Using HPLC to Determination the Composition and Antioxidant Activity of Berberis Vulgaris

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1 Europen Journl of Scientific Reserch ISSN X Vol.29 No.1 (2009), pp EuroJournls Publishing, Inc Using HPLC to Determintion the Composition nd Antioxidnt Activity of Berberis Vulgris Prichehr Hnchi Women Reserch Centre, Biomedicl Unite, Alzhr University Tehrn, Irn E-mil: Tel: ; Fx: Golkho SH Women Reserch Centre, Biomedicl Unite, Alzhr University Tehrn, Irn Abstrct The Berberis vulgris (BV) plnt hs been extensively used s trditionl medicine in some prt of Asin region especilly in Western Asi. The BV fruits is climed lso to hve nti-virl ctivities, nd s tretment for chronic cndidisis, indigestion nd prsites. The present study ws conducted to evlute the ntioxidtive ctivity, nutritionl nd ntinutritionl composition of Berberis vulgris fruit. The ntioxidnt ctivity ws determined by using thiobrbituric cid (TBA method). Absolute ethnol, bsolute methnol nd queous solution (distilled wter) were used s solvent in this study. Result shows significntly differences (P< 0.05). The ntioxidnt ctivity of ethnol extrct ws the highest (27.26 ±1.07 %), followed by BHT (20.29 ±0.23 %), methnol extrct (16.80 ±0.23 %), Vitmin E (6.68 ±0.25 %) nd the lowest ws the queous extrct (6.53 ±0.29 %). Besides ntioxidnt ctivity, Berberis vulgris lso contins nutritionl nd ntinutritionl composition nmely vitmin C, mlic cid nd Tnnin. Vitmin C nd mlic cid lso shows ntioxidtive properties. Vitmin C nd mlic cid lso showed ntioxidtive properties nd ct synergisticlly to ech other s ntioxidnts. The presence of ntinutritionl such s Tnnin which cn ct s medium to prevent nutritionl composition from being bsorbed by the body lso hs been detected. The mount of nutritionl nd ntinutritionl detected in 100g of berberry were μg ± 2.01 of Vitmin C, μg ± 1.12 of Mlic cid nd 20.51μg ± 0.59 of Tnnin. Keywords: Antioxidnt, Berberis vulgris, Thiobrbituric cid, Vitmin C, Mlic cid, Tnnin, HPLC. Introduction Medicinl plnt reserch includes much more thn the discovery of new drugs. This field hs been expnding to lso include such diverse subjects s negotition of power bsed on medicinl plnt knowledge (Grro, 1986) nd the co-evolution of humns nd plnts (Alcorn, 1981). The field lso provides opportunities to study how humn interction with biologicl diversity is influenced by humn psychology, cognition, nd evolution. Therefore the identifiction of ctive plnt chemicls is

2 Using HPLC to Determintion the Composition nd Antioxidnt Activity of Berberis Vulgris 48 n essentil component of modern phrmcognosy nd medicl effects re not necessrily restricted to single plnt chemicl. The biologicl ctivity nd clinicl vlue of the whole plnt, s in medicinl herblism, is lso being pursued (Psqule, 1984). The importnce of identifiction of ctive plnt chemicls does not limit itself to locl Mlysin plnts but lso sprks n inquisitive knowledge to foreign plnts. This effort leds to study non-ntive plnt clled Berberis vulgris which predominntly found in Northern Asi nd Europe (Chevllier, 2001). The determintion of nutritionl nd nti-nutritionl compounds in Berberis vulgris or berberry fruits is very importnt s this fruit in prt of dily diet of certin people. Their presence nd reltive rtio, in fct, cn ffect the chemicl nd sensoril chrcteristics of the mtrix (e.g., ph, totl cidity, microbil stbility, sweetness, globl cceptbility) nd cn provide precious informtion on food wholesomeness or on how to optimize some selected technologicl processes (Romero, et l 1992). The nutritionl vlues such s vitmin components, ntioxidnt compounds in this plnt might be invluble for treting diseses s more secrets within this plnt re yet to be discovered. Hnchi et l 2005 reported tht Berberis vulgris exhibited vry degree of