Antimalarial, antimycobacterial and cytotoxic limonoids from Chisocheton siamensis.

Abstract

Five limonoids isolated from the seeds of Chisocheton siamensis were tested for their antimalarial activity against Plasmodium falciparum, antimycobacterial activity against Mycobacterium tuberculosis and cytotoxic activity against NCI-H187 (human small cell lung cancer), KB (oral human epidermal carcinoma) and MCF-7 (breast cancer) cancer cell lines. All limonoids (1-5) showed inhibitory effect against Plasmodium falciparum with [IC.sub.50] values ranging from 2.06 to 6.31 [micro]g/ml. Only azadiradione (2) exhibited strong inhibitory effect against Mycobacterium tuberculosis with the MIC of 6.25 [micro]g/ml. Compounds 1-4 also showed cytotoxic activity against NCI-H187, KB and MCF-7 cancer cell lines and dysobinine (1) had the highest activity with [IC.sub.50] of 1.67, 3.17 and 2.15 [micro]g/ml, respectively.

[c] 2008 Elsevier GmbH. All rights reserved.

Keywords: Chisocheton siamensis; Meliaceae; Limonoids; Antimalarial activity; Antimycobacterial activity; Cytotoxic activity

Introduction

Meliaceae plants are known to be rich sources of limonoids. A number of limonoids have been isolated from several genera of Meliaceae and some of these exhibit antimalarial (Lee et al., 2007; Saewan et al., 2006), cytotoxic (Awang et al., 2007; Takeya et al., (1996), antiprotozoal (Khalid et al., 1998), and entifeedant (Koul et al., 2003; Nihei et al., 2002) activities. Chisocheton siamensis or "Ta Suea" in a local Thai name is one of the Meliaceae plants, which is found in northern part of Thailand. More than 10 limonoids have been isolated from this genus (Awang et al., 2007; Sarmah et al., 2003; Yadav et al. 1999; Gunning et al., 1994; Bordoloi et al., 1993). There are, however, no experimental reports on the screening of bioactive compounds from this plant. In the course of our on going search for bioactive compounds from natural sources (Laphookhieo et al., 2006, 2007), acetone hexane axtract (1:1; v/v) of the seeds of C. siamensis exhinited antimalarial activity against Plasmodium falciparum and antimycobacterial activity against Mycobacterium tuberculosis. Also, this crude extract exhibited cytotoxic activity against human small cell lung cancer (NCI-H187), oral human epidermal carcinoma (KB) and breast cancer (MCF-7) cell lines. In this paper, we describe on the isolation and biological activity evaluation of the crude extract and pure limonoids isolated from the seeds of C. siamensis.

Material and method

General

The [.sup.1]H and [.sup.13]C NMR spectra were recorded using a 300 MHz Bruker FTNMR Ultra Shield spectrometer. Chemical shifts were recorded in parts per million ([delta]) in [DCl.sub.3] with tetramethysilane (TMS) as the internal reference. Quick column chromatography (QCC) and column chromatography (CC) were carried out on silica gel 60 H (Merck, 5-40 [micro]m) and silica gel 100 (Merck, 63-200 [micro]m), respectively. Precoated plates of silica gel 60 [F.sub.254] used for the analytical procedure.

Plant material

Seeds of C. siamensis were collected from Queen Sirikit Garden, Mae Rim District, Chiang Mai Province, northern Thailand, in Febrauary 2006. The botanical identification was made by Dr. Prachaya Srisanga, botanist at Queen Sirikit Garden. Voucher specimens are deposited in the Herbarium of Queen Sirikit Garden, Mae Rim District, Chiang Mai, Thailand.

Extraction and isolation

Seeds of C. siamensis (600g) were extracted with acetone-hexane (1:1;v/v) over the period of 3 days at room temperature. The mixture was filtered and concentrated under reduced pressure to provide the crude extract (62.21 g). This crude extract was subjected to QCC (column size: 12 x 16 cm) over silica gel and eluted with a gradient of EtOAc-hexane to afford 13 factions (A1-A13). Fractions A2-A4 (4.0g) upon standing at room temperature yielded dysobinin (1) (3.30 g). Epoxyzadiradione (4) (227.0 mg) was obtained from fraction A6 (576.9 mg) after repeated CC with 25% EtOAc-hexane. Purification of fraction A8 (1.01 g) by CC with 25% EtOAc-hexane gave 6[alpha]-acetoxyepox-yazadiradione (5) (17.2 mg) and azadiradione (2) (31.2 mg). Mahonin (3) (18.2 mg) was obtained from fraction A10 (65.5 mg) by repeated CC using 5% EtOAc-[CH.sub.2][Cl.sub.2] as eluent.

