J Nat Med (2014) 68:743–747 DOI 10.1007/s11418-014-0854-8
NOTE
Phenanthrene and phenylpropanoid constituents from the roots of Cymbidium Great Flower ‘Marylaurencin’ and their antimicrobial activity Kazuko Yoshikawa • Chihiro Baba • Kanako Iseki • Takuya Ito • Yoshinori Asakawa Sachiko Kawano • Toshihiro Hashimoto
•
Received: 4 April 2014 / Accepted: 16 June 2014 / Published online: 16 July 2014 Ó The Japanese Society of Pharmacognosy and Springer Japan 2014
Abstract Two new phenanthrenes, and one new phenylpropanoid, named ephemeranthoquinone C (1), and marylaurencinols C (2) and D (3), were isolated from the roots of Cymbidium Great Flower ‘Marylaurencin’, respectively. These structures were determined on the basis of 2D NMR experiments. The compounds were tested for antimicrobial activity against Bacillus subtilis, Klebsiella pneumoniae, and Trichophyton rubrum. Keywords Cymbidium Great Flower ‘Marylaurencin’ Orchidaceae Ephemeranthoquinone Marylaurencinol Antimicrobial activity Trichophyton rubrum
Introduction Species of the Orchidaceae are rich sources of aromatic compounds, such as simple benzene and bibenzyl derivatives, as well as monomeric, dimeric, and triphenanthrenes. Some of these compounds have been reported as having cytotoxic, immunoregulatory, spasmolytic, antimicrobial, anti-allergic, and anti-inflammatory activities [1]. In the course of our study of bioactive substances from the Orchidaceae, we previously reported four new phenanthrene derivatives, ephemeranthoquinone B, marylaurencinols A and B, and marylaurencinoside A, together with six known K. Yoshikawa (&) C. Baba K. Iseki T. Ito Y. Asakawa T. Hashimoto Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-Cho, Tokushima 770-8514, Japan e-mail: [email protected] S. Kawano Kawano-Mericlone Co. Ltd., Wakimachi, Tokushima 779-3604, Japan
phenanthrenes and the antibacterial activity of the fresh roots, and also novel glucopyranosyloxybenzyl derivatives of 2-benzylmalic acid and eucomic acid, marylaurencinosides D and E, respectively, along with eight known aromatic compounds from the fresh flowers of the well-known cultivated cymbidium, Cymbidium Great Flower ‘Marylaurencin’ [2, 3]. We continued the search for new antimicrobial metabolites of the roots. The MeOH extract was partitioned into an AcOEt-H2O mixture to afford AcOEtand H2O-soluble portions. The resulting AcOEt extract showed antimicrobial activity against Bacillus subtilis at 18.75 lg/mL. Further separation of the EtOAc-soluble portion by ordinary-phase and reversed-phase silica gels afforded two new phenanthrenes (1, 2), and one new phenylpropanoid (3), along with two known phenanthrens, 2,4,7,8-tetramethoxy-3-hydroxy-phenanthrene (4) [4], and calanquinone A (5) [5], shown in Fig. 1. This paper describes the isolation, purification, and structural elucidation of 1–3 determined primarily by extensive NMR experiments, and their antimicrobial activities.
Results and discussion Ephemeranthoquinone C (1) was obtained as an amorphous powder. The molecular formula was determined as C17H16O6 with an [M]? peak at m/z 316.0946 by HR-EIMS in conjunction with NMR data analysis. The IR spectrum of 1 revealed absorption bands due to a hydroxy group (3063 cm-1), conjugated carbonyl groups (1670, 1639 cm-1), and aromatic rings (1595, 1452 cm-1). The UV spectrum supported the presence of the aromatic ring (kmax 244, 285 nm). In the 1H-NMR spectrum, a pair of ortho-coupled protons at d 7.81 (d, J = 8.5 Hz), and 7.86 (d, J = 8.5 Hz), a long-range coupled aromatic proton at d
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Fig. 1 Chemical structures of compounds 1–5
OCH3
OH
OH
O OCH3
H3CO OH
HO O
H3CO
OCH3
OCH3
HO
H3CO
H3CO
1
OCH3
2 OCH3
4
OAc
O O
OH H3CO
H3CO
OCH3
O OAc
H3CO
5
H3CO
3 OH
6.99 (br s), one oxymethine at d 4.65 (dd, J = 8.4, 5.8 Hz), three methoxy groups at d 3.61 (s), 4.07 (s), and 4.08 (s), two of which were aromatic substituted, and methylene protons at d 3.13 (dd, J = 17.5, 8.4 Hz) and 3.40 (dd, J = 17.5, 5.8 Hz) were observed. The 13C-NMR spectrum also showed the presence of two carbonyl carbons (d 192.6, 201.3), one methylene (d 46.8) and oxymethine carbon (d 82.3) each, and 10 sp2 hybridized carbons (d 101.9, 120.4, 120.5, 130.9, 132.1, 133.4, 136.3, 139.3, 141.3, 150.6). The COSY spectrum showed two partial structures, indicated by thick lines in Fig. 2. The HMQC correlations were analyzed via an HMBC spectrum that showed correlations of H2-2 to C-1, and C-4; H-8 to C-6, C-7, and C-8a; H-9 to C-4b, C-10a; H-10 to C1, C-4a, and C-10a, and a methoxy group at d 3.61 to C-3; a methoxy group at d 4.08 to C-5; and a methoxy group