S. Shanthi et al. / Journal of Pharmacy Research 2014,8(4),486-488 Research Article Available online through ISSN: 0974-6943 http://jprsolutions.info Estimation of flavonoids in (Linn) JACQ. by HPLC analysis

S. Shanthi*1, S. Seethalakshmi2, D. Chamundeeswari1, P.K. Manna3, X. Fatima Grace1, S. Latha1 1Faculty of Pharmacy, Sri Ramachandra University, Chennai, Tamil Nadu, India 2 Sri Ramachandra Medical College and Research Institute, Chennai, Tamil Nadu,India 3Department of Pharmacy, Annamalai University, Tamil Nadu,India

Received on:18-03-2014; Revised on: 26-03-2014; Accepted on:01-04-2014

ABSTRACT The Dodonaea viscosa () is traditionally used in the treatment of inflammation, swelling, rheumatism, pain, snake-bite and for wounds. The therapeutic efficacy of this plant is attributed to the chemical constituent flavonoids present in the plant. The present study is aimed to detect and quantify the flavonoids present in the of Dodonaea viscosa using High Performance Liquid Chromatography (HPLC). Total phenolic content of the extracts were determined by Folin cio-calteu method. The HPLC system used was Linchosorb Octadecylsilyl silica (ODS) column with Methanol–Water–Phosphoric acid (100:100:1) as the mobile phase and was detected at 270nm. The ethanolic extract showed maximum quantities (7.28, 5.64, 3.42mg/gm) of flavonoid (Rutin, Quercetin and Kaemferol) when compared to ethyl acetate extract (6.38, 5.26, 1.32mg/gm) respectively. Rutin, Quercetin and Kaempferol were identified and quantified in the ethanolic and ethyl acetate extract. This study provides the suitable method for the quantification of flavonoids present in the plant Dodonaea viscosa which will be helpful in authentication of the plant. This study also helps to develop the monograph for this plant.

KEYWORDS: Dodonaea viscosa, Flavonoids, HPLC, rutin.

1. INTRODUCTION Dodonaea viscosa is an evergreen or small , occurring 13,14-diene and sakunaretin13. Isorhamnetin-3-rhamnosyl galactoside almost throughout India. It is popularly known as Sinatha in Hindi isolated from aerial parts showed blood sugar lowering effects. 1 and Velari in Tamil . The plant is used as herbal remedy for Penduletin, quercetin, isorhamnetin and doviscogenin were isolated inflammation, swelling, rheumatism; wound healing in burns, skin from the flowering apex14-15. infections and pain2,3 . A poultice of leaves is used in gout, rheumatism and snake-bite. The plant is also used as a laxative and febrifuge4. It Flavonoids are natural antioxidants produced as secondary plant contains bioflavonoids which are biologically active in improving metabolites, and are therefore widely distributed in , vegetables, blood circulation and strengthening capillaries5. A paste of leaves and many plant-based products. The plant Dodonaea viscosa con- with of Phaseolus mungo is applied on animal bone fractures tains numerous flavonoids which are responsible for its therapeutic for bone setting6. An embrocation of leaves is applied to sprains and efficacy. So, the present study is carried out to detect the flavonoids bruises. The bark is employed in astringent baths and fomentations. present in the extracts of Dodonaea viscosa leaves and to estimate The alcoholic extract of the Dodonaea viscosa showed good inhibi- the same by High Performance Liquid Chromatography. To the best tion against Staphylococcus aureus, Staphyococcus smegmalis and of our knowledge this is the first report on quantification of the fla- Candida albicans7. It is reported to possess antiulcer, wound heal- vonoids in Dodonaea viscosa leaves by HPLC method. ing, antioxidant, anti-inflammatory, analgesic and antipyretic activi- ties8-11.The aerial parts contain a prenylated flavonoid, Viscosol12. The plant is reported to contain 5, 6, 4’-trihydroxy-3,7-dimethoxy fla- 2. MATERIALS AND METHODS vones, hautriwaic acid, ent-15,16-epoxy-3ß,8a-dihydroxy-9a-H-labda- 2.1. Plant material: *Corresponding author. Fresh leaves of Dodonaea viscosa were collected from Alagarkovil S. Shanthi, hills, Madurai district, Tamil Nadu, India and it was authenticated by Lecturer, Department of Pharmacognosy, Prof.P. Jayaraman, Plant Anatomical Research Centre, Chennai. The Faculty of Pharmacy, Sri Ramachandra University, voucher specimen was prepared and kept in the department. The Porur, Chennai – 600 116 collected leaves were shade dried, coarsely powdered and used for Tamil Nadu,India the phytochemical study.