ntioxidnt properties Recently (Mznh, et l, 1999) hd shown tht ntioxidnt cpcities vried ccording to the plnt extrcts nd the comprison between different solvents for the extrction method could be done especilly between wter nd orgnic solvents to investigte which extrct hd the highest ntioxidnt ctivities. The identifiction of certin lkloids nd phenolic compounds in Berberis vulgris somehow provide n lterntive method for medicine nd remedies s mny studies hve proved to llevite certin ilments in treting liver cncer, gll bldder problems, kidney stones, menstrul pins nd etc (Hnchi et l, 2008). All these problems hve led to the development of new drugs derived from plnts, which is believed to be sfer nd more effective (Wrgovich, 1999). The development of drugs from plnts continues with the reserch of screening on herbs (Vickers nd Zollmn, 1999). This shows tht herbl medicine hs creted gret del of public interest. The objective of this study ws to Using HPLC to determintion the composition nd ntioxidnt ctivity of Berberis vulgris. Mteril nd Methods The fruits of Berberis vulgris were purchsed from Irnin mrket in Kul Lumpur. The fruits were bought in the dried form nd were prepred to be extrcted. Preprtion of Plnt Extrct Brberry fruit extrct ws prepred ccording to method modified Shms et l. 310 g BV fruit tht ws free from fungus, bcteri nd ny other plnt diseses were selected rndomly. The fruits were dried in n oven (Memmert SLM 400, GmbH, Germny) for 3 dys t constnt temperture of 65 C. The fruits were cut into smll pieces nd were grounded into fine powder using dry grinder. The grounded smples were sieved to get uniform prticle size, then were kept in ir-tight continer nd stored for further extrction. The smple ws extrcted by boiling in wter for minutes t temperture of 75 C.Wter nd ethnol were dded in the rtio of 1:10 nd stirred t 250 rpm in n orbitl shker (Unimx 1010, Heidolph Instruments GmbH & Co.KG,Germny) for 1 h t room temperture. The extrct ws then seprted from the residue by filtrtion through Whtmn No.1 filter pper. The remining residue ws re-extrcted twice, nd then the two extrcts were combined. The residul solvent of ethnolic extrct ws removed under reduced pressure t 50 C using rotry evportor (BÜCHI Rotvpor R-200, Germny) until thick syrup ws collected. The thick syrup ws evported completely using freeze drying system (FreeZone 77520, LABCONCO, USA) for the determintion of totl ntioxidnt ctivity nd phenolic content.

3 49 Prichehr Hnchi nd Golkho SH Absolute Methnol nd Aqueous Extrctions For bsolute methnol nd queous extrctions, the extrction ws done ccording to Endrini (2002) with slight modifiction. The extrction process ws crried out by soking bout 100 g of dried powder in 1000 ml bsolute methnol for 24 hours. The superntnt ws filtered using Whtmn filter pper no. 1. The superntnt ws then evported t 45 C under reduced pressure nd subsequently irdried. The bove steps were repeted to obtin queous extrction. Determintion of Antioxidnt Activity The method of Ottolenghi (1959) ws used to determine the TBA vlues of the smples. In brief, mixture of 4 mg of Berberis vulgris extrcts, BHT nd α- tocopherol cette (vitmin E) in 4 ml bsolute ethnol, 4.1 ml of 2.52% linoleic cid in bsolute ethnol, 8 ml of 0.05 M phosphte buffer (ph 7.0), nd 3.9 ml of distilled wter ws plced in vil with screw cp nd then plced in shking wter bth (100 rpm) t 40 C in the drk for 24 hours. A control ws set up by replcing the smple with 4 ml ethnol. After the incubtion period, 2 ml of 20% trichlorocetic cid (TCA) solution nd 2 ml thiobrbituric cid (TBA) solution were dded to 1 ml of smple solution. The mixture ws plced in boiling wter bth for 10 minutes. After cooling, it ws centrifuged t 3,000 rpm for 10 minutes nd the bsorbnce of the superntnt ws mesured t 532 nm. The ntioxidnt ctivities