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Bioactive DOPA melanin isolated
and characterised from a marine
actinobacterium Streptomyces sp.
MVCS6 from Versova coast
ab
a
P. Sivaperumal , K. Kamala & R. Rajaram
b
a
Central Institute of Fisheries Education, ICAR-Deemed
University, Off Yari Road, Versova, Andheri (W), Mumbai 400 061,
Maharashtra, India
b
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Department of Marine Science, Bharathidasan University,
Tiruchirappalli 620 024, Tamil Nadu, India
Published online: 18 Dec 2014.
To cite this article: P. Sivaperumal, K. Kamala & R. Rajaram (2014): Bioactive DOPA
melanin isolated and characterised from a marine actinobacterium Streptomyces sp. MVCS6
from Versova coast, Natural Product Research: Formerly Natural Product Letters, DOI:
10.1080/14786419.2014.988712
To link to this article: http://dx.doi.org/10.1080/14786419.2014.988712
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Natural Product Research, 2014
http://dx.doi.org/10.1080/14786419.2014.988712
SHORT COMMUNICATION
Bioactive DOPA melanin isolated and characterised from a marine
actinobacterium Streptomyces sp. MVCS6 from Versova coast
P. Sivaperumalab*, K. Kamalaa and R. Rajaramb
a
Downloaded by [Central Inst Fisheries Ed ] at 21:41 19 December 2014
Central Institute of Fisheries Education, ICAR-Deemed University, Off Yari Road, Versova, Andheri (W),
Mumbai 400 061, Maharashtra, India; bDepartment of Marine Science, Bharathidasan University,
Tiruchirappalli 620 024, Tamil Nadu, India
(Received 7 July 2014; final version received 10 November 2014)
The melanin pigment produced from Streptomyces sp., MVCS6 was isolated and
dihydroxyphenyalanine (DOPA) melanin compound was biochemically identified and
spectroscopically characterised (ultraviolet and FT-IR). DOPA melanin showed a
promising activity as an antibacterial natural product against 12 pathogenic bacteria from
hospital isolations, particularly, against Pseudomonas aeruginosa RMMH7 (inhibition
zone of 18 ^ 0.02 at 30 mg/disc, and MIC of 10 ^ 0.02 mg/mL) and Vibrio
parahaemolytics RMMH12 (inhibition zone of 15 mm ^ 0.03 at 30 mg/disc, and MIC of
14 ^ 0.02 mg/mL). Moreover, in vitro evaluation of reducing power (Ascorbic Acid
Equivalent (160 mg/mL)), DPPH radical-scavenging (89%), NO-scavenging (72%) and
lipid peroxidation activities (89.6%) were determined. Cytotoxicity of DOPA melanin
against cervical cancer cell line showed a dose–response activity, and IC50 value was found
to be 300 mg/mL. These results would open the way to propose Streptomyces sp. MVCS6 as
a promising source of bioactive eumelanin with therapeutic potential in medicine.
Keywords: melanin; FT-IR; antibacterial; antioxidant; cytotoxicity activity
1. Introduction
Almost half of the antibiotics discovered up to 2002 are derivatives of microbial natural products,
making them the primary source from which new medicines are discovered (Berdy 2005).
However, over the decades, efforts for the discovery of pharmaceutically relevant compounds
have been shifted to secondary metabolites that are produced by microbes. Melanins are a broad
class of darkly pigmented macromolecules formed by oxidative polymerisation of phenols and/or
indolic compounds, they widely exist in animals, plants and microorganisms. The primary
*Corresponding author. Email: [email protected]
q 2014 Taylor & Francis
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2
P. Sivaperumal et al.
precursor of melanins generated from 3,4-dihydroxyphenyalanine (DOPA) by phenoloxidases are
referred to as eumelanins. Actinobacteria have long been described as capable of producing these
dark brown/black coloured substances in culture media. These coloured melanins or melanoid
pigments development in the actinobacteria is important and is not only useful in taxonomic
studies but also plays a significant role in improving their survival and competitiveness. Melanins
have been reported to act as ‘armour’ and function in the protection of actinobacteria against
environmental stress such as ultraviolet (UV) radiation and temperature extremes (Butler & Day
1998). In general, actinobacteria are a prolific source of secondary metabolites and the vast
majority of these compounds are derived from the single genus Streptomyces (Pathom-Aree et al.
2006). It has radioprotective and antioxidant properties that can effectively protect the living
organisms from UV radiation (Vinarov et al. 2002). Moreover, the production of melanin from
Streptomyces species (Dastager et al. 2006; Quadri & Agsar 2012), Actinoalloteichus
sp. (Manivasagan et al. 2013b) and Brevundimonas sp. (Surwase et al. 2013) has been described
very well. The melanin showed a broad spectrum of biological roles, including antioxidant activity
(Goncalves & Pombeiro-Sponchiado 2005), antibacterial activity (Manivasagan et al. 2013b),
liver protecting activity (Sava et al. 2003), radio protective activity (Dadachova et al. 2007), etc.
