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1.  Standardization and quality control parameters of Dashanga Kwatha ghana tablet: An Ayurvedic formulation 
Herbal medicines have a long therapeutic history and are still serving many of the health needs of a large population of the world. However, the quality control and quality assurance still remains a challenge because of the high variability of chemical components involved. Herbal drugs, singularly and in combinations, contain numerous compounds in complex matrices in which no single active constituent is responsible for the overall efficacy. This creates a challenge in establishing quality control standards and standardization of finished herbal drugs. Many preparations have been mentioned in Ayurvedic text books for the treatment of Urdhwaga Amlapitta (non-ulcer dyspepsia). Dashanga Kwatha is one such known formulation. In this study, Dashanga Kwatha was converted into tablet form to increase the shelf life, make it easy to dispense, for dose fixation, etc. The Dashanga Kwatha Ghana tablet was subjected to organoleptic analysis, phytochemical analysis, and qualitative analysis to detect the presence of various functional groups, and to high performance thin layer chromatography (HPTLC) examination by optimizing the solvent systems. The investigation revealed the presence of tannins, mucilage, ascorbic acid, alkaloids, saponins, glycosides, flavonoids and carbohydrates mainly.
PMCID: PMC3157108  PMID: 21897642
Dashanga Kwatha; decoction; quality control; standardization; tablet
2.  Pharmacognostical and analytical study of Tulsi-Amla-Yasti Ghrita 
Ayu  2012;33(2):274-278.
Tulasi Amla Yashti Ghrita is an Ayurvedic formulation, which is beneficial in the management of the side effects of Head and Neck Malignancies induced by Radiotherapy and Chemotherapy. A pharmacognostical study involving both the macroscopic and powder microscopy of raw drugs of Tulasi Amla Yashti Ghrita and a physicochemical analysis of the finished product were carried out, to evaluate the quality of the formulation. The specific gravity of the formulation was 0.9130 and pH was 3.5. Thin layer chromatography (TLC) and high performance thin layer chromatography (HPTLC) were carried out after organizing the appropriate solvent system, in which five spots were distinguished in TLC and nine spots in HPTLC. Most of the Rf values for the spots observed were identical. The observations could be considered to be the reference standards in future studies.
PMCID: PMC3611632  PMID: 23559803
Chemotherapy; high performance thin layer chromatography; radiotherapy; Tulasi Amla Yashti Ghrita
3.  Pharmacognostic Standardization, Physico- and Phytochemical Evaluation of Amaranthus Spinosus Linn. Root 
Amaranthus spinosus Linn. (Amaranthaceae) is found throughout India. This tree species has been of interest to researchers because it is a medicinal plant employed in the Indian traditional system of medicine. Pharmacognostic standardization; physico-and phytochemical evaluation of the roots of Amaranthus spinosus was carried out, to determine its macro-and microscopical characters, and also some of its quantitative standards. Microscopical studies were done by using the trinocular microscope. Total ash, water-soluble ash, acid-insoluble ash, sulfated ash values, and alcohol-and water-soluble extractive values were determined for physico-chemical evaluations. A preliminary phytochemical screening was also done to detect different phytoconstituents. Microscopically, the root showed cork, cortex, stellar region, and calcium oxalate crystals. Powder microscopy showed anamalous secondary growth in between the xylem vessels and Calcium Oxalate crystals in the cortex region. Total ash was approximately three times more than acid insoluble and water soluble ash. The ethanol soluble extractive was approximately the same as the water soluble extractive. Thin Layer Chromatography (TLC) of the Petroleum-ether extract using Benzene : Ethyl acetate (6 : 1), showed six spots. In the chloroform extract, using Benzene : Ethyl acetate (4 : 1) nine spots were seen, and in the ethanol extract, using Chloroform: Methanol (93 : 7), only four spots were observed, using Iodine vapor as a viewing medium. Phytochemically, the root exhibited terpenes, alkaloids, glycosides, and sugars. These findings might be useful to supplement information with regard to its identification parameters, which are assumed significant in the way of acceptability of herbal drugs, in the present scenario, which lacks regulatory laws to control the quality of herbal drugs.
PMCID: PMC3159276  PMID: 21897662
Amaranthus spinosus Linn.; pharmacognostic standardization; physicochemical evaluations
4.  Pharmacognostical and Preliminary physicochemical evaluation of Triphaladi granules – A polyherbal Ayurvedic formulation 
Ayu  2013;34(3):288-293.
Triphaladi Kwatha, a polyherbal Ayurvedic formulation, is recommended by Chakradatta and Yogaratnakara in the management of Prameha which has resemblance with type 2 diabetes mellitus. The present study deals with development of pharmacognostical and preliminary pharmaceutical profile of Triphaladi granules. The pH (5% aqueous extract) was 6.0, water-soluble extract 48.66% w/w, alcohol-soluble extract 33.91% w/w, ash value 5.97% w/w, and loss on drying at 105°C was 6.53% w/w. High performance thin layer chromatography were carried out after organizing appropriate solvent system in which maximum nine spots were distinguished and few of the Rf values were identical in the alcoholic extract.