were clculted s below: % Antioxidnt Activity = (OD control - OD smple /OD control ) X 100 % In which OD smple is the opticl density of smple nd OD control is opticl density of control. Determintion of Nutritionl nd Antinutritionl Component Stndrds of nutritionl nd nti-nutritionl for scorbic cid, mlic cid nd tnnin (Sigm Chemicls, Co, USA) were used. The smple preprtion techniques must be done before using the HPLC for the purpose of eliminting contmintion nd to get the best outcome of the experiment. Smple preprtion of Vitmin C determintion ws prepred with mixture of KH 2 PO 4, methnol nd TBAOH with rtio of 97:3:0.05. The following smple preprtion for Tnnin ws used. Smples of berberry fruits were dissolved in 100ml of mixture of 100 volumes of 10% methnol nd 0.1 volume of phosphoric cid. Different steps were used to prepre Mlic cid. About 3 grms of berberry smples were diluted into 3 ml of distilled or purified wter. Therefore the concentrtions must be 50:50, v/v. Determintion of Vitmin C, Mlic cid nd Tnnin Hewlett Pckrd series 1100 consist of Hewlett Pckrd 1049A progrmmble electrochemicl detector, degsser G1322A, UV/ visible detector ws used to detect Vitmin C, Mlic cid nd Tnnin. The method of Abdulnbi et l, 1997 with slight modifiction ws used to determine Vitmin C using HPLC. The content of orgnic cid of Mlic cid cn be determined by using method from Rebecc, (2000). Menwhile the presence of Tnnin is dopted from Hgermn et l., 1992 with slight chnges. This method is prticulrly useful for seprting the constituents of tnnic cid in simple isocrtic system. Better resolution cn be obtined with reversed phse system especilly if grdient HPLC is vilble. Typicl run tkes bout 30 minutes to ensure elution of ll peks lthough longer runs could lso be necessry if smple hd very high moleculr weight Tnnin. The conditions for Vitmin C, Mlic cid nd Tnnin re listed in Tble 1

4 Using HPLC to Determintion the Composition nd Antioxidnt Activity of Berberis Vulgris 50 Sttisticl Anlysis The results obtined were nlyzed using one-wy ANOVA for men differences. The Sttisticl Pckge for Socil Science for windows version (12.0) ws used to nlyze the dt. The (p<0.05) ws considered significnt nd dt from detection of nutritionl nd ntinutritionl were expressed s men ± stndrd devition (SD). Results Determintion of Antioxidnt Activity of Berberis vulgris The results of determintion of ntioxidnt ctivity, Tble 2 shows ethnol extrct of BV ws the highest mong the other extrcts which ws bout (27.26 ± 1.07 %) nd this percentge lso exceeds the percentge of ntioxidnt ctivity of BHT nd Vitmin E followed by BHT (20.29 ± 0.23 %), methnol extrct (16.80 ±0.23 %), Vitmin E (6.68 ± 0.25 %) nd the lowest ws the queous extrct (6.53 ± 0.29 %). Determintion of Nutritionl nd ntinutritionl components of Berberis vulgris The presence of Vitmin C in the berberry fruits ws detected t 225 nm t min. Comprisons were mde between the stndrd chromtogrph nd the smples chromtogrph. All the retention time lie t the sme time. The stndrd chromtogrph hd bigger re compred to the smple re. Lrger re indictes tht the stndrd hd lrger mount of Vitmin C compred to the smples. Little interferences were detected in the chromtogrph due to the contminnts nd interferences. The HPLC profile of queous Berberis vulgris showed significnt presence of Vitmin C Retention time for stndrd Mlic cid ws similr to the retention time in the smple. Both stndrd nd smple detected t min. Few interferences were detected in the chromtogrph due to the contminnts nd interferences. The presence of Mlic cid ws detected t 225 nm. Antinutritionl composition of Tnnin ws detected t 350 nm nd the retention time of min. Both the stndrd nd smple chromtogrph did not show ny sign of interference. Nutritionl nd ntinutritionl quntifiction were clculted from the curve generted