SCREENING OF PHYTOCHEMICAL AND ANTIBACTERIAL ACTIVITIES OF LEAVES OF SELECTED MEDICINAL FORM Al-KHUMS, LIBYA *Salem Mohamed Edrah 1, Fouzy Alafid2, 1Chemistry Department, Science College, El- Mergeb University, Al-Khums Libya *Author & Corresponding Author: [email protected], Tel: +218924748909 2Institute of Organic Chemistry and Technology, Faculty of Chemical Technology, University of Pardubice, Czech Republic

Abstract: Human beings benefit from medicinal plants to make life healthy and free from disease. Medicinal properties of plants perform them effective to halt or remedy disease. The present study was to evaluate the phytochemical constituents and antibacterial activity assessment of Crude extract (aqueous and ethanolic) leaves of three medicinally significant plants (Zygophyllum, visnaga and Rhus tripartita). Qualitative screening of phytochemical components such as Tannins, Phlobatanins, Saponins, Flavonoids, Steroids, Alkaloids, Anthraquinones, Coumarins, Terpenoids and Cardiac Glycosides were present in both extracts whilst steroids not present in all aqueous extracts of these plants and Anthraquinones present only in the Rhus tripartita in aqueous and ethanolic extracts. Whereas the Quantitive screening of phytochemical components percentage’s yields of Zygophyllum, Ammi visnaga and Rhus tripartita were 33%, 26% and 29% respectively, while the percentage’s yields of alkaloids were found as 2.4, 8 and 8.5% correspondingly, and the percentage’s yields of saponins were found as 7.6, 6 and 3.2% respectively. And the percentage’s yields of flavonoids were 9.6, 10 and 8.7% individually. Furthermore, the biological importance of these compounds together with the use of these plants in traditional medicine also encouraged us to investigate it for antibacterial activity. For this determination, the crude extracts (Aqueous and Ethanolic) of the leaves were used. The antibacterial activity of the crude extracts of the tree plants leaves was assessed by Disc Diffusion Method, which showed effective activity against Gram-positive bacterial strains (Staphylococcus aureus and Staphylococcus epidermis) and Gram-negative bacterial strains (Klebsiella pneumonia and Escherichia coli). What is more, the crude extracts of leaves exhibited effective inhibition as the comparison with the used standard Antibiotics.

Keywords: phytochemicals, “Zygophyllum”, “Ammi visnaga (L.)Lam”, Rhus tripartita, Antibacterial Activity.

1. Introduction: Since a long while of time, various plants were used as a provenance of medicines, plants were used for the healing of illnesses without knowledge about the chemical ingredients that influence and their method of action. The biological activity of extracts traditionally used plants is still proper owing toward the presence of microbial resistance to several of antibiotics and the occurrence of fatal infection. A wide variety of medicinal plants is used by African healers for the preparation of treatments to eliminate gastrointestinal parasites (McGaw et al.; 2000.). “Zygophyllum album L” (Z. album) is common perennial herbaceous plants grown in Libya. It’s used as antiseptic, stimulant, carminative (Meng et al.; 2002). “Ammi visnaga (L) LAM.” (A. visnaga) obligates its ancestries in the warm climate of the Mediterranean, Likewise indigenous plants prevalent in North African countries (Tutin, 1968; Batanouny, 1999; Bueno, et al, 2006, Chevallier, 1996). Moreover, A. visnaga were used in the treatment of respiratory conditions such as bronchitis, asthma, and cough for mild angina treatment (Farnsworth, 2001; Anonymous, 2007). And found typically useful for the recovery of wound healing, psoriasis, poisonous bites and inflammation conditions (Abdelfattah, et al.; 1983, Valkova, et al 2004). “Rhus tripartita” (R. tripartita) was used to treat gastrointestinal and inflammatory conditions (El-Mokasabi, 2014). Medicinal plants constituents own a rich source of active phytochemicals for instance Tannins, flavonoids, Glycosides, Anthraquinones and other constituents which are necessary to show specific beneficial properties in several diseases (Murphy, et al, 2003, Caballero-George et al., 2002).

2. Material and Methods: 2.1 Plant Collection: The fresh healthy leaves of Z. Album, A. visnaga, and R. tripartita taxonomically identified by Department of Biology Science College Alkhums El-Mergeb University Alkhums Libya, Samples of plants were collected during April-Maye 2016 from Alkhums Region and then washed by tap water subsequently by distilled water and dried in shadow for three days and finishing drying in an oven at 50 0C. The samples of dried leaves were pulverized into the fine powder using a grinder consequently packed in sterilized dark polyethene bags.

