NPC 2011 Vol. 6 No. 3 349 - 352 Natural Product Communications The Quaternary Indole Alkaloids from Two Sumatran Lerchea species Dayar Arbaina, Nova Syafnia, Friardia, Deddi Prima Putraa, Ismiarni Komalab, Kentaro Yamaguchic and Yoshinori Asakawab a Faculty of Pharmacy, Andalas University, Kampus Limau Manis, Padang 25163, West Sumatra, Indonesia b Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan c Faculty of Pharmaceutical Sciences at Kagawa, Tokushima Bunri University, Sanuki-city, Kagawa 769-2193, Japan d.arbain48@yahoo.com Received: January 16th, 2011; Accepted: February 7th, 2011 The Sumatran forest plants Lerchea cf. bracteata and L. parviflora were found to contain alkaloids and their extract showed siginificant activity toward some testing pathogenic microbes. Isolation work on L.cf. bracteata yielded known quaternary alkaloid N(b)-methylantirhine (2) while L. parviflora gave 5,6-dihydroflavopereirine (3). Keywords: Lerchea cf. bracteata, Lerchea parviflora, N(b)-methylantirhine, 5,6-dihydroflavopereirine. Lerchea spp. (Rubiaceae) is an endemic and endangered plant of Sumatra, Indonesia. There are 19 species of this genus so far reported exist in the world and only 8 of them found in Indonesia, 2 species in Java and 6 species in Sumatra [1]. One of these, Lerchea bracteata Val. a small tree attaining 2 m was found during a field trip in the late 1980 in the Anai Reserved forest, West Sumatra. Later on, the only alkaloid detected on this plant was isolated and identified as the new quaternary alkaloid named lercheine (1) [2]. As a continuation of our study on the Sumatran Rubiaceous plants [3], during an inventory of Sumatran forest plants in Kerinci Seblat National Park (TNKS) in Sumatra in 2006, special attention was given toward the presence of this family. In this way, two alkaloid-bearing plants Lerchea parviflora North. and another species which looked similar to that of Lerchea bracteata [2] previously investigated but could not identified completely were found. This second species, looked similar except shorter as well as smaller and its methanolic extract also showed totally different TLC patterns compared to that of Lerchea bracteata [2]. This second Lerchea species was temporarily identified as Lerchea cf. bracteata. No traditional value has been recorded so far on these two species; however, people in the region occasionally used it as poultice for pain relieving or treatment of skin infection. During the preliminary work, the ethanolic extract of these two plants showed strong inhibition toward 5 6 H 21 9 N 2 N 20 3 15 N H 12 N H H 16 HO 2 1 OH H 14 H 19 17 18 the growth of some common pathogenic microbes Staphylococcus aureus (Sa, ATTC: 6538), Bacillus subtilis (Bs, clinically isolated), Pseudomonas aeruginosa (Pa, ATTC: 1542), Klebsiella (Kl, clinically isolated), Micrococcus luteus (Ml, ATCC: 9342), Escherichia coli (Ec, ATTC:8739), Pseudomonas aeruginosa (Pa, ATTC: 1542), Staphylococcus epidermidis (Se, ATTC) but did not showed activity towards the testing fungi Trichophyton mentagrophytes (Tm, ATTC: 5431) and Candida albicans (Ca, ATTC: 10231) [4]. Inhibition Diameter (ID) and Minimum Inhibition Concentration (MIC) of extracts and fractions are as shown in Table 1 and Table 2. Lerchea cf. bracteata Val. is a small woody shrub with the height of up to 1m. Extraction and isolation work on this species using air dried material was unsuccesful, only a trace of crude polar alkaloids were found and no sign of the presence of alkaloids in the semi-polar fraction. Repeated extraction by using the fresh aerial parts then column chromtographed in the usual way led to isolation of the only detected major quaternary indole alkaloid 350 Natural Product Communications Vol. 6 (3) 2011 Arbain et al. Tabel 1: Growth Inhibition Assay of MeOH extracts of Lerchea cf. bracteata and L. parviflora and their fractions. __________________________________________________________________________________________________________________________________________________________________________ Species Extract/fraction Bs Diameter of inhibition (DI) zone (mm, 5 mg/disk) Ec Pa Kl Sa Ml Tm Ca _____________________________________________________________________________________________________________________________________________________________________________ L. parviflora MeOH` Hexane EtOAc n-BuOH L. bracteata MeOH Hexane EtOAc n-BuOH 6.0 5.0 7.5 8.5 9.5 6.0 8.5 6.0 9.5 - 5.0 5.0 10.5 8.5 8.5 6.5 10.5 7.5 7.5 14.5 7.5 14.0 6.0 9.5 - 6.5 6.0 9.0 15.5 8.5 7.5 11.5 6.5 12.0 15.5 7.5 11.5 6.5 8.5 - - - _ _______________________________________________________________________________________________________________________________________________________________________________ Table 2: Minimum Inhibitory Concentration (MIC) of MeOH extract of Lerchea spp and its fractions. Species Extract/ MIC (μg/mL) Pa Ec Se Sa MeOH Hexane EtOAc n-BuOH MeOH 5 2.5 5 10 20 20 20 5 20 20 20 2.5 2.5 20 20 20 2.5 10 20 20 Hexane EtOAc n-BuOH 10 20 20 20 5 20 5 5 20 20 10 20 fraction L. parvifloraa L.cf. bracteata Bs (Bacillus subtilis), Ec (Escherichia coli), Pa (Pseudomonas aeruginosa), Kl (Klebsiella sp), Sa (Staphylococcus aureus), Ml (Micrococcus luteus), and fungi; Tm (Thrichophyton mentagrophytes) and Ca (Candida albicans). Se Table 3: NMR data of N(b)-methylantirhine (2) (CD3OD, 500 MHz). _____________________________________________________________________________________________________________________ No.C δC(ppm) δH (ppm), multipilcity, J (Hz) ___________________________________________________________ __________________________________________________________ 2. 3. 5. 128.32 64.59 64.77 6. 18.84 7. 8. 9. 10. 11. 12. 13 14. 105.77 127.37 118.53 123.71 120..98 112.70 138. 48 24.48 15. 16. 17. 31.36 46. 82 60.34 18. 118.53 19. 20 140.26 46. 82 21. 22. 64.77 37.85 5.17, d(br), J3a-14b 12.9 3.80, dd, J 5α− 5β12.3, J 5α−6α 6.5 3.94, ddd, J 5β−5α 12.3, J 5β−6β 12.3, J 5β−6α 6.0 3.28, m, J 6α−6β 17.0, J 6α−5β 12.3, J 6α−5α 12.0 3.18, dd, J 6β−6α 17.0, J 6β−5β 6.0 7.40, ddd, J 9-12 0.9, J 9-111.9, J 9-10 8.2 7.18, ddd, J 10-9 0.9, J 10-11 7.0, J 10-9 8.2 7.09, dd, J 11-9 0.9, J 11-10 7.0, J 11-12 7.9 7.50, ddd, J 12-9 0.9, J 12-10 1.9, J 12-11 7.9 2.79, d (br), J 14a-14b 13.6 2.21, ddd, J 14β-14α10.4, J H14 β -H3β 13.6, J H14 β -H15 β 8.2 2.19, m 2.67, m 1H 3.61, ddd, J gem 13.3, J 3.4, 13.3 3.51, ddd, J gem 13.3, J 2.0, 5.7 5.28, ddd, J trans 16.2 J gem 1.7, J 18-20 0.7 5.24, ddd, J cis 10,0, J gem 1.7, J 18-20 0.7 5.78, ddd, J trans 16.2, J cis 10.0, J 19-20 9.6 2.01, d (br), J 16α -16β  16.0 2.37, m, J16β -16α 16.0, J 16β -17α 10.0, J 16β -17β 4.9 3.77, d, 2H, H17, J 4.9 3.30, s, Me _____________________________________________________________________________________________________________________ from n-butanolic fraction in a low yield which was identified as N(b)-methylantirhine (2). Spectroscopic data as well as optical rotation of this compound also looked very similar to that of lercheine [2] except that instead of detecting an ethyl functional group like in lercheine, in this compound a vinyl group was clearly observed. Table 4: NMR data of 5,6-dihydroflavopereirine (3) (CD3OD,500 MHz). _____________________________________________________________________________________________________________________ No.C δC δH, multipilcity, coupling (200 MHz) [12] J (Hz) ______________________________________________________________________________________________________________________ 2. 3. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 18. 19. 20. 21. 124.98 142.03 56.41 19.27 117.66 125.16 112.37 126.53 121.09 120.23 140.25 120.51 145.21 13.64 26.36 140.43 143.25 4.88, 2H, m 3.42, m 7.46, 1H, dd, J 8.0,, 0.8 7.71, 1H, ddd, J 8.3, 8.0, 0.8 7.36, 1H, ddd, J 8.3, 8.0, 0.8 7.67, 1H, dd, J 8.3,0.8 8.07, 1H, d, J 8.0 Hz 8.36, 1H, dd, J 8.0, 2.0 1.38, 3H, t, J 7.6 2.85, 2H, q, J 7.6 2.85, 2H, q, J 7.6 4.98, 2H, m 7.82, d, J 8 7.26, t, J 8 7.45, t, J 8 7.64, d, J 8 8.33, J 8 1.38, t, J 7.5 1.38, t, J 7.5 2.88, q, J 7.5 9.01, 1 H, s ___________________________________________________________________________________________________________________ Unfortunately the COSY, HMBC, HSQC correlation did not clearly showed the exact correlation of position of protons and carbon atoms in the molecule. Surprisingly, single crystal