Fractionation and structural identification of antibiotic activity substances from Streptomyces herbaricolor HNS 2-2

Four groups of antibiotic activity compounds were separated and purified from the ethyl acetate fraction of a broth culture of Streptomyces herbaricolor HNS2-2. Trace analyses were performed and anti-microbial activities were determined. The antibiotic activity compounds were identified as quercetin dehydrate 1, p-hydroxyphenyl 2, 4’-hydroxyflavanone 3, and 3-hydroxyflavone 4 based on spectroscopic data. Compounds 1 4 showed significant antimicrobial activities against Staphylococcus aureus and the tobacco mosaic virus in vitro, suggesting their potential agricultural and medical applications.


INTRODUCTION
Filamentous actinomycetes, especially the members of the genus Streptomyces, are very important sources of different natural metabolites.These organisms possess a broad range of biological activities, such as antibiotic (e.g, streptomycin and oxytetracycline), antitumor (e.g., bleomycin and mitomycin), antifungal (e.g., nystatin and antimycin A), anti-tubercular (aprazamycin B and thiolactomycin), as well as pesticide (e.g., jinggangmycin and liuyangmycin) [1][2][3].Streptomyces can also produce immunomodulators (e.g., tacrolimus and pimecrolimus), organic acids, amino acids, vitamins, steroids, and enzymes, as well as inhibit enzymatic activities [4,5].Some can engage in different types of biological activities at the same time, or in special biological activities by metabolic regulation [6][7][8].Previous studies on the biologi-cal activities of Streptomyces are diverse.However, they are still important potential resources for discovering novel biologically active sources.
In the present study, the ethyl acetate extract of the broth culture of Streptomyces herbaricolor HNS2-2 showed significant antimicrobial activity against Staphylococcus aureus and the tobacco mosaic virus (TMV) in vitro [9,10].The isolation, purification, and structural identification of the antibiotic activity compounds in the extract were reported.

Microorganisms
S. herbaricolor HNS2-2 was isolated in our laboratory from a soil sample obtained from the Shenlongjia Nature Reserve in Sichuan province, China.The strain was maintained in 40% glycerol microtubes at -20˚C.The identity of S. herbaricolor was confirmed based on the morphology results of scanning electronic microscopy, culture characteristics, chemotaxonomic tests, and 16S rRNA analysis [9].The other microorganisms used in the present study were S. aureus and TMV. S. aureus was maintained on nutrient agar slants at 4˚C, and a crude extract of TMV was prepared and stored in our laboratory.

Culture Conditions for S. herbaricolor HNS2-2 and Preparation of Crude Antibiotic Activity Substances
The culture medium for S. herbaricolor HNS2-2 was the same as that reported in literature [9].First, the inoculum prepared by 6d-old S. herbaricolor HNS2-2 on a Gause No. 1 agar slant was inoculated in 300 mL Erlenmeyer flasks, which contained 30 mL of seed medium.The flasks were placed in a rotary shaker at 28˚C and incubated at 180 rpm for 18 h.About 3 mL of seed-incubated liquid (inoculation number = 6%) was trans-ferred into a 500 mL Erlenmeyer flask containing 100 mL of fermented liquid medium (medium volume = 20%).After 7 d of growth at 28˚C on a rotary shaker at 220 rpm, the crude fermentation broth was acidized with HCl to pH 3.5 at 55˚C for 2 h, and thoroughly filtered to separate the broth filtrate and mycelial cake.The broth supernatant was concentrated five times in an evapo-concentration decompressor at 55˚C.The extract containing crude antibiotic activity substances was obtained by extraction and evapo-concentration.
Fractions 48 -57 were recrystallized in acetone and separated by Sephadex LH-20 eluted with acetone.The products were collected to obtain pure compound 3.
The chemical structures of the pure compounds were confirmed via 1 H NMR and 13 C NMR. Tetramethylsilane (TMS) was used as the internal standard; CDCl 3 and acetone-d6 were the solvents.The NMR spectra were compared with relevant references, and the identities of the compounds were further confirmed.

Antimicrobial Activity Test
The method of trace analyses and determination was that compounds from the isolation of antibiotic activity substances were tested for in vitro activity against S. aureus by the cup-plate method and inhibition zone determination, keep the positive compound, give up the negative compound, step by step.A 75% alcohol solution was used as a positive control, and aseptic normal saline was used as a negative control.
The antimicrobial activities of the pure compounds were determined as follows.The compounds were initially dissolved in acetone at a concentration of 1% (diluted 100 times) and mixed with equal volumes of TMV crude extract for 30 min.Stramonium was then introduced into the mixer via sap inoculation.The inhibiting effects of the compounds on Stramonium were recorded and compared.The compounds were also tested for in vitro activity against S. aureus by tube broth culture and spread plate colony calculation.Aseptic normal saline and acetone were used as negative controls.

Fractionation and Structural Identification of Antibiotic Activity Substances
Based on 1 H NMR and 13 C NMR data, the following structural information were obtained.

Pure Compound 1
Yellow powder, 1 H NMR (500 MHz, (CD 3 ) 2 CO):  H The 1 H NMR and 13 C NMR data as well as the chemical structure were identical with those of quercetin dehydrate [11,12].Hence, the pure compound was considered to be a monomer of quercetin dehydrate.
The 1 H NMR and 13 C NMR data as well as the chemical structure of the compound were identical with those of p-hydroxyphenyl [13], the chemical structure of which is given in Figure 1(b).

Antimicrobial Activity of the Pure
Compounds against S. aureus and TMV Table 1 shows that the inhibition of pure compound 1 was stronger than those of the other pure compounds, the inhibition rate of lesion wan 89.12%.Nevertheless, pure compound 2 exhibited destructive effects on Stramonium, resulting in death.
Figure 2 shows that the compounds exhibited anti-S.aureus activity.The number of S. aureus in a petri dish was obviously lower in the test group (left) than the control group(upper right was aseptic normal saline negative controls and lower right was acetone negative controls).The number of S. aureus in a petri dish from pure compound 1 to 4 were (12.5 ± 0.50) CFU/petri dish, (3.00 ±

DISCUSSION
The current study showed that S. herbaricolor could produce the flavonoids quercetin dehydrate, p-hydroxyphenyl, 4'-hydroxyflavanone, and 3-hydroxyflavone, and the compounds have significant bioactivities.These report has never been previously described.
More than 4000 varieties of flavonoids have been identified in nature, especially in plants (e.g., fruit, vegetables, grains, bark, roots, stems, flowers, tea, and wine) [17].Flavonoids have a wide range of functions.They play important roles as signal molecules for the induction of nod genes [18][19][20], serve as precursors for the production of major phytoalexins during plant-microbe interactions [21,22], and inhibit pathogen attack [23,24].However, flavonoids, being naturally produced by plants, are very complex.Some are also produced in very low quantities that cannot meet human requirements.Obtaining sufficient amounts of natural flavonoids will lead to the depletion of natural resources.
Actinomyces species have the advantages of rapid growth, precisely controllable growth condition, relatively easy to be genetically manipulated, abundant supply of substrates, and applicable in the mature liquid submerged fermentation technique, etc. Flavonoid production in the microorganism starter S. herbaricolor is feasible and can overcome the above mentioned problems.Further research on the determination, anabolism, and fermentation of S. herbaricolor antibiotic activity flavonoids will benefit their industry-scale production and application.

Table 1 .
Antimicrobial activity of the pure compounds against TMV.
*The Stramonium died after sap inoculation; ** Different letters showed significant difference at 0.05 level.