Journal of Pharmacy Research Vol.8 Issue 4.April 2014 486-488 S. Shanthi et al. / Journal of Pharmacy Research 2014,8(4),486-488 2.2. Preparation of Extracts: 3. RESULTS The powdered leaves were extracted with Petroleum ether, Chloro- form, Ethyl acetate and Ethanol successively by maceration process Table: 1 - Evaluation of Total Phenolic content by Folin Cio-calteau for 72 hrs. The extract was concentrated under reduced pressure and method was used for further study. Sl.No Extract Total phenolic content (mg GAE gm-1 ) 2.3. Evaluation of Total phenolic content16: The ethanolic and ethyl acetate extract which showed the presence 1 Ethanol 51.08 ±0.53 of phenolic compounds in preliminary phytochemical study were se- 2 Ethyl acetate 35.06 ± 0.18 lected for the determination of total phenolic content using Folin Each value represents mean ± SEM of triplicates Cio-calteau method. The extracts were dissolved in Ethanol (70%) at Table: 2. Quantification of flavonoids in ethanol and ethyl acetate 1mg/ml and Gallic acid was used as a standard. 0.5ml of the diluted extract of Dodonaea viscosa extract was mixed with 2.5ml of Folin-Ciocalteau reagent (10%) and 2.5ml of 7.5% Sodium carbonate. It was then incubated at 50°C for Sl.No Extract Chemical Amount mg/gm 30min. The absorbance was measured at 740nm in UV spectropho- constituent of extract tometer. Phenolic compounds present in the extract were determined 1 Ethanol Rutin 7.28±0.05 by calibration curve method and the results were expressed in mg Quercetin 5.64±0.06 Gallic acid equivalents per gm of extract (Table 1). Kaempferol 3.42±0.08 2 Ethyl acetate Rutin 6.38±0.12 2.4. Determination of flavonoid content by HPLC17,18 Quercetin 5.26±0.05 Kaempferol 1.32±0.09

Flavonoid standards Each value represents mean ± SEM of triplicates Rutin, quercetin and kaempferol was purchased from Merck. Purity was verified by using triplicate injections of standards into HPLC.

Sample preparation for HPLC analysis: The ethanolic and ethyl acetate extracts were refluxed with the mix- ture (78ml) of alcohol, water and Hydrochloric acid (50:20:8) for 135 minutes. 20ml of methanol (70%) was added and sonicated for 30 minutes. The extract was filtered and the volume was made upto 100ml with Methanol. An aliquot (5ml) was filtered through a small column of C-18 Silica and the column was eluted with 4ml of metha- nol. The volume of the eluate was completed to 10ml with methanol and it was used for HPLC analysis. Fig.no:1. HPLC chromatogram of Ethanol extract of leaves of High-performance liquid chromatography (HPLC) Dodonaea viscosa HPLC analysis were performed in a Shimadzu apparatus equipped with SPD-M10A Diode array detector using reverse phase column (Linchosorb RP-18, 25cm x 5mm) at room temperature. Elution was done using the mixture of Methanol, water and Phosphoric acid (100:100:1). The flow rate was about 1ml/min and peaks were de- tected at 270nm. All chemicals used in analysis were of HPLC grade and were purchased from Merck. The standards were dissolved in methanol at 1mg/ml and analyzed in the same elution. 10µl of sample and standards were injected for analysis. The average amounts of flavonoids were calculated from the peak areas of HPLC Chromato- gram from the triplicate sample.

