Secondary Metabolites with Anti-Inflammatory Activities from an Actinobacteria Herbidospora daliensis

Bioassay-guided fractionation of extracts derived from solid cultures of a Herbidospora daliensis originating from Taiwan led to the isolation of five new compounds, for which we propose the name herbidosporadalins A–E (1–5), one isolated for the first time, herbidosporadalin F (6), together with two known compounds (7 & 8). 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 anti-inflammatory activities were also evaluated. Of these isolates, herbidosporadalin A (1), B (2), F (6) and G (8) showed NO inhibitory activity, with IC50 values of 11.8 ± 0.9, 7.1 ± 2.9, 17.8 ± 1.7, and 13.3 ± 6.5 μM, stronger than the positive control quercetin (IC50 = 36.8 ± 1.3 μM). To the best of our knowledge, this is the first report on 3,4-seco-friedelane metabolites (5, 6 & 8) from the genus Herbidospora.


Introduction
Actinobacteria are a very special group of microorganisms that mainly grow in soil and can also be found in oceans, streams, lake water or sediment, animals, plants, nodules, compost, hot springs, geothermal and other environments. Its morphology is as varied as that of fungi, and it has structures such as substrate hyphae, aerial hyphae, spore, spore chain and sporangium in its life history. However, it resembles prokaryotes-like bacteria in physiological structure, so it has been mistaken for fungi for a long time, and some people regard it as a microorganism between bacteria and fungi-boundary microorganisms. Actinobacteria themselves have the ability to produce a variety of physiologically active products [1][2][3][4][5][6][7]. Therefore, it plays a key role in the pharmaceutical industry and the food industry. Our team has also isolated and collected actinobacteria resources from all over Taiwan and various environments over the years. In addition to the common Streptomyces, we have also discovered many new species from different environments throughout Taiwan. The goal is to isolate new compounds from new species, with the hope of discovering special compounds from these new species.
Our team has collected more than 1500 Taiwan native actinobacteria in the past 20 years. They have collected soil samples from various environments in Taiwan, including paddy fields, vegetable gardens, orchards, weeds, hot springs, culture pond sediments, lake sediments, mangroves, and wetland. After appropriate pretreatment, the actinobacteria were isolated by serial dilution and selective medium (HV agar). In the past, our team published 5 new native species in IJSEM [8][9][10][11], one of which was Herbidospora daliensis, which was isolated from the sediments of lakes in central Taiwan [11]. According to liensis, which was isolated from the sediments of lakes in central Taiwan [11]. According to the database literature search, the past research of Herbidospora genus focused on the classification of molecular biology [12][13][14]. There are few studies on natural chemistry [15]. Recently, through the anti-inflammatory activity-screening platform, it was found that the Herbidospora daliensis strain is an actinomycete with strong anti-inflammatory activity. Our research is unique and original compared with the literature [1][2][3][4][5][6][7], and the components secreted by the bacteria are worthy of in-depth research and discussion.
Careful examination of the constituents and the anti-inflammatory principles of this material has led to the isolation and characterization of five other new ones, two of which contain coumarin moiety: herbidosporadalin A (1) and herbidosporadalin B (2), and herbidosporadalin C (3), herbidosporadalin D (4), and herbidosporadalin E (5), as well as three known compounds (Figure 1). The structures of these compounds were determined through spectral analyses (Figures 2 and 3). The structural elucidation of 1-6 and the anti-inflammatory of the isolates are described herein.

Results and Discussion
Scheme 1. The possible biosynthetic pathway of 2.
Compound 8, m.p. 211-212 • C, was obtained as a white, amorphous solid. The molecular formula was deduced to be C 32 H 56 O 3 from its NMR spectral data. Ester carbonyl and hydroxyl groups were indicated by absorption bands at 1752 and 3400 cm −1 in the IR spectrum, respectively. The 13 C NMR spectrum of 7 (in CDCl 3 ) indicated the presence of 32 signals, seven corresponding to quaternary, four to methine, 12 to methylene and nine to methyl carbons on the basis of the DEPT experiment. The spectral features indicated that 7 has a similar molecular framework to 5. The absence of a signal corresponding to a methylene group around δ C 37.4/δ H 2.25 (C-2), the presence of a CHOH group at δ H 4.07/δ C 71.8 (C-2), and a terminal ethyl group [δ H 0.77 (3H,t,J = 7.5 Hz,1.12/1/34 (2H,m,] in 8 was substituted for a vinyl group [δ H 5.60 (dd,J = 17.4,10.8 Hz,4.90 (dd,J = 10.8,1.1 Hz,4.88 (dd,J = 17.4,1.1 Hz,] in 5. The 1 H-and 13 C-NMR (Table 2) (8) [23].
In summary, Actinobacteria have been accepted as a big microbial bank that can be expected to provide a wide variety of structurally unique and biologically potent natural metabolites. In continuation of our previous chemical and biological investigations of microorganism-generated metabolites, a new Actinobacteria strain, identified as Herbidospora daliensis, isolated from a sediment soil sample, was ascertained to be able to produce bioactive metabolites during its solid fermentation according to our systematic screening program. The secondary metabolites of Herbidospora genus have rarely been studied in the past. The strain H. daliensis in this study has only been reported by our team for one component in the past [15]. After modification of the fermentation conditions, we obtained 8 components from the ethyl acetate active layer, 5 of which were new compounds, and the skeleton of the compounds covered benzofuran, coumarin, biphenyl and 3,4-seco-friedelane metabolites. These components were first discovered from the genus Herbidospora, which has chemical taxonomic significance. These results suggest that Herbidospora has distinct and diverse metabolites that arise under different fermentation conditions and soil-derived collections. It may therefore be possible to find more new bioactive natural products by searching the Herbidospora species under a special eco-environment.

