Alkaloidal Fraction and Antibacterial Assay of Bryophyllum Pinnatum

Transcription

1 International Journal of Innovative Biosciences Research 5(4):13-23, Oct.-Dec., 2017 SEAHI PUBLICATIONS, ISSN: Alkaloidal Fraction and Antibacterial Assay of Bryophyllum Pinnatum 1 Akpan, M. M., 1 Andrewessien, M. U. and 2 Danladi, B 1 Department of Microbiology, University of Uyo, Akwa Ibom State, Nigeria 2 Pharmacognosy Department, University of Uyo, Akwa Ibom State, Nigeria ABSTRACT Extract from the leaves of Bryophyllum pinnatum was screened for its phytochemical component, alkaloid isolation and antimicrobial activities using standard Pharmacognosy and Microbiological techniques. The leaves of Bryophllum pinnatum contain tannins, terpenes, alkaloids, flavonoids, cardiac glycosides and phlobatanins. The alkaloidal fraction obtained was tested against some Gram-positive organisms which included Staphylococcus aureus, Streptococcus pneumonia, Streptococcus pygenes, Corynebacterium diphtheraee and some Gram-negative organisms which include Escherichia coli, Klebsiella Pneumoniae, Haemophilus influenza and Pseudomonas aeruginosa. The sensitivity pattern of all the organisms tested varried with their zone of inhibition in relationship with concentrations. The concentrations used were 50mg/ml, 100mg/ml and 150mg/ml. The isolates were more susceptible at the concentration of 100mg/ml and 150mg/ml. The bioactive chemical constituent of the leaf and the high level of potency on sputa isolates confirms its ethnobotanical uses. Keywords: Bryophyllum pinnatum, ethnobotanical, antimicrobial activities INTRODUCTION The case of drug resistant is so alarmed in recent days. Most bacteria become drug resistant in several ways, drug resistant mechanism is not confined to a single class of drug. Different bacteria may use different mode of resistant mechanism to resist a particular chemotherapeutic agent (Prescott et al., 2005). Microorganism in some cases resist by mutation while some change their permeability to the antimicrobial agent used. Transposons according to Jeremy, (1990) are suspected to play vital role in development of drug resistance plasmid by promoting the involvement of gene transportation between different plasmid. Resistance also arises when specific targets enzymes are modified so that the organism is no longer susceptible to chemotherapeutic agent (Jeremy, 1990). In some third world country and some developing countries where there is no drug dispensers, local chemist practitioners who are not trained dispense incomplete dose of different antimicrobial agent while some patient on their own abuse the antimicrobial agent by not taking complete dose. Many factors lead to the emergence of multidrug resistance. The ability of pathogens to resist commonly used chemotherapeutic agent lead to a search for plant for the treatment of diseases caused by some pathogens. Bryophyllum pinatum is one of the plants used locally for the treatment of cough and other diseases. B. pinaatum is a perennial herb found in India, Nigeria and in tropical and subtropical region, it has hollow stem, red or green in colour, about 4-6 ft in height, thick or muscular leaves, simple leaves are found in the lower part while the upper part complex joint leaves 6 to 7 in opposite pattern Jaiswai (2015). B. pinatum is good for the treatment of raise blood pressure, liver diseases, arthritis, inflammation, kidney stone, diarrhea, oliguria, wounds, urinary problems, piles, jaundice and cholera (Jaiswai, 2015). Iwu (1993) reported the present of compounds like saponins, flavonoid, xanthones, anthraquinones, bryollin A and B. Research revealed that Bryophyllum pinatum is generally used as a local remedy for ear-ache, cough, diarrhea, dysentery, abscesses, ulcers, insect bites, epilepsy, heart problem, arthritis, and dysmenorrheal (Adesanya, 2005). Serveral reports are also obtained on the antimicrobial activity of Bryophllum pinnatum extract. Akinsulire et al., 2007 reported the varied efficacy of B.pinatum 13