Identification and quantification of Flavonoids: The retention time and peak area of ethanolic and ethyl acetate ex- Fig.no:2. HPLC chromatogram of Ethyl acetate extract of leaves of tract was compared with the HPLC data of authentic sample for the Dodonaea viscosa identification and quantification of flavonoids ( table 2 and fig 1-3). Journal of Pharmacy Research Vol.8 Issue 4.April 2014 486-488 S. Shanthi et al. / Journal of Pharmacy Research 2014,8(4),486-488 2. Anonymous, The Wealth of India – First supplement series (Raw materials), Vol-III ; D-I., CSIR, New Delhi, 2002, pp-29. 3. L.V.Asolkar, K.K.Kakkar, O.J.chakre . Second supplement to Glossary of Indian medicinal with active principles, part-I (A-K), CSIR, New Delhi, 1998, pp-280. 4. R.N.Chopra, S.L.Nayar, I.C.Chopra. Glossary of Indian me- dicinal plants, CSIR. 1956, pp-100. 5. Nadkarni.KM, Nadkarni.A. Indian Materia Medica,Vol- I.,Bombay popular prakashan, Bombay, 1982, pp-457. 6. Kritikar.KR, Basu.BD. Indian Medicinal Plants, Vol-I, Interna- tional book distributors, Dehradun, India, 1995, 641-643. Fig.no:3 . HPLC chromatogram of Flavonoid standard 7. Getie.M, Gebrearian.T. Reitz, Hohne.C ,Huschka.C 4. DISCUSSION: ,Schmidtke.M ,Abate.A , Neubert.RHH. Evaluation of the anti- microbial and anti-inflammatory activities of the medicinal Total Phenolic content plants, Dodonaea viscosa, Rumex nervosus and Rumex Total amount of phenol in the ethyl acetate extract was found to be abyssinicus, Fitoterapia., 2003, 74.,139-143 35.06 ± 0.18 mg GAE/gm of extract and in ethanol extract was 51.08 8. Veerapur.VP, BadigerAM, JoshiSD, NayakVP, Shastry.CS ±0.53 mg GAE/gm of extract. Antiulcerogenic activity of various extracts of Dodonaea viscosa (L) jacq.leaves., Indian .J.Pharm.Sci., 2004, 66.,407- Quantification of flavonoids 411. The flavonoids were identified in ethanolic and ethyl acetate extracts 9. Mrudula patel, MaeveM. Coogan. Antifungal activity of by comparing the retention time in HPLC chromatograms of extract Dodonaea viscosa, J of Ethnopharmacol., 2008, 118(1), 173- with the standards run in the same condition. The retention time 2.2, 6.. 4 and 8.3 were identified as rutin, quercetin and kaempferol 10. Amabeoku, G.J,Eagles,P, Scott,G, springfield,E, Pand mayeng.I respectively by comparing with standards. The amount of flavonoids . Analgesic and antipyretic effects of was calculated from the peak area of HPLC chromatogram of ethanolic and Salvia Africana-lutea. J of Ethnopharmacol., 2001, and ethyl acetate extract. The results showed that the flavonoid content 75:117-124. is more in ethanolic extract when compared to ethyl acetate extract. 11. N.M.Khalil, J.S.Serotto, M.P.Manfron, Anti-inflammatory and The flavonoids which occur both in the Free State and as glycosides acute toxicity of Dodonaea viscosa, , Fitoterapia, 2006, 478- are the largest group of naturally occuring phenols. This group is 480. known for its anti-inflammatory, antiallergic, antithrombotic and 12. Rachel mata, Jose Luis Contreras, Daniel Crisanto, Chemical vasoprotective effects for the inhibition of tumor promotion and as a studies on Mexican plants used in traditional medicine,New protective for the gastric mucosa. These effects have been attributed secondary metabolite from D.viscosa. J Nat Products, 1991, to the influence of flavonoids on arachidonic acid metabolism. 54: 913-917. 13. Van Heerden.F.R, Vijijoen A.M, Van wyk B.E, The major fla- 5. CONCLUSION: vonoids of D.viscosa, Fitoterapia., 2000, 71(5): 602-604. Many flavonoid containing plants are used as diuretic (e.g: buchu 14. Ramachandran.N, Subramanian.AG, Sankara.S . Isorhamnetin and broom), antispasmodic (e.g: liquorice and parsley), some have and quercetin glycosides from Dodonaea viscosa and antitumour, antibacterial and antifungal activities. In the present study, emarginatus., Indian.J.Chem., 1975, 13: 639-640. the flavonoids present in the plant Dodonaea viscosa was identified 15. Sachdev.K, Kulshreshtha.D.K, Viscosol A C-3’prenylated and quantified by HPLC method. The study reveals that this plant is flavonoid from Dodonaea viscosa., Phytochem., 2008, 25: a rich source of flavonoids such as rutin, quercetin and kaempferol 1967-1969. with significant therapeutic importance. 16. Singleton VL, Rossi JL. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am J Enol 6. ACKNOWLEDGEMENT: Viticult., 1965, 16:1 44-58. The authors are thankful to the management of Sri Ramachandra 17. Brás H. de Oliveira, Tomoe Nakashima, José D. de Souza University for their constant help and support. The authors are also Filho,and Fabiano L. Frehse, HPLC Analysis of Flavonoids in thankful to A to Z Pharmaceuticals, Chennai for their help in Eupatorium littorale, J. Braz. Chem. Soc., 2001, 12: 243-246. conducting the HPLC study. 18. L. Tao, Z.-T. Wang, E.-Y. Zhu, Y.-H. Lu,, D.-Z. Wei, HPLC analysis of bioactive flavonoids from the rhizome of Alpinia 7. REFERENCES: officinarum , South Af j Bot., 2006, 72: 163 – 166. 1. Anonymous, The Wealth of India – Raw materials, Vol-III; D- E. (1953) CSIR, New Delhi, 1953, pp -97.

Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.8 Issue 4.April 2014 486-488