at d 4.07 to C-7. NOE correlations were observed between H2-2 (d 3.13, 3.40) and 3-OCH3 (d 3.61), between 7-OCH3 (d 4.07) and H-8 (d 6.99), and between H-8 and H-9 (d 7.81). These results confirmed that C-1 and C-4, C-5 and C-7, and C-6 are substituted by carbonyl groups, methoxy groups, and a hydroxyl group, respectively. Furthermore, the presence of one remaining methoxyl was established at C-3 position by an important NOESY correlation between 5-OCH3 (d 4.08) and 3-OCH3 (d 3.61). On the basis of the data obtained, the structure of 1 was determined as 6-hydroxy-3,5,7-trimethoxy-2,3-dihydrophenanthrene-1,4-dione. Marylaurencinol C (2) was obtained as an amorphous powder. The molecular formula was determined as C16H16O5 with an [M]? peak at m/z 288.0992 by HREIMS, requiring nine degrees of unsaturation. The IR spectrum of 2 exhibited strong absorption bands at 3348, 3215, 1616, 1582, 1501, 1452, 1329, 1236, and 1094 cm-1, indicating the absence of carbonyl groups comparable to those in 1. The 1H-NMR spectrum showed the presence of two methylene protons [d 2.49 (2H, m), 2.57 (2H, m)], two
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Fig. 2 COSY, selected HMBC and NOESY correlations of 1
methoxy groups [d 3.55 (s), 3.82 (s)], and a pair of orthocoupled protons [d 6.64 (d, J = 8.7 Hz), 6.69 (d, J = 8.7 Hz)], an isolated aromatic proton [d 6.82 (br s)], in addition to three hydroxyl groups [d 8.19 (s), 8.70 (s), and 8.77 (s)]. The 13C-NMR spectrum also revealed the presence of two methoxy carbons (d 55.9, 61.2), two methylene carbons (d 22.3, 29.5), and 12 aromatic carbons, and the absence of carbonyl carbons (Table 1). Therefore, 2 was deduced to have a pentasubstituted 9,10-dihydrophenanthrene derivative. The COSY and HMQC spectra exhibited the presence of partial structures, as shown in Fig. 3. The positions of the substituents in 2 were also confirmed by analysis of the HMBC and NOESY spectra. The negative sign of the CD band at 226 nm is diagnostic of the M helicity of the biaryl chromophore [6]. Thus, the structure of 2 was elucidated to be 3,5,6-trihydroxy-2,4dimethoxy-9,10-dihydrophenanthrene. Marylaurencinol D (3), obtained as an amorphous powder, was assigned the molecular formula C25H30O9, shown at m/z 474.1885 by its HR-EIMS. The 13C-NMR and HMQC spectra showed the signals of three methoxy groups (d 55.8, 56.0, 57.1), two oxymethylene carbons (d
J Nat Med (2014) 68:743–747 Table 1 1H- and
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13
Marylaurencinol C (1) Position
dC
1
201.3 (s)
2
46.8 (t)
Table 2 1H- and
C-NMR spectral data for 1 and 2
13
C-NMR spectral data for 3 in CDCl3
Marylaurencinol D (3)
Marylaurencinol D (2)
dH (J Hz)
dC
dH (J Hz)
Position
dC
dH (J Hz)
108.3 (d)
6.82 (br s)
1
83.2 (d)
4.42 (d, 5.8)
3.13 (dd, 17.5, 8.4)
147.4 (s)
2
81.6 (d)
4.49 (ddd, 6.2, 5.8, 4.0)
3
63.7 (t)
4.05 (dd,11.8, 6.2)
3.40 (dd, 17.5, 5.8) 82.3 (d)
4
192.6 (s)
143.8 (s)
10
129.5 (s)
4a
136.3 (s)
118.3 (s)
20
109.6 (d)
4b 5
133.4 (s) 141.3 (s)
121.0 (s) 145.9 (s)
30 40
150.7 (s) 145.7 (s)
6
139.3 (s)
146.4 (s)
50
114.1 (d)
6.88 (d, 8.5)
7
150.6 (s)
116.5 (d)
6.64 (d, 8.7)
60
120.8 (d)
6.84 (dd, 8.5, 1.5)
8
101.9 (d)
115.3 (d)
6.69 (d, 8.7)
100
148.6 (s)
8a
120.4 (s)
200
146.8 (s)
9
130.9 (d)
2.57 (2H,m)
300
110.0 (d)
2.49 (2H,m)
00
4
130.9 (s)
400 a
134.1 (d)
6.57 (d, 15.8)
400 b
121.7 (d)
6.16 (dt, 15.8, 6.8)
400 c
65.2 (t)
4.69 (d, 6.8)
10
120.5 (d)
10a
132.1 (s)
4.65 (dd, 8.4, 5.8)
4.22 (dd,11.8, 4.0)
3
6.99 (br s)
125.5 (s) 7.81 (d, 8.5) 7.86 (d, 8.5)
22.3 (d) 29.5 (d) 130.5 (s)
2-OCH3 3-OCH3
137.7 (s)
55.9 (q) 58.0 (q)
3.82 (s)
3.61 (s)
4-OCH3
61.2 (q)
6.93 (d, 1.5)
6.86 (d, 1.5)
4.71 (d, 6.8)
3.55 (s)
5-OCH3
59.5 (q)
4.08 (s)
500
119.8 (d)
6.88 (dd, 8.5, 1.5)
7-OCH3
56.4 (q)
4.07 (s)
600
117.8 (q)
6.91 (d, 8.5)
3-OH
8.70 (s)
1-OCH3
57.1 (q)
3.28 (s)
5-OH 6-OH
8.19 (s) 8.77 (s)
30 -OCH3 200 -OCH3
55.8 (q) 56.0 (q)
3.84 (s) 3.87 (s)
3-Ac
20.7 (q), 170.6 (s)
1.97 (s)
21.0 (q), 170.9 (s)
2.10 (s)
1 in CDCl3. 2 in DMSO-d6
00
4 c-Ac
Fig. 3 COSY, selected HMBC and ROESY correlations of 2
63.7, 65.2), two oxymethine carbons (d 81.6, 83.2), and 14 sp2 hybridized carbons, in addition to two acetyl groups (d 20.7, 170.6 and d 21.0, 170.9). The 1H-NMR spectrum of 3 showed the typical spin systems of two 1,3,4-substituted benzene rings (Table 2). The gross structure of 3 was determined by the same strategy as 2. The COSY and HMBC correlations exhibited the presence of guaiacylglycerol (unit A) and coniferyl alcohol (unit B) moieties, as shown in Fig. 4. The substitution patterns of two aromatic
Fig. 4 COSY, selected HMBC and NOESY correlations of 3
