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Biotransformation of patchoulol by Cunninghamella echinulata var. elegans
Fitoterapia 109 (2016) 201–205
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Biotransformation of patchoulol by Cunninghamella echinulata var. elegans Fangfang Xu a,1, Kangsheng Liao a,1, Yuhong Liu b, Zhenbiao Zhang b, Dean Guo c,⁎, Ziren Su b,⁎, Bo Liu a,⁎ a
The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangdong Provincial Academy of Chinese Medical Sciences, Guangzhou 510006, China College of Chinese Medicines, Guangzhou University of Chinese Medicine, Guangzhou 510006, China Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai 201203, China
b c
a r t i c l e
i n f o
Article history: Received 7 November 2015 Received in revised form 31 December 2015 Accepted 4 January 2016 Available online 8 January 2016 Keywords: Biotransformation Patchoulol Cunninghamella echinulata var. elegans X-ray crystallography
a b s t r a c t Biocatalysis of patchoulol (PA) was performed by the fungus Cunninghamella echinulata var. elegans. Eight metabolites (1–8) including four new compounds were obtained, and their structures were elucidated as (5R,8S)-5,8 dihydroxypatchoulol (1), (5R*,9R*)-5,9 dihydroxypatchoulol (2), (6S*, 9S*)-6,9 dihydroxypatchoulol (3), and (4R*)-4 hydroxypatchoulol (4) by spectroscopic analysis. The absolute configuration of 1 was determined by single crystal X-ray diffraction. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Patchoulol (PA, Fig. 1) is a bioactive tricyclic sesquiterpene obtained from the dried aerial part of Pogostemon cablin (Blanco) Benth. (Labiatae), which is commonly known as “Guang-huo-xiang” in traditional Chinese medicine used for the treatment of common cold, nausea and diarrhea [1]. Previous pharmacological studies have revealed PA to be associated with inhibitory activities on neurotoxicity of β-amyloid peptide [2], enhancing cognition in scopolamine-induced learning and memory impairment of mice [3], anti-inflammatory effects in LPS-stimulated RAW 264.7 macrophages and animal models [4–5] and anti-influenza effects in in vitro and in vivo [6–7]. However, the utilization of PA as a herbal medicine is greatly limited by its poor hydrophilicity and low bioavailability [8]. The utmost stability of the patchoulol skeleton and the existence of a sterically hindered hydroxyl group bring about tremendous difficulty for structural modification of PA. Conventional chemical synthesis methods fail to give feasible practice. To overcome this difficulty, we developed a biotransformation method trying to acquire the derivations of PA.
⁎ Corresponding authors. E-mail addresses: [email protected] (D. Guo), [email protected] (Z. Su), [email protected] (B. Liu). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.fitote.2016.01.001 0367-326X/© 2016 Elsevier B.V. All rights reserved.
Biotransformation is a biochemical reaction that is catalyzed by whole cells (microorganisms, plant cells and animal cells) or by isolated enzymes, with the ability of refined region- and stereoselectivity for the oxidation of remote, unactivated C–H sites in a complex skeleton [9]. Many studies have reported that sesquiterpenoids are susceptible to biotransformation by microorganisms [10–12]. Early reports confirmed that PA could be hydroxylated and acetylated by Botrytis cinerea [13], Absidia coerulea and Mucor hiemalis [14]. In our present work, the fungus strain Cunninghamella echinulata var. elegans was found to display the hydroxylated ability to PA. Eight metabolites, four of which have not been reported previously, were isolated and identified in the biotransformation processes of PA.
2. Materials and methods 2.1. General experimental procedures Melting points were measured on an X-5 μ-melting point apparatus. Optical rotations were determined with a Rudolph Autopol I polarimeter in methanol (MeOH). IR spectra were recorded on a Perkin Elmer Spectrum 400 infrared spectrometer. NMR experiments were performed on a Bruker AV-400 spectrometer. Single Crystal Diffractometer was performed on a Bruker Smart 1000 CCD. HPLC was measured on Agilent 1260 chromatograph with evaporative light-scattering detector.
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Fig. 1. Structure of biotransformed products 1–8 of Patchoulol.
2.2. Substance C. echinulata var. elegans ATCC 9245 (American Type Culture Collection) was used. Patchoulol (PA, purity ≥99%) was provided by Professor Su Ziren (Guangzhou University of Chinese Medicine).
2.3. Microorganism and biotransformation of patchoulol by C. echinulata var. elegans Well-developed C. echinulata var. elegans was removed from the surface of Potato Dextrose Agar (Medium), suspended in 5 mL sterilized water, and inoculated in a sterile complex medium (20.0 g Sabourauddextrose, 15.0 g sucrose and 10.0 g peptone, in 1000 mL deionized water, the pH was adjusted to 6.50 using 0.1 N sodium hydroxide). Cultures were grown in twenty four flasks (500 mL medium in 2.0 L) in rotary shakers at 28 °C with shaking speed at 180 rotations per minutes for 48 h. 10 mL patchoulol (25 mg/mL, in methanol) was filtersterilized and added to each flask, and the fermentation continued for a further period of 14 days.
2.4. Isolation and purification of metabolites The cultures were harvested, the broth and mycelia were separated in a Buchner funnel. The mycelia were discarded while the culture broth (12.0 L) was extracted with three equal volumes of ethyl acetate (EtOAc) and concentrated by atmospheric distillation. Then the dark brown residues (2.5 g) were subjected to silica gel column chromatography eluted with a petroleum ether/EtOAc gradient to afford 18 fractions. Fraction 8 (120 mg) was purified using semi-preparative HPLC (MeOH: H2O, 40: 60, flow rate, 3.0 mL/min) to yield compounds 1 and 2 (169 mg, 1:2 = 7.5: 1, tR 10.5 min), 3 (3.3 mg, purity N 90%, tR 13.3 min) and 4 (8.0 mg, purity N90%, tR 16.5 min). Fraction 10 was loaded onto Sephadex LH-20 CC (2.5 cm ∗ 50 cm) and eluted with CHCl3/MeOH (1:1, 300 mL) to give 5 (8.2 mg). Fraction 5 (305 mg) was further purified using semi-prepared HPLC (MeOH: H2O, 70:30, flow rate, 3.0 mL/min) to afford 6 (45 mg, purity N90%, tR 15.4 min), 7 (11 mg, purity N90%, tR 19.0 min) and 8 (12.1 mg, purity N90%, tR 23.7 min).
