A New Iridolactone from Bremeria Erectiloba Wernham (Rubiaceae)

International Journal of Chemical Studies 2018; 6(5): 78-80

Daniel Sendranirina Safidindray Daniel Sendranirina Safidindray, Anne Wadouachi, Amélie Raharisololalao a) Mention Chimie Médicinale, and Léa Herilala Rasoanaivo Faculté de Sciences, Université de Fianarantsoa Madagascar, East Africa Abstract b) Laboratoire de Chimie des From the leaves of a Madagascar endemic Bremeria erectiloba Wernham, a new iridoid 2 4β- Substances Naturelles et Chimie carbomethoxy-6α, 8α-dihydroxy-8β-methyliridolactone and a known triterpene 1 ursolic acid were Organique Biologique isolated and its structure was established by NMR spectroscopic methods. These compounds are (LCSN/COB), Faculté de Sciences, Université d’ Antananarivo, BP described for the first time for this plant. Ankatso, Antananarivo, Madagascar, East Africa Keywords: Rubiaceae, Bremeria erectiloba, triterpene, iridolactone, NMR

Anne Wadouachi Laboratoire de Glycochimie des 1. Introduction Antimicrobiens et des Rubiaceae family contains about 13,143 species and 660 genera in which Mussaenda, Agroressources CNRS UMR Bremeria. Evidence from molecular phylogenies suggests that Rubiaceae family can be Institut de Chimie de Picardie subdivided into three subfamilies (Cinchonoideae, Ixoroideae, and Rubioideae) [1, 2, 3, 4, 5]. CNRS FRE. Université de Picardie [6, 7] Jules Verne, France, Europe About 28 species from Mussaenda were transferred to Bremeria with 18 species endemic of Madagascar whose distinctive characters are given. Bremeria populary known as “fatora” or Amélie Raharisololalao “nofotrakoho”. This species are found in humid to sub-humid evergreen forests [8, 9]. Bremeria Laboratoire de Chimie des genus is met more exclusively in Madagascar and Mascarenes. The juicy of leaves of Substances Naturelles et Chimie Organique Biologique Bremeria erectiloba Wernham is used in traditional medicine Malagasy to treat the (LCSN/COB), Faculté de Sciences, splenomegalie. Not much has been reported on the chemical constituents or biological Université d’ Antananarivo, BP properties of this plant. Bremeria erectiloba Wernham syn. Mussaenda erectiloba belongs to Ankatso, Antananarivo, Madagascar, East Africa the Ixoroideae subfamily in Rubiaceae family. This subfamily is rich of iridoids compounds, 10, 11] which are presented as chemotaxonomic markers [ . The present study has led to the Léa Herilala Rasoanaivo isolation of a new iridoid 4β-carbomethoxy-6α, 8α-dihydroxy-8β-methyliridolactone named Laboratoire de Chimie des bremerine and a known triterpene ursolic acid (Figure 1). Substances Naturelles et Chimie Organique Biologique (LCSN/COB), Faculté de Sciences, Université d’ Antananarivo, BP Ankatso, Antananarivo, Madagascar, East Africa

Ursolic acid bremerine Correspondence Daniel Sendranirina Safidindray Fig 1: Compounds isolated from leaves of Bremeria erectiloba Wernham a) Mention Chimie Médicinale, Faculté de Sciences, Université de Fianarantsoa Madagascar, East 2. Materials and methods Africa 2.1. Plant Material b) Laboratoire de Chimie des The leaves of Bremeria erectiloba Wernham were collected from Ambodiamontana commune Substances Naturelles et Chimie Organique Biologique of Ranomafana district of Ikongo, Region Vatovavy-Fitovinany. The plant was identified to (LCSN/COB), Faculté de Sciences, the herbarium references at Botanical and Zoological Park Tsimbazaza (PBZT, Antananarivo Université d’ Antananarivo, BP Madagascar) and a voucher specimen has been deposited in the “Laboratoire de Chimie des Ankatso, Antananarivo, Substances Naturelles Chimie Organique et Biologique” (LCSN/COB). Madagascar, East Africa ~ 78 ~ International Journal of Chemical Studies

