Secondary Metabolites with Anti-Inflammatory Activities from One Actinobacteria Amycolatopsis taiwanensis

Phytochemical investigation and chromatographic separation of extracts from one new actinobacteria strain Amycolatopsis taiwanensis that was isolated from soil of Yilan township, in the north of Taiwan, led to the isolation of nine new compounds, amycolataiwanensins A–I (1–9, resp.), and one new natural product, namely amycolataiwanensin J (10). The structures of the new compounds were unambiguously elucidated on the basis of extensive spectroscopic-data analysis (1D- and 2D-NMR, MS, and UV) and comparison with literature data. The effect of some isolates on the inhibition of NO production in lipopolysaccharide-activated RAW 264.7 murine macrophages was evaluated. Of the isolates, 3, 5, 7 and 8 exhibited potent anti-NO production activity, with IC50 values of 17.52, 12.31, 17.81 and 13.32 μM, respectively, compared to that of quercetin, an iNOS inhibitor with an IC50 value of 35.94 μM. This is the first report on indole metabolite from the genus Amycolatopsis.


Introduction
Actinobacteria are well known as an outstanding and fascinating source of commercially valuable bioactive compounds, particularly antibiotics. Almost a half of the known microbial bioactive secondary metabolites are derived from actinomycetes, of which more than 70% were obtained from the genus Streptomyces. However, the active ingredients of many new Taiwanese actinobacteria and their mechanisms of actions are still unknown. It is necessary to study on the active compounds by scientific methods from these rare actinobacteria. Actinobacteria are widely distributed in nature. They seem to have unlimited ability to produce secondary metabolites with multiple chemical structures and biological activities, so they have a place in the pharmaceutical industry [1][2][3][4][5]. They are Gram-positive, free-living saprophytic bacteria that exist in soil, water and colonizing plants. The residents of actinobacteria have been identified as one of the main groups of soil populations [4], may vary depending on the type of soil.
Based on our long-term plan for the collection and identification of new species of native actinobacteria in Taiwan, we isolated several new strains from Taiwan soil. A strain named 0345M-7T was isolated from soil sediment samples in Yilan County, Taiwan. It has a unique morphology after observation through an electron microscope [4].
This strain was determined to be Amycolatopsis taiwanensis, (Family: Pseudonocardiaceae) based on their phenotypic and genotypic data [5]. The isolate displayed substrate mycelia, upon which were borne short spore chains. The spore chains were composed of non-motile, smooth-surfaced, oval spores. The genus Amycolatopsis (Lechevalier et al. 1986) contains more than 70 species and its main habitat is soil. Strains of this genus can produce a variety of important antibiotics and secondary metabolites, such as balimycin, dethymicin, rifamycin, vancomycin, and can be used for drug-resistant Staphylococcus, organ transplantation, leprosy, tuberculosis, etc. Although the strains of the genus Amycolatopsis have been researched and developed very early and many times, A. taiwanensis is a new strain isolated from Taiwan, and no relevant research has been carried out. It has the potential to discover new compounds. According to the literature search, it was found that 159 compounds of the genus Amycolatopsis were reported from 8 known species and 18 unknown species [6]. These secondary metabolites are mainly divided into polyphenols, linear polyketides, macrolides, macrolides, thiazolyl peptides, cyclic peptides, glycopeptides, amides and amino derivatives, glycoside derivatives, and enediyne derivatives, and sesquiterpenes. At the same time, they mainly exhibit unique antibacterial, anti-cancer, anti-oxidant, anti-hyperglycemic and enzyme inhibitory activities.
In the course of our search for potential diverse secondary metabolites from natural microbial sources, and to further understanding of the minor metabolites of the genus Amycolatopsis, we examined the EtOAc extract of A. taiwanensis, which showed rich metabolites according to the HPLC fingerprint analysis and inhibitory activity on LPS-induced NO release in RAW 264.7 murine macrophages, as determined by our primary screening. Investigation of the bioactive metabolites of the active EtOAc extract from the microbe A. taiwanensis, fermented by liquid fermentation was investigated. The metabolites investigation guided by the HPLC profile analysis and 1 H-NMR spectrum prescreening led to the isolation of nine new metabolites, amycolataiwanensins A-I (1-9), and one metabolite isolated for the first time from nature sources, amycolataiwanensin J (10) ( Figure 1 and the Supplementary part). The structures of these isolates were established by means of spectral experiments. The isolation, structural elucidation, inhibitory effects of some isolates on nitric oxide (NO) production by RAW264.7 macrophages are described herein.