2.4.1. (5R, 8S)-5, 8 dihydroxypatchoulol (1) and (5R*, 9R*)-5, 9 dihydroxypatchoulol (2) White solid; mp: 148–151 °C; [α]25D + 34.67 (c 0.30, MeOH); IR vmax (film): 3427, 2956, 1458, 1336, 1221, 1021 cm−1; 1H and 13C NMR, see Tables 1 and 2; HREIMS m/z = 254.1876 [M]+ (C15H26O3, calcd for 254.1876). 2.4.2. (6S*, 9S*)-6, 9 dihydroxypatchoulol (3) White solid; mp 144–146 °C; [α]25D + 45.33 (c 0.21, MeOH); IR vmax (film) 3428, 2947, 1627, 1456, 1363, 1203, 1034 cm−1; 1H and 13C NMR, see Tables 1 and 2; HREIMS m/z = 254.1877 [M]+ (C15H26O3, calcd for 254.1876). 2.4.3. (4R*)-4 hydroxypatchoulol (4) White solid; mp 156–158 °C; [α]25D + 85.33 (c 0.18, MeOH); IR vmax (film) 3303, 2908, 1650, 1457, 1361, 1220, 1050 cm−1; 1H and 13C NMR, see Tables 1 and 2; HREIMS m/z = 238.1926 [M]+ (C15H26O2, calcd for 238.1927). 2.4.4. (3R*)-3 hydroxypatchoulol (5) White solid; mp 112–114 °C; [α]25D − 62.33 (c 0.24, MeOH); IR vmax (film) 3351, 2945, 1455, 1366, 1289, 1079 cm−1; 1H and 13C NMR, see Tables 1 and 2; HREIMS m/z = 238.1926 [M]+ (C15H26O2, calcd for 238.1927). 2.4.5. (5R*)-5 hydroxypatchoulol (6) White solid; mp 105–107 °C; [α]25D − 15.76 (c 0.34, MeOH); IR vmax (film) 3502, 2955, 1630, 1455, 1323, 1203, 1090, 974 cm−1; 1H and 13C NMR, see Tables 1 and 2; HREIMS m/z = 238.1929 [M]+ (C15H26O2, calcd for 238.1927). 2.4.6. (8S*)-8 hydroxypatchoulol (7) White solid; mp 156–158 °C; [α]25D − 12.33 (c 0.24, MeOH); IR vmax (film) 3369, 2929, 1464, 1386, 1298, 1033, 977, 931 cm−1; 1H and 13C NMR, see Tables 1 and 2; HREIMS m/z = 238.1925 [M]+ (C15H26O2, calcd for 238.1927). 2.4.7. (9R*)-9 hydroxypatchoulol (8) White solid; mp 157–159 °C; [α]25D − 35.81 (c 0.21, MeOH); IR vmax (film) 3369, 2925, 1454, 1366, 1183, 1054, 986, 822 cm−1; 1H and 13C NMR, see Tables 1 and 2; HREIMS m/z = 238.1926 [M]+ (C15H26O2, calcd for 238.1927).
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Table 1 1 H-NMR data of compounds 1–8. NO 1 2 3 4 5 6 7 8 9 12 13 14 15
2
1.62, m 1.22, m 1.63, m 1.75, m 1.88, m 1.70, m 1.61, m 1.48, s 4.10, dd, J = 4.8, 8.4 Hz 2.15, dd, J = 8.8, 15.2 Hz 1.32, d, J = 15.2 Hz 0.86, d, J = 6.0 Hz 0.85, s 1.03, s, 1.09, s
3
4
1.38, m
1.58, m
1.84, m
1.83, m 1.08, m 1.16, s
1.24, m 2.32, m 1.16, m 2.10, s 0.96, s
1.60, m 1.86, m 1.58, m
2.01, m 1.14, m 1.32, s 1.78, m 1.46, m
– 1.90, m 1.28, m 1.21, s 1.86, m 1.33, m
3.83, br t, J = 4.52 Hz 1.33, s 1.61, m
6a
5
3.92, dd, J = 6.4, 13.2 Hz
3.53, dd, J = 1.1, 9.8 Hz 1.55, m
0.87, d, J = 6.8 Hz 0.79, s 1.13, s 1.22, s
0.97, d, J = 7.8 Hz 1.10, s 1.19, s 1.18, s
1.17, s 1.00, s 1.07, s 1.06, s
2.11, dd, J = 13.6, 6.4 Hz 1.55, m 3.58, ddd, J = 17.0, 11.0, 6.2 Hz 1.78, m 1.51, s 1.27, m, 0.92, m 1.16, s 1.52, m 1.44, m 1.93, m 1.08, m 1.01, d, J = 6.6 Hz 0.90, s 1.09, s 1.09, s
1.52, m
7 1.67. m
1.73, m 1.48, m 1.27, s
1.54, m 1.41, m 1.38, m 1.94, s 1.42, m 1.76, m 1.21, m 1.28, m
1.63, m
4.18, dd, J = 4.4, 8.8 Hz
1.37, m 1.82, m
1.95, d, J = 14.8 Hz 1.13, 2.30, dd, J = 9.2, 14.8 Hz d, J = 3.2 Hz 0.86, s 0.85, d, J = 6.4 Hz 0.82, d, J = 6.8 Hz 0.82, s 0.86, s 1.07, s 1.02, s 1.03, s 1.14, s
8 1.71, m 1.35, m 1.52, m 1.41, m 1.79, m 1.45, m, 1.37, m 1.27,s 2.47, m 1.14, m 4.06, dd, J = 6.0,10.0 Hz 0.82, d, J = 6.8 Hz 0.98, s 1.01, s, 1.08, s
1
H: 400 MHz in CD3OD. a CDCL3.
