Scholarly article on topic 'Antimalarial compounds from Schefflera umbellifera'

Antimalarial compounds from Schefflera umbellifera Academic research paper on "Biological sciences"

CC BY
0
0
Share paper
Academic journal
South African Journal of Botany
OECD Field of science
Keywords
{"Antiplasmodial activity" / Betulin / " ent-kaur-16-en-19-oic acid" / " Plasmodium falciparum " / " Schefflera umbellifera "}

Abstract of research paper on Biological sciences, author of scientific article — X.S. Mthembu, F.R. Van Heerden, G. Fouché

Abstract The organic extract of the leaves of Schefflera umbellifera exhibited good antimalarial activity when tested against the chloroquine-susceptible strain (D10). Bioassay-guided fractionation of the dichloromethane fraction of the dichloromethane/methanol extract yielded an active compound, betulin, which exhibited good antiplasmodial activity with an IC50 value of 3.2µg/ml. The reference compound, chloroquine gave an IC50 value of 27.2ng/ml. Two other compounds were also isolated from the dichloromethane extract namely, 7-hydroxy-6-methoxycoumarin and ent-kaur-16-en-19-oic acid. These two compounds did not exhibit any significant antiplasmodial activity.

Academic research paper on topic "Antimalarial compounds from Schefflera umbellifera"

Available online at www.sciencedirect.com

ScienceDirect

South African Journal of Botany 76 (2010) 82 - 85

www.elsevier.com/locate/sajb

Antimalarial compounds from Schefflera umbellifera

X.S. Mthembua'*, F.R. Van Heerdenb'*, G. Fouchea

a Council for Scientific and Industrial Research, Biosciences, PO Box 395, Pretoria 0001, South Africa b School of Chemistry, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa

Received 5 May 2009; received in revised form 1 July 2009; accepted 31 July 2009

Abstract

The organic extract of the leaves of Schefflera umbellifera exhibited good antimalarial activity when tested against the chloroquine-susceptible strain (D10). Bioassay-guided fractionation of the dichloromethane fraction of the dichloromethane/methanol extract yielded an active compound, betulin, which exhibited good antiplasmodial activity with an IC50 value of 3.2 p,g/ml. The reference compound, chloroquine gave an IC50 value of 27.2 ng/ml. Two other compounds were also isolated from the dichloromethane extract namely, 7-hydroxy-6-methoxycoumarin and ent-kaur-16-en-19-oic acid. These two compounds did not exhibit any significant antiplasmodial activity. © 2009 SAAB. Published by Elsevier B.V. All rights reserved.

Keywords: Antiplasmodial activity; Betulin; ent-kaur-16-en-19-oic acid; Plasmodium falciparum; Schefflera umbellifera

1. Introduction

Schefflera umbellifera is a semi-deciduous tree, widely distributed in Malawi, Mozambique and Zimbabwe as well as in South Africa (Mbambezeli, 2006). This is the only South African member of the genus which grows in warm, tropical regions and is very closely related to Cussonia, even the Xhosa and Zulu common names are the same. According to Palmer and Pitman (1972), the specific epithet umbellifera refers to the umbellate arrangement of the flowers. The genus Schefflera J.R.Forst. & G.Forst. has about 650 species and was named in 1776 by G. and J.R. Forster in honour of J.C. Scheffler of Danzig (Mbambezeli, 2006).

The leaves of S. umbellifera have been used traditionally to treat rheumatism, colic and insanity and for malaria, a bark extract is drunk (Watt and Breyer-Brandwijk, 1962). The Vhavenda people use the roots of this plant as a diuretic and laxative, for bathing, for weaning infants and for malaria, venereal diseases and nausea (Hutchings et al., 1996). Accord-

* Corresponding authors. E-mail addresses: xsmthembu@csir.co.za (X.S. Mthembu), vanheerdenf@ukzn.ac.za (F.R. Van Heerden).

ing to Watt and Breyer-Brandwijk (1962) the bark is used for stomach ulcers and magical purposes. In Tanzania, leaves are used for indigestion while roots are used for fevers and venereal disease, in emetics for nausea and in cold infusions for skin irritation in new-born babies (Hutchings et al., 1996). Root bark decoctions are administered for mental illness (Chhabra et al., 1984). The secondary metabolite characteristic of the Araliaceae family is triterpene glycosides, polyacetylenes, saponins (Gunzinger et al., 1986) and caffeic acid derivatives (Li et al., 2005). Limited information is known about the antimalarial properties of S. umbellifera (Tetyana et al., 2002) and no phytochemical studies of this plant have been reported. For this study, three major compounds were isolated from the active dichloromethane extract.

