Isolation, Purification, and Characterization of Antimicrobial Substances from Endophytic Actinomycetes

Isolation and purification of antimicrobial active substance produced by marine Actinomycetes has been carried out.  Marine sediment samples were obtained from six different places at Banten West Coast.  Isolation was conducted using two pretreatment methods,  acid and heat shock pre-treatment.  A total of 29 Actinomycetes isolates were obtained from the various sediment samples collected, then tested for antimicrobial activity against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC25923, Pseudomonas aeruginosa ATCC27853, Bacillus subtilis ATCC 66923, Candida albicans BIOMCC00122, and Aspergillus niger BIOMCC00134.  Among the isolates, isolate A11 was the most activity to Gram-positive and Gram-negative bacteria, and morphological observation and identification using 16S rRNA showed that the isolate was similar to Streptomyces sp.  Production of active compound from A11 isolate used yeast peptone medium.  Purification of active compounds was carried out using silica-gel-column chromatography and preparative HPLC.  A single peak of active compounds was detected by HPLC, which showed a retention time of 8.35 min and maximum absorbance in UV visible at 210 nm and 274.5 nm respectively.

Endophytic actinomycetes from medicinal plants were rarely reported as compared to those from soil and marine sources. The present results correlate the presence of endophytic actinomycetes in medicinal plants by isolating them from three medicinal plants i.e. Ocimum basillicum, Withania somnifera and Rauvolfia tetraphylla. In the present work surface sterilization method and media was standardized and 32 endophytic actinomycetes were isolated from three medicinal plants. We assessed the efficiency of four different surface sterilization methods and four medium for isolation of endophytic actinomycetes. The method with calcium hypochlorite, sodium hydrogen carbonate and sodium azide (New method) used was more effective in eliminating epiphytic microorganisms. Among four tested media starch casein agar (SCA) found to be best media for the isolation of endophytic actinomycetes. We succeeded in obtaining fast and luxurious growth of pure culture of endophytic actinomycetes on modified ISP-4 (M) (inorganic salt solution agar) in comparison with standard ISP-4(S). Preliminary antibacterial assay of all isolates were tested by confrontation test. The secondary screening of selected isolates were tested by disc diffusion test by using ethyl acetate extract which showed broad spectrum antibacterial activity against test human pathogens. Based on the morphological and phenotypic characters 12 isolates were identified as Streptomyces spp. Among 12 isolates A3 as a representative was characterized by SEM and identified by 16SrRNA analysis as Streptomyces flavoviridis A3WK, it showed significant antibacterial activity against test human pathogen. This is the first report of successful isolation of endophytic actinomycetes from the said medicinal plants, for using newly formulated surface sterilization method and new comparative study on ISP-4 medium.

The polar extract of Mangosteen (Garcinia mangostana) fruit pericarp obtained by cellulase assisted ethanol extraction has strong antioxidant activity, giving an average 2,2 diphenyl-1 pykrylhydrazyl (DPPH) radical scavenging IC50 of 13.9 µg/mL. In order to elucidate the chemical component from this extract that is responsible for the high antioxidant activity, fractionation of the extract should firstly be performed. In this paper we show results of preparative fractionation of the polar extract by two methods, namely preparative Thin Layer Chromatography (PTLC) and preparative High Performance Liquid Chromatography (PHPLC). PTLC used Silica Gel G60 plates, with a hexane:ethyl acetate (6:4) eluent. PHPLC was a reverse phase method, using C18 column and water:acetonitrile gradient elution. 4 fractions from PTLC and 6 fractions from PHPLC were collected and their antioxidant activity analyzed. Both methods gave separated fractions with lower antioxidant activity than the unfractionated original crude extracts, showing that the strong antioxidant activity of Mangosteen pericarp polar extracts maybe due to the concerted synergetic effect of several compounds, rather than a single isolated compound. It also shows the high degree of difficulty in separating mangosteen pericarp polar components having antioxidant activity for further structural analysis.