X-ray diffraction analysis revealed that the skeleton of this compound is different from lercheine (1) [2] where H3 was in the α position while the vinyl group was not attached to the C20 but to C16. This compound was previously found in Hunteria eburnea Pichon [5], Strychnos camptoneura (Loganiaceae) [6], Amsonia elliptica (Apocynaceae) [7], Strychnos usambarensis [8] and from Alstonia angustifolia [9]. Compound (2) showed moderate activity against Plasmodium falciparum (strain K1) in vitro [10] while antirhine itself was first found in Antirhea putaminosa (F. Muell.) Bail. [11]. Another species L. parviflora Val. [12] looked totally different from L. cf. bracteta. The height attained 60 -70 cm with thinner and greener leaves. Extraction and fractionation work on this plant in the usual way gave a brownish gum. As above, the alkaloids containing n-butanolic fraction was chromatographed on silica gel but no pure compunds obtained. Column chromatographic separation on silica gel using an increasing amount of conc. aquaeus ammonia in n-butanol gave major fraction of which showed one spot on TLC. Based on its spectroscopic data particularly 1H NMR COSY, HSQC, HMBC and NOESY this compound was identified as 5,6-dihydroflavopereirine (3) (see the NMR assignment, Table 3). Preparative TLC of more polar fraction gave more of 3 and another more polar one with a slight inadmixture with 3. Alkaloids from Lerchea species Natural Product Communications Vol. 6 (3) 2011 351 Plant material: L. cf. bracteta (DA-RT 2074) was collected in Kerinci Seblat National Park (TNKS) near Kambang, Pesisir Selatan District, West Sumatra, while L. parviflora (DA-RT 6159) was collected in Teluk Kabung Forest near the City of Padang, West Sumatra, Indonesia. Both herbarium specimens were identified by Mr. Rusjdi Tamin and lodged at Herbarium Andalas University (ANDA). Figure 1: ORTEP diagram of N(b)methylantirhine (2). Surprisingly the mass and NMR spectra of these two compounds in deuterated methanol were identical. It was concluded that these two compounds were a quaternary indole alkaloids in the form of (3) and its zwitter-ionic form (3a) which was identical to that of the one synthesized by Lounasmaa et al. which they named 5,6-dihydroflavopereirine (3) following the preferred biogenetic numbering system of Corynanthe alkaloids as in geissoschizine (4) [13]. Compound (3) was previously isolated from Strychnos usambarensis Gilg [14] originally named 6,7-dihydroflavopereirine as it was considered as a quinolizine derivative alkaloid (3b) [14]. 6 9 10 7 5 7 8 1 12 13 N H OH Cl N 11 3 21 4 20 2 14 N 15 19 N H 6 N N H 18 3 3a 6 9 5 10 8 7 N 11 12 13 3b 1 4 21 20 3 2 N H H 14 18 19 15 Extraction and isolation: Fresh aerial parts of L. cf. bracteta (2.5 Kg) was macerated with MeOH for 2 days. After separation of solvent the process was repeated twice more. The combined methanolic extracts were evapolated in vacuo to give 55.4 g thick brownish gum. This thick extract (55 g) was disssolved in MeOH (500 mL) and water (500 mL) was added then fractionated in turn with hexane (3 x 1 L), EtOAc ( 3 x 1 l) and n-butanol (4 x 1 L) respectively and evaporated in vacuo to give fractions of hexane (11.0 g), EtOAc (6.4 g) and n-butanol (18.7 g) and water (19 g) respectively. The n-butanolic fraction (12.5 g) was preadsorbed on SiO2 (12.5 g) then eluted with n-butanol (1 l) then with the organic layer of admixture of n-butanol-AcOH-water (4:1:5) to give only one series of fractions which gave (+)-ve Dragendorff test. The combined fractions were evaporated and recrystallized from MeOH to give colorless plates (115 mg) of N(b)methylantirhine (2). MP: 164-166ºC. [α]D: +172 (c 0.013, MeOH). IR (KBr): 2963, 1643, 1575, 1476, 1458, 1437,1323, 1262, 1199, 1171, 1118, 972, 851, 809, 776, 733, 619 cm-1. UV λmax (ΜeΟΗ) nm (log ε): 231 (2.19), 229 (1.91), 312 (1.60). 1 H NMR (500 MHz) and 13C (125 MHz): Table 3 (based on COSY, HSQC, HMBC and NOE experiments). MS (CI, 70 eV): m/z (%) = 311 (CI,M+1, C20H26N2O with loss of HCl by thermal Hoffman elimination from C20H27ClN2O). 