Secondary Metabolites with Antimycobacterial Activities from One Actinobacteria: Herbidospora yilanensis

The cultivation of one actinobacteria strain, Herbidospora yilanensis, was isolated from sediment samples collected from Yilan County City in Taiwan, resulting in the isolation of five previously undescribed compounds: herbidosporayilanensins A–E (1–5), and four compounds isolated from nature for the first time: herbidosporayilanensins F–I (6–9). Their structures were elucidated by spectroscopic analyses, including 1D- and 2D-NMR experiments with those of known analogues, and on the basis of HR-EI-MS mass spectrometry, their antimycobacterial activities were also evaluated.


Introduction
Actinobacteria have the ability to produce a variety of physiologically active products, so they play a very important role in the food industry, pharmaceutical industry, and environmental protection. Our team has also separated and collected actinobacteria resources from all over Taiwan and various environments over the years, except for common chains. In addition to molds, there are many rare genera. Based on the concept of "new species and new compounds", it is hoped that special compounds can be found from these new species. In recent years, studies have also found that these new species of actinobacteria can produce many active secondary metabolites. In order to further explore the efficacy of different strains of actinobacteria and expand the application range of actinobacteria, this study focused on a strain from central Taiwan, and the new strain H. yilanensis isolated from the sediments of northern rivers and lakes is very novel and worthy of in-depth research and discussion.
Actinobacteria are widely distributed in nature, and they are very useful in the pharmaceutical industry due to their seemingly unlimited capacity to produce secondary metabolites with diverse chemical structures and biological activities [1][2][3]. They are Gram positive, free-living saprophytic bacteria that are widely distributed in soil, water, and colonizing plants. Actinobacteria inhabitants have been identified as one of the major groups of

