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PURIFICATION AND IDENTIFICATION OFBIOACTIVE ANGUCYCLINONES FROMStreptomyces matensis BG5, ISOLATEDFROM THE RHIZOSPHERE OF Rosa indicaL.Imran Sajid a b , Khaled A. Shaaban b & Shahida Hasnain aa Department of Microbiology and Molecular Genetics, University ofthe Punjab, Quaid-e-Azam Campus, Lahore, Pakistanb Institute of Organic and Biomolecular Chemistry, University ofGöttingen, Göttingen, GermanyAccepted author version posted online: 29 Jun 2012.Version ofrecord first published: 07 Dec 2012.

To cite this article: Imran Sajid , Khaled A. Shaaban & Shahida Hasnain (2013): PURIFICATION ANDIDENTIFICATION OF BIOACTIVE ANGUCYCLINONES FROM Streptomyces matensis BG5, ISOLATED FROMTHE RHIZOSPHERE OF Rosa indica L., Preparative Biochemistry and Biotechnology, 43:1, 22-32

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PURIFICATION AND IDENTIFICATION OF BIOACTIVEANGUCYCLINONES FROM Streptomyces matensis BG5,ISOLATED FROM THE RHIZOSPHERE OF Rosa indica L.

Imran Sajid,1,2 Khaled A. Shaaban,2 and Shahida Hasnain1

1Department of Microbiology and Molecular Genetics, University of the Punjab,Quaid-e-Azam Campus, Lahore, Pakistan2Institute of Organic and Biomolecular Chemistry, University of Gottingen,Gottingen, Germany

& A newly isolated strain Streptomyces sp. BG5 was investigated for the production ofbioactive compounds. The strain exhibited broad-spectrum activity against an array of nine testorganisms including gram-positive bacteria, gram-negative bacteria, and fungal and microalgalpathogens, along with a moderate cytotoxic response (28.9% mortality) in a microwell cytotoxicityassay against the brine shrimp Artimia salina. The morphological, physiological, and biochemicalcharacterization of the Streptomyces sp. BG5 strongly suggested it to be a member of the genusStreptomyces. The nucleotide sequence of 16S rRNA gene (1433 pb) of the Streptomyces sp.BG5 (Gene bank accession number EU301836) exhibited high similarity (98%) with Strepto-myces matensis. The large-scale fermentation of Streptomyces sp. BG5 and subsequent extrac-tion, isolation, and purification of the crude extract afforded three pure compounds. The structuresof these compounds were identified as ochromycinone (1a), emycin D (2), and 1-acetyl-b-carbolin(3), based on nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and by compari-son with reference data from the literature.

Supplemental materials are available for this article. Go to the publisher’s online edition ofPreparative Biochemistry and Biotechnology to view the supplemental file.

Keywords angucyclinones, emycin D, 1-acetyl-b-carbolin, ochromycinone, Streptomycesmatensis BG5

INTRODUCTION

Microbial natural products have served us well in combating infectiousdiseases and have revolutionized the medicine. The increased developmentof resistance to the conventional antibacterial, antifungal, and antitumor

Address correspondence to Imran Sajid, Department of Microbiology and Molecular Genetics,University of the Punjab, Quaid-e-Azam Campus, Lahore 54590, Pakistan. E-mail: imran.mmg@pu.edu.pk

Preparative Biochemistry & Biotechnology, 43:22–32, 2013Copyright # Taylor & Francis Group, LLCISSN: 1082-6068 print/1532-2297 onlineDOI: 10.1080/10826068.2012.693897

