Development of pharmacognostical parameters and estimation of quercetin using HPTLC in leaves of Nelumbo nucifera Gaertn

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1 PHCOG J ORIGINAL ARTICLE Development of pharmacognostical parameters and estimation of quercetin using HPTLC in leaves of Nelumbo nucifera Gaertn Reshma Jain, 1 and Sadhana Rajput* 2 1,2 Pharmacy Department, The Maharaja Sayajirao University of Baroda, Gujrat, INDIA 1 Research Scholar, Pharmaceutical Quality Assurance Laboratory, Centre of Relevance and Excellence in Novel Drug Delivery System, Pharmacy Department, G. H. Patel Building, Donor s Plaza, The Maharaja Sayajirao University of Baroda, Fatehgunj, Vadodara, Gujarat, India Professor, Pharmaceutical Quality Assurance Laboratory, Centre of Relevance and Excellence in Novel Drug Delivery System, Pharmacy Department, G. H. Patel Building, Donor s Plaza, The Maharaja Sayajirao University of Baroda, Fatehgunj, Vadodara, Gujarat, India Submission Date: ; Accepted Date: ABSTRACT Introduction: The leaves of Nelumbo nucifera Gaertn. (Nymphaceae) are reported to have great medicinal value. The main objective of the study is to establish the pharmacognostical and phytochemical evaluation of N. nucifera leaves. Morphoanatomy of leaves of this plant was studied in order to establish its complete profile to aid in its identification and avoid confusion in taxanomic species. These were established using light microscopy, WHO recommended physicochemical and phytochemical procedures. Methods: The leaves were first dried and the dried plant material was then subjected to size reduction to obtain coarse powder using grinding mill. Powder was examined for macroscopical, microscopical, physiochemical and preliminary phytochemical investigation as well as quantification of quercetin in hydro alcoholic extract of N. nucifera by HPTLC. Results and Conclusions: Some of the diagnostic features of the leaves are the presence of covering trichomes, fibers, epidermis, and collenchyma cells. The phytochemical screening reveals the presence of alkaloids, carbohydrates, amino acids and flavonoids. The total flavonoids and phenolic content in the hydro alcoholic extract of N. nucifera are ± 0.02 %w/w and ± 0.02 %w/w respectively. The HPTLC analysis data indicated that the collected N. nucifera leaves contain 2.5 ± %w/w quercetin of the total hydro alcoholic extract.validated HPTLC method developed can be used as a tool for standardization of leaves in different formulations using quercetin as a marker. Keywords: HPTLC, Nelumbo nucifera, pharmacognosy, phytoconstituents, quercetin. INTRODUCTION Nelumbo nucifera Gaertn. (Nymphaceae: Kamal) is a perennial aquatic herb with orbicular, peltate leaves, *Corresponding author. Sadhana Rajput Professor, Pharmaceutical Quality Assurance Laboratory, Centre of Relevance and Excellence in Novel Drug Delivery System, Pharmacy Department, G. H. Patel Building, Donor s Plaza, The Maharaja Sayajirao University of Baroda, Fatehgunj, Vadodara, Gujarat, India Phone No , Fax No sjrajput@gmail.com reshmajain_pharmacy@yahoo.com DOI: /pj pinkish or white flowers, spongy torous fruits, commonly found in tanks, lakes and marshy places, through out India. [1] All parts of N. nucifera have been used for various medicinal purposes, including the leaves, flowers, stamens, embryos, rhizomes, and seeds, have been used as traditional medicines and have pharmacological properties, including hepatoprotective, anti-oxidant activity, antipyretic effects, and prevention of atherosclerosis and fatty liver. [2] In particular, rhizomes, leaves and seeds showed antioxidant and hepatoprotective activities, anti-diabetic and anti-inflammatory effects of rhizomes, anti-hyperlipidemic effects of leaves, antifertility effects of seeds were also reported. [3] The leaves are known for diuretic and astringent properties, and are used to treat fever, sweating, and strangury and as a styptic. [4] Phcog J Dec 2012 Vol 4 Issue 34 31

