In vitro Antibacterial and Cytotoxic Activities of a Brown Antibiotic Metabolite
from a Strain of Actinomycetes
Md. Akteruzzaman choudury,
Md. Aziz Abdur Rahman
Md. Abdul Gafur
The research work was conducted to investigate the in vitro antibacterial and
cytotoxic activities of a antibiotic metabolite. The chloroform (CHCl3) extract of
Yeast extract-broth media (acidic) of an Actinomycetes strain yielded a brown,
amorphous antibiotic pigment Di- (2-ethyl hexyl)-phthalate [AK2]. The antibiotic
and the extract exhibited significant antibacterial activity against both gram
positive and gram negative organisms. The zones of inhibition produced by the
test materials were found to be between 10 to 22mm. The Minimum inhibitory
concentration (MIC) values of the antibiotic were determined against six strains of
bacteria and were found to be between 128 to 256μg ml‾1. The cytotoxic activity
of the antibiotic and CHCl3 extract was done by brine shrimp lethality bioassay
and the LC50 (median lethal concentration) values were 15.8 and 12.5μg ml‾1
respectively, calculated by extrapolation from graph.
The Actinomycetes and in particular the genus Streptomyces have been identified as one of the most potent sources for the production of various antibiotics which are used therapeutically (Weese and Smith, 1975) among these streptomycin, kanamycin and neomycin etc. are most important (Goodman and Gilman, 1975). Based on this concept, the field of research for newer antibiotic was broadened. As a part of our continuing studies (Biswas et al., 2000; Azam and Jabbar, 1999), metabolites produced by micro-organisms obtained from soil samples, collected throughout Bangladesh and we isolated a strain of Actinomycetes and Streptomyces species (Holt et al., 1994) from soil collected in the region of Rajshahi.
From the CHCl3 extract of the yeast extract-glucose-broth culture filtrate of the organism, an antimicrobial agent AK2 was isolated by preparative thin layer chromatographic technique (PTLC) (Egon and Stahl, 1969) and was identified as Di-(2-ethyl hexyl)-phthalate by spectral analysis.
This paper reported the antimicrobial and cytotoxic activities of the CHCl3 extract and the antibiotic pigment AK2.
Materials and Methods
Collection of soil sample: The organism was isolated from a soil sample,
collected from Upashahar, Rajshahi, Bangladesh at a depth of 1m during July
1999 using crowded plate technique (Hammond and Lambert, 1978). The isolated
organism was identified as Streptomyces species by its morphological
and biochemical characteristics such as carbon utilization test, amino acid
utilization test, growth characteristic in milk, potato-agar, cellulose etc.
(Holt et al., 1994).
Production of antibiotic: The Actinomycetes was cultured in yeast extract broth media at pH 6 (optimum pH) and after 11 days of incubation at 37.5±0.5°C (optimum temperature) the culture filtrate was extracted with CHCl3. The CHCl3 fraction thus obtained was evaporated to give a brownish solid mass [A mixture of 66mg antibiotic was obtained from 1L of culture filtrate]
Isolation and characterization of the compound: A brown colored compound was isolated by PTLC technique from a crude CHCl3 extract using C2H5-O-C2H5 and CHCl3 (3:1) solvent system. The isolated compound was named as AK2. For the characterization of AK2 physical, chemical and spectroscopic method (1H-NMR, 13C-NMR, 1H-1H COSY and mass spectroscopy) were utilized (Akteruzzaman, 2000).
Antimicrobial assay: The standard test micro organisms were collected
from the Institute of Nutrition and Food sciences, university of Dhaka and ICDDRB
Dhaka Bangladesh. The antibacterial activity of AK2 and CHCl3
extract was determined against six gram positive and ten gram negative bacteria
by standard disc diffusion method (Barry, 1980; Beur et al., 1966). Kanamycin
disk (30μg disc-1) was used as standard. The MIC of the antibiotic
AK2 was determined against six test organisms by simple in vitro
visual method serial tube dilution technique (Reiner, 1982).
Cytotoxic activity: The cytotoxic activity of the CHCl3 extract and AK2 was determined by brine shrimp lethality bioassay (Mayer et al., 1982; Mclaughlin and Anderson, 1988).