2.2. General experimental procedures MHz): δ (ppm), DEPT Q (CD3OD, 125.78 MHz) of 2 are 1H and 13C NMR spectra were recorded on a Bruker Varian given in table 1. 600 NMR instrument with a 600.19/125.78 MHz operating 1 frequency using CD3OD solutions with Tetramethylsilane Compound 1: white powder; δ(ppm) H NMR (600.19 MHz, (TMS) as internal standard. Chemical shifts are reported in CD3OD): 5.49 (1H,br.s, H-12), 3.45 (1H,dd, J=5.7, 9.9 Hz, H- ppm and δ scale with the coupling constants given in Hz. 3), 2.63 (1H,d, J=11.2 Hz, H-18), 1.24 (3H,s, H-23), 1.23 Column chromatography was carried out using silica gel 60 (3H,s, H-27), 1.05 (3H,s, H-25), 1.03 (3H,s, H-26), 1.00 (Merck 70 to 230 Mesh ASTM 0.063-0.200 mm). Thin Layer (3H,br.s, H-29), 0.96 (3H,s, H-30), 0.90 (3H,s, H-24). Chromatography (TLC) was carried out on silicagel 60 F254 δ(ppm) DEPT Q (125.78 MHz, CD3OD) : 180.0 (C-28), 139.3 (Merck). (C-13), 125.7 (C-12), 78.2 (C-3), 55.9 (C-5), 53.6 (C-18), 48.1 (C-9), 48.1 (C-17), 42.6 (C-14), 40.0 (C-19), 39.5 (C- 2.3. Extraction and isolation 20), 39.5 (C-8), 39.2 (C-1), 37.7 (C-4), 37.5 (C-22), 37.3 (C- The dried powder (500 g) was kept for maceration with 80% 10), 33.6 (C-7), 33.1 (C-21), 28.8(C-23), 28.2 (C-2), 25.0 (C- aqueous ethanol (2000 mL) at room temperature for 6 days. 16), 24.0 (C-27), 23.7 (C-11), 21.4 (C-30), 18.8 (C-6), After concentration under reduced pressure. The percentage 17.5(C-29), 17.5 (C-26), 16.6 (C-24), 15.7 (C-25). yield of extract was found to be 6.83%. The hydro-alcoholic extract (32.43 g) was suspended in water at 40°C and then 3. Results and discussion partitioned sequentially using hexane and ethyl acetate, The phytochemical screening subjected to detect the presence furnishing hexanic (0.53 g), ethyl acetate (8.56 g) and of some secondary metabolites following standard procedure aqueous (23.12 g) extracts respectively. showed presence of terpenic compounds (triterpenes and The ethyl acetate extract was first subjected to fractionation iridoids), phenolic compounds (leucoanthocyanins, using silica gel column. Elution with hexane, then gradually coumarins) polyphenols, catechictanins, the polysaccharides increasing polarity with ethyl acetate (100:0 → 0:100) and and saponins in ethanolic extract from the leaves of a ethyl acetate-methanol (100:0 → 0:100). 488 fractions were Bremeria erectiloba. collected and combined as usual using TLC monitoring. Compound 1 was obtained as white amorphous from ethyl Combined fractions were purified. Compound 1 (6 mg, RF= acetate extract. The 1H and 13C NMR spectroscopic data of 1 0.75, Dichloromethane/MeOH 3/7) was obtained as white was in agreement with those reported for ursolic acid (3β- amorphous from fractions 142-145 eluted by hexane/AcOEt hydroxyurs-12-en-28-oic acid) [12] previously isolated from 80/20 and compound 2 (10.34 mg, RF= 0.30, AcOEt/MeOH Mussaenda pubscens, Mussaenda hainanensis, Mussaenda 9/1) was isolated as white powder from fractions 278-284 frontosa [13,14] 1 eluted by AcOEt. Data spectral of NMR H (CD3OD, 600.19

