Phytochemical analysis of Hibiscus caesius using high performance liquid chromatography coupled with mass spectrometry Quratul Ain1, Muhammad Naveed1, Abdul Samad Mumtaz*1, Muhammad Farman2, Iftikhar Ahmed3 and Nauman Khalid4* 1 Department of Plant Sciences, Quaid-i-Azam University, Islamabad, Pakistan Department of Chemistry, Quaid-i-Azam University, Islamabad, Pakistan 3 National Institute for Genomics and Advanced Biotechnology (NIGAB), National Agricultural Research Centre (NARC), Park Road, Islamabad, Pakistan 4 Graduate School of Agricultural and Life Science, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, Japan 2 Abstract: Various species in genus Hibiscus are traditionally known for their therapeutic attributes. The present study focused on the phytochemical analysis of a rather unexplored species Hibiscus caesius (H. caesius), using high-pressure liquid chromatography coupled with mass spectrometry (HPLC-MS). The analysis revealed five major compounds in the aqueous extract, viz. vanillic acid, protocatechoic acid, quercetin, quercetin glucoside and apigenin, being reported for the first time in H. caesius. Literature suggests that these compounds have important pharmacological traits such as anticancer, anti-inflammatory, anti-bacterial and hepatoprotective etc. however, this requires further pharmacological investigations at in vitro and in vivo scale. The above study concluded the medicinal potential of H. caesius. Keywords: H. caesius, Phytochemicals, medicinal value, HPLC-MS. INTRODUCTION Hibiscus sabdariffa commonly known as sorrel or roselle is extensively studied plant; literature bears a wealth of information about its phytochemistry and pharmacological effects. Anthocyanins, flavonoids and polyphenols are the major chemical constituents of flowers of Hibiscus sabdariffa (Lin et al., 2007) contributing to its practical medicinal effects. The calyx drink is rich in ascorbic acid, carotene, calcium, riboflavin, iron and niacin which are nutritionally important (Yadong et al., 2005). The petals are the potential resource of antioxidizing agents as anthocyanins and ascorbic acid (Prenesti et al., 2007). Various biologically active compounds are reported in H. tiliaceus (Chen et al., 2006), H. rosasinensis (Gauthaman et al., 2006; Gilani et al., 2005). Hibiscus rosasinensis, grown as ornamental plant is reported to have favorable effects in heart diseases especially in ischemic disease (Gauthaman et al., 2006). However, Hibiscus caesius is a poorly studied species. The compounds isolated from H. sabdariffa could be a source of its therapeutic and pharmacological properties (Ali et al., 2005). In the Indian Ayurvedic literature, different parts of this plant have been suggested as a cure for complaints for instance hypertension, pyrexia, liver disorders and as an antidote to poisoning chemicals (Agoreyo et al., 2008). Various nerve and heart disorder, high blood pressure and calcified arteries have also been treated with this plant (Asolkar et al., 1992). Hibiscus pigments reduce the blood viscosity thus decreasing frequent occurrence of liver inflammation, necrosis and leucocyte infiltration (Kong et al., 2003). Phytochemical investigation is vital to make appreciable use of medicinal plants. Current study was conducted to explore the phytochemistry of relatively unexplored species of Hibiscus using HPLC-MS and comparing it with a well-studied species H. sabdariffa which have several documented health benefits e.g., Hypoglycaemic (Agoreyo et al., 2008; Sini et al., 2011), Hypolipidaemic (Farombi and Ige 2007), hypocholestrodemic (Lin et al., 2007), antioxidant (Akim et al., 2011; Mohd-Esa et al., 2010), antimicrobial (Fullerton et al., 2011; Zhang et al., 2011), antilithic (Laikangbam and Damayanti Devi 2012; Woottisin et al., 2011), anticancerous (Lin et al., 2012) and diuretic. MATERIALS AND METHODS Plant material and extraction Two species of the genus Hibiscus (H. caesius and H. sabdariffa) were selected from botanical garden of Quaidi-Azam University Islamabad and its taxonomic status were verified from Department of Plant Sciences, Quaide-Azam University, Islamabad, Pakistan. Three samples of each species were taken for the analysis. The petals and sepals of the flowers were collected and shade dried for 10 days and ground to fine powder using mortar and pestle. 