rings were confirmed by the following NOEs, H-20 (d 6.93)/OMe (d 3.84), and H-300 (d 6.86)/OMe (d 3.87). Two phenylpropanoid units in 3 were connected through an
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Table 3 Antimicrobial activity against Trichophyton rubrum of 1–5 Compd
Inhibition zone (mm)
Extracta,b
10.7
Ephemeranthoquinone C (1)b
12.7
Marylaurencinol C (2)c
15.7
Marylaurencinol D (3)
b
–
Compound 4 (4)b
–
Calanquinone A (5)b
–
Ketoconazoled
16.7
a
EtOAc-soluble portion of MeOH extract
b
100 lg/disk (mm)
c
10 lg/disk (mm)
d
5 lg/disk (mm)
ether bond at C-2 and C-100 by the HMBC correlation from H-2 (d 4.49) to C-100 (d 148.6). Furthermore, the HMBC correlations from the aliphatic methoxy group at d 3.28 (s) to C-1 (d 83.2), from H2-3 (d 4.05, 4.22) at the acylation site to carbonyl carbon at d 170.6, and from H2-400 c (d 4.69, 4.71) at the acylation site to carbonyl carbon at d 170.9 could confirm the presence of the methoxy group at C-1, and two acetyl groups at C-3 and C-400 c, respectively. Thus, from the above findings, the structure of 3 was concluded to be as shown in Fig. 1. The isolated compounds (1-5) were tested for antimicrobial activity against B. subtilis, K. pneumonia, and T. rubrum. Compounds 1, 2, 3, and 5 exhibited weak activities against B. subtilis with MICs of 29.7, 26.0, 79.1, and 59.7 lM, respectively, and 0.36 lM for the positive control, ampicillin. However, none was active against K. pneumonia. On the other hand, compounds 1 and 2 showed antifungal activity against T. rubrum (Table 3). It is interesting to note that 2 showed half the activity of ketoconazole. In conclusion, our current chemical investigation yielded two new and two known phenanthrene derivatives, and a new phenylpropanoid derivative, some of which revealed the antimicrobial activity of this plant. This is the first report of phenanthrene derivatives showing antimicrobial activity against T. rubrum from a natural source [7], and therefore it could be lead to the development of new topical antifungal agents.
spectrophotometer, respectively. NMR spectra were recorded on a Varian UNITY 600 spectrometer. The chemical shifts are given as d (ppm) in CDCl3 or DMSO-d6 solution, using tetramethylsilane (TMS) as the internal standard. NMR experiments included COSY, HMQC, HMBC, and ROESY. Coupling constants (J values) are given in Hz. HR-FAB-MS was measured on a JEOL JMS700 MStation. For HPLC column chromatography, COSMOSIL 5C18-AR-II (Nacalai Tesque, Inc., Kyoto, Japan, 20 mm i.d. 9 250 mm) was used. TLC was performed on pre-coated silica gel 60F254 (Merck). Spots were detected by examining plates sprayed with p-anisaldehyde/H2SO4/ MeOH reagent followed by heating on a hot plate. Plant material Cymbidium Great Flower ‘Marylaurencin’ (Ministry of Agriculture, Forestry and Fisheries of Japan, seed registration No. 2841) was cultivated and harvested in February 2012 at Kawano Mericlone Co., Ltd. (Tokushima prefecture, Japan), and identified by one of the authors (S. K.). A voucher specimen (TB 5430) has been deposited in the Herbarium of the Department of Pharmacognosy, Tokushima Bunri University, Tokushima, Japan. Extraction and isolation Fresh roots (8.0 kg) were extracted with MeOH at room temperature for 1 week and concentrated in vacuo. The MeOH extract was partitioned into an EtOAc-H2O mixture to afford EtOAc- and H2O-soluble portions. The EtOAc extract (42 g) was subjected to silica gel column chromatography with n-hexane–EtOAc-MeOH (10:1:0–0:1:10) to afford eight fractions (Fr.1-8). Fr. 3 (5.69 g) was chromatographed further over silica gel column chromatography using n-hexane–EtOAc (7:3) to afford five subfractions (Fr. 3-A-E). Fr. 3-C was purified by RP-HPLC (60 % aq. MeOH) to yield 4 (20 mg). Fr. 4 (4.49 g) was further subjected to silica gel column chromatography with nhexane–EtOAc (6:4) and separated again using RP-HPLC (70 % aq. MeOH) to furnish 1 (72 mg), 2 (380 mg) and 3 (17 mg). Fr. 5 (1.15 g) was subjected to silica gel column chromatography with n-hexane–EtOAc (6:4) and purified using RP-HPLC (55 % aq. MeOH) to furnish 5 (14.5 mg). Ephemeranthoquinone C (1)
Experimental General Optical rotation was performed on a JASCO P-1030 digital polarimeter. IR and UV spectra were measured on a JASCO FT/IR 410 and Shimadzu UV-1650PC
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An amorphous powder; ½a25 D -0.55 (c 1.3, CHCl3); UV (MeOH) kmax (log e) 244 (4.10), 285 (4.10) nm; FT-IR (film) mmax 3063, 2993, 1670, 1639, 1595, 1452, 1246 cm-1; CD (MeOH) nm (De):285 (?0.50), 245 (?1.08); 1H- and 13C-NMR data, see Table 1; HR-EIMS m/z 316.0946 (calcd for C17H16O6, 316.0947).
