Journal of Ethnopharmacology 64 (1999) 277 282

Short communication

Antibacterial and antifungal activity of small protein of Indigofera oblongifolia leaves

M. Umar Dahot
Enzyme and Fermentation Biotechnology Research Laboratory, Department of Biochemistry, Institute of Chemistry, Uni6ersity of Sindh, Jamshoro, Pakistan Received 13 December 1997; received in revised form 26 June 1998; accepted 21 July 1998

Abstract Four fractions from the leaves of Indigofera oblongifolia were obtained on Sephadex G-25 column chromatography. A single bands of these fractions were detected on Poly-acrylamide SDS gel electrophoresis. An antibacterial action of small protein/peptide was tested against Escherichia coli, Klebsiella aerogenes, Kl. pneumoniae, Staphyllococcus aureus, and Bacillus subtilis. Peptide 3 showed strong inhibitory activity against B. subtilis and Aspergillus niger but clear zone of inhibition was also noted against A. fumigatus. Peptide 4 showed signicant inhibitory zone against Kl. pneumoniae, S. aureus, B. subtilis, A. fumigatus, A. niger and A. a6us. 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Antibacterial; Antifungal; Small protein; Indigofera oblongifolia; leaves; Chromatography; Electrophoresis

1. Introduction Several small proteins or peptides with antifungal activity have been isolated from plants (Robert and Seletrennikoff, 1986; Hejgaard et al., 1992; Terras et al., 1992) and are believed to be involved in a defence mechanism against phytopathogenic fungi by inhibiting fungal growth through diverse molecular modes, such as binding to chitin or premeabilizing fungal membranes or

cell wall. Another strategy followed by plants to thwart invaders is based on the localized production of antimicrobial low molecular weight secondary metabolites known as phytoalexins (Van Etten et al., 1989; Maher et al., 1994). Moreover, the synthesis of many presumed defence related protein is induced when plants are confronted with pathogens (Linthorst, 1991). In this country numerous studies have been carried out to extract natural product for screen-

0378-8741/99/$ - see front matter 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S0378-8741(98)00136-6

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Table 1 Protein content and antimicrobial activity of rude aqueous extract of I. oblongifolia leaves Antibacterial activity E. coli Negative Antifungal activity Aspergillus niger ++Positive Protein content, 1.60 mg/ml.

Kl. aerogenes Negative A. fumigatus Trace

Kl. Pneumoniae Trace A. a6us +Positive

S. aureus Trace P. expansum Negative

B. subtilis ++Positive

ing antimicrobial activity but attention has not been focused to isolate small protein/peptide for biological activity from Pakistani shrubs, plants and seeds. This paper reports the rst systematic attempt to isolate small protein/ peptide from the leaves of Indigofera oblongifolia possessing antibacterial and antifungal activity.

2.2. Preparation of soluble extract

I. oblongifolia leaves were dried at room temperature and were ground into ne powder with mortar and pestle. The dried powder of leaves (50 g) was defatted with 150 ml of diethyl ether at room temperature (28C) for 8 h and ltered with Whatman no. 1. The residue was extracted with 33 ml cold distilled water and centrifuged (Kubota refrigerated centrifuge, Japan) at 6000 rpm. The supernatant was transferred to 100 ml volumetric ask and this procedure was repeated twice and volume was made up to the mark with distilled water. Small proteins/peptides were precipitated by the addition of two fold acetone. These isolated precipitates were dissolved in 0.2 M acetic acid and dialyzed overnight in the same.

2. Materials and methods

2.1. Materials
I. oblongifolia Forsk belongs to Fabaceae or Papilionaceae (previously known as Leguminoceae) family which is locally known as Jhil. This shrub is 0.91.8 m high. Branches are numerous, stout and woody. Leaves are imparipinnate and petiole is 6 12 mm long with small owers. Pod are numerous and contains 6 8 seeds per pod. It is distributed throughout the plains of Pakistan, India, Sri Lanka, Jordan, Yemen, Bahrain, Egypt, Sudan, Senegal, Angola and Nigeria (Jafari, 1960). The leaves of I. oblongifolia were collected during June July 1995 from the plants from Village Qaim Baber, District Hyderabad, Pakistan. The plant was identied at the Department of Botany, Shah Abdul Latif University Khairpur and a voucher specimen SM 1130 has been deposited in the Departments Herbarium. Sephadex G 25 and Commassie brilliant blue R-250 were obtained from Sigma. Acetic acid, diethyl ether, SDS, acrylamide, bisacrylamide, ammonium persulphate were purchased from E. Merck, Germany.

2.3. Separation on Sephadex G-25

The dialyzed sample was subject to a column of Sephadex G-25 (1.5137 cm) and eluted with 0.2 M acetic acid. Fraction of 4.5 ml each was collected on fraction collector (Eyla Fraction Collector, Japan) at a ow rate of 48 ml/h.