2.5. Crystallographic data for (5R, 8S)-5, 8 dihydroxypatchoulol (1) C15H26O3·H2O, M = 272.37, monoclinic, a = 16.02474 (18) Å, b = 7.27737 [8] Å, c = 12.83472 [14] Å, β = 102.5606 [10], V = 1460.94 [3] Å3, T = 293 [2], space group C2 (no. 5), Z = 4, μ (Cu Kα) = 0.707. Data was collected using a crystal of size ca. 0.6 × 0.4 × 0.3 mm3 on Bruker Smart 1000 CCD diffractometer. A total of 13,562 reflections measured for 20.42 b 2θ b 143.38, − 19 ≤ h ≤ 16, − 8 ≤ k ≤ 8 and −15 ≤ l ≤ 15, 2781 unique (Rint = 0.0235) which were used in all calculations. The final wR2 was 0.0754 (all data) and R1 was 0.0277 (N2 sigma (I)). Crystallographic data of 1 had been deposited in the Cambridge Crystallographic Data Centre as a supplementary publication (1, CCDC 1430962).
2.6. GC–MS analysis GC–MS analysis was carried out on an Agilent 6890–5975 GC–MS system (Agilent, USA). Chromatographic separation was achieved on a 5% phenyl methyl siloxane HP-5MS capillary column (30 m × 0.25 mm, 0.25 μm). The oven temperature was set initially at 100 °C followed by a gradient of 15 °C/min up to 180 °C (held for 1 min), then heated at a rate of 10 °C/min up to 210 °C (held for 2 min), finally achieved to 230 °C by a gradient of 5 °C/min. Nonsplit Table 2 13 C-NMR data of compounds 1–8.
13
No.
PA
1
2
3
4
5
6a
7
8
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
75.3 32.6 28.4 28.1 43.5 24.1 39.1 24.4 28.8 37.6 40.1 18.5 20.7 26.8 24.3
77.0 30.9 26.9 34.1 75.9 31.9 46.6 66.6 34.5 45.5 41.4 14.6 15.6 27.1 25.3
79.6 24.6 24.3 42.0 76.5 35.2 35.8 38.3 75.5 45.2 416 14.5 15.8 29.0 26.6
77.9 30.1 27.4 27.9 52.5 66.3 48.0 32.4 76.3 43.0 39.9 19.4 18.1 27.7 24.7
77.9 29.9 36.0 76.8 – 39.6 40.7 24.8 25.4 44.7 40.6 24.8 19.3 28.0 24.4
77.9 43.0 72.8 37.9 45.4 25.2 40.2 26.9 29.3 41.1 38.6 15.4 21.6 27.3 25.3
75.8 31.9 23.1 34.6 76.8 34.8 39.6 23.8 29.9 43.6 39.2 14.2 15.1 27.2 24.6
75.7 32.9 29.7 29.3 44.3 16.6 46.8 66.7 40.7 39.6 41.9 19.0 21.2 27.0 25.1
76.4 33.5 25.6 28.8 37.0 29.3 40.8 36.8 69.5 44.5 41.3 19.1 16.8 28.0 24.8
C: 100 MHz in CD3OD. a CDCL3.
injection (0.5 μL) was conducted and helium was used as carrier gas of 1.0 mL/min. The spectrometer was set in electron-impact (EI) mode, the ionization energy was 70 eV, the scan range was 50–500 amu and the scan rate was 0.25 s per scan. The inlet, ionization source temperatures were 230 °C and 250 °C, respectively. 3. Results and discussion 3.1. Biotransformation of patchoulol Eight metabolites (1–8, Fig. 1), including four new compounds, were obtained from the biotransformation of PA by the fungus C. echinulata var. elegans. Their structures were elucidated by spectroscopic analysis (1D, 2D NMR and MS). Compound 1 was recrystallized from the mixture of Compounds 1 and 2, had the molecular formula C15H26O3, as established by a HREIMS ion at m/z 254.1876 [M]+ (calcd for C15H26O3, 254.1876), corresponding to three degrees of unsaturation. The IR spectrum exhibited absorption band for hydroxy groups (3427 cm−1). The 1H NMR spectrum showed four methyl group signals [δH 0.86 (3 H, d, J = 6.0, H12), 0.85 (3 H, s, H-13), 1.03 (3 H, s, H-14) and 1.09 (3 H, s, H-15)], one oxygenated methine signal [δH 4.10 (1 H, dd, J = 8.4, 4.8 Hz, H-8) and a series of aliphatic methylene or methine multiplets. The 13C NMR spectrum, in combination with DEPT experiment, resolved 15 carbon resonances attributable to four methyls, three sp3 methines (one oxygenated), four sp3 methylenes and four sp3 quaternary carbons (including two oxygenated ones). The three degrees of unsaturation required that 1 was tricyclic system. The spectral data of 1 were similar to those of the known co-isolated compound 5R*-hydroxypatchoulol (6) isolated from the fermentation of Botrytis cinerea and PA [13], except for the presence of one more hydroxyl group and the absence of one methine carbon, indicating that 1 was the hydroxylated derivative of 6. HMBC correlations from the hydroxymethine protons δH 4.10 (H-8) to 45.5 (C-10), 41.4 (C-11) and 46.6 (C-7) suggested that the hydroxyl group was located at C-8. The planar structure of 1 was further established by detailed interpretation of its 2D NMR data (Fig. 2). The structure including absolute configuration (AC) of 1 was confirmed by a single crystal X-ray crystallographic analysis using anomalous scattering of Cu Kα radiation (Fig. 3). Therefore, compound 1 was determined as shown. Compounds 1 and 2 were obtained as crystalline mixture. The two compounds could not be separated chromatographically, but the NMR spectra illustrated the difference, the ratio between 1 and 2 was 7.5:1.