J Nat Med (2014) 68:743–747
Marylaurencinol C (2) An amorphous powder; ½a25 D ?0.58 (c 0.35, CHCl3/MeOH 1:1); UV (MeOH) kmax (log e) 229 (4.05), 272 (3.99), 309 (3.96) nm; FT-IR (film) mmax 3348, 3215, 1616, 1582, 1501, 1452, 1329, 1236, 1094 cm-1; CD (MeOH) nm (De): 226 (–7.90); 1H- and 13C-NMR data, see Table 1; HREIMS m/z 288.0992 (calcd for C16H16O5, 288.0998).
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(3.9 % PDA; Nissui, Japan) and incubated at 28 °C for 5 days. A stock solution of crude extracts or samples was prepared at 10 mg/mL in DMSO and impregnated circular paper disks (8 mm; Toyo Roshi Kaisha, Ltd., Japan) were placed on the surface of each PDA plate. Then the plates were incubated at 28 °C for 5 days and inhibition zone diameters (IZD) were measured in millimeters. Ketoconazol (LKT Laboratories, Inc., USA) was used as a reference reagent for the bacterial strains.
Marylaurencinol D (3) An amorphous powder; ½a25 D –2.94 (c 0.7, CHCl3); UV (MeOH) kmax (log e) 214 (3.96), 266 (4.17) nm; FT-IR (film) mmax 3420, 1730, 1600, 1515, 1252, 1097, 1047 cm-1; 1H- and 13C-NMR data, see Table 2; HREIMS m/z 474.1885 (calcd for C25H30O9 [M]?, 474.1890). Antimicrobial assay B. subtilis NBRC 3134 and K. pneumoniae NBRC 3512 were used for testing antimicrobial activity. These strains were tested using micro dilution assays and MIC values were determined. Bacterial strains were inoculated on YP agar plates [0.5 % peptone (Becton, Dickinson and Co., USA), 0.3 % yeast extract (Becton, Dickinson and Co.), 0.1 % MgSO47H2O and 2 % agar (Nacalai Tesque, Japan)] and incubated at 37 °C (B. subtilis) and 27 °C (K. pneumoniae) for 12 h. A stock solution of crude extracts or samples was prepared at 10 mg/mL in DMSO and further diluted to varying concentrations in 96-well plates which contained microbial strains incubated in YP medium. Each plate was further incubated at 37 °C (B. subtilis) and 27 °C (K. pneumoniae) overnight and ampicillin (Nacalai Tesque) was used as a reference reagent for the bacterial strains. T. rubrum NBRC 5807 was also tested using disk-diffusion assays. Bacterial strains were inoculated onto potato dextrose agar (PDA) plates
References 1. Kova´cs A, Vasas A, Hohmonn J (2008) Natural phenenthrenes and their biological activity. Phytochemistry 69:1084–1110 2. Yoshikawa K, Itou T, Iseki K, Baba T, Imagawa H, Morita H, Kawano S, Asakawa Y, Hashimoto T (2012) Phenanthrene derivatives from Cymbidium Great Flower ‘Marylaurencin’ and their biological activities. J Nat Prod 75:605–609 3. Yoshikawa K, Okahuji M, Ito T, Asakawa Y, Kawano S, Hashimoto T (2014) Two novel aromatic glucosides, marylaurencinosides D and E from the fresh flower of Cymbidium Great Flower ‘Marylaurencin’. J Nat Med 68:455–458 4. Reisch J, Bathory M, Novak I, Szendrei K (1970) Structure and biochemistry of natural phenanthrene derivatives. Herba Hung 9:43–48 5. Lee CH, Chang FP, Yen MH, Yu D, Liu YN, Bastow KF, MorrisNatschke SL, Wu YC, Lee KH (2009) Cytotoxic Phenanthrenequinones and 9,10-dihydrophenenthrenes from Calanthe arisanensis. J Nat Prod 72:210–213 6. Gawron´ski J, Grycz P, Kwit M, Rychlewska U (2002) Circular dichroism of 9,10-dihydrophenanthrene derivative reveals both the absolute configuration and conformation: a novel approach to Mislow’s helicity rule. Chem Eur J 8:4210–4215 7. Zacchino SA, Lo´pez SN, Pezzenati GD, Furla´n RL, Santecchia CB, Mun˜oz L, Giannini FA, Rodrı´guez AM, Enriz RD (1999) In vitro evaluation of antifungal properities of phenylpropanoids and related comounds acting againt dermatophytes. J Nat Prod 62:1353–1357
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NOTE
Phenanthrene and phenylpropanoid constituents from the roots of Cymbidium Great Flower ‘Marylaurencin’ and their antimicrobial activity Kazuko Yoshikawa • Chihiro Baba • Kanako Iseki • Takuya Ito • Yoshinori Asakawa Sachiko Kawano • Toshihiro Hashimoto
•
Received: 4 April 2014 / Accepted: 16 June 2014 / Published online: 16 July 2014 Ó The Japanese Society of Pharmacognosy and Springer Japan 2014
Abstract Two new phenanthrenes, and one new phenylpropanoid, named ephemeranthoquinone C (1), and marylaurencinols C (2) and D (3), were isolated from the roots of Cymbidium Great Flower ‘Marylaurencin’, respectively. These structures were determined on the basis of 2D NMR experiments. The compounds were tested for antimicrobial activity against Bacillus subtilis, Klebsiella pneumoniae, and Trichophyton rubrum. Keywords Cymbidium Great Flower ‘Marylaurencin’ Orchidaceae Ephemeranthoquinone Marylaurencinol Antimicrobial activity Trichophyton rubrum