Biological assays

Antimalaria assay

Antimalarial activity was evaluated against the parasite Plasmodium falciparum ([K.sub.1], multidrug resistant), using the method of Trager and Jensen (1976). Quantitative asessment of in vitro malarial activity was determined by means of the microculture radioisotope technique based on the method described by Desjardins et al. (1979). The inhibitory concentration ([IC.sub.50]) represented the concentration that caused 50% reduction in parasite growth, which was indicated by the in vitro uptake of [sup.3 H]-hypoxanthine by P. falciparum. The standard compound was dihydroartemisinine ([IC.sub.50] 0.00125 [micro]/ml).

Antimycobacterial assay

Antimycobacterial activity was evaluated against M. tuberculosis [H.sub.37]Ra employing the Microplate Alamar Blue Assay (MABA) (Collins and Franzblau, 1997). The reference drugs were rifampicin, Kanamycin and isoniazid and the minimum inhibitory concentration (MIC) values were summarized in Table 1.

Table 1. Biological activity of crude extract and pure compounds

Compound                        Cytotoxic activity (I[C.sub.50],
                                     [micro]g/ml KB (a)

                              K[B.sup.(a)]  NCI-H187 (b)   MCF7 (c)

Crude extract                    5.43           2.78         5.33

Dysobinin (1)                    3.17           1.67         2.15

Azadiradione (2)                 9.38           6.44         7.13

Mahonin (3)                    Inactive        15.61        18.42

Epoxyazadiradione (4)           12.87           7.54         4.68

6[alpha]-                      Inactive       Inactive     Inactive
Acctoxyepoxyazadiradione (5)

Ellipticine                      0.217          0.592        0.738

Doxorubicin                      0.096          0.023        0.149

Kanamycin                          --           --           -

Rifampicin                         --           --           -

Isoniazid                          --           --           -

Dihydroartemisinine                --           --           -

Compound                              Antimalarial  Antimycobacterial
                                      activity (d)  activity (c) (MIC,
                                      (I[C.sub.50,  [micro]g/ml)
                                      [micro]g/ml)

Crude extract                            0.784          100.00

Dysobinin (1)                            2.06            200.00

Azadiradione (2)                         2.91              6.25

Mahonin (3)                              2.92             50.00

Epoxyazadiradione (4)                    3.18             25.00

6[alpha] Acctoxyepoxyazadiradione        6.31            200.00
(5)

Ellipticine                               --               -

Doxorubicin                               --               -

Kanamycin                                 --              1.25

Rifampicin                                --              0.019

Isoniazid                                 --              0.052

Dihydroartemisinine                      0.00125            -

(a) KB = Oral human epidermal Carcinoma.
(b) NCI-H187 = human small cell lung cancer.
(c) MCF-7 = Breast cancer.
(d) Against Plasmodium falciparum.
(e) Against Mycobacterium tuberculosis.

Cytotoxic assay

The procedures for cytotoxic assay were performed by the sulphorhodamine B (SRB) assay (anti-KB and MCF-7) and colorimetric method (anti-NCI-H187) as described by Skehan et al. (1990). In this study, three cancer cell lines, MCF-7 (breast cancer), NCI-H187 (human, small cell lung cancer) and KB (oral human epidermal carcinoma) were used. Ellipticine and doxorubicin were the reference substances in this study and the [IC.sub.50] values are summarized in Table 1.

Results and discussion

The seeds of C. siamensis were extracted with acetone-hexane (1:1; v/v) for 3 days provided a crude extract. Bioassay-guided investigation of this extract resulted in the isolation and identification of five bioactive limonoids (1-5, Fig. 1). Compound 1 was isolated as a major constituent in this study (3.30g; 5.30% compared to the crude extract).

[FIGURE 1 OMITTED]

