Phytochemical analysis and antimicrobial activity of the extract of leaves of fleabane

Phytochemical analysis of the extracts from the leaves of the plant fleabane (Conyza sumatrensis) revealed the presence of some substances such as tannins, flavonoids, saponins, steroids and glycosides. The presence of these substances is an indicator of the pharmacological property as well as the nutritive value of the plant leaves. Antimicrobial tests showed that the leaves extract is not sensitive towards the bacteria Pseudomonas aureginosa, staphylococcus aureus, Bacillus spp and Escherichia coli, but inhibited the growth of the fungus Aspergilus niger. @ JASEM With the phenomenal growth in world population and the attendant depletion in food and drugs, mankind has to grapple with these problems by looking for alternative sources for these essentials of life. Consequently, the search for and use of drugs and dietary supplements derived from plants have accelerated in recent years. Though plants play significant roles in metabolic activities such as photosynthesis and respiration, the choice for food and drugs is predicated on the presence of photochemicals (Nsi and Dyegh, 2004). These are chemical substances present in plants and include alkaloids saponins, glycosides, tannins, flavonoids and steroids. The importance of these phytochemicals cannot be overemphasized as some have been used for the treatment and control of diseases (Iwu et al; 1999, Ayandele and Adebiyi, 2007) while others are known to inhibit the growth of microorganisms (Okerelu and Ani, 2001; Alinor, 2006, Farnsworth, 1996 and Ogbeche et al; 1997). Conyza sumatrensis commonly called broadleaf fleabane is a dicotyledonous herb of the asteraceae family occurring widely in Nigeria especially in the Niger Delta region and also in central Kenya. It is an erect, hairy, annual herb upto 120 cm high with sessile and deeply serrated leaves. The most prominent uses of conyza summatresis in Nigeria include treatment of stomach disorder and facial pimples. It also serves as a good source of food for the fowls. It is the success recorded in the treatment of facial pimples with extract from the leaves of conyza sumatrensis that has prompted the investigation into the phytochemical constituents as well as the antimicrobial activities of the plant. By this approach, we hope to develop an effective but cheap and readily available antifungal product for use. MATERIALS AND METHODS The plant was collected from Choba, a village in Rivers State of Nigeria, thoroughly washed with water and 25g of the fresh leaves were steam distilled. The steam volatile fraction was obtained by extraction with n-hexane (50mlx3), washed with water (100mlx2) and dried over anhydrous sodium sulphate. Evaporation of the n-hexane solvent gave 1.38g of crude extract. The ethanol extract was obtained by soaking the leaves in a round bottom flask containing 200ml of ethanol for forty-eight hours with shaking occasionally. The ethanol extract was filtered through 110 mm Whatman filter paper and evaporated to give 4.28g crude extract. Thin layer chromatography (TLC) were run on silica gel 254 and eluted with 4 toluene: 1 ethylacetate. All reagents and solvents used were purified before use. Photochemical analysis The phytochemical analysis of the plant was carried out by the standard methods provided by Odebiyi and Ramstard, 1978 and Waterman (1993). Test for tannins (a) 1cm of freshly prepared 10% KOH was added to 1cm of each of the extracts and observed for dirty white precipitate. (b) 2 drops of 5% Fe Cl3 was added to 1cm of the extracts and observed for green precipitate. Test for saponins (frothing test). 2 cm of each extract in a test tube was vigorously shaken for two minutes and observed for persistent foaming. Test for Flavonoids To 3cm of each extract was added 1cm NaOH and observed for yellow colouration. Salkwoski’s for test steroids 5 drops of concentrated H2SO4 was added to 1cm of each extract and observed for red colouration. Fehling’s test for glycosides. 10 cm of 50% H2SO4 was added to 1 cm of the extract in a test tube. The mixture was heated in a Phytochemical analysis and antimicrobial activity of the extract of leaves of fleabane (Conyza sumatrensis) * Corresponding author: Jack, I.R. 64 boiling water-bath for 15 minutes. 10 cm of fehling’s solution was added and the mixture was boiled and observed for brick red precipitate. Test for alkaloids 1 cm of HCl was added to 3 cm of each extract in a test tube. The mixture was heated for 20 minutes, cooled and filtered. 