Introduction Species of the Orchidaceae are rich sources of aromatic compounds, such as simple benzene and bibenzyl derivatives, as well as monomeric, dimeric, and triphenanthrenes. Some of these compounds have been reported as having cytotoxic, immunoregulatory, spasmolytic, antimicrobial, anti-allergic, and anti-inflammatory activities [1]. In the course of our study of bioactive substances from the Orchidaceae, we previously reported four new phenanthrene derivatives, ephemeranthoquinone B, marylaurencinols A and B, and marylaurencinoside A, together with six known K. Yoshikawa (&) C. Baba K. Iseki T. Ito Y. Asakawa T. Hashimoto Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-Cho, Tokushima 770-8514, Japan e-mail: [email protected] S. Kawano Kawano-Mericlone Co. Ltd., Wakimachi, Tokushima 779-3604, Japan
phenanthrenes and the antibacterial activity of the fresh roots, and also novel glucopyranosyloxybenzyl derivatives of 2-benzylmalic acid and eucomic acid, marylaurencinosides D and E, respectively, along with eight known aromatic compounds from the fresh flowers of the well-known cultivated cymbidium, Cymbidium Great Flower ‘Marylaurencin’ [2, 3]. We continued the search for new antimicrobial metabolites of the roots. The MeOH extract was partitioned into an AcOEt-H2O mixture to afford AcOEtand H2O-soluble portions. The resulting AcOEt extract showed antimicrobial activity against Bacillus subtilis at 18.75 lg/mL. Further separation of the EtOAc-soluble portion by ordinary-phase and reversed-phase silica gels afforded two new phenanthrenes (1, 2), and one new phenylpropanoid (3), along with two known phenanthrens, 2,4,7,8-tetramethoxy-3-hydroxy-phenanthrene (4) [4], and calanquinone A (5) [5], shown in Fig. 1. This paper describes the isolation, purification, and structural elucidation of 1–3 determined primarily by extensive NMR experiments, and their antimicrobial activities.
Results and discussion Ephemeranthoquinone C (1) was obtained as an amorphous powder. The molecular formula was determined as C17H16O6 with an [M]? peak at m/z 316.0946 by HR-EIMS in conjunction with NMR data analysis. The IR spectrum of 1 revealed absorption bands due to a hydroxy group (3063 cm-1), conjugated carbonyl groups (1670, 1639 cm-1), and aromatic rings (1595, 1452 cm-1). The UV spectrum supported the presence of the aromatic ring (kmax 244, 285 nm). In the 1H-NMR spectrum, a pair of ortho-coupled protons at d 7.81 (d, J = 8.5 Hz), and 7.86 (d, J = 8.5 Hz), a long-range coupled aromatic proton at d
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Fig. 1 Chemical structures of compounds 1–5
OCH3
OH
OH
O OCH3
H3CO OH
HO O
H3CO
OCH3
OCH3
HO
H3CO
H3CO
1
OCH3
2 OCH3
4
OAc
O O
OH H3CO
H3CO
OCH3
O OAc
H3CO
5
H3CO
3 OH
6.99 (br s), one oxymethine at d 4.65 (dd, J = 8.4, 5.8 Hz), three methoxy groups at d 3.61 (s), 4.07 (s), and 4.08 (s), two of which were aromatic substituted, and methylene protons at d 3.13 (dd, J = 17.5, 8.4 Hz) and 3.40 (dd, J = 17.5, 5.8 Hz) were observed. The 13C-NMR spectrum also showed the presence of two carbonyl carbons (d 192.6, 201.3), one methylene (d 46.8) and oxymethine carbon (d 82.3) each, and 10 sp2 hybridized carbons (d 101.9, 120.4, 120.5, 130.9, 132.1, 133.4, 136.3, 139.3, 141.3, 150.6). The COSY spectrum showed two partial structures, indicated by thick lines in Fig. 2. The HMQC correlations were analyzed via an HMBC spectrum that showed correlations of H2-2 to C-1, and C-4; H-8 to C-6, C-7, and C-8a; H-9 to C-4b, C-10a; H-10 to C1, C-4a, and C-10a, and a methoxy group at d 3.61 to C-3; a methoxy group at d 4.08 to C-5; and a methoxy group at d 4.07 to C-7. NOE correlations were observed between H2-2 (d 3.13, 3.40) and 3-OCH3 (d 3.61), between 7-OCH3 (d 4.07) and H-8 (d 6.99), and between H-8 and H-9 (d 7.81). These results confirmed that C-1 and C-4, C-5 and C-7, and C-6 are substituted by carbonyl groups, methoxy groups, and a hydroxyl group, respectively. Furthermore, the presence of one remaining methoxyl was established at C-3 position by an important NOESY correlation between 5-OCH3 (d 4.08) and 3-OCH3 (d 3.61). On the basis of the data obtained, the structure of 1 was determined as 6-hydroxy-3,5,7-trimethoxy-2,3-dihydrophenanthrene-1,4-dione. Marylaurencinol C (2) was obtained as an amorphous powder. The molecular formula was determined as C16H16O5 with an [M]? peak at m/z 288.0992 by HREIMS, requiring nine degrees of unsaturation. The IR spectrum of 2 exhibited strong absorption bands at 3348, 3215, 1616, 1582, 1501, 1452, 1329, 1236, and 1094 cm-1, indicating the absence of carbonyl groups comparable to those in 1. The 1H-NMR spectrum showed the presence of two methylene protons [d 2.49 (2H, m), 2.57 (2H, m)], two