All limonoids (1-5) isolated from the seeds of C. siamensis were tetranortriterpenoids, which contained and [alpha], [beta]-unsaturated ketone on ring A and a [beta]-furan moiety at C-17 or ring D. The (1) H NMR signals of [alpha] [beta]-unsaturated ketone were observed at ca. [[sigma].sub.H] 7.10 and 5.90 for H-1 and H-2, respectively, (each doublet with J value ca. 10Hz), while a [beta]-furan (1)H NMR signals resonated at ca. [sigma].sub.H]7.45, 7.35 and 6.25 identified to H-23, H-21, and H-22, respectively. In compound 1 two additional oxymethine at [[sigma].sub.H] 5.31 (1H, br d, j = 9.6 Hz) and 5.20 (1H, br s, H-7) together with two acetoxymethyl groups at [[sigma].sub.H] 2.06 (3H, s, 6-OCOC[H.sub.3]) and 2.01 (3H, s, 7-OCOC[H.sub.3]) were observed, in (1)H NMR spectrum, which were located at C-6 and C-7, respectively. Moreover, an olefnic proton was also observed at [[sigma].sub.H] 5.33 (1H, br s) and identified to H-15. In case of compounds 3 and 5, they also contained two acetyl groups at C-6 and C-7 as compound 1. The major different (1) H NMR signals were found only on ring D. Both of them (3 and 5) showed an additional carbonyl carbon at C-16, [[sigma.sub.c] 204.0 for 3 and [[sigma].sub.c] 207.8 for 5. The H-15 of compound 3 resonated at [[sigma].sub.H] 5.87 (s), whereas compound 5 appeared at [[sigma].sub.H] 3.88 (s). This result implied that 3 contained a double bond at C-14 and C-15, while compound 5 possessed an epoxy instead. The last two compounds, 2 and 4, were also characterized by comparison (1) H NMR spectral data to those of 3 and 5, respectively. Compounds 2 and 4 differed from compounds 3 and 5 only on ring B. Compounds 2 and 4 had a one acetyl group, which was located at C-7 (4.54, br s (H-7)/2.09 (7-OCOC[H.sub.3] for 2 and 4.70, br s (H-7)/2.00 (7-OCOC[H.sub.7]. Therefore, Compounds 1-5 were characterized as dysobinin (1) (Suresh et al., 1997), azadiradione (2) (Saewan et al., 2006), mahonin (3) (Siddiqui et al., 2000), epoxyazadiradione (4) (Singh et al., 1976), and 6[alpha]-acetoxyepoxyazadiradione (5) (Kadota et al., 1990). In addition, the comparison of NMR spectral data with reported compounds was also done.

As summarized in Table 1, the crude extract and pure limonoids (1-5) were evaluated for their antimalarial activity against P. falciparum, antimycobacterial activity against M. tuberculosis and cytotoxic activity against NCI-H187 (human lung cancer), KB (oral human epidermal carcinoma) and MCF-7 (breast cancer) cancer cell lines. The crude extract showed strong inhibitory effect against P. falciparum with I[C.sub.50] 0.784 [micro]g/ml and weak inhibitory effect against M. tuberculosis with MIC 100 [micro]g/ml. Also, the crude extract exhibited strong cytotocix activity against NCI-H187, KB, MCF-7 cancer cell lines with I[C.sub.50] 2.78, 5.43 and 5.33 [micro]g/ml, respectively. In case of pure compounds, limonoids 1-5 exhibited moderate inhibitory effect against P. falciparum with the I[C.sub.50] of 2.06, 2.91, 2.92, 3.18 and 6.31 [micro]g/ml, respectively (Table 1). All compounds were also found to be active with antimycobacterial activity against M. tuberculosis and only azadiradione (2) showed strong inhibitory effect with the MIC value of 6.25 [micro]g/ml. In case of cytotoxic activity, all limonoids showed inhibitory effects against all three cancer cell lines, except 6[alpha]-acetoxyepoxyazadiradione (5) which was found to be inactive with all three cancer cell lines (Table 1). Dysobinin (1) exhibited strong activity against NCI-H187, KB and MCF-7 cancer cell lines with the I[C.sub.50] values of 3.17, 1.67 and 2.15 [micro]g/ml, respectively. It is interesting to note that the structural differences of all limonoids, 1-5, are only at ring B and D. Two acetyl groups on ring B and also the double bond on ring D of limonoid 1 play an important role in the antimalarial activity. Slightly decreasing activity of limonoid 5 probably due to the loss of double bond on ring D and the methylene carbon at C-16 was oxidized to carbonyl functionality. In case of cytotocix activity, both of diacetate groups on ring B and double bond on ring D are crucially important for the cytotocix activity.

In summary, dysobinin (1), a limonoid, was isolated as a major component from the seeds of C. siamensis. This compound was also most effective against malarial parasite and all three cancer cell lines, while azaridadione (2) was most effective against M. tuberculosis compared with all isolated limonoids.

Acknowledgments

We thank the Bioresources Research Network, National Center for Genetic and Engineering and Biotechnology (grant number BRN 003 G-49) and Mae Fah Luang University for financial support. special thanks are address to Dr. Prachaya Srisanga, botanist at Queen Sirikit Garden, Mae Rim, Chiang Mai for plant collection and identification. We are also indebted to the Bioassay Research Facility of BIOTEC (Thailand) for bioactivity tests.

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W. Maneerat (a), S. Laphookhieo (a), *, A. Koysomboon (b), K. Chantrapromman (c)

(a) School of Science, Mac Fah Luang University, Tasud, Muang, Chiang Rai 57100, Thailand

(b) Faculty of Arts and Science, Prince of Songkla University, Suratthani 84100, Thailand

(c) Institute of Research and Development, Walailak University, Thaiburi, Thasala, Nakhon Si Thammarat 80160, Thailand

* Corresponding author. Tel.: [phone]; fax: [phone].

E-mail addresses: [e-mail], [e-mail] (S.Laphookhieo).

0944-7113/$-see front matter (C) 2008 Elsevier GmbH. All rights reserved.

doi:10.1016/j.phymed.2008.05.004