Table 1: 1H, DEPT Q and HMBC NMR data of compound 2

Position 휹1H (ppm) 휹13C (ppm) HMBC 1 -- 175.7 -- 3 3.84 (12.60, 6.00) 3.97 (12.00, 6.00) 63.9 C-1, C-4, C-5, C-11 4 2.84 50.9 C-3, C-5, C-6, C-11 5 2.69 50.2 C-1, C-3, C-4, C-6, C-7, C-9, C-11 6 4.39 76.5 C-5, C-7, C-8 7 1.70 (12.00, 6.00) 2.40 (12.00, 6.00) 49.5 C-6, C-8, C-9 C-5, C-6, C-7, C-10 8 -- 79.6 -- 9 2.76 (6.00, 1.80) 61.9 C-1, C-4, C-5, C-6, C-7, C-8 10 1.28 25.3 C-7, C-8, C-9 11 -- 175.9 -- OMe 3.67 51.9 C-11 Coupling constant in Hz are given in parentheses

Compound 2 was isolated from the ethyl acetate extract. The The 13C NMR data along with HMBC clearly corroborated 1 H NMR spectrum in CD3OD showed signals for one tertiary the proposed structure. The correlations from protons H-3 (훿 methyl at 훿 1.28 (3H, s), one carbomethoxy at 훿 3.67 (3H, s), 3.84 and 3.97) with C-1 (훿 175.7), C-4 (훿 50.9), C-5 (훿 50.2) four methine protons at 훿4.39 (1H, m, H-6), 2.84 (1H, m, H- and C-11 (훿 175.9) and of OCH3 (훿 3.67) with C-11 4), 2.76 (1H, dd, J = 6.00 and 1.80 Hz, H-9) and 2.69 (1H, m, demonstrated that the carboxyl is methoxylated. In addition, H-5), two methylene groups at 훿2.40 (1H, dd, J = 12.00 and the correlations between H-7 (훿 1.70 and 2.40) with C-5 (훿 6.00 Hz, Hβ-7), 1.70 (1H, dd, J = 12.00 and 6.00 Hz, Hα-7), 50.2), C-6 (훿 76.5), C-7 (훿 49.5), C-8 (79.6), C-9 (훿 61.9) and 3.97 (1H, dd, J = 12.60 and 6.00 Hz, He-3), 3.84 (1H, dd, C-10 (훿 25.3) corroborated the partial structure of 2 as similar [15, 16] 12.00 and 6.00 Hz, Ha-3). to those of iridolactone . The COSY and HSQC correlations indicate that proton in β These findings were also supported by 13C chemical shift position at 2.40/49.5 is correlated to proton at 4.39/76.5 itself values analyzed for 2 in comparison with those observed for correlates to proton at 2.69/50.2 that itself correlates to proton shanzilactone and shanzilactone acetate model compounds at 2.76/61.9. These protons are in β positions. The proton used to establish the structure. But, there are little differences. 2.84/50.9 is in α position that it has a great value coupling Conﬁrmation of the stereochemistry of its stereogenic centers constant with the proton in α position at 3.84/63.9.The C was achieved by analysis of J values and comparison with 13C NMR spectrum showed 11 carbon signals which were shown NMR literature data, especially those for chiral centers at C-5, by DEPT Q experiments as 2 primary, 2 secondary, 4 tertiary C-6, C-8, and C-9 which indicated the α-conﬁguration of the and 3 quaternary carbons. hydroxyl group at C-6 and C-8. The trans junction between