10 g powder was transferred to airtight container and kept at -20oC before analysis. The weighed powdered samples were subjected to acid hydrolysis by adding 2M HCl (Wako Pure Chemical Industries Ltd., Osaka, Japan) and boiled for 3 hr at 100oC. Acid is used to convert cellulose or starch to sugar and to separate the sugar moieties from aglycone part to analyze them *Corresponding author: e-mail: nauman_khalid120@yahoo.com; asmumtaz@qau.edu.pk Pak. J. Pharm. Sci., Vol.28 No.5, September 2015, pp.1625-1629 1625 Phytochemical analysis of Hibiscus caesius using high performance liquid chromatography separately. The crude extract was filtered using Whatman No.1 filter paper. Sample preparation for UV spectroscopy UV spectroscopy plays an essential role in identification of many plant constituents, and frequently used to screen crude plant extracts for the presence of phytochemicals. For colorless compounds, measurements were made in the range 200-400nm and for colored compounds at 200700nm. The UV spectra of the isolated compounds were recorded on UV-Visible spectroscope. The analysis was done using Spectrophotometer model no. UV-1700 (E) 23 OCE, UV 1700 Pharmaspec, Shimadzu Corporation. High-performance liquid chromatography (HPLC-MS) The dried filter papers were dissolved in methanol and refiltered with double filter paper and samples for UV spectroscopy were prepared by diluting with MeOH (Wako Pure Chemical Industries Ltd., Osaka, Japan). Solvent extraction with ethyl acetate (Wako Pure Chemical Industries Ltd., Osaka, Japan) was done for the separation of non-polar compounds and the polar compounds remained in the aqueous layer. The aqueous layer was then extracted with n-butanol (Wako Pure Chemical Industries Ltd., Osaka, Japan) for anthocyanidins. The n-butanol was evaporated using rotary evaporator at 38ºC. LC separation of the extracts were performed on an Agilent 1200 series LC system coupled with Chem station for LC, 3-D system Rev.B.01.03 [2004]. DAD detected peaks at three different wavelengths 254, 320 and 370nm. The volume of injected sample was 5 µL, and elution was performed at ambient temperature. The gradient program starting from 10% CH3CN (eluent B, Wako Pure Chemical Industries Ltd., Osaka, Japan) organic solvent in 90% double distilled H2O (eluent A) aqueous solvent. Compounds were separated on HPLC column (4.6 × 150mm stainless-steel column packed with Agilent Eclipse XRD C-18 (5 µm). The compound appeared at tR of 7.0min on HPLC chromatogram. This tR corresponds to the eluent composition consisting of 10% CH3CN in H2O indicating its hydrophobic character and giving an evidence for the presence of phenolic acid type moiety. The DAD was observed at three different wavelengths i.e., 254, 320 and 380nm. The UV spectrum (fig. 1a) showed single band pattern at 260 nm. The λmax value and also the shape of UV-visible spectrum indicated the presence of vanillic acid type moiety. The ESI-MS of compound displayed in positive mode [M+H] + (fig. 1b) showed the signal at TIC time 7.1 min. The molecular ion peak exhibited at m/z=527.1 a.m.u, which was also the base peak referring to most stabilized fragment formed by the addition of a molecule of glucose, an acetyl xylose molecule and an atom of sodium. The molecular mass was inferred to be 526 a.m.u and the compound was identified as vanillyl - 4 - [O-glucosyl] - O- acetylxyloside. The λmax value and also the shape of UV-visible spectrum indicated the presence of vanillic acid type moiety when compared to literature (Yi et al., 2000). Fig. 1(a): DAD plot of vanillyl-4-[O-glucosyl]-Oacetylxyloside (b) Mass spectrum and structure of vanillyl-4-[O-glucosyl]-O-acetylxyloside. RESULTS HPLC-MS Profile The aqueous extract of H. caesius was analyzed by reverse phase HPLC. Using 60 min, 10-80% organic solvent CH3CN. UV absorbance was observed at 254, 320 and 370 nm. The identification of compounds were inferred on the basis of comparing retention time (tR), TIC, UV absorption spectrum and mass fragmentation pattern with reference literature (Marbey et al., 1970). Graph was plotted between tR and concentration of solvent B (CH3CN). Different molecules were eluted at different tR. During tR 0-10 min., phenolic acid type moieties were eluted followed by various secondary metabolites glycosides and towards the end aglycones were eluted. 