Results and Discussion
Compound 1 was isolated as oil. The HR-EI-MS spectrum gave a molecule ion [M] + at m/z 215.0948, consistent with a molecular formula of C 13 H 13 NO 2 . UV spectrum showed maximum absorption at 241 (4.20), 264 (4.09) and 310 (3.98) nm, indicating the presence of an indole skeleton [7]. Its IR spectrum revealed NH absorption at 3320 cm −1 . Analysis of the 1 H NMR spectrum of 1 revealed four typical mutually coupling aromatic protons of indole alkaloid at δ H 7.08 (1H, td, J = 8.0, 1.2 Hz, H-7), 7.11 (1H, td, J = 8.0, 1.2 Hz, H-6), 7.26 (1H, dd, J = 8.0, 1.2 Hz, H-8), 7.84 (1H, dd, J = 8.0, 1.2 Hz, H-5) and one NH group at δ H 7.66 (1H, br s, exchangeable with D 2 O). According to the molecular formula, the degree of unsaturation can be calculated to be 8. After deducting the two rings and four double bonds of indole, there are also 2 remains. In addition to the carbon spectrum, there is a conjugated carbonyl signal at δ C 183.99. It is speculated that the branched chain forms a ring (C ring). It can be confirmed from the fact that the methylene signal on the carbonyl α carbon is δ H 2.63 (2H, s) in a lower magnetic field. In addition, the two carbon absorption signals on the pyrrole ring of indole, a particularly low magnetic field and a high magnetic field, are presumably affected by the electronic resonance of the carbonyl group. Therefore, one end of the branch is connected to a carbon with a higher magnetic field (δ C 95.15) through a carbonyl carbon. The carbon with a lower magnetic field (δ C 160.87) should also be connected to an oxygen and be affected by the carbonyl group to achieve such a low magnetic field. Observing the carbon spectrum, there is also a quaternary carbon connected to oxygen. It is obvious that the other end of the branch chain is connected to indole with this oxygen. There is also a single peak integrated into 6H in the 1 H NMR, δ H 1.57 (6H, s, CH 3 -5 , 6 ), which is presumed to be two methyl groups with the same signal, and the position is on the quaternary carbon (δ C 86.2) connected to oxygen. The HMBC 3 J-correlations ( Figure 2) from δ H 2.63 (CH 2 -3 ) to δ C 95.2 (C-3) and one weak 4 J-correlations δ H 1.57 (6H, s, CH 3 -5 , 6 ) to δ C 160.9 (C-2), verify the junction of the 2,2-dimethyldihydropyrano ring to the indole moiety at C-2, and 3. The other key correlations of HMBC are illustrated in Figure 2. Based on the above data, the structure of 1, named amycolataiwanensin A, was elucidated as 2,2-dimethyl-2,3-dihydropyrano [2,3b]indol-4(9H)-one, which was further confirmed by 13  The IR spectrum of 2 displayed an absorption for an OH group (3500 cm −1 ) and a C = O group (1740 cm −1 ). The 1 H-and 13 C-NMR, COSY, HMBC, and NOESY data ( Figure 3) established the structure of 2 as 12-acetoxy-11-hydroxyacora-3-ene.