by plotting the pek re of ech stndrd versus smple concentrtion. The highest mount ws Vitmin C μg ± 2.01, followed by Mlic cid μg ±1.12 nd Tnnin with μg ± 0.59 (Tble. 3). Discussion Antioxidnt Activity of Berberis vulgris All the extrctions were significntly different in terms of ntioxidnt ctivity. Both the BHT nd nturl ntioxidnt Vitmin E ct s comprison stndrd. The methnol extrction showed second highest nd queous extrction cme in lst with vlues of ±0.23 % nd 6.53 ±0.29 % respectively. Bsed on the results obtined, it ws highly possible tht severl compounds of different polrity my contribute to the totl phenolic content of the extrcts. Methnol extrcts my include phenolic nd hydroxy-phenolic compounds with cids, lcohols, sugrs or glycosides, s reported by Kim nd Prtt (1993). Mny reserchers hd shown tht high totl phenols content increses ntioxidnt ctivity nd there ws liner correltion between phenolic content nd ntioxidnt ctivity (Motleb et l, 2005). The ntioxidnt ctivity of phenolics ws minly due to their redox properties, which llow them to ct s reducing gents, hydrogen donors nd singlet oxygen quenchers (Mrj et l., 1999). The TBA test is colorimetric technique in which the bsorbnce of red chromogen formed between TBA nd mlondildehyde is mesured (Rhee, 1978). The first step in lipid oxidtion is the bstrction of hydrogen tom from ftty cid nd subsequent oxygen involvement gives peroxy

5 51 Prichehr Hnchi nd Golkho SH rdicl. Peroxide then grdully decomposes to lower moleculr compounds; minly mlondildehyde (Ledwozyw et l., 1986) during oxidtion process nd mlondildehyde cn be determined by TBA method. At low ph nd high temperture, mlondildehyde binds TBA to form red complex tht cn be mesured t 532 nm (Ottolenghi, 1959). Generlly, the ntioxidnts suppress the hydrogen tom bstrction from ftty cid, which leds to the decrese of hydroperoxide formtion. It is well known tht phenolic compounds ct s hydrogen donors in tht rection mixture nd therefore, the formtion of hydroperoxides were decresed (Frg et l., 1989). From the result, the ntioxidnt ctivity of ethnol extrction of BV ws the highest mong ll the extrcts, lso mybe due to its phenolic content. Recently, Frg et l. (2003) reported tht, ddition of extrcted phenolic compound from olive oil, to sunflower oil t 400-ppm level, exhibited remrkble ntioxidnt ctivity nd were superior to tht of BHT in retrding sunflower oil oxidtive rncidity. The results lso hd indicted tht different solvents used my hve different effects on ntioxidnt ctivities. Nutritionl nd Antinutritionl Components of Berberis vulgris In this studies, only fully ripen fruits were used for the detection of Vitmin C, Mlic cid nd Tnnin. This becuse during the ripening process mny fruits undergo chrcteristic chnges tht, in generl led to n increse in nutritive vlue. However, some importnt nutrients tend to decrese when fruits become fully ripe. This depends upon the mechnism of the ripening process such s climte, temperture, sunlight nd mny more. These fctors must be tken into ccount becuse the ripening cn ffect the nutritionl nd ntinutritionl composition. For exmple the dvnced ripeness cused the Vitmin C content to decrese, most due to its ntioxidnt function when ripening bsorb high mounts of oxygen s result of increse rte of cell respirtion, the physiologic chnge in climtic fruits (Tünk et l, 1993). The presence of nutritionl compositions such s Vitmin C nd Mlic cid nd lso ntinutritionl composition of Tnnin hs been detected. The highest mount ws Vitmin C μg ± 2.01, followed by Mlic cid μg ±1.12 nd Tnnin with μg ± The results of Vitmin C nd Mlic cid hd higher men nd stndrd devition becuse common processing techniques, like dry nd especilly wet heting, extrcting with wter, nd etc sometimes hve unintended dverse effects on the nutritionl qulity of the fruits