2.4. Determination of Protein

The absorbance at 280 nm was measured to monitor the protein during chromatography separation. The protein content of water extract was measured by the method of Lowry et al. (1951), using bovine serum albumin as standard.

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Fig. 1. Separation of I. Oblongifolia leaves small protein/peptide on Sephadex G-25.

2.5. Electrophoresis
The homogeneity of pooled fractions were checked by SDS disc gel electrophoresis by applying 50 vl of denatured sample on the surface of the gel. Power supply was adjusted at 5 mA current per tube for 80 min. The gel was stained with Coomassie blue R 250 and destained with methanol:acetic acid: water (30:60:10 V/V) as reported by Davis (1964), Hames and Rickwood (1986).

2.6. Antibacterial acti6ity

The cultures of bacteria (Escherichia coli ATCC
Table 2 Purication of small protein from I. oblongifolia leaves Purication steps Crude Dialyzed Sephadex G 25 Fraction-1 Fraction-II Fraction-III Fraction-IV Volume (ml) Total Protein (mg) 160.00 108.30 64.98 32.82 13.43 14.24 4.49 Yield (%)

100.0 10.0

100.00 67.69 40.61 20.51 8.39 8.90 2.81

11229, Klebsiella aerogenes ATCC 5409, Kl. pneumoniae ATCC 6071, Staphyllococcus aureus ATCC 6538, and Bacillus subtilis ATCC 1813) grown overnight at 37C were used for testing the antibacterial activity from different fractions separated on Sephadex G-25 and aqueous extract of I. oblongifolia leaves. The anti-bacterial activity was checked by seed plate method as reported by Rashed et al. (1990). In this technique meat extract nutrient medium containing 1.5% agar was adjusted to pH 7.0, distributed in 40 ml quantity in screw capped bottles and sterilized. The bacterial culture was then added aseptically to the agar medium at 45C, mixed well and poured immediately in sterilized petri-plates. After hardening, 6 mm diameter well were cut into agar and 100 vl of the I. oblongifolia leave extract and fractions were placed in these wells. The plates were incubated at 37C and observations were made after 2472 h. The zone inhibition produced by the plant fractions were compared with zone inhibition produced by commercial standard antibiotics disc such as Tarvid and Enaxbid (20 vg/ml).

40.5 18.0 31.5 45.0

2.7. Antifungal acti6ity

Antifungal activity was tested against Aspergillus niger A25717, A. fumigatus A9381, A. a6us A15197 and P. expansum CMI 39761. The diffu-

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2.8. Determination of minimal inhibitory concentration (MIC)

The minimum inhibitory concentration was determined by serial dilution in the same type of agar, with concentration ranging from 5, 10, 25, 50, 75 and 100 vg/ml. The inoculum was prepared from fresh overnight broth culture in nutrient dextrose broth. Plates were incubated for 24 h at 37C and 96 h at 28C for antibacterial and antifungal activity respectively.

3. Results Table 1 show the results of protein content, anti-bacterial and antifungal activity from the water extract of I. oblongifolia leaves. Separation of I. oblongifolia sample on Sephadex G-25 column chromatography showed four optical density peaks (Fig. 1). The small proteins/ peptides associated with these optical density peaks were termed as AP1, AP2, AP3 and AP4 in the order, in which they were eluted from the column and their protein content is shown in Table 2. The homogeneity of pooled fractions was checked by SDS polyacrylamide disc gel electrophoresis and were found homogeneous by showing single band (Fig. 2). Antibacterial/antifungal activity of AP1,AP2, AP3 and AP4 fraction is shown in Table 3. The minimum inhibitory concentration of isolated fractions against bacterial and fungal species is shown in Table 4.

Fig. 2. Electrophoresis pattern of crude and puried fractions of I. Oblongifolia leaves.

sion plate method was used to test I. oblongifolia leave fractions with slight modication as reported by Terras et al. (1995). In this technique, 0.1 ml of the fungal spore suspension (grown for 3 days in 10 ml of nutrient Dextrose agar) was thoroughly mixed with 20 ml of melted Sabouraud dextrose agar and poured into sterilized petri plates. When the agar was set, 5 wells of 6 mm diameter were made on each of the seeded plate. These holes were lled with 100 vl of the testing sample. All these experiments were performed in duplicate. The petri plates were incubated at 28C for 7 8 days. All the culture plates were examined after 24 96 h and the results are tabulated. The zone inhibition produced by the plant fractions were compared with zone produced by the standards (nystatin and griseofulvin 20 vg/ml).