2.7 High performance liquid chromatography (HPLC) and related techniques: 2.7.2. Isolation of impurities by (semi)preparative HPLC

Isolation of recombinant hirudin by preparative high-performance liquid chromatography

Microorganisms associated with medicinal plants are of interest as the producers of important bioactive compounds. To date, the diversity of culturable endophytic actinomycetes associated with medicinal plants is in its initial phase of exploration. In this study, 42 endophytic actinomycetes were isolated from different organs of seven selected medicinal plants. The highest number of isolates (n = 22, 52.3%) of actinomycetes was isolated from roots, followed by stems (n = 9, 21.4%), leaves (n = 6, 14.2%), flowers (n = 3, 7.1%), and petioles (n = 2, 4.7%). The genus Streptomyces was the most dominant among the isolates (66.6%) in both the locations (Dampa TRF and Phawngpuii NP, Mizoram, India). From a total of 42 isolates, 22 isolates were selected for further studies based on their ability to inhibit one of the tested human bacterial or fungal pathogen. Selected isolates were identified based on 16S rRNA gene analysis and subsequently the isolates were grouped to four different genera; Streptomyces, Brevibacterium, Microbacterium, and Leifsonia. Antibiotic sensitivity assay was performed to understand the responsible antimicrobials present in the isolates showing the antimicrobial activities and revealed that the isolates were mostly resistant to penicillin G and ampicillin. Further, antimicrobial properties and antibiotic sensitivity assay in combination with the results of amplification of biosynthetic genes polyketide synthase (PKS-I) and non-ribosomal peptide synthetase (NRPS) showed that the endophytic actinomycetes associated with the selected medicinal plants have broad-spectrum antimicrobial activity. This is the first report of the isolation of Brevibacterium sp., Microbacterium sp., and Leifsonia xyli from endophytic environments of medicinal plants, Mirabilis jalapa and Clerodendrum colebrookianum. Our results emphasize that endophytic actinomycetes associated with medicinal plants are an unexplored resource for the discovery of biologically active compounds.

BackgroundBrazilian propolis type 6 (Atlantic forest, Bahia) is distinct from the other types of propolis especially due to absence of flavonoids and presence of other non-polar, long chain compounds, but presenting good in vitro and in vivo antimicrobial activity. Several authors have suggested that fatty acids found in this propolis might be responsible for its antimicrobial activity; however, so far no evidence concerning this finding has been reported in the literature. The goals of this study were to evaluate the antibacterial activity of the main pure fatty acids in the ethanolic extract and fractions and elucidate the chemical nature of the bioactive compounds isolated from Brazilian propolis type 6.MethodsBrazilian propolis type 6 ethanolic extract (EEP), hexane fraction (H-Fr), major fatty acids, and isolated sub-fractions were analyzed using high performance liquid chromatography (HPLC), high resolution gas chromatography with flame ionization detection (HRGC-FID), and gas chromatography-mass spectrometry (GC-MS). Three sub-fractions of H-Fr were obtained through preparative HPLC. Antimicrobial activity of EEP, H-Fr, sub-fractions, and fatty acids were tested against Staphyloccus aureus ATCC 25923 and Streptococcus mutans Ingbritt 1600 using minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC).ResultsEEP and H-Fr inhibited the growth of the microorganisms tested; nevertheless, no antimicrobial activity was found for the major fatty acids. The three sub-fractions (1, 2, and 3) were isolated from H-Fr by preparative HPLC and only sub-fraction 1 showed antimicrobial activity.Conclusiona) The major fatty acids tested were not responsible for the antimicrobial activity of propolis type 6; b) Sub-fraction 1, belonging to the benzophenone class, was responsible for the antimicrobial activity observed in the present study. The identification of the bioactive compound will improve the development of more efficient uses of this natural product.

