Thin-layer Chromatographic Identification Research Articles

Establishment and Optimization of Quality Standards for the Traditional Tibetan Medicine Preparation of Liuwei Nengxiao Pills

To establish quality standards for Liuwei Nengxiao pills, to optimize the quality control method, and to provide references for the quality control of Liuwei Nengxiao pills. Chebula, dried ginger, and Tibetan liqueur root in Liuwei Nengxiao pills of different batch numbers were analyzed by thin layer chromatography (TLC). Then, the content of chrysophanol in the preparation was determined by high performance liquid chromatography (HPLC). Furthermore, a series of methodological validation, including the investigation of the linear relationship, precision, stability, and reproducibility and sample recovery test, were performed to verify the reliability of the results. The TLC identification method was easy to perform and demonstrated high specificity, clear spots, and good separation effect. In addition, the negative controls showed no interference. The HPLC method showed high accuracy. The results of methodological validation showed that the peak area of chrysophanol had a good linear relationship (r2=1.0) in the range of 0.06-0.80 μg, presenting good precision (with the relative standard deviation being lower than 2.0%), good stability and reproducibility (with the relative standard deviation being lower than 1.0%), and an average recovery rate of 100.8%. TLC and HPLC are easy to perform, showing high accuracy and reproducibility. The quality standards established are scientific, reasonable, stable, and feasible, providing references for the quality control of Liuwei Nengxiao pills.

Study of Quality Standards of Xiao’er Qingre Enema Preparation in Hospitals

Objective: To establish the quality standards for the preparation of Xiao’er Qingre enema in hospitals. Method:Thin-layer chromatography (TLC) was used to identify Radix Isatidis and Glycyrrhizae in the prescription. The content of (R,S)-Goitrin was determined by high-performance liquid chromatography (HPLC). Results: In TLC identification, there was no interference between the negative controls of Radix Isatidis and glycyrrhizae, and the spots were well-separated. The HPLC results of (R,S)-Goitrin showed a good linear relationship between the peak area and concentration within the range of 0.476–95.2 μg·mL-1 (r = 0.9999). Conclusion: The TLC and HPLC methods established in this experiment are simple, reproducible, and specific, making them suitable for the quality control Xiao’er Qingre enema preparation in hospitals.

Studies on anatomy, physico-chemical and thin-layer chromatography of rhizome, root and leaf of Dracaena trifasciata (Prain) Mabb

Dracaena trifasciata (Prain) Mabb., commonly known as mother-in-law’s tongue, used in the treatment of various ailments such as ulcer, jaundice, skin itches, urinary diseases, asthma, cough, snake and insect bites in folklore medicine. In the present investigation, anatomical, physico-chemical and thin-layer chromatographic identification of rhizome, root and leaf has been studied in detail and provided diagnostic key to identify the original drug from the adulterant(s).

Quality evaluation of Psoraleae Fructus based on QAMS method and TLC identification

The present study established a determination method of Psoraleae Fructus by quantitative analysis of multi-components by the single marker(QAMS) and further improved the thin-layer chromatography(TLC) method. The QAMS method was established by UPLC with psoralen as the internal marker, and the content of psoralenoside, isopsoralenoside, psoralen, and isopsoralen was simultaneously determined. As revealed by the comparison with results of the external standard method, the QAMS method was accurate and feasible. According to the current quality standards of Psoraleae Fructus, the TLC method was further optimized and improved, and bakuchiol was added for identification based on the original TLC method with psoralen and isopsoralen as indicators. This study provides a reference for improving the quality control method of Psoraleae Fructus.

Microscopic investigations and pharmacognostic techniques for the standardization of the fruits of Rosa laxa Retz.

Rosa laxa Retz., a shrub belonging to the family Rosaceae, is widely distributed in the northern foothills of the Tianshan Mountains in Xinjiang, China. The fruits of R. laxa (FRL) has antibacterial, hypolipidemic, and antioxidant effects. In this study, FRL was subjected to pharmacognostic identification of its source, morphology, microscopic characteristics, and physicochemical properties. The microscope showed that the cross-sectional features of FRL were obvious, and the FRL powder contained vessel, parenchyma cells, exocarp cells, pollen grains, and cluster crystals. Scanning electron microscopy (SEM) analysis results show that numerous villi and many small particles (particle size of 5-50 μm) were observed in the FRL powder, and there are many gullies on the surface of the particles. In addition, the secondary metabolites of FRL were characterized via ultraviolet-visible (UV-Vis) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and thin-layer chromatography (TLC). Results showed that FRL contains various secondary metabolites, including flavonoids, phenolic acids, glycosides, and tannins. Water as the extraction solvent had the highest extraction rate and the contented of total flavonoids was 2.88 mg/g, and the contented of total polyphenols was 54.89 mg/g. Moreover, TLC identification revealed that it contains catechin and tiliroside. These parameters of FRL, which are reported herein, are important to the development of the pharmacognostic standards, as well as in the identification and quality control of FRL.

