Iraqi J Pharm Sci, Vol.24(1) 2015 Iraqi Tribulus 68 Phytochemical Investigation and Antioxidant Activity of Iraqi Tribulus terrestris Nabaa M. Ibrahim * and Enas J. Kadhim *,1 * Department of Pharmacognosy and Medicinal Plant , College of Pharmacy,University of Baghdad,Baghdad,Iraq. Abstract The aim of the present study was to characterize the Iraqi Tribulus terrestris for the presence of biologically active phyto-chemicals using methanolic extracts of the plant (aerial parts) by Gas Chromatography –Mass spectrometry (GC/MS), while the mass spectra of the compounds found in the extract was matched with the National Institute of Standards and Technology (NIST) library , in addition to study the antioxidant activity of plant extract , results confirmed the presence of therapeutically potent compounds in the Iraqi Tribulus terrestris extract predominantly alkaloids, flavonoids, saponins, tannins and terpenoids. Antioxidant potential of Iraqi Tribulus terrestris herbal preparations was evaluated by determination of blood glutathione, serum ascorbic acid and serum superoxide dismutase in rats. The obtained results demonstrated that T. terrestris preparations possess a significant antioxidant activity. Keywords: Iraqi Tribulus terrestris, Phytochemical investigation, Anti-oxidant activity. دراست المكوناث الكيمائيت والفعاليت المضادة لالكسدة لنباث ذقن الشيخ العراقي نبأ محمد ابراهيم * و ايناس جواد كاظم ،*1 * فزع العمالٍز َالىباحاث الطبٍت ،كلٍت الصٍذلت ، جامعت بغذاد ، بغذاد ، العزاق . الخالصة المُاد الكٍمٍائٍت الفعالت المُجُدة فً االجشاء الٍُائٍت للمسخخلص الكحُلً لىباث عزالً شخٍصالٍذف مه ٌذي الذراست ٌُ ح َمماروت الىخائج مع الىخائج GC/MS مطٍاف الكخلت-الفصل الكزَماحُجزافً للغاساث ٌسمى )دله الشٍخ( اَ شمشك باسخخذام طزٌمت ( باالضافت الى دراست الفعالٍت المضادة لالكسذة لمسخخلص الىباث . NISTالمثبخت فً المعٍذ الُطىً للمعاٌٍز َالخكىُلُجٍا) شُفاث كٍمٍائٍت محذدة لذ حمج على المسخخلص المٍثاوُلً ان عملٍت الكشف الىُعً الخمٍٍذي لالٌضاث الثاوٌُت المخخلفت مه لبل ك .لالجشاء الٍُائٍت مه الىباث َاشارث الىخائج ان ٌذي االجشاء ححخُي على الملٌُذاث, فالفٍىٌُذاث، صابُوٍاث , مُاد دباغٍت َ حزبٍىاث الىخائج ان المسخخلص الكحُلً لذًٌ فعالٍت حضمىج ٌذي الذراست اٌضا الكشف عه الفعالٍت المضادة لالكسذة لىباث الشمشك َاظٍزث .ةجٍذة ضذ االكسذ نباث ذقن الشيخ العراقي ، الدراست الكيميائيت ،الفعاليت المضادة لألكسدة .:الكلماث المفتاحيت Introduction Tribulus terrestris (Family: Zygophyllaceae ) is a perennial creeping herb widely distributed in Iraq. it is regarded as an aphrodisiac in addition to its beneficial claims on various ailments such as urinary tract infections, inflammations, oedema and ascites (1) . In Iraq T. terrestris(figure-1) is used in folk medicine as tonic, aphrodisiac, analgesic, astringent, stomachic, anti- hypertensive, diuretic, lithon- triptic and urinary anti- infective (2) .The aphrodisiac property of this plant extract was examined in rats (3) . Administration of Tribulus extract to humans and animals improves libido and spermatogenesis (4) .Clinical studies showed that this plant improved reproductive function, including increased concentration of hormones such as estradiol, with testosterone being very slightlyinfluenced, thereby improving reproductive function, libido and ovulation (5) . Free radicals and reactive oxygen species are generated in living cells as a result of different biochemical and physiological processes, they are the causative agents for many chronic diseases, such as cancer, diabetes, aging and other degenerative diseases in humans due to oxidative damage of proteins, lipids and DNA (6) . Plants are the valuable sources of natural products for maintaining human health, more than 80%of population across the world use traditional medicine including compounds derived from medicinal plants. Therefore, such plant should be investigated to better understand their properties, safety and efficiency (7) . The aim of this work was to investigate the chemical constituents and antioxidant activity of methanolic extract of aerial parts of a newly studied plant widely