The 1 H-NMR spectrum exhibited signals for a trisubstituted olefinic proton [δ H 5.30 (br s, H-3)], an oxymethylene at δ H 3.93/4.05 (each d, J = 11.0 Hz, CH 2 -12), and the four methyl groups include three singlet methyl groups δ H 1.24 (s), 1.58 (s), 2.08 (s) and a doublet methyl group at δ H 0.82 (d, CH 3 -14). From the carbon spectrum ( 13 C-NMR) and DEPT, because of the appearance of one quaternary carbon at δ C 171.2 (ester) and one primary carbon at δ C 20.9 (CH 3 ), it can be seen that there is an acetoxy group. In addition, there are two quaternary carbons δ C 44.70 and 74.85 (of which δ C 74.35 is the oxygen-containing quaternary carbon). Calculating the degree of unsaturation, subtracting a double bond and a carbonyl group, there is 2 left, so it can be determined that there are two more rings in this structure. According to the above characteristic spectrum information and reference data, [8] it can be known that the NMR information of this structure are very similar to the spectrum of a known compound 12-acetoxy-11-hydroxyacora-4-ene, so it is inferred that this compound It is also belonged to acorane backbone compound. According to the signal of HMBC, the structure is similar to the known compound 12-acetoxy-11-hydroxyacora-4-ene, [8] while the signal of NOESY explains the difference in stereo orientation: (1) The H-2 has NOE correlation signals with H-3 and H-14 in compound 2. (2) In the similar compound 12-acetoxy-11-hydroxyacora-4-ene, its H-6 has NOE correlation signal with H-13 and H-5. Therefore, the structure and steric orientation of compound 2 were proved. The above spectroscopic data proves that the structure is correct, and this new compound is named amycolataiwanensin B.
Compound 3 had the dehydrated molecular ion peak [M-H 2 O] + at m/z 330.1831 (HR-EI-MS), as analyzed for C 20 H 28 O 5 . The IR spectrum of 3 exhibited the presence of an OH group at 3400 cm −1 and a CO moiety at 1695 cm −1 . The UV absorptions (λ max 229.0 and 256.0 nm) confirmed an aromatic system. Seven IHD were determined from the molecular formula, 13 C-NMR spectrum, and DEPT. Further spectral data (Tables 1 and 2) and comparison with reference compounds [9] established the structure of 3 as 6α,7α,11trihydroxy-3-oxoferrugiol. The 1 H-NMR spectrum of 3 indicated the presence of an iPr group (δ H 1.27 (d) and 1.28 (d), and 2.99 (sep)) attached to the benzene ring, two phenol groups (δ 4.85 (s), 5.81 (s)), another three Me groups (δ H 1.21 (s), 1.33 (s), and 1.45 (s)) attached to a quaternary carbon, two OCH groups (δ H 4.96 (d, 5.0) and 4.44 (dd, 11.5, 5.0)), the signal of five substituted benzene ring at 6.81 (s, H-14). According to the 13 C-NMR and DEPT, the benzene ring δ C 125.33, 130.44, 139.96, 142.42, 132.91 and 119.70 composed of 6 olefinic carbons; there is a carbonyl group at δ C 219.66, and two oxygen-containing tertiary carbons at δ C 68.3 (C-7), and 74.3 (C-6). Based on the above information and combining the above characteristic spectra data, the compound 3 with the abietane skeleton can be identified. The H-1β at δ H 3.03 (m) is the result of the displacement of the low magnetic field due to the influence of the hydroxyl group on C-11. In order to determine the structure and the position of each functional group, continue with two-dimensional nuclear magnetic resonance spectroscopy (HSQC, HMBC) and NOESY experiments. According to the key information of HMBC: (1) H-18 and H-19 are correlated to δ C 219.66, so it is determined that the carbonyl group is located at the position of C-3; (2) H-14 is only related to δ C 68.26, so the two tertiary hydroxyl carbons can be