Comprehensive analysis of components in Chinese medicines derived from Apocynum venetum and Poacynum pictum in Xinjiang based on HPLC fingerprint and chemometrics

This study established high-performance liquid chromatography(HPLC) fingerprints of Chinese medicines derived from Apocynum venetum and Poacynum pictum in Xinjiang and explored their composition differences with the combination of content determination, similarity analysis, cluster analysis and principal component analysis. The HPLC conditions included Phenomenex Kinetex C_(18) column(4.6 mm ×100 mm, 2.6 μm), acetonitrile-0.01% trifluoroacetic acid aqueous solution as mobile phase, gradient elution, flow rate of 0.6 mL·min~(-1), detection wavelength of 281 nm and column temperature of 25 ℃. The content of chlorogenic acid, quercetin-3-O-sophoroside, rutin, hyperin, isoquercitrin, trifolin and astragalin was determined in 31 batches of medicinal materials, and fingerprint research and chemometric analysis were performed with Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine(Version 2004 A) and SPSS 21.0. In the Chinese Pharmacopoeia 2020, the quality of Apocyni Veneti Folium is controlled by character identification, microscopic identification, thin layer chromatography identification and quantitative determination of hyperin. There were 21 common peaks of A. venetum and P. pictum in the HPLC fingerprints, 5 of which were identified as chlorogenic acid, hyperin, isoquercitrin, trifolin and astragalin, with their content also determined. Except for 3 batches of medicinal materials, the similarity of other 28 batches was higher than 0.83, indicating good similarity. Two categories were formed in the cluster analysis based on content determination, which showed that some differences existed in similarities between different regions of Xinjiang. The medicinal materials were ranked by quality with principal component analysis, and the results indicated that the top 15 all came from northern Xinjiang. The quality difference of A. venetum and P. pictum had a correlation with the place of origin. This study provides a reference for the analysis and evaluation of A. venetum and P. pictum from different habitats and the selection of introduction and cultivation areas.

Improvement researches on quality standard of Anemarrhenae Rhizoma and its raw processed products

This study is to improve the quality standard and supply the scientific basis for Anemarrhenae Rhizoma and its raw processed products. Steroidal saponin including timosaponin BⅡ, timosaponin AⅢ and flavonoids including neomangiferin and mangiferin were selected as the indicative components. Silica gel G thin layer chromatography(TLC) and polyamide TLC were used to detect the two types of compounds, respectively. The contents of timosaponin BⅡ and timosaponin AⅢ were determined by HPLC-ELSD and the content of neomangiferin, mangiferin and isomangiferin were determined by HPLC-UV. Moisture, total ash and acid insoluble ash were determined according to Chinese Pharmacopoeia(2015 edition). And 80% ethanol was selected as the solvent and the content determination of total extract were determined. The fingerprints of Anemarrhenae Rhizoma and its raw processed products were established by HPLC-UV and HPLC-ELSD. The results showed that the methods of TLC and HPLC have been successfully stablished. There are 2 and 3 peaks which have been identified by HPLC-ELSD and HPLC-UV, respectively. The HPLC fingerprint methods are specific and can be used to identify and quality control for Anemarrhenae Rhizoma and its raw processed products in the mass. Comparing to Chinese Pharmacopoeia(2015 edition), the TLC identification and content determination were revised and the total extract determination and HPLC fingerprints were added in the present study. Our results can be used as the scientific basis of quqlity control for Anemarrhenae Rhizoma and its raw processed products.

TLC-MS identification of alkaloids in Leonuri Herba and Leonuri Fructus aided by a newly developed universal derivatisation reagent optimised by the response surface method.