PMCID: PMC3902596  PMID: 24501525
High performance thin layer chromatography; Pharmacognosy; Prameha; Triphaladi granules
5.  Pharmacognostical and phytochemical studies of Curcuma neilgherrensis (Wight) leaf - A folklore medicine 
Ayu  2012;33(2):284-288.
Curcuma neilgherrensis Wight is a folk medicinal plant used in the management of diabetes mellitus. The leaves of this herb are said to be successful in managing high blood glucose levels. This study is aimed at assessing the scientific appraisal of C. neilgherrensis in the course of pharmacognostical characters and phytochemical parameters, as these are not yet been done. Pharmacognostic study mainly covered the macroscopic and microscopic features of the leaves including powder microscopy, and revealed the presence of trichomes, spiral vessels etc. Phytochemical parameters such as pH, total ash value, water-soluble extract and MeOH extract values were assessed in the preliminary physicochemical screening. Qualitative analysis revealed the existence of certain chemical constituents such as flavonoids, tannins, organic acids and saponin glycosides. The crude extract of leaves was subjected to TLC and HPTLC for the separation of components.
PMCID: PMC3611638  PMID: 23559805
Curcuma neilgherrensis; HPTLC; pharmacognosy; phytochemistry; TLC
6.  Analytical profile of Brahmi Ghrita: A polyherbal Ayurvedic formulation 
Ayu  2012;33(2):289-293.
Brahmi Ghrita, a polyherbal Ayurvedic formulation is recommended in the management of various psychological disorders like Unmada, Apasmara and Graharogas. The present study deals with the pharmacognostical identification of ingredients of Brahmi Ghrita and its physico-chemical analysis. Pharmacognostical study containing both macroscopic and powder microscopy of raw drug revealed the quality and genuineness of all the constituents of Brahmi Ghrita. Organoleptic features of coarse powder made out of the crude drugs were within the standards prescribed. Acid value was 0.16075, saponification value 184.17, Refractive Index value 1.467 at room temperature, Iodine value 26.715, Specific gravity at room temperature was 0.9133. HPTLC was carried out after organizing appropriate solvent system in which maximum 9 spots were distinguished and most of the Rf values were identical in alcoholic extract which shows the presence of certain definite constituents in Brahmi Ghrita.
PMCID: PMC3611637  PMID: 23559806
Brahmi Ghrita; HPTLC; pharmacognosy; physico-chemical analysis
7.  Anti-inflammatory activity and qualitative analysis of different extracts of Maytenus obscura (A. Rich.) Cuf. by high performance thin layer chromatography method 
To perform aqueous ethanol soluble fraction (AESF) and dichloromethane extract of aerial parts of Maytenus obscura (A. Rich.) Cuf. using high performance thin layer chromatography (HPTLC) and to test anti-inflammatory activity of these extracts.
HPTLC studies were carried out using CAMAG HPTLC system equipped with Linomat IV applicator, TLC scanner 3, Reprostar 3, CAMAG ADC 2 and WIN CATS-4 software were used. The anti-inflammatory activity was tested by injecting different groups of rats (6 each) with formalin in hind paw and measuring the edema volume before and 1 h later formalin injection. Control group received saline i.p. The extracts treatment was injected i.p. in doses of 100 and 200 mg/kg 1 h before formalin administration. Indomethacin (30 mg/kg) was used as standard.
The results of preliminary phytochemical studies confirmed the presence of protein, lipid, carbohydrate, phenol, flavonoid, saponin, triterpenoid, alkaloid and anthraquinone in both extracts. Chromatography was performed on glass-backed silica gel 60 F254 HPTLC plates with the green solvents toluene: ethyacetate: glacial acetic acid (5:3:0.2, v/v/v) as mobile phase. HPTLC finger printing of AESF revealed major eight peaks with Rf values in the range of 0.28 to 0.80 and the dichloromethane revealed major 11 peaks with Rf values in the range of 0.12 to 0.76. The purity of sample was confirmed by comparing the absorption spectra at start, middle and end position of the band. Treatment of rats (i.p.) with AESF and dichloromethane in doses of 100 and 200 mg/kg inhibited singnificantly (P<0.05, n=6) formalin-induced inflammation by 50%, 55.9%, 45.5%, and 51.4%, respectively.
HPTLC finger printing of AESF and dichloromethane of Maytenus obscura revealed eight major spots for alcoholic extracts and nine major spots for dichloromethane extracts. These HPTLC profiles may be of great usefulness in the quality control of herbal products containing these extracts. The anti-inflammatory activity of both extracts also revealed the medicinal importance of these extracts. The plant can be further explored for the isolation of phytoconstituents having anti-inflammatory activity.
PMCID: PMC3819484
Maytenus obscura; Phytochemical screening; Finger print; Anti-inflammatory; HPTLC
8.  Pharmacognostical studies of leaves of Combretum albidum G. Don 
Ancient Science of Life  2013;32(4):187-192.
Combretum albidum Don belonging to family Combretaceae is an unexplored medicinal plant in the Indian medicinal system. According to ethnobotanical information, the leaves are used in the treatment of peptic ulcer and its fruits are used in diarrhoea and dysentery. Stem bark is used in the treatment of jaundice and skin diseases. The problem encountered in standardisation of this medicinal plant is its identification by source.
