New lignan esters from Alyxia schlechteri and antifungal activity against Pythium insidiosum

Fitoterapia 91 (2013) 39–43

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New lignan esters from Alyxia schlechteri and antifungal activity against Pythium insidiosum Uraiwan Sriphana a, Yordhathai Thongsri b, Pispong Ardwichai c, Kitisak Poopasit c, Chularut Prariyachatigul b, Sontaya Simasathiansophon d, Chavi Yenjai c,⁎ a

Department of Science and Technology, Faculty of Liberal Arts and Science, Roi-Et Rajabhat University, Selapoom, Roi-Et 45120, Thailand Department of Microbiology, Faculty of Associated Medical Sciences, Centre for Research and Development of Medical Diagnosis Laboratories, Khon Kaen University 40002, Thailand c Natural Products Research Unit, Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand d Department of Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand b

a r t i c l e

i n f o

Article history: Received 4 July 2013 Accepted in revised form 8 August 2013 Available online 28 August 2013 Keywords: Alyxia schlechteri Pythium insidiosum Lignans Alyterinates A−C

a b s t r a c t Three new lignan esters, alyterinates A–C (1−3), as well as 10 known compounds were isolated from the roots of Alyxia schlechteri. Antifungal activity against Pythium insidiosum of all lignan derivatives was evaluated using disk diffusion assay. P. insidiosum is not a true fungus since its cell walls do not contain ergosterol as usual fungi, so the antifungals available now are not effective. From activity testing, it was found that compounds 3, 4 and 5 could inhibit the mycelia growth of P. insidiosum. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Alyxia schlechteri, belonging to the family Apocynaceae, is a traditional Thai medicinal plant. It is widely distributed in the northeast of Thailand. The stems and roots of this climbing plant are used to treat fainting, heart failure and abdominal discomfort. The leaves and fruits are also used to reduce fever. Alyxia reinwardtii, one of the species which found in Thailand, contains phenolic compounds, coumarins, lignans, and iridoid glycosides [1–4]. In continuation of our research on bioactive compounds from medicinal plants [5], we have intensively screened the natural compounds against Pythium insidiosum [6]. P. insidiosum is not a true fungus because its cell walls do not contain ergosterol which is the target for antifungal agents and in addition, it is closely

⁎ Corresponding author. Tel.: + 66 4320 2222 41x12243; fax: + 66 4320 2373. E-mail address: [email protected] (C. Yenjai). 0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.08.005

related to diatoms and golden-brown algae [7]. This organism causes pythiosis which is found in animals such as dogs, horses, calves, and cats [8]. It was first reported in humans in Thailand and since then, other cases have been reported in tropical and subtropical areas world-wide [9]. Since P. insidiosum is a fungus like organism, neither antifungal drugs nor vaccines now available are effective in eliminating the organism. Thus the search for new compounds for the treatment of pythiosis from natural sources is still necessary. 2. Experimental 2.1. General experimental procedures Melting points were determined on a SANYO Gallenkamp (UK) melting point apparatus and are uncorrected. UV spectra were measured on an Agilent 8453 UV–Visible spectrophotometer (Germany). IR spectra were recorded as KBr disks or thin films, using a Perkin Elmer Spectrum One FT-IR spectrophotometer (UK). The NMR spectra were recorded

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on a Varian Mercury plus spectrometer (UK) operating at 400 MHz (1H) and at 100 MHz (13C). Mass spectra were determined on a Micromass Q-TOF 2 hybrid quadrupole timeof-flight (Q-TOF) mass spectrometer with a Z-spray ES source (Micromass, Manchester, UK). Thin layer chromatography (TLC) was carried out on a MERCK silica gel 60 F254 TLC aluminum sheet. Column chromatography was done with silica gel 0.063– 0.200 mm or less than 0.063 mm. Preparative layer chromatography (PLC) was carried out on glass supported silica gel plates using silica gel 60 PF254. All solvents were routinely distilled prior to use. 2.2. Plant material The roots of A. schlechteri were collected in September 2012 from Phu Wiang National Park, Khon Kaen Province, Thailand. The plant was identified by Prof. Dr. Pranom Chantaranothai, Faculty of Science, Khon Kaen University. A botanically identified voucher specimen (KKU0012012) was deposited at the herbarium of the Department of Chemistry, Faculty of Science, Khon Kaen University, Thailand.