2 However, there are no reports on the detail Pharmacognostical features of the leaves. Hence, the present investigation is an attempt in this direction and includes morphological and anatomical evaluation, determination of physico-chemical constants and preliminary phytochemical screening of different extracts of N. nucifera. Quantitative estimation of quercetin is important for current research and a TLC method for detection of flavonoids has been reported in literature. [5] However HPTLC method for quantification of quercetin from N. nucifera has not been reported in literature. Densitometric HPTLC has been widely used for the phytochemical evaluation of the herbal drugs, due to its simplicity and minimum sample clean up requirement. Plant material MATERIAL AND METHODS The leaves of Nelumbo nucifera Gaertn were collected locally from Vadodara in August 2010 and authenticated by herbal specimen at Botany Department, M. S. University of Baroda, Vadodara. The fresh plant material was used for the macroscopy and microscopy. The leaves were first dried in shade and than dried in an oven at C for 1 hrs. The dried plant material was then subjected to size reduction to obtain coarse powder using grinding mill. Chemicals and instruments Charged Coupled Device (CCD, Lawrence and Mayo) camera attached with compound microscope, solvents viz. petroleum ether, toluene, chloroform, ethyl acetate, acetone, ethanol (80%) and various reagents like phloroglucinol, glycerin, hydrochloric acid, chloral hydrate were procured from India Scientific, India. Folin Ciocaluteu reagent was procured from Sigma Chemical Co. (St. Louis, MO, USA) Pharmacognostical studies Macroscopy The plant was macroscopically examined for shape of leaves, apex, base, margin etc. [6] Microscopy Microscopic studies were done by preparing a thin hand section of midrib and lamina region of N. nucifera Garten leaf. The section was stained by phloroglucinol and conc. hydrochloric acid. Powder (# 60) of the dried leaf was used for the observation of powder microscopic characters. [7,8] Physicochemical parameters Physicochemical parameters were determined as per Ayurvedic Pharmacopoeia of India. Moisture content, total ash value, acid insoluble ash value, alcohol soluble extractive value and water soluble extractive value were determined. [8,9] Preliminary phytochemical studies The powder of dried leaves was subjected to continuous soxhlet extraction with various organic solvent such as petroleum ether, toluene, chloroform, ethyl acetate, acetone and methanol respectively. After concentration and drying of each extract in vacuum desiccator, identification of phytoconstituents was carried out using chemical test and thin layer chromatography method by different detecting reagents. [9 11] Estimation of total flavonoid content (I) Aluminum chloride colorimetric method The aluminum chloride colorimetric method was modified from the procedure reported by Woisky and Salatino [12] Quercetin was used to make the calibration curve. Ten milligrams of quercetin was dissolved in 80% ethanol and then diluted to 25, 50 and 100 µg/ml. The diluted standard solutions (0.5 ml) were separately mixed with 1.5 ml of 95% ethanol, 0.1 ml of 10% aluminum chloride, 0.1 ml of 1 M potassium acetate and 2.8 ml of distilled water. After incubation at room temperature for 30 min, the absorbance of the reaction mixture was measured at 415 nm with a Shimadzu UV-1700 spectrophotometer. The amount of 10% aluminum chloride was substituted by the same amount of distilled water in blank. Similarly 1000 µg/ml of sample solution was reacted with aluminum chloride for determination of flavonoid content as described in above procedure. (II) 2, 4-Dinitrophenylhydrazine colorimetric method The current method was modified from the procedure described by Nagy and Grancai. [13] (±)-Naringenin was used as the reference standard. Twenty milligrams of (±)-naringenin was dissolved in methanol and then diluted to 500, 1000 and 2000 µg/ml. One milliliter of each of the diluted standard solutions was separately reacted with 2 ml of 1% 2, 4-dinitrophenylhydrazine reagent and 2 ml of methanol at 50 C for 50 min. After cooling to room 32 Phcog J Dec 2012 Vol 4 Issue 34

