectively. Total ash, acid insoluble ash, water soluble ash content, loss on drying and extractability in methanol for A. pedunculata stem barks were 20.42% w/w, 8.35% w/w, 25.56% w/w, 15.67% w/w and 13.42% w/w. HPTLC profile of A. pedunculata leaf extract showed 10 peaks (Rf values;-0.01, 0.11, 0.32, 0.39, 0.43, 0.57, 0.69, 0.77, 0.85, 0.88) whereas, stem bark extract also showed 10 peaks (Rf values;-0.06, 0.02, 0.06, 0.17, 0.34, 0.48, 0.61, 0.71, 0.82, 0.97) for solvent system; n-hexane: ethyl acetate: chloroform in 2: 6: 2 ratio. The above parameters can be considered as a preliminary tool in detection and contrast of raw materials excluding the counterfeit and substandard raw materials in Ayurveda manufacturing.

The rapid, accurate and sensitive HPTLC method was used for quantification of flavonoids in Syzygium cumini (Linn.), a medicinally important plant of family Myrtaceae. The present analysis was done using five different solvents, ethanol, methanol, ethyl acetate, chloroform and water. The leaf extraction was done using sonication method. HPTLC analysis of leaf extracts in S. cumini responded well with good quantified amount of targeted flavonoid compounds in all the studied solvents. The plant leaf extract of S. cumini revealed the presence of four types of flavonoids compounds, Quercetin, Rutin, Ellagic acid and Kaempferol. Out of the five solvent used, it was evident that ethanolic leaf extract indicates highest quantity of Quercetin (0.19394±0.05µg/mL), Rutin was move in aqueous extract (0.04731±0.05µg/mL) whereas ethyl acetate extract exhibited highest content for Ellagic acid (0.02492±0.03µg/mL) and Kaempferol (0.02521±0.05µg/mL). The HPTLC method provides a good resolution for flavonoid compound. Separation was performed on silica gel 60 F254 HPTLC plate with Ethyl acetate: Formic acid: Acetic acid: Water in the proportion of 10:0.5:0.5:1.3 (v/v) as a mobile phase. The antimicrobial activity was carried out using five different microorganisms, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, and Salmonella typhi. The sonicated ethanolic leaf extract indicated high level of antimicrobial activities against Candida albicans, E. coli and S. aureus.

Mentha  piperita  and  Citrus sinensis  was an aromatic plants well
known for its fragrance and distinct aroma.  In this prospective study
to evaluate the chromatogram detection of ethanol extract of  Mentha
piperita  and  Citrus sinensis  with standard flavonoid marker eriocitrin
by HPTLC technique.  HPTLC chromatogram was developed in
ethanolic extract of  Mentha  piperita, Citrus  sinensis  and standard
flavonoid marker eriocitrin by using Toluene-Ethyl acetate-Formic
acid-Methanol  (3:6:1.6:0.4) as mobile phase. The identified bands of
compounds 1-9 in the ethanolic extracts of  Mentha  piperita  and
Citrus sinensis  was confirmed under the TLC scanner with the
standard marker eriocitrin at 254nm.

Analysis of pharmaceutical and natural compounds and newer drugs is commonly used in all the stages of drug discovery and development process. HPTLC is an advanced type of planar chromatography used extensively in the recent years for fingerprinting of medicinal plants, products and for screening lichen substances. Major advantages include precise sample application, wide choice of stationary phases, simultaneous processing of standards, simultaneous scanning in different light sources and excellent documentation facility. It is more sensitive and possible to run more samples in a short period of time, by using small amount of solvent. It is one of the sophisticated instrumental techniques based on the full capabilities of thin layer chromatography. In the present review brief discussion is presented with regard to type of instrument used in HPTLC, its entire methodology and how this technique is better than TLC. This concise information consists of salient points showing immense use in the practical analysis of HPTLC. This article consists of compilation of 63 formulations in total, out of which 38 containing one active substances and 25 containing dual active substances are shown in separate Tables (Table 1 and 2). The names of products, solvent system, details of plates to be used and details of quantitation showing densitometric evaluation are shown against each formulation.

