Bioactive glycosides from the African medicinal plant Boerhavia erecta L.

Natural Product Research

2015, Volume 29, Issue 20

Editorial Board

Editor-in-Chief:

Armandodoriano Bianco - Dipartimento di Chimica, Università degli Studi di Roma "La Sapienza" , Piazzale Aldo Moro 5, 00185 Roma, Italy

Editors:

Søren Rosendal Jensen-DTU Chemistry, Department of Chemistry, Emeriti, Organic Chemistry, DK-2820 Lyngby, Denmark

Marcello Nicoletti-Facolta di Farmacia e Medicina, Università degli Studi di Roma "La Sapienza", Piazzale Aldo Moro 5, 00185 Roma, Italy

Editorial Office:

Alessandro Venditti -Dipartimento di Chimica, Università degli Studi di Roma "La Sapienza" , Piazzale Aldo Moro 5, 00185 Roma, Italy

Associate Editors:

D. J. Aitken- University Blaise-Pascal-Clermont-Ferrand, France

B Charlwood- Universidade Federal de Alagoas, Campus Universitario, Brazil P. Ciminiello - Università di Napoli "Federico II", Italy

M. E. Kuehne- University of Vermont, USA

L. N. Mander- The Australian National University, Canberra, Australia K. Mori- University of Tokyo, Japan

I. Ninomiya- Kobe Pharmaceutical University, Kobe, Japan R. J. Parry- Rice University, Houston, Texas

O. Sterner- Lund University, Sweden

J Stöckigt- Johannes Guttenberg University, Germany

C. Szantay - Technical University of Budapest and Hungarian Academy of Sciences, Budapest, Hungary

Editorial Board: G. Appendino-Italy Y. Asakawa- Japan M. Ballero- Italy Y. Ban- Japan E. Block- USA M. Bruno-Italy

H. Budzikiewicz- Germany S. D. Burke- USA

L. Castedo- Spain G. Cimino- Italy G. A. Cordell- USA B. Danieli- Italy S. Danishefsky- USA V. De Feo- Italy I. Fleming- UK B. Fraser-Reid- USA O. R. Gottlieb- Brazil L. Gunatilaka- USA G. M. Halpern- China S. Hanessian- Canada J. R. Hanson- UK T. Higa- Japan

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E. Rodriguez- USA M. Rueffer- Germany D. S. Rycroft- UK A. I. Scott- USA B. Sener- Turkey M. Serafini- Italy L. -Y. Sheen - Taiwan D W. Slocum- USA G. Solladie- France

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W. C. Taylor- Australia G. Topcu- Turkey M. Tori- Japan B. M. Trost- USA I. Ugi- Germany

R. Verpoorte- Netherlands W. Voelter- Germany H. Wagner- Germany P. G. Waterman- Australia J. D. White- USA

E. Winterfeldt- Germany K. R. Zeller- Germany

Natural Product Research

2015, Volume 29, Issue 20 Contents

A new highly oxygenated daphnane diterpene esters from the flower buds of Daphne genkwa Hai-Long Jiang, Rui Wang, Jian-Yong Li & Yan-Ping Shi

pages 1878-1883

A new prenylated biflavonoid from the leaves of Garcinia dulcis Arun Saelee, Souwalak Phongpaichit & Wilawan Mahabusarakam pages 1884-1888

New sesquiterpenoid derivatives from Solanum septemlobum with cytotoxicities Lei Zhang, Hai-Qing Lin, Gui-Sheng Li, Xi-Dian Yue & Sheng-Jun Dai pages 1889-1893

The metabolic profile of lemon juice by proton HR-MAS NMR: the case of the PGI Interdonato Lemon of Messina Nicola Cicero, Carmelo Corsaro, Andrea Salvo, Sebastiano Vasi, Salvatore V. Giofré, Vincenzo

Ferrantelli, Vita Di Stefano, Domenico Mallamace & Giacomo Dugo pages 1894-1902

Two new eudesmane-type glucopyranosides from the fruits of Daucus carota L. Jin-Ke Xu, Xiang-Hong Lu, Wei Zhu, Bing-Xian Yang, Lin Zhang & Jing-Kui Tian pages 1903-1908