Materials and Methods:
The pharmacognostical studies were carried out in terms of organoleptic, macroscopic, microscopic, physicochemical, florescence and phytochemical analysis. Physicochemical parameters such as total ash, moisture content and extractive values are determined by World Health Organization guidelines. The microscopic features of leaf components are observed with Nikon lab photo device with microscopic units.
Macroscopically, the leaves are simple, obovate in shape, acuminate apex, entire margin and smooth surface. Microscopically, the leaves showed a large vascular strand that consists of thick walled xylem elements mixed with xylem fibres and phloem which is present in a thin layer along inner and outer portions of xylem. External to the xylem occur a thin line of sclerenchyma. Powder microscopy revealed glandular trichomes in the adaxial epidermal peelings also shows the non-glandular trichomes fairly common in powder and epidermis with anisocytic stomata. Vessels elements are narrow, long, cylindrical and dense multi-seriate bordered pits. Xylem fibres are thin and long, with thick walls, which are lignified. Preliminary phytochemical screening showed the presence of carbohydrate, glycoside, saponin, flavonoid, phytosterols and phenolic compounds.
The results of the study can serve as a valuable source of pharmacognostic information as suitable standards for identification of this plant material in future investigations and applications.
PMCID: PMC4078467  PMID: 24991065
Combretum albidum; fluorescence analysis; macroscopy; microscopy; physicochemical; phytochemical
9.  A comparison of the cytotoxic potential of standardized aqueous and ethanolic extracts of a polyherbal mixture comprised of Nigella sativa (seeds), Hemidesmus indicus (roots) and Smilax glabra (rhizome) 
Pharmacognosy Research  2010;2(6):335-342.
A decoction (hot-water extract) comprised of Nigella sativa (seeds), Hemidesmus indicus (roots), and Smilax glabra (rhizome) has been reported to prevent chemically-induced hepatocarcinogenic changes in rats and to exert significant cytotoxic effects on human hepatoma (HepG2) cells. However, the decoction used in previous studies to determine cytotoxicity was not standardized. Further, during preparation of pharmaceuticals for clinical use, it is more convenient to use an ethanolic extract. Therefore this study was carried out to (a) develop standardized aqueous and ethanolic extracts of the plant mixture (N. sativa, H. indicus, and S. glabra) used in the preparation of the original decoction, and (b) compare the cytotoxic effects of these two extracts by evaluating cytotoxicity to the human hepatoma (HepG2) cell line.
Aqueous and ethanolic extracts have been standardized by evaluating organoleptic characters, physicochemical properties, qualitative and quantitative analysis of chemical constituents, and analysis of High Performance Liquid Chromatography (HPLC) and Thin Layer Chromatography (TLC) profiles. Cytotoxic potentials of the above standardized extracts were compared by evaluating their effects on the survival and overall cell activity of HepG2 cells by use of the 3-(4, 5-dimethylthiazol-2yl) -2, 5 – biphenyl tetrazolium bromide (MTT) and Sulphorhodamine B (SRB) assays.
Results from MTT and SRB assays demonstrated that both extracts exerted strong dose-dependent in vitro cytotoxicity to HepG2 cells. The standardized aqueous extract showed a marginally (though significantly, P<0.05) higher cyotoxic potential than the ethanolic extract. Thymoquinone, an already known cytotoxic compound isolated from N. sativa seeds was only observed in the standardized ethanolic extract. Thus, compounds other than thymoquinone appear to mediate the cytotoxicity of the standardized aqueous extract of this poly-herbal preparation.
It may be concluded that results obtained in the present study could be used as a diagnostic tool for the correct identification of these aqueous or ethanolic extracts and would be useful for the preparation of a standardized pharmaceutical product that may be used in the future for clinical therapy of hepatocellular carcinoma.
PMCID: PMC3111691  PMID: 21713135
Cytotoxicity; Hemidesmus indicus; MTT and SRB assays; Nigella sativa; Smilax glabra; Standardization
10.  Comparison of liquid-liquid extraction-thin layer chromatography with solid-phase extraction-high-performance thin layer chromatography in detection of urinary morphine 
Journal of Biomedical Research  2011;25(5):362-367.
Liquid-liquid extraction-thin layer chromatography (LLE-TLC) has been a common and routine combined method for detection of drugs in biological materials. Solid-phase extraction (SPE) is gradually replacing the traditional LLE method. High performance thin layer chromatography (HPTLC) has several advantages over TLC. The present work studied the higher efficiency of a new SPE-HPTLC method over that of a routine LLE-TLC method, in extraction and detection of urinary morphine. Fifty-eight urine samples, primarily identified as morphine-positive samples by a strip test, were re-screened by LLE-TLC and SPE-HPTLC. The results of LLE-TLC and SPE-HPTLC were then compared with each other. The results showed that the SPE-HPTLC detected 74% of total samples as morphine-positive samples whereas the LLE-TLC detected 48% of the same samples. We further discussed the effect of codeine abuse on TLC analysis of urinary morphine. Regarding the importance of morphine detection in urine, the present combined SPE-HPTLC method is suggested as a replacement method for detection of urinary morphine by many reference laboratories.
