Preliminary Phytochemical and Antimicrobial Analyses of the Leaves of Nigerian Bignoniaceae Juss

Full Length Research

Preliminary Phytochemical and Antimicrobial Analyses of the Leaves of Nigerian Bignoniaceae Juss.

By G.E.Ugbabe1*, A.E. Ayodele2, G. A. Ajoku3, O. F. Kunle1, I. Kolo3 & J.I.Okogun1.

1Medicinal Plant Research and Traditional Medicine (MPR & TM) Department National Institute for Pharmaceutical Research and Development,Idu-Industrial Area, P.M.B 21, Garki-Abuja,Nigeria.

2Botany and Microbiology Department University of Ibadan. Ibadan, Nigeria

3Microbiology, Human Virology and Biotechnology (MHV & BT) Department National Institute for Pharmaceutical Research and Development,Idu-Industrial Area, P.M.B 21, Garki-Abuja, Nigeria.

Accepted 8th July 2010

The phytochemical and antimicrobial analysis of the leaves of ten species of the Nigerian Bignoniaceae Viz. Crescentia cujete Linn, Jacaranda mimosifolia D. Don., Kigelia africana (Lam.) Benth, Markhamia tomentosa (Benth.) K. Schum., Newbouldia laevis Seem., Spathodea campanulata P. Beauv., Stereospermum acuminatissimum K. Schum., Stereospermum kunthianum Cham., Tabebuia rosea (Berthol.)DC. and Tecoma stans (Linn.) H.B. & K. were carried out. Crescentia cujete, Jacaranda mimosifolia, Tabebuia rosea and Tecoma stans are introduced species while the others are introduced to Nigeria. The phytochemical analysis included the screening of leaf extracts of the species for secondary metabolites such as carbohydrates, tannins, phlobatannins, anthraquinone, saponin, flavonoids, alkaloids, sterols, resins, and phenolic nucleus. The results show the presence of sterols, flavonoids, terpenes, tannins, resins, carbohydrates and phenolic nucleus in all the species. Whereas Phlobatanins and Alkaloids remained absent in all the species. Antimicrobial screening was carried out using 2.0_mg/ml of successive extracts (hexane, ethyl acetate, methanol and water) of each species. The test organisms were Pseudomonas aeruginosa, Escherichia coli, Candida albicans, Bacillus subtilis, Staphylococcus aureus and Salmonella typhi, which were clinical isolates. Results were taken after 24hours to record the growth (no activity) or no growth (activity) of the test organism on each of the extracts. The antimicrobial screening shows that all the hexane extracts exhibited activity on B. subtilis and S. aureus and the hexane extract of C. cujete had activity on all the test organisms. The hexane extract has more activity on the test organisms followed by the ethylacetate extracts while the water extract has the least activity on the test organisms. On the other hand, in the ethylacetate extract only C. cujete had activity on all test organisms; and the ethylacetate extracts of N. laevis and S. acuminatissimum had no activity at all on all test organisms. In the methanol extracts C. cujete, J. mimosifolia, N. laevis, S. acuminatissimum, and T. stans had activity on P. aeruginosa.

Key Words: Bignoniaceae, Phytochemical screening, Antimicrobial, Clinical isolates, Nigeria.