3 temperature, the reaction mixture was mixed with 5 ml of 1% potassium hydroxide in 70% methanol and incubated at room temperature for 2 min. Then, 1 ml of the mixture was taken, mixed with 5 ml of methanol and centrifuged at 1,000 g for 10 min to remove the precipitate. The supernatant was collected and adjusted to 25 ml. The absorbance of the supernatant was measured at 495 nm. Similarly, 5 mg/ml sample solution was reacted with 2, 4-dinitrophenylhydrazine for determination of flavonoid content as described in above procedure. Total phenolic content The total phenolic content of the extracts were determined by the Folin-Ciocalteau method. [14] 10 mg extract per 10 ml of methanol was filtered with whatman No. 1 paper. 1 ml of the sample was added to 25 ml of 0.2 N Folin-Ciocalteau reagent and placed for 5 minutes. 4 ml of 20% of Na 2 CO 3 were then added and the total volume made up to 25 ml using distilled water. The above solution was then kept for incubation at room temperature for 30 min. Absorbance was measured at 765 nm using Shimadzu UV-1700 spectrophotometer. Gallic acid ( µg/ml) was used to produce standard calibration curve. The total phenolic content was expressed in mg of gallic acid equivalents/g of extract. Fluorescent studies of powder drugs A pinch of dried powdered plant material was taken in a clean test tube with about 10 ml of solvent like acetone, benzene, petroleum ether chloroform, ethanol, glacial acetic acid, HCl, HNO 3, methanol and distilled water. All the tubes were shaken well and incubated for about 30 min. The colors of the drug solutions thus obtained were observed for their characteristic color reaction under the visible light (fluorescent tube) and ultra violet light (UV 366 nm) and were recorded by comparing with a standard color chart [15] ( Table-5) Quantification of quercetin in hydro alcoholic extract of N. nucifera leaves by high performance thin layer chromatography Reagents and standards Methanol, Toluene, ethyl acetate and formic acid were procured from Merck, Quercetin was procured from Sigma Aldrich, Bangalore, India. Preparation of hydro alcoholic extract of N. nucifera About 100 gm of the powder was defatted with petroleum ether, taken in a Soxhlet extractor and extracted with hydro alcohol. (Ethanol: Water, 80:20) The solvent was recovered by distillation. The residue was concentrated to the semi solid mass and lyophilized to obtain the dry extract powder. The same was stored in the desiccator for further experiment and analysis. Preparation of quercetin standard solution A stock solution of standard quercetin (100 μg/ml) was prepared by transferring 1 mg of quercetin, accurately weighed, into a 10 ml volumetric flask, dissolving in methanol. Preparation of sample solution Accurately weighed 100 mg of dried hydro alcoholic extract of N. nucifera was transferred to a 100 ml volumetric flask, initially dissolving in 80 ml of methanol. It was then sonicated for 10 minutes and the contents of the flask were filtered through whatman No. 1 paper (Merck, Mumbai, India). The final volume of the solution was made up to 100 ml with methanol to get stock solution containing 1.0 mg/ml. Instrumentation and chromatographic conditions HPTLC was performed on 10 cm 10 cm aluminum backed plates coated with silica gel 60F254 (Merck, Mumbai, India). Standard solution of quercetin and sample solution were applied to the plates as bands 6.0 mm wide, 30.0 mm apart, and 10.0 mm from the bottom edge of the same chromatographic plate by use of a Camag (Muttenz, Switzerland) Linomat V sample applicator equipped with a 100 μl Hamilton (USA) syringe. Ascending development to a distance of 80 mm was performed at room temperature (28 ± 2 C), with toluene: methanol: ethyl acetate: formic acid, 5: 1: 4: 0.2 (v/v/v), as mobile phase, in a Camag glass twin-trough chamber previously saturated with mobile phase for 20 min. After development, the plates were dried with a hair dryer and sprayed with 10% AlCl 3 then scanned at 366 nm with a Camag TLC Scanner with WINCAT software, using the deuterium lamp. The method was validated according to the ICH guidelines. Calibration curve of quercetin A stock solution of standard quercetin (100 μg/ml) was prepared in methanol. Different volume of stock solution 1, 5, 10, 15, 20 and 25 μl, were spotted on to TLC plate to obtain concentration 100, 500, 1000, 1500, 2000 and 2500 ng/spot of quercetin, respectively. The data of peak areas plotted against the corresponding concentrations. Phcog J Dec 2012 Vol 4 Issue 34 33

limit of quantification as per ICH guidelines.