The present paper deals with development and standardization of HPTLC method used for quantification of vasicine and vasicinone in Adhatoda vasica commonly known as Vasaka, a plant of the family Acanthaceae. Today, vasaka is the most widely known herb and widely used in the treatment for a variety of respiratory ailments including cough, bronchitis and asthma. Both vasicine and vasicinone are two major alkaloids of A. vasica, which are known to possess biological activities like respiratory stimulant, bronchodilator and hypotensive activity. Hence, a need to develop and standardize a chromatographic method for quantification of vasicine and vasicinone, for better standardization of Adhatoda vasica. An attempt has been made to quantify vasicine and vasicinone in A. vasica raw herbs, extracts and formulations by HPTLC method. The lowest detectable limit of vasicine and vasicinone was found up to 1 ng and 25 ng respectively with good resolution and separation of vasicine and vasicinone from other constituents of A. vasica. Further, recovery values of vasicine and vasicinone were found to be about 95-102 %, which shows the reliability and suitability of the method. The validated HPTLC method was found to be simple, reproducible, accurate and precise. The structure of isolated vasicine and vasicinone was characterized and confirmed by various advanced spectroscopic methods.

A HPTLC method for the estimation of Nebivolol (NBV) and Hydrochlorothiazide (HCTZ) has been developed. It
employs aluminum backed silica gel 60 F254 TLC plates, (20 cm × 10 cm, layer thickness 0.2 mm) pre-washed with
methanol and mobile phase comprising of 1,4-dioxane: toluene: triethylamine (5:3:0.1 v/v). The developing solvent was
run up to 80 mm in Camag chamber previously saturated with 10.0 ml of solvent mixture for 30 min. Densitometric
scanning was then performed with Camag TLC Scanner-3 equipped with winCATS (version 1.3.0) at λ max 281 nm. The Rf
values were found to be 0.75 and 0.43 for Nebivolol and Hydrochlorothiazide respectively. The limit of detection and limit
of quantitation were found to be 43ng/spot & 130ng/spot for Nebivolol and 25ng/spot & 76ng/spot for
Hydrochlorothiazide respectively. The % RSD of intra-day variation and inter day variation were 0.54 and 0.41
respectively for NBV and 0.46 and 0.39 for HCTZ respectively. The proposed method can also be used for routine quality
control to accurately determine Nebivolol and Hydrochlorothiazide in bulk and tablet dosage form.

Nowadays, crimes related to forged documents are increasing. Any erasure, addition or modification in the document content always involves the use of writing instrument such as ballpoint pens. Hence, there is an evident need to develop a fast and accurate ink analysis protocol to solve this problem. This study is aimed to determine the discrimination power of high performance thin layer chromatography (HPTLC) technique for analyzing a set of blue ballpoint pen inks. Ink samples deposited on paper were extracted using methanol and separated via a solvent mixture of ethyl acetate, methanol and distilled water (70:
35: 30, v/v/v). In this method, the discrimination power of 89.40% was achieved, which confirm that the proposed method was able to differentiate a significant number of pen-pair samples. In addition, composition of blue pen inks was found to be homogeneous (RSD <2.5%) and the proposed method showed good repeatability and reproducibility (RSD <3.0%). As a conclusion, HPTLC is an effective tool to separate blue ballpoint pen inks.

Piperine, an alkaloid with diverse biological activity commonly occurring in fruits of Piper sp., has high commercial, economical, and medicinal value. In this study densitometric estimation of piperine from fruits of Piper longum L and Piper retrofractum Vahl.. It can be stated from this study that at industrial level P. longum Lin. is more suitable because of its preservation less and high percentage of Piperine(3.26µg/ml) than P.retrofractum Vahl.(0.49µg/ml) pharmacognostical and phytochemical characters of both species is discussed in present study.

Fruits of Embelia ribes Burn. F possesses antifertility activity and is widely used in herbal formulations related to suppress fertility. It is an important component of an ayurvedic contraceptive-Pippalivadi Vati. Medicinal properties of any plant are attributed to the presence of wide array of phytochemicals and minerals. Authentication and identification of plant material for checking adulteration and ensure quality control of plant material is most important problem faced today by herbal industry. In present study preliminary phytochemistry, elemental analysis and chemical fingerprint for fruits of Embelia ribes Burn. F was carried out to address these issues. Priliminary phytochemistry revealed presence of wide range of secondary metabolites like Alkaloids, glycosides, flavanoids, phenolics, sterols, steroids, triterpenoids, saponins and tannins. Elements from plants were analysed by ICP-AES technique. 10 elements viz. Copper, Cadmium, Iron, Zinc, Manganese, Sodium, Lead, Nickel, Calcium and Chromium were analysed. Elemental contents of the plant were found to be less than prescribed permissible limits of WHO. The HPTLC fingerprint effectively separates stable chemical constituents and helps us to identify and authenticate plant material. Chloroform: Ethyl acetate: Formic acid (5:4:1 v/v/v) was used as the mobile phase. Derivatisation was carried out with anisaldehyde sulphuric acid and was best observed at 366nm.