Dragendorff's reagent has low sensitivity and non-specificity for some alkaloids. A new alkaloid derivatisation reagent has been developed and optimised by using a Box-Behnken design method. This new reagent is applicable for structurally diverse natural alkaloids, and is proposed as a universal alkaloid staining reagent. To establish an efficient and sensitive thin-layer chromatography (TLC) identification method for Leonuri Herba and Leonuri Fructus to characterise their differences and similarities. Three key components (bismuth subnitrate, potassium iodide and iodine) in the derivatisation reaction were re-constructed and optimised using a response surface method. Different inorganic acids, essential additives of the staining reaction, were compared by a single-factor experiment design. This newly optimised reagent for alkaloids reported in this study, named the enhanced Dragendorff-Wagner's reagent, is composed of 0.82% bismuth subnitrate, 11.1% potassium iodide and 0.76% iodine in 38% phosphoric acid solution. Validation results indicate that the TLC spot of stachydrine stained with the enhanced Dragendorff-Wagner's reagent had a limit of detection of 2.0 μg, good intra- and inter-plate, and intra- and intra-day precisions with relative standard devition values less than 5.0%, and stability over 90 min. This enhanced Dragendorff-Wagner's reagent was applied for TLC identification of 16 reference alkaloids representing 11 structural skeletons, and two closely related herbs (Leonuri Herba and Leonuri Fructus). This newly enhanced Dragendorff-Wagner's reagent is a universal, effective, and sensitive staining reagent for alkaloids.

SKRINING FITOKIMIA EKSTRAK CARICA PAPAYA RADIX, PIPER ORNATUM FOLIUM DAN NEPHELIUM LAPPACEUM SEMEN ASAL KALIMANTAN SELATAN

Penyakit Diabetes Mellitus merupakan penyakit dengan prevalensi tinggi dan menduduki peringkat 10 besar di dunia termasuk di Indonesia. Permasalahan besar yang dihadapi dunia kesehatan dewasa ini adalah Diabetes Mellitus tidak bisa disembuhkan secara total dengan obat antihiperglikemik konvensional. Penelitian perlu dilakukan dalam hal menelusuri potensi bahan alam yang biasa digunakan oleh masyarakat Kalimantan berdasarkan skrining fitokimia dari beberapa tumbuhan yang secara empiris digunakan sebagai antidiabetes yaitu Carica papaya radix (CPR), Nephelium lappaceum semen (NLS) dan Piper ornatum folium (POF). Tumbuhan obat yang diperoleh dilakukan ekstraksi maserasi dan diidentifikasi golongan senyawa dan profil kromatografinya.Kegiatan penelitian ini meliputi: 1) Pengambilan sampel tumbuhan,2) Pembuatan simplisia uji, 3) Pembuatan ekstrak etanol tumbuhan, 4) Uji kualitatif kandungan kimia tumbuhan, dan 5) Identifikasi secara kromatografi lapis tipis.
 Hasil dari penelitian dipilih dan dilakukan pengujian terhadap 3 ekstrak yaitu Carica papaya radix, Nephelium lappaceum semen dan Piper ornatum folium. Ekstrak Carica papaya radix diduga mengandung senyawa alkaloid (Rf : 0,53), saponin (Rf : 0,93), flavonoid (Rf : 0,38 ; 0,75) dan tanin (Rf : 0,52 ; 0,88). Ekstrak Piper ornatum folium diduga mengandung senyawa saponin (Rf : 0,89), steroid (Rf :0,20 ; 0,42) dan tanin (Rf : 0,52 ; 0,71 ; 0,85). Ekstrak Nephelium lappaceum semen menunjukkan hasil positif terhadap keberadaan senyawa alkaloid (Rf : 0,65 ; 0,82), flavonoid (Rf : 0,83) dan tanin (Rf : 0,79 ; 0,92).

Phytochemical screening and TLC studies of different extracts of Chenopodium album

Chenopodium album (C. album), extensively exploit in folk medicine in various parts of world. The plant possesses various therapeutic properties such as antiphlogistic, mildly laxative, anthelmintic, odontalgic properties and antirheumatic. In the present research, Histological examination, phytochemical screening and Thin layer chromatographic identification of leaf and extracts has been studied extensively and provided diagnostic key to detect the presence of adulterant. It is concluded that our study provides the data which helps to isolate and characterize the different medicinal potential of the C. album such as diuretic, antiparasitic, hepato-protective, laxative and sedative.