distinguished. (3) Because δ H 5.81 (s, OH-12) is correlated to C-12, the two phenols can be distinguished and make sure that δ H 5.81 (s) is connected to C-12. According to the signal from NOESY: (1) H-5 is connected with H-1α and H-18 respectively; (2) H-6 is connected with H-19 and H-20, so it is determined to be in the axial position; (3) H-7 is related to H-6β and H-14, and by its coupling constant (J = 5.0 Hz), it can also be determined that it is in the equatorial position. Compound 3 is a previously undescribed diterpene and was named amycolataiwanensin C.        3 -16, 17), and 3.12 (H-15, COSY cross-peaks with δ H 1.19 and 1.18) suggested that 5 has an iPr group and three Me groups attached to a quatenary C-atom. According to 13 C-NMR and DEPT, in addition to isopropyl and three singlet methyl groups, there is a carbon at δ C 78.24, which is a tertiary carbon connected to oxygen, and δ C 183.25 and 187.31 show quinone group signals. In addition, there are four olefinic carbons, δ C 123.85, 150.56, 145.74 and 145.97. Since the compound is yellow and the UV absorption spectrum shows, coupled with the above-mentioned spectral data ( 1 H-NMR and 13 C-NMR data of known compounds in Reference [10], it can be inferred that compound 4 is a derivative of hydroxybenzoquinone in the abietane skeleton. The C ring is a quinone ring, and H-1β (δ 2.79) is affected by the quinone group of C-11, so the magnetic field is relatively low. From the signal of δ H 3.24 (dd, J = 10.6, 5.7 Hz), it can be seen that this H is in the axial position, and -OH is in the equatorial position. By heteronuclear correlation spectroscopy (HSQC, HMBC) and NOESY to analyze its structural correlation and stereo orientation. According to HMBC's information as following: (1) δ 3.24 is related to C-18 and C-19 respectively, so it is determined that the hydroxyl group is connected to the position of C-3; (2) H-5 is connected to C-3, C-7, and C-3, respectively. C-10, C-18, C-19, C-20 are connected; (3) H-15 is connected with C-12, C-13, C-14 respectively; (4) H-20 is connected with C-1. C-5, C-9, C-10 are related. According to the signal from NOESY: (1) H-5 is related to H-1α, H-3, H-6α, H-7α, and H-18 respectively; (2) H-3 is related to H-5 and H-18 Therefore, it is determined that H-3 is in the axial direction; (3) H-20 is related to H-2β and H-6β; (4) H-2β is related to H-19 and H-20. The structure was proved to be correct, and the new compound was named amycolataiwanensin D.
Compound 6 was isolated as oil. Its molecular formula, C 20 H 30 O 3 , was determined on the basis of the positive HR-EI-MS at m/z 318.2197 [M] + (calcd 318.2195) and was supported by the 1 H, 13 C, and DEPT data. The IR absorption bands of 6 revealed the presence of the COOH (3400 cm −1 for OH; 1699 cm −1 for CO) and a conjugated carbonyl (1670 cm −1 ) functions. According to DEPT plots, there are four primary carbons, seven secondary carbons, three tertiary carbons, and six quaternary carbons. The degree of unsaturation is estimated to be 6. In 13 C-12) respectively. From the above analysis of 1D and 2D spectra, compound 6 is 12(13→15)abeoabietane diterpenes, C-12 is not connected to C-13, and C-15 is reversed to cause the six-ring to seven-ring, geminal dimethyl group is substituted for isopropy. As for its proposal biosynthesis and the stereochemistry, it can be explained by the NOESY spectrum. H-20 and H-19 have a NOESY correlation, which is sufficient to show that -COOH is located in the equatorial direction. The spectra of HMQC and COSY confirm that the compound 6 is 12(13→15)abeo-13-oxo-8(14)-abietene-18-oic acid and designated as amycolataiwanensin F.