2.2 Preparation of extracts: 20 g of each pulverized wild plant leaves powder was dissolved separately in 500ml of appropriate solvents (Distilled water, Ethyl Alcohol) using Soxhlet apparatuses for 8-10 hours. Subsequently, the extracts were filtered through Whatman filter paper No. 1 and the solvents was evaporated by Rotary Vacuum Evaporator and a green, brown and black sticky substance was obtained that was stored in the refrigerator at 4 0C until further use.

2.3 Phytochemical screening: 2.3.1 Qualitative determination of the chemical constituency:

Qualitative phytochemical analyses for each sample alkaloids, saponins, flavonoids and tannins were determined quantitatively as described below. Chemical tests were carried out both on the extracts (aqueous and ethanolic) and on the powdered specimens using standard procedures to identify the constituents (Harbone, 1973.; Trease, et al 1989; Sofowora, 1993; Dietland, 2009). The specific procedure involved for the evaluation of a particular group of chemicals is mention below:

2.3.1.1 Determination of Tannins: 2 ml of water and 2-3 drops of ferric chloride solution were added in 1 ml of crude extracted solution. The blue colour was observed for Gallic tannins and green black for Catecholic tannins (Iyengar, 1995).

2.3.1.2 Determination of Saponins: Foam test: a few amount of crude extracted solution was shaken with little quantity of water. If foam produced persists for ten minutes it indicates the presence of saponins (Roopashree, et al, 2008).

2.3.1.3 Determination of Flavonoids: (Alkaline Reagent Test): Crude extracted solution was treated with few drops of sodium hydroxide solution. Formation of intense yellow colour, which becomes colourless on the addition of dilute acid, indicates the presence of flavonoids (Roopashree, et al, 2008).

2.3.1.4 Determination of Steroids: 4 ml of acetic anhydride was added to a 1g ethanolic crude extract of each sample with 4 ml H2S04. The colour changed from violet to blue or green in some samples indicating the presence of steroids.

2.3.1.5 Determination of Terpenoids: (Salkowski’s test): 10ml of each crude extracted solution was mixed in 4 ml of chloroform and concentrated H2SO4 (6 ml) was carefully added to form a layer. A reddish-brown colouration of the interface was formed to show the presence of terpenoids.

2.3.1.6 Determination of Cardiac glycosides: (Keller-Killani test): 10ml of each crude extracted solution was treated with 4 ml of glacial acetic acid containing 2 drops of ferric chloride solution. This was under-layer with 2 ml of concentrated Sulphuric acid. A brown ring of the interface indicates a deoxy-sugar characteristic of cardenolides. A violet ring may appear below the brown ring, while in the acetic acid layer, a greenish ring may form just gradually throughout the thin layer.

2.3.1.7 Determination of Alkaloids: Alkaloids are basic nitrogenous compounds with definite physiological and pharmacological activity. Alkaloid solution produces white yellowish precipitate when a few drops of Mayer’s reagents are added (Siddiqui, et al, 1997).

2.3.1.8 Determination of Anthraquinones: Borntrager’s test: 1 g of the plant extract was shaken with benzene layer separated and half of its own volume of 10% ammonia solution added. A pink, red or violet colouration in the ammoniacal phase indicated the presence of Anthraquinone.

2.3.2. Quantitative determination of the chemical constituency: Quantitative phytochemical analyses for each sample alkaloids, saponins and flavonoids were determined quantitatively well-known tests procedure available in the literature as described below (Harbone, 1973; Obdoni, et al, 2001; Boham et al 1994):

2.3.2.1 Alkaloids determination: 10g of the fine powder of dried plants were weighed into 500 ml beakers to which 400ml of 10% acetic acid in ethanol was added, covered and allowed to stand for about 4 hr. The filtered extracts were concentrated on a water bath to one-quarter of the original volume. Concentrated ammonium hydroxide was added dropwise to the extracts until complete precipitation. The precipitate was collected and washed with dilute ammonium hydroxide and then filtered. The alkaloid residue was dried to a constant weight and expressed as the percentage (Harbone, 1973).

2.3.2.2 Saponin determination: 10 g of each were put into a conical flask and 50 ml of 20% aqueous ethanol were added. The samples were heated over a hot water bath for 4 h with continuous stirring at about 55°C. The mixture was filtered and the residue re-extracted with another 100 ml 20% ethanol. The combined extracts were reduced to 40 ml over a water bath at about 90°C. The concentrate was transferred into a 250 ml separatory funnel and 10 ml of diethyl ether was added and shaken vigorously. The aqueous layer was recovered while the ether layer was discarded. The purification process was repeated and 30 ml of n-butanol was added. The combined n-butanol extracts were washed twice with 5 ml of 5% aqueous sodium chloride. The remaining solution was heated in a water bath. After evaporation the samples were dried in the oven to a constant weight; the saponins content was calculated as the percentage (Obdoni, et al, 2001).