Selection of the Optimal conditions for analysis of plant active Pharmaceutical Inulin Ingredients by Thin-layer Chromatography to develop the draft for the National Monograph “Inulin”

Inulin is a representative of the class of soluble dietary fibers. One of the stated methods for identification of substances in the monograph “Inulin” of the British Pharmacopoeia 2010 (??) is the method of thin-layer chromatography (TLC), in the State Pharmacopoeia of Ukraine (SPhU) the method of TLC for identification of plant active pharmaceutical ingredients (API) of inulin is absent. The Pharmacopoeia provides only quality requirements of such monosaccharides as “Fructose”, “Anhydrous glucose”, “Glucose, monohydrate”, “Anhydrous lactose”, “Lactose monohydrate”. The experimental studies were conducted using nine herbal substances of inulin obtained from plant sources such as chicory, Jerusalem artichoke and agave and two standard substances of inulin with chicory and dahlia. Identification and determination of related impurities of plant API of inulin using TLC were performed according to the method of the monograph “Inulin” of the BP and the method of determination of monosaccharides given in the SPhU. The optimal conditions for the chromatographic determination have been chosen; validation of the method using indicators of specificity, robustness and precision has been conducted. The validated method for analysis of plant API of inulin using TLC has been proposed to be included into the draft for the monograph “Inulin” of the SPhU in the sections “Identification” and “Test for purity”.

Histological study and thin layer chromatographic profile of stem-bark and leaf of Holoptelea integrifolia (Roxb.) Planch

Holoptelea integrifolia (Roxb.) Planch., a multifaceted drug, extensively used in Ayurveda, Siddha and other various systems of medicine. Leaves and bark are the officinal parts of the plant and used for various ailments. In the present investigation, histological and thin layer chromatographic identification of stem-bark and leaf has been studied in detail and provided diagnostic key to identify the original drug from the adulterant(s).

The establishment of cell suspension culture of sabah snake grass (Clinacanthus nutans (Burm.F.) Lindau)

Clinacanthus nutans (Burm.F.) Lindau is an herbaceous plant that has long been used for traditional medicinal purposes in Asia. It has recently gained popularity as an alternative treatment for cancer. The aim of this study was to establish cell suspension cultures of C. nutans and to identify targeted bioactive compounds in the cultures. Young leaf explants were cultured on Murashige and Skoog medium supplemented with various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin to identify a suitable medium for callus induction and proliferation. Proliferated, friable calluses were cultured in different combinations of plant growth regulators (2,4-D, naphthaleneacetic acid [NAA], picloram, kinetin, and 6-benzylaminopurine) in liquid medium to establish cell suspension cultures. Three cell lines of suspension culture, callus, and intact plant parts were subjected to ethyl acetate extraction followed by thin layer chromatography for identification of selected bioactive compounds. Medium supplemented with 0.25 mg L−1 2,4-D and 0.75 mg L−1 kinetin was found to be optimal for callus induction, whereas supplementation with 0.50 mg L−1 2,4-D was efficient for callus proliferation. Liquid medium supplemented with 0.25 mg L−1 2,4-D and 0.50 mg L−1 NAA produced the highest growth index (2.52). Quercetin, catechin, and luteolin were present together in the callus and cell suspension cultures of C. nutans, but all three compounds were detected separately in young leaves, mature leaves, and stems. This study is the first to report the establishment of cell suspension culture of C. nutans with both cell and callus cultures producing quercetin, catechin, and luteolin.

Hydralazine: A novel chromogenic spray reagent for thin-layer chromatographic analysis of α-cyano ester pyrethroids

A novel chromogenic spray reagent “hydralazine” (phthalazin-1-ylhydrazine) is reported for thin-layer chromatographic (TLC) detection and identification of synthetic pyrethroids cypermethrin, deltamethrin, and λ-cyhalothrin from their dilute commercial formulations ranging from 2.5% to 25%. Alkaline hydralazine forms orange-yellow hydrazones with these insecticides at room temperature and does not require any further treatment. Reaction mechanism is discussed. The necessary TLC parameters like pH and concentration of analyte are also suggested for the efficient detection of these toxic pyrethroid insecticides.

Pharmacognostical, Physicochemical and Preliminary Phytochemical Standardization of Aerial Parts of Onosma bracteatum Wall

Objective: The present study was undertaken to develop the standardization parameters of powdered aerial parts of Onosma bracteatum Wall, family Boraginaceae. Methods: Different parameters such as pharmacognostical, physicochemical, preliminary phytochemical evaluation along with thin layer chromatography for identification of phytoconstituents were studied. Results: On microscopical examination of the aerial parts it showed the presence of oval to polygonal thin walled straight epidermal cells; spiral vessels, a few fibres elongated with blunt tips, long warty, tubercle based unicellular hairs and paracytic stomata. On physicochemical evaluation it was found to contain more amount of polar constituents as the ethanol extractive value was found to be more. Total ash value and acid insoluble ash indicated the presence of inorganic acids and silicaceous matter respectively. Foaming index and swelling index were indicative of saponins and mucilaginous matter present in the aerial parts. On preliminary phytochemical screening and thin layer chromatographic studies it revealed the presence of saponins, flavonoids, phenolic compounds and mucilage. Conclusion: The present work carried out can serve as a purpose for identification, authentication and standardization of the crude drug.

