Tanzania Journal of Health Research Health User's Trust Fund (HRUTF) ISSN: 1821-6404 Vol. 12, Num. 2, 2011

Tanzania Journal of Health Research, Vol. 12, No. 2, April, 2010

ORIGINAL RESEARCH ARTICLE

Cytotoxic and anti-HIV activities of some Tanzanian Garcinia species

J.J. MAGADULA¹* and H.O. SULEIMANI²

¹Institute of Traditional Medicine, Muhimbili University of Health and Allied Sciences, P.O. Box 65001, Dar eS Salaam, Tanzania
²Department of Botany, University of Dar es Salaam, P. Box 3060, Dar es Salaam, Tanzania

* Correspondence: J.J. Magadula; E-mail: magadulajanguj@yahoo.com

Received 1 September 2009
Revised 1 February 2010
Accepted 28 February 2010

Code Number: th10018

Abstract

Cancer and HIV/AIDS remains the greatest public health and humanitarian challenges in the current world’s health sector. For many decades now, millions of lives have been compromised by the two diseases. This study has evaluated ethanol extracts from nine Garcinia plant species collected in Tanzania for their in vitro cytotoxicity against four human cancer cell lines and for anti-HIV activity against HIV-1 viral replication in MT4 cells. Among the tested extracts, the fruit extracts of G. livingstoneii and G. semseii showed moderate to mild cytotoxic activities against A549, DU145, KB and Kbivin human cell lines with 50 % cytotoxic (CC₅₀) values ranging from 5.7-20.0 µg/ml. Furthermore, only fruit extracts of G. livingstonei and G. semseii showed significant anti-HIV-1 activity with EC₅₀ values of 2.25 ± 0.51 and 0.93 ± 0.67 µg/ml respectively. This study has shown the potential of the Garcinia extracts to be the source of possible lead compounds and anti-HIV drug candidates currently needed for the management of HIV/AIDS. Phytochemical screening indicated dominance of phenolic compounds in Garcinia species while isolation of active principles from active fractions will be further undertaken.

Keywords: Garcinia species, Clusiaceae, extracts, cytotoxic, HIV, Tanzania

Introduction

HIV/AIDS and cancer pandemics remain the critical crisis due to both their emergent and long-term development. Cancer cases distribution shows no differences in its epidemiology in all regions of the world whereas, HIV/AIDS have affected the world disproportionately and the greatest burden being in sub-Saharan Africa (WHO, 2008). Currently, no reliable and user friendly treatment can be claimed to combat these diseases. The current anti-HIV drugs that include reverse transcriptase (RT) and protease inhibitors have experienced drug resistance with HIV strains (Boden et al., 1999). On the other hand, the currently available anti-cancer chemotherapy are of limited efficacy on advanced cancer cases hence, there is a demand for more effective cancer treatments. This necessitates the demand for the development of new drugs particularly of plant origin owing to their success as sources of anticancer drugs. The use of plants for managing different diseases has become a common practice since time immemorial with most of the people in the developing world relying on traditional medicines for their primary health care including management of cancer and HIV/AIDS (WHO, 1999).

Plants of the genus Garcinia have been reported in the literature to display both anti-HIV and cytotoxicity activity (Tao et al. 2009). For instance, G. mangostana gave mangostin, a compound that indicated significant HIV-1 protease inhibition effect (Chen et al., 1996) while G. livingstoneii has been reported to produce gutifferone A, being an anti-HIV compound (Gustafson et al., 1992). Ethnomedically, different parts of Garcinia plants have been reported to exhibit many pharmacological effects. Thus, fruits of most species in this genus are edible, among them; those of G. mangostana are famous, while G. gambogia fruits are used in traditional medicine for treating diarrhoea and dysentery (Ambasta, 1986) and the rind of this fruit is rich in hydroxycitric acid (HCA) which has been associated with promoting weight loss, suppressing appetite, and increasing energy levels (Yamada et al., 2007). Other Garcinia species, such as G. indica, have oily seeds yielding more than 15% oil. Fruit extracts from G. kola have been claimed to be effective at stopping Ebola virus replication in laboratory tests and its seeds are also used in folk medicine (Yamaguchi et al., 2000).