1626 Fig. 2(a): DAD plot of protocatechoyl-4-[O-rhamnosyl]O-acetylrhamnoside (b) Mass spectrum and structure of protocatechoyl-4-[O-rhamnosyl]-O-acetylrhamnoside. Pak. J. Pharm. Sci., Vol.28 No.5, September 2015, pp.1625-1629 Quratul Ain et al The compound appeared at tR14.11 min on HPLC chromatogram. This tR corresponds to the eluent composition of 10% CH3CN in H2O indicating its hydrophobic character and giving an evidence for the presence of phenolic acid type moiety. The DAD showed single band pattern at λmax295 nm (fig. 2a). The λmax value and also the shape of the UV indicated the presence of protocatechuic acid type moiety. The molecular ion peak at m/z=489 displayed in positive ion mode [M+H]+ (fig. 2b) at TIC time 14.2 min which was also the base peak referring to most stabilized fragment and the molecular mass was inferred to be 488 a.m.u. [M+H]+. The fragment ion peak atm/z 155 [M+H]+ appeared due to the loss of a rhamnose and an acetyl rhamnose moiety. The structure proposed on the basis of tR, TIC time, DAD plot and mass fragmentation pattern was protocatechoyl-4-[Orhamnosyl]-O-acetylrhamnoside. The λmax value and also the shape of UV-visible spectrum indicated the presence of phenolic acid type moiety when compared to literature (Chen et al., 2012). spectrum indicated the presence of apigenin type moiety when compared to literature (Molyneux et al., 1970). The compound appeared at tR of 34.072min on HPLC chromatogram. This tR corresponds to the eluent composition of 34% CH3CN in H2O indicating its hydrophilic character and giving an evidence for the presence of flavonol type moiety. The DAD was observed at three different wavelengths i.e., 254, 320 and 370 nm. The UV spectrum (fig. 4a) having double band pattern showing band II at 255 nm and band I at 371 nm. The λmax value and also the shape of UV spectrum indicated the presence of quercetin type moiety when compared to literature (Mabry et al., 1970). The ESI-MS of compound displayed in positive mode [M+H]+ (fig. 4a) showed the signal at TIC time 34.4 min. The molecular ion peak appeared at m/z=465.2. The fragment ion peak which was also the base peak appeared at m/z =251.2 due to the loss of one glucose molecule along with one molecule of water and two carbon monoxide molecules [M+Na-glcH2O-2CO]. The molecular mass was inferred to be 464 a.m.u. indicating the presence of Quercetin-7-O-glucoside (fig 4b). Fig. 3(a): DAD plot of apigenin-7-O-acetylxyloside (b) Mass spectrum and structure of apigenin-7-Oacetylxyloside. The compound appeared at tR 33.1 min. on HPLC chromatogram corresponding to the eluent composition of 33% CH3CN in H2O indicating its hydrophilic character and giving an evidence for the presence of flavone type moiety. The DAD observed at three different wavelengths i.e., 254, 320 and 370 nm showed double band pattern (fig. 3a) having peak II at 271 nm and peak I at 355 nm. The λmax value and also the shape of UV spectrum are related to the apigenin type moiety. The ESI-MS of compound displayed in positive mode [M+H] + (fig. 3a) showed the signal at TIC time 33.2 min. The molecular ion peak appeared at m/z=463.2. The fragment ion peak which was also the base peak at m/z =364.7 appeared due to the loss of acetyl 1,3 Xo xylosyl residue along with a molecule of water where ionization occurred by Na+ ion suggesting the presence of apeginine-7-O- acetylxyloside (fig. 3b). The λmax value and also the shape of UV-visible Pak. J. Pharm. Sci., Vol.28 No.5, September 2015, pp.1625-1629 Fig. 4(a): DAD plot of quercetin-7-O-glucoside (b) Mass spectrum and structure of quercetin-7-O-glucoside. The appearance of compound at tR38.8 min corresponding to the eluent composition of 38% CH3CN in H2O indicated its hydrophilic character and giving an evidence for the presence of flavonol type moiety. The DAD was observed at three different wavelengths i.e., 254, 320 and 370 nm. The UV spectrum (fig. 5a) showed double band pattern having band II at 370 nm and band I at 255 nm. The λmax values and also the shape of UV-visible spectrum indicated the presence of quercetin type moiety when compared to literature (Mabry et al., 1970). The molecular ion peak appeared at m/z=303.4 which is also the base peak displayed in positive mode [M+H]+ (fig. 5b) showed the signal at TIC time 39.0 min. The molecular mass was inferred to be 302a.m.u, which indicated the presence of quercetin aglycone. 