Many artificial synthetic colourants have been noticed as undesirable and have potentially harmful
effects; some of them are considered as carcinogenic, responsible for allergenic and adverse
reactions (Keymolen 1998; Duran et al. 2002). Hence, the present investigation focused on DOPA
melanin produced from Streptomyces sp., MVCS6 strain which was isolated and characterised,
and their antibacterial, antioxidant and cytotoxic properties were examined.
2. Results and discussion
2.1. Isolation and identification of marine actinobacteria
A marine actinobacterial strain MVCS6 was isolated from the marine sediment of Versova coast
of Mumbai, India, and this study revealed that the strain produced melanoid pigments (Figure S1
(A)). The strain showed aerial and substrate mycelia (Figure S1(B)) and vegetative hyphae with
an extensively branched mycelium. Its spore chain was retinaculiaperti rectiflexibile with spiny
spore surface morphology (Figure S1(C)). This isolate was tentatively identified as Streptomyces
sp. MVCS6 based on the morphological and it was confirmed by 16S rDNA sequencing. The
sequence was submitted to the Gene Bank in NCBI (http://www.ncbi.nlm.nih.gov/nuccore/
KC292198) and obtained the accession number (KC292198). In this strain Streptomyces sp.,
MVCS6 is phylogenetically close to the genus Streptomyces (Figure S1(D)).
2.2. Treatment with melanin synthesis inhibitors
Melanin synthesis inhibitors can be used to identify the different types of melanins (Rizner &
Wheeler 2003) such as tricyclazole, pyroquilon, thalide and chlobenthiazone and these inhibit
the synthesis of 1,8-dihydroxynaphthalene (DHN) melanin. However, DOPA melanin synthesis
was inhibited by tropolone, kojic acid, 2-mercaptobenzimidazole and diethyldithiocarbamate
(Elliott 1995). In this study, inhibition of melanin production was observed by the test of
Streptomyces sp., MVCS6 grown in Kojic acid (DOPA melanin inhibitor). Moreover, the marine
actinobacterium Streptomyces sp., MVCS6 showed growth in the presence of tricyclazole (DHN
melanin inhibitor), which appeared normal and was similar to control. These results showed that
Streptomyces sp. MVCS6 strain synthesised DOPA melanin (Figure S2(A)).
2.3. Time course of melanin production
Growth and melanin production were determined from the marine actinobacterium
Streptomyces sp. MVCS6. Samples were collected at every 12-h intervals and the time course
Natural Product Research
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was studied up to 168 h in an optimised medium. Maximum melanin production (98.6^ 0.13 mg/
mL) was achieved at 120 h of incubation (Figure S2(B)). The DOPA melanin production built up
gradually during the exponential phase and reached maximum at the commencement of the
stationary phase which was similar to that reported by Wang et al. (2006). However, the melanin
production increased gradually and reached a maximum at 24 h in the case of bacteria (VilasBoas et al. 2005).
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2.4. Spectroscopic studies
The typical melanin UV absorption could be informed at 300 nm, approximately (Figure S3(A)).
This might be due to the presence of many complex conjugated structures in the melanin
molecule (Cockell & Knowland 1999). Hence, the strong UV absorption near 300 nm is often
used to characterise melanins (Ravishankar et al. 1995). The FT-IR spectrum of the melanin was
characterised and a broad absorption band observed at 3399 cm21 may be ascribed to the OZH
stretching (hydroxyl groups), as well as the NZH and OZH of COOH functionalities. The peaks
at 2960 and 2927 cm21 were due to alkanes group CZH stretching due to the presence of
impurities. The peak at 1078 cm21 was assigned to CZN stretching bond related to amines, and
CZO of alcohols and phenols. IR peaks at 1654 and 1647 cm21 were assignable to conjugated
CvO stretch, and aromatic group signals (ArCvC st) at 1457, 1437 and 1420 cm21 were also
present (Figure S3(B)). Suryanarayanan et al. (2004) also described the IR spectrum of melanin
with peaks near 3352.5 cm21 were ascribed to OZH and NZH bonds.
2.5. Determination of antibacterial activity and MIC
Natural products are considered as a significant source of new antibacterial agents. Marine
forms natural products comprise approximately half of the total global biodiversity; large-scale
screening could continue to play an important role in the development of new drugs (Xu et al.