16 17 4 H 3COOC OH Experimental General: Melting points were measured on a Sybron Thermolyne Melting Point Apparatus MP-12615 and are uncorrected, optical rotations were aquired on JASCO P 1030 polarimeter, FT-IR spectra on Perkin Elmer FT-IR Spectrum One spectrophotometer. UV spectra on Shimadzu Spectrophotometer UV-VIS Pharmaspec 1700, the 1H and 13C NMR spectra were measured using Bruker500 (500 MHz for 1H and 125 MHz for 13C) instruments with chemical shift values expressed in δ (ppm) downfiled from TMS as internal standard. Mass spectra were obtained on a JEOL JMS 700 instrument. TLC was carried out using silica gel 60F254 (Merck) and visualized under UV light (254 nm) or sprayed with Dragendorff reagent. Column chromatography was performed on silica gel 60 (0.063-0.200 mm) (Merck). Preparative TLC were done by using silica gel PF254 (Merck). Fresh aerial parts of L. parviflora (1.5 kg) was treated as above to give fractions of hexane (4.5 g), EtOAc (12.5 g), and BuOH (16.1 g). The n-butanolic alkaloid containing fraction (11 g) was chromatographed on silica gel as above but no pure compounds were isolated. The work was repeated by using an increasing amount of conc. aquaeus ammonia solution in n-butanol as a mobile phase to give two major fractions of alkaloids. The less polar factions were combined and recrystallized from methanol to give 210 mg colorless feathery needles which was identified as 6,7-dihydroflavopereirine (3). MP: 246ºC (dec.). IR (KBr): 3022, 1638, 1589, 1557, 1432, 1313, 1286, 1242 cm-1. UV λmax (ΜeΟΗ) nm (log ε): 247 (2.52), 314 (1.82), 392 (1.79). 1 H NMR (500 MHz) and 13C (125 MHz): Table 4 (based on COSY, HSQC, HMBC and NOE experiments). 352 Natural Product Communications Vol. 6 (3) 2011 MS (CI, 70 eV): m/z (%) = 248 (M+1), (C17H15N2 with loss of HCl by thermal Hoffman elimination from C17H16ClN2). The more polar fractions was chromatographed on preparative TLC eluted with 10% aqueous NH4OH in n-butanol which after recrystallization yielded 90 mg of (3) and 110 mg of more polar quaternary indole alkaloid (3b) with a slight inadmixture with 3. Antimicrobial test: Bacterial cultures, namely Echerichia coli (Ec, ATTC:8739), Pseudomonas aeruginosa (Pa, ATTC: 1542), Staphylococcus aureus (Sa, ATTC: 6538), Micrococcus luteus (Ml, ATCC: 9342), Bacillus subtilis (Bs, clinically isolated), Klebsiella (Kl, clinical isolate), and fungi; Candida albicans (Ca, ATTC: 10231) and Thrichophyton mentagrophytes (Tm, ATTC: 5431) were obtained from Biofarma-Bandung Indonesia and grown in Arbain et al. nutrient agar media at 37ºC for bacteria and Sabaroud dextrose agar at room temperature for fungi. Each bacterium and fungus strain was transferred from stored media to fresh media 24 hour before being used as well as for a preculture on nutrient broth media. Assay for antimicrobial activity was performed using agar diffusion methods. A paper disk 5 mm in diameter (Whatman No.3) was used as reservoir [16]. MIC was determined using dilution method in 96-well microtiter plate and nutrient broth used as media [17]. Acknowledgment - Competency Grant from the Directorate General of Higher Education, the Ministry of Education of the Republic of Indonesia (to DA) and Research grant from International Foundation for Science (IFS) and from Organization for Prevention of Chemical Weapons (OPCW) (to DPP) are gratefully acknowledged. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] Kew plant database (Accessed October 2006). Arbain D, Putra DP, Lajis NH, Sargent MV, White AH. (1992) A new quaternary alkaloid from Lerchea bracteata, Journal of Chemical Society Perkin Transaction I, 3039-3042. (and literatures cited therein) Arbain D, Dachriyanus, Sargent MV. (1998) The Alkaloids of Ophiorrhiza blumeana, Journal of Chemical Society Perkin Transaction I, 2537-2540. (and literatures cited therein). Putra DP, Friardi, Arbain D. 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(1980) Procedure for Testing Antimicrobial Agent in Agar media; Theoretical Considerations, in Antibiotics in Laboratory Medicines, Ed. Lorian, The William & Wilkins Co, Baltimore. Lennette TH, Barilows A, Hausler Jr WJ, Shadony HJ. (1991) Manual of Clinical Microbiology (5th ed). American Society for Microbiology, Washington, DC.