was purified by preparative TLC using 10% EtOAc:hexane as developing solvent to furnish 3 (21 mg) and 6 (22 mg). Purification of subfraction F16.4 was performed on a silica gel column and eluted with an isocratic system of 2% MeOH: CH2Cl2 to afford 5 (1.7 g). 2.4. Antifungal activity; disk diffusion assay All purified compounds were dissolved in suitable solvents to a final volume of 100 μl. Then 20 μl of the tested compounds were impregnated on sterilized disks (6.0 mm) (Whatman, England) and placed on the Sabouraud Dextrose Agar (SDA) plate (Oxoid, UK) which had been inoculated with an agar block of P. insidiosum (1 × 1 cm). Plates were kept at room temperature for 2 h in the laminar flow cabinet, then inverted and incubated at 25 °C for 3, 6 and 9 days. Terbinafine, itraconazole (10 mg/100 μl; 20 μl/disk) (Sigma-Aldrich, USA) and a disk with a diluted solvent only were used as control disks. Inhibition of the mycelial growth of P. insidiosum compared with the control was observed and reported as positive antifungal activity [10].

2.3. Extraction and isolation

3. Results and discussion

Air-dried and finely powdered roots (1.9 kg) of A. schlechteri were sequentially extracted at room temperature for three days with EtOAc (2 × 3 l) and MeOH (2 × 3 l). The extracts were evaporated in vacuo to obtain two dry extracts, crude EtOAc (77 g) and crude MeOH (42 g). The crude EtOAc extract (77 g) was subjected to column chromatography on silica gel 60 and subsequently eluted with a gradient of three solvents (hexanes, EtOAc, and MeOH) by gradually increasing the polarity of the elution solvent system. The eluents were collected and monitored by TLC, producing 21 groups of eluting fractions, which were designated as F1 to F21. The solid substance in F6 was recrystallized from EtOAc–hexane to obtain 8 (2.1 g). Fraction F9 was further purified by silica gel column chromatography and eluted with a gradient of the EtOAc–hexane system to yield nine subfractions, F9.1–F9.9. Subfraction F9.2 was purified by preparative TLC using 1:9 EtOAc:hexane as a developing solvent system to yield 13 (0.2 g). Subfraction F9.6 was purified by preparative TLC using 1:19 EtOAc:CH2Cl2 as a developing solvent system to give 12 (0.18 g) and 9 (0.19 g). Subfraction F9.9 was further purified by silica gel column chromatography and eluted with a gradient of the EtOAc–hexane system to yield 10 (1.5 g). Further rechromatography of F12 on silica gel column chromatography using gradient elution of CH2Cl2:MeOH mixtures afforded subfractions F12.1–12.5. Subfraction F12.1 was purified by preparative TLC using 10% EtOAc:CH2Cl2 to yield 11 (13 mg). Subfraction F12.4 was applied to RP-18 column chromatography and eluted with an isocratic system of 50% H2O:MeOH to give 1 (57 mg) and 2 (10 mg). Fraction F14 was further purified by silica gel column chromatography and eluted with a gradient of the EtOAc–hexane system to yield 10 subfractions, F14.1–F14.10. Further purification of F14.1 by silica gel column chromatography and eluting with a gradient of the EtOAc–hexane system yielded 4 (0.19 g). The solid substance in F14.6 was recrystallized from EtOAc–hexane to obtain 7 (0.25 g). Fraction F16 was rechromatographed by silica gel column and eluted with a gradient system of CH2Cl2:EtOAc to furnish five subfractions designated as F16.1–16.5. Subfraction F16.3

3.1. Structural determination The root barks of A. schlechteri, collected in Phu Wiang National Park in September 2012, were air-dried, pulverized, and sequentially extracted with EtOAc and MeOH. The EtOAc extract was subjected to silica gel column chromatography and further purified by chromatographic methods to obtain 13 pure compounds (1−13) (Fig. 1). The structures of 4−13 were assessed by comparison of their spectroscopic data with those reported in the literature and the remaining 1−3 are new lignans. The known compounds include (+)-pinoresinol (4)

Fig. 1. Chemical structures of all compounds.