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Fig. 2. Selected 1H–1H COSY (
) and HMBC (
The NMR spectra of 2 were very similar to those of 1, the major differences came from carbon chemical shifts of C-8 (δc 66.6 in 1, δc 38.3 in 2) and C-9 (δc 34.5 in 1, δc 75.5 in 2), suggesting that 2 was an isomer of 1. In HMBC spectrum, the correlations between the proton signal from δ 3.92 (1 H, dd, J = 13.2, 6.4 Hz, H-9) to 79.6 (C-1), 38.3 (C-8) and 76.5 (C-5) suggested that the hydroxyl group was located at C-9, which was further confirmed by the correlations of HSQC from the protons at δ 3.92 (H-9) to 77.5 (C-9). The relative configuration of 2 was established by NOESY experiment and by comparison of its 1D NMR data with those of PA. The NOE signal between H-9 and H-15 also conformed that these two groups were the same face and were arbitrarily assigned as β. The compound 2 was identified as (5R*, 9R*)-5, 9 dihydroxypatchoulol (2). Compound 3 was also obtained as white solid, the molecular formula C15H26O3 was determined by HREIMS at m/z 254.1877 [M]+ (calcd. 254.1876). IR spectrum showed absorption band at 3428 cm−1 ascribable to hydroxyl groups. The 1H NMR spectrum showed four methyl group signals [δH 0.97 (3 H, d, J = 7.8, H-12), 1.10 (3 H, s, H-13), 1.19 (3 H, s, H-14) and 1.18 (3 H, s, H-15)], two oxygenated methine signals at δ H 3.53 (1 H, dd, J = 9.8, 1.1 Hz, H-9) and 3.83 (1 H, br t, J = 4.52 Hz, H-6). The 13C NMR and DEPT spectra showed 15 carbon resonances attributable to four methyls, five sp3 methines (two oxygenated), three sp3 methylenes, and three sp3 quaternary carbons (including one oxygenated) suggested that compound 3 have the same scaffold with PA. Compared with compound PA, the major differences were the presence of two oxygenated methine groups and the absence of two methylenes (δC 66.3 and 76.3 in 3, δC 24.1 and 28.8 in PA) indicating that 3 was the dihydroxylated derivative of PA. In HSQC spectrum, the correlations of the protons at δH 3.53 (H-9) to C-9 (δc 76.3), suggested that one hydroxyl group was located at C-9, which was further confirmed by the correlations from the proton signal at δH 3.53 (H-9) to 77.9 (C-1), 48.0 (C-7) and 43.0 (C-10) in HMBC spectrum. In HSQC spectrum, the correlations
) correlations of compounds 1–4.
of the protons at δH 3.83 (H-6) to C-6 (δc 66.3), suggested that another hydroxyl group was located at C-6, which was further confirmed by the correlations from the proton signal at δH 3.83 (H-6) to 27.9 (C-4), 39.9 (C-11) and 32.4 (C-8) in HMBC spectrum. The relative configuration of 3 was established by NOESY experiment and by comparison of its 1D NMR data with those of PA. The stereochemistry at C-6 and C-9 was revealed by NOE effects between H-6 and H-12 and between H-9 and H-13. In NOE spectrum, the signal between H-9 and H-13 also conformed that these two groups were the same face and arbitrarily assigned as α.The NOE effects between H-6 and H-12 indicated that the orientation of H-6 was β. The compound 3 was identified as (6S*, 9S*)-6, 9 dihydroxypatchoulol (3). Compound 4 was white solid, the molecular formula C15H26O2 was determined by HREIMS at m/z 238.1926 [M]+ (calcd. 238.1927). IR spectrum showed absorption bands at 3303 cm−1 ascribable to hydroxyl groups. The 1H NMR spectrum showed four methyl group signals [δ 1.00 (3 H, s, H-13), 1.06 (3 H, s, H-15), 1.07 (3 H, s, H-14) and 1.17 (3 H, s, H-12)]. The 13C NMR and DEPT spectra showed 14 carbon resonances attributable to four methyls, one sp3 methine, five sp3 methylenes, and four sp3 quaternary carbons (including two oxygenated), one carbon signal was missing. Compared with PA, the major differences were the presence of one oxygenated quaternary carbon and the absence of one methine group (δC 76.8 in 4, δC 28.1 in PA) indicating that 4 was the hydroxylated derivative of PA. The correlation from δH 1.17 (H-12) to 76.8 (C-4) in HMBC spectrum, and the multiplicity of H3-12 changed from a doublet to a singlet, suggested that the hydroxyl groups was located at C-4. The relative configuration of 4 was established by NOESY experiment and by comparison of its 1D NMR data with those of PA. The NOE signal between H-12 and H-14 also conformed that these two groups were the same face and were arbitrarily assigned H3-12 as β. The compound 4 was identified as (4R*)-4 hydroxypatchoulol. The compounds (3R*)-3 hydroxypatchoulol (5), (5R*)-5 hydroxypatchoulol (6), (8S*)-8 hydroxypatchoulol (7) and (9R*)-9 hydroxypatchoulol (8) were readily identified by comparing their spectroscopic data (IR, 1H-NMR, 13C-NMR, and HREIMS) with those reported in the literature [13]. 3.2. Metabolic profile by GC–MS analysis After 14 days incubation, GC–MS was used to analyze the metabolites production in the broths of the biotransformation of patchoulol by C. echinulata var. elegans, as presented in Fig. 4. The results revealed that eight metabolites as well as patchoulol were detected, compound 6 was the major product, 5, 7 and 8 were minor products, while metabolites 1–4 were in minimum yield. The analysis result was different from the actual mass of isolated products, maybe because of the exclusion of biocatalyzed products in unpurified fractions and the volatility of the products. 4. Conclusions
Fig. 3. Crystal structure of (5R, 8S)-5, 8 dihydroxypatchoulol (1).
In this paper eight metabolites, of which four have not been reported previously, were obtained and identified after incubation by C. echinulata var. elegans. All of them are the hydroxylated derivatives
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Fig. 4. GC–MS chromatogram analysis of biotransformation of patchoulol by C. echinulata var. elegans.
of PA. The highly stereospecific hydroxylation of PA by C. echinulata var. elegans was verified as an effective method for structural modifications, which could be used as a tool for obtaining hydroxylated derivatives of PA to improve its water-solubility. However, the relationship between P450 enzymes and products needs further research. Conflict of interest The authors declare no conflict interest. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 81202398), the Specific Research Construction Project of National Clinical Research Base of TCM (No.JDZX2012010). References [1] Board of Pharmacopoeia of P. R. China, Pharmacopoeia of the People's Republic of China, Chinese Edition 2015 Chemical Industry Press, Beijing, 2015 45 (Part I). [2] X.W. Huang, L. Bai, F.H. Xu, Y.J. Wu, Inhibitory activities of patchouli alcohol on neurotoxicity of β-amyloid peptide, Med. J. Chin. PLA 24 (2008) 338–340. [3] X.W. Huang, R.T. Liu, Q.J. Lu, Patchouli alcohol on memory impairment induced by scopolamine learning and memory function in mice, J. Chin. Med. Mater. 40 (2009) 1431–1433.
