Chapter One Introduction and literature review. Infection diseases are one of the main reasons which cause the death, killing almost

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1 Chapter One Introduction and literature review 1.1 Medicinal plants Infection diseases are one of the main reasons which cause the death, killing almost people everyday [1]. Medicinal plants are used by 80% of the world population as the only available medicines especially in developing countries [2], the use of medicinal plants is very wide spread in many parts of the world because it is commonly considered that herbal drugs are cheaper and safer as compared to synthetic drugs and may be used without or minimum side effects. Plants used for traditional medicine contain a wide range of substances that can be used to treat chronic as well as infectious diseases. Clinical microbiologists have great interest in screening of medicinal plants for new therapeutics [3]. The active principles of many drugs found in plants are secondary metabolites. The antimicrobial activities of plant extracts may reside in a variety of different components, including aldehyde and phenolic compounds[4]. Resistance to antimicrobial agents is emerging in a wide variety of pathogens and multiple drug, resistance is becoming common in divers organisms such as Staphylococcus sp and Salmonella sp[5][6][7]. The appearance of resistant paved the way to the occurrence of infections that are only treated by a limited number of antimicrobial agents the gradual rise in resistance of bacterial and fungal pathogens for antibiotics and antifungals highlights the need to find alternative sources from medicinal plants[8]. 1

2 In this study three medicinal plants were screened for their phytochemical contents and were tested for their antibacterial activity against some types of standard bacteria. 1.2 Trigonella Fenoum-Graeceum Fenugreek, Trigonella foenum-graecum L. is an annual crop from the family Leguminosae Fig(1), which have health potential with the ability to lower blood glucose and cholesterol levels, and hence in the prevention and treatment of diabetes and coronary heart diseases[9]. Due to its strong flavor and aroma, fenugreek in one of such plants whose leaves and seeds are widely consumed in countries as a spice in food preparations, and as an ingredient in traditional medicine. It is rich source of calcium, iron, carotene and other vitamins. Fenugreek seeds have been found to contain protein, vitamin C, niacin, potassium, and diosgenin (which is a compound that has properties similar to estrogen). Other active constituents in fenugreek are alkaloids, lysine and L-tryptophan, as well as steroidal saponins (diosgenin, yamogenin, tigogenin, and neotigogenin).[10] Fig.(1):Trigonella Fenoum-graeceum seeds 1.3 Artemisia herba- alba 2

3 Artemisia herba- alba (Asteraceae) [Fig. 2], commonly known as sheeh, is herb or shrub which distributed in north Africa (Libya), and most of Europe [11], is a chamaeophyte that grows to cm (8 16 in). Leaves are strongly aromatic and covered with fine glandular hairs that reflect sunlight giving a grayish aspect to the shrub. The leaves of sterile shoots are grey. This plant has been used in traditional medicine as antidiabetic (12). Fig. (2): Artemisia herba alba leaves 1.4 Solenostemma argel Solenostemma argel (Argel) [Fig. 3], known locally by the name (Hargel) and scientifically known as Solenostemma argel is a plant in the "Apocynaceae" family. It is indigenous to Africa. The parts used are the leaves and stems, the leaves contain high carbohydrates and protein as well as crude oil, ash, calcium and magnesium [13]. The leaves are used in herbal medicine for the treatment of some diseases such as of liver and kidney and allergies. It treats gastro-intestinal cramps, stomach-aches and urinary tract infections. Argel tea is used for lessening the pains of childbirth and for treating eating disorders since it increases appetite. Fig. (3:) Solenostemma argel leaves 3

4 Chapter Two Materials and Methods 2.1 Plants material Specimens of three plants were collected in a form of seeds (Trigonella foenumgraecum) and leaves (Artemisia herba-alba, Solenostemma argel) from different retail markets of Khartoum in September, The spices were botanically identified at the Department of Botany University of Khartoum. The collected plants were dried under shade at room temperature and ground with a grinder into a powder. 2.2 Preparation of extracts Extracts from the three tested plants were obtained by using cold extraction method (14), 50g of each air-dried powder was taken in a 250 ml conical flask. A hundred ml of (80%ethanol: 20% water) was added. The flask was plugged with cotton wool and then kept on a rotary shaker at rpm for 24 h. After 24 hours, the supernatant was collected and the solvent was evaporated to make the final volume one fourth of the original volume then stored at 4 C in airtight pottles [Fig., 4]. Figure (4): ethanol extracts of the three tested plants 4