Biological Studies
The 8 isolates in sufficient amounts were evaluated by examining their inhibitory effects on LPS-induced inducible nitric oxide synthase (iNOS)-dependent NO production in the murine macrophage cell line RAW 264.7 (Table 3). The inhibitory activity data of the 8 isolated compounds on NO generation by macrophages are shown in Table 3. Compared to quercetin (IC 50 value 36.8 ± 1.3 µM), which was used as the positive control in this study, herbidosporadalins A, B, G & H (1, 2, 6, & 8) exhibited NO inhibitory activity with IC 50 values of 11.8 ± 0.9, 7.1 ± 2.9, 17.8 ± 1.7 & 13.3 ± 6.5, respectively. Compounds 1, 2, 8, & 6 showed about 3, 5, 2 and 3-fold NO inhibitory activities compared to quercetin, respectively. Compounds 3 and 7 showed weak NO inhibitory activity, whereas compounds 4 and 5 displayed no NO inhibitory activities.
From the results of our above tests, the following conclusions can be drawn. Compound 6 (3,4-seco-friedelan analogs, herbidosporadalin F), with an ethyl 3-propanoate at C-10, exhibited more effective inhibition than its analogue, compound 8 (2-hydroxyl-3,4-seco-friedelan-3-oic acid ethyl-ester), with an ethyl-2-hydroxypropanoate group, and compound 5 (herbidosporadalin E), with a vinyl groups at C-5 against LPS-induced NO generation. Compound 5 (herbidosporadalin E), with a double bond between C-4 &23 substituent, exhibited less effective inhibition than its analogue, compounds 6 & 8. The presence of ethyl groups on the C-4 position of the 3,4-seco-friedelans seem to play an important role in anti-inflammatory activity. Furthermore, the RT-PCR analysis in the present study indicated that LPS treatment increased the level of iNOS mRNA expression, and that compounds (1, 2, 6, & 8) inhibited this increase in a concentration-dependent manner. At the highest concentration, none of the compounds tested showed any obvious cytotoxicity toward RAW 264.7 cells. Cytotoxic effects were measured using MTT assay. The high cell viability (>95%) indicated that the inhibitory activities of LPS-induced NO production by active compounds (1, 2, 6, & 8) did not result from its cytotoxicity.

Microorganism, Cultivation, and Preparation of the Actinobacteria Strain
The Actinobacteria, Herbidospora daliensis (0385M-1 T ), was isolated from sediment collected from the Dali area of Taiwan using HVY agar and was then cultured at 45 • C during 7 days. This Actinobacteria was identified by Mrs. Min Tseng, and the specimens (0385M-1 T ) were deposited at the Bioresource Collection and Research Center, Food Industry Research and Development Institute. Strains are maintained on oat agar and spore or mycelial suspensions are harvested with 20% (v/v) glycerol and stored at −20 • C. Mature slant cultures of strain 0385M-1T were inoculated into 500 mL flasks containing 100 mL of seed medium composed of 0.4% glucose, 0.4% yeast extract, and 1% malt extract (pH 7.0). After 4 days of growth at 30 • C on a rotary shaker (200 rpm), an aliquot (2 mL) of the seed culture was tranferred to 500 mL of production medium (Humic acid 1.0 g, KCl 1.7 g, FeSO 4 ·7H 2 O Funding: This work was kindly partially supported by the Food Industry Research and Development Institute (FIRDI) and supported by Ministry of Science and Technology, R.O.C. (MOST-108-2320-B-080-002-and 110-2320-B-080-001-).

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.