PMCID: PMC3596733  PMID: 23554712
morphine detection; liquid-liquid extraction; thin-layer chromatography; solid-phase extraction; high-performance thin layer chromatography
11.  Pharmacognostic standardization of stems of Thespesia lampas (Cav.) Dalz & Gibs 
To establish the standardization parameters for complete pharmacognostic evaluation of stems of Thespesia lampas (T. lampas) (Cav.) Dalz & Gibs (Malvaceae), an important plant in the Indian system of medicine.
Morphological, microscopical, physico-chemical evaluations, florescence analysis of T. lampas stems were investigated and preliminary phytochemical analysis, GC-MS analysis and HPTLC fingerprinting were carried out for qualitative phytochemical evaluation of various extracts of stems of T. lampas.
Chemo-microscopy revealed the presence of lignin, starch grains and calcium oxalate crystals. Physico-chemical evaluation used to determine numerical standards showed a result with total ash (9.03 ± 0.05) % w/w, acid insoluble ash (1.50 ± 0.01) % w/w, water soluble ash (2.51 ± 0.02) % w/w, sulphated ash (7.50 ± 0.01) % w/w, ethanol soluble extractive (0.24 ± 0.02) % w/w, water soluble extractive (0.08 ± 0.01) % w/w, moisture content (6.03 ± 0.05) % w/w and total crude fibre content of stem powder (47.36 ± 0.32) % w/w. Behavior characteristics of the stem powder showed presence of steroids, starch, alkaloid, flavonoids and proteins. Preliminary phytochemical analysis revealed presence of glycosides, phenolic compounds, tannins, steroids, saponins, flavonoids, carbohydrates and proteins. GC-MS analysis showed the presence of fatty acids such as dodecanoic acid, tetradecanoic acid, n-hexadecanoic acid, 9-tetradecenal and HPTLC fingerprinting revealed the presence of β-sitosterol and quercetin in stems of T. lampas.
The pharmacognostic standardization of T. lampas is useful towards establishing standards for quality, purity and sample identification.
PMCID: PMC3609316  PMID: 23569930
Thespesia lampas; Stems; Pharmacognosy; Malvaceae; Physicochemical analysis; Preliminary phytochemical testing; GC-MS analysis; Phelloderm; Periderm; Xylem
12.  Pharmacognostical and physicochemical evaluation of Agasti leaf 
Sesbania grandiflora (L.) Pers., commonly known as Agasti, is widely used in Ayurveda for the treatment of diseases and for processing of various formulations in Rasashastra. It is used for its astringent, antihistaminic, anxiolytic, anticonvulsive and febrifugal activities. Moreover, because of its edible nature, the leaves and pods are used as flavoring items in the cuisine of South India. A detailed investigation of fresh and powder of leaves of Agasti was carried out. The diagnostic characters of this plant include stomatal characters, presence of resins, oil globules, appressed epidermal hair and mucilage cells. Physicochemical studies revealed loss on drying (0.6%), total ash (10.75%), acid insoluble ash (0.045%), alcohol-soluble extractive (21.7%), and water-soluble extractive (30.72%). Preliminary analysis for the presence of various functional groups revealed the presence of alkaloids, saponins, phenols and proteins. Thin layer chromatographic study of the alcoholic extract showed the presence of five, six and seven spots in short UV, long UV and after spraying developing reagent, respectively. The information generated by this particular study will provide relevant pharmacognostical and physicochemical data needed for proper identification and authentication of leaves of this particular species.
PMCID: PMC3059446  PMID: 21455451
Pharmacognostical study; physicochemical study; thin layer chromatography
13.  Pharmacognostical evaluation of Barringtonia acutangula leaf 
Barringtonia acutangula (L.) Gaertn. (Family: Lecythidaceae) is an evergreen tree with simple, alternate leaves, long pendulous racemes, dark scarlet flowers, and ellipsoid to ovoid berries containing one ovoid black seed. The present study deals with a detailed pharmacognostical study on the leaf of the crude drug, B. acutangula. Morphoanatomy of the leaf was studied using light and confocal microscopy and World Health Organization (WHO) guidelines on quality control methods for medicinal plant materials. Literature reveals that the phytoconstituents like tanginol, barrinic acid, and barringenic acid are present in the wood and fruits of this plant. Our preliminary phytochemical studies of the powdered leaves revealed the presence of terpenes, flavanoids, carbohydrates, tannins, steroids, and glycosides. The physico-chemical, morphological, histological parameters, and High Performance-Thin Layer Chromatographic (HPTLC) profile presented in this paper may be proposed as parameters to establish the authenticity of B. acutangula and can possibly help to differentiate the drug from its other species and the pharmacognostic profile of the leaves presented here will assist in standardization viz., quality, purity, and sample identification.
PMCID: PMC3157107  PMID: 21897641
Barringtonia acutangula; high performance thin layer chromatographic profile; pharmacognosy
14.  HPTLC finger print and anti-inflammatory activity of ethanolic extract of different Maytenus species grown in Kingdom of Saudi Arabia 
To evaluate and compare the anti-inflammatory activity and to develop HPTLC fingerprint profile of ethanolic extract of Maytenus obscura (M. obscura) and Maytenus parviflora (M. parviflora).
