Microsoft Word - 1-7 1 | Biology 2015) عام 2(العدد 28المجلد مجلة إبن الھيثم للعلوم الصرفة و التطبيقية Ibn Al-Haitham J. for Pure & Appl. Sci. Vol. 28 (2) 2015 Anti-oxidant Activity of Methanol Extracts of Arum maculatum L. and Physalis peruviana L. Plants Zahraa H. Mohammed Dept. of Biology / College of Education for pure science-(Ibn Al-Haitham)/ University of Baghdad Ruqaya M. Ibraheem Dept. of Biotechnology /College of Science / University of Al-Nahrain Received in :21 /December/2014, Accepted in :2/February/2015 Abstract Total flavonoid contents, reductive ability and radical scavenging activity were studied in the methanol extracts of Arum maculatum L. and Physalis peruviana L. The results revealed that A. maculatum extract had total flavonoids of 535.3±109.9 µg/ml, which was significantly higher than the recorded flavonoids in P. peruviana extract (352.0±12.7 µg/ml). Assessment of reductive ability revealed that both extract were effective in such activity and concentration-dependent. The highest absorbance was found at the concentration 0.64 mg/ml of A. maculatum methanol extract (0.929±0.006), which was significantly higher (P ≤ 0.05) than the corresponding concentration of P. peruviana extract (0.850 ± 0.050) or trolox (0.278±0.010), but the second extract also showed a significant increased absorbance compared to trolox. The results of DPPH radical scavenging activity confirmed those of reductive ability, and again the highest concentration (0.500 mg/ml) of both extracts (A. maculatum and P. peruviana) recorded the best radical scavenging activity (93.33±0.58 and 95.33±2.52%, respectively), which was significantly higher than that of vitamin C (64.67±5.03%). In conclusion, both extracts can be considered as important medicinal plants that have the potential of anti-oxidant and free-radical scavenging activities. Keywords: Arum maculatum , Physalis peruviana , Flavonoids , Anti-oxidant. 2 | Biology 2015) عام 2(العدد 28المجلد مجلة إبن الھيثم للعلوم الصرفة و التطبيقية Ibn Al-Haitham J. for Pure & Appl. Sci. Vol. 28 (2) 2015 Introduction The human body has a complex system of natural enzymatic and non-enzymatic anti- oxidant defenses, which counteract the harmful effects of free radicals and other oxidants. Free radicals are responsible for causing a large number of diseases including cancer, cardiovascular disease, neural disorders, Alzheimer’s disease, mild cognitive impairment, Parkinson’s disease, aging and atherosclerosis [1]. Protection against free radicals can be enhanced by ample intake of dietary anti-oxidants, and there is a substantial evidence indicates that nutrients containing anti-oxidants and medicinal plants or their secondary metabolite are of a major importance in disease prevention; therefore, anti-oxidants are of a great benefit in improving the quality of life by preventing or postponing the onset of degenerative diseases due to oxidative stress [2]. The Oxidative stress results from an imbalance between excessive formation of reactive oxygen species (ROS) and/or reactive nitrogen species and limited anti-oxidant defenses [3]. One of the important anti-oxidants is flavonoids, which represent a range of polyphenolic compounds naturally occurring in plants [4]. Flavonoids are potentially involved in cardiovascular prevention mainly by decreasing oxidative stress and increasing NO bioavailability; therefore the estimation of flavonoid content in plant play important roles in providing protection against ROS [5]. Various in vitro and in vivo methods are used to investigate the anti-oxidant property of samples (diets or plant extracts). In the present investigation two in vitro evaluations (reductive ability and DPPH radical scavenging activity) were adopted. The former is based on the principle of increase in the absorbance of the reaction mixtures. Increase in the absorbance indicates an increase in the anti-oxidant activity. In this method, antioxidant compound forms a colored complex with potassium ferricyanide, trichloro acetic acid and ferric chloride. Increase in absorbance of the reaction mixture indicates the reducing power of the samples [6]. The second method employs DPPH molecule [1, 1-diphenyl-2picrylhydrazyl (α,α-diphenyl-βpicrylhydrazyl], which is characterized as a stable free radical by virtue of the delocalization of the spare electron over the molecule as a whole, so that the molecule does not dimerize, as would be the case with most other free radicals. The delocalization of electron also gives rise to the deep violet color, characterized by an absorption band in ethanol solution. [7]. In this investigation, two plants (Arum maculatum and Physalis peruviana) were selected to assess their anti-oxidant activity because of their wide range of medicinal applications. Arum maculatum L. is a member of the family Araceae, and in north of Iraq is known as "Kardi". The plant is a rich source of important chemical constituents; for instance, alkaloids, saponins, glycosides and lectins [8]. Traditional and folk medicine revealed that A. maculatum is suggested to treat kidney stone disease, colitis, liver disease, rheumatic pains and hyperacidities. Furthermore, the plant substance Ari Tubera has been clinically demonstrated to have an anti-inflammatory activity in the intestinal and respiratory tract [9]. In addition, the plant has been reported to have anti- microbial activity, and the plant lectins have been shown to induce neutrophil migration in vitro [10]. Physalis peruviana L.is a member of the family Solanaceae, and in north of Iraq is known as "Gulewaje". It is eaten freshly and commonly used in folk medicine as anti- microbial, anti-pyretic and immune modulator agent. It is also suggested to treat malaria, cancer, leukemia, hepatitis, rheumatism, asthma, dermatitis and other diseases [11]. Phytochemical studies have isolated a number of compounds from P. peruviana, which confirmed its medical importance such as ticloidine, phygrine, withanolide and viscosalactone [12]. Thus different active agents have been extracted from the plant, with a variety of pharmacological activities, and antiinflammatory and cytostatic activity being the most important. It is also an important source of vitamins A and C [13]. 3 | Biology 2015) عام 2(العدد 28المجلد مجلة إبن الھيثم للعلوم الصرفة و التطبيقية Ibn Al-Haitham J. for Pure & Appl. Sci. Vol. 28 (2) 2015 Material and Methods The Plants Arum maculatum (whole plant) and Physalis peruviana (fruits) were obtained from local markets in Erbil, and classified at the Herbarium of Biology Department (College of Education for Pure Sciences / Ibn Al-Haitham, University of Baghdad). Both plants were supplied as air-dried materials, and they were powdered using a coffee grinder. Preparation of Plant Extract Both plants were extracted with methanol as previously described [14]. Briefly, 50 grams of the plants powder were extracted with 80% methanol (250 ml) at 65°C for 3 hours using the soxhlet apparatus. The extracts solution were concentrated to dryness under reduced pressure in a rotary evaporator to yield dried crude extract, which was frozen at -20°C until use to prepare the required concentrations. Determination of Total Flavonoids Total flavonoids content was spectrophotochemically determined in the methanol extract as rutin (flavonoids standard) equivalent