2.3.2.3 Flavonoids Determination: 20g of the finely powdered plant sample was extracted repeatedly with 200 ml of 80% aqueous methanol at room temperature. The whole solution was filtered through Whatman filter paper No 42 (125 mm). The filtrate was later transferred into a crucible and evaporated to dryness over a water-bath and weighed to a constant weight (Boham et al 1994).

2.4 Anti-bacterial activity assay: The crude extracts of the plants were transported from Department of Chemistry, Science College El-Mergeb University Alkhums Libya to the Department of Microbiology Laboratory at Alkhums Teaching Hospital, Alkhums, Libya. The antibacterial potential of plants crude extracts was studied using the Paper Disc Diffusion Method. A loop-full bacterium (Two Gram-positive bacterial strains (Staphylococcus aureus and Staphylococcus epidermis) and Two Gram-negative bacterial strains (Klebsiella pneumonia and Escherichia coli)) was taken from the stock culture and dissolved in 0.1ml of saline. All the tests were done by placing the disc (6mm diameter) impregnated with (40µl) various crude solvent extracts separately on the Muller Hinton Agar (MHA) surface previously inoculated with 10ml of MHA liquid medium with Gram-positive and Gram-negative bacteria. Standard antibiotic discs (Augmentin, Amoxicillin, Penicillin, Tetracycline, Ciprofloxacin, Ceftriaxone and Cefotaxime) were used as a reference or positive control. And blank discs (impregnated with Ethanol and aqueous without plant extracts, separately) was used as negative control. The plates were then incubated at 370C for 24 hr. to allow maximum growth of the microorganisms. The antibacterial activity of the test samples was determined by measuring the diameter of zone of inhibition expressed in millimetre. The assay was repeated twice and mean of the three experiments was recorded (Barry; Thornsberry; 1985; Bauer et al; 1966).

3. Result and Discussion:

Table 1: Result of the Phytochemical Screening of the plants Leave:

Chemical

Components

Saponins Steroids

Tannins

Alkaloids

Glycosides Flavonoids

Plants Triterpenes Phlobatannins Names Anthraquinons

Aqueous +++ +++ ++ + + - +++ - ++ Extract Z. Album Ethanolic +++ +++ ++ ++ +++ +++ + - ++ Extract Aqueous +++ +++ + ++ + - +++ - ++ Extract + A. visnaga Ethanolic +++ +++ ++ ++ +++ ++ + - +++ Extract Aqueous +++ +++ +++ ++ ++ - +++ + ++ R. tripartita Extract + Ethanolic +++ +++ ++ + +++ +++ + + ++ Extract +++ = Reach, ++ = Moderate, + = Good, - = Absent

The present study conducted on the leaves of plant viz. Z. Album, A. visnaga, and R. tripartita they can be used as medicinal plants estimate the need to evaluate the composition of these plants. As showed in table 1 the chemical analysis confers of active ingredients medicinally. The qualitative estimation of the crude yields of chemical constituents of the plants leave considered revealed that the leaves were almost rich in alkaloids, flavonoids, tannins, cardiac glycosides and saponins. While the steroids are not present in each of aqueous extracts of Z. Album, A. visnaga and R. tripartita, also, Anthraquinones only present in R. tripartita correspondingly not present in the other two plants.

Table 2: Quantitative Estimates of the Phytochemicals in the crude plants extracts Leaves:

Chemical Alkaloids Flavonoids Saponins Percent Yield Components (%) (%) (%) %

Plants Names Z. Album 2.4 9.6 7.6 33 A. visnaga 8 10 6 26 R. tripartita 8.5 8.7 3.2 29

In addition as showed in table 2 quantitative estimates of the phytochemicals in the crude plants extracts Leaves, The percentage’s yields of chemical constituents was found indecent concentration in all leaves of plants possibly will be responsible for their usage in remedy for the treatment of several ailments. While the percentage’s yields of chemical compounds in Z. Album and R. tripartita were 33%, 26% and 29% respectively. And percentage’s yields alkaloids were found as 2.4, 8 and 8.5% correspondingly and saponins were found 7.6, 6 and 3.2% individually. And flavonoids were found 9.6, 10 and 8.7% respectively. As well, the table 2 showed that flavonoids have the greatest percent yield which may be beneficial for sexual stimulation (European J. Scientific Res.; 2008). Furthermore, flavonoids have powerful anticancer activities and inhibited tumour growth (Stauth, 1993; Sharma, et. al. 2007). A study showed that flavonoids could prevent the progress of juices that occasioned in diarrhoea by foremost the intestinal cystic fibrosis transmembrane conductance regulator (Schuier, et. al. 2005). The presence of some chemical constituents such as flavonoids, saponins and alkaloids in Z. Album plant leaves could lead to using it for medication.