INTRODUCTION: Hutchinson and Dalziel (1954) recorded five Bignoniaceae Juss. is a family of trees, shrubs or genera in Nigeria; these are Kigelia Lam., Markhamia lianas and rarely herbs (Watson & Dallwitze, 1992). The Seemann.ex K.Schum., Newbouldia Seemann. ex family is made up of about 100 genera and 800 species Bureau., Spathodea P.Beauv. and Stereospermum. (Watson & Dallwitze, 1992) and it is distributed in the Beside these genera, there are also introduced species tropics and forms an important part of the vegetation such as Crescentia cujete Linn. , Tabebuia rosea (Berthol) (Shashina,1989). A few of the species are found in the D. C, Tecoma stans (Linn) H, B &K. and Jacaranda temperate and sub-tropical regions. Members of the family mimosifolia D.Don. in the country. have showy flowers. R Members of this family are grown mostly for ornamental *Corresponding author: G.E.Ugbabe and medicinal purposes in Nigeria. E-mail: [email protected] Medicinal plants represent a rich source from which antimicrobial agents may be obtained. Plants are used medicinally in different countries and are a source of The standard method as described by Odebiyi many potent and powerful drugs. The interest in the and Sofowora, (1990) and Fadeyi et al, (1989) were scientific investigation of these ten medicinal plants from adopted. The plant extracts were screened for the Nigeria is based on claims of their effective use for presence of carbohydrates, saponin, tannin, sterols, treatment of many diseases. Therefore, research into the alkaloids, flavonoids, anthraquinone, phlobatanin, resins, effects of these medicinal plants is expected to enhance phenolic nucleus, and terpenes. the use of these plants against diseases caused by the test pathogens. However, most of the plants used in folk Antimicrobial activity. medicine have not been screened for their antimicrobial The crude hexane, ethylacetate, methanol and activity. water extracts were each screened for antimicrobial The active principles of many drugs found in activity against clinical isolates from NIPRD clinic using plants are secondary metabolites. Therefore, basic Agar Dilution streak technique (Mitscher et al, 1978). The phytochemical investigation of the extracts for their major test organisms were prepared by incubating them in phyto constituents is also vital. freshly prepared nutrient broth at 37oC for 3hrs. (having approximately 1.25 x 106 – 1.25 x 107). 64mg of the crude extracts were dissolved in 1ml Dimethyl Sulphur Oxide MATERIALS AND METHODS (DMSO) and made up to 2ml with sterile distilled water to This work is based on ten species in the family give a concentration of 32mg/ml of extract. 1ml of the Bignoniaceae. Fresh specimens were collected at different prepared extracts were introduced into 15ml of molten times from the field for the study. Specimens of the family nutrient agar placed in water at 54oC. These were mixed were studied at Forestry Research Institute of Nigeria properly and poured into sterile petri dishes to give a final Herbarium, (FHI) Ibadan, Nigeria; Botany and concentration of 2mg/ml. The dishes were then allowed to Microbiology department, University of Ibadan Herbarium gel and thereafter, the test organisms were inoculated by (UIH) and National Institute for Pharmaceutical Research streaking onto the nutrient agar using a wire loop meant to and Development Herbarium (NIPRDH), Abuja Nigeria. deliver 0.002ul containing approximately 2.5x103-2.5x104 The voucher specimens of the species collected have colony forming units (cfu). The organisms were also been deposited at Ibadan. Duplicates are kept at Ibadan streaked on dishes containing only agar (Origins Viability and NIPRD Abuja (Table 1.) Control) and dishes containing nutrient agar and DMSO, which also served as controls. The petri dishes were then Preparation of plant extracts incubated over night at 37oC. They were then observed for The plant materials were dried at room microbial growth inhibition. Both water and DMSO showed temperature and then powdered using a mortar and no inhibiting effect on the organisms. The two controls pestle. A sample (10g) of each powdered plant material were used in all the tests Viz. control 1 had only the agar was soaked in 100ml distilled water and heated on the and control 2 had agar and DMSO. The controls were set water bath for 10 minutes after which it was filtered and up to show that the agar supports growth and also that the the filtrate used for the phytochemical screening. DMSO does not inhibit the growth of the test organisms.

Phytochemical screening

Table 1. Herbarium specimen numbers

NIPRD HERBARIUM NUMBER S/No. SPECIES 1. Crescentia cujete Linn NIPRD/H/6209 2. Jacaranda mimosifolia D. Don. NIPRD/H/6206 3. Kigelia africana (Lam) Benth. NIPRD/H/6211 4. Markhamia tomentosa (Benth.) K.Schum. NIPRD/H/6203 5. Newbouldia laevis Seem. NIPRD/H/6201 6. Spathodea campanulata P. Beauv. NIPRD/H/6162 7. Stereospermum acuminatissimum K. Schum. NIPRD/H/6202 8. Stereospermum kunthianum Cham. NIPRD/H/6222 9. Tabebuia rosea (Berthol) D.C. NIPRD/H/6178 10. Tecoma stans (Linn.) H.B. & K. NIPRD/H/6163

RESULTS AND DISCUSSIONS: tannins, and terpenes and the absence of alkaloids and The phytochemical screening showed the presence of phlobatannins in all the species (Table 2). carbohydrates, flavonoids, resins, phenolic nucleus,

Glo. J. Agric. Bio. Res. 002

Table 2. Phytochemical Screening of the leaves of the Nigerian Bignoniaceae. S.No SPECIES CHO STE ALK PHT ANT FLA RES PHE SPO TAN TER 1. Crescentia cujete + + - - - + + + + + + 2. Jacaranda + + - - + + + + + + + mimosifolia 3. Kigelia africana + + - - - + + + + + + 4. Markhamia tomentosa + + - - - + + + + + + 5. Newbouldia laevis + + - - - + + + + + + 6. Spathodea + + - - - + + + - + + campanulata 7. Stereospermumacumi + + - - + + + + + + + natissimum 8. Stereospermum + + - - + + + + - + + kunthianum 9. Tabebuia rosea + + - - - + + + + + + 10. Tecoma stans + + - - - + + + - + +