by aluminium chloride colorimetric method [15]. Briefly, the methanol extract (3.2 mg) was dissolved in 5 ml of 50% methanol, followed by addition of 1 ml of 5% (w/v) sodium nitrite solution. After 6 minutes, 1 ml of a 10% (w/v) aluminium chloride solution was added and the mixture was allowed to stand for a further 5 minutes before 10 ml of a 10% (w/v) NaOH solution was added. The mixture was made up to 50 ml with distilled water and mixed well. Then, the absorbance was measured at 450 nm with a spectrometer after 15 min. A similar procedure was applied to six concentrations (2.5, 5, 10, 20, 40 and 80 µg) of rutin in order to plot a standard curve. Reductive Ability The method described by Fua et al. [14] was adopted to evaluate the reductive ability, in which 1 ml of each concentration of the plant extract (0.02, 0.04, 0.08, 0.16, 0.32 and 0.64 mg/ml) was mixed with 1ml of 0.2M phosphate buffer (pH 6.6) and 1.5 ml of 1% potassium ferricyanide, and then incubated at 50°C for 20 minutes. After that, 1 ml of 10% trichloroacetic acid was added to the mixture to stop the reaction. The mixture was centrifuged for 10 minutes at 3000 rpm, and 2.5 ml of the supernatant was mixed with 2 ml of distilled water and 0.5 ml of freshly prepared 0.1% Ferric chloride. By then, the absorbance was measured at 700 nm. The same procedure was applied to trolox (vitamin E) solutions (standards). All tests were done in triplicates. DPPH Radical Scavenging Activity The anti-oxidant activity of plant methanol extract and standard (vitamin C) were assessed on the basis of the radical scavenging effect of the stable DPPH free radical [16]. An aliquot of 0.1 ml of the extract or standard (0.062, 0.125, 0.250 and 0.500 mg/ml) was added to 3.9 ml of DPPH solution in a test tube. After incubation at 37°C for 30 minutes, the absorbance of each solution was determined at 517 nm using spectrophotometer. All measurements were made in triplicates. The ability to scavenge DPPH radical was calculated by the following equation: 100 Standard of Absorbance Sample of Absorbance 1 (%)activity scavenging radical DPPH        Statistical Analysis Data are given as mean ± standard deviation (S.D.), and differences between means were ANOVA (analysis of variance) followed by either LSD (least significant difference) of Duncan test. The analyses were carried out using the statistical package SPSS version 13. 4 | Biology 2015) عام 2(العدد 28المجلد مجلة إبن الھيثم للعلوم الصرفة و التطبيقية Ibn Al-Haitham J. for Pure & Appl. Sci. Vol. 28 (2) 2015 Results and Discussion Methanol extract of A. maculatum was found to have total flavonoids of 535.3 ± 109.9 µg/ml, which was significantly higher than the recorded flavonoids in P. peruviana extract (352.0 ± 12.7 µg/ml) (Table 1). However, in both cases, the extracts can be considered as rich sources of flavonoids. As demonstrated by phytochemical investigations, flavonoids belong to a very vast group of plant secondary metabolites with variable phenolic structures and can be found in fruits, vegetables, grains, bark, roots, stems and flowers, and have a broad spectrum of pharmacological activities including their potential role as anti-cancer agents which give them a significance therapeutic advantage [3,4,5]. In agreement with such scope, a positive correlation had been found between flavonoids-rich diet (from vegetables and fruits) and a lower risk of colon, prostate and breast cancers has been observed. It has also been evident, that a treatment with A. maculatum aqueous extract remarkably lowered the mitotic index in bone marrow cells of Swiss male mice at all exposure times, and in almost all concentrations tested compared