Research Article Qualitative and quantitative phytochemical analy- sis of Heterophragma quadriloculare (Roxb.) K. Schum. leaves BHAVIKKUMAR SATANI1*, VILAS SURANA1, SHAILESH SHAH1, SHRI HARI MISHRA2 1Maliba Pharmacy College, Uka Tarsadia University, Bardoli-Mahuva Road, Bardoli, Gujarat, India. 2Emeritus Professor, Faculty of Pharmacy, The M. S. University of Baroda, Vadodara, Gujarat, India. ABSTRACT Objectives: Heterophragma quadriloculare (Roxb.) K. Schum. (HQ) is belonging to Bignoneace family. This plant has been traditionally utilized as anti-diabetic, antifungal, antiseptic and in skin disease like toe sores and chil- blain. Since, scientific documentation is not well available it was thought of interest to develop scientific data- base for HQ leaves. With this respect we have attempted qualitative and quantitative evaluation of various phy- tochemicals from HQ leaves. Methods: Successive extracts of HQ leaves have been analyzed by qualitative chemical tests and TLC. HPTLC fin- gerprints have also been developed for all extracts. Lupeol and ursolic acid have been identified and simulta- neously estimated in petroleum ether extract of HQ leaves. Amount of secondary metabolites i.e. alkaloids, tan- nins, phenolics and flavonoids, have been estimated using reported methods. Results: Carbohydrates, proteins, aminoacids, fats, alkaloids, steroidal compounds, flavonoids, terpenoids, tan- nins and phenolics were found in HQ leaves. By HPTLC method, 0.50 % and 0.93 % w/w of lupeol and ursolic acid have been observed in HQ leaves respectively. Percentage yield of alkaloid rich fraction was found to be in the range of 0.03-0.78 % w/w at different pH. Alkaloid rich fraction prepared at pH 10 was found to be best as per % yield and TLC profile. HQ leaves have been found to contain 3.14 %, 4.03 % and 4.09 % w/w of tannins, phenolics and flavonoids respectively. Conclusions: This report can create the way for further identification and isolation of phytoconstituents espe- cially alkaloids and terpenoids. Chromatographic fingerprint can also serve the purpose of drug standardization. The work can be further extended for drug discovery and drug development by isolation and characterization of phytoconstituents. Keywords: Heterophragma quadriloculare (Roxb.) K. Schum., Phytochemical profile, Quantitative phytochemical analysis, HPTLC fingerprinting, Alkaloid, Secondary metabolite known as Warras [1]. In India it is found in differ- 1. INTRODUCTION ent regions of Madhya Pradesh, Gujarat, Maha- rashtra, Andhra Pradesh, Karnataka and Tamil Heterophragma is a genus of family Bignoneace. Nadu [2-4]. This plant is utilised as anti-diabetic, Heterophragma quadriloculare (Roxb.) K. antifungal, antiseptic and in skin disease like toe Schum. (HQ) is representative of genus and *Corresponding Author – Bhavikkumar Satani, Maliba Pharmacy College, Uka Tarsadia University, Bardoli-Mahuva Road, Bardoli, Dist-Surat, Gujarat (394350), India. Email: [email protected] Received – 26/02/2016 Accepted – 20/05/2016 18 | Journal of Pharmacy and Applied Sciences | January - June 2016 | Volume 3 | Issue 1 Satani et al/ Phytochemistry of Heterophragma quadriloculare sores and in chilblain [5, 6]. Utility of HQ plant 2. MATERIALS AND METHODS material has been claimed traditionally by many authors but scientific documentation is not yet 2.1. Chromatographic condition for available. So it was thought of interest to develop HPTLC scientific database for HQ leaves. Data on phar- Whenever required separation was performed on macognostic and phytochemical characterization TLC plate precoated with silica gel 60 F254 (E. provide means to authenticate raw material as well Merk, Darmstsdt, Germany). Before sample appli- as their usage. Pharmacognostic work has already cation TLC plates were per-washed with methanol been published [5] but detailed phytochemical and dried in oven at 50 ˚C for 10 min. Samples profiling is yet to be carried out. Hence in present were spotted on TLC plate 15 mm from the bot- work we have attempted qualitative and quantita- tom edge using CAMAG Linomat V semi- tive evaluation of phytochemicals to step ahead in automatic spotter. The TLC plate was developed the direction of drug development and drug dis- in twin trough chamber. The TLC plate was covery. In present work alkaloids have been fo- scanned and analysed by CAMAG TLC Scanner cused which were remained unexplored till now. IV and WinCATS software respectively. Figure 1: Schematic presentation of preparation of Figure 2: Schematic presentation of preparation of alkaloid fractions by basification method (Method-1) alkaloid fractions by acidification method (Method-2) Journal of Pharmacy and Applied Sciences | January - June 2016 | Volume 3 | Issue 1 | 19 Satani et al/ Phytochemistry of Heterophragma quadriloculare 2.2. Preliminary phytochemical screening nm. For visualization of spots, plate was treated of HQ leaves with anisaldehyde sulphuric acid reagent (AS), heated at 110 °C for 5 min and scanned in visible 2.2.1. Preparation of extract from leaves by mode at 540 nm [9, 11]. successive solvent extraction 2.3. Qualitative and quantitative evalua- Dry powdered leaves of HQ (Voucher number: tion of secondary metabolites Pharmacy/HDT/HQ/09-10/01/BS) weighing 40 gms was extracted using soxhlet apparatus with 2.3.1. Identification and simultaneous es- solvents of increasing polarity starting from petro- timation of lupeol and ursolic acid leum ether followed by toluene, chloroform, ethyl acetate, acetone, methanol and water. Each time Lupeol and ursolic acid were simultaneously iden- before extracting with new solvent, the powdered tified in petroleum ether extract by co-TLC using leaf material was air dried. All extracts were con- aluminium backed precoated TLC plate of silica centrated by distilling the solvent followed by dry- gel 60 F254. A mixture of petroleum ether: ethyl ing on water bath. Consistency, colour, appear- acetate: toluene (7:2:1, v/v/v) was used as mobile ance of the extracts and their percentage yield phase and plate was treated with anisaldehyde sul- were recorded [7, 8]. phuric acid reagent for visualisation of band of phytoconstituents. 2.2.2. Preliminary phytochemical evalua- Lupeol and ursolic acid have also been simulta- tion of extracts by chemical test neously estimated in petroleum ether extract by All successive extracts of leaves were subjected to HPTLC method. Separation was performed on various qualitative chemical tests using reported aluminium backed plates precoated silica gel 60 methods to determine the presence and/or absence F254. The TLC plate was developed in twin trough of metabolites viz., alkaloids, glycosides, carbo- chamber saturated with petroleum ether: ethyl ace- hydrates, proteins and amino acids, phytosterols, tate: toluene (7:2:1, v/v/v) . The TLC plate was tannins and phenolics, terpenoids etc [7, 8]. dried, sprayed with anisaldehyde sulphuric acid reagent and analyzed at 580 nm. Amount of lupeol 2.2.3. Preliminary phytochemical evalua- and ursolic acid was calculated from peak area in tion of extracts by TLC terms of % w/w of dry leaves [12]. All successive extracts were subjected to thin 2.3.2. Qualitative and quantitative evalua- layer chromatographic studies using reported me- tion of alkaloids thods to verify presence of various phytoconstitu- ents like alkaloids, glycosides, carbohydrates, pro- Alkaloid fractions were prepared using pH depen- teins and amino acids, phytosterols, phenolics and dant method. Procedures adopted are given in Fig- terpenoids [9, 10]. ure 1 and Figure 2 [13]. Alkaloids are compound having nitrogen atom in 2.2.4. HPTLC fingerprinting of successive heterocyclic ring and/or out of heterocyclic ring. extracts of HQ Hence it was our first step to confirm the presence All successive extracts and methanol extract were of nitrogen in separated fraction. Lassaigne test dissolved in appropriate solvents and filtered. was performed for detection of nitrogen in each Clear solutions having concentration of 2 mg/ml fraction [14]. Further, each fraction was tested were used for HPTLC fingerprinting. Precoated with dragendorff’s reagent, mayer’s reagent, hag- HPTLC plate (10 x 10 cm) of silica gel 60 F 254 ger’s reagent and wagner’s reagent for presence of was used. The plate was developed with toluene: alkaloids [7, 8]. ethyl acetate (9:1, v/v) as mobile system. Air dried developed plate was scanned at 254 nm and 365 20 | Journal of Pharmacy and Applied Sciences | January - June 2016 | Volume 3 | Issue 1 Satani et al/ Phytochemistry of Heterophragma quadriloculare TLC profile of each alkaloid fraction was devel- 3. RESULT AND DISCUSSION oped on precoated silica gel 60 F254 TLC plates using chloroform: methanol (8.5:0.5, v/v) as m o- 3.1. Preliminary phytochemical screening bile phase and dragendorff’s reagent as a visualiz- of HQ leaves ing agent [9]. 3.1.1. Preparation of extract from leaves by Table 1: Preliminary phytochemical profile for leaves successive solvent extraction of HQ Powdered leaves of HQ were subjected to succes- Successive Colour Consistency % extract Yield sive solvent extraction (except water extract, (w/w) which was prepared by decoction). The different Petroleum Greenish Sticky mass 1.76 extracts obtained are recorded in Table 1 with Ether black their % yield, colour, and consistency. Toluene Green Sticky mass 0.38 Chloroform Green Sticky mass 0.2 3.1.2. Preliminary phytochemical evalua- Ethyl Acetate Blackish Sticky mass 0.48 green tion of extracts by chemical test Acetone Green Sticky