Structure Elucidation of Compounds
Compound 1 was isolated as an optically active oil with α] 25 D : + 10.6 (c 0.071, CHCl 3 ). The HR-EI-MS gave an [M] + ion peak at m/z 410.2667 (calcd 410.2666), consistent with a molecular formula of C 23 H 38 O 6 . The IR absorption bands suggested the presence of a COOH and OH (2500-3400 cm −1 ), a terminal double bond (1664, 858 cm −1 ), and a CO (1698 cm −1 ) group. The 13 C-NMR (DEPT) spectrum of 1 (Table 1) exhibited 23 signals: four Me, eight CH 2 (one being oxygenated and one exocyclic double bond), and four CH groups (two being oxygenated), as well as five quaternary C-atoms, including a C=O function (δ C 182.6). Five indices of hydrogen deficiency (IHD) were determined from the molecular formula. After subtracting one double bonds and carboxyl groups, the remaining unsaturation degree is 3, and then the three typical methyl groups at δH 1.25 (s), 1.22 (s), and 0.59 (s) in 1H-NMR, it was deduced that compound 1 is a diterpenoid containing two six-membered labdane-type structures, and the basic structure of labdane is a C20 compound, so it was deduced that it contains a three-carbon derivative functional groups, which contain the two methyl signals δH 1.39 (s) and 1.35 (s).
From the 13 C-NMR spectrum at δ C 108.4, a signal was found and confirmed by DEPT as a quaternary carbon. Furthermore, the HMBC spectrum showed that H-21/H-23 were correlated with C-22, along with no COSY correlations between H-21 and H-23, and established that two methyl groups are located at C-22 (Figure 1). The quaternary carbon (δ C 108.4) had such a low field that it must be a conjugated double bond, but this quaternary carbon did not have other conjugated double bonds and was connected to two methyl groups. Therefore, it was deduced that this quaternary carbon is an acetonide functional group with two oxygens. In combination with the HMBC correlations of H-15, H-21, and H-23 to C-22, this led to the establishment of acetonide being located on C-14 and C-15 instead of the C-12/C-13 or C-13/C-14 position. From the HSQC spectrum, it can be seen that the proton signal corresponding to δ C 74.4 is δ H 3.56 (1H, d, J = 10.5 Hz), and it has an HMBC correlation with C-9, C-11, and C-13. It was inferred that δ C 74.4 is C-12, and that C-12 is connected to a hydroxyl group (-OH). In addition, δ C 73.9 has an HMBC correlation with H-12, H-14, and H-16 and from the IR, it was found that there was an OH signal at 3418 cm −1 , so it was inferred that δ C 73.9 is C-13, which is connected to a hydroxyl group (-OH), and δ H 1.25 (s) is correlated to C-12, C-13, and C-14, so this methyl group was deduced as being located on C-13. From the NOESY results, H-11 is correlated to H-20, it can be confirmed that C-20 is in the axial. The presence of NOESY correlations between H-11 and H-20, between H-18 and H-3/5, and the absence between H-18 and H-20 indicated that Me-18 was α-orientated at C-4 in equatorial ( Figure 2).
A major difference was that a 4-acetylcyclohex-1-en-1-yl group at C-12 [δ H 2.07/2.  C-7), which is a conjugated carbonyl group. UV absorption at 231 nm showed that it should be adjacent to the benzene ring. The carbon signal at δ C 175.29 (C-11) is a characteristic signal of the γ-lactone, which forms a five-ring lactone with the remaining signals at δ C 69.19 (C-9), 31.06 (C-10), and 41.97 (C-8). This is the framework of C6-C5 type. Compound 6 showed dextrorotatory optical activity with α] 25 D = +6.0 (c 0.02, CHCl 3 ). By comparing the reference to the R-configuration of (R)-(+)-4-(3-Methoxyphenyl)methylbutyrolactone [12] ( α] 20 D = +5.5, CHCl 3 ), the absolute configuration at C-8 should be tentatively proposed as R. The 1 H-and 13 C-NMR (Table 2), COSY (Figure 2), NOESY (Figure 3), HSQC and HMBC (Figure 2) experiments confirmed the structure as (R)-4-(benzo[d][1,3]dioxole-5-carbonyl)dihydrofuran-2(3H)-one, and was designated herbidosporayilanensin F. Compound 6 was first isolated from a natural source, though it has never been synthesized [12].  0712)). The IR spectrum showed absorption bands for a hydroxyl group at 3422 cm −1 and a butyrolactone group at 1769 cm −1 , along with a resonance signal in the 13 C-NMR spectrum at δ C 195.0. Moreover, the 13 C-NMR spectrum, in combination with DEPT and HSQC experiments, showed signals for two oxymethines at δ C 85.9 (C-5), 68.8 (C-4), one methylene at δ C 39.5 (C-3), and one ethyl at δ C 9.9 (C-7 and 21.5 (C-6), respectively. All the above data indicated that 8 was a dihydrofuran-2-one derivative (= butyrolactone)). The 1 H NMR data of 8 (Table 2)  With aid of HMBC and NOESY, the relative configuration could be inferred, and it was further seen that H-4 and H-5 are in a cis relationship. The laevorotatory optical activity of 8 indicated the C-4 hydroxyl group in R-configuration [14,15], and the chemical shifts of H-4 (ca. δ H 4.55) and 7-Me (ca. δ H 1.34) of 8 were also similar to those of related analogues. The NOESY correlations were observed for H-4/H-5, indicating that they were on the same side of the molecular plane, assumed to have an R-orientation. Therefore, the absolute configuration of C-4 and C-5 of 8 was deduced to be the (4R,5R) configuration [14,15]. This compound has been synthesized in the literature [16], but it was isolated for the first time as a natural product. Based on the spectral evidence, the structure of 8 was elucidated as (4R,5R)-5ethyl-4-hydroxydihydrofuran-2(3H)-one, named herbidosporayilanensin H, which was further confirmed by 1 H-and 13 C-NMR chemical shifts (Tables 1 and 2), and the HMBC, 1 H, 1 H-COSY, and NOESY correlations are shown in Figure 2.  [14,15]. No NOESY contacts were observed for H-4/H-5, once it has been indicating that they were on the opposite side of the molecular plane, assumed to have a (4R,5S)-orientation instead of a (4R,5R) in 8. Thus, the structure of 8 was established, as shown in Figure 1 and was named herbidosporayilanensin I.
The biological activities of the isolates present in sufficient amounts (1-7) in compounds from fermented broth were filtered to separate the mycelium and culture broth. The culture broth was repeatedly extracted three times with EtOAc. The EtOAc-soluble fraction of the fermented broth of the actinobacteria H. yilanensis was tested in vitro against M. tuberculosis strain H 37 Rv. The antimycobacterial activity data are shown in Table 3. The clinically used antimycobacterial agent, ethambutol, was used as a positive control. The results of the antimycobacterial activities indicated that herbidosporayilanensin A (1), herbidosporayilanensin A (2), and herbidosporayilanensin F (6) exhibited more potent antimycobacterial activities against M. tuberculosis strain H 37 Rv in vitro, showing MIC values of 16.6, 19.2, and 18.2 µM, respectively, than did the clinical drug, ethambutol (MIC 6.25 µg/mL). The results showed moderate antimycobacterial activity, indicating that 3 and 4 had MIC values of 40.8 and 50.6 µg/mL, respectively. Compounds 5 and 7 showed no antimycobacterial activities. When comparing the two benzenoids (6-7), the presence of ketone groups on the 7 position of the side chain in 6 was seen to play an important role in antimycobacterial activity.