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the two rings and β configuration [12, 13]. According to these Mussaendeae (Rubiaceae). American Journal of Botany. assumptions the new iridoid adopts the 5 and 8 R 2005; 92: 544-557. configurations, 6 and 9 S conﬁgurations, not similar to those 7. Davis AP, Razafimandimbison SG, Andriambololonera iridoid models. S. Nomenclatural changes in the genus Bremeria These data spectral lead to identified compound 2 as 4β- (Rubiaceae). Blumea. 2011; 56:4-5. carbomethoxy-6α, 8α-dihydroxy -8β-methyliridolactone 8. Andriambololonera S. Ireo toetra famantarana ny named bremerine. Selected correlations COSY observed in Mussaenda. Ravintsara. 2004; 2:20-21. compound 2 are given in figure 2. 9. Shatz GE. Generic tree Flora of Madagascar. London: Royal. Botanical Gardens Kew; St Louis: Missouri Botanical Garden. 10. Daiane M, Cecilia VN. 2015. Secondary Metabolites from Rubiaceae Species. Molecules. 2001; 20:13422- 13495. 11. Bolzani VS, Yong MCM, Furlan M, Cavalheiro AJ, Araujo AR, Silva DHS et al. Secondary Metabolites from Brazilian Rubiaceae Plant Species: Chemotaxonomical and Biological Significance. Recent Research Developments in Phytochemistry. 2001; 5:19-3. 12. Chao-Min Wang, Hsiao-Ting Chen1, Zong-Yen Wu, Yun-Lian Jhan, Ching-Lin Shyu, Chang-Hung Chou. Supplementary Materials: Antibacterial and Synergistic Activity of Pent acyclic Triterpenoids isolated from Alstonia scholar. Molecules. 2016; 21:139. 13. Ke Yuan, Wen-Tao Qiao, Ming-Wen Yin. Phytochemical

Studies of Mussaenda hainanensis Merr. Asian Journal Fig 2: Selected COSY correlations observed in bremerine of Chemistry. 2009; 21(9):7138-7142. (4β-carbomethoxy-6α, 8α-dihydroxy-8β-méthyliridolactone) 14. Zhou Zhonglin, Sun Jiyan, Pan Liming; Wang Hui. Studies on chemical constituents from Mussaenda 4. Conclusion pubescens Ait. F Journal of Guangdong Pharmaceutical The present study reports to the isolation and identification of University, 2017, 2. a new iridoid and a known triterpene, these compounds are 15. Vidyalakshmi KS, Vasanthi Hannah R, Rajamanickam isolated for the first time of Bremeria erectiloba Wernham. GV. Ethnobotany, Phytochemistry and Pharmacology of The existence of Bremeria erectiloba Wernham to the Mussaenda species (Rubiaceae). Ethnobotanical Leaflets. Ixoroideae subfamily in Rubiaceae family is verified because 2008; 12:469-475. iridoids compounds are presented as chemotaxonomic 16. Biswanath D, Sudhan D, Santanu M, Shiho A, Narico S, markers of this subfamily. Yoshihiro H. Chemical constituents of Mussaenda uncana. Indian Journal of Chemitry. 2005; 44:2362-2366. 5. References 1. Davis AP, Govaerts R, Bridson DM, Ruhsam M, Moat J, Brummitt NA. A global assessment of distribution, diversity, endemism, and taxonomic effort in the Rubiaceae. Ann. Mo. Bot. Gard. 2009; 96:68-78. 2. Robbrecht E, Manen J. The Major Evolutionary Lineages of the Coffee Family (Rubiaceae, Angiosperms). Combined Analysis (n DNA and cp DNA) to infer the Position of Coptosapelta and Luculia, and Supertree Construction Based on rbcL, rps16, trnL-trnF and atpB- rbcL Data. A New Classification in Two Subfamilies, Cinchonoideae and Rubioideae. Systematics and Geography of Plants. 2006; 76:85-146. 3. Bremer B, Andreasen K, Olsson D. Subfamilial and tribal relationships in the Rubiaceae based on rbcL sequence Data. ANN. Missouri BOT. Gard. 1995; 82:383-397. 4. Bremer B, Robbrecht E, Puff C, Smets E. Phylogenetic studies within Rubiaceae and relationships to other families based on molecular Data. Opera Bot. Belg. 1996; 7:33-50. 5. Bremer B, Jansen RK, Bengt O, Maria B, Henrik L, KI- JOONG K. More Characters or More Taxa for a Robust Phylogeny Case Study from the Coffee Family (Rubiaceae). Syst. Biol. 1999; 48(3):413-435. 6. Alejandro GD, Razafimandimbison SG, Liede-Schumann S. Polyphyly of Mussaenda inferred from ITS and TrnT- F data and its implication for generic limits in

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