$The fresh leaves are green, leathery, brittle and membranous, petioles of the aerial leaves are erect and stout. Odour is distinct and fracture is fibrous (Figure 1). N.$

4 Reshma Jain, et al.: Pharmacognostical parameters and HPTLC method Validation of developed HPTLC method The developed method was validated in terms of linearity, precision, accuracy, specificity, limit of detection and limit of quantification as per ICH guidelines.[16] RESULTS AND DISCUSSION The morphological studies revealed that it is compound, opposite, bipinnate leaf with oblong to orbicular shape, cm in diameter and mucoranate apex. The fresh leaves are green, leathery, brittle and membranous, petioles of the aerial leaves are erect and stout. Odour is distinct and fracture is fibrous (Figure 1). N. nucifera Garten revealed the presence of lower and upper epidermis, xylem and phloem, vascular bundle, mesophyll, large cavities in the center and small cavities in the periphery, branch trichomes and collenchyma (Figure 2). The powder Figure 1. Leaves of Nelumbo nucifera. Figure 3. Powder microscopy of N.nucifera leaves (a) Calcium oxalate crystals (b) lamina (c) Anomocytic Stomata (d) branch trichome. icroscopy of the leaves revealed the presence of fibers, m xylem, phloem, epidermal cell, anomocytic stomata and covering trichome (Figure 3). The proximate analysis result show that the moisture content, total ash value, acid insoluble ash value, alcohol soluble extractive value and water soluble extractive value were 4.62 ± 0.33%, 4.26 ± 0.15%, 2.31 ± 0.09%, 7.84 ± 0.27%, and 8.71 ± 0.18% respectively (Table 1). Successive solvent extractions were shown in percentage of yield along with physical appearance. The percentage yield values for petroleum ether, toluene, chloroform, ethyl acetate, acetone and methanol were 4.42%, 3.17%, 2.44%, 2.19%, 5.71% and 8.36% respectively (Table 2). All extract were then subjected to study chemical nature of the drug (Table 3). Table 1. Evaluation of leaves of Nelumbo nucifera Garten. Parameters Moisture content Total ash value Acid insoluble ash Alcohol soluble extract Water soluble extract Value obtained on dry weight basis (% w/w)* 4.62 ± ± ± ± ± 0.18 *Average of three reading ± SEM on of leaves of Nelumbo nucifera Garten. Table 2. Successive solvent extraction of leaves of Nelumbo nucifera Garten. Figure 2. Transverse Sections of N.nucifera leaves (a) Midrib (b) Lower part of mid rib (c) lamina with rosette crystals (d) cortex with xylem vessels. 34 Solvent used Color & consistency Petroleum ether Toluene Chloroform Ethyl acetate Acetone Green solid mass Dark green solid mass Blackish green residue Brown solid mass Dark Brown semisolid mass Average extrac tive values on dry wei ght basis (% w/w) Phcog J Dec 2012 Vol 4 Issue 34