Key: CHO=Carbohydrate PHE=Phenolic nucleus + = presence STE=Sterols SPO=Saponin - = absence ALK=Alkaloids TAN=Tannin PHT=Phlobatanin TER=Terpenes ANT=Antraquinone FLA=Flavonoids RES=Resins

The antimicrobial screening showed that all the hexane ethylacetate extract of C. cujete had activity on all test extracts (Table 3) exhibited activity on B. subtilis and S. organisms; and the ethylacetate extracts of N. laevis and aureus and the hexane extract of C. cujete had activity on S. acuminatissimum had no activity at all on all test all the test organisms. The hexane extract had more organisms. In the methanol extracts (Table 5), C. cujete, activity, followed by the ethylacetate extracts (Table 4) J. mimosifolia, N. laevis, S. acuminatissimum, and T. while the water extract (Table 6) had the least activity on stans had activity on P. aeruginosa. the test organisms. On the other hand only the

Table 3. Antimicrobial Screening of the hexane extracts of the leaves of the Nigerian Bignoniaceae S.No Species Ps Bs Ca Ec Sa St 1. Crescetia cujete + + + + + + 2. Jacarada mimosifolia - + + + + + 3. Kigelia Africana - + + + + + 4. Markhamia tomentosa - + + + + + 5. Newboudia laevis - + + - + - 6. Spathodea campanulata - + - - + - 7. Stereospermum - + - - + + acuminatissimum 8. Stereospermum kunthianum + + + + + - 9. Tabebuia rosea + + + + + - 10 Tecoma stans - + + - + - Control 1 ------Control 2 ------

Key: Ps = Pseudomonas aeruginosa + = Activity = absence of growth Bs = Bacillus subtilis - = No activity = presence of growth Ca = Candida albicans Ec = Escherichia coli Sa = Staphylococcus aureus St = Salmonella typhi

Glo. J. Agric. Bio. Res. 003

Table 4. Antimicrobial Screening of the Ethylacetate extracts of the leaves of the Nigerian Bignoniaceae S.No Species Ps Bs Ca Ec Sa St

1. Crescetia cujete + + + + + + 2. Jacaranda mimosifolia - + + + + + 3. Kigelia Africana + + + + + - 4. Markhamia tomentosa - + + + + + 5. Newbouldia laevis ------6. Spathodea campanulata + - - - + + 7. Stereospermun ------acuminatissimum 8. Stereospermum kunthianum - + + + + - 9. Tabebuia rosea + + + + + - 10 Tecoma stans - + + + + + Control 1 ------Control 2 ------

Table 5. Antimicrobial Screening of the Methanol extracts of the leaves of the Nigerian Bignoniaceae

S.No Species Ps Bs Ca Ec Sa St 1. Cresentia cujete + + - - - + 2. Jacarada mimosifolia + + + + - - 3. Kigelia Africana + + - - + - 4. Markhamia tomentosa - + + - + - 5. Newbouldia laevis + - + - + - 6. Spathodea campanulata - + + - + - 7. Stereospermum + - - - + - acuminatissimum 8. Stereospermum kunthianum - + - + + + 9. Tabebuia rosea - + + - + - 10 Tecoma stans + + + + + + Control 1 ------Control 2 ------

Key: Ps = Pseudomonas aeruginosa + = Activity = absence of growth Bs = Bac illus subtilis - = No activity = presence of growth Ca = Candida albicans Ec = Escherichia coli Sa = Staphylococcus aureus St = Salmonella typhi

The results obtained indicated the existence of antimicrobial extracts had schozontocidal activity while the water extract had compound in the various extracts of these plants and showed a no activity. Binutu and Lajubutu (1994) reported that the good correlation between the reported uses of these plants in methanolic extracts of leaves and stem bark of J. mimosifolia, traditional medicine against infectious diseases. For example the T.stans, T. rosea and C. cujete have antimicrobial activity on inhibition of E. coli by some of the plant extracts such as hexane, both gram positive and gram-negative bacteria and fungi. This ethyl acetate and methanol extracts of J. mimosifolia and S. has also been shown to be true in the present study. Adesanya kunthianum; hexane and ethyl acetate extracts of K. africana, M. et al (1994) isolated four pyrazole (withasomnine, 4’-hydroxy- tomentosa, and T. rosea; ethyl acetate and methanol extracts of withasomnine, newbouldine and 4’-hydroxynewbouldine) from T. stans justifies their use in the treatment of diarrhea and the root bark of N. laevis. The cytotoxic and anticancer activity of dysentery in traditional medicine. the ethanol extracts of N. laevis leaves were reported by Azuine Makinde et al (1987) reported the alcoholic extract of et al (1995) to have antineoplastic activity. In contrast to their the leaves of S. campanulata antiplasmodial activity to be more findings the phytochemical screening of N. laevis leaves contain effective in treating the early infection than the established one no anthraquinone and alkaloids in the present study. This could and they also reported in 1988 that the hexane and chloroform be due to the environment where the collections were made.