2 drops of Wagner’s reagent was added to 1 cm of the filtrate and observed for reddish brown precipitate. Antimicrobial screening The antimicrobial activities of the crude extract were screened (Moore and Winston, 1996) against four bacteria namely Pseudomonas aeruginosa, staphylococcus aureus, bacillus spp and Escherichia coli and two fungi; Aspergilus niger and Candida albicans. The nutrient agar medium was prepared and sterilized in an autoclave at 121C and 1.1N pressure for 15 minutes and allowed to cool and solidity in Petri dishes to a depth of 4 mm (Ekpendu et al 2001). Pure cultures of the microorganisms were separately used to inoculate the Petri dishes and incubated at 37C for 24 hours for bacteria and at room temperature for72 hours for fungi. RESULTS AND DISCUSSION The photochemical analysis of conyza sumatrensis revealed the presence of tannins, flavonoids, saponins, steroids and glycosides in both the nhexane and ethanol extracts (Table 1). Table 1: Phytochemical Analysis of the Extracts of Fleabane Active principle Test n-hexane extract ethanol extract Tannins FeCl3 + + Saponins Frothing test + + Flavonoids NaOH + + Steroids Salkwoski + + Glycosides Fehling’s test + + Alkaloids Wagner’s test Key = + is present is absent Table 2A: Percentage yield of n-hexane and ethanol extracts Extract Wt of extract (g) Mean (g) X % Yield Degree of Freedom Standard deviation Confidence interval of mean test at 95% n-hexane 0.80 0.82 0.81 3.2 3 0.01 0.81 + 0.021 Ethanol 4.0 – 4.3 4.30 17.2 3 0.02 4.3 + 0.032 Table 2B Variable ratio (Ftest) for n-hexane and ethanol extract Standard deviation Standard deviation Degree of Freedom F (Calculated) F values at 95% Observation n-hexane Ethanol 2.1 3.18 No significant difference Table 3: TLC results for n-hexane and ethanol extracts Extract Solvent System No. of Components Rf value ranges (cm) Mean x Rf value (cm) Standard deviation (cm) Degree of freedom Confidence interval of mean test at 95% Intensity n-hexane 4 – Toluence 1 Ethl-acetate 1 0.80 – 0.81 0.81 0.01 3 0.081 + 0.016 F Ethanol -do3 0.64 – 0.66 0.49 – 0.51 0.36 – 0..39 0.65 0.50 0.38 0.066 0.006 0.013 3 3 3 0.65 + 0.1 0.51 + 0.01 0.38 + 0.02 V..I M.I V.I Key F = Faint V.I = Very intense M.I = Moderately intense TABLE 4: Antimicrobial Analysis Concentration S/no Micro organism 20 mg/ml 50 mg/ml 1. Psendomonas aeruginosa 2. Staphylococcus aureus 3. Bacillus spp 4. Escherichia coli 5. Candida Albicans 6. Aspergilus niger ++ ++ Key: = Resistant ++ = Sensitive Phytochemical analysis and antimicrobial activity of the extract of leaves of fleabane (Conyza sumatrensis) * Corresponding author: Jack, I.R. 65 The presence of these phytochemicals is an indicator that the plant can be a potential source of precursors in the development of synthetic drugs. Alkaloids were however absent and this suggests that the plant may not be toxic. This evidence is corroborated by the fact that the plant provides a ready source of food for the domestic fowls. Relatively, low yields pf extracts were obtained for both solvents, i.e. 3.2% for n-hexane and 17.3% for ethanol (Tables 2A and 2B). Since steam distillation of the plant and subsequent extraction with n-hexane gave much lower yields, it may be presumed that the components are not very steam volatile. Statistical analysis of tables 2 and 3 however gives an indication of the precision of the measurements obtained for both solvents. Table 2B confirms that there is no significant difference in the precision of weight of extract obtained from the two solvents. The faint intensity of the spot having Rf values of 0.81 on T L C after staining with iodine vapour suggests that the component is either saturated or aromatic (Table 3). The artificial culture medium used for the antimicrobial test showed that the ethanol extract from conyza sumatrensis has no antimicrobial activity against Pseudomonas aureqinosa, Staphylococcus aureus, Bacillus SPP and Escherichia coli: When tested on two fungi namely Candida albicans and Aspergilus niger however, the culture medium showed sensitivity towards the latter by inhibiting its growth at moderate and high concentrations of 20mg/ml and 50mg/ml respectively (Table 4). This inhibition gives credence to the fact that conyza sumatrensis is an antifungal plant and hence can be used in the treatment of ailment due to this fungus. REFERENCES Alinor, L. J., (2006). Preliminary photochemical and antimicrobial activity of Garcinia cola. J. Chem Soc. Nig. 32 (2) 41-47 Ayandele, A. and Adebiyi, A.O. (2007). Antimimcrobial screening of extracts of Olax Subscorpiodes African J. Biotech. Vol. 6 No. 7 868-870 Ekpendu, J.O.E., Ekundayo, O and Laokso, L. (2001). Preparation of nutrient agar medium. J. Chem. Soc. Nig. Vol. 26 No. 2 194. Farnsworth, N.R. (1994). Biological and photochemical screening of plants. J. Pharm. Sciences 55, 225-276 Iwu, M.W; Duncan, A.R. and Okunji. C. O. (1999). New antimicrobials of plant origin. Janick J. Educ. 457-462 Moore, W. and Winston, A. (1996). Laboratory manual for organic microscope approach Pub. McGraw Hill Co. Inc USA Nsi,E.W. and Dyegh, A.H. (2004). Preliminary photochemical screening and antimicrobial activity of terminalia Avicennioides, Int. J. Sc & Tech. No. 3 Vol. 2 42-44 Odebiyi, E.O and Ramstard, A. H. (1978). Investigation photochemical screening and antimicrobial screening of extracts of Tetracarpidium conophorum J. Chem Soc. Nig. 26 1 Ogbeche, A. K; Ajayi, G.O. and Onyenta, P. (1997). Antimicrobial activities of the 

