Research Article INTRODUCTION. O. G. Bhusnure 1 *, V. S. Pangave 1, S. B. Gholve 1, P. S. Giram 2, M. S. Attar 3 ABSTRACT

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1 Research Article Qualitative phytochemical screening and development of high-performance thin layer chromatography fingerprint profile of Andrographis paniculata (Leaf) O. G. Bhusnure 1 *, V. S. Pangave 1, S. B. Gholve 1, P. S. Giram 2, M. S. Attar 3 ABSTRACT Objectives: To develop HPTLC method for the determination of Andrographolide in Andrographis paniculata (Leaf). Materials and Methods: A simple, rapid, selective, and quantitative high-performance thin layer chromatography (TLC) method has been developed for the determination of Andrographolide in A. paniculata (Leaf). The alcoholic extract of A. paniculata samples was applied on TLC aluminum plate pre-coated with Silica gel60 F 254 and developed using chloroform:methanol:formic acid (7:2:0.5) v/v/v as a mobile phase. The plate was sprayed (derivatized) with sulfuric acid reagent followed by heating at 1100C for 3 min, and detection and quantification were carried out densitometrically using an ultraviolet detector at a wavelength of 235 nm. Conclusion: A simple, rapid, selective, and quantitative high-performance thin layer chromatography (TLC) method has been developed for the determination of Andrographolide in A. paniculata (Leaf). KEY WORDS: Andrographis paniculata (Leaf), Andrographolide, High-performance thin layer chromatography, Kalmegh, King of bitter INTRODUCTION Plants are a naturally gifted tool and the extractions, characterization of active compounds from medicinal plants have resulted in the discovery of new drugs with high therapeutic values. [1] Herbal extracts can act in a synergistic manner within the human body and provide unique therapeutic properties with minimal or no undesirable side effects. [2] Phytochemistry is the subject that deals with chemicals derived from plants. There are a large number of secondary metabolic compounds found in plants. Different phytoconstituents of herbal products are safer than synthetic medicine and beneficial in the treatment of diseases caused by free radicals, and it also protects the body from tissue injury. Photochemical comprise primary and secondary compounds. Chlorophyll, proteins and common sugars are included in primary constituents, and secondary compounds are terpenoid, alkaloids, and phenolic compounds. High-performance thin layer Access this article online Website: jprsolutions.info ISSN: chromatography (HPTLC) and high-performance liquid chromatography, both emerged efficient tools for phytochemical evaluation, and enable the analysis of several samples simultaneously. [3-6] It reduces both time and cost of analysis. The same plate can be visualized in several ways, and individual spots can be quantitatively determined by densitometry in a specific track, called a fingerprint. [7-9] Figure 1 shows the image of Andrographis paniculata wall belonging to family Acanthaceae, commonly known as Kalmegh is one of the widely used medicinal herbs. It is an important drug in the ancient system of medicine. Whole plant has a wide range of pharmacological activity. Andrographolide is used as a standard to analyze Kalmegh. Kalmegh is used mainly for liver disorders and jaundice. A decoction or infusion of leaves is used in general debility and dyspepsia and a tincture of the root as a tonic, stimulant, and aperients. The macerated leaves and juice, together with carminative spices such as cardamom, clove, and cinnamon, may be made into pills and prescribed for gripe and other 1 Department of Quality Assurance, Channabasweshwar Pharmacy College, Latur, Maharashtra, India, 2 Department of Pharmacology, Channabasweshwar Pharmacy College, Latur, Maharashtra, India, 3 Department of Pharmachemistry, Channabasweshwar Pharmacy College, Latur, Maharashtra, India *Corresponding author: O. G. Bhusnure, Department of Quality Assurance, Channabasweshwar Pharmacy College, Latur, Maharashtra, India. omprakashbhusnure@gmail.com Received on: ; Revised on: ; Accepted on: Journal of Pharmacy Research Vol 12 Issue