and wildly distributed in our country Iraq. 1 Corresponding author E-mail: dr.Enasjawad@yahoo.com Received: 8/3/2015 Accepted: 18 /5/2015 Iraqi J Pharm Sci, Vol.24(1) 2015 Iraqi Tribulus 69 Plant material and Methods The aerial part of Tribulus terrestris(Family: Zygophyllaceae) was collected from Kirkuk, a city in the north of Iraq, 236 kilometers (147 mi) north of Baghdad. The plant was authenticated by the National Herbarium at Abu-Graib, the plant leaves were dried in the shade for several days at room temperature and then grinded as powder and weighed. Figure(1):Iraqi Tribulus terrestris The experimental work is divided into • The experimental preliminary phytochemical screening of various secondary metabolites like alkaloids, flavonoids, steroids, tannins, saponins, anthraquinioin and terpenoids in the plant. • Extraction of different active constituents. • GC-MS analysis of methanolic extract of the plant. • Investigation of the antioxidant activity of methanolic extract of aerial parts of plant Preliminary qualitative phytochemical analysis Chemical tests were carried out using the methanolic extracts from plant using standard procedures to identify the active constituents (8- 10) : Test for alkaloids Alcoholic extract (10 ml) was stirred with 5 ml of 1% HCL on a steam bath. Mayer’s (1.35gm mercuric chloride in 60ml water + 5gmpotassium iodide in 10ml water )and Wagner’s reagents (1.27g of iodine and 2g of potassium iodide in 100ml of water) were added, white and reddish brown color precipitate respectively, were taken as evidence for the presence of alkaloids. Test for flavonoids Lead acetate test: Lead acetate 10% (1 ml) solution was added to 5ml of alcoholic extract, the formation of a yellowish- white precipitate was taken as a positive test for flavonoids. Tests for steroids Liebermann-Burchard test: Extract (3ml) was treated with chloroform, acetic anhydride and drops of sulphuric acid was added. The formation of dark pink or red color indicates the presence of steroids. Test for tannins Plant material (10mg) in 10ml distilled water was filtered, and then the filtrate (3ml) + 3ml of FeCl3 solution (5%w/v) were mixed. The formation of a dark green or blue black precipitate was considered an indication for the presence of tannins. Tests for anthraquinones Borntrager’s test: Alcoholic extract of 3ml was shaken with 3 ml of benzene, filtered and 5 ml of 10% ammonia solution was added to the filtrate. The mixture was shaken and the development of a pink, red or violet color in the ammonical (lower) phase indicates the presence of free anthraquinoin. Test for terpenoids Alcoholic extract (2ml) was dissolved in chloroform (2ml) and evaporated to dryness. Concentrated sulphuric acid (2ml) was then added and heated for about 2 min. A grayish color was considered an indication for the presence of terpenoids. Preparation of extract Shade-dried coarsely powdered aerial parts of Tribulus plant was defatted with hexane for 24 hours then allowed to dry at room temperature. The defatted plant material was extracted with 85% methanol in soxhlet apparatus until complete exhaustion. The alcoholic extract was evaporated under reduced pressure at a temperature not exceeding 40C • to give a dark-brown residue designated as a crude extract. Animals Healthy adult 30 male mice weighing 120- 150gm were used in this study. The animals had free access to a standard commercial diet and water; they were kept in rooms maintained at 25-27°C. The animals were divided randomly into three groups; each group consisted of ten male mice: Group 1: Received 100 mg/Kg body wt. of 85 % methanolic extract of the plant. Group 2: Received 50 mg/Kg body wt. of85 % methanolic extract of the plant. Group 3: Served as control group and received only 2% gum acacia (0.2ml). Iraqi J Pharm Sci, Vol.24(1) 2015 Iraqi Tribulus 70 The extracts were suspended in distilled water using Tween 20, and the dose was orally administered once daily for 4 weeks. At the end of treatment, blood samples were collected centrifuged and serum was separated for the determination of the following: 1- Blood glutathione content according to the method described by Beutler (11) . 