With the phenomenal growth in world population and the attendant depletion in food and drugs, mankind has to grapple with these problems by looking for alternative sources for these essentials of life.Consequently, the search for and use of drugs and dietary supplements derived from plants have accelerated in recent years.Though plants play significant roles in metabolic activities such as photosynthesis and respiration, the choice for food and drugs is predicated on the presence of photochemicals (Nsi and Dyegh, 2004).These are chemical substances present in plants and include alkaloids saponins, glycosides, tannins, flavonoids and steroids.The importance of these phytochemicals cannot be overemphasized as some have been used for the treatment and control of diseases (Iwu et al;1999, Ayandele andAdebiyi, 2007) while others are known to inhibit the growth of microorganisms (Okerelu and Ani, 2001;Alinor, 2006, Farnsworth, 1996 andOgbeche et al;1997).Conyza sumatrensis commonly called broadleaf fleabane is a dicotyledonous herb of the asteraceae family occurring widely in Nigeria especially in the Niger Delta region and also in central Kenya.It is an erect, hairy, annual herb upto 120 cm high with sessile and deeply serrated leaves.
The most prominent uses of conyza summatresis in Nigeria include treatment of stomach disorder and facial pimples.It also serves as a good source of food for the fowls.It is the success recorded in the treatment of facial pimples with extract from the leaves of conyza sumatrensis that has prompted the investigation into the phytochemical constituents as well as the antimicrobial activities of the plant.By this approach, we hope to develop an effective but cheap and readily available antifungal product for use.

MATERIALS AND METHODS
The plant was collected from Choba, a village in Rivers State of Nigeria, thoroughly washed with water and 25g of the fresh leaves were steam distilled.The steam volatile fraction was obtained by extraction with n-hexane (50mlx3), washed with water (100mlx2) and dried over anhydrous sodium sulphate.Evaporation of the n-hexane solvent gave 1.38g of crude extract.The ethanol extract was obtained by soaking the leaves in a round bottom flask containing 200ml of ethanol for forty-eight hours with shaking occasionally.The ethanol extract was filtered through 110 mm Whatman filter paper and evaporated to give 4.28g crude extract.Thin layer chromatography (TLC) were run on silica gel 254 and eluted with 4 toluene: 1 ethylacetate.All reagents and solvents used were purified before use.

Photochemical analysis
The phytochemical analysis of the plant was carried out by the standard methods provided by Odebiyi andRamstard, 1978 andWaterman (1993).
Test for tannins (a) 1cm 3 of freshly prepared 10% KOH was added to 1cm 3 of each of the extracts and observed for dirty white precipitate.(b) 2 drops of 5% Fe Cl 3 was added to 1cm 3 of the extracts and observed for green precipitate.

Test for saponins (frothing test).
2 cm 3 of each extract in a test tube was vigorously shaken for two minutes and observed for persistent foaming.

Test for Flavonoids
To 3cm 3 of each extract was added 1cm 3 NaOH and observed for yellow colouration.
Salkwoski's for test steroids 5 drops of concentrated H 2 SO 4 was added to 1cm 3 of each extract and observed for red colouration.
Fehling's test for glycosides.10 cm 3 of 50% H 2 SO 4 was added to 1 cm 3 of the extract in a test tube.The mixture was heated in a 64 boiling water-bath for 15 minutes.10 cm 3 of fehling's solution was added and the mixture was boiled and observed for brick red precipitate.
Test for alkaloids 1 cm 3 of HCl was added to 3 cm 3 of each extract in a test tube.The mixture was heated for 20 minutes, cooled and filtered.2 drops of Wagner's reagent was added to 1 cm 3 of the filtrate and observed for reddish brown precipitate.

Antimicrobial screening
The antimicrobial activities of the crude extract were screened (Moore and Winston, 1996) against four bacteria namely Pseudomonas aeruginosa, staphylococcus aureus, bacillus spp and Escherichia coli and two fungi; Aspergilus niger and Candida albicans.The nutrient agar medium was prepared and sterilized in an autoclave at 121 0 C and 1.1N pressure for 15 minutes and allowed to cool and solidity in Petri dishes to a depth of 4 mm (Ekpendu et al 2001).Pure cultures of the microorganisms were separately used to inoculate the Petri dishes and incubated at 37 0 C for 24 hours for bacteria and at room temperature for72 hours for fungi.

RESULTS AND DISCUSSION
The photochemical analysis of conyza sumatrensis revealed the presence of tannins, flavonoids, saponins, steroids and glycosides in both the nhexane and ethanol extracts (Table 1).

Table 1 :
Phytochemical Analysis of the Extracts of Fleabane

Table 2A :
Percentage yield of n-hexane and ethanol extracts

Table 3 :
TLC results for n-hexane and ethanol extracts