Preliminary phytochemical screening was done and HPTLC studies were carried out using CAMAG HPTLC system equipped with Linomat IV applicator, TLC scanner 3, Reprostar 3, CAMAG ADC 2 and WIN CATS-4 software. The anti-inflammatory activity was tested by injecting different groups of rats (6 each) with formalin in hind paw and measuring the edema volume before and 1 h after formalin injection. Control group received saline i.p. The extract treatment was injected i.p with doses of 200 and 400 mg/kg 1 h before formalin administration. Indomethacin (30 mg/kg) was used as standard.
Treatment of rats (i.p.) with M. obscura and M. parviflora in doses of 200 and 400 mg/kg inhibited significantly (P<0.05) formalin-induced inflammation by 55.9%, 63.2% and 77.9%, 82.4%, respectively. Preliminary phytochemical studies were done which confirmed the presence of protein, lipid, carbohydrate, phenol, flavonoid, saponin, triterpenoid, alkaloid and anthraquinone. Chromatography was performed on glass-backed silica gel 60 F254 HPTLC plates with the solvent system: Toluene: ethylacetate: glacial acetic acid (5:2:0.1, v/v/v) as mobile phase. HPTLC finger printing of M. obscura revealed major 8 peaks with Rf values in the range of 0.27 to 0.77 and the M. parviflora revealed maximum 9 peaks with Rf values in the range of 0.17 to 0.76. The purity of sample was confirmed by comparing the absorption spectra at start, middle and end position of the band.
HPTLC of M. parviflora revealed 8 major spots and 9 spots for M. obscura. HPTLC finger printing of ethanolic extract of M. obscura and M. parviflora may become potential tool for checking authenticity of these species. It may help in quality control against adulterant and act as a biochemical marker for these medicinally important plants in the pharmaceutical industry and plant systematic studies. The anti-inflammatory potential of these plants also reveals its medicinal importance. It might be further explored for the isolation of phytoconstituents having anti-inflammatory potential.
PMCID: PMC4027328
Maytenus obscura; Maytenus parviflora; Phytochemical screening; Finger print; Standardization; HPTLC
15.  Simultaneous HPTLC determination of strychnine and brucine in strychnos nux-vomica seed 
A simple, sensitive, and specific thin layer chromatography (TLC) densitometry method has been developed for the simultaneous quantification of strychnine and brucine in the seeds of Strychnos nux-vomica.
Materials and Methods:
The method involved simultaneous estimation of strychnine and brucine after resolving it by high performance TLC (HPTLC) on silica gel plate with chloroform–methanol–formic acid (8.5:1.5:0.4 v/v/v) as the mobile phase.
The method was validated as per the ICH guidelines for precision (interday, intraday, intersystem), robustness, accuracy, limit of detection, and limit of quantitation. The relationship between the concentration of standard solutions and the peak response was linear within the concentration range of 50–1000 ng/spot for strychnine and 100–1000 ng/spot for brucine. The method precision was found to be 0.58–2.47 (% relative standard deviation [RSD]) and 0.36–2.22 (% RSD) for strychnine and brucine, respectively. Accuracy of the method was checked by recovery studies conducted at three different concentration levels and the average percentage recovery was found to be 100.75% for strychnine and 100.52% for brucine, respectively.
The HPTLC method for the simultaneous quantification of strychnine and brucine was found to be simple, precise, specific, sensitive, and accurate and can be used for routine analysis and quality control of raw material of S. nux-vomica and several unani and ayurvedic formulations containing this as an ingredient.
PMCID: PMC3341717  PMID: 22557924
HPTLC; method development; strychnine; brucine; validation
16.  Standardization of the finished product: Habbe Irqun Nisa - A Unani anti-inflammatory formulation 
Ancient Science of Life  2012;32(1):38-44.
Habb (Pill) is one of the important dosage forms of Unani system of medicine. A number of effective formulations are manufactured in form of Habb because of its various advantages. Out of these, Habbe Irqun Nisa (HI) is a popular anti-inflammatory formulation used in the treatment of Warame Mafasil (arthritis) and Irqun Nisa (sciatica). Nowadays, with increased incidence of these diseases many non-steroidal anti-inflammatory drugs (NSAIDs) are being used in their treatment. Owing to the adverse effects of these drugs, the use of herbal medicines is seen as a better alternative. The basic requirement for the development of Unani system of Medicine is the standardization of single and compound drugs. HI is mentioned in National Formulary of Unani Medicne and selected for the present study.
Materials and Methods:
HI was prepared manually with the powder of crude drugs, passed through sieve no. 100 and mixed with 1% w/w of gum acacia in mucilage form. It was then dried at 60°C for 90 min and then tested for its standardization on different physicochemical parameters, e.g. organoleptic properties, pH values, moisture content, ash values, friability, hardness, weight variation, disintegration time, and thin layer chromatography (TLC).
Results and Conclusion:
The data evolved from this study will make it a validated product and will help in the quality control of other finished products in future research.
PMCID: PMC3733206  PMID: 23929993
Anti-inflammatory; Habb; standardization; Unani system of medicine
17.  Phytochemical analysis of ethanolic extract of Dichrostachys Cinerea W and Arn leaves by a thin layer chromatography, high performance thin layer chromatography and column chromatography 
Ancient Science of Life  2013;32(4):227-233.