38g of sea salt was weighted, dissolved in one liter of distilled water, filtered off and was kept in a small tank. Artemia salina Leach (brine shrimp eggs) was added to the divided tank. Constant oxygen supply was provided and temperature (37±1°C) was maintained for 48hr to hatch and mature the shrimp as nauplii (larvae).
1mg of each sample (crude CHCl3 extract and AK2) were initially dissolved in 200μl of dimethyl sulfoxide (DMSO) to get a concentration of 5μg μl-1. Forty clean vials were taken for the 4 samples in five concentrations (two vials for each concentration) and two vials also taken for control test for each sample. 5ml sea water containing 10 brine shrimp nauplii was given to each of the 5 vials and specific volume of samples were transferred from the stock solutions to the vials to get final concentration of 5, 10, 20, 40 and 80μg ml-1. Control vials contain 5ml of seawater and same volume of DMSO as in the sample vials. After 24hr the number of survivals in each vial was counted. The percentage of mortality of the brine shrimp was calculated for each concentration and the median lethal concentration (LC50) values were determined (Goldstein, 1974).
Results and discussion
A Sreptomyces species was isolated from soil sample, collected at Rajshahi, Bangladesh. This local species was subjected to antibiotic production using yeast extract glucose agar media. The CHCl3 soluble portion of the extract yielded a brown antibiotic pigment AK2.The antibiotic was characterized as Di-(2-ethyl hexyl)-Phthalate (Fig. 1) on the basis of its spectral data (Akteruzzaman, 2000).
|| Structure of Di-(2-ethyl hexyl)-Phthalate
The compound and the extract showed remarkable antibacterial activity against
both gram positive and gram negative bacteria in comparison with standard Kanamycin.
The zone of inhibition produced by the test materials was observed between 10
to 13mm and 15 to 22mm for CHCl3 extract (500μg disc-1)
and AK2 (500μg disc-1) respectively (Table
||Antibacterial activities of CHCl3 extract, AK2
and Kanamycin standard.
The MIC value of AK2 against six organisms were found to be 128 to 256μg ml-1 (Table 2). The cytotoxic activity of the CHCl3 extract and AK2 was determined by brine shrimp lethality bioassay and the results are presented in Table 3.
||The MIC values of the antibiotic AK2 against six
||Table 3:Results of the brine shrimp lethality bioassay of
CHCl3 extract and antibiotic AK2.
The mortality was determined by counting survivors at the end of the exposure
period (24hr) and the control mortality was adjusted by using Abbotts
formula (Abbott, 1925). As shown in Table 3 the mortality
rate of nauplii was increased with the increase of concentration of each sample.
The LC50 was determined by extrapolation from graph (Logarithm of
concentration versus percentage of mortality, Fig. 1) and
the value were 12.5 and 15.8μg ml-1 for CHCl3 extract
and AK2 respectively (Fig. 2).
||Determination of LC50 of CHCl3 extract
and Pure compound AK2 against brine shrimp nauplii.
From the anti bacterial results it is evident that the crude CHCl3 extract showed moderate antibacterial activity. Whereas compound AK2 showed significant antibacterial activity but was less potent than that of standard Kanamycin.
From the results of cytotoxic activity it may be concluded that the CHCl3 extract and compound AK2 were toxic to the brine shrimp nauplii and they are biologically and pharmacologically active. However, the CHCl3 extract was comparatively more cytotoxic than compound AK2. It may be due to the presence of any synergistic compound(s) in the crude CHCl3 extract other than compound AK2
The results of the this study demonstrate that the local Streptomyces species particularly its metabolite AK2 possesses strong antibacterial and cytotoxic activity. This is in agreement with our previous finding on anti shigella activity of this species (Gafur et al.,1991).
In recent years the pathogenic organisms are gaining resistance to existing
antimicrobial agents hence the search for new, safe and more effective antibiotics
against these organisms is a pressing need. Thus the findings of this investigation
and pervious investigation on other genus (Anisuzzaman et al., 2001)
would give valuable support to make clinical trial as well as toxicity studies
of the isolated antibacterial and cytotoxic metabolites to get a more potent
The authors wish to thank the Institute of Nutrition and food science, Dhaka University, Bangladesh and ICDDRB, Dhaka Bangladesh for supplying the test organisms. The authors also like to thank Dr. Naoki Sugimoto, National Institute of Health Science, Tokyo, Japan for spectral analysis.