to controls [17]. The authors justified their findings on the basis of high flavonoid contents acting in synergism with other constituents. In addition, flavonoids are known to inhibit lipid-peroxidation and platelet aggregation, and they exert these effects due to their anti-oxidant potential in scavenging free radicals [18]. Such potentials and high content of flavonoids encouraged further to assess the anti-oxidant activity of both extracts. Assessment of reductive ability revealed that both extract were effective in such activity, in which it was concentration-dependent. The highest absorbance was observed at the concentration 0.64 mg/ml of A. maculatum methanol extract (0.929 ± 0.006), which was significantly higher (P ≤ 0.05) than the corresponding concentration of P. peruviana extract (0.850 ± 0.050) or trolox (278 ± 0.010), but the second extract also showed a significant increased absorbance compared to trolox. However, in both extract, the increase was almost three-folds higher than that of trolox (positive control) (Table 2). The results of DPPH radical scavenging activity confirmed those of reductive ability, and again the highest concentration (0.500 mg/ml) of both extracts (A. maculatum and P. peruviana) recorded the best radical scavenging activity (93.33 ± 0.58 and 95.33 ± 2.52%, respectively), which significantly higher than that of vitamin C (64.67 ± 5.03%) (Table 3). The presented results suggest the antioxidant and free-radical scavenging activities of both extracts, which even exceeded the positive control values of trolox (vitamin E) and vitamin C. It is possible to explain that in the ground of high flavonoid contents, and as presented earlier, flavonoids of natural resources represent important anti-oxidant chemical constituents of plants [3,4,5]. With respect to P. peruviana, interest in the anti-oxidant properties of its fruits is also shared by recent investigations, and some of the medicinal properties of the plant have been associated with the anti-oxidant capacity of polyphenols [19, 20]. However, the anti-oxidant activity of P. peruviana has been suggested to be not a property of a single phytochemical compound, but due to the synergistic effect of different antioxidants existing in the fruits [21]. In a further investigation, hot aqueous and methanolic extracts P. peruviana were also evaluated for anti-oxidant activities, and the authors concluded that ethanol extract possessed good anti-oxidant activities, and the highest activity was observed in 95% ethanol extract, and in addition, this extract demonstrated a strong superoxide anion scavenging activity and xanthine oxidase inhibitory effect [12]; conclusions that are also favored by the present results. The A. maculatum plant is the less studied, but it showed better results than those obtained for P. peruviana, and the presented antioxidant property of the extract was higher. This is possibly might be associated with the higher concentration of flavonoids. The conclusin is that A. maculatum and P. peruviana are a rich source of flavonoids, which lend both plants to have important anti-oxidant and free-radical scavenging properties. However, the presented evidences were based on in vitro experiments, 5 | Biology 2015) عام 2(العدد 28المجلد مجلة إبن الھيثم للعلوم الصرفة و التطبيقية Ibn Al-Haitham J. for Pure & Appl. Sci. Vol. 28 (2) 2015 and therefore in vivo evaluations are certainly required to explore the medicinal applications of the two plants especially in the fields of antioxidant and anti-inflammatory potentials. References 1. Chatterjee, S., Chatterjee, S., Dey, K.K. and Dutta, S. (2013). Study of antioxidant activity and immune stimulating potency of the ethnomedicinal plant, Cassia alata (L.) Roxb. Med. Aromat. Plants, 2: 4. 