1627 Phytochemical analysis of Hibiscus caesius using high performance liquid chromatography CONCLUSION The current study focused on the phytochemical analysis of the unexplored species H. caesius concluding that the phytochemicals in H. caesius are of great pharmacological value, which may present a basis for the development of new drugs. REFERENCES Fig. 5(a): DAD plot of Quercetinaglycone (b) Mass spectrum and structure of quercetinaglycone. DISCUSSION The compounds: Vanillic acid, protocatechoic acid, quercetin, quercetin glucoside, apigenin identified in the current study have previously been identified in the extracts of H. sabdariffa and found to be the cause of its pharmacological importance (Kong et al., 2003; Lin et al., 2012). Quercetin, quercetin glucoside and apigenin has been identified by HPLC-MS in Hibiscus esculentus and confirmed by NMR (Sini et al., 2011). Apigenin has been found naturally in a number of plants (Lepley et al., 1996) and is reported in literature to have pharmacological activities like anti-carcinogenic and anti-inflammatory (Birt et al., 1997; Galati et al., 1999). Phenolic acids have been found in many plant species playing important role like anti-inflammatory activity (Chiang et al., 2003; Fernandez et al., 1998). Protocatechoic acid has been previously reported in Hibiscus sabdariffa and found in medicinal herbs contributing to their antibacterial and anti-tumor activities (Chao and Yin 2009; Lin et al., 2003). Vanillic acid has been found in Hibiscus tiliaceus and has shown anti-inflammatory properties (Narender et al., 2009); also reported in other plants of medicinal value for example in Amburana cearensis (Leal et al., 2011). Phytochemical screening and structural analysis identified anthocyanin responsible for anticancer, antiinflammatory, hepatoprotective activities of Hibiscus sabdariffa. The activities of these compounds may be associated theoretically with the H. caesius, which may be further tested through pharmacognosy in the laboratory. 1628 Agoreyo FO, Agoreyo BO and Onuorah MN (2008). Effect of aqueous extracts of Hibiscus sabdariffa and Zingiber officinale on blood cholesterol and glucose levels of rats. Afr. J. Biotechnol., 7: 3949-3951. Akim A, Ling LC, Rahmat A and Zakaria ZA (2011). Antioxidant and anti-proliferative activities of Roselle juice on Caov-3, MCF-7, MDA-MB-231 and HeLa cancer cell lines. Afr. J. Pharm Pharmacol., 5: 957-965 Ali BH, Wabel NA and Blunden G (2005). Phytochemical, pharmacological and toxicological aspects of Hibiscus sabdariffa L. A review. Phytother. Res., 19: 369-375. Asolkar LV, Kakkar KK and Chakre OJ (1992). Second Supplements to Chopra, Glossary of Indian Medicinal Plants with Active principles Part 1(A-K). Council of Scientific and Industrial Research, New Delhi, India. pp.1965-1981. Birt DF, Mitchell D, Gold B, Pour P and Pinch HC (1997). Inhibition of ultraviolet light induced skin carcinogenesis in SKH-1 mice by apigenin, a plant flavonoid. Anticancer Res., 17: 85-91. Chao CY and Yin MC (2009). Antibacterial effects of roselle calyx extracts and protocatechuic acid in ground beef and apple juice. Foodborne Pathog. Dis., 6: 201206. Chen JJ, Huang SY, Duh CY, Chen IS, Wang TC and Fang HY (2006). A new cytotoxic amide from the stem wood of Hibiscus tiliaceus. Planta. Med., 72: 935-938. Chen HJ, Inbaraj BS and Chen BH (2012). Determination of phenolic acids and flavonids in Taraxacum formosanum Kitam by Liquid ChromatographyTandem Mass Spectrometry coupled with a postcolumn dertivatization technique. Int. J. Mol. Sci., 13: 260-285. Chiang LC, Ng LT, Chiang W, Chang MY and Lin CC (2003). Immunomodulatory activities of flavonoids, monoterpenoids, triterpenoids, iridoid glycosides and phenolic compounds of Plantago sp. Planta. Med., 69: 600-604. Farombi EO and Ige OO (2007). Hypolipidemic and antioxidant effects of ethanolic extract from dried calyx of Hibiscus sabdariffa in alloxan-induced diabetic rats. Fundam. Clin. Pharmacol., 21: 601-609. Fernandez MA, Saenz MT and Garcia MD (1998). Antiinflammatory activity in rats and mice of phenolic