Study on the chemistry and antimicrobial activity of psychotria reevesii wall. (rubiaceae)

628 Journal of Chemistry, Vol. 45 (5), P. 628 - 633, 2007 Study on the Chemistry and Antimicrobial Activity of Psychotria reevesii Wall. (Rubiaceae) Received 28 August 2006 Phan Minh Giang, Ha Viet Son, Phan Tong Son Laboratory of Chemistry of Natural Products, College of Natural Science, VNU Hanoi Summary The first chemical investigation on Vietnamese medicinal plant Psychotria reevesii Wall. (Rubiaceae) led to the isolation and structural determination of
-sitosterol and stigmasterol as a mixture, 1-octacosene, and asperglaucide from n-hexane- and CHCl3-soluble fractions of MeOH extract from the aerial parts of P. reevesii. Phytochemical screening based on color reactions, HPLC analysis, and NMR spectroscopy revealed the concentration of condensed tannins in EtOAc- and n-BuOH soluble fractions. The high accumulation of tannins may be responsible for the antibacterial activities of the polar fractions against Staphylococcus aureus, Pseudomonas aeruginosa, Shigella sonnei, and Shigella flexneri. However, they did not exhibit any inhibitory effect against Escherichia coli, Candida albicans, and Candida stellatoides. Keywords: Psychotria reevesii; Rubiaceae; asperglaucide; antibacterial activity; antifungal activity. I - Introduction Psychotria reevesii Wall. (syn. Psychotria rubra (Lour.) Poir.) of the family Rubiaceae is a medicinal plant known as Lau or Bo chat in Vietnam [1, 2]. P. reevesii is a plant of 1 - 9 m high, widely distributed in Vinh Phu, Thai Nguyen, Lang Son,... The roots and leaves of P. reevesii (Radix et Folium Psychotriae Rubrae) are used in the treatment of throat inflammation, dysentery, and rheumatic fever; leaves are also used externally to cure wounds. This paper deals with the chemical study and the investigation of antimicrobial activity of the aerial parts of P. reevesii. II - Experimental General Melting points were recorded on a Boetius micromelting point apparatus without correction. Optical rotations were measured on a Union Giken PM-101 digital polarimeter. Infrared (IR) spectra were recorded on an Impact 410-Nicolet FT-IR spectrometer. Electron impact mass spectra (EIMS) were recorded at 70 eV on a Hewlett Packar 5989B spectrometer. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AV 500 spectrometer. High performance liquid chromatography (HPLC) was run on Dionex HPLC equipment with a Photodiode array detector and an automated sample injector (injection volume 10 µl). Analytical HPLC was performed on an YMC HPLC analytical column (J’sphere ODS-H80, 150×4.6 mm I.D., S-4 µm, 8 nm) using gradient elution from 20% to 100% MeOH in H2O in 25 min., and MeOH in 5 min. at a flow rate of 1 ml/min. Column chromatography (open CC and flash FC) was performed on silica gel Merck (63 - 100 µm). Thin-layer chromatography (TLC) was performed on Aluminium precoated sheets 629 (silica gel Merck, 60 F254). Spray reagent vanilin/H2SO4 1% and UV light at