The leaves of Dichrostachys cinerea are used as laxative, diuretic, painkiller. It is also used in the treatment of gonorrhoea, boils, oedema, gout, veneral diseases and nasopharyngeal affections, etc.
Materials and Methods:
The Phytochemical investigation of ethanolic extract of D. cinerea leaves were performed by standard chemical tests, thin layer chromatography (TLC) by using various solvent systems, and by high performance liquid chromatography (HPTLC). Two compounds were isolated by column chromatography and one of the compounds was identified by various spectral studies.
Preliminary phytochemical screening of ethanolic extract of D. cinerea leaves showed the presence of Carbohydrates, proteins, Glycosides, Saponins, Tannins, Aminoacids and Terpenoids. The TLC and HPTLC fingerprint of ethanolic extract were studied and various fractions were isolated by column chromatography and one of the fraction contain β-amyrin glucoside which was confirmed by Infra Red[IR] Spectroscopy, 1H-Nuclear Magnetic Resonance (NMR), C-13 NMR and Mass spectroscopic (MS) studies.
PMCID: PMC4078474  PMID: 24991072
Column chromatography; Dichrostachys cinerea; high performance thin layer chromatography; leaves extract; phytochemical evaluation
18.  Secondary metabolite credentials of Evolvulus alsinoides by high performance thin layer chromatography (HPTLC) 
Journal of Biomedical Research  2012;26(4):295-302.
Plants and plant-based products are the bases of many modern pharmaceuticals that are current in use today for various diseases. The aim of the study was to investigate the biochemical constituents and high performance thin layer chromatography (HPTLC) finger printing of the ethanolic extract of Evolvulus alsinoides. Phytochemical screening was done by standard procedures and HPTLC method was also established to analyze alkaloids, flavonoids and phenolic compounds from the ethanolic extract of Evolvulus alsinoides. Preliminary phytochemical screening showed that ethanol extracted more secondary metabolites than other solvents. HPTLC fingerprinting analysis showed the presence of various alkaloids, flavonoids and phenols (quercetin) in the ethanolic extract. It can be concluded that Evolvulus alsinoides may serve as a source of potent antioxidants that may be used in the prevention of various diseases such as cancer, diabetes and cardiovascular diseases due to the presence of phenolic compounds. HPTLC finger print of Evolvulus alsinoides may be useful in the differentiation of the species from adulterants and act as a biochemical marker for this medicinally important plant in the pharmaceutical industry and plant systematic studies.
PMCID: PMC3596747  PMID: 23554763
Evolvulus alsinoides; high performance thin layer chromatography (HPTLC); secondary metabolites; convolvulaceae
19.  Morpho-anatomical and physicochemical studies of Fumaria indica (Hausskn.) Pugsley 
To study morpho-anatomical characters and physicochemical analysis of Fumaria indica (F. indica) (Hausskn.) Pugsley, (Fumariaceae), an important medicinal plant used extensively for treating a variety of ailments in various system of indigenous medicine.
Evaluation of the different parts of the plant was carried out to determine the morpho-anatomical, physicochemical, phytochemical and HPTLC fingerprinting profile of F. indica and other WHO recommended methods were performed for standardization.
Morpho-anatomical studies showed compound and pinnatifid leaf, 4 to 6 cm in length, linear and oblong in shape and anomocytic arrangement of stomata, thin walled parenchymatous cells, scattered, sclerenchymatous, capped vascular bundles and radiating medullary rays. Physicochemical studies showed foreign matter 0.2%, loss on drying 6.8%, total ash 16.77%, alcohol and water soluble extractives 8.92% and 20.26%, respectively, sugar 17.75%, starch 22.97% and tannins 2.37%. Phytochemical evaluation revealed the presence of carbohydrate, alkaloids, flavonoids, saponins, tannins and sterol. Thin layer chromatography was carried out with different solvents and the best solvent system was chloroform and methanol in 80:20 ratio and revealed 12 spots with different Rf value under UV light 366λ.
The results of the study can serve as a valuable source of information and provide suitable standards for identification of this plant material for future investigations and applications.
PMCID: PMC3609232  PMID: 23569856
Morpho-anatomy; Transverse sections; HPTLC; Stomata; Xylem; Physicochemical
20.  Diffusible component from the spore surface of the fungus Aspergillus fumigatus which inhibits the macrophage oxidative burst is distinct from gliotoxin and other hyphal toxins 
Thorax  1997;52(9):796-801.