Microorganism, Cultivation, and Preparation of the Actinobacteria Strain
The actinobacteria, Herbidospora yilanensis (0351M-12 T ), was isolated from sediment collected from the northern area of Taiwan using HVY agar and was then incubated at 45 • C for 7 days. This actinobacteria was identified by Mrs. Min Tseng, and the specimens (0351M-12 T ) were deposited at the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI). The strain was maintained on oatmeal agar, and the spores or mycelia suspension were harvested with 20% (v/v) glycerol and stored at −20 • C. A mature slant culture of strain 0351M-12 T was inoculated into a 500 mL flask containing 100 mL of the seed medium consisting of 0.4% glucose, 0.4% yeast extract, and 1% malt extract (pH 7.3). After growing at 30 • C for 4 d on a rotary shaker (200 rpm), the aliquots (2 mL) of the seed culture were transferred into a 500 mL flask containing 200 mL of production medium (Humic acid 1.0 g, Na 2 HPO 4 0.5 g, KCl 1.7 g, MgSO 4 7H 2 O 0.05 g, FeSO 4 7H 2 O 0.01 g, CaCO 3 0.02 g, yeast extract 1.0 g, Agar 20.0 g, dist. water 1.0 L, pH 7.4). After 18 days cultivation at 30 • C temperature on a rotary shaker (200 rpm), the culture filtrates were obtained by filtering through filter paper.

Isolation and Characterization of Secondary Metabolites
Fermented broth (3 l) was filtered to separate the mycelium and culture broth. The culture broth was repeatedly extracted three times with EtOAc. The EtOAc layers were combined and dried to give fraction-soluble EtOAc (25.2 g).

Antitubercular Activity Assay
The in vitro antitubercular activity of each tested compound (1-7) was evaluated using Mycobacterium tuberculosis H 37 Rv. Middlebrook 7H10 agar was used to determine the MICs, as recommended by the proportion method [17]. Briefly, each test compound was added to Middlebrook 7H10 agar supplemented with OADC (oleic acid-albumin-dextrosecatalase) at 50-56 • C by serial dilution to yield a final concentration of 100 to 0.8 µg/mL. Ten milliliters of each concentration of test compound-containing medium was dispensed into plastic quadrant Petri dishes. Several colonies of a test isolate of M. tuberculosis were selected to make a suspension with Middlebrook 7H9 broth and used as the initial inoculum. The inoculum of test isolate of M. tuberculosis was prepared by diluting the initial inoculum in Middlebrook 7H9 broth until turbidity was reduced to the equivalent of the McFarland no. 1 standard. Final suspensions were prepared by adding Middlebrook 7H9 broth and preparing 10 −2 dilutions of the standardized bacterial suspensions. After solidification of the Middlebrook 7H10 medium, 33 µL of the 10 −2 dilution of the standardized bacterial suspensions was placed on each quadrant of the agar plates. The agar plates were then incubated at 35 • C with 10% CO 2 for 2 weeks. The minimal inhibitory concentration (MIC) is the lowest concentration of test compounds that completely inhibits the growth of the test isolate of M. tuberculosis, as detected by the unaided eye.

Conclusions
Herbidospora yilanensis is a new strain isolated from the sediment of rivers in northern Taiwan. It is very novel and worthy of further study. This strain has been published in the International Journal of Systematic and Evolutionary Microbiology (IJSEM). Based on the concept of "new species and new compounds", it is expected that special compounds will be found from these new strains. The current study was conducted on the biologically active metabolites of the EtOAc soluble fraction of by the actinomycetes H. yilanensis. This led to the isolation of five previously undescribed compounds, namely, herbidosporayilanensin A-E (1-5), and four compounds isolated from nature for the first time, namely, herbidosporayilanensins F-I (6-9) (Figure 1). The structure of these isolates was determined by spectroscopic experiments. The EtOAc soluble fraction from the H. yilanensis fermentation broth was tested in vitro against Mycobacterium tuberculosis strain H37Rv. See Table 3 for the anti-mycobacterial activity data. The clinically used anti-mycobacterial agent ethambutol was used as a positive control. The results of anti-mycobacterial activity showed that the herbidosporayilanensins A (1), B (2), and F (6) showed more effective resistance to Mycobacterium tuberculosis strain H37Rv in vitro with MIC values of 16.6, 19.2, and 18.2 µM, respectively, than did the clinical drug, ethambutol (MIC 6.25 µg/mL). Compounds 5 and 7 did not show anti-mycobacterial activity. When comparing the two benzene analogues (6 and 7), it was observed that the presence of the ketone group at position 7 of the side chain in 6 played an important role in the anti-mycobacterial activity.
In previous surveys, there have been many reports on the activity of actinomycete metabolites in the literature, but there are some reports of active natural products against Mycobacterium tuberculosis in vitro. Therefore, it is still worth continuing to study the active substance against different Mycobacterium tuberculosis strains.