Fifteen 13 C-NMR signals and the HR-EI-MS confirmed the molecular formula C 15 H 26 O 2 of 10. Analysis of its IR spectrum suggested that 10 contained OH (3299 cm −1 ) moiety. The three IHD (from the DEPT experiment), the 13 C-NMR data, and the molecular formula indicated that 10 is a sesquiterpene. Further spectral data established the structure of 10 as (2S*,3S*,6R*)-3,6,8,8-tetramethyloctahydro-1H-3a,7-methanoazulene-2,6-diol (3αhydroxycedrol). The 1 H-NMR shows that δ H 0.94 (d) is a doublet methyl group attached to a tertiary carbon. At δ H 1.00 (s), 1.23 (s), and 1.32 (s), there are three singlet methyl groups on the quaternary carbon. Among them, δ H 1.23 (s) and 1.32 (s) are located in the lower magnetic field because they are connected to the hydroxyl group. From 13 C-NMR, DEPT, and HSQC plots, there are two carbons attached to oxygen at δ C 72.84 and 81.43, 72.84 belongs to the quaternary carbon, and δ C 81.43 belongs to the tertiary carbon. Calculating the degree of unsaturation, because there is no double bond or carbonyl carbon, it can be proposed this compound is a tricyclic ring. Based on the above spectral data, it is speculated that the compound should be a cedrane skeleton. After comparing with the reference data, and comparing with the 1 H-NMR and 13 C-NMR of the known compound cedrol [15], there is only one more oxygen-containing tertiary carbon. It can be confirmed by the signal (δ H 3.58 (1H, ddd, J = 15.5, 10.5, 5.5 Hz, H-3); δ C 81.4) appeared on the 1 H-NMR & 13 C-NMR. Continue to perform two-dimensional heteronuclear correlation spectroscopy (HSQC, HMBC) and NOESY to further determine the structural relevance and stereo orientation of 10. The 1 H signal at δ H 1.45 (H-2)/δ H 1.36 (CH 2 -4) and δ H 0.94 (CH 3 -12) showed a two and three-bond connectivities with C-3 (δ C 81.4) in the HMBC plot (Figure 2), which suggested that the second OH group at C-3. According to the signals from NOESY, determine the relative configuration of the C-3 and C-8 hydroxyl groups: (1) Compound 3 exhibited the HMBC correlation: H-3/12-Me, and judging from the split pattern (ddd), H-3 is located in the β-axial position; (2) 15-CH 3 is correlated to H-9α and H-9β, so it is judged that 15-CH 3 is equatorial. The structure was proved to be correct, and compared with the literature, it was confirmed that this was a compound discovered for the first time in nature, named amycolataiwanensin J.
NO is a mediator in the inflammatory response involved in host defense. In the course of our search for potential diverse secondary metabolites from natural fungal sources, and to further understanding of the bioactive metabolites of the genus Amycolatopsis, we examined the EtOAc extract of A. taiwanensis, which showed inhibitory activity on LPSinduced NO release production in RAW 264.7 murine macrophages, as determined by our primary screening (approximately 95% inhibition at a concentration of 10 µg/mL). Investigation of the bioactive metabolites of the active EtOAc extract from the titled material A. taiwanensis, led to the isolation of ten new compounds. Due to the small quantity of isolated compound (1), we evaluated the inhibitory effects of amycolataiwanensins B-J (2-10, resp.) on the production of NO induced by LPS. The inhibitory activity data of the 10 isolated compounds on NO generation by macrophages are shown in Table 3. (e) Among the abietane diterpene analogues, compound 3 (with (abietane with 3-isopropylbenzene-1,2-diol unit in C ring) exhibited more effective inhibition than its analogue, compound 4 (abietane with hydroxybenzoquinone unit in C ring), compound 5 (with 1-phenylethan-1-ol moiety in C ring) and compound 6 (with 7,7-dimethylcyclohept-2-en-1-one moiety in C ring. (f ) Among the aromatics analogues, compound 7 (simple aromatic with 9-acetoxydihydrosyringenin) displayed better inhibition than its analogue, compound 8 (abietane with hydroxybenzoquinone unit in C ring), compound 5 (with 1-phenylethan-1-ol moiety in C ring) and compound 9 (with 7,7-dimethylcyclohept-2-en-1one moiety in C ring. (g) Furthermore, the RT-PCR analysis in the present study indicated that LPS treatment increased the level of iNOS mRNA expression, and that compounds 3, 5, 8 and 9 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. (h) 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 3, 5, 8, and 9 were not resulted from its cytotoxicity.

Microorganism
Amycolatopsis taiwanensis (0345M-7 T ) was used throughout this study, and deposited at Bioresource Collection and Research Center (BCRC), Food Industry Research and Development Institute (FIRDI). This actinobacteria was identified by Min Tseng., and specimens (0345M-7 T ) deposited at the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI).