for exmple the het tretment reportedly lters the chemicl nture nd decreses the nutritionl qulity of berberry fruits. HPLC is highly sensitive method of detection nd quntifiction of ny chemicls in prticulr smple using ultrviolet nd visible bsorbnce. However this method suffers from interferences. For exmple contminnts in the Vitmin C smple chromtogrm indicte the presence of contminnts tht cn interfere with the peks displyed by the chromtogrph, which led to high stndrd devition. All in ll, slightest presence of foreign mterils or contminnts cn ffect the outcome of experiment. Other fctors tht cn cuse error in detection nd quntifiction re with the selection of mobile phse nd solvents. Only selected mobile phse nd solvents with the right mounts hve been recommended becuse they yield higher recoveries. For exmple Vitmin C is highly sensitive to fctors such s light, het, nd ph. A slight chnge in mobile phse, solvents nd temperture during the detection cn give flse result to the chromtogrph tht would led to chnge in retention time. Further more the differences in solvent refrective index cuse n unstble chromtogrphic bseline. Selected solvents such s methnol nd cetonitrile were used becuse they were found to be very stble nd comptible with the reversed-phse HPLC. Conclusion From these results it cn be concluded tht, BV hs n ntioxidnt ctivity t different degrees in different extrction solvents The ntioxidnt ctivity of bsolute ethnol extrction ws significntly higher nd exceeds stndrd ntioxidnt such s BHT nd Vitmin E. The presences of nutritionl nd

6 Using HPLC to Determintion the Composition nd Antioxidnt Activity of Berberis Vulgris 52 ntinutritionl composition were lso detected using high performnce liquid chromtogrphy (HPLC). From the result it cn be concluded tht BV fruits contin rich mount of Vitmin C, followed by Mlic cid nd Tnnin. The mount of Vitmin C provides bulk chrcteristic of ntioxidnt ctivity in the fruit. Menwhile Mlic cid ct synergisticlly with Vitmin C to increse the ntioxidnt ctivity. The nutritionl composition of Tnnin ws detected. The rich mount of Vitmin C mkes Berberis vulgris fruit good source of Vitmin C for dily consumptions. Tble 1: Condition for HPLC seprtion of Vitmin C, Mlic cid nd Tnnin. Prmeters Vitmin C Mlic cid Vitmin E Column Genesis C18, 5μm, 4.6 X 150mm Allure Orgnic Acids, 5μm Merck Hibr, lichrosolv RP X 300mm 5μ Mobile Phse 0.1M KH 2 PO 4 / methnol/ TBAOH 1000mM phosphte buffer, A 0.2% H 3 PO 4 in wter; B: 0.2% (97:3:0.05) ph 2.5 H 3 PO 4 in methnol Flow rte 1.0 ml/min 0.5 ml/min 1.0 ml/min Detection UV 225nm UV 225nm UV 350nm Tble 2: Antioxidnt ctivity of Berberis vulgris extrcts, BHT nd Vitmin E using Thiobrbituric cid method (TBA). Smple Absolute Ethnol extrct Methnol extrct Aqueous extrct BHT Vitmin E Vlues with the sme letter () were significntly (p< 0.05) different between the smples. Antioxidnt Activities (%) (Men ± SD) ± ± ± ± ±0.25 Figure 1: Antioxidnt ctivity of methnol nd ethnol of extrctions of Berberis vulgris s mesured by the TBA method. Absorbnce vlue represents triplictes of different smples nlyzed. Vlues with sme letter re significntly different (p< 0.05) between smples Antioxidnt Activity (%) queous methnol ethnol BHT Vit E 5 0 queous methnol ethnol BHT Vit E smple