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agents has been challenged by the discovery of many new candidates.[1] Inthis perspective, microbial secondary metabolites remain the most impor-tant source for discovery of new and potential drug molecules.[2] A largenumber of bacterial and fungal species have been investigated for the pro-duction of bioactive compounds; however, actinomycetes, the filamentousgram-positive bacteria, are still considered to be the major producers ofpharmaceuticals, agricultural pesticides, and veterinary medicine. Themembers of the genus Streptomyces have continued to provide a larger num-ber and wider variety of new antibiotics than any other genus, suggestingthat a substantial number of Streptomyces species or strains with novel anti-biotic productivity exist in nature. These bacteria produce about 75% ofcommercially and medically useful antibiotics.[2,3] Moreover, approximately60% of the antibiotics developed for agricultural use are isolated from Strep-tomyces spp.[4] In the search for antibiotics, Streptomyces strains have been iso-lated from various types of soils, including rice paddy, lake mud and water,deciduous forest, tropical forest, wasteland, and cave soils.[5–10] Their natu-ral habitat soil is nutritionally, biologically, and physically complex and vari-able, demanding their fast adaptation. As a consequence, they are able toperform a broad range of metabolic processes and to produce an immensediversity of bioactive secondary metabolites. Additionally, theplant-associated streptomycetes, specifically endophytes and those isolatedfrom rhizospheres of different plants, have shown enormous potential toproduce novel compounds, which are useful for the control of bacterialand fungal pathogens.[11–13]

The angucyclinones antibiotics have been isolated from several strainsof Streptomyces; they exhibit a broad spectrum of pharmacological proper-ties, including antiviral, antifungal, antitumor, and enzyme inhibitoryactivity.[14,15] This group of antibiotics is, after the tetracyclines and anthra-cyclines, the third class of natural antibiotics featuring a carbotetracyclicskeleton.[16] They include naturally occurring quinines[17] having an angu-lar tetracyclic framework, which are believed to be biosynthesized from adecaketide derivative.[18] Ochromycinone (1a), first isolated by Bowieand Jhonson[19] from the strain of Streptomyces, is one of the simple and use-ful angucyclinones; it has been reported to exhibit selective activity againstHelicobacter pylori, the major cause of stomach ulcers and duodenal ulcer,with low activity against other common bacteria.[20]

In this study we isolated an actinomycete strain designated asStreptomyces sp. BG5 from a rhizospheric soil sample collected from the rosefields at province Punjab, Pakistan. In prescreening studies the strainStreptomyces sp. BG5 exhibited a promising broad-spectrum antimicrobialprofile and an impressive metabolic fingerprint by showing differentcolored spots on thin layer chromatography (TLC) plates after stainingwith spraying reagents (Ehrlich’s reagent and anisaldehyde=sulfuric acid)

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and by high-performance liquid chromatography–mass spectroscopy(HPLC-MS) analysis. This suggested it to be a talented candidate for thepreparative screening. We reported here the isolation, characterization,and taxonomic status of the strain Streptomyces sp. BG5, along with the fer-mentation, isolation, purification, and structure elucidation of its activemetabolites.

EXPERIMENTAL

Isolation and Identification of Streptomyces sp. BG5

The Streptomyces sp. BG5 was isolated from the soil sample collectedfrom the rhizosphere of rose plant (Rosa indica L.) on Casein-KNO3 agar(glycerol 10 g, KNO3 2 g, casein 0.3 g, NaCl 2 g, K2HPO4 2 g, MgSO4 � 7H2OO 0.05 g, CaCO3 0.02 g, FeSO4 � 7H2O 0.01 g, agar 18 g in 1 L, cyclohexi-mide 50mg=ml as an antifungal agent). The purified culture of Streptomycessp. BG5 was characterized morphologically, microscopically, biochemically,and physiologically and by 16S rRNA gene sequencing. Cultural character-istics were observed during the incubation at 28�C for 21 days on M2