acids isolated from Scrophularia frutescens. J. Pharm. Pharmacol., 50: 1183-1186. Pak. J. Pharm. Sci., Vol.28 No.5, September 2015, pp.1625-1629 Quratul Ain et al Fullerton M, Khatiwada J, Johnson JU, Davis S and Williams LL (2011). Determination of antimicrobial activity of sorrel (Hibiscus sabdariffa) on Escherichia coli O157:H7 isolated from food, veterinary and clinical samples. J. Med. Food., 14: 950-956. Galati G, Chan T, Wu B and O'Brien PJ (1999). Glutathione-dependent generation of reactive oxygen species by the peroxidase-catalyzed redox cycling of flavonoids. Chem. Res. Toxicol., 12: 521-5. Gauthaman K, Saleem M, Thanislas P, Prabhu V, Krishnamoorthy K, Devaraj N and Somasundaram J (2006). Cardioprotective effect of the Hibiscus rosa sinensis flowers in an oxidative stress model of myocardial ischemic reperfusion injury in rat. BMC Compl. Alter. Med., 6: 32. Gilani AH, Bashir S, Janbaz KH and Shah AJ (2005). Presence of cholinergic and calcium channel blocking activities explains the traditional use of Hibiscus rosasinensis in constipation and diarrhoea. J. Ethnopharmacol., 102: 289-294. Kong JM, Chia LS, Goh NK, Chia TF and Brouillard R (2003). Analysis and biological activities of anthocyanins. Phytochem., 64: 923-933. Laikangbam R and Damayanti Devi M (2012). Inhibition of calcium oxalate crystal deposition on kidneys of urolithiatic rats by Hibiscus sabdariffa L. extract. Urol. Res., 40: 211-218. Leal LK, Pierdona TM, Goes JG, Fonseca KS, Canuto KM, Silveira ER, Bezerra AM and Viana GS (2011). A comparative chemical and pharmacological study of standardized extracts and vanillic acid from wild and cultivated Amburana cearensis A.C Smith. Phytomedicine., 18: 230-233. Lepley DM, Li B, Birt DF and Pelling JC (1996). The chemo preventive flavonoid apigenin induces G2/M arrest in keratinocytes. Carcinogenesis, 17: 2367-2375. Lin HH, Chan KC, Sheu JY, Hsuan SW, Wang CJ and Chen JH (2012). Hibiscus sabdariffa leaf induces apoptosis of human prostate cancer cells in vitro and in vivo. Food Chem., 132: 880-891. Lin TL, Lin HH, Chen CC, Lin MC, Chou MC and Wang CJ (2007). Hibiscus sabdariffa extract reduces serum cholesterol in men and women. Nutr Res., 27: 140-145. Lin WL, Hsieh YJ, Chou FP, Wang CJ, Cheng MT and Tseng TH (2003). Hibiscus protocatechuic acid inhibits lipopolysaccharide-induced rat hepatic damage. Arch. Toxicol.,77: 42-47. Mabary JJ, Markham KR and Thomas MB, The systematic identification of flavonoids. (1970), Springer-Verlag, Berlin-New York. Heidelberg. Mohd-Esa N, Hern FS, Ismail A and Yee CL (2010). Antioxidant activity in different parts of roselle (Hibiscus sabdariffa L.) extracts and potential exploitation of the seeds. Food Chem., 122: 1055-1060. Molyneux RJ, Waiss A., and Haddon WF (1970). Oxidative coupling of apigenin. Tetrahedron, 26: 1409-1416. Narender K, Kumar D and Kumar V (2009). Antinociceptive and Anti-Inflammatory activity of Hibiscus tiliaceus leaves. Int. J. Pharm. Phytochem. Res., 1(1): 15-17. Prenesti E, Berto S, Daniele PG and Toso S (2007). Antioxidant power quantification of decoction and cold infusions of Hibiscus sabdariffa flowers. Food Chem., 100: 433-438. Sini JM, Umar IA and Inuwa HM (2011). The beneficial effect of the extract of Hibiscus sabdariffa calyces in Alloxan diabetic rats: Hypoglycaemic and Hypolipidaemic activities. J. Med. Plant Res., 5: 21822186. Woottisin S, Hossain RZ, Yachantha C, Sriboonlue P, Ogawa Y and Saito S (2011). Effects of Orthosiphon grandiflorus, Hibiscus sabdariffa and Phyllanthus amarus extracts on risk factors for urinary calcium oxalate stones in rats. J. Urol., 185: 323-328. Yadong QI, Chin KL, Malekian F, Berhane M and Gager J (2005). Biological characteristics, nutritional and medicinal value of roselle, Hibiscus sabdariffa. Agricultural Research and Extension Center No.604, pp.1-2. Yi JH, Zhang GL and Li BG (2000). Two glycosides from the stem bark of Tetracentron sinense. Phytochemistry, 53: 1001-1003. Zhang M, Hettiarachchy NS, Horax R, Kannan A, Praisoody MDA, Muhundan A and Mallangi CR (2011). Phytochemicals, antioxidant and antimicrobial activity of Hibiscus sabdariffa, Centella asiatica, Moringa oleifera and Murraya koenigii leaves. J. Med. Plants Res., 5: 6672-6680. Pak. J. Pharm. Sci., Vol.28 No.5, September 2015, pp.1625-1629 1629