Endophytic actinomycetes from medicinal plants produce a wide diversity of secondary metabolites (SM). However, to date, the knowledge about endophytes from Brazil remains scarce. Thus, we analyzed the antimicrobial potential of 10 actinomycetes isolated from the medicinal plant Vochysia divergens located in the Pantanal sul-mato-grossense, an unexplored wetland in Brazil. Strains were classified as belonging to the Aeromicrobium, Actinomadura, Microbacterium, Microbispora, Micrococcus, Sphaerisporangium, Streptomyces, and Williamsia genera, through morphological and 16S rRNA phylogenetic analyzes. A susceptibility analysis demonstrated that the strains were largely resistant to the antibiotics oxacillin and nalidixic acid. Additionally, different culture media (SG and R5A), and temperatures (28 and 36°C) were evaluated to select the best culture conditions to produce the active SM. All conditions were analyzed for active metabolites, and the best antibacterial activity was observed from metabolites produced with SG medium at 36°C. The LGMB491 (close related to Aeromicrobium ponti) extract showed the highest activity against methicillin-resistant Staphylococcus aureus (MRSA), with a MIC of 0.04 mg/mL, and it was selected for SM identification. Strain LGMB491 produced 1-acetyl-β-carboline (1), indole-3-carbaldehyde (2), 3-(hydroxyacetyl)-indole (4), brevianamide F (5), and cyclo-(L-Pro-L-Phe) (6) as major compounds with antibacterial activity. In this study, we add to the knowledge about the endophytic community from the medicinal plant V. divergens and report the isolation of rare actinomycetes that produce highly active metabolites.

A method using preparative and analytical high-performance liquid chromatography (HPLC) is proposed for the assay of 25-hydroxyvitamin D3 (25-OH-D3) in human plasma. A constant volume (0.2--2.0 ml) of a plasma sample was saponified. The unsaponifiable matter was first applied to preparative HPLC using a column of the straight-phase type (Zorbax SIL) in order to separate a 25-OH-D3 fraction from lipophilic concomitants giving ultraviolet-absorbing noise. Then, the separated 25-OH-D3 fraction was applied to analytical HPLC using a column of the reversed-phase type (Zorbax ODS) in order to measure the content of 25-OH-D3 from the peak height. This is a revised method from Jones (1978): Clin. Chem., 24, 287--298). The results showed that the clean-up procedure by the first preparative HPLC was successfully performed because the peak corresponding to 25-OH-D3 on the chromatogram of the second analytical HPLC was not disturbed by any other interfering peaks. Moreover, recovery through the whole procedure was satisfacotry (about 100%) and the procedures of saponification and isolation of the unsaponifiable matter diminished the overload to the columns. These are the revised points of Jones' method. When two determinations were performed on 12 samples of plasma taken from normal adults in October, the values were 22.6 +/- 4.8 and 21.0 +/- 3.6 (mean +/- SD) ng/ml, respectively.

A novel approach to auto‐purification of a wide variety of organic compounds is described. It is based on normal‐phase (NP) gradient high performance liquid chromatography (HPLC), performed on a 2 × 15 cm cyano column hyphenated with atmospheric pressure chemical ionization (APCI) source, and a single quad mass spectrometer (MS). A commercially available preparative HPLC–MS system, equipped with mass‐directed fraction collection capabilities, has been successfully used for NP purification of neutral, acidic, and basic pharmacologically active compounds. Samples of 10–100 mg were chromatographed in gradients of methanol in hydrophobic organic solvents, and collected using generic chromatographic, detection and fraction collection experimental parameters. “Smart” collection—one component–one collection vessel—with 90–95% recovery, was achieved by controlling injection volume and gradient slope and by adding acetic acid and diethylamine to the mobile phase to keep peak elution volumes below 50 mL. Auto‐collection of a solute was based on the main ion in its APCI–MS spectrum. The technique described has been also successfully used for chiral preparative HPLC applications and purification of non‐UV‐active compounds.