Two new chalcones from Shuteria sinensis

Zhuya Yang, Xiaoxia Ma, Wenhong Tan, Libin Zhou, Xinying Zhuang, Shude Yang, Zigang Qian & Zhihong Zhou

pages 1909-1913

Butyrolactones derivatives from the fermentation products of a plant entophytic fungus Penicillium oxalicum Lin Yuan, Wenzhong Huang, Kun Zhou, Yuede Wang, Wei Dong, Gang Du, Xuemei Gao, Yinhai Ma & Qiufen Hu

pages 1914-1919

Three new secoiridoid glycosides from the rhizomes and roots of Gentiana scabra and their anti-inflammatory activities

Wei Li, Wei Zhou, Sohyun Kim, Jung-Eun Koo, Yuna Kim, Young-Sang Koh, Sang Hee Shim, Jin Yeul Ma &Young Ho Kim

pages 1920-1927

Study of quantitative and qualitative variations in essential oils of Sicilian Rosmarinus officinalis L. Teresa Tuttolomondo, Giacomo Dugo, Giuseppe Ruberto, Claudio Leto, Edoardo M. Napoli, Nicola Cicero,Teresa Gervasi, Giuseppe Virga, Raffaele Leone, Mario Licata & Salvatore La Bella

pages 1928-1934

Sesquiterpene-rich volatile constituents of Ipomoea obscura (L.) Ker-Gawl. Rajesh K. Joshi

pages 1935-1937

Mass cultivation of various algal species and their evaluation as a potential candidate for lipid production Nadia Sharif, Neelma Munir, Faiza Saleem, Farheen Aslam & Shagufta Naz

pages 1938-1941

Chemical composition and variability of the volatile components from inflorescences of Cirsium species Małgorzata Kozyra, Marek Mardarowicz & Joanna Kochmańska

pages 1942-1944

Chemotypes of essential oil of unripe galls of Pistacia atlantica Desf. from Algeria Ibrahim Sifi, Nadhir Gourine, Emile M. Gaydou & Mohamed Yousfi

pages 1945-1949

Analysis of essential oils from Voacanga africana seeds at different hydrodistillation extraction stages: chemical composition, antioxidant activity and antimicrobial activity

Xiong Liu, Dongliang Yang, Jiajia Liu & Na Ren pages 1950-1953

Bioactive glycosides from the African medicinal plant Boerhavia erecta L.

Ari S. Nugraha, Adama Hilou, Nicholas Vandegraaff, David I. Rhodes, Rachada Haritakun & Paul A. Keller pages 1954-1958

Sony George, S Ajikumaran Nair, Ramaswamy Venkataraman & Sabulal Baby pages 1959-1962

HPLC simultaneous determination of arbutin, chlorogenic acid and 6′-O-caffeoylarbutin in different parts

of Vaccinium dunalianum Wight

Xu-Lu Luo, Na Li, Min Xu, Hong-Tao Zhu, Peng He, Yong Ding, Ping Zhao & Ying-Jun Zhang pages 1963-1965

Bioactivity and chemical composition of the essential oil from the leaves of Guatteria australis A.St.-Hil Carlos Alberto Theodoro Siqueira, Alessandra Freitas Serain, Aislan Cristina Rheder Fagundes

Pascoal, Nathalia Luiza Andreazza, Caroline Caramano de Lourenço, Ana Lúcia T. Góis Ruiz, João Ernesto de Carvalho, Ana Cláudia Oliveira de Souza, Juliana Tonini Mesquita, Andre Gustavo Tempone & Marcos José Salvador

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Bioactive glycosides from the African

medicinal plant Boerhavia erecta L.

Ari S. Nugrahaa, Adama Hiloub, Nicholas Vandegraaf fc, David I. Rhodesac, Rachada Harit akund & Paul A. Kellera

a

School of Chemist ry, Universit y of Wollongong, Nort hf ields Avenue, Wollongong, New Sout h Wales 2522, Aust ralia b

Départ ement de Biochimie–Microbiologie, UFR/ VT, Universit é de Ouagadougou, Burkina Faso 03, Af rica

c

Avexa Limit ed, 576 Swan St reet , Richmond, Vict oria 3121, Aust ralia

d

Nat ional Cent re f or Genet ic Engineering and Biot echnology (BIOTEC), Nat ional Science and Technology Development Agency (NSTDA), 113 Phaholyot hin Road, Klong1, Klong Luang, Pat humant hani 12120, Thailand

Published online: 20 Feb 2015.

To cite this article: Ari S. Nugraha, Adama Hilou, Nicholas Vandegraaf f , David I. Rhodes, Rachada Harit akun & Paul A. Keller (2015): Bioact ive glycosides f rom t he Af rican medicinal plant Boerhavia erect a L. , Nat ural Product Research: Formerly Nat ural Product Let t ers, DOI:

10. 1080/ 14786419. 2015. 1013470

To link to this article: ht t p: / / dx. doi. org/ 10. 1080/ 14786419. 2015. 1013470

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SHORT COMMUNICATION

Bioactive glycosides from the African medicinal plant

Boerhavia erecta

_L.

Ari S. Nugrahaa, Adama Hiloub*, Nicholas Vandegraaffc, David I. Rhodesa,c, Rachada Haritakund and Paul A. Kellera*

a

School of Chemistry, University of Wollongong, Northfields Avenue, Wollongong, New South Wales 2522, Australia;bDe´partement de Biochimie – Microbiologie, UFR/VT, Universite´ de Ouagadougou, Burkina Faso 03, Africa;cAvexa Limited, 576 Swan Street, Richmond, Victoria 3121, Australia;dNational Centre for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Phaholyothin Road, Klong1, Klong Luang, Pathumanthani 12120, Thailand

(Received 14 November 2014; final version received 26 January 2015)

Phytochemical studies of the previously unexplored stem ofBoerhavia erectafrom

Burkina Faso, resulted in the isolation of an unreported glycoside 4,

2,3-dihydroxypropylbenzoate-3-O-b-[400

-methoxy] glucuronide as well as seven known glycosides (1–3, 5–8). The major isolate5and 8indicated a significant inhibition against HIV integrase (IC5010 and 22mg/mL, respectively). The extracts and isolates were also tested for anti-malarial activity, but insignificant activity was observed.

Keywords:African medicinal plant;Boerhavia erecta; glycosides; anti-HIV integrase activity; anti-malarial activity

1. Introduction

Burkina Faso is a country rich with medicinal plants including prospective medical sources of theBoerhavia erectaLinnaeus species; the aerial component of this plant is used in traditional medicine for the treatment of nervous malaria in infants, seizures in children, generalised oedema, dyspnoea, difficult delivery, haematuria, as an anti-convulsant, diuretic or spasmolytic (Hilou et al.2004). Previous studies on the extract of the leaves ofB. erecta revealed some activities, including anti-microbial (Perumal Samy et al.1999) and anti-malarial effects (Hilou et al.2004,2006; Stintzing et al.2004). This led to the isolation of sterols and betanin (Miralles et al.,1988; Stintzing et al.2004). The previous investigation was driven mainly by the isolation of phenolic compounds from the leaves of the plant for anti-oxidant studies (Petrus et al.2012). Here, we report for the first time the results of the stem bark of this plant and the bioactive constituents of polar fraction of this plant.

q2015 Taylor & Francis

*Corresponding author. Email:keller@uow.edu.au

Natural Product Research, 2015

http://dx.doi.org/10.1080/14786419.2015.1013470

2. Result and discussion

The non-polar alkanes and long chain fatty acids of the crude extract were removed by applying solid liquid (hexane, dichloromethane) back extraction. From the polar fraction, short normal phase column chromatography produced a major fraction, which was subjected to preparative HPLC., isolating the following eight compounds (Figure 1): 3-methoxybenzoic acid 4-O-b -glucoside1(Liu et al.2006), 3-methoxyacetophenone 4-O-b-glucopyranoside2(De Rosa et al.

1996), isorhamnetin-3-O-rutinoside-7-O-b-glucopyranoside3(Aquino et al.1987),

quercetin-3-O-rutinoside 5, quercetin-3-O-b-glucopyranoside 6, kaemferol-3-O-rutinoside 7 (Hamzah & Lajis1998), and isorhamnetin-3-O-rutinoside8. Compounds5,6and8were previously reported from the leaves of the plant (Petrus et al. 2012), and are spectroscopically identical to that reported. Importantly, compounds1 – 3and7were isolated from this plant for the first time in this study.

In addition, for the previously unreported compound, we propose the structure4(Figure 2). The HR-ESI-MS analysis of4indicated a peak atm/z409.1107 ([Mþ_Na]þ_{), assigned to the}

molecular formula of C17H22O10Na. The NMR spectral analysis suggested a molecular structure

that was similar to a reported glycerola-D-glucuronide carboxylic acid (Cai et al.2011) and its ester9(Maggi et al.2009). The1H and13C NMR spectral analysis indicated a singlet peak atd 3.46 and 60.8 ppm, respectively, assigned to a methoxy group; this was in contrast to the corresponding ester OMe in9with values assigned in the1H and13C NMR spectra ofd3.66 and 52.8 ppm, respectively (Maggi et al. 2009). This is particularly telling with the 8.0 ppm difference in the OMe in the13C NMR spectrum, with the methoxy ester more upfield compared with the methoxy substituent, as expected. gHMBC spectral analysis of4 indicated a proton-carbon correlation between the methoxy and proton-carbon peak atd83.0 (assigned to C400

of the sugar moiety). In addition, a 3-bond correlation between H400

and the carbonyl was evident. Thus, the molecular structure for 4 is proposed as 2,3-dihydroxypropyl-benzoate 3-O-b-[400

-methoxy] glucuronide. The position of the benzoic ester carbonyl peak in the13C NMR spectrum was consistent across all three derivatives (,168 ppm).

Preliminary studies on the crude extract revealed significant inhibition activity against HIV integrase in which ‘fraction B’ (the extract residue) showed a higher activity than the supernatant (fraction A). Further assays on the major constituents, compounds5and8, indicated moderate and less activity compared with the original extract (Table 1). The isolates were also tested against P. falciparum K1, which showed no significant activity (Table 1). The isolated compounds showed less inhibition relative to the fractions with the HIV integrase activity testing. This suggests a synergic mechanism playing an important role in the bioactivity. Fractions, sub-fractions and compounds1 – 8were inactive againstP. falciparumK1 (Table 1).

OCH₃

O R₁

OR₂

O

OH O OR₂

OH R₃O

R₁

R₁=OH, R₂ = β-glucoside, 1

R₁=CH₃, R₂ = β-glucoside, 2 R1=OCH3, R2 = rutinoside, R3 = β-glucoside, 3

R1=OH, R2 = rutinoside, R3 = H, 5 R1=OH, R2 = β-glucoside, R3 = H, 6 R1=H, R2 = rutinoside, R3 = H, 7 R1=OCH3, R2 = rutinoside, R3 = H, 8

Figure 1. Molecular structure of glycosides isolated from stem of Boerhavia erecta. Compound9was isolated fromV. hookeriana(Maggi et al.2009).

2 A.S. Nugrahaet al.

Quercetin has been reported to inhibit HIV-infected cells (Tang et al.1994), which were later found to have diverse modes of action including protease (PR), integrase (IN) and anti-transcriptase (RT) activities. Quercetin and rutin revealed inhibition against HIV-1 protease with IC50values of 34 and 28mg/mL, respectively, although methoxylation or glucosylation at 3,7,40

reduced its activity (Xu et al. 2000). Quercetin was a weak HIV-1 RT inhibitor (IC50 150 –

200mg/mL) possibly due to structural planarity, which weakens its intercalating properties (Tan et al.1991); however, glucosylation at 30

increased the activity (IC50 15mg/mL). Quercetin

possessed anti-integrase activity with IC50of 4mg/mL; Raghavan et al.1995).

O O OH O O O OH HO OH OMe 1 1'

1'' O O

OH O O O OMe HO OH OH 1 1' 1'' 4 9 O O OH O O O OH HO OH OH 1 1' 1''

1_{H NMR} δ 3.46 ppm

13_{C NMR} δ 60.8 ppm

1_{H NMR} δ 3.66 ppm

13_{C NMR} δ 52.8 ppm 4''

key 3-bond gHMBC correlations indicated by double-headed arrows

13_{C NMR} δ 173.0 ppm 13_{C NMR} δ 171.9 ppm

H

13_{C NMR} δ 168.0 ppm 13_{C NMR} δ 167.9 ppm

13_{C NMR} δ 168.1 ppm

10

13_{C NMR} δ _{177.7 ppm}

Figure 2. Proposed structure of 4and the known a-D-glucuronide carboxylates 9and 10. Key NMR

assignments are indicated (single-headed arrows) while key HMBC correlations (double-headed arrows) are also shown. For a larger set of correlations for4, see Supporting Information.

Table 1. Anti-HIV integrase and anti-plasmodial activities (IC50 in mg/mL) of the extracts and pure compounds isolated fromB. erecta.

Anti-HIV integrase activity Anti-malarial activity

Entry IC50(mg/mL) Activity IC50(mg/mL)

Crude extract ,1.6 na na

Fraction A 16 inactive –

Fraction B ,0.4 inactive –

Fraction B1 ,10 na na

Fraction B2 ,46 na na

Fraction B4 .50 na na

5 ,10 inactive –

8 ,22 inactive –

Notes: na, data not available. No activity. Compounds1–4,6and7were inactive againstP. falciparumK1 and were not tested against HIV integrase. Anti-malarial testing, final conc. of samples: 10mg/mL, negative control: 0.1% DMSO. IC50of positive control: Dihydroartemisinine¼2.20 nM, Mefloquine¼0.0310mM.

Natural Product Research 3

3. Conclusions

A previously unreported derivative of the glycerol a-D-glucuronide class of compounds (4) was isolated from the stem of B. erecta, which was proposed as 2,3-dihydroxypropyl-benzoate 3-O-b-[400

-methoxy] glucuronide. Moderate anti-HIV integrase activity was shown by the major constituents (5 and 8). The anti-malarial testing against P. falciparum K1 indicated none of the purified compounds possessed anti-plasmodial activity. However, the major compounds 5 and 8 were previously reported to have anti-malarial activity against

P. falciparum (FCR3, cycloguanil-resistant from Gambia) (Murakami et al. 2001).

Therefore, these compounds show significant differences in their P. falciparum inhibitory activity between different strains.

Supplementary material

Experimental details relating to this article are available online; 1H, 13C, COSY, gTOCSY, HSQC, HMBC, HR-ESI-MS and MS/MS spectra of compounds4.

Acknowledgements

ASN thanks the Australian Government for an ADS (AusAid) Scholarship and the University of Wollongong for University Postgraduate Scholarships.

References

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Cai WH, Matsunami K, Otsuka H, Shinzato T, Takeda Y. 2011. A glycerol a-D-glucuronide and a megastigmane glycoside from the leaves ofGuettarda speciosa L. J Nat Med. 65:364– 369. doi:10.1007/ s11418-010-0484-8.

De Rosa S, De Giulio A, Tommonaro G. 1996. Aliphatic and aromatic glycosides from the cell cultures of Lycopersicon esculentum. Phytochem. 42:1031 – 1034. doi:10.1016/0031-9422(96)00083-0.

Hamzah AS, Lajis N. 1998. Chemical constituents ofHedytois herbacea. Biodiv Environ Conserv Asean Rev. 2:1 – 5. Hilou A, Nacoulma OG, Guiguemde TR. 2006.In vivoantimalarial activities of extracts fromAmaranthus spinosusL.

andBoerhaavia erectaL. in mice. J Ethnopharmacol. 103:236– 240. doi:10.1016/j.jep.2005.08.006.

Hilou A, Piro J, Nacoulma GO. 2004. Physicochemical parameters in the stability of betacyanins fromAmaranthus spinosusL. (Amaranthaceae) andBoerhaavia erectaL. (Nyctagynaceae). J Soc Ouest-Afric Chim. 9:57 – 70. Liu P, Guo H, Tian Y, Wang Q, Guo D. 2006. Benzoic acid allopyranosides from the bark ofPseudolarix kaempferi.

Phytochem. 67:1395– 1398. doi:10.1016/j.phytochem.2006.05.017.

Maggi A, Taskova R, Gotfredsen CH, Bianco A, Jensen SR. 2009. Chemical markers inVeronicasect. Hebe. III. Syst Ecol. 37:731 – 736. doi:10.1016/j.bse.2009.11.001.

Miralles J, Noba K, Ba AT, Gaydou EM, Kornprobst JM. 1988. Chemotaxonomy in Nyctagynaceae family: sterols and fatty acids from the leaves of three Boerhaavia species. Biochem Syst Ecol. 16:475 – 478. doi:10.1016/0305-1978 (88)90047-6.

Murakami N, Mostaqul HM, Tamura S, Itagaki S, Horii T, Kobayashi M. 2001. New anti-malarial flavonol glycoside fromHydrangeae dulcisfolium. Bioorg Med Chem Lett. 11:2445– 2447. doi:10.1016/S0960-894X(01)00467-X. Perumal Samy R, Ignacimuthu S, Raja DP. 1999. Preliminary screening of ethnomedicinal plants from India.

J Ethnopharmacol. 66:235 – 240. doi:10.1016/S0378-8741(99)00038-0.

Petrus AJA, Hemalatha SS, Suguna S. 2012. Isolation and characterisation of antioxidant phenolic metabolites of

Boerhavia erectaL. leaves. J Pharm Sci Res. 5:1856 – 1861.

Raghavan K, Buolamwini JK, Fesen MR, Pommier Y, Kohn KW, Weinstein JN. 1995. Three-dimensional quantitative structure-activity relationship (QSAR) of HIV integrase inhibitors: a comparative molecular field analysis (CoMFA) study. J Med Chem. 38:890 – 897. doi:10.1021/jm00006a006.

Stintzing FC, Kammerer D, Schieber A, Adama H, Nacoulma OG, Carle R. 2004. Betacyanins and phenolic compounds fromAmaranthus spinosusL. andBoerhavia erectaL. Zeitschrift fuer Naturforschung, C. J Biosci. 59:1 – 8. Tan GT, Pezzuto JM, Kinghorn AD. 1991. Evaluation of natural products as inhibitors of human immunodeficiency virus

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Tang R, Chen K, Cosentino M, Lee KH. 1994. Apigenin-7-O-b-D-glucopyranoside, an anti-HIV principle from

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Xu HX, Wan M, Dong H, But PPH, Foo LY. 2000. Inhibitory activity of flavonoids and tannins against HIV-1 protease. Biol Pharm Bull. 23:1072 – 1076. doi:10.1248/bpb.23.1072.

Natural Product Research 5

This art icle m ay be used for research, t eaching, and privat e st udy purposes. Any subst ant ial or syst em at ic reproduct ion, redist ribut ion, reselling, loan, sub- licensing, syst em at ic supply, or dist ribut ion in any form t o anyone is expressly forbidden. Term s & Condit ions of access and use can be found at ht t p: / / w w w.t andfonline.com / page/ t erm s-and- condit ions

SHORT COMMUNICATION

Bioactive glycosides from the African medicinal plant

Boerhavia erecta

_L.

Ari S. Nugrahaa, Adama Hiloub*, Nicholas Vandegraaffc, David I. Rhodesa,c, Rachada Haritakund and Paul A. Kellera*

a

School of Chemistry, University of Wollongong, Northfields Avenue, Wollongong, New South Wales 2522, Australia;bDe´partement de Biochimie – Microbiologie, UFR/VT, Universite´ de Ouagadougou, Burkina Faso 03, Africa;cAvexa Limited, 576 Swan Street, Richmond, Victoria 3121, Australia;dNational Centre for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Phaholyothin Road, Klong1, Klong Luang, Pathumanthani 12120, Thailand (Received 14 November 2014; final version received 26 January 2015)

Phytochemical studies of the previously unexplored stem ofBoerhavia erectafrom Burkina Faso, resulted in the isolation of an unreported glycoside 4, 2,3-dihydroxypropylbenzoate-3-O-b-[400

-methoxy] glucuronide as well as seven known glycosides (1–3, 5–8). The major isolate5and 8indicated a significant inhibition against HIV integrase (IC5010 and 22mg/mL, respectively). The extracts and isolates were also tested for anti-malarial activity, but insignificant activity was observed. Keywords:African medicinal plant;Boerhavia erecta; glycosides; anti-HIV integrase activity; anti-malarial activity

1. Introduction

Burkina Faso is a country rich with medicinal plants including prospective medical sources of theBoerhavia erectaLinnaeus species; the aerial component of this plant is used in traditional medicine for the treatment of nervous malaria in infants, seizures in children, generalised oedema, dyspnoea, difficult delivery, haematuria, as an anti-convulsant, diuretic or spasmolytic (Hilou et al.2004). Previous studies on the extract of the leaves ofB. erecta revealed some activities, including anti-microbial (Perumal Samy et al.1999) and anti-malarial effects (Hilou et al.2004,2006; Stintzing et al.2004). This led to the isolation of sterols and betanin (Miralles et al.,1988; Stintzing et al.2004). The previous investigation was driven mainly by the isolation of phenolic compounds from the leaves of the plant for anti-oxidant studies (Petrus et al.2012). Here, we report for the first time the results of the stem bark of this plant and the bioactive constituents of polar fraction of this plant.

q2015 Taylor & Francis

*Corresponding author. Email:keller@uow.edu.au

2. Result and discussion

The non-polar alkanes and long chain fatty acids of the crude extract were removed by applying solid liquid (hexane, dichloromethane) back extraction. From the polar fraction, short normal phase column chromatography produced a major fraction, which was subjected to preparative HPLC., isolating the following eight compounds (Figure 1): 3-methoxybenzoic acid 4-O-b -glucoside1(Liu et al.2006), 3-methoxyacetophenone 4-O-b-glucopyranoside2(De Rosa et al.

1996), isorhamnetin-3-O-rutinoside-7-O-b-glucopyranoside3(Aquino et al.1987),

quercetin-3-O-rutinoside 5, quercetin-3-O-b-glucopyranoside 6, kaemferol-3-O-rutinoside 7 (Hamzah & Lajis1998), and isorhamnetin-3-O-rutinoside8. Compounds5,6and8were previously reported from the leaves of the plant (Petrus et al. 2012), and are spectroscopically identical to that reported. Importantly, compounds1 – 3and7were isolated from this plant for the first time in this study.

In addition, for the previously unreported compound, we propose the structure4(Figure 2). The HR-ESI-MS analysis of4indicated a peak atm/z409.1107 ([Mþ_Na]þ_{), assigned to the}

molecular formula of C17H22O10Na. The NMR spectral analysis suggested a molecular structure

that was similar to a reported glycerola-D-glucuronide carboxylic acid (Cai et al.2011) and its ester9(Maggi et al.2009). The1H and13C NMR spectral analysis indicated a singlet peak atd

3.46 and 60.8 ppm, respectively, assigned to a methoxy group; this was in contrast to the corresponding ester OMe in9with values assigned in the1H and13C NMR spectra ofd3.66 and 52.8 ppm, respectively (Maggi et al. 2009). This is particularly telling with the 8.0 ppm difference in the OMe in the13C NMR spectrum, with the methoxy ester more upfield compared with the methoxy substituent, as expected. gHMBC spectral analysis of4 indicated a proton-carbon correlation between the methoxy and proton-carbon peak atd83.0 (assigned to C400

of the sugar moiety). In addition, a 3-bond correlation between H400

and the carbonyl was evident. Thus, the molecular structure for 4 is proposed as 2,3-dihydroxypropyl-benzoate 3-O-b-[400

-methoxy] glucuronide. The position of the benzoic ester carbonyl peak in the13C NMR spectrum was consistent across all three derivatives (,168 ppm).

Preliminary studies on the crude extract revealed significant inhibition activity against HIV integrase in which ‘fraction B’ (the extract residue) showed a higher activity than the supernatant (fraction A). Further assays on the major constituents, compounds5and8, indicated moderate and less activity compared with the original extract (Table 1). The isolates were also tested against P. falciparum K1, which showed no significant activity (Table 1). The isolated compounds showed less inhibition relative to the fractions with the HIV integrase activity testing. This suggests a synergic mechanism playing an important role in the bioactivity. Fractions, sub-fractions and compounds1 – 8were inactive againstP. falciparumK1 (Table 1).

OCH₃ O R₁ OR₂ O OH O OR₂ OH R₃O

R₁

R₁=OH, R₂ = β-glucoside, 1

R₁=CH₃, R₂ = β-glucoside, 2 R1=OCH3, R2 = rutinoside, R3 = β-glucoside, 3

R1=OH, R2 = rutinoside, R3 = H, 5

R1=OH, R2 = β-glucoside, R3 = H, 6

R1=H, R2 = rutinoside, R3 = H, 7

R1=OCH3, R2 = rutinoside, R3 = H, 8

Figure 1. Molecular structure of glycosides isolated from stem of Boerhavia erecta. Compound9was isolated fromV. hookeriana(Maggi et al.2009).

2 A.S. Nugrahaet al.

Quercetin has been reported to inhibit HIV-infected cells (Tang et al.1994), which were later found to have diverse modes of action including protease (PR), integrase (IN) and anti-transcriptase (RT) activities. Quercetin and rutin revealed inhibition against HIV-1 protease with IC50values of 34 and 28mg/mL, respectively, although methoxylation or glucosylation at 3,7,40

reduced its activity (Xu et al. 2000). Quercetin was a weak HIV-1 RT inhibitor (IC50 150 –

200mg/mL) possibly due to structural planarity, which weakens its intercalating properties (Tan et al.1991); however, glucosylation at 30

increased the activity (IC50 15mg/mL). Quercetin

possessed anti-integrase activity with IC50of 4mg/mL; Raghavan et al.1995).

O O OH O O O OH HO OH OMe 1 1'

1'' O O

OH O O O OMe HO OH OH 1 1' 1'' 4 9 O O OH O O O OH HO OH OH 1 1' 1''

1_{H NMR} δ 3.46 ppm 13_{C NMR} δ 60.8 ppm

1_{H NMR} δ 3.66 ppm 13_{C NMR} δ 52.8 ppm

4''

key 3-bond gHMBC correlations indicated by double-headed arrows

13_{C NMR} δ 173.0 ppm 13_{C NMR} δ 171.9 ppm

H

13_{C NMR} δ 168.0 ppm 13_{C NMR} δ 167.9 ppm

13_{C NMR} δ 168.1 ppm 10

13_{C NMR} δ _{177.7 ppm}

Figure 2. Proposed structure of 4and the known a-D-glucuronide carboxylates 9and 10. Key NMR assignments are indicated (single-headed arrows) while key HMBC correlations (double-headed arrows) are also shown. For a larger set of correlations for4, see Supporting Information.

Table 1. Anti-HIV integrase and anti-plasmodial activities (IC50 in mg/mL) of the extracts and pure compounds isolated fromB. erecta.

Anti-HIV integrase activity Anti-malarial activity

Entry IC50(mg/mL) Activity IC50(mg/mL)

Crude extract ,1.6 na na

Fraction A 16 inactive –

Fraction B ,0.4 inactive –

Fraction B1 ,10 na na

Fraction B2 ,46 na na

Fraction B4 .50 na na

5 ,10 inactive –

8 ,22 inactive –

Notes: na, data not available. No activity. Compounds1–4,6and7were inactive againstP. falciparumK1 and were not tested against HIV integrase. Anti-malarial testing, final conc. of samples: 10mg/mL, negative control: 0.1% DMSO. IC50of positive control: Dihydroartemisinine¼2.20 nM, Mefloquine¼0.0310mM.

3. Conclusions

A previously unreported derivative of the glycerol a-D-glucuronide class of compounds (4) was isolated from the stem of B. erecta, which was proposed as 2,3-dihydroxypropyl-benzoate 3-O-b-[400

-methoxy] glucuronide. Moderate anti-HIV integrase activity was shown by the major constituents (5 and 8). The anti-malarial testing against P. falciparum K1 indicated none of the purified compounds possessed anti-plasmodial activity. However, the major compounds 5 and 8 were previously reported to have anti-malarial activity against

P. falciparum (FCR3, cycloguanil-resistant from Gambia) (Murakami et al. 2001). Therefore, these compounds show significant differences in their P. falciparum inhibitory activity between different strains.

Supplementary material

Experimental details relating to this article are available online; 1H, 13C, COSY, gTOCSY, HSQC, HMBC, HR-ESI-MS and MS/MS spectra of compounds4.

Acknowledgements

ASN thanks the Australian Government for an ADS (AusAid) Scholarship and the University of Wollongong for University Postgraduate Scholarships.

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