2 methanolic, squeezed, aqueous and palm wine extract on S. aureus, B. subtilis, E. coli, P. aeruginosa, Shigella flexneri, Salmonella typhi, Citrobacter sp and C. albicans. In this work, B. pinatum potency is tested mainly on sputa isolates to confirm its remedy for treatment of cough. MATERIALS AND METHODS The leaves of B. pinatum were collected from Ediene Ikono, in Ikono L.G.A of Akwa Ibom State and were identified in the herbarium centre in the Department of Botany and Ecological Studies, University of Uyo, Uyo. Extraction: The leaves were dried in the oven at 40 o C to dryness. Then it was ground to powder using pestle and mortar. One kilogram of the leaves was weighed out and macerated with 70% ethanol for 72 hours. The extract was separated from the marc by filtration. The filtrate was concentrated to dryness in the vacuo at 39±05 o C and the weight of the extract yield 30g. Phytochemical Screening: The leaves extract was screened for the following bioactive substances; alkaloids, saponin, tannins, phlobatannins, anthraquinones, cardiac glycosides, flavonoids, and terpenes using standard procedure as described by Sofowora (2006); Salehi et al., (1992) and Trease and Evans (1989). Partitioning: The extract was partitioned successfully between 200ml of water and n-hexane, chloroform, ethyl acetate and butanol. The fraction was concentrated in vacuo to yield 5g, 3g, 1g, and the aqueous respectively. Thin layer chromatography was carried out using the ethanol extracts of fine fractions. The leaves extract was further subjected to alkaloidal partitioning according to the procedure. Harbone (1988) modified below. 14

3 LEAF EXTRACT 1kg of powdered plant material extracted with 70% ethanol for 72 hours then concentrated in vacuum at 40 0 C Marc Ethanol Extract (g) Partitioned between equal volumes of water and n- hexane Aqueous phase N-hexane phase (Concentrated) Partition with Chloroform Chloroform phase (Concentrate) Aqueous phase Partition with Ethylacetate Aqueous phase Ethylacetate phase (Concentrated) Partition with Butanol Aqueous phase (Concentrated) Butanol phase (Concentrated) Fig.1: Flow diagram of partitioning of ethanolic plant extract 15

4 Chloroform fraction in B. pinatum contained alkaloid and was partitioned three times using chloroform. The organic fraction was discarded. The acidic aqueous fraction phase was made basic by adding ammonia hydroxide until it turned red litmus to blue. It was further partitioned using chloroform three times. The organic phase was pooled together and thin layer chromatography (TLC) was carried out using chloroform and methanol 9:1 (Trease and Evans, 1989). The plates were viewed under ultraviolet (UV) light. Sports were seen, the plates were cut into two, the first part was sprayed with Dragendoff s reagent, and it showed orange colour which indicate the presence of alkaloid in the mid spot. Four grams of the chloroform fraction was subjected to column chromatography to purify the alkaloid. This was absorbed unto silica gel by titrating it with half of it volume of silica gel to a fine powder. Gravity column chromatography method was used. The column used was 80cm and 3cm in diameter. The column was packed using sample dry park method. It was packed to 2/3 with silica gel and n-hexane added by tapping it gradually. The column was loaded using the dry method. Table 1: Elution and collection of Fractions N-Hexane Chloroform Methanol Volume used Test tube number A total of 30 fractions were collected. The fractions were concentrated in a vacuum and analyzed by TLC as described by Trease and Evans (1989). The solvent system used was chloroform and methanol in the ratio 9:1 and detection was done by spraying with Dragendoff s spray reagent. The orange sport shown on the spray confirmed the presence of alkaloid in purified stage in B. pinatum as shown in Table 1. Preparation of concentrations: Sample dilution method was used C 1 V 1 = C 2 V 2 Collection of Samples Microbial isolates used were collected from Front Line Laboratory and University of Uyo Health Centre. They were isolated from sputa samples collected from patient. The isolates were reconfirmed by carrying out the following test: Gram s staining, catalase test, spore test, motility test, gelatin liquefaction test, Simon s citrate test, methyl red MV, Voges proskauer (VP) test, oxidase test, urase test and carbohydrate fermentation test. They were subculture and stocked for the antibacterial assay. Antibacterial Assay The antibacterial activity test of the alkaloidal fraction was carried out using agar-disc diffusion technique described by Prescott et al., (2005). The plates were labelled and inoculated by spread plate technique with 18 hours old test organisms. The concentrations of the alkaloids used were 50mg/ml, 100mg/ml and 150mg/ml, sterile 6mm paper disc previously prepared were impregnated with various concentrations of the alkaloids and placed on the seeded plates (Akpan, et al., 2011). The plates were incubated at 37 o C for 18 hours. The result were observed and recorded. The zones of inhibition were measured with in millimeter with the aid of a ruler. NCCLS standard was used for the interpretation of result. 16