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Biotransformation of patchoulol by Cunninghamella echinulata var. elegans Fangfang Xu a,1, Kangsheng Liao a,1, Yuhong Liu b, Zhenbiao Zhang b, Dean Guo c,⁎, Ziren Su b,⁎, Bo Liu a,⁎ a
The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangdong Provincial Academy of Chinese Medical Sciences, Guangzhou 510006, China College of Chinese Medicines, Guangzhou University of Chinese Medicine, Guangzhou 510006, China Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai 201203, China
b c
a r t i c l e
i n f o
Article history: Received 7 November 2015 Received in revised form 31 December 2015 Accepted 4 January 2016 Available online 8 January 2016 Keywords: Biotransformation Patchoulol Cunninghamella echinulata var. elegans X-ray crystallography
a b s t r a c t Biocatalysis of patchoulol (PA) was performed by the fungus Cunninghamella echinulata var. elegans. Eight metabolites (1–8) including four new compounds were obtained, and their structures were elucidated as (5R,8S)-5,8 dihydroxypatchoulol (1), (5R*,9R*)-5,9 dihydroxypatchoulol (2), (6S*, 9S*)-6,9 dihydroxypatchoulol (3), and (4R*)-4 hydroxypatchoulol (4) by spectroscopic analysis. The absolute configuration of 1 was determined by single crystal X-ray diffraction. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Patchoulol (PA, Fig. 1) is a bioactive tricyclic sesquiterpene obtained from the dried aerial part of Pogostemon cablin (Blanco) Benth. (Labiatae), which is commonly known as “Guang-huo-xiang” in traditional Chinese medicine used for the treatment of common cold, nausea and diarrhea [1]. Previous pharmacological studies have revealed PA to be associated with inhibitory activities on neurotoxicity of β-amyloid peptide [2], enhancing cognition in scopolamine-induced learning and memory impairment of mice [3], anti-inflammatory effects in LPS-stimulated RAW 264.7 macrophages and animal models [4–5] and anti-influenza effects in in vitro and in vivo [6–7]. However, the utilization of PA as a herbal medicine is greatly limited by its poor hydrophilicity and low bioavailability [8]. The utmost stability of the patchoulol skeleton and the existence of a sterically hindered hydroxyl group bring about tremendous difficulty for structural modification of PA. Conventional chemical synthesis methods fail to give feasible practice. To overcome this difficulty, we developed a biotransformation method trying to acquire the derivations of PA.
⁎ Corresponding authors. E-mail addresses: [email protected] (D. Guo), [email protected] (Z. Su), [email protected] (B. Liu). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.fitote.2016.01.001 0367-326X/© 2016 Elsevier B.V. All rights reserved.
Biotransformation is a biochemical reaction that is catalyzed by whole cells (microorganisms, plant cells and animal cells) or by isolated enzymes, with the ability of refined region- and stereoselectivity for the oxidation of remote, unactivated C–H sites in a complex skeleton [9]. Many studies have reported that sesquiterpenoids are susceptible to biotransformation by microorganisms [10–12]. Early reports confirmed that PA could be hydroxylated and acetylated by Botrytis cinerea [13], Absidia coerulea and Mucor hiemalis [14]. In our present work, the fungus strain Cunninghamella echinulata var. elegans was found to display the hydroxylated ability to PA. Eight metabolites, four of which have not been reported previously, were isolated and identified in the biotransformation processes of PA.
2. Materials and methods 2.1. General experimental procedures Melting points were measured on an X-5 μ-melting point apparatus. Optical rotations were determined with a Rudolph Autopol I polarimeter in methanol (MeOH). IR spectra were recorded on a Perkin Elmer Spectrum 400 infrared spectrometer. NMR experiments were performed on a Bruker AV-400 spectrometer. Single Crystal Diffractometer was performed on a Bruker Smart 1000 CCD. HPLC was measured on Agilent 1260 chromatograph with evaporative light-scattering detector.
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Fig. 1. Structure of biotransformed products 1–8 of Patchoulol.
2.2. Substance C. echinulata var. elegans ATCC 9245 (American Type Culture Collection) was used. Patchoulol (PA, purity ≥99%) was provided by Professor Su Ziren (Guangzhou University of Chinese Medicine).
2.3. Microorganism and biotransformation of patchoulol by C. echinulata var. elegans Well-developed C. echinulata var. elegans was removed from the surface of Potato Dextrose Agar (Medium), suspended in 5 mL sterilized water, and inoculated in a sterile complex medium (20.0 g Sabourauddextrose, 15.0 g sucrose and 10.0 g peptone, in 1000 mL deionized water, the pH was adjusted to 6.50 using 0.1 N sodium hydroxide). Cultures were grown in twenty four flasks (500 mL medium in 2.0 L) in rotary shakers at 28 °C with shaking speed at 180 rotations per minutes for 48 h. 10 mL patchoulol (25 mg/mL, in methanol) was filtersterilized and added to each flask, and the fermentation continued for a further period of 14 days.
2.4. Isolation and purification of metabolites The cultures were harvested, the broth and mycelia were separated in a Buchner funnel. The mycelia were discarded while the culture broth (12.0 L) was extracted with three equal volumes of ethyl acetate (EtOAc) and concentrated by atmospheric distillation. Then the dark brown residues (2.5 g) were subjected to silica gel column chromatography eluted with a petroleum ether/EtOAc gradient to afford 18 fractions. Fraction 8 (120 mg) was purified using semi-preparative HPLC (MeOH: H2O, 40: 60, flow rate, 3.0 mL/min) to yield compounds 1 and 2 (169 mg, 1:2 = 7.5: 1, tR 10.5 min), 3 (3.3 mg, purity N 90%, tR 13.3 min) and 4 (8.0 mg, purity N90%, tR 16.5 min). Fraction 10 was loaded onto Sephadex LH-20 CC (2.5 cm ∗ 50 cm) and eluted with CHCl3/MeOH (1:1, 300 mL) to give 5 (8.2 mg). Fraction 5 (305 mg) was further purified using semi-prepared HPLC (MeOH: H2O, 70:30, flow rate, 3.0 mL/min) to afford 6 (45 mg, purity N90%, tR 15.4 min), 7 (11 mg, purity N90%, tR 19.0 min) and 8 (12.1 mg, purity N90%, tR 23.7 min).