2. Materials and methods

2.1. Plant material

Plant material was collected at Mariepskop, Mpumalanga, on the road towards the top above an electrical substation. This area was a forest with a well-drained, rocky soil and humus clay. A voucher specimen deposited at the South African National Biodiversity Institute (SANBI) was identified as S. umbellifera (Sond.) Baill.

0254-6299/$ - see front matter © 2009 SAAB. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.sajb.2009.07.019

2.2. Extraction and isolation of compounds

The wet leaves (6.00 kg) were dried in an oven at 60 °C for overnight. 2.90 kg was recovered as a dry matter and finely ground. 2.2 kg of the dried, ground leaves were extracted using methanol/dichloromethane (1:1) for 12 h, filtered and reduced to dryness using a rotovaporator. Methanol/dichloromethane extract was then partitioned between hexane, dichloromethane and water. The dichloromethane layer was dried and applied to a silica column, chromatographed using silica gel (0.0400.063 mm), eluted with 5% acetone/hexane followed by 100% acetone. This yielded eighteen fractions and some of these fractions were later combined on the basis of their TLC profiles. The first fraction (3.20 g) was further fractionated using flash silica with 2% EtOAc/hexane as the mobile phase and afforded a white pure compound 1 (23 mg, 0.72% yield). The second fraction (0.66 g) was further fractionated on flash silica gel using 40% EtOAc/hexane as the mobile phase and yielded a pure white crystalline compound 3 (16 mg, 2.24% yield). The third fraction (0.10 g) was also further chromatographed on flash silica using 5% acetone/dichloromethane as mobile phase and yielded a pure bright blue fluorescing compound 2 (6 mg, 6% yield).

The structures of all three compounds (Fig. 1) were determined using 1H NMR and 13C NMR data recorded on a Varian 400 MHz Unity spectrometer at the CSIR. All compounds were dissolved in either deuterated chloroform or methanol and the chemical shifts were recorded in ppm, tetramethylsilane (TMS) was used as an internal standard and spectra were recorded at room temperature. The mass spectral data were obtained from a coupled HPLC-UV/MS instrument with a triple pole Quattro LC Micro mass spectrometer which was set to operate both in the ESI and ESI+ modes. High-resolution mass spectra (HREIMS) of compounds were obtained from the University of Witwaters-rand using a VG 70SEQ HRMS instrument, operating mainly on EI+ mode using 8 kV as standard ionization energy. A Perkin Elmer 241 polarimeter at 589 nm using a sodium lamp as a light

Fig. 1. Structures of compounds isolated from S. umbellifera.

source and a cell with path length of 1 dm was used in all experiments. These experiments were done using either methanol or chloroform as solvent depending on the solubility of the compound. Melting points were determined using a Reichert Koffler hotstage apparatus and are uncorrected.

2.3. Description of the in vitro antimalarial assay

Compounds or extracts were assayed against Plasmodium falciparum strain, namely chloroquine-sensitive (CQS) (D10). Continuous in vitro cultures of asexual erythrocyte stages of P. falciparum were maintained using the method of Trager and Jensen (1976). The parasites were maintained at a 5% haema-tocrit with RPMI 1640 (Biowhittaker) medium supplemented with Albumax II (lipid rich bovine albumin) (GibcoBRL) (25 g/l), hypoxanthine (44 mg/l), HEPES [N-(2-hydroethyl)-piperazine-N'-(2-ethansulfonic acid)] (Sigma Aldrich) (50 mg/l). The cultures were incubated at 37 °C in an atmosphere of 93% N2, 4% CO2 and 3% O2.

Quantitative assessment of in vitro antiplasmodial activity was determined via a parasite lactate dehydrogenase (pLDH) assay (Makler et al., 1993). Chloroquine (CQ) was used as a reference drug in all experiments and the 50% inhibitory concentration (IC50) values were obtained using a non-linear dose-response curve fitting analyses via Graph Pad Prism v.4.0 software.

3. Results and discussion

3.1. Identification and characterization of isolated compounds

Compound 1 was obtained as a white crystalline solid, crystallized using an acetone/hexane solvent mixture. This compound is not UV active but shows an intense pinkish colour after spraying with vanillin on the TLC plate. The mass spectrum of compound 1 has an M+ peak at m/z 302.4011, which corresponds to a molecular formula of C20H30O2 and some distinct fragment peaks at m/z 288 due to [M-CH2]+, m/z 242 which corresponds to a loss of both a carboxyl and a methyl group. After careful inspection of the NMR data the compound was identified as ent-kaur-16-en-19-oic acid and the structure confirmed by comparison of the 1H and 13C NMR data with literature values (Buckingham, 1996; Lobitz et al., 1997).