Background: Uraria picta Desv. is one of the ingredient of ten herbs formulation called Dashmoola. Rhoifolin is an active compound which has been isolated from the aerial parts of Uraria picta and has been established as marker compound for quality evaluation of this species. In the present time of global resurgence in traditional and alternative health care systems, the quality assurance of medicinal plants is very essential. Since, WHO and herbal pharmacopoeia are also emphasizing on quality standardization of medicinal plants in terms of active ingredients, the present study was aimed to assess the variation in rhoifolin content in aerial parts of Uraria picta collected from eight locations of Madhya Pradesh for its quality standardization and selection of superior chemotype. Methods: The powdered samples of aerial parts were extracted by soxhlet extraction in methanol  for  4  hours on water bath. Extracts were quantified for rhoifolin content using Ethyl acetate: Formic acid: Glacial acetic acid: Water (10: 1.1: 1.1: 2.6) mobile phase and a CAMAG HPTLC system equipped with Linomat V applicator, TLC scanner, WIN CATS software Version 1.4.8 and Hamilton Syringe (100 µL).  Results: Rhoifolin content varied from 0.039% to 0.195% among the aerial part samples of eight locations corresponding to seven agroclimatic regions of the state. The highest content was found in aerial parts of Pandado, Sehore (0.25%) followed by Kapoornala, Chhindwara (0.167%) and Pamakhedi, Khandwa (0.152%) respectively and lowest content in plant sample of  Navali, Mandsaur (0.039%). Conclusions: The study provided significant variation  in rhoifolin content in aerial parts of Uraria picta. The accession collected from Pandado, Sehore emerged out of best quality on the basis of rhoifolin content and superior chemotype too.

Gas chromatography mass spectroscopic investigation of methanolic extract of Pluchea lanceolata – a semi-arid land plant is investigated using GCMS-QP 2010 Plus while the mass spectra of the compounds found in the extract are matched with the standard library of NIST. Maximum % area are found for hexadecanoic acid (19.93 %) with Rt = 24.598 min. and Ethanol, 2-(vinyloxy)- (16.46%) with Rt = 6.974 min. for stem and callus respectively. A simple HPTLC method is used for the qualitative determination of daidzein. The samples are dissolved in methanol and development is carried out in Camag twin trough chamber saturated with mobile phase consisting of Toluene: Ethyl acetate: Acetone: Formic acid (20:4:2:1v/v/v/v). Spectrodensitometric scanning is performed by TLC scanner III (CAMAG) in absorbance mode. The system are found to give compact spot for daidzein (Rf = 0.21 ± 0.03). The data of the results of stem and callus of Pluchea lancolata revealed that the samples also contain daidzein with maximum Rf = 0.20 & 0.21 respectively which is a potent bone healer compound. Data obtained could be useful in the identification and authentication of traditional drug for the international acceptance.

Background: Urtica dioica L. is a common Himalayan species which produces allergenic substances causing oedema and inflammation in humans. It has become a source of folk medicine for the treatment of many diseases. The leaves and roots both are used internally as a blood purifier and diuretic and an infusion of the plant is used for nasal and men-strual haemorrhage, diabetes, rheumatism, eczema, anaemia, hair loss, as an expectorant and antidiarrhoeal. Present study includes pharmacognostic evaluation, antioxidant activity and HPTLC analysis of Urtica dioica L. Methods: Pharmacognostic evaluation of aerial part of U. dioica has been performed as per Indian pharmacopoeia. In-vitro anti-oxidant evaluation of U. dioica has been performed using DPPH free radical scavenging activity. Ferulic acid, a poten-tial phenolic antioxidant present in this species, has been studied through HPTLC. Results: U. dioica hydro-alcoholic extract shows positive results for antioxidant activity with IC50 value of 88.33 ± 2.88 μg/ml. Standard ascorbic acid showed IC50 value of 2.8 ± 0.62 μg/ml. Ferulic acid was identified at Rf 0.61 ± 0.01 and quantified to 0.73% in this spe-cies through CAMAG HPTLC analysis. Conclusion: The pharmacognostical parameters reported can be considered as quality standards of U. dioica in herbal industry. Hydro-alcoholic extract of U. dioica showed positive in vitro antioxi-dant activity. Presence of phenolic compound suggests that antioxidant activity may be due to ferulic acid content.
Keywords: Antioxidant Activity; DPPH; Ferulic Acid; HPTLC; U. Dioica

A high-performance thin-layer chromatography (HPTLC) method for the simultaneous quantitative determination of gallic acid, van-illic 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, van-illic 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, specific , 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.