medium (malt extract 10 g, yeast extract 4 g, glucose 4 g, in 1 L distilledwater). For cell shape, arrangement of hyphae, length of hyphae, branch-ing pattern, and chains of arthrospores, 7-day incubated cultures at 28�Cwere observed under the microscope (Leica 4000 DM). The biochemicaland physiological characterization includes determination of optimalgrowth temperature, formation of melanin, utilization of nine differentsugars as carbon source, utilization of organic acids, utilization of oxalates,hydrolysis of urea, and hemolysis. For 16S rRNA gene sequencing,high-molecular weight chromosomal DNA of the isolate was prepared fromM2 grown mycelia following the methods described by Kieser et al.[21] Poly-merase chain reaction (PCR) amplification of the 16S rRNA gene of strainswas performed using two primers: P1, 50-AGAGTTTGATCATGGC-30, andP2, 50-TACCTTGTTACGACTT-30. Approximately 300 ng genomic templateDNA was used with 150 pmol of each primer per 50 mL of reaction volume.Amplification was performed in an automated thermocycler (EppendorfMastercycler) using 1 U Pfu DNA polymerase (Fermentas) and the recom-mended buffer system according to the following amplification profile: 1,94�C, 5 min; 2, 94�C, 1 min; 3, 55�C, 1 min; 4, 72�C, 2 min; 5, 72�C,5 min; 30 cycles. The PCR product was analyzed by agarose gel electro-phoresis and the 1.5-kb DNA fragment was purified by QIAquick gel extrac-tion column (catalogue number 28704, Qiagen, Inc.); the sequencing wasdone using dye-terminator chemistry with Mega-BACE 1000=4000 DNAautomated sequencers (Amersham Bioscience). The obtained 16S rRNAgene sequence data was analyzed using advance BLAST search program

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at the NCBI website: http://www.ncbi.nlm.nih.gov/BLAST. The nucleotidesequence data were deposited to the gene bank, and the gene bankaccession number was obtained.

Fermentation, Isolation, and Purification of the ActiveCompounds

The Streptomyces sp. BG-5 was cultivated as a 20-L shaking culture usingM2 medium (malt extract 10 g, yeast extract 5 g, glucose 5 g, in 1 L tapwater), and the pH was adjusted to 7.8 with 2 N HCl and 2 N NaOH. Themedium was dispensed in eighty 1-L Erlenmeyer flasks with inflections, eachcontaining 250 mL of the M2 broth. The flasks were covered with cottonplugs and were autoclaved (Autoclave: Albert Dargatz Autoclave, volume119 L, working temperature 121�C, working pressure 1.2 kg=cm2). Each ofthe flasks was inoculated separately by an agar plug cut from a well-grownplate of the Streptomyces sp. BG5. The fermentation was carried out at95 rpm on the linear shaker (Infors AG (CH 4103 Einbach) type ITE, labora-tory shaker IKA-shaker type S50 (max. 6000 Upm), for 7 days at 28�C.

The obtained dark pinkish culture broth was harvested and filteredover celite by filter press (Schenk Niro 212 B40) to separate the myceliumfrom the liquid phase. The mycelia cake was extracted with ethyl acetate (3times, 2 L each) and acetone (1 time, 1 L). The water phase was extractedby adsorption on Amberlite XAD-16 resin in a large-size glass column(100� 5 cm) and finally eluted with methanol (4 L). The solutions (ethylacetate 6 L, acetone 1 L, and methanol 4 L) having bioactive metaboliteswere recycled by rotary evaporator (Rotavapor R152), affording 2.60 gcrude extract from the cell mass and 1.75 g crude extract from the waterphase. The mycelial extract (2.60 g) was fractionated on a silica gel column(30 g, 1.5� 50 cm) using a CH2Cl2:MeOH gradient (100% CH2Cl2 (1 L);5% MeOH (0.5 L), 10% MeOH (0.5 L), 20% MeOH (0.5 L), 30% MeOH(0.5 L), 40% MeOH (0.5 L), and 50% MeOH (1 L)) resulting into two mainfractions based on thin-layer chromatography (TLC). Fraction I was ident-ified as fats (0.74 g; Rf¼ 0.95 (CH2Cl2)) based on TLC and sprayingreagents. Fraction II was followed by further purification using preparativeTLC (CH2Cl2) and Sephadex LH-20 (Lipophilic Sephadex, AmershamBiosciences Ltd.; purchased from Sigma-Aldrich Chemie, Steinheim,Germany) in a 86� 2.0 cm column eluted with CH2Cl2:40% MeOH, afford-ing ochromycinone (1a; 28 mg; Rf¼ 0.55 (cyclohexane=50% EtOAc)) inpure form. Similarly, fractionations of water-phase extract on silica-gel col-umn chromatography resulted in two main fractions. Fraction I was alsoidentified as fats (5.1 mg; Rf¼ 0.96 (CH2Cl2)) based on TLC and sprayingreagents, while fraction II was subjected to further purifications usingPTLC (CH2Cl2=5% MeOH) and Sephadex LH-20 (CH2Cl2=40% MeOH),