Sunaryanto R, Marwoto B (2010) Marine Actinomycetes screening of Banten West Coast and their antibiotics purification. Biodiversitas 11: 176-181. Isolation and purification of active compounds produced by marine Actinomycetes has been carried out. Marine sediment samples were obtained from six different places at Anyer, Banten West Coast in October 20, 2007. Isolation was carried out using two methods pretreatments, acid treatment and heat shock treatment. A total of 29 Actinomycetes isolates were obtained from the various sediment samples collected, then tested for antimicrobial test against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC25923, Pseudomonas aeruginosa ATCC27853, Bacillus subtilis ATCC 66923, Candida albicans BIOMCC00122 and Aspergillus niger BIOMCC00134. Identification of potential isolate was carried out using 16S rRNA. Purification of active compound was carriedout using silica gel column chromatography and preparative HPLC. Result of this research showed that isolate A11 produced the most active compound against Gram-positive and Gram-negative bacteria. Morphology and identification test using 16S rRNA gen showed that isolate A11 is Streptomyces sp. Production of active compound from isolate A11 used yeast peptone medium. The single peak of active compound was detected by HPLC and showed retention time on 8.35 min and maximum absorbance UV visible of antibiotic was 210 nm and 274.5 nm. Active purified compound showed inhibition activity to Gram-positive and Gram-negative bacteria. Minimum inhibitory concentration (MIC) to E. coli ATCC 25922 was 27 μg/mL, P. aeruginosa ATCC 27853 68.7 μg/mL, S. aureus ATCC 2592380.2 μg/mL, and B. subtilis ATCC 66923 73.7 μg/mL.Key words: marine Actinomycetes, isolation, screening, antimicrobial activity, minimum inhibitory concentration

Actinomycetes is the main source of antibiotics and endophytic actinomycetes from medicinal plants has considerable potential as like the host. The aim of this research is to identify rare actinomycetes isolated from Neesia altissima and to screen their antagonistic activity against diarrhea-causing bacteria in order to find new potential secondary metabolites. Samples of N. altissima were collected from mount Halimun-Salak National Park. Endophytic actinomycetes were isolated from roots of N. altissima by surface sterilization method. Screening of antagonistic activity was conducted against five diarrhea-causing bacteria such as Bacillus cereus ATCC 10876, Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 25241, Shigella flexneri ATCC 12022, and Staphylococcus aureus ATCC 25923 by using diffusion disc methods. The endophytic actinomycete showed in vitro antibacterial activity against four diarrhea-causing bacteria, except the B. cereus ATCC 10876. The phylogenetic tree generated from 16S rRNA sequence showed that sequence of endophytic actinomycetes isolates nested in the clade belonging to the genus Nonomuraea. Sequence of UICC B-94 formed a monophyletic clade with N. jabiensis strain A4036 and N. rubra strain AC 615. Therefore, it is named as Nonomuraea sp. strain UICC B-94.

Endophytic microorganisms are rich sources for drug discovery. Isolation of actinomycetes from the surface-sterilized tissues of 12 medicinal plants in Hainan, China, was carried out using different media of American Type Culture Collection (ATCC) 172 agar, Gauze’s No. 2 agar, glucose-asparagine agar, humic acid-vitamin agar, and starch-casein-mineral salts agar. Of the 280 isolates recovered, 154 were from roots, 73 from stems and 53 from leaves. Bioactivity test of crude fermentation extracts were performed on all the isolates. About 41.1% of the extracts showed activity against liver cancer cell SMMC-7721, 19.3% against Candida albicans ATCC10231, and 10.0% against Staphylococcus aureus ATCC 51650. In addition, metabolites of nine isolates inhibited caspase 3, a protein related to neurodegenerative diseases, and three inhibited protein tyrosine phosphatase 1B (PTP1B), a protein related to diabetes. Based on their phenotypic and genotypic characteristics, endophytic actinomycetes were classified to actinobacterial genera including Streptomyces , Micromonospora , Nocardia, Nonomuraea and Amycolatopsis spp. The high bioactivity percentage, broad bioactivities and the novel taxa of the isolated endophytic actinomycetes presented their potential in pharmaceutical utilization. Key words: Filamentous actinomycetes, medicinal plants, antimicrobial activity, anti-tumor cell activity, protein tyrosine phosphatase 1B, caspase 3.

The isolation and purification of glucoraphanin from broccoli seeds by solid phase extraction and preparative high performance liquid chromatography

The zopiclone was obtained in a 99.5% pure form, and in good yield by preparative scale high performance liquid chromatography. Purification was from a complex reaction mixture. A reversed phase preparative scale HPLC column is used. This work demonstrates how kilogram quantities of pure material can be conveniently obtained using preparative HPLC, even when the components in a mixture are difficult to resolve.