2- Serum superoxide dismutase activity, the method was carried out according to the pyrogallol method of Marklund (12) . 3- Serum ascorbic acid was estimated by the method of Jagota (13) . GC-MS analysis Instruments and chromatographic conditions GC-MS analysis was carried out on GC- MS-QP2010 Shimadzu system comprising a gas chromatographinterfaced to a mass spectrometer instrument employing the following conditions : column VF-5MS fussed silica capillary column (30.0m x 0.25mm x 0.25μm, composed of 5% phenyl/95% dimethylpolysiloxane), operating in electron impact mode at 70ev; helium (99.999%) was used as carrier gas at a constant flow of 1. ml/min and an injection volume of 0.5μl was employed (split ratio of 10:1) injector temperature 240 0C ion-source temperature 200 C ₒ . The oven temperature was programmed from 100 C ₒ (isothermal for 3 min), with an increase of 10C ₒ /min, to 240 C ₒ , ending with a 9min isothermal at 270 C ₒ . Mass spectra were taken at 70ev; a scan interval of0.5 seconds and fragments from 40 to 440Da. Total GC running time is 30min. Results and Discussion The results of preliminary qualitative phytochemical of Iraqi Tribulus terrestris are given in table-1.The results of preliminary phytochemical screening of plant extracts showed the presence of alkaloids, flavonoids, tannins, saponins and terpenoids and the absence of steroids and anthraquinoin. Many researchers reported that the concentration of secondary metabolites are varying from plant to plant belong to the same genus and even in the different parts of the same plant (14) , this is due to many factors like environmental heterogeneity, since the effect of environmental heterogeneity is highly scale- dependent. It may create high niche diversity and hence allow species to coexist at a large spatial scale (15) , also the high complexity and heterogeneity of soil, like(soil structure, texture and depth, moisture retention characteristics, aeration) create a big variation in the chemical constituents even in the same country (16) . Identification of components by GC-MS: Interpretation on mass spectrum of GC- MS was done using the database of National Institute of standard and Technology (NIST) having more than 62,000 patterns. The mass spectrum of the unknown component was compared with the spectrum of the known components stored in the NIST library . The results of GC-MS analysis led to the identification of number of compounds from the methanol extract of Iraqi Tribulus plant. GC-MS chromatogram showed 46peaks, indicating the presence of 46 compounds (figure-2) and (table- 2).many of these components reported in this plant for the first time like monoterpene [example Terpineol] sesquiterpenes: [2,3,8,8-Tetramethyltricyclo- 2ene], [1 ,4 – dimethyl – 8 - isopropylidenetricyclodecane, alkaloids like - (3-Methoxy-2-pyrazinyl)-2-methyl-1-propanol and Thiazole, Saturated fatty acid[example Myristic acid]Coumaran and many phenolic compounds, Coumaric acid, Linoleic acid, Arachidic acid and oleic acid. Table(1): Phytochemical screening of Tribulus extract Terpenoids Anthraquinoin Saponins Tannins Steroids Flavonoids Alkaloids + - + + - + + +, - represent presence and absence of phytoconstituents respectively. Iraqi J Pharm Sci, Vol.24(1) 2015 Iraqi Tribulus 71 Figure(2): GC-MS Chromatogram of methanolic extract of Iraqi Tribulus terrestris Table(2): Phytocomponents identified in the methanolic extracts ofTribulus terrestris Peak# R.Time Area% Name 1 4.168 0.56 3-Penten-2-one, 4-methyl- 2 4.257 0.39 1-Butanol 3 4.534 0.24 Hexanal dimethyl acetal 4 4.981 0.68 Glycerin 5 5.592 0.97 Trimethylsilylmethanol 6 6.092 1.54 4-Hexenoic acid, 2-(phenylsulfonyl)-, methyl ester, (E)- 7 6.626 1.50 Coumaric acid 8 7.081 12.75 2-Hexanol, 2-methyl- 9 8.300 2.29 Archidic acid 10 8.552 0.30 2-Pentanone, 4-hydroxy- 