U. Sriphana et al. / Fitoterapia 91 (2013) 39–43

[14,15]. Furthermore, the NOESY experiment revealed a correlation between H-8 and 8′ which indicated that these hydrogens were cofacial. This compound showed that [α]26 D + 40.3 (c 0.001, CHCl3) [11,16]. Thus, the structure of 1 was assigned as (7S*,8S*,8′R*)-4,4′-dihydroxy-3,3′-dimethoxy7,9′-epoxy-9-methoxycarbonyl-8,8′-lignan which was named alyterinate A. Compound 2 was found as a pale yellow oil. It was assigned the molecular formula C21H22O7 as determined from its quasimolecular ion peak at m/z 409.1205 [M + Na]+ in the HRESIMS, corresponding to 11° of unsaturation. The IR spectrum showed the hydroxyl group at 3382 cm−1, and the carbonyl group at 1723 cm−1. The 1H NMR spectrum showed two ABX systems in the aromatic region. Two phenolic protons displayed at δ 5.69 (OH-4′) and 5.64 (OH-4) in the 1H NMR spectrum (Table 1). The broad singlet signal at δ 6.44 (H-7′) correlated with carbon at δ 122.9 (C-7′) in the HMQC spectrum which indicated the double bond moiety. In addition, the 13C NMR spectrum showed a signal at δ 137.0 which was assigned as C-8′. The correlations between H-7′ and C-8 (δ 58.4), C-2′ (δ 110.9), C-6′ (δ 121.6), and C-9′ (δ 70.2) were seen in the HMBC experiment (Fig. 2). The proton at δ 5.21 (d, J = 8.0 Hz, H-7) coupled with the proton at δ 3.68 (d, J = 8.0 Hz, H-8) in the COSY spectrum and J7,8 value (8.0 Hz) indicated the 7,8-trans configuration. The HMBC spectrum exhibited H-7 correlating with C-2 (δ 108.5), C-6 (δ 119.3), C-9 (δ 171.7), and C-8′ (δ 137.0). Two doublet signals at δ 4.91 and 4.77 (each 1H, d, J = 13.2 Hz) were assigned as H-9′, which correlated with C-9′ (δ 70.2) in the HMQC spectrum. The NOESY experiment revealed a correlation between H-8 and 7′ which indicates the Z geometry of the double bond [17]. The HMBC spectrum showed the methoxy proton at δ 3.78 correlated

[11,12], (+)-medioresinol (5) [13], (+)-syringaresinol (6) [13], (+)-6-(4-hydroxy-3-methoxyphenyl)-3,7-dioxabicyclo [3.3.0]octan-2-one (7) [11], coumarin (8), 8-hydroxycoumarin (9), 5-hydroxycoumarin (10), 7-hydroxycoumarin (11), 4-hydroxybenzaldehyde (12), and vanillin (13). Compound 1 was found as a colorless solid, mp. 114–115 °C. It was assigned the molecular formula C21H24O7 as determined from its quasimolecular ion peak at m/z 411.1418 [M + Na]+ in the HRESIMS, corresponding to 10° of unsaturation. The IR spectrum showed the hydroxyl group at 3429 cm−1, and the carbonyl group at 1726 cm−1. The 1H NMR spectrum displayed two ABX systems in the aromatic region. Two singlet signals at δ 5.59 and 5.52 were assigned as two hydroxyl protons (Table 1). The 1H-1H COSY spectrum showed the spin system in the aliphatic region, representing H-7/H-8/H-8′, H-8′/H-9′ and H-8′/H-7′. In the HMQC spectrum, correlation of the proton at δ 5.23 (d, J = 7.2 Hz, H-7) and C-7 (δ 82.3) was observed. The methine proton at δ 3.09 (t, J = 7.2 Hz, H-8) correlated with carbon at δ 55.9 (C-8), and methylene protons at δ 4.08 (dd, J = 8.8, 6.8 Hz, H-9′a) and 3.79 (dd, J = 8.8, 7.2 Hz, H-9′b) correlated with carbon at δ 72.9 (C-9′) in the HMQC spectrum. The multiplet signal at δ 2.90 was assigned as H-8′ while the methylene protons at H-7′ showed two doublet of doublets pattern at δ 2.76 (J = 13.6, 5.6 Hz) and 2.55 (J = 13.6, 11.2 Hz). The HMBC spectrum exhibited H-7 correlating with C-2 (δ 108.3), C-6 (δ 118.7) and C-9 (δ 172.5) (Fig. 2). The correlations of methylene protons at H-7′ with C-8 (δ 55.9), C-9′ (δ 72.9), C-1′ (δ 131.3), C-2′ (δ 113.1) and C-6′ (δ 121.3) were also seen in the HMBC spectrum. In this spectrum, correlation between the methoxy protons at δ 3.70 with carbonyl carbon (C-9, δ 172.5) confirmed the existence of the ester group. The J7,8 value (7.2 Hz) indicated the 7,8-trans configuration

Table 1 1 H NMR (400 MHz) and

13

C NMR (100 MHz) spectral data of compounds 1–3 (δ in ppm).

Position

1 (CDCl3)

1 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′

133.4 108.3 146.5 145.2 114.3 118.7 82.3 55.9 172.5 131.3 111.3 146.4 144.1 114.4 121.3 34.5

2 (CDCl3) δH (J in Hz)

δC sa d s s d d d d s s d s s d d t

8′ 9′

44.2 d 72.9 t

OCH3 OCH3-ester OH-4 OH-4′

55.9 51.7

a

41

6.88, m

6.85, m 6.84, m 5.23 d (7.2) 3.09, t (7.2)

6.65, s

6.85, 6.66, 2.76, 2.55, 2.90, 4.08, 3.79, 3.87, 3.70, 5.59, 5.52,

m d (6.5) dd (13.6, 5.6) dd (13.6, 11.2) m dd (8.8, 6.8) dd (8.8, 7.2) s s br s br s

Multiplicities were deduced from DEPT and HMQC experiments.

δC 131.7 108.5 146.6 145.6 114.3 119.3 82.6 58.4 171.7 129.1 110.9 146.4 145.1 114.5 121.6 122.9

δH (J in Hz) s d s s d d d d s s d s s d d d

137.0 s 70.2 t 55.9 52.3

3 (CDCl3)

6.92, br s

6.89, d 6.89, d 5.21, d 3.68, d

(8.0) (8.0) (8.0) (8.0)

6.66, s

6.89, d (8.0) 6.67, d (8.0) 6.44, br s

4.91, d (13.2) 4.77, d (13.2) 3.89, s 3.78, s 5.64, br s 5.69, br s

δC 132.5 108.7 146.7 145.5 114.5 119.3 83.4 63.9 172.1 127.5 112.6 146.5 144.8 114.3 122.8 40.1

δH (J in Hz) s d s s d d d d s s d s s d d t

82.9 s 77.7 t 55.9 52.1

6.98, s

6.85, d 6.85, d 5.21, d 3.17, d

(8.0) (8.0) (7.2) (7.2)

6.77, s

6.85, d 6.73, d 2.93, d 2.85, d

(8.0) (8) (13.6) (13.6)

3.99, d (9.2) 3.73, d (9.2) 3.87, s 3.75

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U. Sriphana et al. / Fitoterapia 91 (2013) 39–43

with C-9 (δ 171.7), which confirmed the ester moiety. Thus, the structure of 2 was assigned as (7S*,8S*,7′Z)-4,4′-dihydroxy-7′,8′dehydro-3,3′-dimethoxy-7,9′-epoxy-9-methoxycarbonyl-8,8′-lignan which was named alyterinate B. Compound 3 was found as a colorless solid, mp. 120–122 °C. It was assigned the molecular formula C21H24O8 as determined from its quasimolecular ion peak at m/z 427.1367 [M + Na]+ in the HRESIMS, corresponding to 10° of unsaturation. The IR spectrum showed the hydroxyl group at 3478 cm−1, and the carbonyl group at 1721 cm−1. The 1H NMR spectrum at the aromatic region showed two ABX systems as compounds 1 and 2. The characteristic proton at H-7 (δ 5.21) showed a doublet signal with J = 7.2 Hz the same as 1 and 2. The proton at H-8 (δ 3.17) also showed a doublet signal (J = 7.2 Hz) which correlated with H-7 in the COSY experiment. Thus, the configuration at positions C-7 and 8 was a trans configuration. The HMQC spectrum showed the correlation of H-7 with C-7 (δ 83.4) and H-8 with C-8 (δ 63.9). The HMBC spectrum showed the correlation of H-7 with C-1 (132.5), C-2 (108.7), C-6 (119.3), C-8 (63.9), and C-9 (172.1) (Fig. 2). The 13C NMR spectrum showed oxygenated carbon at δ 82.9 which was assigned as the C-8′. The signals at δ 2.93 (d, J = 13.6 Hz) and 2.85 (d, J = 13.6 Hz) were assigned as the methylene protons at H-7′. These methylene protons at H-7′ correlated with C-1′ (127.5), C-2′ (112.6), C-6′ (122.8), and C-8′ (82.9) in the HMBC spectrum. Another methylene protons at H-9′ showed two doublet signals (J = 9.2 Hz) at δ 3.99 and 3.73. The geminal couplings at H-7′ and H-9′ and optical rotation [α]23 D − 11.3° (c = 0.01, MeOH) were nearly the same as (−)-olivil [18]. Therefore, the structure of 3 was assigned as (7S*,8S*,8′ S*)-4,4′,8′-trihydroxy-3,3′-dimethoxy-7,9′-epoxy-9-methoxycarbonyl-8,8′-lignan which was named alyterinate C.