Glo. J. Agric. Bio. Res. 004

Table 6. Antimicrobial Screening of the Water extracts of the leaves of the Nigerian Bignoniaceae

S.No Species Ps Bs Ca Ec Sa St 1. Crescentia cujete ------2. Jacarada mimosifolia - - + - + - 3. Kigelia Africana - - - - + - 4. Markhamia tomentosa ------5. Newbouldia laevis ------6. Spathodea campanulata ------7. Stereospermum ------acuminatissimum 8. Stereospermum kunthianum ------9. Tabebuia rosea ------10 Tecoma stans + + - - + + Control 1 ------Control 2 ------

In conclusion, the study is a preliminary evaluation of the Azuine, M.A; Ibrahim, K; Enwerem, M; Gamaniel, K and phytochemical screening and antimicrobial activity of the Wambebe, (1995). Cytotoxic and anti-cancer activity of N. successive extracts of the leaves in the family Bignoniaceae. laevis in mice. West Afr J Pharm and Drug Res. 11:72-79. The results obtained and the previous work done on some of Binutu, O.A; and Lajubutu B.A (1994). Antimicrobial potentials of these plants indicated that these plants have the potentials to some plant species of the Bignoniaceae family. Afr J Med generate novel metabolites. The extracts with anticandidal and Med Sci : 23(3)269-273. activity (e.g. hexane, ethylacetate, methanol and water extracts Fadeyi, MG; Adeoye, AE and Olowokudejo, JD (1989). of J. mimosifolia; hexane, ethylacetate and methanol extracts of Epidermal and Phytochemical studies with genus of Boerhvia M. tomentosa, T. rosea and T. stans; Hexane and ethylacetate (Nyetangiaceae) Crude Drug Res. 29:178-184. extracts of K. africana; hexane extracts of N. laevis and S. Hutchinson, J. and Dalziel, J. M (1954). Flora of West Tropical acuminatissimum; Ethyl acetate extract of S. kunthianum African. Vol. II. Part 1. Crown Agents for Oversea methanol extract of S. campanulata) could result in the discovery Government and Administrations London P. 383 – 388. of novel anticandidal agents. Similarly, the plants demonstrating Makinde, J.M; Adesogan E.K and Amusan, O.O (1987). The broad spectra of activity may help to discover chemical classes chizontocidal activity of S. campanulata leaf extracts on of antibiotics that could serve as agents for the maintenance of Plasmodium beghei beghei in mice. Phytotherapy Res health. Further work will be carried out to determine the minimum 1(2):65-68. inhibitory concentration of the active extracts before isolation and Makinde JM, Amusan OG, Adesogan EK (1988). The elucidation of the structure of the most active compounds. Antimalarial Activity of Spathodea campanulata stems bark extract on Plasmodium berghei berghei in mice. Plant ACKNOWLEDGMENT: Medica: 122-125. Mitscher, L; Leu, R.P; Bathola, M.S; Wu, W and Beal, J.L (1978). We would like to acknowledge the following people, the DG Antimicrobial agents from higher plants. Introduction Rational NIPRD, Dr. Inyang for providing the enabling environment for and methodology. Llyodia. 35:157-166. this work, Mrs. Ibrahim Jemilat for helping in the collection of C. Odebiyi A, Sofowora EA (1990). Phytochemical Screening of cujete leaves, Mr. Yusuf and Mr. Femi the Technologists of M & Nigerian plants. Part III.Lloydia 41: 234-246. BT Dept. who provided materials to work with and all M.P.R & Shashina, G. (1989). Savanna Plants. An Illustrated Guide. T.M Dept. N.I.P.R.D Abuja who have contributed in one way or Macmillan P. 157- 158. the other to make this work a success. Watson, L. and Dallwitze, M. J.(1992).The Families of Flowering Plants. Description, Illustration, Identification and Information REFERENCES Retrieval Version P.1- 3. Adesanya, S.A; Nia, R; Fantaine, C and Pais, M (1994). Pyrozole alkaloids from N. laevis. Phytochemistry. 3(4):1053- 1055.

Glo. J. Agric. Bio. Res. 005