5 RESULT/DISCUSSION The phytochemical screening, Table2, revealed the bioactive constituent of Bryophyllum pinatum leaves which includes tannins, alkaloids, cardiac glycosides, flavonoids, terpenes, and plobatanins, this conforms to the work of Iwu (1993).The present of saponins and tannins highlight its it uses for skin diseases. Cardiac glycosides gave an insight to its for heart problems. Plate 1 shows the present of alkaloids isolated from other bioactive component by partitioning which confirm the preliminary phytochemical test. The sensitivity test revealed varied sensitivity of isolated. Fig 2 to fig 9 the organisms were more sensitive at higher concentration with varied percentage. The sensitive of the isolates to the alkaloided fraction conforms to the work of Akinsulire (2007). The plant leaves exhibit broad spectrum activity as its sensitivity level cut across gram-negative and Gram-positive organism. The antibacterial activities observed gave baseline information on the usage of B. pinatum leaves for the treatment of cough. The sensitivity pattern observed also revealed that the leave of B. pinnatum can be used for the treatment of diseases associated with the organisms tested. B. pinnatum can be used by pharmaceutical companies to produce antibacterial drug for the treatment cough associated with these organism. CONCLUSION B. pinnatum has chemical component for treatment of cough and may be use for treatment of other diseases associated with isolates used in these research. It also confirm the local use of the plant leaves for treatment of cough and associated diseases. Table 2: Result of Phytochemical Screening of Bryophyllum pinatum Constituent Test Interference Tannins ferric chloride test +++ Saponin Frothing test - Alkaloids Dragendoff's test ++ Anthaquinones Picric-Acid test ++ Flavonoids Benzene-ammonia test - Terpenes Magnesium metal in acid test +++ Phobatannins Moleschott's test ++ Cardiac glycoside Keller-kiliani test +++ Lieberman's test +++ Salkouiskites

6 Plate 1:Purified alkaloid plate of leave extract sprayed with Drogindoff s reagent after partitioning. Key: P. B = Partitioned Bryophyllum pinnatum. 18

7 Fig. 2: Sensitivity Pattern of S. pneumonia Fig. 3: Sensitivity Pattern of S. aureus 19

8 Fig. 4: Sensitivity Pattern of H. infudenza Fig. 5: Sensitivity Pattern of K. pneumonia 20

9 Fig. 6: Sensitivity Pattern of E. coli Fig. 7: Sensitivity Pattern of S. pyogenes 21

10 Fig. 8: Sensitivity Pattern of P. aeruginos Fig. 9: Sensitivity Pattern of C. diphtheria 22

11 REFERENCES Adesanya, S. A. (2005). In Theories and Realitiees. Inaugural Lectures Series 181, published by Obafemi Awolowo University Press Ltd. Lle-lfe, Osun State, Nigeria. Akinsulire, O. R., Aibinu, I. E., Adenipekum, T., Adelowotan, T. and Odugbemi, T. (2007). In vitro antimicrobial activity of crude extracts from plants Bryophyllum pinnatum and Kalanchoe crenata. African Journal of Traditional, Complementary and Alternative Medicines, 4(3) Akpan, M. M., Odeomena, S. C., Nwachukwu, C. N. and Danladi, B (2011) Antimicrobial Assessment of Etharolic Extract of Costus afer leaves. Asian J. of plant Sc. and Res. 2(3) Harborne, J. B. (1988). Phytochemical Methods, A Guide to Modern Technique of Plant Analysis. Oxford Press, London, Pp Iwu, M. M. (1993). In Handbook of African Medicinal Plants. Published by RC Press Inc., Florida USA. Jaiswai, S. (2015) Health benefit of Bryophyllum for Kidney and Urinary disorders Jeremy W. D. (1990). Causes of variation. In Jeremy, W. D. (ed.) Moleular Genetics of Bacteria. New York; John Willey, Pp Prescott, L. M., Harley, I. R. and Klein, D. A (2005). Microbiology (7 th ed.) McGraw-Hill Companies, Inc. America, New York Pp Salehi, S. M., Aynechi, Y., Amin, G. H. and Mahnoodi, Z. (1992). Survey of Iranian plants for Saponins, Alkaloids. Tannins IV Daru 2: Sofowora, A. (2006). Medicinal Plant and Traditional Medicine in African. Spectrum Book limited, Ibadan, Nigeria, Pp Trease, A., Evans, W. C. (1989). Pharmacognosy. 14 th edition. India W. C. Saunders Company Limited. 23