366 nm were used for visualization. Plant Material The aerial parts of P. reevesii were collected in June 2000 in province Thai Nguyen, Northern Vietnam. The plant was identified by Dr Nguyen Hoanh Coi, Military Institute of Pharmaceutical Control and Research, Hanoi, Vietnam. Extraction and Isolation The aerial parts of P. reevesii were dried at 40 - 50oC, then powdered, and extracted with 70% EtOH in H2O at room temperature for five times (each time for three days). Successive fractionation of the concentrated EtOH extract between H2O and solvents of increasing polarities gave the following corresponding soluble fractions: n- hexane- (PH, 0.2 g, 0.04% on the basis of the dried material), CHCl3- (PC, 1.7 g, 0.3%), EtOAc- (PE, 116 g, 20.4%), and n-BuOH- soluble fractions (PB, 17 g, 3%). The n-hexane- soluble fraction (0.15 g) was subjected to silica gel CC eluting with 0 - 15% EtOAc in n-hexane to give a mixture of
-sitosterol and stigmasterol (20 mg), which were determined by comparison of their 1H- and 13C-NMR data with those of an authentic sample. The CHCl3-soluble fraction (1.5 g) was fractionated on a silica gel column eluting with n-hexane-EtOAc-(CH3)2CO (19:1:0 - 1:2:1) to give 14 fractions. Recrystallization of the precipitated solid from fraction 1 with CHCl3 gave 1-octacosene (1) (15 mg). Fraction 8 was purified by a silica gel CC (n-hexane-EtOAc-(CH3)2CO, 5:5:1), followed by recrystallization in CHCl3 to give asperglaucide (2) (10 mg). 1-Octacosene (1): white needles, m.p. 30 - 32oC. Rf = 0.82 (silica gel TLC, n-hexane- EtOAc-(CH3)2CO, 5:2:1). IR (KBr): max (cm-1) 3070, 2920, 2860, 1637, 1464, 1375, 995, 909, 723. EIMS: m/z (%) 392 (M+., C28H56, < 0.1), 223 (9.3), 209 (4.2), 195 (5.9), 181 (7.6), 167 (10.2), 153 (14.4), 139 (21.2), 125 (39.8), 111 (62.7), 97 (100), 83 (86.4), 69 (73.7), 57 (96.6), 55 (77.1). 1H-NMR (supported by 1H-1H COSY) (CDCl3, ppm):  5.81 (1H, ddt, J = 10 Hz, 17 Hz, 7 Hz, H-2), 4.99 (1H, dd, J = 17 Hz, 2 Hz, H-1a), 4.92 (1H, dd, J = 10 Hz, 2 Hz, H-1b), 2.04 (2H, dt, J = 7 Hz, 7.5 Hz, 2H-3), 1.38 (2H, m, 2H-4), 1.25 (46H, m, 2H-5
2H-27), 0.88 (3H, t, J = 7 Hz, 28-CH3). 13C-NMR (supported by DEPT 135, DEPT 90, and HMQC) (CDCl3, ppm):  139.3 (d, C-2), 114.1 (t, C-1), 33.8 (t, C-3), 29.7, 29.6, 29.5, 29.4, 29.2, 28.9 (all t, C-4
C-26), 22.7 (t, C- 27), 14.1 (q, C-28). Asperglaucide (2): white needles, m.p. 184 - 188oC (Lit. m.p. 185 - 187oC [3]), []30D -45 (c = 1.0, CHCl3). R f= 0.59 (silica gel TLC, n- hexane-EtOAc-(CH3)2CO, 5:2:1). IR (KBr): max (cm-1) 3315, 3070, 3030, 2950, 2921, 2850, 1726, 1661, 1630, 1600, 1532, 1450, 1380, 1370, 1261, 1055, 745, 703, 604. EIMS: m/z (%) 444 (M+., C27H28O4N2, 1.5), 384 (1.1), 368 (0.5), 353 (3.9), 323 (3.4), 311 (6.2), 293 (2.0), 269 (12.8), 253 (11.5), 252 (66.4), 232 (7.8), 225 (15.8), 224 (51.1), 194 (1.7), 190 (2.0), 176 (4.2), 172 (11.7), 131 (8.5), 105 (100), 91 (9.7), 77 (18.4), 60 (2.0). 