Thin-layer chromatographic quantification of three alkaloid compounds in Sophora alopecuroides and TLC—bioautography for screening antioxidant components

This study aims at establishing a thin-layer chromatographic (TLC) scanning method for the determination of three alkaloids in Sophora alopecuroides and preliminary screening of antioxidant active components in S. alopecuroides with TLC—bioautography technology. The alkaloids in S. alopecuroides were identified by silica gel thin-layer chromatography; the expansion agent was toluene— acetone—methanol—ammonia—water (8:3:0.2:0.5:8), and the chromogenic agent was modified bismuth potassium iodide solution, sophoridine, matrine, and sophocarpine in S. alopecuroides by TLC scanning at 500 nm. The linear ranges were 0.4152–2.4912 μg for sophordine, 0.4245–2.5470 μg for matrine, and 0.4101– 2.4606 μg for sophocarpine, with correlation coefficients of 0.9939, 0.9956, and 0.9975. Ultraviolet (UV) method was used to determine the total scavenging activity of the 2,2-diphenyl-1-picrylhdrazyl (DPPH) free radical. The best TLC identification condition was selected, and the antioxidant activity of DPPH was screened wit...

Thin-layer chromatographic identification of flavonoids and phenolic acids contained in cosmetic raw materials

ABSTRACTPolyphenols, widespread in the kingdom of plants, are targeted in numerous chemical, biochemical, and clinical studies. Flavonoids make a particularly interesting group of polyphenols due to their diverse biological activities. In both in vivo and in vitro studies, they strongly demonstrate antioxidant, anti-inflammatory, anticancer, and anti-aggregation activities, and they are also known for tightening the capillary blood vessels. Due to their remarkable biological activities, flavonoids find a wide spectrum of practical applications and the botanical materials rich in flavonoids are omnipresent in traditional medicines, phytotherapy, pharmacotherapy, and cosmetics. Particularly attractive biological properties and, hence, considerable valor of the cosmetics are attributed to such flavonoids, as rutin, quercetin, naringenin, catechin, catechin hydrate, and apigenin. In our study, we selected a simple and cost-friendly thin-layer chromatographic technique to identify the aforementioned flavonoids, and also caffeic, ferulic, gallic, and coumaric acids, in the commercially available cosmetic raw materials (such as extracts derived from red wine, grape skins, pomegranate peels and juice, green tea, etc.) Quercetin and rutin proved to be the two flavonoids most frequently encountered in the examined cosmetic raw materials. The results of such investigations provide the phenolics-oriented evaluation of the quality of the cosmetic raw materials and, hence, better judgment on the applicability thereof, e.g., as components of specific cosmetics prepared to treat various skin problems.

Finger Printing of the Anatomical Markers, HPTLC Profile and Heavy Metals Content in the Leaves of Aristolochia indica Linn.

Background: Aristolochia indica Linn is now an endangered medicinal plant belonging to the family Aristolochiaceae. Many ethono-botanically important species of Aristolochia were found used in the traditional forms of medicine for the treatment of various illnesses. Aim: The present study concerns the microscopic, fluorescent, powder, quantitative microscopic characteristics of the leaves of Aristolichia indica Linn and its physico-chemical standards. Materials and Methods: Morpho-histological profile, Highperformance thin-layer chromatographic (HPTLC) finger print profile, and heavy metals content of the leaves of Aristolichia indica Linn. Results: The presence of camptodromous pinnate venation with pentagonal shaped areoles with linear veinlet endings, non-glandular hooked trichomes, amphistomatic and anomocytic stomata, small palisade ratio, small stomatal index were the anatomical features characteristics of the taxon. Physico-chemical evaluation of the leaves gave moisture content of 10.5%, total ash 12.7%, acid insoluble ash 1.9%, acid soluble ash 10.8%. Highperformance thin-layer chromatographic (HPTLC) identification of marker compound (Aristolochic acid I or (AAI)) in methanol extract of leaves was carried out. The developed HPTLC Chromatogram had revealed nine phytoconstutents in extract of leaf sample. The Rf value for Aristolochic acid I (C17H11NO7) was found to be 0.41 and densitometric scanning had shown λmax at 318 nm for the marker compound. The content of marker constituent (AA I) present in shade-dried leaves of Aristochia indica Linn was estimated as 0.049%. The Flame atomic absorption spectrometric determination of elements had shown appreciable amounts of the elements such as Fe (0.5442 mg/g), Zn (0.026.82 mg/g), Ni (0.008 mg/g,) and Cu (0.002 mg/g) by dry weight of the leaves of Aristolichia indica. Conclusion: The present study suggests that the delineated characteristics of the leaves of A. indica, could tag as the identifying parameters to substantiate and authenticate the raw drugs from the spurious/adulterants materials and could also be effectively used for the regulatory perspectives and quality assessment of Aristololic acid Ι in the medicinal formulation/finished products. The presence of important mineral elements inside this plant showed that it could be a nutritious plant and important to the human health.