Compound 2, was isolated as a pure bright blue fluorescing powder. Electro-spray mass spectrometry data gave a molecular ion signal M+ at m/z 192, corresponding to a molecular formula of C10H8O4 and a distinct fragment peak at m/z 121 due to [M-C3H2O2]+. This compound was identified as 7-hydroxy-6-metho-xycoumarin, also known as scopoletin. NMR data corresponded with the reported data (Sun et al., 2006).

Compound 3 was obtained as a white crystalline solid using flash chromatography and was crystallized from an ethyl ace-tate/hexane mixture. This compound showed a very intense pinkish colour upon spraying the TLC plate with vanillin. Compound 3 was identified as betulin [lup-20(29)-en-3ß,28-diol] with a molecular formula of C30H50O2. The mass spectra also showed very intense fragments at m/z 411 due to a [M-CH2 OH]+, m/z 426 due to [M-OH]+ and fragment peaks at m/z 235,

202, 193 which are characteristic of fragmentation patterns of a lupine-type of compound with an angular hydroxyl methylene group. NMR data of compound 3 compares very well with authentic betulin (Mahato and Kunda, 1994; El Deeb et al., 2003).

3.2. In vitro antimalarial activity of compounds isolated from S. umbellifera

The dichloromethane/methanol extract of the leaves of S. umbellifera exhibited good antimalarial activity (IC50 5.0 ^g/ml) when tested against the chloroquine-susceptible strain (D10). This extract was then partitioned between hexane, dichloromethane and water and these fractions tested for antimalarial activity. Only the dichloromethane fraction exhibited potent antiplasmodial activity against the chloroquine-sensitive strain (D10) with an IC50 of 3.7 ^g/ml. This result is regarded as potent based on the criteria set by the Novel Drug Discovery Platform (NDDP) (potent< 5 ^g/ml; Clarkson et al. (2004). The dichloromethane fraction was further fractionated and because of limited plant material three major compounds were targeted. From the three compounds isolated, only betulin (compound 3) exhibited significant in vitro antimalarial activity (3.2 ^g/ml) against the P. falciparum chloroquine-susceptible strain (D10). This activity compares well to that of the dichloromethane fraction (Table 1) and therefore betulin can be considered as an active ingredient. Although isolation and biological activity of betulin is well published, no information is known regarding its isolation from S. umbellifera.

Although antimalarial activity of betulin has been reported, limited information is known about the antimalarial activity of S. umbellifera. According to Ziegler et al., 2004, betulin was isolated from several plant families such as Rhamnaceae (Ziziphus vulgaris) and Labiatae (Zataria multiflora) that have been tested against P. falciparum strains and showed moderate activity (IC50 < 12 ^g/ml and< 27 ^M, respectively).

Badisa et al. (2002) and Henry et al. (2006) isolated betulin from a Tanzanian plant, Uapaca nitida and reported it to be inactive at 500 ^g/ml. However, Monte et al. (1988), reported betulin to inhibit P. falciparum strains with an IC50 of 12 ^g/ml. According to literature studies conducted, antiplasmodial activity of ent-kaur-16-en-19-oic acid has not been reported previously. It should also be noted that betulin does not exhibit significant antimalarial activity except when it is converted into betulinic acid. Antimalarial activity of betulinic acid against

Table 1

Antiplasmodial activity of the dichloromethane fraction of S. umbellifera, ent-kaur-16-en-19-oic acid (1), scopoletin (2) and betulin (3) against chloroquine-susceptible strain (D10).

Extract/compound Antiplasmodial activity, D10 IC50 (^g/ml)

CH2Cl2/MeOH extract 5.0

CH2Cl2 fraction 3.7

Compound 1 32.2

Compound 2 28.2

Compound 3 3.2

Chloroquine 27.2 ng/ml

chloroquine-sensitive strains (T9-96) was reported to be 25.9 p.g/ml whereas no activity was demonstrated by betulin (500 ^g/ml) (Yogeeswari and Sriram, 2005).

The results of this study confirms the traditional use of the plant S. umbellifera and bioassay-guided fractionation of the dichloromethane fraction of the dichloromethane/methanol extract yielded an active compound, betulin, which exhibited good antiplasmodial activity against the chloroquine-suscepti-ble strain D10, with an IC50 value of 3.2 ^g/ml. Considering that a large percentage of South African plants have not been investigated chemically or pharmacologically, they remain a potential source of leads for drug development.

Acknowledgements

We gratefully acknowledge the National Research Foundation (South Africa) and the Council for Scientific and Industrial Research for the financial support.