BACKGROUND: The fungus Aspergillus fumigatus, whose spores are present ubiquitously in the air, causes a range of diseases in the human lung. A small molecular weight (< 10 kD) heat stable toxin released from the spores of clinical and environmental isolates of A fumigatus within minutes of deposition in aqueous solution has previously been described. A key effect of the toxin was to inhibit the oxidative burst of macrophages as measured by superoxide anion release. It was hypothesised that the toxin was one of the commonly found A fumigatus hyphal toxins such as gliotoxin. This inhibitor may be an important factor which allows the fungus to colonise the lung. METHODS: The spore derived inhibitor was shown to inhibit the respiratory burst of rat alveolar macrophages, as measured by the generation of superoxide anion. Samples of the spore diffusate were subject to reversed phase high performance liquid chromatography (HPLC), thin layer chromatography (TLC), high performance thin layer chromatography (HPTLC), or organic extraction followed by TLC or HPLC to identify the presence of gliotoxin, fumagillin, helvolic acid, fumigaclavine-C, and aurasperone-C. Commercially obtained preparations of the toxins gliotoxin, fumagillin and helvolic acid and extracts enriched for fumigaclavine-C and aurasperone-C were used as internal and external standards and in the respiratory burst measurements. RESULTS: Gliotoxin, fumagillin, helvolic acid, fumigaclavine-C, and aurasperone- C were not detected in spore derived diffusate using PHLC or TLC. Using extraction procedures with solvents known to extract gliotoxin from A fumigatus culture supernatants, no gliotoxin was detected in the spore derived diffusate. Commercial gliotoxin, fumagillin, and helvolic acid or extracts enriched for fumigaclavine-C and aurasperone-C did not inhibit the oxidative burst of macrophages. CONCLUSIONS: The hypothesis that the spore derived toxin is one of the toxins derived from hyphae such as gliotoxin, helvolic acid, fumagillin, fumigaclavine-C, or aurasperone-C is not proved. The spore toxin may exert its effect through its ability to diffuse rapidly into the lung lining fluid, diminish the macrophage oxidative burst, and play a part in allowing A fumigatus to persist in the lung and manifest its well known pathogenic effects. 

PMCID: PMC1758635  PMID: 9371210
21.  Determination of gallic acid in Phyllanthus emblica Linn. dried fruit powder by HPTLC 
Emblica (Phyllanthus emblica L.), an euphorbiaceous plant, is widely distributed in subtropical and tropical areas of India, China and Indonesia. The fruits possess antimicrobial, antioxidant, anti-inflammatory, analgesic and antipyretic properties. In the current article a new, simple, sensitive, selective, precise, and robust high-performance thin-layer chromatographic (HPTLC) method was developed and validated for the determination of gallic acid in dried fruit powder of Phyllanthus emblica.
Materials and Methods:
The quantitative determination of gallic acid was performed on TLC aluminium plates pre-coated with silica gel 60F-254 as the stationary phase. The linear ascending development was carried out in a twin trough glass chamber saturated with a mobile phase consisting of toluene: ethyl acetate: formic acid: methanol (3:3:0.8:0.2) at room temperature (25 ± 2°C). Camag TLC scanner III was used for spectrodensitometric scanning and analysis, in the absorbance mode, at 278 nm.
The linear regression analysis data for the calibration plots showed good linear relationship with r2 = 0.99977 in the concentration range of 40 – 240 ng spot—1, with respect to the peak area. According to the guidelines of the International Conference on Harmonization (ICH), the method was validated for precision, accuracy, and recovery.
Statistical analysis of the data showed that the method was reproducible and selective for the estimation of gallic acid.
PMCID: PMC3147091  PMID: 21814441
Phyllanthus emblica; HPTLC fingerprinting; gallic acid
22.  Marker based standardization of polyherbal formulation (SJT-DI-02) by high performance thin layer chromatography method 
Preparation of highly standardized herbal products with respect to chemical composition and biological activity is considered to be a valuable approach in this field. SJT-DI-02 polyherbal formulation was successfully developed at our institute and filed for patent at Mumbai patent office.
The present work was marker based standardization of patented, novel and efficacious polyherbal formulation namely SJT-DI-02 for the treatment of diabetes. The SJT-DI-02 was comprised of dried extracts of rhizomes of Acorus calamus, leaves of Aegle marmelose, fruits of Benincasa hispida, roots of Chlorophytum arendinaceum, seeds of Eugenia jambolana, leaves of Ocimum sanctum, pericarp of Punica granatum, seeds of Tamarindus indica. Selected plants were collected, dried and extracted with suitable solvents. The formulation was prepared by mixing different fractions of extracts.
Materials and Methods:
For successful and best standardization, first of all selection and procurement was carried out. Selection is done on the basis of therapeutic efficacy and amount of the marker present in the particular plant part. At the time of procurement side by side phytochemical screening and estimation of phytoconstituents was carried out. After completion of preliminary screening using characterized markers, we tried to develop best TLC systems using selected solvent composition. Finally well-developed TLC systems were applied in HPTLC. In the present study polyherbal formulation was standardized by using different four markers. TLC Densitometric methods were developed using HPTLC for the quantification of these marker compounds. Solvent systems were optimized to achieve best resolution of the marker compounds from other components of the sample extract. The identity of the bands in the sample extracts were confirmed by comparing the Rf and the absorption spectra by overlaying their UV absorption spectra with those of their respective standards. The purity of the bands due to marker compounds in the sample extracts were confirmed by overlaying the absorption spectra recorded at start, middle and end position of the band in the sample tracks. After conforming all these things fingerprints were developed for all three formulations which will be act as authentification and quality control tool.
% w/w of asarones is 3.61, % w/w of marmelosin is 4.60, % w/w of gallic acid is 10.80 and % w/w of lupeol is 4.13. The method was validated in terms of linearity, precision, repeatability, limit of detection, limit of quantification and accuracy. In well-developed mobile phase system linearity was found to be in the range of 0.983-0.995, % recovery was found to be in the range of 97.48-99.63, % RSD for intraday and interday was found to be 0.13- 0.70 and 0.32 -1.41 and LOD and LOQ was found to be in the range of 0.15- 0.61 and 0.45 -1.83 microgram per ml.