2. Zhu, X.X., Yang, L., Li, Y.J., Zhang, D., Chen, Y., Kostecká, P., Kmoníčková, E. and Zídek, Z. (2013). Effects of sesquiterpene, flavonoid and coumarin types of compounds from Artemisia annua L. on production of mediators of angiogenesis. Pharmacol. Rep., 65: 410- 420. 3. Grassi, D., Desideri, G., Croce, G., Tiberti, S., Aggio, A. and Ferri, C. (2009). Flavonoids, vascular function and cardiovascular protection. Curr. Pharm., 15: 1072-1084. 4. Çirak, C., Radušien, J., Janulis, V., Ivanauskas, L., Çamaş, N. and Kemal, A. (2011) Phenolic constituents of Hypericum triquetrifolium Turra (Guttiferae) growing in Turkey: variation among populations and plant parts. Ayan Turk. J. Biol., 35: 449456. 5. Grassi, D., Desideri, G. and Ferri, C. (2010). Flavonoids: antioxidants against atherosclerosis. Nutrients, 2: 889-902. 6. Jayaprakash, G.K., Singh, R.P. and Sakariah, K.K. (2001). Antioxidant activity of grape seed extracts on peroxidation models in-vitro. J. Agric. Food Chem., 55: 1018-1022. 7. Rengasamy, K.R., Aderogba, M.A., Amoo, S.O., Stirk, W.A. and Van Staden, J. (2013). Potential antiradical and alpha-glucosidase inhibitors from Ecklonia maxima (Osbeck) Papenfuss. Food Chem., 141: 1412-1415. 8. Bedalov, M. and Kupfer, F. (2005). Studies on the genus Arum (Araceae). – Bull. Soc. Neuchâteloise Sci. Nat. 128: 43-70. 9. Nikolov, S. (ed.) (2006). Specialized Encyclopedia of medicinal plants in Bulgaria. “Bulgarian encyclopedia” Sofia . 10. Alencar, V.B., Alencar, N.M., Assreuy, A.M., Mota, M.L., Brito, G.A., Aragao, K.S., Bittencourt, F.S., Pinto, V.P., Debray, H., Ribeiro, R.A. and Cavada, B.S. (2005). Pro- inflammatory effect of Arum maculatum lectin and role of resident cells. Int. J. Biochem. Cell. Biol., 37: 1805-1814. 11. Ramar, K., Ayyadurai, V. and Arulprakash, T. (2014). In vitro shoot multiplication and plant regeneration of Physalis peruviana L. an important medical plant. Int. J. Curr. Microbiol. App. Sci., 3: 456-464. 12. Zhang, Y.J., Deng, G.F., Xu, X.R., Wu, S., Li, S. and Li, H.B. (2013). Chemical components and bioactivities of cape gooseberry (Physalis peruviana). Int. J. Food Nutr. Saf., 3: 15-24. 13. Chang, J.C., Lin, C.C., Wu, S.J., Lin, D.L., Wang, S.S., Miaw, C.L. and Ng, L.T. (2008). Antioxidant and hepatoprotective effects of Physalis peruviana extract against acetaminophen-induced liver injury in rats. Pharm. Biol., 46: 724-731. 14. Fua, W., Chena, J., Caia, Y., Leia, Y., Chenb, L., Peic, L., Zhoua, D., Lianga, X. and Ruana, J. (2010). Antioxidant, free radical scavenging, antiinflammatory and hepatoprotective potential of the extract from Parathelypteris nipponica (Franch. et Sav.) Ching. J Ethnopharmacol., 130: 521-528. 15. Sakanaka, S., Tachibana, Y. and Okada, Y. (2005). Preparation and antioxidant properties of extracts of Japanese persimmon leaf tea (kakinohacha). Food Chem., 89: 569575. 16. Sanja, S.D., Sheth, N.R., Patel, N.K., Dhaval, P. and Biraju, P. (2009). Characterization and evaluation of antioxidant activity of Portulaca oleracea. Int. J. Pharm. Pharm. Sci., 1: 74- 84. 17. Nabeel, M., Abderrahman, S. and Papini, A. (2008). Cytogenetic effect of Arum maculatum extract on the bone marrow cells of mice. Caryologia, 61(4):383-387. 6 | Biology 2015) عام 2(العدد 28المجلد مجلة إبن الھيثم للعلوم الصرفة و التطبيقية Ibn Al-Haitham J. for Pure & Appl. Sci. Vol. 28 (2) 2015 18. Sandhar, H.K., Kumar, B. Prasher, S., Tiwari, P., Salhan, M. and Sharma, P. (2011). A review of phytochemistry and pharmacology of flavonoids. Internationale Pharmaceutica Sciencia, 1: 25-41. 