Abbott, W.S., 1925.
A method of computing the effectiveness of an insecticide. J. Econ. Entomol., 18: 265-267.CrossRef | Direct Link |
Akteruzzaman, C., 2000.
Study on Actinomyces
species and its metabolites. M.Sc. Thesis, Department of Pharmacy, University of Rajshahi, Bangladesh.
Anisuzzaman, A.S.M., N. Sugimoto, G. Sadik and M.A. Gafur, 2001.
Sub-acute toxicity study of 5-Hydroxy-2(Hydroxy-Methyl) 4H-pyran-4 One, isolated from Aspergillus fumigatus
. Pak. J. Biol. Sci., 4: 1012-1015.CrossRef | Direct Link |
Zafrul Azam, A.T.M. and A. Jabbar, 1999.
Antibacterial and cytotoxic activities of red antibiotic pigments from a Penicillium
strain. J. Asiat. Soc. Bangladesh Sci., 25: 267-274.
Barry, A.L., 1980.
Procedures for Testing Antimicrobial Agents in Agar Media. In: Antibiotic in Laboratory Medicine, Lorian, V. (Ed.). Williams and Wilkins Co., Baltimore, USA., pp: 1-23
Bauer, A.W., W.M.M. Kirby, J.C. Sherris and M. Turck, 1966.
Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol., 45: 493-496.CrossRef | PubMed | Direct Link |
Biswas, M.H.U., A.R.M.R. Amin, M.A. Isalm, C.M. Hasan and K.R. Gustafson et al
Monocillinols A and B, novel fungal metabolites from a Monocillium
sp. Tetrahedron Lett., 41: 7177-7180.CrossRef | Direct Link |
Stahl, E., 1969.
Thin Layer Chromatography: A Laboratory Handbook. 2nd Edn., Springer, New York, USA
Gafur, A, A. Jabbar and I. Muhammad, 1991. In vitro
activities of metabolites from two local Streptomyces
species. Dhaka Univ. Stud. Part E, 6: 103-109.
Goldstein, A., L. Aronow and S.M. Kalkan, 1974.
Principle of Drug Action. 2nd Edn., Wiley Biochemical Health Publication, New York, pp: 376-381
Goodman, L.S. and A. Gilman, 1975.
Streptomycin, Gentamycin and other Aminoglycosides. 5th Edn., Macmillan Publishing Co. Inc., New York, pp: 1167-1180
Hammond, S.M. and P.A. Lambert, 1978.
Antimicrobial Actions. Edward Arnold Ltd., London, pp: 8-9
Meyer, B.N., N.R. Ferrigni, J.E. Putnam, L.B. Jacobsen, D.E. Nichols and J.L. Mclaughlin, 1982.
Brine shrimp: A convenient general bioassay for active plant constituents. Planta Med., 45: 31-34.CrossRef | Direct Link |
Reiner, R., 1982.
Detection of Antibiotics Activity: Antibiotics an Introduction. Roche Scientific, Switzerland, pp: 21-25
Weese, W.C. and I.M. Smith, 1975.
A study of 57 cases of actinomycosis over a 36-year period: A diagnostic failure with good prognosis after treatment. Arch. Internal Med., 135: 1562-1568.CrossRef | Direct Link |
Holt, J.G., N.R. Kreig, P.H.A. Sneath, J.T. Staley and S.T. Williams, 1994.
Bergey's Manual of Determinative Bacteriology. 9th Edn., Lippincott Williams and Wilkins, Baltimore, USA., ISBN-13: 9780683006032, Pages: 787Direct Link |
McLaughlin, J.L. and J.E. Anderson, 1988.
Brine shrimp and crown gall tumors: Simple bioassay for the discovery of plant antitumour agents. Proceedings of the NIH Workshop on Bioassays for Discovery of Antitumor and Antiviral Agents from Natural Sources, October 18-19, 1988, Bethesda, pp: 22-24