2 stomach ailments in infants. The leaves and roots also find use as an adjunct in the treatment of diabetes, malaria, cholera dysentery, enteritis, gastritis, pneumonia, pyelonephritis, diarrhea, and even rabies. The plant contains bitter glucosides: Andrographolide, neo andrographolide, panaculoside, flavonoids, andrographonin, panicalin, apigenin 7,4dimethyl ether4; diterpenoids1,4deoxy11 oxo andrographolide; 14deoxy1,12didehydroandrographolide, 14deoxyandrographolide, neoandrographolide, and andrographolide. MATERIALS AND METHODS Plant Material The A. Paniculata leaf powder was purchased from Genius Nature Herbs Pvt. Ltd, Coimbatore, India. Preliminary Phytochemical Analysis [7] The obtained extracts were subjected to phytochemical testing [Figure 2] according to the standard test. [4,5] Test for Alkaloids Test substance shaken with few drops of 2N HCI. Aqueous layer formed, decanted and to which one or two drops of Mayer s reagent added. Formation of white precipitate indicates the presence of alkaloids. Test for Triterpenoids The substance was warmed with Tin and Thionyl chloride. Purple coloration indicates the presence of Triterpenoids. Test for Flavonoids To the substance of alcohol, a few magnesium turnings and few drops of concentrated HCI were added and boiled for 5 min. Red coloration shows the presence of flavonoids. Test for Tannins The substance mixed with basic lead acetate solution. Formation of white precipitate indicates the presence of Tannins. Test for Quinones To the test substance, sodium hydroxide was added. Blue-green or red color indicates the presence of Quinone. Test for Protein To the test solution, the biuret reagent is added. The blue reagent turns violet in the presence of protein. Test for Sugars The substance was mixed with equal volume of Fehling s A and B solutions, heated on a water bath. Formation of red color is the indication of the presence of sugar. Test for Gum To the substance, add few ml of water and shake well. Formation of swells and adhesives indicates the presence of gum. It is concluded that tannin, alkaloid, flavonoids, and triterpenoid are present in A.P leaf [Table 1 and Figure 2]. Test for Steroids 1 g of substance was dissolved in a few drops of acetic acid, acetic anhydride, warmed, and cooled under tap water and drop of sulfuric acid were added along the sides of the test tube. Presence of green color shows the positive test for steroids. Table 1: Phytochemical evaluation of A.P Test Tannin Alkaloid Flavonoids Triterpenoid Result Figure 1: Image of Andrographis paniculata 744 Journal of Pharmacy Research Vol 12 Issue

1 2 3 4 Figure 2: Preliminary phytochemical analysis. (1) Test for steroids. (2) Test for flavonoids. (3) Test for sugars.

2 mm thick) used was obtained from E. Merck Ltd. (Mumbai, India). Reference standard andrographolide procured from Otto Chemie Pvt. Ltd, Mumbai.

methanol for 30 min by sonication extraction method. The extracts were filtered by Whatman filter paper and makeup to 10 ml in a volumetric flask.

3 Figure 2: Preliminary phytochemical analysis. (1) Test for steroids. (2) Test for flavonoids. (3) Test for sugars. (4) Test for protein Chemicals Analytical grade; alcohol, toluene, ethanol, formic acid, chloroform, methanol, and sulfuric acid are used. TLC pre-coated plate with Silica gel 60 F254 (10 10 cm; 0.2 mm thick) used was obtained from E. Merck Ltd. (Mumbai, India). Reference standard andrographolide procured from Otto Chemie Pvt. Ltd, Mumbai. Experimental Figure 3: Alcoholic extract of samples before derivatization under ultraviolet 254 nm Sample preparation About 500 mg of coarsely powdered plant samples were extracted with 5 ml absolute methanol for 30 min by sonication extraction method. The extracts were filtered by Whatman filter paper and makeup to 10 ml in a volumetric flask. Standard preparation About 10 mg of standard andrographolide dissolved in 10 ml of absolute Methanol. Chromatography Figure 4: Alcoholic extract of samples before derivatization under ultraviolet 366 nm Equipment A Cammag (Switzerland) HPTLC system equipped with a sample applicator Linomat V, Twin trough glass Chamber (20 10 cm) with SS lid, TLC Scanner IV, and Wincats an integrated Software (Switzerland). Procedure TLC Aluminum pre-coated plate with Silica gel60 GF254 (20 10 cm) was used with chloroform: methanol:formic acid (7:2:0.5) V/V/V as the mobile phase. Alcoholic extract of samples and andrographolide standard solution applied on the plate using Linomat V applicator. Cammag twin trough glass chamber (20 10 cm) with SS lid was used for development of TLC plate. The twin trough glass chamber was saturated with mobile phase for 20 min. TLC plate was developed to 8 cm distance above the position of the sample application. The plate was removed from the chamber and air dried at room temperature. This plate was sprayed (derivatized) with methanolic sulfuric acid reagent followed by heating at 1100C for 3 min and HPTLC fingerprint profile was snapped by Camag Reprostar III, before derivatization under ultraviolet 254 nm, 366 nm and after Journal of Pharmacy Research Vol 12 Issue

Figure 5: High-performance thin layer chromatography chromatogram of Andrographis paniculata leaf powder 1 µl (540 nm) Peak Start Max End Area RF H RF H % RF H A % 1 0.000 0.0000 0.003 0.0541 29.70 0.

4 Figure 5: High-performance thin layer chromatography chromatogram of Andrographis paniculata leaf powder 1 µl (540 nm) Peak Start Max End Area RF H RF H % RF H A % Figure 6: Identification of andrographolide Peak Start Max End Area RF H RF H % RF H A % Figure 7: Quantification of andrographolide (R 254) 746 Journal of Pharmacy Research Vol 12 Issue