4. Discussion and conclusions It is clearly noted that aqueous extract of I. oblongifolia leaves posses signicant antimicrobial activity against gram positive, gram negative and fungal species. Fraction AP2 is slightly active against B. subtilis and A. fumigatus but fractions (AP3) was found highly active by producing clear zones of inhibition against B. subtilis and A. niger as shown in Table 3. Moreover, fraction AP4 was found moderately active against bacteria such as Kl. pneumoniae, S. aureus, B. subtilis and fungi including A. niger, A. fumigatus and A. a6us.

M.U. Dahot / Journal of Ethnopharmacology 64 (1999) 277282 Table 3 Antimicrobial activity of peptides isolated and puried from I. oblongifolia against bacteria and fungi Antibacterial activity Fraction E. coli P-1 Negative P-2 Negative P-3 Negative P-4 Negative Tarivid Trace Enaxbid ++Positive Antifungal activity Fraction P-1 P-2 P-3 P-4 Nystatin Griseofulvin A. fumigatus Negative +Positive +Positive +Positive Negative +Positive

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Kl. aerogenes Negative Negative Negative Negative Negative Negative A. niger Negative Negative +++Positive ++Positive ++Positive ++++Positive

Kl. pneumoniae Negative Negative Negative +Positive Negative +++Positive A. a6us Negative Negative Negative ++Positive Trace ++Positive

S. aureus Negative Negative Negative +Positive ++Positive +++Positive P. expansum Negative Negative Negative Negative ++Positive +++Positive

B. subtilis Negative Trace ++++Positive +Positive +++Positive ++++Positive

Positive, Trace, 2 mm inhibitory zone; +, 5 mm inhibitory zone; ++, 10 mm inhibitory zone; +++, 15 mm inhibitory zone; ++++, 20 mm inhibitory zone

Fraction AP1 did not show any activity against bacteria and fungi. E. coli, Kl. aerogenes and P. expansum were found ineffective against aqueous extract as well as all fractions of I. oblongifolia leaves. The antimicrobial effect, expressed as MIC in the serial dilution broth method, most of the organisms tested were not inhibited by concentration of up 100 vg/ml with fraction AP1 and AP2.
Table 4 Minimal inhibitory concentration of Indigofera oblongifolia leaves fractions in the serial dilution method Test organisms MIC fractions (mg/ml) P-1 Bacteria E. coli Kl. aerogenes Kl. pneumoniae S. aureus B. subtilis Fungi A. fumigatus A. niger A. a6us P. expansum P-2 P-3 P-4

\100 \100 \100 \100 \100 \100 \100 \100 \100

\100 \100 \100 \100 75100 50100 \100 \100 \100

\100 \100 \100 \100 510 5075 1020 \100 \100

\100 \100 5075 5075 5075 5075 1530 2550 \100

The inhibition of micro-organism growth is most pronounced for B. subtilis and A. niger with Fraction AP3 of I. oblongifolia leaves (Table 4). However, fraction AP4 exhibit a weak antibacterial but moderate antifungal activity. This observation provide strong circumstantial evidence that antifungal small proteins/peptides play an important role in a plants antimicrobial defence system (Terras et al. 1995). To our knowledge, this study is the rst report on purication and characterization of antimicrobial small protein/peptide from I. oblongifolia leaves in this country. This study provides considerable scope in exploiting the local indigenous resources for isolation of antibacterial/antifungal peptides or smaller proteins. Further work is under progress in this Laboratory for the determination of amino acid composition of active small protein/peptide, and results will be reported in near future.

Acknowledgements This work was supported by Pakistan Science Foundation through Research Project S-SU/ Chem-272.

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M.U. Dahot / Journal of Ethnopharmacology 64 (1999) 277282 genic tobacco plants with suppressed level of preformed phenyl propanoid products. Proceedings National Academy of Science USA 91, 7802 7806. Rashed, A., Khan, M.R., Khalid, N., 1990. Antibacterial activity of some medicinal plants and seeds. Pakistan Journal of Biochemistry 23, 55 62. Robert, W.K., Seletrennikoff, C.P., 1986. Isolation and partial characterization of two antifungal proteins from barley. Biochemica et Biophysica Acta 880, 161 170. Terras, F.R.G., Schoofs, H.M.E., De Bolle, M.F.C., et al., 1992. Analysis of two novel classes of antifungal proteins from radish (Raphanus sati6aus L.) seeds. Journal of Biological Chemistry 267, 15301 15309. Terras, F.R.G., Eggermont, K., Kovaleva, V., et al., 1995. Small cysteine rich antifungal proteins from radish: Their role in host defence. The Plant Cell 7, 573 588. Van Etten, H.D., Matthews, D.E., Matthews, P.S., 1989. Phytoalexin detoxication importance for pathogenicity and practical implications. Annual review of Phytopathology 27, 143 164.

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