1H-NMR (supported by 1H-1H COSY and HMBC) (CDCl3, ppm):  7.71 (2H, d, J = 8.5 Hz, H-3, H-7), 7.52 (1H, t, J = 8.5 Hz, H-5), 7.43 (2H, t, J = 8.5 Hz, H-4, H-6), 7.26 (2H, m, H-5, H-9), 7.26 (3H, m, H-6, H-7, H-8), 7.15 (3H, m, H-6, H-7, H-8), 7.07(2H, d, J = 8.3 Hz, H-5, H-9), 6.76 (1H, d, J = 7.5 Hz, NH-1a), 6.0 (1H, d, J = 8.5 Hz, NH-1a), 4.76 (1H, m, H-2), 4.34 (1H, m, H-2), 3.92 (1H, dd, J = 11 Hz, 5 Hz, H-1b), 3.81 (1H, dd, J = 11 Hz, 4.5 Hz, H-1a), 3.21(1H, dd, J = 13.5 Hz, 6 Hz, H- 3b), 3.06 (1H, dd, J = 13.5 Hz, 8.5 Hz, H-3a), 2.75 (2H, m, 2H-3), 2.02 (3H, s, CH3COO-). 13C-NMR (supported by DEPT 135, DEPT 90, HMQC, and HMBC) (CDCl3, ppm):  170.8 (s, CH3COO-), 170.3 (s, C-1), 167.1 (s, C-1), 136.7 (s, C-4), 136.6 (s, C-4), 133.7 (s, C-2), 131.9 (d, C-5), 129.3 (2d, C-5, C-9), 129.1 (2d, C-5, C-9), 128.8 (2d, C-4, C-6), 128.7 (2d, C-6, C-8), 128.6 (2d, C-6, C-8), 127.2 (d, C-7), 630 127.1 (2d, C-3, C-7), 126.8 (d, C-7), 64.6 (t, C-1), 54.99 (d, C-2), 49.5 (d, C-2), 38.4 (t, C- 3), 37.4 (t, C-3), 20.8 (q, CH3COO-). Preparation of test fractions PE1 and PE2 from fraction PE The EtOAc-soluble fraction PE (5 g) was washed several times with EtOAc. After removing the insoluble material, toluene was added to the soluble part up to a volume corresponding to a 1/1 (EtOAc-toluene, v/v). The soluble fraction, which was separated from insoluble PE2 fraction, was concentrated under reduced pressure at 50oC to give the PE1 fraction. Antibacterial and Antifungal Assay Staphylococcus aureus ATTC 29213, Staphylococcus aureus BN, Pseudomonas aeruginosa ATTC 27853, Shigella sonnei BN, Shigella flexneri BN, Escherichia coli ATCC 25922, Candida albicans BN, and Candida stellatoides BN were used for the assay. The disk diffusion method (8 mm filter papers) was used for the preliminary screening [4]. Gentamycin and mycostatin were used as reference antibiotics. III - Results and Discussion The dried aerial parts of P. reevesii Wall. (Rubiaceae) were extracted with 70% EtOH in H2O at room temperature to give an EtOH extract. Then, the extract was subjected to the fractionation between H2O and solvents of increasing polarities to afford the corresponding n-hexane- (PH), CHCl3- (PC), EtOAc- (PE), and n-BuOH-soluble (PB) fractions. Phytochemical screening of soluble fractions of the MeOH extract from P. reevesii The phytochemical analysis was carried out to detect the main classes of phytochemical constituents in n-hexane-, CHCl3-, EtOAc-, and n-BuOH-soluble fractions using the characteristic color reactions [5]. The results were summarized in the table 1. Table 1: Phytochemical screening of soluble fractions from P. reevesii Reagent Soluble fraction 1 2 3 4 5 6 7 8 9 10 Main class of photochemical PH – – – – – – – + – Violet Phytosterol PC – – – + – – + – – Violet Polyphenol PE – – + + + + + – – Red Flavonoid, tannin PB – – + + + + + – – Red Flavonoid, tannin –: negative reaction; +: positive reaction Reagents: 1: Mayer; 2: Dragendorff; 3: Shinoda; 4: Diazo; 5: H2SO4; 6: NaOH; 7: FeCl3; 8: Liebermann-Burchardt; 9: formation of foams with NaOH or HCl; 10: Vanilin/H2SO4. It is clear, that the phytosterols were detected in n-hexane-soluble fraction, and polyphenols were found in CHCl3-soluble fraction. The high concentration of tannins, which were detected in EtOAc- and n-BuOH- soluble fractions, may