Thus High performance thin layer chromatography (HPTLC) methods were developed and validated in terms of linearity, precision, repeatability, limit of detection, limit of quantification and accuracy. The methods were rapid, sensitive, reproducible and economical. It does not suffer any positive or negative interference due to common other component present in the formulation and would also serve as a tool for authentication of herbal products containing marmelosin, gallic acid, lupeol and asarones. Thus this work provides standardized and therapeutically active polyherbal formulations for the different ailments.
PMCID: PMC4097936  PMID: 25035642
Asarones; gallic acid; HPTLC; lupeol; marmelosin; SJT-DI-02
23.  A validated high performance thin layer chromatography method for determination of yohimbine hydrochloride in pharmaceutical preparations 
Pharmacognosy Magazine  2013;9(33):4-8.
Yohimbine is an indole alkaloid used as a promising therapy for erectile dysfunction. A number of methods were reported for the analysis of yohimbine in the bark or in pharmaceutical preparations.
Materials and Method:
In the present work, a simple and sensitive high performance thin layer chromatographic method is developed for determination of yohimbine (occurring as yohimbine hydrochloride) in pharmaceutical preparations and validated according to International Conference of Harmonization (ICH) guidelines. The method employed thin layer chromatography aluminum sheets precoated with silica gel as the stationary phase and the mobile phase consisted of chloroform:methanol:ammonia (97:3:0.2), which gave compact bands of yohimbine hydrochloride.
Linear regression data for the calibration curves of standard yohimbine hydrochloride showed a good linear relationship over a concentration range of 80–1000 ng/spot with respect to the area and correlation coefficient (R2) was 0.9965. The method was evaluated regarding accuracy, precision, selectivity, and robustness. Limits of detection and quantitation were recorded as 5 and 40 ng/spot, respectively. The proposed method efficiently separated yohimbine hydrochloride from other components even in complex mixture containing powdered plants. The amount of yohimbine hydrochloride ranged from 2.3 to 5.2 mg/tablet or capsule in preparations containing the pure alkaloid, while it varied from zero (0) to 1.5–1.8 mg/capsule in dietary supplements containing powdered yohimbe bark.
We concluded that this method employing high performance thin layer chromatography (HPTLC) in quantitative determination of yohimbine hydrochloride in pharmaceutical preparations is efficient, simple, accurate, and validated.
PMCID: PMC3647393  PMID: 23661986
Accuracy; dietary supplements; high performance thin layer chromatography; precision; selectivity; yohimbine
24.  Solicitation of HPLC and HPTLC Techniques for Determination of Rutin from Polyalthia longifolia Thwaites 
Pharmacognosy Research  2014;6(3):234-239.
Polyalthia longifolia Thwaites is an important traditional plant in India. Rutin, an active constituent has been reported to possess good amount of pharmacological as well as therapeutic potential.
The aim of the present study was to find out by analytical techniques how much percentage of rutin is present in the plant leaves’ ethanolic extract by analytical techniques.
Materials and Methods:
Shade dried leaves of Polyalthia longifolia were subjected to cold ethanolic extraction followed by monitoring the isolated rutin high-pressure liquid chromatography (HPLC) and high performance thin layer chromatography (HPTLC) after carrying out preliminary phytochemical screening.
Extraction yield was found to be 13.94% w/w. Phytochemical screening of the extract showed the presence of flavonoids, steroids, diterpenoids, alkaloids, saponins, tannins and phenolic compounds and mucilage. From the Rf value, the ethanolic extract was found to be having constituent identical to rutin. By HPTLC and HPLC the amount of rutin was found to be 11.60% w/w and 4.03% w/v, respectively.
The active constituent isolated was found to be equal to rutin.
PMCID: PMC4080504  PMID: 25002804
High-pressure liquid chromatography; High performance thin layer chromatography; Polyalthia longifolia; Rutin; Thin layer chromatography
25.  Physico-chemical standardization of Sitopaladi churna 
Ancient Science of Life  2012;31(3):107-116.
Standardization of a compound Ayurvedic formulation is a critical and essential issue to be considered in assuring the therapeutic efficacy and safety and to rationalize their use in the health care. Sitopaladi churna is a reputed polyherbal formulation of Ayurveda. It is prescribed for the treatment of pleurodynia, intercostal neuralgia, cold, cough associated with bronchitis, pneumonia, tuberculosis, viral respiratory infection, and in pharyngeal and chest congestion.
The present study aimed at physico-chemical standardization of in-house and two marketed brands of Sitopaladi churna.
Materials and Methods:
In our investigation, in-house churna and two commercial brands of Sitopaladi churna were standardized based on powder microscopy, physico-chemical evaluations, thin layer chromatography (TLC) and high performance thin layer chromatography (HPTLC) finger printing as per standard procedures.
The set parameters were sufficient to evaluate the churna based on various physico-chemical parameters.
The data evolved can be adopted for laying down the standards for the manufacturing units of Sitopaladi churna.
PMCID: PMC3530334  PMID: 23284216
High performance thin layer chromatography; physico-chemical; Sitopaladi churna; standardization

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