7 53 Prichehr Hnchi nd Golkho SH Tble 3: Composition of nutritionl nd ntinutritionl in 100g of Berberis vulgris fruits Smple Amount in μg/100g (%) (Men ± SD) Vitmin C ± 2.01 Mlic cid ± 1.12 Tnnin ± 0.59 References [1] Abdulnbi AA, Emhemed AH, Hussein GD & Bics PA (1997). Determintion of ntioxidnt vitmins in tomtoes. Food Chem 60 : [2] Alcorn, J. B Hustec Noncrop Resource Mngement: Implictions for Prehistoric Rin Forest Mngement. Humn Ecology 9(4): [3] Chevllier A. (2001.) Encyclopedi of Medicinl Plnts. Revised Edition. Sydney, Austrli: Dorling Kindersley. [4] Edrini, S Chemicl constituents nd biologicl ctivities of Lwsoni inermis nd Strobilnthes crispus. PhD Thesis. University Putr Mlysi. Mlysi. [5] Frg, R.S., Bdei, A.Z. & El-Broty, G.S. (1989). Influence of thyme nd clove essentil oils on cottonseed oil oxidtion. Journl of the Americn Oil Chemistry Society 66: [6] Frg, R.S., El-Broty, G.S. & Bsuny, A.M. (2003). The influence of phenolic extrcts obtined from the olive plnt (cvs. Picul nd Kronkii), on the stbility of sunflower oil. Interntionl Journl of Food Science & Technology 38(1): [7] Grro, Lind C. (1986). Intrculturl Vrition in Folk Medicl Knowledge: A Comprison Between Curers nd Noncurers. Americn Anthropologist 88: [8] Hgermn, A. E., C. T. Robbins, Y. Weersuriy, T. C. Wilson, nd C. Mcrthur Tnnin chemistry in reltion to digestion. J. Rnge Mng. 45: [9] Hnchi P, Fuzih O., Motlleb G. (2008). Evlution of Sodium, Potssium nd poptotic cells in the liver of heptocrcinogenic rts treted with Berberis vulgris. Irnin Journl of Bsic Medicl science 11(2); [10] Lunert. E. Edible nd Medicinl Plnts. (1981) Covers plnts in Europe. Hmlyn. [11] Ledwozyw, A., Michlk, J., Stepien A. nd Kdziolk, A. (1986). The reltionship between plsm triglycerides, cholesterol, totl lipids nd lipid peroxidtion products during humn therosclerosis. Clin. Chim. Act. 155: [12] Ledwozyw, A., Michlk, J., Stepien A. nd Kdziolk, A. (1986). The reltionship between plsm triglycerides, cholesterol, totl lipids nd lipid peroxidtion products during humn therosclerosis. Clin. Chim. Act. 155: [13] Mrj, P.K., Anu, I.H., Heikki, J.V., Jussi-Pekk, R., Klevi, P., Tytti S.K. nd Mrin, H. (1999). Antioxidnt ctivity of plnt extrcts contining phenolic compounds. Journl of Agriculturl nd Food Chemistry 47: [14] Morris, K. (1998). Tckling thorny issue of herbl medicines worldwide. The Lncet 352:1190 [15] Mznh, I., Teoh, S.L. nd Loh, S.P. (1999). Determintion of totl ntioxidnt ctivity of selected locl medicinl plnts. In: Proceeding Herbs n Interntionl Conference nd Exhibitions 9-11 Nov (Mines, Seri Kembngn, Mlysi). pp Mlysin Agriculture Reserch nd Development Institute (MARDI). Ministry of Agriculturl, Mlysi [16] Motlleb, G., Hnchi, P., Ku, SH., Fuzih, O., Asmh, R. Evlution of phenolic content nd totl ntioxidnt ctivity in Berberis vulgris fruit extrct. Journl of Biologicl Science. 5(5): [17] Ottolenghi, A. (1959). Interction of scorbic cid nd mitochondril lipids. Achives of Biochemistry nd Biophysics 79: [18] Psqule, A.D. (1984). Phrmcognosy: the oldest modern science. Journl of Ethnophrmcology 11:1-16.

8 Using HPLC to Determintion the Composition nd Antioxidnt Activity of Berberis Vulgris 54 [19] Rebecc Wittrig.(2000) Verify Fruit Juice Qulity from Orgnic Acid Profiles. [20] Rhee, K.S. (1978). Minimiztion of further lipid peroxidtion in the distilltion 2-thiobrbituric cid test of fish nd met. Journl of Food Science 43: [21] Romero Rodriguez, M.A., Vzquez Oderiz, M.L., Lopez Hernndez, J. nd Siml Lozno, J., (1992). Determintion of vitmin C nd orgnic cids in vrious fruits by HPLC. Journl of Chromtogrphic Science 30 11, 1992 pp [22] Sccni, G., Gherrdi, S., Trifirò, A., Soresi Bordini, C., Clz, M. nd Freddi, C, (1995). Use of ion chromtogrphy for the mesurement of orgnic cids in fruit juices. Journl of Chromtogrphy , pp [23] Shms, F., A. Ahmdini nd R. Khosrokhvr, Antihistminic nd nticholinergic ctivity of brberry fruit (Berberis vulgris) in the guine - pig ileum. Journl of Ethnophrmcology, 64 : [24] Tünk, R. Seniz,V. Őzdemir, N nd Süzen O, (1993) Chnge in Chlorophyll, cretenoids nd lycopene in tomtoes in reltion to temperture Act Hdl pg 862 [25] Vickers, A nd Zollmn, C. (1999). Herbl medicine. British Medicl Journl 336 (26): [26] Wrgovich, M.J. (1999). Nutrition nd cncer: the herbl revolution. Current Opinion in Clinicl Nutrition nd Metbolic Cre. 2(5):