2004). In this study, purified DOPA melanin showed antibacterial activity against 12
human bacterial pathogens with respect to the standard 30 mg/disc streptomycin, and the results
are given in Table S1(A). The maximum zone of inhibition was found to be 18 ^ 0.02 mm
(30 mg/disc) for Pseudomonas aeruginosa RMMH7 and 15 ^ 0.03 mm (30 mg/disc) for
Vibrio parahaemolytics RMMH12, whereas it was 21 ^ 0.01 and 18 ^ 0.02 mm for the
standard streptomycin. Similarly Manivasagan et al. (2013b) reported that purified melanin
showed maximum zone of inhibition 18 ^ 0.01 mm (30 mg/disc) and 25 ^ 0.02 mm (50 mg/
disc) for B. subtilis and 20 ^ 0.01 mm (30 mg/disc) and 29 ^ 0.03 mm (50 mg/disc) for
Escherichia coli. Moreover, the MIC value of purified melanin showed good activity against
12 pathogenic bacteria (Gram positive and Gram negative) from hospital isolations,
particularly against P. aeruginosa RMMH7 (10 ^ 0.02 mg/mL) and V. parahaemolytics
RMMH12 (14 ^ 0.02 mg/mL).
2.6. Antioxidant activity
All antioxidant assays were performed at 0.2 –1 mg/mL concentrations. The results presented
in Figure S4(A) clearly shows that the melanin exhibited an interesting radical scavenging
activity (89%) at 1 mg/mL. When DPPH radicals encounter a proton-donating substrate such as
an antioxidant, the radicals were scavenged and the absorbance was reduced (Shimada et al.
1992). The melanin showed a moderate nitric oxide-scavenging activity in a dose dependent
manner (Figure S4(B)). The highest scavenging activity (72%) was observed at 1 mg/mL
and ascorbic acid was used as a positive control for comparison. Reducing power of melanin
(Figure S4(C)) suggested that it is likely to contribute significantly towards the observed
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P. Sivaperumal et al.
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antioxidant effect. Vimala et al. (2007) observed a direct correlation between antioxidant
activities and reducing power of certain melanins. Lipid peroxidation of the purified melanin
showed a potent activity (89.6%) at 1 mg/mL concentration (Figure S4(D)). These results
suggest that DOPA melanin compound isolated from Streptomyces sp., MVCS6 has a prominent
percentage of inhibition, consistent with increasing concentration. However, the antioxidant
activities have been ascribed to various mechanisms, along with prevention of chain initiation,
binding of transition metal ion catalysts, disintegration of peroxides, anticipation of continued
hydrogen abstraction, reductive capacity and radical scavenging (Diplock 1997).
2.7. Cytotoxic activity
There are only a few studies available to determine that the cytotoxic effects of biologically
synthesised secondary metabolites. In this study in-vitro cytotoxic activity of purified melanin
against vero cell lines and cervical cancer cell lines (HeLa) was determined by using the MTT
assay method. The IC50 value of purified melanin against HeLa cells was determined at
300 mg/mL concentrations (Figure S5(F)). Purified melanin inhibits the viability of the HeLa
cancer cell lines in a dose-dependent manner and the cell proliferation capacity significantly
decreased with increasing dose of melanin (100 –600 mg/mL). The HeLa cells treated with
purified melanin at 300 mg/mL concentration for 24 h showed considerable morphological
changes, which are distinctive features of apoptotic cells, such as defeat of membrane
integrity, cell shrinkage and reduced cell density (Figure S5(C),(D)). Moreover, the positive
control of chemotherapy doxorubicin was studied at different concentrations (0 –40 mM) and
IC50 value also observed against HeLa cells (5G). Compared with purified DOPA melanin, the
positive control of doxorubicin showed maximum morphological changes, cell contraction and
very less cell density (5E). However, there were no morphological changes in vero cell lines
treated with purified melanin (Figure S5(A),(B)). Recently Madhusudhan et al. (2014) have
observed that 100% of total mortality was shown in the highest concentration (64 mg/mL) of
soluble melanin and 95% mortality was observed in insoluble melanin. In addition,
Manivasagan et al. (2013a) reported that Nocardiopsis sp. MBRC-1 derived AgNPs has
considerable cytotoxic effect against in vitro HeLa cancer cell lines and it has 200 mg/mL
(IC50) concentrations.
3. Conclusion
The present study found that the DOPA melanin produced marine actinobacteria Streptomyces
sp. MVCS6 has antibacterial, antioxidant and cytotoxic activities. Consequently, melanins are
potential targets for antimicrobial drug discovery and for preventing cancer. In conclusion the
active DOPA melanin is less toxic to cells and possesses antioxidant property which could be
studied further by in-vivo animal model studies. Interestingly, microbial melanins could
influence the outcome of antimicrobial and anticancer chemotherapy.
Supplementary material
Experimental details relating to this article are available online, alongside Figures S1 – S5 and
Table S1.
Acknowledgements
The authors are grateful to the Director CIFE, ICAR-Deemed University, Mumbai, for providing facilities
to carry out this study and thankful to Dr. B.B. Nayak, PHT department, CIFE, for giving valuable
suggestions.
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