ORIGINAL RESEARCH PAPER

Antioxidant properties of dihydroherbimycin A from anewly isolated Streptomyces sp.

Hung Bae Chang Æ Jae-heon Kim

Received: 8 August 2006 / Revised: 29 November 2006 / Accepted: 1 December 2006 /Published online: 6 January 2007� Springer Science+Business Media B.V. 2007

Abstract During antioxidant screening using

1,1-diphenyl-picrylhydrazyl (DPPH) and a lipid

peroxidation assay, a streptomycete strain was

found to produce herbimycin A and dihydroher-

bimycin A as antioxidants in the culture filtrate.

These molecules were identified by using spectral

analyses, including infrared, ultraviolet, mass spec-

trum, and nuclear magnetic resonance assays. In

the DPPH radical-scavenging assay, dihydroher-

bimycin A exhibited more potent antioxidant

activity (IC50, 1.3 lM) than a-tocopherol (IC50,

2.7 lM) that was used as a reference compound. In

the lipid peroxidation assay, both herbimycin A

and dihydroherbimycin A demonstrated antioxi-

dant activities of 61% and 72%, respectively, at

100 lg/ml, while a-tocopherol exhibited an activity

of 93% at the same concentration. Therefore,

dihydroherbimycin A might have the potential to

be developed into a new therapeutic agent.

Keywords Antioxidant � Dihydroherbimycin A �Herbimycin A � Streptomyces sp.

Introduction

Since oxidative stress caused by reactive oxygen

species (ROS) plays an important role in the

development of various diseases, such as the

Alzheimer and Parkinson diseases, the screening

of new microbial metabolites that have antioxi-

dant activities has been the major area of focus of

many researches that aim to develop new drugs

(Adelman et al. 1989; Amstad and Cerutti 1990).

Most antioxidants can be categorized into two

groups based on their origin. The first group

includes synthetic antioxidants such as tert-buty-

lhydroxytoluene, tert-butylhydroxyquinone, and

propylgallate (Black 2002). The second group

includes vitamins and their derivatives such as

ascorbic acid (Bendich et al. 1986), a-tocopherol

(Fukuzawa et al. 1981), and flavonoid (Fugimoto

et al. 1986). In addition, various products, such as

carazostatin (Kato et al. 1993), thiazostatin (Shin-

do et al. 1989), and benzastatin (Kim et al. 1996)

that are produced by micro-organisms, have been

discovered. However, there is a demand for

screening new metabolites that are less expensive

and have fewer side effects (Aoyama et al. 1982).

Therefore, we also attempted to discover new

antioxidants from streptomycetes that are con-

sidered the natural treasure house of secondary

metabolites and found antioxidants from the

culture filtrate of a Streptomyces strain. The

chemical and spectroscopic analyses were carried

H. B. ChangBio Polytechnic College, 315-1, Chaewoon-ri,Ganggyeong-eub, Nonsan-Si 320-905, Korea

J.-h. Kim (&)Department of Microbiology, Institute of BasicSciences, Dankook University, Cheonan 330-714,Koreae-mail: jaehkim1@dankook.ac.kr

123

Biotechnol Lett (2007) 29:599–603

DOI 10.1007/s10529-006-9288-z

out to determine the molecular structures of the

active compounds that were purified in our

experiment. We had first described their antican-

cer activities and cytotoxic effects (Chang et al.

2006). In this study, we will describe the structure

and chemical properties of the antioxidants that

were identified as herbimycin A and dihydroher-

bimycin A. Of these, dihydroherbimycin A

showed high radical scavenging activity and was

a potent inhibitor of lipid peroxidation.

Materials and methods

Antioxidant preparation and its identification

The cultivation of bacterium Streptomyces sp.

AO-0511 and purification of antioxidants have

already been described (Chang et al. 2006).

Spores were inoculated into five 500 ml Erlen-

meyer flasks each of which contained 100 ml seed

culture medium (yeast extract 1 g, beef extract

1 g, N–Z amine A 2 g, glucose 10 g, CaCO3 1 g,

and deionized water 1 l) and shaken at 240 rpm

for 36 h at 28�C. The seed culture (400 ml) was

transferred to 30 l jar fermenter containing 15 l

producing medium (corn starch 5 g, glucose 30 g,

glycerol 15 g, soybean meal 5 g, peptone 5 g,

CaCO3 1 g, antifoamer 0.5 g, and deionized water

1 l). The fermentation was carried out at 28�C for

5 days while stirring at 200 rpm. Culture filtrate

(10 l) obtained by centrifugation was mixed with

5 l ethyl acetate. The ethyl acetate layer was

evaporated to yield a crude preparation that was

dissolved in methanol. Then, the antioxidants

were purified by Sephadex LH-20 (Sigma)