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which yielded two pure components: emycin D (2; 12.8 mg, Rf¼ 0.55(CHCl3=5% MeOH)) and 1-acetyl-b-carbolin (3; 19.8 mg; Rf¼ 0.9(CHCl3=10% MeOH).

Determination of Antimicrobial Activity and Cytotoxicity of theStrain Streptomyces sp. BG5

The crude extract obtained from 1-L shaking culture of the strainStreptomyces sp. BG5 was screened for its antimicrobial activity and cytotoxicity.The antimicrobial activity was determined by disc diffusion bioassay[22,23]

against a panel of nine test organisms, including Staphylococcus aureus, Bacillussubtilis, Streptomyces viridochromogenes (Tu57), Escherichia coli, Candida albicansand Mucor miehei, Chlorella sorokiniana, Chlorella vulgaris, and Scenedesmus sub-spicatus. Paper disks with diameter of 9 mm were impregnated with 40mLof crude extract solution (crude extract 1mg=mL dissolved in CH2Cl2=MeOH,1:1); the disks were dried under sterile conditions and were placed on thesurface of test plates. The cytotoxicity of the crude extract was determinedagainst brine shrimp (Artimia salina) using a modified microwell cytotoxicityassay[24] as described by Sajid et al.[22]

Spectroscopic Studies

Mass Spectra. The purified fractions were analyzed by electron-impactmass spectroscopy (EI MS) at 70 eV with Varian MAT 731, Varian 311A,AMD-402; ESI MS with Quattro Triple Quadruple mass spectrometerFinigan MAT-Incos 50; and ESI-MS LCQ (Finnigan) and ESI-HRMS withperflurokerosine as standard.

Nuclear Magnetic Resonance (NMR) Spectroscopy. 1H-NMR spectra weremeasured by Varian Unity 300 (300 MHz), Bruker AMX 300 (300 MHz),and Varian Inova 500 (499.8 MHz). Coupling constants (J) are in Hz. 13CNMR spectra were measured by Varian Unity 300 (75.5 MHz) and VarianInova 500 (125.7 MHz). Chemical shifts (d) were measured relative to tetra-methylsilane (TMS) as internal standard.

RESULTS AND DISCUSSION

Taxonomic Studies

The strain Streptomyces sp. BG5 produced pink to reddish substratemycelium and whitish aerial mycelium, along with significantly visiblesoluble pigments that diffuse into the surrounding culture medium. It pro-duced round colonies that were hard and embedded into the agar medium

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with a diameter of 4 mm and exhibited characteristic actinomycetes fila-mentous structure under the microscope (Figure 1, A and B). In physio-logical characterization the strain exhibited growth in the temperaturerange of 20�C to 37�C with an optimum at 28�C. The production of mel-anin was indicated by the appearance of a dark blue to black color in theagar medium. Among the nine different sugars tested as carbon sources,the strain BG5 exhibited positive growth on seven different sugars; however,it could not grow on raffinose and sucrose. Additionally, the isolate exhib-ited positive results in utilization of organic acids, utilization of oxalates,urea hydrolysis, and negative hemolysis tests (Table 1). The comparisonof these characteristics with those of actinomycete species described inBergey’s Manual of Systematic Bacteriology[25] strongly suggested that the iso-late BG5 belongs to the genus Streptomyces. The nucleotide sequence of1433 bp (accession number EU301836) of the 16S rRNA gene of theStreptomyces sp. BG5 was determined in both strands. The alignment of thissequence through matching with reported 16S rRNA gene sequences in thegene bank showed high similarity (98%) to Streptomyces matensis.