was found that three of them 3, 4 and 5 showed clearing zones with diameters of 16.0, 16.1 and 13.3 mm, respectively. Among lignan esters 1–3, compound 3 which contains a hydroxyl group at C-8′ showed good activity against P. insidiosum. The results showed convincingly that a hydroxyl group at C-8′ seems to have an important role against P. insidiosum. Comparing the activity of lignan derivatives 4–6, it was found that 4 showed stronger activity than 5. It is suggested that the methoxy group at C-5 appeared to be detrimental for the activity. In the case of compound 6 which contains an additional methoxy group at C-5′ no activity against P. insidiosum was displayed. The results indicated that the methoxy group led to a dramatic loss of the inhibition potency. The antifungal activities of these three compounds have been attributed to showing higher activity compared to the tested control. The amount of the test samples at 6–7 mg was less than the control (10 mg) and could inhibit the mycelial growth of P. insidiosum. It is indicated that these compounds are likely to be more effective and useful for the development of anti-P. insidiosum agents. 4. Spectroscopic data of compounds 4.1. Alyterinate A. (1) Colorless solid; mp. 114−115 °C; [α]26 D + 40.3 (c 0.001, CHCl3); UV (CHCl3) λmax (log ε) 243 (3.44), 281 (3.06) nm; IR (KBr) νmax 3429, 2938, 1726, 1605, 1513, 1432, 1269, 1154, 1030, 815, 785 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) spectroscopic data, see Table 1; HRESIMS m/z 411.1418 [M + Na]+ (calcd. for C21H24O7Na 411.1412). 4.2. Alyterinate B. (2)

3.2. Biological activity All isolated compounds were evaluated for antifungal activity against P. insidiosum using disk diffusion assay [10]. P. insidiosum is not a true fungus because its cellular membranes do not contain steroid, ergosterol as usual fungi. Thus, the antifungal agents available now such as terbinafine and itraconazole are not effective to treat the disease. However, itraconazole and terbinafine are the antifungal drugs which are used now [19,20], so we choose these drugs for use as positive controls. The antifungal activity results are shown in Table 2. It

Pale yellow oil; [α]23 D + 14.66 (c 0.001, CHCl3); UV (CHCl3) λmax (log ε) 239 (4.26), 274 (4.26) nm; IR (KBr) νmax 3382, 2933, 1723, 1608, 1598, 1514, 1432, 1272, 1162, 1119, 1032, 807, 771 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) spectroscopic data, see Table 1; HRESIMS m/z 409.1205 [M + Na]+ (calcd. for C21H22O7Na 409.1256). 4.3. Alyterinate C. (3) Colorless solid; mp. 120−122 °C; [α]25 D − 11.3 (c 0.001, MeOH); UV (CHCl3) λmax (log ε) 242 (4.81), 280 (3.84) nm; IR (KBr) νmax 3478, 3320, 2937, 1721, 1607, 1514, 1434, 1274, 1203, 1123, 1031, 978, 822, 771 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) spectroscopic data,

Table 2 Antifungal activity of compounds against P. insidiosum.

Fig. 2. Key HMBC and NOESY correlations for alyterinates A–C (1–3).

Compound

Concentration (μg/μl)

Inhibition zone (mm.)

Interpretationa

3 4 5 Terbinafine Itraconazole

73 76 65 100 100

16.0 16.1 13.3 – –

A A A IA IA

a

Interpretation; A = Active, IA = Inactive.

U. Sriphana et al. / Fitoterapia 91 (2013) 39–43

see Table 1; HRESIMS m/z 427.1367 [M + Na]+ (calcd. for C21H24O8Na 427.1361). Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments We thank the Center of Excellence for Innovation in Chemistry (PERCH-CIC), Office of the Higher Education Commission, Ministry of Education and the National Research University Project of Thailand through the Advanced Functional Materials Cluster of Khon Kaen University. The Thailand Research Fund (TRF) through the Royal Golden Jubilee Ph.D. Program (Grant no. PHD/0071/2551), DBG 5080010 TRF Grant is gratefully acknowledged. Appendix A. Supplementary data 1

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