5 Figure 8: Test for protein derivatization [Figures 3-5]. The plate was scanned before derivatization using Camag TLC Scanner IV at wavelength 235 nm. Wincats an integrated Software was used for the detection as well as for the evaluation of data. RESULTS AND DISCUSSION The preliminary phytochemical screening of A. paniculata leaves showed the presence of proteins, amino acids, alkaloids, flavonoids, glycosides, steroids, tannins and triterpenoid, Quinones, and Gum. The various mobile phases tried, the mobile phase containing chloroform: methanol:formic acid (7:2:0.5) v/v/v and the active principle andrographolide resolved as a dark gray color band at Rf very efficiently from the other components in alcoholic extract of A. paniculata wall (Leaf). Sharp peaks of andrographolide (standard and samples) were obtained when the plate was scanned at wavelength 235 nm [Figure 6]. Quantity of andrographolide found in samples was obtained automatically through graph [Figure 7]. Quantity of andrographolide found is mg in 500 mg of a drug sample in Kalmegh. No significant change of Rf or response to andrographolide was observed by comparing previous study down on HPTLC. [10-15] Calibration Results Area calibration for andrographolide at 235 nm [Figure 8]. CONCLUSION A suitable HPTLC method was developed for the identification andrographolide in plant sample using the stationary phase: TLC Silica gel 60 F254 and mobile phase: Chloroform:methanol:formic acid (7:2:0.5 v/v/v). After the application of bands using CAMAG Linomat 5, the plate was developed in the solvent system up to 70 mm in CAMAG twin trough chamber. A spectrum was recorded using CAMAG Scanner TLC IV, and the ʎmax was found to be 235 nm. After scanning the plate at 235 nm, the Rf value (0.72) of the andrographolide in plant sample and the amount was calculated with the help of CAMAG Vision CATS software, and it was mg in 500 mg of plant sample. ACKNOWLEDGMENT The work was supported by Anchrome Laboratory, Mumbai. Our special thanks to Management Committee members and My Guide Dr. Bhusnure O.G. and Dr. Thonte S.S., Principal, Channabasweshwar Pharmacy College, Latur, for their constant inspiration, moral support, and facility provided for this research. REFERENCES 1. Dhiman AK. Ayurvedic Drug Plants. New Delhi: Daya Publishing House; p Husain A, Virmani OP, Popli SP, Misra LN, Gupta MM, Srivastava GN, et al. Dictionary of Indian Medicinal Plant. Lucknow: CIMAP; p Seasotiya L. Phytochemical evaluation and HPTLC fingerprint profile of Cassia fistula. Int J Adv Pharm Biol Chem 2014;3: Brindha P, Sasikala B, Purushottaman KK. Pharmacognostic studies on Merugan kizhangu. BMEBR. Bull Med Ethnobot 1981;3: Edeoga HO, Okwu DE, Mbaebie BO. Phytochemical constituents of some Nigerian medicinal plants. Afric J Biotechnol 2005;4: Pawar RK, Singh KC. Development and validation of HPTLC method for determination of andrographolide from Andrographis paniculata (Whole plant). Int J Chem Res 2010;1: Dwivedi D, Thanwar M. A phytochemical investigation on Andrographis paniculata. J Chem Pharm Res 2015;7: Agarwal VS. Drugs Plants of India. Vol 1. Ludhiana: Kalyani Publishers: p Anubhuti P, Parul S. Identification and standardization of some herbal drugs, their extracts and marketed formulations by Journal of Pharmacy Research Vol 12 Issue

6 HPTLC Fingerprinting. Indo Am J Pharm Res 2011;6: Sushma GS, Devi BA, Madhulatha CH, Kumar KU, Harathi P, Subramaniam NS. Preliminary phytochemical screening and HPTLC fingerprinting of leaf extracts of Ficus nervosa Heyne ex Roth. J Chem Pharm Res 2013;5: Gowda JS, Veerabhadrappa SB. Study of in vitro antioxidant activity and HPTLC fingerprint of quercitin in Cassia auriculata L. Asian J Plant Sci Res 2013;3: Hemmalakshmi S, Priyanga S. Phytochemical screening and HPTLC fingerprinting analysis of ethanolic extract of Erythrina variegata. flowers Int J Pharm Pharm Sci 2016;8: Nile SH, Park SW. HPTLC densitometry method for simultaneous determination of flavonoids in selected medicinal plants. Front Life Sci 2014;8: Gallo FR, Multari G, Federici E, Palazzino G, Giambenedetti M, Petitto V, et al. Chemical fingerprinting of Equisetumarvense L. using HPTLC densitometry and HPLC. Nat Prod Res 2011;25: Gallo FR, Multari G, Federici E, Palazzino G, Nicoletti M, PetittoV. The modern analytical determination of botanicals and similar novel natural products by the HPTLC finger print approach. In: Atta-ur-Rahman, editor. Studies in Natural Products Chemistry. Bioactive Natural. Products. Vol. 37. Oxford: Elsevier; p Journal of Pharmacy Research Vol 12 Issue

DEVELOPMENT AND VALIDATION OF HPTLC METHOD FOR THE DETERMINATION OF ANDROGRAPHOLIDE IN KALMEGH NAVAYAS LOHA AN AYURVEDIC FORMULATION

International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 3, Issue 2, 2011 Research Article DEVELOPMENT AND VALIDATION OF HPTLC METHOD FOR THE DETERMINATION OF ANDROGRAPHOLIDE IN