5 Table 3. Chemical examination of various extracts of leaves of Nelumbo nucifera Garten. Constituents Extract P T C A M Alkaloids + Carbohydrates + + Proteins & amino acids Saponin Fixed oil/ Fat Gums/ mucilage Flavonoids + + Phenolic The preliminary phytochemical studies revealed that petroleum ether fraction contain fixed oil and fats, toluene fraction also contains fixed oils, chloroform fraction contain carbohydrate and saponins, acetone fraction contains carbohydrate, saponin and flavanoids and phenolics, while methanolic fraction contain carbohydrate, proteins and amino acids, saponin, phenolic and flavonoids (Table 3). The extract showing presence of phytoconstituents of interest was further subjected to TLC (Table 4). Total flavonoid and phenolic content in the hydro alcoholic extract of N. nucifera were ± 0.02% and ± 0.02% respectively (Table 5). The fluorescence character of powdered drug plays a vital role in the determination of quality and purity of the drug material. In the present study, powder treated with various reagents shows characteristic fluorescence at 254 nm and 366 nm wavelength (Table 6). Solvent system Used Toluene: Ethyl acetate (93:7) Ethyl acetate: Methanol: Water: (100:13.5:10) Chloroform: Methanol: Formic acid (10:0.3:0.1) Table 4. TLC Screening of various crude drug extract of Nelumbo nucifera leaves Garten. Detection Reagent Observation Inference P T C A M VS reagent Red/Yellow/Brown/ Essential + + Blue-green Oil AS reagent Pink/green Essential + + Oil AS reagent Red/Yellow/Brown/ Bitter Principle + + Blue-green VS reagent Blue Saponin NP/PEG/ and Yellow colour Flavonoid + + UV 10% AlCl 3 Yellow colour Anthrone LB Dark green Phytosterol + reagent (P = Petroleum ether extract; T = Toluene extract; C = Chloroform extract; A = Acetone extract; M = Methanol extract, VS = Vanillin sulphuric acid, AS = Anisaldehyde sulphuric acid, LB = Libermann Burchard, + Indicates presence of constituents. Indicates absence of constituents) Table 5. Total flavonoids and phenolic content in the hydroalcoholic extract of N.nucifera leaves. Sample Flavonoid content (%w/w)* Total phenolic content (%w/w)* By AlCl 3 method By 2,4 DNPH method Total Flavonoid content N.nucifera extract 3.61 ± ± ± ± 0.02 *Results were presented as mean ± SD (n = 3). Table 6. Fluorescence analysis of N. nucifera leaf powder under day light and ultra violet (UV) radiation. Sr.No. Treatment Fluorescence Day light UV light 1. Powder as such Light green Bright green 2. Powder + 1N NaOH (Aq.) Brown Greenish black 3. Powder+ 1 N NaOH (MeOH) Light green Dark green 4. Powder+ 1 N HCl (Aq.) Reddish yellow Green 5. Powder + Iodine (N/50) Dark green Black 6. Powder + 50% H 2 SO 4 Brownish orange Black 7. Powder + 50 % HNO 3 Reddish yellow Dark green Phcog J Dec 2012 Vol 4 Issue 34 35

Quantification of quercetin in hydro alcoholic extract of N. nucifera leaves by HPTLC The mobile phase consisting of toluene: methanol: ethyl acetate: formic acid (5: 1: 4: 0.

6 Quantification of quercetin in hydro alcoholic extract of N. nucifera leaves by HPTLC The mobile phase consisting of toluene: methanol: ethyl acetate: formic acid (5: 1: 4: 0.2, v/v/v) gave a sharp peak at R f : Well-defined spots were obtained when the chamber was saturated with mobile phase for 20 min at room temperature. The TLC plate was visualized under UV light at 254 nm. Photographs of a TLC plates after chromatography of quercetin standard and a hydro alcoholic extract of the dried leaves of N. nucifera were shown in Figure 4. The identity of the quercetin bands in sample chromatograms were confirmed by comparison of the chromatogram obtained from the sample with that obtained from the reference standard solution (Figure 5 a & b) and by comparing retention factors of quercetin from sample and standard solutions. The peak corresponding to quercetin from the sample solution had same retention factor as that from the quercetin standard (R f : 0.45) Table 7. Summary of validation parameters of quercetin for the developed HPTLC method. Sr. No. Parameter Results 1. Linearity Precision Repeatability of measurement 0.71% Repeatability of application 0.85% Intraday % Interday % 3. Range of linearity ng/spot 4. Limit of quantification (LOQ) Limit of detection (LOD) Accuracy % 7. Specificity Specific Validation of HPTLC method for estimation of quercetin in leaves of N. nucifera The summary of all validation parameters for the developed HPTLC method for quercetin are listed in Table 7. CONCLUSION Figure 4. HPTLC plates of N.nucifera derivatised by 10%AlCl 3. Figure 5. (a) Chromatogram of Standard quercetin (b) Chromatogram of hydro alcoholic extract of N. nucifera. Establishing standards is an integral part of establishing the correct identity and quality of a crude drug. The majority of the information on the identity, purity and quality of the plant material can be obtained from its macroscopy, microscopy and physiochemical parameters. As there is no record on detail pharmacognostical work on leaves of N. nucifera. The present work is undertaken to establish some pharmacognostical standards. With various biological activities, flavonoids are reagrded as key candidate compounds for evaluating the quality of propolis products. However, the convenient colorimetric method utilizing aluminum chloride reaction to determine flavonoids contents was proved to be specific only for flavones and flavonols, while another colorimetric method utilizing 2, 4-dinitrophenylhydrazine reaction was specific for flavanones. A rapid, simple, accurate and specific HPTLC method for quantitative estimation of quercetin present in the hydro alcoholic extract of leaves of N. nucifera has been developed and validated. The data could be used as a QC standard. The method used in this work resulted in good peak shape and enabled good resolution of quercetin from other constituents of the plant material. Because recovery ( ) was close to 100%, there was no interference with the quercetin peak from other constituents present in the plant. 36 Phcog J Dec 2012 Vol 4 Issue 34