2.4.1. (5R, 8S)-5, 8 dihydroxypatchoulol (1) and (5R*, 9R*)-5, 9 dihydroxypatchoulol (2) White solid; mp: 148–151 °C; [α]25D + 34.67 (c 0.30, MeOH); IR vmax (film): 3427, 2956, 1458, 1336, 1221, 1021 cm−1; 1H and 13C NMR, see Tables 1 and 2; HREIMS m/z = 254.1876 [M]+ (C15H26O3, calcd for 254.1876). 2.4.2. (6S*, 9S*)-6, 9 dihydroxypatchoulol (3) White solid; mp 144–146 °C; [α]25D + 45.33 (c 0.21, MeOH); IR vmax (film) 3428, 2947, 1627, 1456, 1363, 1203, 1034 cm−1; 1H and 13C NMR, see Tables 1 and 2; HREIMS m/z = 254.1877 [M]+ (C15H26O3, calcd for 254.1876). 2.4.3. (4R*)-4 hydroxypatchoulol (4) White solid; mp 156–158 °C; [α]25D + 85.33 (c 0.18, MeOH); IR vmax (film) 3303, 2908, 1650, 1457, 1361, 1220, 1050 cm−1; 1H and 13C NMR, see Tables 1 and 2; HREIMS m/z = 238.1926 [M]+ (C15H26O2, calcd for 238.1927). 2.4.4. (3R*)-3 hydroxypatchoulol (5) White solid; mp 112–114 °C; [α]25D − 62.33 (c 0.24, MeOH); IR vmax (film) 3351, 2945, 1455, 1366, 1289, 1079 cm−1; 1H and 13C NMR, see Tables 1 and 2; HREIMS m/z = 238.1926 [M]+ (C15H26O2, calcd for 238.1927). 2.4.5. (5R*)-5 hydroxypatchoulol (6) White solid; mp 105–107 °C; [α]25D − 15.76 (c 0.34, MeOH); IR vmax (film) 3502, 2955, 1630, 1455, 1323, 1203, 1090, 974 cm−1; 1H and 13C NMR, see Tables 1 and 2; HREIMS m/z = 238.1929 [M]+ (C15H26O2, calcd for 238.1927). 2.4.6. (8S*)-8 hydroxypatchoulol (7) White solid; mp 156–158 °C; [α]25D − 12.33 (c 0.24, MeOH); IR vmax (film) 3369, 2929, 1464, 1386, 1298, 1033, 977, 931 cm−1; 1H and 13C NMR, see Tables 1 and 2; HREIMS m/z = 238.1925 [M]+ (C15H26O2, calcd for 238.1927). 2.4.7. (9R*)-9 hydroxypatchoulol (8) White solid; mp 157–159 °C; [α]25D − 35.81 (c 0.21, MeOH); IR vmax (film) 3369, 2925, 1454, 1366, 1183, 1054, 986, 822 cm−1; 1H and 13C NMR, see Tables 1 and 2; HREIMS m/z = 238.1926 [M]+ (C15H26O2, calcd for 238.1927).
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Table 1 1 H-NMR data of compounds 1–8. NO 1 2 3 4 5 6 7 8 9 12 13 14 15
2
1.62, m 1.22, m 1.63, m 1.75, m 1.88, m 1.70, m 1.61, m 1.48, s 4.10, dd, J = 4.8, 8.4 Hz 2.15, dd, J = 8.8, 15.2 Hz 1.32, d, J = 15.2 Hz 0.86, d, J = 6.0 Hz 0.85, s 1.03, s, 1.09, s
3
4
1.38, m
1.58, m
1.84, m
1.83, m 1.08, m 1.16, s
1.24, m 2.32, m 1.16, m 2.10, s 0.96, s
1.60, m 1.86, m 1.58, m
2.01, m 1.14, m 1.32, s 1.78, m 1.46, m
– 1.90, m 1.28, m 1.21, s 1.86, m 1.33, m
3.83, br t, J = 4.52 Hz 1.33, s 1.61, m
6a
5
3.92, dd, J = 6.4, 13.2 Hz
3.53, dd, J = 1.1, 9.8 Hz 1.55, m
0.87, d, J = 6.8 Hz 0.79, s 1.13, s 1.22, s
0.97, d, J = 7.8 Hz 1.10, s 1.19, s 1.18, s
1.17, s 1.00, s 1.07, s 1.06, s
2.11, dd, J = 13.6, 6.4 Hz 1.55, m 3.58, ddd, J = 17.0, 11.0, 6.2 Hz 1.78, m 1.51, s 1.27, m, 0.92, m 1.16, s 1.52, m 1.44, m 1.93, m 1.08, m 1.01, d, J = 6.6 Hz 0.90, s 1.09, s 1.09, s
1.52, m
7 1.67. m
1.73, m 1.48, m 1.27, s
1.54, m 1.41, m 1.38, m 1.94, s 1.42, m 1.76, m 1.21, m 1.28, m
1.63, m
4.18, dd, J = 4.4, 8.8 Hz
1.37, m 1.82, m
1.95, d, J = 14.8 Hz 1.13, 2.30, dd, J = 9.2, 14.8 Hz d, J = 3.2 Hz 0.86, s 0.85, d, J = 6.4 Hz 0.82, d, J = 6.8 Hz 0.82, s 0.86, s 1.07, s 1.02, s 1.03, s 1.14, s
8 1.71, m 1.35, m 1.52, m 1.41, m 1.79, m 1.45, m, 1.37, m 1.27,s 2.47, m 1.14, m 4.06, dd, J = 6.0,10.0 Hz 0.82, d, J = 6.8 Hz 0.98, s 1.01, s, 1.08, s
1
H: 400 MHz in CD3OD. a CDCL3.