The study was carried out to develop a validated HPTLC method based on ICH guidelines for the quantification of embelin from medicinally important plant Embelia ribes Burm.F. Methanolic extract of the dried fruit of the plant was sonicated and used for the analysis. CAMAG Linomat 5 and CAMAG TLC scanner was used for HPTLC studies. Chromatography was performed on aluminium-backed silica gel 60F254 E. MERCK KGaA HPTLC plates of 250 μm thickness were used. Chloroform: ethyl acetate: formic acid (5:4:1 v/v/v) was used as the mobile phase and derivatization was carried out with anisaldehyde sulphuric acid. The method was developed and validated for specificity, linearity, range, accuracy and precision. Linearity was established in the range of 0.1 to 0.5 µg when plotted against their respective peak area. LOD and LOQ values were found to be 0.46 µg/mL and 1.39 µg/mL respectively. The External Standard Method and linearity graph was used for quantification analysis. The concentration of embelin in dried fruits of the plant is found to be 1.657µg/mg or 0.1657%. The statistical analysis conclusively establishes statistical fitness of the method. It is a specific, sensitive and reproducible HPTLC method for the quantification of embelin from Embelia ribes Burm.F.

Siddha system of medicine is one of the oldest Indian system of medicine known to the mankind. Several indigenous siddha formulations formulated and hypothesized by ancient siddha practitioners are still used for clinical management of several dreadful metabolic disorders in humans like diabetes mellitus. Global market for siddha formulations seems remarkable but the need of the hour is to ensure the standard of the preparation. Starting from raw material to finished product the quality and genuinity of the product as to be ascertained. Considering the global need the modern standardization method adopted for identity, purity and shelf life of the preparation. Different techniques has been followed to analyze the purity of the raw materials it includes microscopic, macroscopic, Physical, chemical and biological method of analysis. Hence the main aim of the present investigation is to standardize the novel formulation Pungampoo Chooranam (PPC) as per AYUSH guideline and to reveal the property of the formulation to the scientific community for better understanding about the standards of the formulation. The results obtained from the physicochemical evaluation revels that the total ash value of PPCwas found to 8.01 %. In which the water soluble ash was 4.75% and acid insoluble ash was 0.69%. Similarly loss on drying value at 105 o C was fond to be 9.24% respectively. The water soluble extractive value of PPC was found to be 26.46 % whereas alcohol soluble extractive value is 29.35 %. n-hexane soluble extractive value of the test drug PPC was found to be 19.26%. Preliminary TLC analysis of the sample PPC emits fluorescence indicates the presence of fluorescent emitting compound. The results of HPTLC analysis of the sample PPC reveals the presence of 14 prominent peaks corresponds to 14 different compound's with Rf value ranging from 0.01 to 0.93 with percentage area of 0.82 to 18.52%. From the results of the present investigation it was concluded that the formulation PPC is highly stable and also possess biologically significant phytocomponents which is may be used as an ailment for treating various disease

Chemical diversity among the plant species provides great opportunity for identification of new and elite sources for further bio-prospection and also plays an important role in plant conservation. Centella asiatica (L.) Urban (Apiaceae) is claimed to possess various healing effects and antioxidant
properties. However, there has been very less focus on investigation of chemotypic variations of C. asiatica found in different geographical zones of the country. In order to conserve elite accessions of C. asiatica as it is an industrially valuable herb and over exploited from wild, thus, different
distinct accessions of C. asiatica from central zones of India were compared in relation to the levels of triterpenoid saponins. Physico-chemical parameters were evaluated in all the accessions. The metabolites investigated include
madecassoside, asiaticoside and its sapogenin, asiatic acid. The three triterpenoids of interest were analyzed and quantified by HPTLC. A comparison and evaluation of the triterpenoid content in these accessions are reported. The highest concentration of asiaticoside and madecassoside
was found in the CA-15 accession that was asiaticoside (1.35 per cent), madecassoside (2.15 per cent) and asiatic acid was found higher in CA-17 (0.29 per cent). It can be concluded that the geographical conditions (soil type and altitude) of CA-15 and CA-17 are comparatively favourable for 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 central zones of India in comparison to other geographical areas, and
lays a foundation for the conservation of this plant from over exploitation.
Keywords: Asiatic acid, Asiaticoside, Madecassoside, Chemotypes, Centella asiatica, HPTLC, Conservation