11 8.800 0.19 Nonanal dimethyl acetal 12 9.049 0.48 1-Hexanol, 2-ethyl 13 9.242 0.77 Linoleic acid 14 9.341 0.74 Silane, [2-(2-methoxyethoxy)ethoxy]trimethyl- 15 9.729 0.32 Propanoic acid 16 9.821 0.81 Myristic acid 17 10.923 0.27 (R)-(-)-2,2-Dimethyl-1,3-dioxolane-4-methanol 18 10.982 0.26 1,4-dimethyl-8-isopropylidenetricyclodecane 19 12.323 0.35 Cyclohexanol, 1-methyl-4-(1-methylethylidene)- 20 13.767 38.72 Stearic acid 21 14.219 0.27 (2-Benzyloxy-2-oxiran-2-ylethoxy)-t-butyldimethylsilane 22 14.585 0.30 Cyclononasiloxane, octadecamethyl- Iraqi J Pharm Sci, Vol.24(1) 2015 Iraqi Tribulus 72 Table(2): Contained phytocomponents identified in the methanolic extracts of Tribulus terrestris The groups treated with alcoholic extracts of aerial part of Iraqi Tribulus showed a significant increase in blood glutathione level, serum superoxide dismutase activity and serum ascorbic acid level comparing with control group Table( 3): Mean blood glutathione content, serum ascorbic acid and serum superoxide dismutase among group of rats treated alcoholic extracts of T. terrestris. Group (2) Group (1) Control group Tested parameter 4.15 ± 0.21* 8.53 ± 0.317* 1.92 ± 0.052 Blood glutathione (mg/ gm Hb) 17.5 ± 1.03 * 30.2 ± 2.06* 10.23 ± 0.6 Superoxide dismutase (μg/ml ) 6.54 ± 1.03* 12.09 ± 0.46* 3.23 ± 0.43 Ascorbic acid (μg/ml ) Group (1): treated with100 mg/Kg body wt. of 85 % methanolic extract of the plant Group (2): treated with 50 mg/Kg body wt. of 85 % methanolic extract of the plant. * Significantly different from control value. Discussion In much of the developing countries, 70– 95% of the populationrely on traditional medicines for primary care, and between70% and 90% of populations in industrialized world use traditional medicines under the titles “complementary”, “alternative”,or “nonconventional” (17) . Plants have formed the basis for traditional medicinal systems for thousands of years, with the first records dating from about 2600 BC in Mesopotamia. Traditional knowledge of medicinal plants has always guided the search for new cures. In spite of the advent of modern high throughput drug discovery and screening techniques, traditional knowledge systems have given clues to the discovery of valuable drugs. In the present study, methanolic extract of the Iraqi Tribulus terrestriswas analyzed for the first time for the presence of different secondary metabolites which could be of medicinal & economic value and study antioxidant activity of crude extract of this Iraqi plant. The comparison of the mass spectrum with the NIST database library gave more than 90% match as well as a confirmatory compound structure match. This work will help to identify the compounds, which may be used in body products, drugs, pharmaceutical and therapeutic value since Peak# R.Time Area% Name 23 14.641 0.07 N-Cbz-glycylglycine p-nitrophenyl ester 24 15.099 0.62 Phenylethyl Alcohol 25 15.465 1.59 3,7,11,15-Tetramethyl-2-hexadecen-1-ol 26 15.816 0.58 Dodecanal 27 16.134 0.68 D-Mannotridec-6-ene-1,2,3,4,5-pentaol 28 16.361 0.42 Heptacosanoic acid, methyl ester 29 16.544 0.23 Heptanoic acid, 3-buten-1-yl ester 30 16.874 0.55 Thiazole, 4-ethyl-2,5-dimethyl 31 17.409 0.33 Methyl(methyl 3,4-di-O-methyl-.alpha.-D- mannopyranoside)uronate 32 17.409 2.24 2-Pentadecanone, 6,10,14-trimethyl- 33 18.322 0.15 Ethanone, 1-phenyl-2-(phenylsulfonyl)- 34 18.891 0.48 Z-25-Tetratriaconten-2-one 35 19.136 12.43 Hexadecanoic acid, methyl ester 36 19.303 0.46 2,3,8,8-Tetramethyltricyclo-2ene 37 19.734 0.18 Phenol, 3,5-bis(1,1-dimethylethyl) 38 20.515 0.99 Methyl 10-methyl-hexadecanoate 39 20.932 0.22 1,4-dimethyl-8-isopropylidenetricyclodecane 40 21.089 0.03 Hexanedioic acid, bis(2-ethylhexyl) ester 41 21.709 0.2 Diethyl Phthalate 42 21.961 1.9 Benzofuran, 2,3-dihydro- 43 23.241 3.59 Octadecanoic acid, methyl ester 44 23.762 1.73 9-Octadecenoic acid, methyl ester 45 24.231 0.77 3-Methoxy-2-pyrazinyl)-2-methyl-1-propanol 46 25.211 0.43 Oleic acid Iraqi J Pharm Sci, Vol.24(1) 2015 Iraqi Tribulus 