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Fig. 2 COSY, selected HMBC and NOESY correlations of 1
methoxy groups [d 3.55 (s), 3.82 (s)], and a pair of orthocoupled protons [d 6.64 (d, J = 8.7 Hz), 6.69 (d, J = 8.7 Hz)], an isolated aromatic proton [d 6.82 (br s)], in addition to three hydroxyl groups [d 8.19 (s), 8.70 (s), and 8.77 (s)]. The 13C-NMR spectrum also revealed the presence of two methoxy carbons (d 55.9, 61.2), two methylene carbons (d 22.3, 29.5), and 12 aromatic carbons, and the absence of carbonyl carbons (Table 1). Therefore, 2 was deduced to have a pentasubstituted 9,10-dihydrophenanthrene derivative. The COSY and HMQC spectra exhibited the presence of partial structures, as shown in Fig. 3. The positions of the substituents in 2 were also confirmed by analysis of the HMBC and NOESY spectra. The negative sign of the CD band at 226 nm is diagnostic of the M helicity of the biaryl chromophore [6]. Thus, the structure of 2 was elucidated to be 3,5,6-trihydroxy-2,4dimethoxy-9,10-dihydrophenanthrene. Marylaurencinol D (3), obtained as an amorphous powder, was assigned the molecular formula C25H30O9, shown at m/z 474.1885 by its HR-EIMS. The 13C-NMR and HMQC spectra showed the signals of three methoxy groups (d 55.8, 56.0, 57.1), two oxymethylene carbons (d
J Nat Med (2014) 68:743–747 Table 1 1H- and
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13
Marylaurencinol C (1) Position
dC
1
201.3 (s)
2
46.8 (t)
Table 2 1H- and
C-NMR spectral data for 1 and 2
13
C-NMR spectral data for 3 in CDCl3
Marylaurencinol D (3)
Marylaurencinol D (2)
dH (J Hz)
dC
dH (J Hz)
Position
dC
dH (J Hz)
108.3 (d)
6.82 (br s)
1
83.2 (d)
4.42 (d, 5.8)
3.13 (dd, 17.5, 8.4)
147.4 (s)
2
81.6 (d)
4.49 (ddd, 6.2, 5.8, 4.0)
3
63.7 (t)
4.05 (dd,11.8, 6.2)
3.40 (dd, 17.5, 5.8) 82.3 (d)
4
192.6 (s)
143.8 (s)
10
129.5 (s)
4a
136.3 (s)
118.3 (s)
20
109.6 (d)
4b 5
133.4 (s) 141.3 (s)
121.0 (s) 145.9 (s)
30 40
150.7 (s) 145.7 (s)
6
139.3 (s)
146.4 (s)
50
114.1 (d)
6.88 (d, 8.5)
7
150.6 (s)
116.5 (d)
6.64 (d, 8.7)
60
120.8 (d)
6.84 (dd, 8.5, 1.5)
8
101.9 (d)
115.3 (d)
6.69 (d, 8.7)
100
148.6 (s)
8a
120.4 (s)
200
146.8 (s)
9
130.9 (d)
2.57 (2H,m)
300
110.0 (d)
2.49 (2H,m)
00
4
130.9 (s)
400 a
134.1 (d)
6.57 (d, 15.8)
400 b
121.7 (d)
6.16 (dt, 15.8, 6.8)
400 c
65.2 (t)
4.69 (d, 6.8)
10
120.5 (d)
10a
132.1 (s)
4.65 (dd, 8.4, 5.8)
4.22 (dd,11.8, 4.0)
3
6.99 (br s)
125.5 (s) 7.81 (d, 8.5) 7.86 (d, 8.5)
22.3 (d) 29.5 (d) 130.5 (s)
2-OCH3 3-OCH3
137.7 (s)
55.9 (q) 58.0 (q)
3.82 (s)
3.61 (s)
4-OCH3
61.2 (q)
6.93 (d, 1.5)
6.86 (d, 1.5)
4.71 (d, 6.8)
3.55 (s)
5-OCH3
59.5 (q)
4.08 (s)
500
119.8 (d)
6.88 (dd, 8.5, 1.5)
7-OCH3
56.4 (q)
4.07 (s)
600
117.8 (q)
6.91 (d, 8.5)
3-OH
8.70 (s)
1-OCH3
57.1 (q)
3.28 (s)
5-OH 6-OH
8.19 (s) 8.77 (s)
30 -OCH3 200 -OCH3
55.8 (q) 56.0 (q)
3.84 (s) 3.87 (s)
3-Ac
20.7 (q), 170.6 (s)
1.97 (s)
21.0 (q), 170.9 (s)
2.10 (s)
1 in CDCl3. 2 in DMSO-d6
00
4 c-Ac
Fig. 3 COSY, selected HMBC and ROESY correlations of 2
63.7, 65.2), two oxymethine carbons (d 81.6, 83.2), and 14 sp2 hybridized carbons, in addition to two acetyl groups (d 20.7, 170.6 and d 21.0, 170.9). The 1H-NMR spectrum of 3 showed the typical spin systems of two 1,3,4-substituted benzene rings (Table 2). The gross structure of 3 was determined by the same strategy as 2. The COSY and HMBC correlations exhibited the presence of guaiacylglycerol (unit A) and coniferyl alcohol (unit B) moieties, as shown in Fig. 4. The substitution patterns of two aromatic
Fig. 4 COSY, selected HMBC and NOESY correlations of 3
rings were confirmed by the following NOEs, H-20 (d 6.93)/OMe (d 3.84), and H-300 (d 6.86)/OMe (d 3.87). Two phenylpropanoid units in 3 were connected through an
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J Nat Med (2014) 68:743–747
Table 3 Antimicrobial activity against Trichophyton rubrum of 1–5 Compd