2.5. Crystallographic data for (5R, 8S)-5, 8 dihydroxypatchoulol (1) C15H26O3·H2O, M = 272.37, monoclinic, a = 16.02474 (18) Å, b = 7.27737 [8] Å, c = 12.83472 [14] Å, β = 102.5606 [10], V = 1460.94 [3] Å3, T = 293 [2], space group C2 (no. 5), Z = 4, μ (Cu Kα) = 0.707. Data was collected using a crystal of size ca. 0.6 × 0.4 × 0.3 mm3 on Bruker Smart 1000 CCD diffractometer. A total of 13,562 reflections measured for 20.42 b 2θ b 143.38, − 19 ≤ h ≤ 16, − 8 ≤ k ≤ 8 and −15 ≤ l ≤ 15, 2781 unique (Rint = 0.0235) which were used in all calculations. The final wR2 was 0.0754 (all data) and R1 was 0.0277 (N2 sigma (I)). Crystallographic data of 1 had been deposited in the Cambridge Crystallographic Data Centre as a supplementary publication (1, CCDC 1430962).
2.6. GC–MS analysis GC–MS analysis was carried out on an Agilent 6890–5975 GC–MS system (Agilent, USA). Chromatographic separation was achieved on a 5% phenyl methyl siloxane HP-5MS capillary column (30 m × 0.25 mm, 0.25 μm). The oven temperature was set initially at 100 °C followed by a gradient of 15 °C/min up to 180 °C (held for 1 min), then heated at a rate of 10 °C/min up to 210 °C (held for 2 min), finally achieved to 230 °C by a gradient of 5 °C/min. Nonsplit Table 2 13 C-NMR data of compounds 1–8.
13
No.
PA
1
2
3
4
5
6a
7
8
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
75.3 32.6 28.4 28.1 43.5 24.1 39.1 24.4 28.8 37.6 40.1 18.5 20.7 26.8 24.3
77.0 30.9 26.9 34.1 75.9 31.9 46.6 66.6 34.5 45.5 41.4 14.6 15.6 27.1 25.3
79.6 24.6 24.3 42.0 76.5 35.2 35.8 38.3 75.5 45.2 416 14.5 15.8 29.0 26.6
77.9 30.1 27.4 27.9 52.5 66.3 48.0 32.4 76.3 43.0 39.9 19.4 18.1 27.7 24.7
77.9 29.9 36.0 76.8 – 39.6 40.7 24.8 25.4 44.7 40.6 24.8 19.3 28.0 24.4
77.9 43.0 72.8 37.9 45.4 25.2 40.2 26.9 29.3 41.1 38.6 15.4 21.6 27.3 25.3
75.8 31.9 23.1 34.6 76.8 34.8 39.6 23.8 29.9 43.6 39.2 14.2 15.1 27.2 24.6
75.7 32.9 29.7 29.3 44.3 16.6 46.8 66.7 40.7 39.6 41.9 19.0 21.2 27.0 25.1
76.4 33.5 25.6 28.8 37.0 29.3 40.8 36.8 69.5 44.5 41.3 19.1 16.8 28.0 24.8
C: 100 MHz in CD3OD. a CDCL3.
injection (0.5 μL) was conducted and helium was used as carrier gas of 1.0 mL/min. The spectrometer was set in electron-impact (EI) mode, the ionization energy was 70 eV, the scan range was 50–500 amu and the scan rate was 0.25 s per scan. The inlet, ionization source temperatures were 230 °C and 250 °C, respectively. 3. Results and discussion 3.1. Biotransformation of patchoulol Eight metabolites (1–8, Fig. 1), including four new compounds, were obtained from the biotransformation of PA by the fungus C. echinulata var. elegans. Their structures were elucidated by spectroscopic analysis (1D, 2D NMR and MS). Compound 1 was recrystallized from the mixture of Compounds 1 and 2, had the molecular formula C15H26O3, as established by a HREIMS ion at m/z 254.1876 [M]+ (calcd for C15H26O3, 254.1876), corresponding to three degrees of unsaturation. The IR spectrum exhibited absorption band for hydroxy groups (3427 cm−1). The 1H NMR spectrum showed four methyl group signals [δH 0.86 (3 H, d, J = 6.0, H12), 0.85 (3 H, s, H-13), 1.03 (3 H, s, H-14) and 1.09 (3 H, s, H-15)], one oxygenated methine signal [δH 4.10 (1 H, dd, J = 8.4, 4.8 Hz, H-8) and a series of aliphatic methylene or methine multiplets. The 13C NMR spectrum, in combination with DEPT experiment, resolved 15 carbon resonances attributable to four methyls, three sp3 methines (one oxygenated), four sp3 methylenes and four sp3 quaternary carbons (including two oxygenated ones). The three degrees of unsaturation required that 1 was tricyclic system. The spectral data of 1 were similar to those of the known co-isolated compound 5R*-hydroxypatchoulol (6) isolated from the fermentation of Botrytis cinerea and PA [13], except for the presence of one more hydroxyl group and the absence of one methine carbon, indicating that 1 was the hydroxylated derivative of 6. HMBC correlations from the hydroxymethine protons δH 4.10 (H-8) to 45.5 (C-10), 41.4 (C-11) and 46.6 (C-7) suggested that the hydroxyl group was located at C-8. The planar structure of 1 was further established by detailed interpretation of its 2D NMR data (Fig. 2). The structure including absolute configuration (AC) of 1 was confirmed by a single crystal X-ray crystallographic analysis using anomalous scattering of Cu Kα radiation (Fig. 3). Therefore, compound 1 was determined as shown. Compounds 1 and 2 were obtained as crystalline mixture. The two compounds could not be separated chromatographically, but the NMR spectra illustrated the difference, the ratio between 1 and 2 was 7.5:1.