5 3 Method of identification 3.1 Alkaloids [Mayer s test] 1.36 mg of mercuric chloride dissolved in 60 ml and 5 mg of potassium iodide were dissolved in 10 ml of distilled water respectively. These two solvents were mixed and diluted to 100 ml using distilled water. To one ml of acidic aqueous solution of samples few drops of reagent was added. Formation of white or pale precipitate showed the presence of alkaloids. 3.2 Flavonoids In a test tube containing 0.5 ml of alcoholic extract of the samples, 5 to 10 drops of diluted HCl and small amount of Zn or Mg were added and the solution was boiled for few minutes. Appearance of reddish pink or dirty brown colour indicated the presence of flavonoids. 3.3 Glycosides A small amount of alcoholic extract of samples was dissolved in 1ml water and then aqueous sodium hydroxide was added. Formation of a yellow colour indicated the presence of glycosides. 3.4 Cardiac glycosides [Keller killiani s test] About 100 mg of extract was dissolved in 1ml of glacial acetic acid containing one drop of ferric chloride solution and 1ml of concentrated sulphuric acid was added. A brown ring obtained at the interface indicated the presence of a de oxy sugar characteristic of cardenolides. 5

6 3.5 Steroids [Salkowski s test] About 100 mg of dried extract was dissolved in 2 ml of chloroform. Sulphuric acid was carefully added to form a lower layer. A reddish brown colour at the interface was an indicative of the presence of steroidal ring. 3.6 Saponins A drop of sodium bicarbonate was added in a test tube containing about 50 ml of an aqueous extract of sample. The mixture was shaken vigorously and kept for 3 min. A honey comb like froth was formed and it showed the presence of saponins. 3.8 Phenols [Ferric Chloride Test] To one ml of alcoholic solution of sample, 2 ml of distilled water followed by a few drops of 10% aqueous ferric chloride solution were added. Formation of blue or green color indicated the presence of phenols. 3.9 Tannin [Lead acetate test] In a test tube containing about 5ml of an aqueous extract, a few drops of 1% solution of lead acetate was added. Formation of a yellow or red precipitate indicated the presence of tanine. 4 Antibacterial Assay 4.1Culture media Nutrient broth This medium contained ( peptic digest of animal tissue 5.00g, yeast extract 1.50g, beef extract 1.50g and sodium chloride 5.00g [HiMedia Laboratories Pvt.Ltd- Mumbai , India]. It was prepared by dissolving 13 grams of the medium in one liter of distilled water. The ph of the medium was adjusted to 7.4 and 6

7 the medium was then distributed into screw capped bottles, 5 ml each and sterilized by autoclaving at 121 C for 15 minutes Nutrient agar (oxoid, England) The medium contained lab- lemco powder (1.0 g), yeast extract (2.0 g), peptone (5.0 g) and agar no.3 (15.0 g)-( HiMedia Laboratories Pvt.Ltd-Mumbai , India). Twenty eight grams of dehydrated medium were dissolved in one liter of distilled water, ph was adjusted to 7.4. The dissolved medium was sterilized by autoclaving at 121 C for 15 minutes. 4.2 Test of the extracts for antimicrobial activity The cup-plate agar diffusion method was adopted to assess the antibacterial activity of the prepared extracts(15). Two milliliters of a standardized bacterial stock suspension ( cfu\ml) were thoroughly mixed with 200 ml of a sterile molten nutrient agar. Twenty ml aliquots of the inoculated nutrient agar were distributed in sterile Petri dishes. The agar was left to set and four cups (10 mm in diameter) were cut using a sterile cork Borer (no.4) in each plate and the agar discs were removed using a sterile wire loop. Each cup was filled with 100µ of one of four extract concentration using microtiter pipette and the extract was allowed to diffuse at room temperature for 2 h. Two replicates were carried out for each extract against each of the bacterial organisms. The plates were then incubated at 37 C for 18 h and simultaneously, in separate Petri dishes a cup was made for each organism. After incubation, the diameters of the growth inhibition zones were measured and the average values were tabulated. 7

8 Chapter Three Results and Discussion 4.1 Moisture and ash contents The moisture contents and ash contents of the tested were measured and the results Plants Moisture content% Ash content% Trigonella foneum were varied slightly from previous studies (13). The amount of moisture in all samples was in the range of %. The highest level of moisture content was detected in Trigonella foneum seeds. Artemisia herba- alba and Solenostemma argel leaves showed high amount of ash content as compared with Trigonella foneum seeds (Table 1). Table(1) the moisture content% and ash content% of three tested plants 8