Inhibition zone (mm)
Extracta,b
10.7
Ephemeranthoquinone C (1)b
12.7
Marylaurencinol C (2)c
15.7
Marylaurencinol D (3)
b
–
Compound 4 (4)b
–
Calanquinone A (5)b
–
Ketoconazoled
16.7
a
EtOAc-soluble portion of MeOH extract
b
100 lg/disk (mm)
c
10 lg/disk (mm)
d
5 lg/disk (mm)
ether bond at C-2 and C-100 by the HMBC correlation from H-2 (d 4.49) to C-100 (d 148.6). Furthermore, the HMBC correlations from the aliphatic methoxy group at d 3.28 (s) to C-1 (d 83.2), from H2-3 (d 4.05, 4.22) at the acylation site to carbonyl carbon at d 170.6, and from H2-400 c (d 4.69, 4.71) at the acylation site to carbonyl carbon at d 170.9 could confirm the presence of the methoxy group at C-1, and two acetyl groups at C-3 and C-400 c, respectively. Thus, from the above findings, the structure of 3 was concluded to be as shown in Fig. 1. The isolated compounds (1-5) were tested for antimicrobial activity against B. subtilis, K. pneumonia, and T. rubrum. Compounds 1, 2, 3, and 5 exhibited weak activities against B. subtilis with MICs of 29.7, 26.0, 79.1, and 59.7 lM, respectively, and 0.36 lM for the positive control, ampicillin. However, none was active against K. pneumonia. On the other hand, compounds 1 and 2 showed antifungal activity against T. rubrum (Table 3). It is interesting to note that 2 showed half the activity of ketoconazole. In conclusion, our current chemical investigation yielded two new and two known phenanthrene derivatives, and a new phenylpropanoid derivative, some of which revealed the antimicrobial activity of this plant. This is the first report of phenanthrene derivatives showing antimicrobial activity against T. rubrum from a natural source [7], and therefore it could be lead to the development of new topical antifungal agents.
spectrophotometer, respectively. NMR spectra were recorded on a Varian UNITY 600 spectrometer. The chemical shifts are given as d (ppm) in CDCl3 or DMSO-d6 solution, using tetramethylsilane (TMS) as the internal standard. NMR experiments included COSY, HMQC, HMBC, and ROESY. Coupling constants (J values) are given in Hz. HR-FAB-MS was measured on a JEOL JMS700 MStation. For HPLC column chromatography, COSMOSIL 5C18-AR-II (Nacalai Tesque, Inc., Kyoto, Japan, 20 mm i.d. 9 250 mm) was used. TLC was performed on pre-coated silica gel 60F254 (Merck). Spots were detected by examining plates sprayed with p-anisaldehyde/H2SO4/ MeOH reagent followed by heating on a hot plate. Plant material Cymbidium Great Flower ‘Marylaurencin’ (Ministry of Agriculture, Forestry and Fisheries of Japan, seed registration No. 2841) was cultivated and harvested in February 2012 at Kawano Mericlone Co., Ltd. (Tokushima prefecture, Japan), and identified by one of the authors (S. K.). A voucher specimen (TB 5430) has been deposited in the Herbarium of the Department of Pharmacognosy, Tokushima Bunri University, Tokushima, Japan. Extraction and isolation Fresh roots (8.0 kg) were extracted with MeOH at room temperature for 1 week and concentrated in vacuo. The MeOH extract was partitioned into an EtOAc-H2O mixture to afford EtOAc- and H2O-soluble portions. The EtOAc extract (42 g) was subjected to silica gel column chromatography with n-hexane–EtOAc-MeOH (10:1:0–0:1:10) to afford eight fractions (Fr.1-8). Fr. 3 (5.69 g) was chromatographed further over silica gel column chromatography using n-hexane–EtOAc (7:3) to afford five subfractions (Fr. 3-A-E). Fr. 3-C was purified by RP-HPLC (60 % aq. MeOH) to yield 4 (20 mg). Fr. 4 (4.49 g) was further subjected to silica gel column chromatography with nhexane–EtOAc (6:4) and separated again using RP-HPLC (70 % aq. MeOH) to furnish 1 (72 mg), 2 (380 mg) and 3 (17 mg). Fr. 5 (1.15 g) was subjected to silica gel column chromatography with n-hexane–EtOAc (6:4) and purified using RP-HPLC (55 % aq. MeOH) to furnish 5 (14.5 mg). Ephemeranthoquinone C (1)
Experimental General Optical rotation was performed on a JASCO P-1030 digital polarimeter. IR and UV spectra were measured on a JASCO FT/IR 410 and Shimadzu UV-1650PC
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An amorphous powder; ½a25 D -0.55 (c 1.3, CHCl3); UV (MeOH) kmax (log e) 244 (4.10), 285 (4.10) nm; FT-IR (film) mmax 3063, 2993, 1670, 1639, 1595, 1452, 1246 cm-1; CD (MeOH) nm (De):285 (?0.50), 245 (?1.08); 1H- and 13C-NMR data, see Table 1; HR-EIMS m/z 316.0946 (calcd for C17H16O6, 316.0947).