19. Liu, H., Qiu, N., Ding, H., and Yao, R. (2008). Polyphenols contents and antioxidant capacity of 68 Chinese herbals suitable for medical or food uses. Food Res. Int., 41: 363−370. 20. Vijaya Kumar Reddy, C., Sreeramulu, D., and Raghunath, M. (2010). Antioxidant activity of fresh and dry fruits commonly consumed in India. Food Res. Int., 43: 285−288. 21. Wu, S. J., Ng, L. T., Huang, Y. M., Lin, D. L., Wang, S. S., Huang, S. N., and Lin, C. C. (2005). Antioxidant activities of Physalis peruviana. Biol. Pharm. Bull., 28: 963−966. Table No.(1):Total flavonoid content in the methanol extract of Arum maculatum and Physalis peruviana. P ≤ Mean ± S.D. (µg/mg) Plant Methanol Extract 0.05 535.3 ± 109.9 Arum maculatum 352.0 ± 12.7 Physalis peruviana TableNo.(2): Reductive ability of Arum maculatum and Physalis peruviana methanol extracts. Plant Concentration (mg/ml) Absorbance (Mean ± S.D.) Positive control (Trolox) Arum maculatum Physalis peruviana 0.02 C0.109±0.002f A0.504±0.014f B0.446±0.027f 0.04 C0.117±0.002de A0.602±0.010e B0.491±0.022e 0.08 C0.138±0.008d A0.655±0.024d B0.565±0.041d 0.16 B0.164±0.015c A0.715±0.006c A0.704±0.013c 0.32 C0.230±0.022b A0.846±0.006b B0.744±0.014b 0.64 C0.278±0.010a A0.929±0.006a B0.850±0.050a Different upper case letters: Significant difference (P ≤ 0.05) between means of columns. Different lower case letters: Significant difference (P ≤ 0.05) between means of rows. Table No.(3): DPPH radical scavenging activity in Arum maculatum and Physalis peruviana methanol extract. Plant Concentration (mg/ml) Anti-oxidant activity (mean ± S.D.; %) Positive control (Trolox) Arum maculatum Physalis peruviana 0.062 C39.00±1.73d A72.33±0.58d B69.33±1.53d 0.125 B46.00±4.58bc A80.00±1.00c A76.67±2.08c 0.250 B50.33±6.81b A83.00±1.00b A88.33±2.08b 0.500 B64.67±5.03a A93.33±0.58a A95.33±2.52a Different upper case letters: Significant difference (P ≤ 0.05) between means of columns. Different lower case letters: Significant difference (P ≤ 0.05) between means of rows. 7 | Biology 2015) عام 2(العدد 28المجلد مجلة إبن الھيثم للعلوم الصرفة و التطبيقية Ibn Al-Haitham J. for Pure & Appl. Sci. Vol. 28 (2) 2015 Arum كاردي الفعالية المضادة لالكسدة للمستخلصات الكحولية لنباتي maculatum L. كولة وازهوPhysalis peruviana L. زھراء حسين محمد جامعة بغداد.)/ابن الھيثم(قسم علوم الحياة/كلية التربية للعلوم الصرفة رقية محمد ابراھيم جامعة النھرين.-العلومقسم التقانة االحيائية/كلية ٢/٢/٢٠١٥:، قبل البحث في ٢٠١٤/ ٢١/١٢استلم البحث في: الخالصة درس المحتوى الفالفوني والقابلية االختزالية وفعالية كنس الجذور الحرة في المستخلص الكحولي للنباتين كاردي Arum maculatum L. كولة وازهوPhysalis peruviana L. أظھرت النتائج امتالك مستخلص .A. maculatum مايكروغرام/مل، بحيث كان مرتفع معنويا مما ھو عليه في مستخلص 535.3±109.9محتوى فالفوني كلي P. peruviana 12.7±352.0) مايكروغرام/مل). واتضح عند تقدير القابلية االختزالية بأن كال المستخلصين كانا ملغم/مل للمستخلص 0.64 مؤثرين في تلك الفعالية ومرتبطا على التركيز. لقد لوحظت أعلى امتصاصية في التركيز ما ھو عليه في مستخلص ) مp ≤ 0.05) والتي كانت مرتفعة معنويا ((A. maculatum 0.006±0.929 الكحولي لنبات P. peruviana لكن كانت امتصاصية المستخلص (0.278±0.010) او عقار ترولكس (0.850±0.050لنفس التركيز ،( معززة لتلك المالحظة DPPHالثاني ھي االخرى مرتفعة معنويا مقارنة بالترولكس. جاءت نتائج كنس الجذور الحرة لمادة .P و A. maculatum ملغرام/مل) لكال المستخلصين ( (0.500جل اعلى تركيزفي القابلية االختزالية، بحيث س peruviana) على التوالي)، والتي كانت 95.33±2.52%و 93.33±0.58) أفضل النتائج في كنس الجذور الحرة ، دان الى نباتين ). يمكن االستنتاج بأن كال المستخلصين يعوC )64.67 ± 5.03%مرتفعة معنويا مما ھو عليه في فيتامين ذو اھمية طبية من ناحية القابلية االختزالية وفعالية كنس الجذور الحرة. .Arum maculatum , Flavonoids , Physalis peruviana , Anti-oxidantالكلمات المفتاحية: