BIOLOGICAL ACTIVITIES OF 13 , 28-EPOXYOLEANANE TRITERPENE SAPONINS FROM TWO PERUVIAN MYRSINACEAE

Two known 13,28-epoxy-oleanane triterpene saponins (1) and (2), were isolated from the 95% ethanolic extract of the roots of Myrsine coriaceae and Myrsine andina. Their structures were deduced by combined spectral analysis and chemical evidences based on data reported in the literature. Compounds 1 and 2 were evaluated in vitro against different cellular models such as Mycobacterium tuberculosis, Leishmania amazonensis axenic amastigotes, six human cancer cell lines (Hs683, T98G, U251, HT29, MCF7, SKMEL28) and two murine cell lines (CT26 and B16F10). Compound 1 was found to exhibit antileishmanial activity (IC = 16 μg/mL) 50 whereas compound 2 was inactive (IC > 50 μg/mL). Furthermore, compound 1 exhibited 50 stronger inhibition activity on human cancer cells (IC = 15 μg/mL) and on murine cell lines 50 (IC = 10 μg/mL) than compound 2 (IC > 82 and 42 μg/mL, respectively). As the only 50 50 difference between 1 and 2 is due to a substitution of an aldehyde group by a hydroxymethyl moiety, these results showed the crucial role of the aldehyde function at C-30 for the cytotoxicity. In contrast, none of the tested compounds revealed activity against M. tuberculosis.


INTRODUCTION
Infectious diseases such as leishmaniasis and tuberculosis (TB), commonly being considered as neglected diseases, are prevalent in Third World countries.Because most of the affected population live in developing countries and cannot afford existing drugs, both diseases have been ignored by pharmaceutical industry.Leishmaniasis is a parasitic protozoal disease caused by parasites belonging to the genus Leishmania, occurring in subtropical and tropical regions.An estimated 12 million people are infected worldwide in 88 countries with an annual incidence 1,2 of about 2-3 million.Clinically, leishmaniasis occurs in visceral, cutaneous and mucocutaneous forms, 90% of the latter occurring in Bolivia, Brazil and Peru.It is a major public health problem in tropical and subtropical regions for which development of drug resistance by the parasites has worsened this problem.In the absence of effective vaccines, 3 chemotherapy still plays a critical role in treating this disease.As far as TB is concerned, this is a common and in some cases deadly infectious disease, caused by an important intracellular pathogen Mycobacterium tuberculosis.This disease causes 2 million deaths per year worldwide, with 98% occurring in developing countries.One of the 4,5 highest incidence rates in the Americas occurs in Peru.Current standard treatments for tuberculosis include antibacterial drugs such as Rifampin and Isoniazid, which require between six to twelve month-therapies to fully eliminate Mycobacterium from the body.As very few treatments are available, there is an urge to discover new treatments with less toxicity and low manufacturing costs.Cancer, responsible for 7.6 million deaths worldwide in 2008 with approximately 70% 6 occurring in low-and middle-income countries, represents another major health issue.In such 7a-d 8 countries.including Peru, some plant extracts are used to combat various types of cancers and provide evidence that plants represent a major reservoir within which we may identify 9a-c novel anticancer drugs.
In the course of our investigations on compounds with pharmacological properties and since it has been shown that the 13,28-epoxy bridge in the saponin skeleton was putatively assigned as 10 crucial for antileishmania activity, we carried out a literature search in order to find saponins showing the same structural features as the Maesabalides.These compounds, possessing a 11 13,28-epoxy-oleanane bridge and isolated previously from Maesa balansae (Myrsinaceae), 12 13 have been shown to possess potent and highly speci?c in vitro and in vivo antileishmania 14 activity.Vermeersch et al., (2009) reviewed all those saponins from Myrsinaceae, Primulaceae, Aceraceae and Icacinaceae families possessing a completely saturated pentacyclic triterpene skeleton and a 13,28-epoxy bridge, in which C(28) was a methylene or a hydroxymethylene group, excluding the C(28) carbonyl derivatives.For all the studied plants, a clear correlation was found between the presence of close analogue 13,28-epoxy-oleanane triterpene saponins and a potent and selective antileishmania activity.Furthermore, Aegicerin, an oleanane-type pentacyclic triterpene skeleton with this 13,28-epoxy bridge, has shown a consistently high level of activity against a large panel of both sensitive and resistant strains of 15 M. tuberculosis .In this article, we report the isolation and structure elucidation of two known triterpenoid saponins, Ardisiacrispin B (1) and Ardisicrenoside A (2), previously isolated from several 16,17 Myrsinaceae plants.Both compounds were found to possess the 13,28-epoxy bridge 12 13 previously demonstrated as crucial for antileishmanial activity in vitro and in vivo.We evaluated their activity in vitro against Leishmania amazonensis axenic amastigotes alongside other pathogenic cellular model, e.g.Mycobacterium tuberculosis, as well as two mouse and six human cancer cell lines.
To the best of our knowledge, this is the ?rst report of the isolation of saponins from the Myrsine andina (Mez.)Pipoly and Myrsine coriacea (Sw.) R. Br. ex Roem.& Schult

EXPERIMENTAL General
Optical rotations were measured on a Perkin-Elmer 241 polarimeter with a sodium lamp (k = 589 nm) in a 1 cm microcell.The structures of the isolated compounds were identified by nuclear magnetic resonance (NMR; Bruker Avance 500 equipped with a TBI z-gradient 5 mm

Extraction and isolation
The air-dried and powdered roots of Myrsine andina and Myrsine coriacea (500 g and 300 g, respectively) were exhaustively macerated with 95% EtOH (3 x 3 L) at room temperature to yield 29.1 g and 52.8 g of crude extract after evaporation of the solvent in vacuo, respectively.Both residues were dissolved in the least amount of MeOH and the solution was diluted with tenfold amount of diethyl-ether to precipitate 2 g and 2.3 g of crude saponin mixture, respectively, and then partitioned with n-BuOH saturated with H O (3 x 500 mL).The     con? rmed the absolute con? guration of the aglycone.Based on these findings, the aglycone was identified as 3β ,16α dihydroxy-13β ,28-epoxyoleanane.

C
The ring protons of the monosaccharide residues were assigned starting from the anomeric protons by means of the COSY, TOCSY, HSQC, and HMBC NMR plots (table 1), and the sequence of the oligosaccharide chains was obtained from the HMBC and NOESY experiments.All the protons within each spin system were delineated using COSY with the structures lead to the identification of the four monosaccharide units as β -D-glucose (Glc x2), one α -L-arabinose (Ara), and one α -L-rhamnose (Rha).The COSY and TOCSY spectra confirmed the presence of the one α -rhamnopyranosyl (Rha) from their typical pattern in the 23,24 COSY spectrum and the α -configuration of this latter was also confirmed by observation of NOESY correlations between δ (Rha-1), δ (Rha-3), and δ (Rha-5).This information was was equatorial, thus possessing an α -configuration in the C form.respectively.Two methine carbons bearing oxygen were found at δ 88.8 and 76.9 ppm.The C structural assignment was initiated from the long-range coupling networks observed between the methyl protons and the adjacent carbons from the HMBC experiments.Extensive NMR analysis (table 1) showed that the aglycone was an oleanane skeleton with an oxygen bridge between Agly-13 (δ 86.3) and Agly-28 (δ 77.7).This was confirmed by comparison of the The ring protons of the monosaccharide residues were assigned starting from the anomeric protons by means of the COSY, TOCSY, HSQC, and HMBC NMR plots (table 1), and the sequence of the oligosaccharide chains was obtained from the HMBC and NOESY experiments.All the protons within each spin system were delineated using COSY with the aid hydrolysate of 2 in comparison with reference sugars, NMR data of related structures lead to the identification of the four monosaccharide units as β -D-glucose (Glc x2), one α -Larabinose (Ara) and one α -L-rhamnose (Rha).The COSY and TOCSY spectra confirmed the presence of the one α -rhamnopyranosyl (Rha) from their typical pattern in the COSY 23,24 spectrum and the -configuration of this latter was also confirmed by observation of NOESY correlations between Rha-1, Rha-3, and Rha-5'.This information was confirmed by the Rha-1 non-splitting pattern of rhamnose unit and the three-bond HMBC correlations from H-Rha-1 to Rha-3 and Rha-5 indicated that its anomeric H-atom was equatorial, thus possessing an - 1 25 configuration in the C form. the Glc . 1 13 The H-and C-NMR signals of 2 assigned from 2D-NMR spectra were almost superimposable on those of 1, except for the disappearance of the signals corresponding to the aldheyde group (δ 207.3, C-30) replaced by a hydroxymethyl function (δ 65.7) (table 1).

C C
On the basis of the above results, the structure of 2 was elucidated as 3-β -Oα -lrhamnopyranosyl (1→ 2)-β -D-glucopyranosyl (1→ 3)-[β -D-glucopyranosyl (1→ 2)]-β -d-arabinopyranosyl}-16α -hydroxy-30-hydroxymethyl-13β ,28-epoxy-oleanane.Compounds 1 and 2 were evaluated in vitro against different cellular models such as 15 16 Mycobacterium tuberculosis , Leishmania amazonensis axenic amastigotes , mouse peritoneal macrophages, six human cancer cell lines (Hs683, T98G, U251, HT29, MCF7, SKMEL28) and two murine cell lines (CT26 and B16F10) (table 3).µg/mL) than compound 2 (IC > 82 and 42 µg/mL, respectively).As the only difference 50 between 1 and 2 is due to a substitution of an aldehyde group by a hydroxymethyl moiety, these results showed the crucial role of the aldehyde function at C-30 for the cytotoxicity.The possibility remains that this growth inhibitory activity is related to compound 1 and 2 mediated cytotoxic effects on the parasites' host cell itself.One possible explanation previously raised to explain the cytotoxicity of such saponins on macrophages is that saponins are amphiphilic and induce the formation of micelles, which can be easily taken up by  Literature reports that the presence of a carbonyl group at the C-16 position of a triterpene would be crucial to exhibit anti-mycobacterial activity.For example, protoprimulagenin A, bearing a hydroxyl moiety at C-16, displays no antibacterial activity whereas aegicerin, having almost the same structure but with a carbonyl group at C-16, exhibits marked 15 antibacterial activity against a large number of resistant Mycobacterium strains.

CONCLUSIONS -
In conclusion, the structures of two known 13,28-epoxy-oleanane triterpene saponins (1, 2) isolated from two Peruvian Myrsinaceae, Myrsine coriacea and Myrsine andina, were elucidated.This is the first report of saponins isolation from these plants.The present study demonstrated that compound 1 displayed significant anti-tumor activities towards a broad spectrum of human cancer cells.Furthermore, we demonstrated the crucial role of the aldehyde function at C-30 for the cytotoxicity.Further experiments should be carried out in order to investigate their ability to induce apoptosis in different cancer cell lines.These results may be useful in the search for new compounds for cancer prevention and therapy.

18 Compounds 1
MHz) and C NMR (125 MHz) data of 1 and 2 in C D N. The assignments were based on DEPT, H , 5 5 1 H-COSY, TOCSY, NOESY, HMQC and HMBC experiments.b Not determined.Bioassays and 2 were evaluated against L. amazonensis axenic amastigotes in vitro and 15 against M. tuberculosis in vitro .Two independent experiments were carried out to determine the in vitro growth inhibitory activity of compounds 1 and 2 in cancer cell lines.This growth inhibitory activity was determined by means of the sulforhodamine B (SRB) assay method 19a-b; 20a-d and the MTT colorimetric assay, respectively, as detailed previously.RESULTSExtraction of the roots of Myrsine andina (Mez.)Pipoly and Myrsine coriacea (Sw.) R. Br. ex Roem.& Schult and puri?cation of the extract as described in the experimental section yielded compounds 1 and 2. The elucidation of their structures was performed mainly by 500 MHz NMR analysis, including 1D-and 2D-NMR (COSY, TOCSY, NOESY, HSQC, HMBC), and mass spectrometry.Compound 1 was obtained as a white crystalline powder with molecular formula C H O 53 86 22, which was determined by analysis of the MS data.Its positive ESI-MS showed the + pseudomolecular ion peak at m/z 1097 [M + Na] .This was con? rmed from its negative ion -ESI-MS that showed the quasimolecular ion peak at m/z 1073 [M -H] .Other fragment ion -peaks were observed at m/z 927 [(M -H) -146] , 765 [(M -H) -146 -162] , 603 [(M -H) ---146 -162 -162] , and 471 [(M -H) -146 -162 -162 -132] corresponding to the successive loss of one deoxyhexosyl, two hexosyl and one pentosyl moieties, respectively.Of the 53 carbons in the C-NMR spectrum (pyridine-d ), 30 were assigned to the triterpenoid 5 skeleton and 23 to the oligosaccharide moiety.Among the 30 carbons of the triterpene 13 skeleton in the C-NMR spectrum, 6 were assigned to the methyl carbons at δ 16.

13 aid
of TOCSY and NOESY spectra.After assignments of the protons, the C-NMR resonances of each sugar unit were identified by HSQC and further confirmed by HMBC.HPTLC of the acid hydrolysate of 1 in comparison with reference sugars, NMR data of related11,21 δ (Rha-1) non-splitting pattern of rhamnose unit and the three-bond HMBC C correlations from δ (Rha-1) to δ (Rha-3) and δ (Rha-5) indicated that its anomeric H

13 of
TOCSY and NOESY spectra.After assignments of the protons, the C-NMR resonances of each sugar unit were identified by HSQC and further confirmed by HMBC.HPTLC of the acid 11, 21

4 3 The
relatively large J values of the Glc (7.5-7.8),indicated a β anomeric orientation for H-1,H-2 26 have been shown to exhibit antibacterial activity against Mycobacterium tuberculosis, this was not the case for saponins 1 or 2 (IC > 25 µM). 50

Table 1 .
NMR Spectroscopic data of aglycon and sugar moieties of compounds 1 and 2.