73 many components isolated from this plant reported for the first time, also the present study results were confirmed the traditional uses of this plant as an antioxidant, anti- inflammatory, antispasmodic agent due to different secondary metabolites constituents like flavonoids, essential oil, alkaloids , saponins and others . The characteristic antioxidant properties of T.terrestris may cause significant increase in blood glutathione level, serum superoxide dismutase activity and serum ascorbic acid level. Based on the results obtained in this study, it could be said that T.terrestris plant powder contains chemical constituents of pharmacological and nutritional significance. However, it is recommended that further work be carried out to isolate and purify the bioactive constituents in this plant powder using various extraction solvents with a view to characterizing their molecular structure, formula, weight as well as evaluating their safety or otherwise (toxicity) for human and other animal use. Acknowledgment Many thanks to College of Pharmacy in University of Baghdad for their support and our deepest gratefulto AL-Mustansiriya University .College of Science, Department of Chemistry for their help in running GC-MS and many thanks to College of Science in Baghdad University for their help in estimating antioxidant activity of our plant. References 1. Gauthaman K, Ganesan A. The hormonal effects of Tribulus terrestris and its role in the management of male erectile dysfunction – an evaluation using primates, rabbit and rat. Phytomedicine 2008; 15:44. 2. S. H. Majeed, and M. J. Mahmood. Herbs and Medicinal Plants in Iraq between Traditional Medicine and Scientific Research. 1st Ed. Baghdad:Dar Al- Thaowra for Publishing., 1988, p. 40. 3. Anand R, Patnaik GK, Kulshreshtha DK, Dhawan BN. Activity of certain fractions of Tribulus terrestris fruits against experimentally induced urolithiasis in rats. Ind J ExpBiol 1994;32:548–552. 4. Koumanov F, Bozadjieva E, Andreeva M, Platonva E, Ankov V, Clinical trial of Tribestan. Experiment Med 1982; 1:2–4. 5. Gauthaman K, Adaikan PG, Prasad RN. Aphrodisiac properties of Tribulus terrestris extract (Protodioscin) in normal ancastrated rats. Life Sci 2002; 71: 1385- 1396. 6. Harman D., Aging: phenomena andtheories. Ann NY AcadSci, 1998;854:1-7. 7. Ogbole O. O., Gbolade A. A, AjaiyeobaE. O. , Ethno-botanical Survey of Plants used in Treatment of Inflammatory Diseases in Ogun State of Nigeria. European Journal of Scientific Research, , 2010;43 (2): 183- 191 8. Kokate C. K., Gokhale S. B., Purohit A. P. A Text book of Pharma-cognosy. 29th ed., Nirali Prakashan, 2009, p. 635. 9. Harborne J.B. Phytochemical Methods, A Guide to Modern Techniques of Plant Analysis.1st ed. London: Chapman and Hall; New York, 1973, p.278. 10. Sarker S. D., Latif Z., Gray A. I. Natural Products Isolation. 2 nd ed. Humana Press, Totowa, New Jersey, 2005, p .515. 11. Beutler E, Duron O and Kellely B (1963). Improved methods for determination of blood glutathione.Journal of Laboratory and Clinical Medicine, 61: 882-888. 12. Marklund S and Marklund G. Involvement of the Superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase.Eur. J. Biochem., 1974; 47: 469-474. 13. .Jagota S and Dani H. A new colorimetric technique for estimation of Vitamin C. Biochemistry, 1982;127: 178-182. 14. Abdul K. K., Palwasha A., Ayeesha M., Safdar Ali K. ,Rasool B.T.: Response of plant parts and age on the distribution of secondary Metabolites on plants found in Quetta. Pak J Bot 2009; 41(5): 2129-35. 15. Pausas J. G.1 , Austin M.: Patterns of plant species richness in relation to different environments: An appraisal. Journal of Vegetation Science 2001; 12: 153-166. 16. Karlovsky P.: Secondary Metabolites in Soil Ecology. Volume 14,1st ed., Springer-Verlag Berlin, Heidelberg ,2008, 293p. 17. Robinson, M.M., Zhang, X. The World Medicines Situation Traditional Medicines: Global Situation, Issues and Challenges. WHO Press, World Health Organization. 2011.