column chromatography (8 · 50 cm) using ace-

tone as the eluent. The active fractions showed

two separate activity peaks (I and II). The peak

I-concentrate was applied to a silica gel (Sigma)

column (2 · 25 cm) using chloroform/methanol

(10:0.2, v/v) as elution solvent. The active frac-

tions were combined and concentrated to give the

antioxidant I (32 mg). For the peak II-concen-

trate, two silica gel column chromatographies

were carried out in succession using chloroform/

methanol (10:2, v/v) as the first eluent and

chloroform/methanol (10:0.2, v/v) as the second

eluent. The active effluents were combined and

concentrated to give the antioxidant II (10 mg).

The two purified antioxidants were subjected

to various spectroscopic analyses in order to

determine their chemical structures and the

results were compared with previously published

data using Antibase 2005 (Laatsch 2005). The

compounds obtained were either yellowish (anti-

oxidant I) or white powders (antioxidant II) that

were highly soluble in acetone and ethyl acetate

but not in water and n-hexane. The infrared (IR)

spectra were measured using a Bruker Vector 22

spectrophotometer with KBr pellet sample and

the ultraviolet (UV) spectra were measured using

a Hewlett Packard HP 8453 spectrophotometer.

The nuclear magnetic resonance (1H-NMR, 13C-

NMR) spectra were measured using a Bruker

DPX-400 using tetramethylsilane as the initial

standard and electrospray ionization (ESI)-mass

analysis was carried out using Hewlett Packard

HP 5989B spectrophotometer. The molecular

weight of antioxidant I was found to be 597

Table 1 Physico-chemical properties of the purified antioxidantsa

Antioxidant I Antioxidant II

Appearance Yellowish powder White powderSolubility Soluble (acetone, ethyl acetate)

Insoluble (water, n-hexane)Soluble (acetone, ethyl acetate)

Insoluble (water, n-hexane)uvkmax (nm) 292 254, 310

IR vmaxKBrcm–1 3448, 3370, 2934, 1735, 1697, 1648, 1077 3400, 3230, 2975, 1710, 1651, 1640, 1100

ESI-MS(m/z)

597 (M + Na)+ 577 (M + H)+

Antioxidant I referred to the purified herbimycin A and antioxidant II to the purified dihydroherbimycin A in thisexperimenta Antibase 2005 was used for identification of the purified antioxidant I and II

600 Biotechnol Lett (2007) 29:599–603

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(M + Na)+, while that of antioxidant II was 577

(M + H)+ or 1152, suggesting that antioxidant II

could be present in a dimeric form (Table 1).

Based on the combination of the UV, IR, and

ESI-mass spectra data and 1H- and 13C-NMR

spectral data, we found that antioxidant I was

identical to herbimycin A, and antioxidant II was

considered to be its reduced form, dihydroherbi-

mycin A (Fig. 1).

Antioxidant activity assay

To measure the antioxidant activity of the sam-

ples during the screening and purification steps,

we performed a modified DPPH (1,1-diphenyl-

picrylhydrazyl) scavenging assay (Bendich et al.

1986). The DPPH radical reacts with a suitable

reducing agent and loses its characteristic violet

colour stoichiometrically with the number of

electrons consumed, which is measured at

517 nm. The 0.1 sample dissolved in ethanol was

added to 0.9 ml DPPH in ethanol (150 lM). The

absorbance was recorded at 517 nm at intervals of

30 s for 10 min.

The inhibition of lipid peroxidation was also

determined. Different concentrations of the sam-

ples were added to the rat liver microsome. Lipid

peroxidation was initiated by adding 0.1 ml 4 mM

FeSO4 to 0.5 ml of mixed rat liver microsome

suspension (pH 7.4) followed by the addition of

0.1 ml 2 mM ascorbic acid. After 30 min at 37�C,

0.1 ml reaction mixture was mixed with 0.25 ml

10% (w/v) trichloroacetic acid in a new tube. After

10 min, the tubes were centrifuged and the super-

natant was separated and mixed with 0.25 ml

0.67% (w/v) thiobarbituric acid in 2 M HCl. The

mixture was then held at 100�C for 45 min to

complete the reaction. After cooling, the intensity

of coloured complex formed was determined at

532 nm using malondialdehyde as a standard. In

the presence of antioxidants (100 lM), the amount

of thiobarbituric acid reactive substances

(TBARS) was expected to decrease, the percent-

age inhibition of lipid peroxidation was calculated

as the antioxidant activity.