lead to “non-specific” biological activities of P. reevesii in many bioassay systems. We got further evidences for the presence of tannins in the EtOAc- and n-BuOH-soluble fractions since it correlated chromatographically with a broad “hump” eluting over the polar/moderately polar region of the HPLC chromatograms (figures 1 and 2). NMR methods also proved the concentration of condensed tannins in the EtOAc- and n- BuOH-soluble fractions. After fractionation of these soluble fractions on silica gel, the obtained fractions were collected on the basis of major spots on TLC, which showed characteristic 1H- and 13C-NMR signals (data not shown) for catechin moieties. 631 -500 -250 0 250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 mAU min ANAL #19 L3 UV_VIS_1 1 - 1.673 2 - 6.780 3 - 7.940 4 - 10.1675 - 11.173 6 - 12.867 7 - 13.593 WVL:210 nm -500 -250 0 250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 mAU min ANAL #20 L4 UV_VIS_1 1 - 0.333 2 - 1.660 3 - 9.253 4 - 9.893 5 - 10.540 6 - 11.060 7 - 11.673 8 - 12.3279 - 13.333 10 - 14.440 11 - 15.967 12 - 22.233 13 - 3 WVL:210 nm Figure 1: HPLC Profile of the EtOAc-soluble Fraction from P. reevesii Figure 2: HPLC Profile of the n-BuOH-soluble Fraction from P. reevesii Susceptibility of bacteria and fungi to soluble fractions from P. reevesii The antibacterial and antifungal activities of the tannin-containing fractions PE and PB, together with the test fractions PE1 and PE2 were evaluated in this study. PE1 and PE2 were prepared from PE on the basis of the solubility of compounds in PE in different solvent systems. The data in table 2 showed the noticeable activities of PE, PB, PE1, and PE2 against S. aureus strains. In the case of P. aeruginosa, the activity was improved from PE (0 mm) to the test fractions PE1 (11.3 mm) and PE2 (11.8 mm). However, the activities against S. flexneri and S. sonnei were decreased in case of PE2, showing the specific concentration of active compounds against Shigella strains in PE1. It is noticeable that all the test fractions did not exhibit any inhibitory effect against Escherichia coli, Candida albicans, and Candida stellatoides. Isolation and structure determination of compounds 1 and 2 The CHCl3-soluble fraction was subjected to repeated column chromatography on silica gel and recrystallization to give compounds 1 and 2. Compound 1 was isolated as a white needle. The IR spectrum showed the presence of a vinyl Table 2: Susceptibility of bacteria and fungi to soluble fractions from P. reeversii Diameter of inhibition zone (mm) No. Organism PEa) PBa) PE1 a) PE2 a) 1 Staphylococcus aureus ATTC 29213 11.8 13.7 15.7 11.4 2 Staphylococcus aureus BNc) 12.3 13.4 15.5 11.3 3 Pseudomonas aeruginosa ATTC 27853 0b) 12.4 11.3 11.8 4 Shigella sonnei BNc) 8.5 12.8 10.7 0b) 5 Shigella flexneri BNc) 9.7 10.9 11.6 0b) 6 Escherichia coli ATCC 25922 0b) 0b) 0b) 0b) 8 Candida albicans BNc) 0b) 0b) 0b) 0b) 9 Candida stellatoides BNc) 0b) 0b) 0b) 0b) a) 3 mg/disk; b) 0 means no visible zone of inhibition; c) from Bach Mai Hospital patients. 