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Fig. 2. Selected 1H–1H COSY (
) and HMBC (
The NMR spectra of 2 were very similar to those of 1, the major differences came from carbon chemical shifts of C-8 (δc 66.6 in 1, δc 38.3 in 2) and C-9 (δc 34.5 in 1, δc 75.5 in 2), suggesting that 2 was an isomer of 1. In HMBC spectrum, the correlations between the proton signal from δ 3.92 (1 H, dd, J = 13.2, 6.4 Hz, H-9) to 79.6 (C-1), 38.3 (C-8) and 76.5 (C-5) suggested that the hydroxyl group was located at C-9, which was further confirmed by the correlations of HSQC from the protons at δ 3.92 (H-9) to 77.5 (C-9). The relative configuration of 2 was established by NOESY experiment and by comparison of its 1D NMR data with those of PA. The NOE signal between H-9 and H-15 also conformed that these two groups were the same face and were arbitrarily assigned as β. The compound 2 was identified as (5R*, 9R*)-5, 9 dihydroxypatchoulol (2). Compound 3 was also obtained as white solid, the molecular formula C15H26O3 was determined by HREIMS at m/z 254.1877 [M]+ (calcd. 254.1876). IR spectrum showed absorption band at 3428 cm−1 ascribable to hydroxyl groups. The 1H NMR spectrum showed four methyl group signals [δH 0.97 (3 H, d, J = 7.8, H-12), 1.10 (3 H, s, H-13), 1.19 (3 H, s, H-14) and 1.18 (3 H, s, H-15)], two oxygenated methine signals at δ H 3.53 (1 H, dd, J = 9.8, 1.1 Hz, H-9) and 3.83 (1 H, br t, J = 4.52 Hz, H-6). The 13C NMR and DEPT spectra showed 15 carbon resonances attributable to four methyls, five sp3 methines (two oxygenated), three sp3 methylenes, and three sp3 quaternary carbons (including one oxygenated) suggested that compound 3 have the same scaffold with PA. Compared with compound PA, the major differences were the presence of two oxygenated methine groups and the absence of two methylenes (δC 66.3 and 76.3 in 3, δC 24.1 and 28.8 in PA) indicating that 3 was the dihydroxylated derivative of PA. In HSQC spectrum, the correlations of the protons at δH 3.53 (H-9) to C-9 (δc 76.3), suggested that one hydroxyl group was located at C-9, which was further confirmed by the correlations from the proton signal at δH 3.53 (H-9) to 77.9 (C-1), 48.0 (C-7) and 43.0 (C-10) in HMBC spectrum. In HSQC spectrum, the correlations
) correlations of compounds 1–4.
of the protons at δH 3.83 (H-6) to C-6 (δc 66.3), suggested that another hydroxyl group was located at C-6, which was further confirmed by the correlations from the proton signal at δH 3.83 (H-6) to 27.9 (C-4), 39.9 (C-11) and 32.4 (C-8) in HMBC spectrum. The relative configuration of 3 was established by NOESY experiment and by comparison of its 1D NMR data with those of PA. The stereochemistry at C-6 and C-9 was revealed by NOE effects between H-6 and H-12 and between H-9 and H-13. In NOE spectrum, the signal between H-9 and H-13 also conformed that these two groups were the same face and arbitrarily assigned as α.The NOE effects between H-6 and H-12 indicated that the orientation of H-6 was β. The compound 3 was identified as (6S*, 9S*)-6, 9 dihydroxypatchoulol (3). Compound 4 was white solid, the molecular formula C15H26O2 was determined by HREIMS at m/z 238.1926 [M]+ (calcd. 238.1927). IR spectrum showed absorption bands at 3303 cm−1 ascribable to hydroxyl groups. The 1H NMR spectrum showed four methyl group signals [δ 1.00 (3 H, s, H-13), 1.06 (3 H, s, H-15), 1.07 (3 H, s, H-14) and 1.17 (3 H, s, H-12)]. The 13C NMR and DEPT spectra showed 14 carbon resonances attributable to four methyls, one sp3 methine, five sp3 methylenes, and four sp3 quaternary carbons (including two oxygenated), one carbon signal was missing. Compared with PA, the major differences were the presence of one oxygenated quaternary carbon and the absence of one methine group (δC 76.8 in 4, δC 28.1 in PA) indicating that 4 was the hydroxylated derivative of PA. The correlation from δH 1.17 (H-12) to 76.8 (C-4) in HMBC spectrum, and the multiplicity of H3-12 changed from a doublet to a singlet, suggested that the hydroxyl groups was located at C-4. The relative configuration of 4 was established by NOESY experiment and by comparison of its 1D NMR data with those of PA. The NOE signal between H-12 and H-14 also conformed that these two groups were the same face and were arbitrarily assigned H3-12 as β. The compound 4 was identified as (4R*)-4 hydroxypatchoulol. The compounds (3R*)-3 hydroxypatchoulol (5), (5R*)-5 hydroxypatchoulol (6), (8S*)-8 hydroxypatchoulol (7) and (9R*)-9 hydroxypatchoulol (8) were readily identified by comparing their spectroscopic data (IR, 1H-NMR, 13C-NMR, and HREIMS) with those reported in the literature [13]. 3.2. Metabolic profile by GC–MS analysis After 14 days incubation, GC–MS was used to analyze the metabolites production in the broths of the biotransformation of patchoulol by C. echinulata var. elegans, as presented in Fig. 4. The results revealed that eight metabolites as well as patchoulol were detected, compound 6 was the major product, 5, 7 and 8 were minor products, while metabolites 1–4 were in minimum yield. The analysis result was different from the actual mass of isolated products, maybe because of the exclusion of biocatalyzed products in unpurified fractions and the volatility of the products. 4. Conclusions
Fig. 3. Crystal structure of (5R, 8S)-5, 8 dihydroxypatchoulol (1).
In this paper eight metabolites, of which four have not been reported previously, were obtained and identified after incubation by C. echinulata var. elegans. All of them are the hydroxylated derivatives
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Fig. 4. GC–MS chromatogram analysis of biotransformation of patchoulol by C. echinulata var. elegans.
of PA. The highly stereospecific hydroxylation of PA by C. echinulata var. elegans was verified as an effective method for structural modifications, which could be used as a tool for obtaining hydroxylated derivatives of PA to improve its water-solubility. However, the relationship between P450 enzymes and products needs further research. Conflict of interest The authors declare no conflict interest. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 81202398), the Specific Research Construction Project of National Clinical Research Base of TCM (No.JDZX2012010). References [1] Board of Pharmacopoeia of P. R. China, Pharmacopoeia of the People's Republic of China, Chinese Edition 2015 Chemical Industry Press, Beijing, 2015 45 (Part I). [2] X.W. Huang, L. Bai, F.H. Xu, Y.J. Wu, Inhibitory activities of patchouli alcohol on neurotoxicity of β-amyloid peptide, Med. J. Chin. PLA 24 (2008) 338–340. [3] X.W. Huang, R.T. Liu, Q.J. Lu, Patchouli alcohol on memory impairment induced by scopolamine learning and memory function in mice, J. Chin. Med. Mater. 40 (2009) 1431–1433.
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