9 Artemisia herba- alba Solenostemma argel Phytochemical screening Aerial parts of S. argel, A.herba-alba and seeds of T. fengoum- graceum plants were successively extracted with ethanol 80%. The crude extracts were photochemical tested and the three plants showed some variation in their composition. Very few amounts of alkaloids were found in each plant whereas the other phytoconstituents were greater [Table 2]. The phytochemical screening revealed the presence of flavonids, glycocides, cardic glycosides, phenols, tannins and terpenids in S. argel and this result agreed with other studies (14). In case of T. fenoum graceum the phytochemical screening showed highest content of phenolic,flavinoids and revealed the presence of other constituents, earlier reports have shown that ethanol extract had the same phytochemical contents (15). The A. herba alba showed the same results with the other two tested plants (16) [Table 2]. Table(2) : The comparison of the phytochemicals composition for the three tested plants: Trigonella foneum, Solenostemma argel and Artemisia herba-alba 9

10 Phytochemical tests Trigonella fenum gream Solenostemma argel Artesimia herbaalba Alkaloids [Mayer, s test] Flavonoids + _ + Glycosides Cardic glycosides [Killiani, s test] Steroids [Salkowski, s test]. Phenols [Ferric Chloride test ] Tannin, s[lead acetate test] Terpenoid Antibacterial Activity The ethanolic extract of T. foneum graceum was adjusted for it, s antibacterial activity against Escherichia coli and Pseudomonas aevregoma as agram negative bacteria and the obtained zone of inhibition was found 2 mm with the Escherichia coli and it exhibited no action with Pseudomonas aevregoma bacteria. 10

11 On the hand, the antibacterial activity of T. foneum showed a considerable effect on gram positive bacteria Staphylococcus aureus, Bacillius subtilis which obtained 5-6 mm and 5 mm zone of inhibition, respectively [Table 3 and Fig. 5]. Table(2):T he measurement of inhibition zone diameter (mm) of the ethanolic extract of Trigonella foenum- graceum against tested bacteria. Concentration E.c P.a B.s S.t mg/ml _ _ _ 6 5 *E.C = Escherichia coli B.s = Bacillus subtilis P.s = Pseudomonas aevregoma S.t = Staphylococcus aureus Other papers carried the same study but they have used another solvent and other methods of extraction showed some difference in results (8)(9)(10). Some studies demonstrate wide spectrum antibacterial activity against gram negative and gram positive bacteria, for both aqueous and ethanol extracts from fenugreek seed Trigonella foenum-graecum (16). Seed purchased from Pakistan was extracted into either water or ethanol, and then used to make antibiotic discs, which prevented bacterial growth in zones surrounding each disc. Zones cleared of bacterial growth ranged in size from 12 to 21 mm, and exhibited a direct dose response relationship when different concentrations of the extracts were used. However, ethanol extracts from fenugreek seed purchased from India 11

12 did not inhibit growth of either bacteria or yeast (18). In our experiments, we examined Local fenugreek seed, none of concentrations that we examined possessed this activity. However, other study have shown that chemical composition can vary among different fenugreek originating from different countries of the world. This variation could be attributed to genetics and environmental response of plants to production of phytochemicals (19). The antibacterial activity which was obtained from the ethanolic extract of A. herbaalba showed activity against both gram negative and gram positive bacteria [Table 4 and Fig. 6]. Table(4): T he measurement of inhibition zone diameter (mm) of the ethanolic extract of Artemisia herba alba against tested bacteria *E.C = Escherichia coli B.s = Bacillus subtilis P.s= Pseudomonas aevregom S.t= Staphylococcus aureus Concentration E.c P.s B.s S.t mg/ml The activity of the extract against gram positive bacteria was high as compared with the activity against the gram negative one. This results agreed with other study which had shown that only the essential oil was active against some gram negative and gram positive bacteria, 12

13 in this study the activity was perfumed by using the crude extraction which may cause a decrease in the activity because the active principle component can be found in one fraction (20). The activity of crude ethanolic extraction of S.argel exhibit a weak zone of inhibition against Bacillus subtilis,staphylococcus aureus and no action against E.coli, P. aevregoma [Table(5) and Fig(7)]. Table(5) T he measurement of inhibition zone diameter (mm) of the ethanolic extract of Solenostemma argel against tested bacteria Concentration E.c P.a B.s S.t mg/ml -1 5 _ 2 4 _ 10 _ 2 3 _ 15 _ 2 4 _ E.C = Escherichia coli B.s = Bacillus subtilis P.s= Pseudomonas aevregoma S.t= Staphylococcus aureus This results may attributed to different constituents found in the crude extract and the type of solvent used, and this results agreed with other report (21).Earlier study had shown that only one fraction had exhibited antibacterial activity against both types of bacteria.. 13