References

Buckingham, J., 1996. CD-ROM Dictionary of Natural Products. CRC Press. Badisa, R.B., Chaudhuri, S.K., Pilarihou, E., Walker, E.H., 2002. Licamichaux-iioic-A and -B acids — two ent-kaurene diterpenoids from Licania michauxii. Natural Product Letters 16, 39-45. Chhabra, S.C., Uiso, F.C., Mshiu, E.N., 1984. Phytochemical screening of Tanza-

nian medicinal plants. Journal of Ethnopharmacology 11, 157-179. Clarkson, C., Maharaj, V.J., Crouch, N.R., Olwen, M., Grace, O.M., Pillay, P., Matsabisa, M.G., Bhangwandin, N., Smith, P.J., Folb, P.I., 2004. In vitro antiplasmodial activity of medicinal plants native to or neutralized in South Africa. Journal of Ethnopharmacology 92, 177-191. El Deeb, K.S., Al-Haidari, R.A., Mossa, J.S., Ateya, A.M., 2003. Phytochemical and pharmacological studies of Maytenus forsskaoliana. Saudi Pharmaceutical Journal 11, 184-191. Gunzinger, J., Msonthi, J.D., Hostettmann, K., 1986. Moluscicidal saponins from

Cussonia spicata. Phytochemistry 25, 2501-2503. Henry, G.E., Adams, L.S., Rosales, J.C., Jacobs, H., Heber, D., Seeram, N.P., 2006. Kaurene diterpenes from Laetia thamnia inhibit the growth of human cancer cells in vitro. Cancer Letters 244, 190-194. Hutchings, A., Lewis, G., Scott, A.H., Cunningham, A.B., 1996. Zulu medicinal

plants. An inventory. InUniversity of Natal Press, pp. 221-222. Li, Y., But, P.P.H., Ooi, V.E.C., 2005. Antiviral activity and the mode of action of caffeolyquinic acids from Schefflera heptaphylla (L.). Antiviral Research 68, 1-9.

Lobitz, G.O., Tamayo-Castillo, G., Merfort, I., 1997. Diterpenes and sesquiter-

penes from Mikania banisteriae. Phytochemistry 46, 161-164. Mahato, S.B., Kunda, A.P., 1994.13C NMR spectra of pentacyclic triterpenoids —

a compilation and some salient features. Phytochemistry 37, 1517-1575. Makler, M.T., Ries, J.M., Williams, J.A., Bancroft, J.E., Piper, R.C., Gibbins, B.L., Hinrichs, D.J., 1993. Parasite Lactate dehydrogenase as an assay for Plasmodium falciparum drug sensitivity. American Journal of Tropical Medicine and Hygiene 48, 739-741. Mbambezeli, G., http://www.plantzafrica.com/frames/plantsfram.htm, Kirstenbosch Botanical Garden, February 2005. Accessed on the 18 December 2006. Monte, F.J.Q., Dantas, E.M.G., Braz, F.R., 1988. New diterpenoids from Croton

argrophylloides. Phytochemistry 27, 3209-3212. Palmer, E., Pitman, N., 1972. Trees of southern Africa, covering all known indigenous species in the Republic of South Africa. South-West Africa, Botswana, Lesotho & Swaziland, vol. 1-3. Balkema, A.A, Cape Town. Sun, L., Fu, W., Ren, J., Xu, L., Bi, K., Wang, M., 2006. Cytotoxic constituents

from Solanum lyratum. Archives of Pharmacal Research 29, 135-139. Tetyana, P., Prozesky, E.A., Jäger, A.K., Meyer, J.J.M., Van Staden, J., 2002. Some medicinal properties of Cussonia species used in traditional medicine. South African Journal of Botany 68, 51-54.

Trager, W., Jensen, J.B., 1976. Human malaria parasites in continuous cultures. Science 193, 673-675.

Watt, J.M., Breyer-Brandwijk, M.G., 1962. The medicinal and poisonous plants of Southern and Eastern Africa, 2nd ed. E & S Livingston Ltd, Edinburgh, p. 117. and London.

Yogeeswari, P., Sriram, D., 2005. Betulinic acid and its derivatives: a review on their biological properties. Current Medicinal Chemistry 12, 657-666.

Ziegler, H.L., Franzyk, H., Sairafianpour, M., Tabatabai, M., Tahrani, D., Bagherzadeh, K., Hagerstrand, H., Staerk, D., Jaroszewski, J.W., 2004. Erythrocyte membrane modifying agents and the inhibition of Plasmodium falciparum growth: structure-activity relationships for betulinic acid analogues. Biorganic and Medicinal Chemistry 12, 119-127.

Edited by J Van Staden