Cultivation and Preparation of the Fungal Strain
The actinobacteria, Amycolatopsis taiwanensis (0345M-7 T ), was isolated from a sediment collected from the northern area of Taiwan, by using HVY agar, and was then incubated at 45 • C for 7 days. This actinobacteria was identified by one of the authors (Mrs. Min Tseng), and specimens (0345M-7 T ) 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 harvest with 20% (v/v) glycerol and stored at −20 • C. A mature slant culture of strain 0345M-7 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 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 21 days cultivation at 30 • C temperature on a rotary shaker (500 rpm), the culture filtrates (10 L) were obtained by filtering through filter paper.

Determination of NO Production and Cell Viability Assay
Mouse macrophage cell line (RAW 264.7) was obtained from Bioresource Collection and Research Center (BCRC 60001) and cultured at 37 • C in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (Gibco), 4.5 g/L glucose, 4 mM glutamine, penicillin (100 units/mL), and streptomycin (100 µg/mL) in a humidified atmosphere in a 5% CO 2 incubator. The cells were treated with 10, 25, 50 µM natural products in the presence of 1 µg/mL LPS (lipopolysaccharide, Sigma-Aldrich, St. Louis, MO, USA) for 20 h. The concentration of NO in culture supernatants was determined as nitrite, a major stable product of NO, by Griess reagent assay [16], and cell viabilities were determined using the MTT assay as described previously [17].

Conclusions
Actinobacteria have the ability to produce a variety of physiologically active products, so they play a very important role in the food and pharmaceutical industries. Over the years, our team has also separated and collected actinomycetes resources from all over Taiwan and various environments. In addition to common Streptomyces, there are also many rare species of actinobacteria, and there are many new species. Based on the concept of "new species and new compounds", it is expected that special compounds can be found from these new strains. 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 actinomycetes, therefore, this project uses one new species of actinobacteria that have not been studied in the past, and they are cultured, extracted, purified and identified with high-level and highly active anti-inflammatory compounds with solid rice, in order to improve the research of actinobacteria in my country level, and develop health products related to actinobacteria. Under the support of the Ministry of Economic Affairs, the Bioresource Collection and Research Center at Food Industry Research and Development Institute, has been dedicated to the research work on the collection, separation and preservation of bio-resource research in the past few years, and has constructed a complete indigenous strains resource bank in Taiwan. The applicant analyzed the active constituents from it, and obtained more than sixty active new compounds isolated from red yeast rice, endophytes, actinobacteria, and mushrooms, among which many new compounds have anti-cancer and anti-inflammatory effects. Most of them have been published [18][19][20][21][22][23]. According to those findings, we have proved that the exploration of bioactive compounds on indigenous strains in Taiwan is a research-worthy topic. Actinobacteria are well known as an outstanding source of commercially valuable bioactive compounds, particularly antibiotics. Many microbial bioactive metabolites are derived from actinomycetes (Streptomyces sp.).
However, the metabolites of many new Taiwanese actinobacteria and their mechanisms of actions are still unknown. It is necessary to study on the bioactive by scientific methods from these rare actinobacteria. In summary, we have isolated and characterized nine undescribed derivatives, amycolataiwanensins A-J from an actinobacteria strain Amycolatopsis taiwanensis that was isolated from soilt of Yilan township, in the north of Taiwan. The relative configurations of new isolates were determined by comparing their optical activities with related derivatives and NOESY plots. Amycolataiwanensins C, E, H & I showed inhibitory activities against LPS-induced NO production in RAW 264.7.
The discovery of indole, sesquiterpenes, diterpenes, and chromenes derivatives from actinobacteria pointed toward the potential of endophytic or associated Amycolatopsis taiwanensis as alternative producer of indole, sesquiterpenes, diterpenes, and chromenes derivatives. The current results may encourage further investigations on the chemistry and bioactivity of flavan metabolites. These results also suggest that Amycolatopsis 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 Amycolatopsis species under special eco-environment. For the sake of better understanding the distribution of flavan acid analogs, the actinobacteria of the tilted research material and other special strains are worth examining for the presence of these secondary metabolites.