Results and discussion

On comparing the antioxidizing activities of

dihydroherbimycin A that was purified in this

experiment with other authentic antioxidants

and antibiotics by using the DPPH scavenging

assay, dihydroherbimycin A was observed to

exhibit potent scavenging activity (IC50, 1.3 lM);

thus it was found to be more effective than

ascorbic acid, a-tocopherol, novobiocin, and

rifamycin O (ansamycin group antibiotics). On

the other hand, herbimycin A showed very low

activity (IC50 > 100 lM) (Table 2). As shown in

Fig. 1, dihydroherbimycin A is the reduced form

of herbimycin A and possesses an additional

hydrogen to be transferred to DPPH; thus,

dihydroherbimycin A is considered to be a

strong DPPH radical scavenger.

The antioxidant activities were also confirmed

by a lipid peroxidation assay. The degree of lipid

peroxidation was assayed by estimating the

amount of TBARS with a previously described

method with slight modifications (Cheeseman

1993). As shown in Table 3, herbimycin A and

ONH

O

H2NCOO

CH3

O

CH3

OCH3CH3

CH3

H3CO

H3COH3CO

OHNH

O

H2NCOO

CH3

OH

CH3

OCH3CH3

CH3

H3CO

H3COH3CO

Antioxidant I (Herbimycin A) Antioxidant II (Dihydroherbimycin A)

Fig. 1 Structures of antioxidant I (herbimycin A)and antioxidant II (dihydroherbimycin A) produced byStreptomyces sp. AO-0511

Table 2 DPPH scavenging activity of herbimycin A, di-hydroherbimycin A, and other compounds

Compound IC50 (lM)a

Ascorbic acid 4.2a-Tocopherol 2.7Novobiocin 14Rifamycin O 91.2Herbimycin Ab >100Dihydroherbimycin Ac 1.3

a Antioxidant concentrations at which the colour intensityof the 150 lM DPPH solution reduced to 50% of theoriginal colour intensityb, c Herbimycin A and dihydroherbimycin A were thepurified antibiotics in this experiment

Biotechnol Lett (2007) 29:599–603 601

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dihydroherbimycin A demonstrated an inhibitory

activity of 61% and 72%, respectively, against

lipid peroxidation in the radical-generating

system of the rat liver microsome. These values

were slightly low; however, they presented potent

suppressive effects when compared with 93%

inhibitory activity of a-tocopherol.

Herbimycin A is an inhibitor of tyrosine

kinase (Uehara et al. 1989) and, accordingly, it

shows versatile biological effects such as the

induction of apoptosis of some cancer cells

(Mancini et al. 1997) and the inhibition of

inflammatory responses (Ogino et al. 2004).

With regard to the reactive oxygen species

(ROS), herbimycin A indirectly decreased the

cellular level of the superoxide radical as a

result of tyrosine kinase inhibition (Yang et al.

2000). However, there are contradictory reports

stating that herbimycin A increased hydroxyl

radical or ROS levels (Benchekroun et al. 1994;

Mancini et al. 1997).

With regard to dihydroherbimycin A, except

for its cytotoxicity, there is little information on

its effect on the cellular ROS level (Lin et al.

1988). It should be mentioned that benzoquinone

ansamycin antibiotics such as herbimycin A and

geldanamycin are reductively activated for the

hydroxyl radical formation (Benchekroun et al.

1994). Benzoquinone ansamycins can also be

reduced to hydroquinone ansamycins by quinone

oxidoreductase, which is more active in the

inhibition of heat-shock protein 90 (Guo et al.

2006). In the present study, dihydroherbimycin A

had much higher DPPH scavenging activity than

herbimycin A, its oxidized form. Thus, hydroqui-

none ansamycins appears to have enhanced bio-

logical activities as compared to benzoquinone

ansamycin. Therefore, to the best of our knowl-

edge, the strong antioxidant activity of dihydro-

herbimycin A described in the present study has

not been reported elsewhere. In our previous

study, dihydroherbimycin A was reported to be

heat stable and showed a strong inhibitory effect

with moderate cytotoxicity on the lung cancer

cells and leukaemia cells (Chang et al. 2006).

These results suggest that dihydroherbimycin A

could be a good candidate for developing a new

therapeutic agent.

Acknowledgement This work was supported by a grantof joint research organization of the Industry andBiopolytechnic College.

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