Structure Elucidation of the Purified Compounds

The large-scale fermentation of Streptomyces sp. BG5 was performed as a20-L shaking culture using M2 medium on a linear shaker for 7 days. Afterharvesting, cell separation, and solvent extraction, 2.60 g and 1.75 g crudeextracts were obtained from the mycellial cake and liquid phase, respect-ively. The components of the crude extracts were purified using silica gelcolumn, preparative TLC, and gel exclusion chromatography, yieldingthree pure compounds identified as ochromycenone (1a), 28 mg, emycinD (2), 12.8 mg, and 1-acetyl-b-carbolin (3), 19.8 mg (Figure S1, supportinginformation).

FIGURE 1 (a) Colonies of Streptomyces matensis BG5 on M2 agar. (b) Microscopic image (X 500) of Strep-tomyces matensis BG5 (color figures available online).

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Compound 1a was isolated as a yellow solid with medium polarity fromsubfraction II of the mycelial extract, using PTLC (CH2Cl2) followed bycolumn chromatography on Sephadex LH-20 (CH2Cl2=40%MeOH). Itchanges coloration to red when treated with 2 N NaOH, indicating a peri-hydroxy quinine, and gave no color change with anisaldehyde=sulfuricacid spraying reagent. The 1H and 13C NMR as well as mass spectrometry(Figures S2–S5, supporting information), and searching in AntiBase[26]

resulted in the angucyclinone ochromycinone (1a) (Figure 2), which wasfurther confirmed by comparison with authentic sample spectra and datafrom literature. Ochromycinone (1a) is a simple angucyclinone that is

TABLE 1 Cultural and Physiological Characteristics of Streptomyces sp. BG5

Growth pattern Well grown, smoothSubstrate mycelium Pink, reddishAriel mycelium Off-whitishGrowth temperature range 20–37�CProduction of melanin þSoluble pigments þUtilization of organic acids þUtilization of oxalates þHydrolysis of urea þHemolysis �Carbon source utilizationD-Glucose þD-Fructose þL-Arabinose þSucrose �D-Fructose þD-Mannitol þRaffinose �D-Galactose þSoluble starch þGlycerol þ

Note. þ, Positive; –, negative.

FIGURE 2 Chemical structure of ochromycinone (1a, 1b), emycin D (2), and 1-acetyl-b-carbolin (3).

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reported to exhibit selective anti-Helicobacter pylori activity, and with lowactivity against other common bacteria.[19,27] Its methyl ether rubiginoneBl (1b) was isolated and reported from Streptomyces griseorubiginosusQ144-2. Rubiginone Bl (1b) is effective in potentiating the cytotoxicity ofVCR against VCR-resistant P388 leukemia in vitro and in vivo.[28]

Compound 2 was isolated as a pale-yellow solid with medium polarityfrom subfraction II of the water-phase extract, after being subjected to PTLCand column chromatography on Sephadex LH-20 (CH2Cl2=40%MeOH).TLC of compound 2 showed no color change when treated with 2 N NaOHas well as anisaldehyde=sulfuric acid after heating. Based on 1H- and13C-NMR (Figures S6–S9, supporting information), indicating the missingof the two carbonyl moieties of compound 1a in case of 2, as well as the massdifference (Dm=z¼ 2), indicating the rearranging of ring C in compound 2,by searching in AntiBase,[26] emycin D (2) was deduced (Figure 2), whichwas further confirmed by comparison with literature data.[27,29] Emycin D(2) seems to be an unrearranged form of the ring C in ochromycinone(1a) and was isolated previously from Streptomyces cellulosae.[27,29]