632 N H H NH H O O CH2OAc 1H-1H COSY HMBC N H H NH H O O 2 1 2 3 4 5 6 7 8 9 1' 2' 3' 4' 5' 6' 7' 8' 9' 1'' 2'' 3'' 4'' 5'' 6'' 7'' 1'a 1''a CH2OAc N H NH O O O CH3 O m/z 77 m/z 224 m/z 91 m/z 252 m/z 105 group (max 1637, 995, 909 cm-1). The 1H- and 13C-NMR spectroscopic data showed a vinyl group [H 5.81 (ddt, J = 10 Hz, 17 Hz, 7 Hz), 4.99 (dd, J = 17 Hz, 2 Hz), and 4.92 (dd, J = 10 Hz, 2 Hz); C 139.3 (d), 114.1(t)], a long aliphatic hydrocarbon chain [H 2.04 (dt, J = 7 Hz, 7.5 Hz), 1.38 (m), 1.25 (m)], and a terminal methyl group [H 0.88 (t, J = 7 Hz), C 14.1 (q)]. Thus structure of 1 was deduced to be a natural 1-ankene. The molecular formula of 1 was revealed to be 392 (M+., C28H56) by EIMS spectrum, leading to the structure of 1 as 1- octacosene. Figure 3: Chemical Structure of Asperglaucide (2) Compound 2 was isolated as white needles. The molecular formula of 2 was determined to be 444 (M+., C27H28O4N2) by EIMS spectrum. The IR spectrum showed the presence of amide (max 3315, 1661, and 1630 cm-1) and ester (max 1726 and 1261 cm-1) functional groups, and aromatic rings (max 1600, 1532, and 1450 cm-1). The 1H- and 13C-NMR spectroscopic data exhibited the presence of two secondary amide groups [H 6.76 (d, J = 7.5 Hz), 6.0 (d, J = 8.5 Hz); C 171.8 (s), 167.7 (s)], three monosub- stituted benzene rings, an acetyloxymethyl group [H 3.92 (dd, J = 5 Hz, 11 Hz), 3.81 (dd, J = 4.5 Hz, 11 Hz), 2.02 (s); C 64.6 (t), 170.8 (s), 20.8 (q)], and two -CH2-CH(NH-)- groups. Two main structural fragments were constructed on the basis of the 1H-1H COSY spectrum and they were connected to the amide centers and benzene rings using HMBC correlations (Fig. 4). Finally the sign and value of the optical rotation were conclusive for the stereochemistries at C-2 and C-2 [6]. Thus the structure of 2 was deduced to be asperglaucide, an antiallergic compound previously isolated from a Euphorbia species [6], Pteris multifida Poir. (Pteridaceae) [7], and from the fungus Aspergillus glaucus [6]. EIMS fragmentation of 2 (Fig. 5) is in full agreement with this structure. Figure 4: 1H-1H COSY and Selected HMBC Correlations of 2 Figure 5: EIMS Fragmentations of 2 Acknowledgements: This work was supported by the International Foundation for Science (IFS, Stockholm, Sweden) through a Research Grant to Phan Minh Giang and the Basic Research Program in Natural Sciences of Vietnam. 633 References 1. Do T. L., Medicinal Plants and Herbal Remedies of Vietnam, Science and Techniques, Hanoi (1991). 2. Vo V. C., Dictionary of Vietnamese Medicinal Plants, Medicine, Ho Chi Minh City (1997). 3. McCorkindale N. J., Baxter R. L., Roy T. P., Schields H. S., Stweart R. M., Hutchinson S. A., Tetrahedron, 34, 2791 - 2795 (1978), and references cited herein. 4. Machado T. B., Pinto A. V., Pinto M. C. F. R., Leal I. C. R., Silva M. G., Amaral A. C. F., Kuster R. M., Netto-dosSantos K. R., Int. J. Antimicrob. Agents, 21, 279 - 284 (2003). 5. Harborne J. B., Phytochemical Methods, second edition, Chapman and Hall, New York (1984). 6. Lu H., Hu J., Zhang L. X., Tan R. X., Planta Med., 65, 586 - 587 (1999).