Thin-layer Chromatographic Identification of Chinese Propolis Using Chemometric Fingerprinting

Poplar tree gum has a similar chemical composition and appearance to Chinese propolis (bee glue) and has been widely used as a counterfeit propolis because Chinese propolis is typically the poplar-type propolis, the chemical composition of which is determined mainly by the resin of poplar trees. The discrimination of Chinese propolis from poplar tree gum is a challenging task. To develop a rapid thin-layer chromatographic (TLC) identification method using chemometric fingerprinting to discriminate Chinese propolis from poplar tree gum. A new TLC method using a combination of ammonia and hydrogen peroxide vapours as the visualisation reagent was developed to characterise the chemical profile of Chinese propolis. Three separate people performed TLC on eight Chinese propolis samples and three poplar tree gum samples of varying origins. Five chemometric methods, including similarity analysis, hierarchical clustering, k-means clustering, neural network and support vector machine, were compared for use in classifying the samples based on their densitograms obtained from the TLC chromatograms via image analysis. Hierarchical clustering, neural network and support vector machine analyses achieved a correct classification rate of 100% in classifying the samples. A strategy for TLC identification of Chinese propolis using chemometric fingerprinting was proposed and it provided accurate sample classification. The study has shown that the TLC identification method using chemometric fingerprinting is a rapid, low-cost method for the discrimination of Chinese propolis from poplar tree gum and may be used for the quality control of Chinese propolis.

Pharmacognostic and HPTLC Fingerprint Profile of the Root of Aristolochia indica Linn. and Quantification of the Marker Compound

Introduction: Root of Aristolochia indica Linn. has long been used as an oxytoxic agent to aid women in child birth and as abortifacient in Indian folk medicine. It is also one of the ingredients in some traditional Ayurveda medicinal preparations. Aims: The present work has been designed to delineate the pharmacognostic profile of the root of Aristolochia indica Linn and the High-performance thin-layer chromatographic (HPTLC) identification of the active compound and its quantitative estimation in the herbal sample. Materials and Methods: Macroscopic, microscopic evaluation, powder analysis, fluorescence standards of the root of Aristolochia indica Linn and its HPTLC fingerprint profile. Results: Pharmacognostic profile of the root investigated revealed the transverse section possessing somewhat circular outline with tissue organization as outer thin walled cork layers, narrow cortex, and inner cortical cells with groups of stone cells. Secondary xylem tissues were fissured to form narrow strips, wide medullary rays with greater quantities of parenchyma, ray cells with rich deposition of starch. Vessels were solitary and occluded with tyloses and starch grains with 'Maltese cross' were the characteristic features of the taxon. HPTLC method was developed for the estimation of the marker constituent, Aristolochic Acid I (AAI) in dried root sample. Chloroform: Methanol (6:2v/v), was used as mobile phase to separate the analyte. The Rf value for Aristolochic Acid I (C17H11NO7) was found to be 0.53. Calibration plot was established showing the dependence of Original Research Article European Journal of Medicinal Plants, 4(9): 1113-1124, 2014 1114 response on the amount chromatographed. Linearity was found to be in the concentration range of 100 to 500ng/spot for AAI with the correlation coefficient value r=0.998. The result showed that the content of marker compound (AAI) in dried root of Aristochia india Linn was 0.082%. Conclusions: The results of the present study suggest that, the documented morphological descriptors, delineated anatomical markers and developed HPTLC methods are complementary characteristics, which could be effectively used for the identification and authentication of the root of Aristolochia indica Linn.