7 ACKNOWLEDGEMENTS The authors are thankful to Botany Dept., Faculty of Science, The M. S. University of Baroda for their technical support. REFERENCES 1. La Cour B, Molgaard P, Yi Z. Traditional Chinese medicine in treatment of hyperlipidaemia. J. Ethnopharmacol., 1995; 46(2): Sohn DH, Kim YC, Oh SH, Park EJ, Li X, Lee BH, et al. Hepatoprotective and free radical scavenging effects of Nelumbo nucifera. Phytomed. 2003; 10(2 3): Mukherjee PK Sah D J Pal M, Saha BP. Studies on the antiinflammatory activity of rhizomes of Nelumbo nucifera. Planta Med. 1997; 63: Hu M, Skibsted LH. Antioxidative capacity of rhizome extract and rhizome knot extract of edible lotus. Food chem. 2002; 76: Quality Standards of Indian Medicinal Plants, Vol. 3, Published by Indian council of Medicinal Research, New Delhi, 2005; Jain RA Shukla SH. Pharmacognostic Evaluation and Phytochemical Studies on Stem of Clitoria ternatea linn. PHCO J.2011; 3 (24): Mukherjee PK, Quality Control of Herbal Drugs, Business Horizon Pharmaceutical Publishers, New Delhi pp , Wallis T.E., Practical Pharmacognosy, 6th ed. (J. & A. Churchill Ltd., London) , , Khandelwal KR. Practical Pharmacognosy Technique and Experiments. 13th ed. Nirali Prakashan, Pune, , The Indian Pharmacopoeia. Vol. II. The Controller of Publications, New Delhi, A-53, A-54; A-89, Evans WC. Trease and Evans. Pharmaconosy. 15th ed. W.B. Sounders Company Ltd, London. 224, 230, 336, 541 5, Woisky R. and Salatino A. Analysis of propolis: some parameters and procedures for chemical quality control. 1998; J. Apic. Res. 37: Nagy, M. and Grancai, D. Colorimetric determination of flavanones in propolis. Pharmazie. 1996; 51: Sigleton V L, Rossi Jr. J A. Colorimetry of Total Phenolics with Phosphomolybdic-Phosphotungstic Acid Reagents. American J. Enol. Viticult. 1965: 16(3): Chase CR and Pratt RJ. Flourescence of powdered vegetables drugs. Ind. Journal of Experimental Biology. 1949; 33(6): I.C.H. Guidelines on Analytical Method Validation, In: Proc. Int. Convention on Quality for the Pharmaceutical Industry, Toronto, Canada, Sept Phcog J Dec 2012 Vol 4 Issue 34 37

Research Article GALLIC ACID AND FLAVONOID ACTIVITIES OF AMARANTHUS GANGETICUS

ISSN 2395-3411 Available online at www.ijpacr.com 238 Research Article GALLIC ACID AND FLAVONOID ACTIVITIES OF AMARANTHUS GANGETICUS G. Jyoti Jain 1* and S. Ramachandra Setty 2 1 Department of Pharmacology,