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15 Fig(5) The zones of inhibition of the Trigonella foneum -graceum extract against the four type of bacteria 15

16 16

17 Fig(6) The zones of inhibition of the Artemisia herba-alba extract against the four type ofbacteria 17

18 18

19 Fig(4) The zones of inhibition of the S. olenostemma argel extract against the four type of bacteria References [1] Anonymous, Centre of Disease control and prevention. Preventing emerging infectious diseases. Astrategy for the 21 st century,2000, pp

20 [2]Hashim H, Kamali EL, Mohammed Y, Antibacterial activity and phytochemical screening of ethanolic extracts obtained from selected Sudanese medicinal plants. Current Research Journal of Biological Science, [3] Periyasamy A, Rajkumar, Mahalingam K, Phytochemical screening and antimicrobial activity from five Indian medicinal plants against human pathogens. Middle-East Journal of Scientific Research, 5(6): [4]. Lai PK, Roy J, Antimicrobial and chemo preventive properties of herbs and spices. Current Medicinal Chemistry, 11: [5] Ahmed I and Beg AZ, Antimicrobial and phytochemical studies on 45 indian medicinal plants againt multi-drug resistant human pathogens. J Ethnopharmacol, 2001, 74, [6] Threlfall EJ.Frost JA and Ward LR, Increasing spectrum of resistance in multiresistant Samonella typhimurium, Lancet, 1996, 343, [7] Cowan MM, Plant products as antimicrobial agents, Clin Microbial Rev, 1999, 12, [8]. Erdogrul OT, Antibacterial activities of some plant extracts used in folk medicine. Pharmaceutical Biology, 40: [9] Karim,A,M. Nouman, S, Munir Sattar,2011. Pharmacology and phytochemistry of Pakistani herbs and herbal drugs used for treatement of diabetes.int.j, Pharmacol.,7: [10]Tarka, P,Antimicrobial studies of methanol and ethanol extracts (Soxhlet extraction) of Fenugreek (Trigonella Foenum)SLC, S College of pharmacy JNTUH, Hyderabad,

21 [11]Jafri. S and Ei Gadi. A. (1983) Asteraceae. In :Flora of Libya 107. Al- Fateh University, Tripoli Libya. [12] 2-Al Shamaony, L., Al Khazraji, S.M. and Twaji, H.A.A (1994). Hypoglycemic effect of Artemisia herba alba._. Effect of a valuable extract on some blood parameter in diabetic animals. Journal of ethanopharmacology 43: [13] Murwan, K. El-Kheir, S. (2010). Chemical Composition, Minerals, Protein Fractionation, and Anti-nutrition Factors in Leaf of Hargel Plant (Solenostemma argel) Euro. Journals Publishing, Inc., 43, pp [14]Yogesh Mahida and JSS Mohan * 2007 Screening of plants for their potential antibacterial activity against Staphylococcus and Salmonella spp.v6(4).pp [15] Kavanagh, F. (1972). Analytical Microbiology. Vol.II. F.Kavanagh (Ed), Academic Press, New York and London. PP.11. [16] Dr. Jamal A. ElturbiG,Najib M. SufyaG,Aisha M. EllafiG 2007 (Phytochemical Investigation of Artemisia Herba Alba Asteraceae) [17]Bhatti, M. A., Khan, M. T. J., Ahmed, B. and Jamshaid, M Antimicrobial activity of Trigonellafoenum-graecum seeds. Fitoterapia 67: [18]De, M., De, A. K. and Banerjee, A. B Antimicrobial screening of some Indian spices. Phytother. Res. 13: (19)Taylor, W. G., Zulyniak, H. J., Richards, K. W., Acharya, S. N., Bittman, S. and Elder, J. L Variation in diosgenin levels among 10 accessions of fenugreek seeds produced in western Canada. J. Agric. Food Chern. 50:

22 [(20]) J. Yashphe, I. Feuerstein, S. Barel and R. Segal The Antibacterial and Antispasmodic Activity of Artemisia herba alba Asso. II. Examination of Essential Oils from Various Chemotypes 1987, Vol. 25, No. 2, Pages (doi: / ) [21] FATEN K. ABDO ELHADY *,A.G.HEGAZI **, NAGWA ATA ** AND MONA L.ENBAAWY *** (Studied for determining antimicrobial activity of solenostemma argel (DEL) HAYNE 2- extraction with chloroform, methanol in different proportions (c):pp [22]Tharib,S.M., S. EL-Migirab and G.B.Veitch, Apreliminary investigation of the potential antimicrobial activity of Solenostemma argel.int.j.crude Drug Res.,24(2):

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