Table 3 Results of the antibiotics and Z. Album, A. visnaga, and R. tripartita. Crude extracts against pathogenic bacteria:

Bacteria spp. Klebsiella Escherichia Staphylococcus Staphylococcus Plants pneumonia Coli Aureus Epidermis Extracts & (mm) (mm) (mm) (mm) Antibiotics Aqueous 7 8 6 8 Z. Album Extract Ethanolic 8 10 7 7 Extract Aqueous 7 9 9 10 A. visnaga Extract Ethanolic 11 8 8 9 Extract Aqueous 8 6 8 6 R. tripartita Extract Ethanolic 10 8 10 8 Extract Augmentin R R R R Amoxicillin R R R R Penicillin S S R R Tetracycline S S R R Ciprofloxacin R S S S Ceftriaxone R S S S Cefotaxime R S S S R = Resistant, S = Sensitive, (mm) = millimeters

Antibacterial activities of aqueous and ethanolic extracts of the three plants are presented in Table 3 The crude plant extracts have important inhibitors against bacterial and it was among 6 to 11 mm. In addition, the A. visnaga ethanolic extract has greatest inhibition zone 11mm against Klebsiella pneumonia, while the plant's aqueous extracts have moderate inhibition zones. As well as in comparison to the antibiotics was highly susceptible to the pathogens bacteria. As a result could be the A. visnaga which known as a traditional medicinal plant which was utilized in the treatment of bronchial asthma and coronary diseases in the ancient and present time and could be also for that reason played an important role nowadays as meaningful raw material for the pharmaceutical industry (Sitting, 1988; Kleeman, 1999).

3. Conclusion: In spite of, there is no sufficient knowledge for detailed and phytochemical estimations on Libyan plants such Z. Album, A. visnaga, and R. tripartita are reported. In the present study, these plants were thoroughly investigated for their phytochemical screening and antibacterial activity. Since the results of the phytochemical analysis, we can conclude that the leaves of Z. Album, A. and R. tripartita are important sources of significant phytochemicals which are in accord with their use in the traditional remedy.

4. Acknowledgment: The authors acknowledge their profound gratitude to the Chemistry Department Science College, Al-Khums, El-Mergeb University Al-Khums, Libya and highly indebted to the Department of Microbiology at Al-khums Teaching Hospital, Alkhums, Libya, for providing the facilities for this research work. The authors also are would like to thank Institute of Organic Chemistry and Technology, Faculty of Chemical Technology, University of Pardubice, the Czech Republic for their valuable suggestion and for providing the invaluable help during the research work.

References:

Abdelfattah, A.; M.N. Aboulenein,; G.M. Wassel and B. Elmenshawi. 1983. Preliminary report on the therapeutic effect of in Psoriasis, Dermatologica, 167: 109-110. Barry A.L. and Thornsberry C. Susceptibility tests: Disc diffusion test procedures. In: Manual of Clinical Microbiology (Eds). Lennette Balows, EH; W.J. Hauster and H.J. Shadomy; 1985; American Society for Microbiology, Washington, DC; 978-987. Batanouny, K.H. 1999. Wild medicinal plants in Egypt: an inventory to support conservation and sustainable use. Palm Press, Egypt, ISBN 977 5089 24 7. Bauer AW, WM Kirby, JC Sherries and M Tuck.; (1966), Antibiotic susceptibility testing by a standardized disc diffusion method, American Journal of Clinical Pathology; 45:493- 496. Boham, B. A. and R. Kocipai-Abyazan (1994).; Flavonoids and condensed tannin from leaves of Hawaiian Vccinium vaticulatum and V. calycinium. Pacific Sci. 48:458-463. Bueno, E., A. Juan and M.B. Crespo.; 2006, Lactotypification of three endemic taxa of Ammi L. () from the archipelago of the Azores. Anales de Jardin Botánico de Madrid,; 63: 31-33. Caballero-George C, Vanderheyden PM, De Bruyne T, Shahat AA, Van den HeuveH, Solis PN, Gupta MP, Claeys M, Pieters L, Vauquelin G, Vlietinck AJ.; 2002; In Vitro Inhibition of [3H]-Angiotensin II Binding on the Human AT1 Receptor by Proanthocyanidins from Guazuma ulmifolia Bark. Planta Medica.; 68(12):1066–71. Chevallier, A.; 1996, The Encyclopedia of Medicinal Plants; Dorling Kindersley, London, ISBN 9-780751- 303148. Dietland, M. S.; (2009); Test for cyanogenic compounds in Plants‖, Handbook on Chemical Ecology,( Simple Field and Laboratory Exercises), Part 1, Springer, New York, pp. 69-73 El-Mokasabi F.; 2014; The State of the Art of Traditional Herbal Medicine in the Eastern Mediterranean Coastal Region of Libya; Middle-East J Sci. Res.; 21 (4):575–82. Farnsworth, N.R., ed. NAPRALERT database. 2001; Chicago, IL, University of Illinois at Chicago, 9- February 2001 production, (an online database available directly through the University of Illinois at Chicago. Harbone, J.B. (1973). Phytochemicals methods in: J.B. Harborne (Ed).; A guide to modern techniques of plant analysis. Chapman and Hall, London. Pp279. Kleeman, A., J. Engel, B. Kutsher and D. Reichert. 1999; Pharmaceutical Substances Syntheses Patents, Applications, Thieme; Stuttgart. Iyengar, M.A. (1995).; Study of Crude Drugs.; 8th ed., Manipal Power Press. McGaw, L. J., Jäger, A. K. and van Staden, J. 2000.Antibacterial, anthelmintic and ant amoebic activity in South African medicinal plants, Journal of Ethnopharmacology 72: 247- 263. Meng, X. L., Riordan, N. H., Casciari, J. J., Zhu, Y., Zhong, J., Gonzlez, M. J., MirandaMassari, J. R. and Riordan, H. D. (2002).; Effects of a high molecular mass Convolvulus arvensis extract on tumour growth and angiogenesis. P. R. Health Science Journal 21: 323-328. Murphy KJ, Chronopoulos AK, Singh I, Francis MA, Moriarty H, Pike MJ, et al. 2003; Dietary flavanols and procyanidin oligomers from cocoa (Theobroma cacao) inhibit platelet function. Am J Clin Nutr.; 77(6):1466–73. Obdoni, B. O. and P. O. Ochuko; (2001).; Phytochemical studies and comparative efficacy of the crude extracts of some Homostatic plants in Edo and Delta States of Nigeria; Global J. Pure Appl. Sci. 8b:203-208. Roopashree T. S., R. Dang, R. H. S. Rani and C. Narendra (2008).; Antibacterial activity of antipsoriatic herbs: Cassia tora, Momordica charantia and Calendula officinalis. International J. App. Res. Nat.l Prod. 1: 20-28. Schuier, M., H. Sies, B. Illek and H. Fischer (2005).; "Cocoa-related flavonoids inhibit CFTR-mediated chloride transport across T84 human colon epithelia". J. Nutr. 135: 2320–5 Schum K.; (Commelinaceae). (2008); Assessment of Sexual Stimulant Potential of Total Flavonoids Extracted from Leaves of Palisota Hirsuta Thunb.,; European J. Scientific Res. 22: 533-538. Sharma, V. K., A. Bhattacharya, A. Kumar and H. K Sharma (2007).; Health Benefits of Tea Consumption. Tropical J. Pharmaceutical Res. 6: 785-792. Siddiqui, A. A. and M. Ali (1997),; Practical Pharmaceutical chemistry. I St. Ed., CBS Publishers and Distributors, New Delhi, Pp. 126-131. Sitting, M.; 1988, Pharmaceutical Manufacturing Encyclopedia. Noyes Publications, USA. Sofowora, A. (1993).; Medicinal plants and Traditional medicine in Africa. Spectrum Books Ltd, Ibadan, Nigeria; p. 289. Stauth, D. (1993). Studies force new view on biology of flavonoids", Eurek Alert.; Adapted from a news release issued by Oregon State University. URL accessed. Trease, G. E. and W.C. Evans (1989),; Pharmacognosy. 11th edn.; Brailliar Tiridel Can, McMillian Publishers. Tutin, T.G. 1968. In: Flora Europaea, vol. 2. (T.G. Tutin, V.N. Heywood, N.A. Burges, D.M. Moole, D.H. Valentine, S.M. Walters, D.A. Webb, Eds.), Cambridge University, Cambridge, p. 205 Valkova, S., M. Trashlieva and P. Christova. 2004. Treatment of vitiligo with local khellin and UVA: Comparison with systemic PUVA. Clin. Exp. Dermatol. 29: 180-184 WHO; monographs on selected medicinal plants, 2007, WHO Library Cataloguing in Publication Data., Vol 3.; pp23-32.