As a pale yellow solid, compound 3 was obtained from the same subfrac-tion II of emycin D (2), using PTLC (DCM=5% MeOH) followed by columnchromatography on Sephadex LH-20 (CH2Cl2=40%MeOH). Compound 3has a blue fluorescence under long ultraviolet (UV), and gave a yellowish-green color reaction with anisaldehyde=sulfuric acid. The 1H-NMR spec-trum of compound 3 in CDCl3 showed a broad singlet acidic proton at d10.30 (1H), and two o-coupled doublets each of 1-H at d 8.54 and 8.16,which may belong to a heteroaromatic ring due to their small coupling con-stants (3J¼ 5.1 Hz). An additional 1,2-disubstituted aromatic ring wasobserved due to the presence of four multiplet protons at d 8.15 (5-H),7.62 (7,8-H) and 7.31 (6-H). In the aliphatic region, it showed a singletof an aromatic bounded methyl group at d 2.96, which could be presentin peri position to the carbonyl group, or may be present as NCH3. TheEI and ESI-MS spectra determined the mass of 3 as 210 Da (Figures S10–S12, supporting information). Based on the revealed 1H-NMR and MS dataalong with the search in AntiBase,[26] 1-acetyl-b-carbolin (3) was deduced(Figure 2), which was further confirmed by comparison with the authenticsample that is frequently isolated in our laboratory.[30]

Antimicrobial Activity and Cytotoxicity

The Streptomyces sp. BG5 exhibited promising antimicrobial activityagainst an array of nine test organisms including gram-positive bacteria suchas Staphylococcus aureus, Bacillus subtilus, and Streptomyces viridochromogens(Tu57), gram-negative bacteria such as Escherichia coli, fungal pathogenssuch as Candida albicans and Mucor miehei, and microalgal strains such as

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Chlorella vulgaris and Chlorella sorokiana; however, there was no antimicrobialactivity against Scendesmus subspicatus (Table 2). Additionally, in cytotoxicitytesting, the crude extract obtained from the Streptomyces sp. BG5 exhibited28.9% mortality against Artimia salina (Table 2).

CONCLUSION

The preparative screening of isolate Streptomyces matensis BG5 yieldedthree pure compounds, which were identified based on mass spectrometry(ESI-MS), 1H- and 13C NMR analysis, and database search as ochromyci-none (1a), emycin D (2), and 1-acetyl-b-carbolin (3). The study demon-strates the enormous potential of rhizospheric Streptomyces isolates toproduces unique compounds, and it can be inferred from the results thatthis ecological niche may contain unique Streptomyces strains and should becontinuously explored for the isolation of lead antimicrobial compounds.

ACKNOWLEDGMENTS

We are thankful to Prof. Dr Hartmut Laatsch, Institute of Organic andBiomolecular Chemistry, University of Gottingen, Germany, for providingfermentation, NMR, and MS analysis facilities. Financial support of thiswork by a grant from the Higher Education Commission (HEC) of Pakistanunder IRSIP is gratefully acknowledged.

SUPPORTING INFORMATION AVAILABLE

Workup procedure scheme of the isolated compounds from Streptomycessp. BG5, and NMR and MS spectra of ochromycinone (1a), emycin D (2),and 1-acetyl-b-carbolin (3).

TABLE 2 Antimicrobial Activity and Cytotoxicity of the Extractfrom Streptomyces sp. BG5

Test organism Zone of inhibition (mm)

Staphylococcus aureus 16Bacillus subtilus 20Streptomyces viridochromogens (Tu 57) 27Escherichia coli 19Candida albicans 27Mucor miehei 11Chlorella vulgaris 12Chlorella sorokiana 12Scendesmus subspicatus 0Percent mortality (Artimia salina) 28.9

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