J Nat Med (2014) 68:743–747
Marylaurencinol C (2) An amorphous powder; ½a25 D ?0.58 (c 0.35, CHCl3/MeOH 1:1); UV (MeOH) kmax (log e) 229 (4.05), 272 (3.99), 309 (3.96) nm; FT-IR (film) mmax 3348, 3215, 1616, 1582, 1501, 1452, 1329, 1236, 1094 cm-1; CD (MeOH) nm (De): 226 (–7.90); 1H- and 13C-NMR data, see Table 1; HREIMS m/z 288.0992 (calcd for C16H16O5, 288.0998).
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(3.9 % PDA; Nissui, Japan) and incubated at 28 °C for 5 days. A stock solution of crude extracts or samples was prepared at 10 mg/mL in DMSO and impregnated circular paper disks (8 mm; Toyo Roshi Kaisha, Ltd., Japan) were placed on the surface of each PDA plate. Then the plates were incubated at 28 °C for 5 days and inhibition zone diameters (IZD) were measured in millimeters. Ketoconazol (LKT Laboratories, Inc., USA) was used as a reference reagent for the bacterial strains.
Marylaurencinol D (3) An amorphous powder; ½a25 D –2.94 (c 0.7, CHCl3); UV (MeOH) kmax (log e) 214 (3.96), 266 (4.17) nm; FT-IR (film) mmax 3420, 1730, 1600, 1515, 1252, 1097, 1047 cm-1; 1H- and 13C-NMR data, see Table 2; HREIMS m/z 474.1885 (calcd for C25H30O9 [M]?, 474.1890). Antimicrobial assay B. subtilis NBRC 3134 and K. pneumoniae NBRC 3512 were used for testing antimicrobial activity. These strains were tested using micro dilution assays and MIC values were determined. Bacterial strains were inoculated on YP agar plates [0.5 % peptone (Becton, Dickinson and Co., USA), 0.3 % yeast extract (Becton, Dickinson and Co.), 0.1 % MgSO47H2O and 2 % agar (Nacalai Tesque, Japan)] and incubated at 37 °C (B. subtilis) and 27 °C (K. pneumoniae) for 12 h. A stock solution of crude extracts or samples was prepared at 10 mg/mL in DMSO and further diluted to varying concentrations in 96-well plates which contained microbial strains incubated in YP medium. Each plate was further incubated at 37 °C (B. subtilis) and 27 °C (K. pneumoniae) overnight and ampicillin (Nacalai Tesque) was used as a reference reagent for the bacterial strains. T. rubrum NBRC 5807 was also tested using disk-diffusion assays. Bacterial strains were inoculated onto potato dextrose agar (PDA) plates
References 1. Kova´cs A, Vasas A, Hohmonn J (2008) Natural phenenthrenes and their biological activity. Phytochemistry 69:1084–1110 2. Yoshikawa K, Itou T, Iseki K, Baba T, Imagawa H, Morita H, Kawano S, Asakawa Y, Hashimoto T (2012) Phenanthrene derivatives from Cymbidium Great Flower ‘Marylaurencin’ and their biological activities. J Nat Prod 75:605–609 3. Yoshikawa K, Okahuji M, Ito T, Asakawa Y, Kawano S, Hashimoto T (2014) Two novel aromatic glucosides, marylaurencinosides D and E from the fresh flower of Cymbidium Great Flower ‘Marylaurencin’. J Nat Med 68:455–458 4. Reisch J, Bathory M, Novak I, Szendrei K (1970) Structure and biochemistry of natural phenanthrene derivatives. Herba Hung 9:43–48 5. Lee CH, Chang FP, Yen MH, Yu D, Liu YN, Bastow KF, MorrisNatschke SL, Wu YC, Lee KH (2009) Cytotoxic Phenanthrenequinones and 9,10-dihydrophenenthrenes from Calanthe arisanensis. J Nat Prod 72:210–213 6. Gawron´ski J, Grycz P, Kwit M, Rychlewska U (2002) Circular dichroism of 9,10-dihydrophenanthrene derivative reveals both the absolute configuration and conformation: a novel approach to Mislow’s helicity rule. Chem Eur J 8:4210–4215 7. Zacchino SA, Lo´pez SN, Pezzenati GD, Furla´n RL, Santecchia CB, Mun˜oz L, Giannini FA, Rodrı´guez AM, Enriz RD (1999) In vitro evaluation of antifungal properities of phenylpropanoids and related comounds acting againt dermatophytes. J Nat Prod 62:1353–1357
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