In the course of our ongoing search for bioactive extracts/compounds from natural sources, nine Garcinia species growing in Tanzania have been screened for their cytotoxic and anti-HIV-1 viral replication activities. This paper reports the evaluation of crude extracts of nine Garcinia plants against HIV-1 NL4−3 viral strain on MT-4 cells and cytotoxic activities on four human cancer cell lines as well as the phytochemical screening of extracts aiming to establish the classes of compounds responsible for the biological activities noted in this study.

Materials and Methods

Collection of plant materials

The plant materials were collected from different parts in Tanzania (Table 1) and identified by Mr. Haji O. Suleimani of the Department of Botany, University of Dar es Salaam. They include Garcinia bifasculata N. Robson, G. buchananii Bak., G. edulis Exell. G. ferrea Pierre, G. huillensis Welw. ex Oliv., G. kingaensis Engl., G. livingstonei T. Anderson, G. semseii Verdc and G. volkensis. The voucher specimens are deposited in the Herbarium at the Department of Botany, University of Dar es Salaam, Tanzania.

Table    1:  Localities of some Garcinia plant species in Tanzania

Preparation of crude extracts

Twelve grams of the specified part of each dried plant material were soaked in ethanol (300 ml) for 48 hours at room temperature. The ethanol extracts were filtered and evaporated under vacuum on a rotary evaporator. The crude extracts (20 mg) were dissolved in DMSO (5 ml) for bioassay.

Anti-HIV assay on MT4 cells

An HIV-1 infectivity assay previously described was used in the experiments (Huang et al., 2004), was performed at the Natural Products Research Laboratories, University of North Carolina, USA. A 96-well microtiter plate was used to set up the HIV-1 viral replication assay. HIV-1 at a multiplicity of infection (MOI) of 0.01 was used to infect MT4 cells. Culture supernatants were collected on day 4 post infection for P24 assay using an ELISA kit from ZeptoMetrix Corporation (Buffalo, New York). If a test sample inhibited virus replication and was not toxic, its effects were reported in the following terms: CC₅₀, the concentration of test sample that was toxic to 50% of the mock-infected cells; EC₅₀, the concentration of the test sample that was able to suppress HIV replication by 50%; and the Selectivity Index (SI), the ratio of the IC₅₀ to EC₅₀. Azidothymidine (AZT) was used as a positive control. The definition of the anti-HIV activity used: EC₅₀ < 0.5 µg/ml - strong activity; 0.5-5.0 µg/ml – moderate activity; 5.0-10 µg/ml – mild activity and EC₅₀ > 10 µg/ml – inactive.

Cytotoxicity assay

Cytotoxicity assay was performed at the Natural Products Research Laboratories, University of North Carolina, USA. The in vitro cytotoxicity screening was conducted by measuring toxicity against cancer cells using NIH-NCI protocol (Grever et al., 1992; Alley et al., 1988). In this study four cancer cells, A549 (lung adenocarcinoma), DU145 (prostate carcinoma), KB (nasopharyngeal carcinoma) and Kbvin (vincristine-resistant nasopharyngeal) were used. The definition of the cytotoxicity used was; CC₅₀ < 1.0 µg/ml – high cytotoxicity; CC₅₀ 1.0-10.0 µg/ml – moderate; CC₅₀ 10.0-20.0 µg/ml – mild cytotoxicity; and CC₅₀ > 20 µg/ml – non-cytotoxic.

Test for flavonoids, alkaloids, tannins, saponins and steroids

To test for alkaloids, tannins, flavonoids, steroids and saponins, methods developed by Trease & Evans (1983) and Harbourne (1983) were used. An amount 0.3 g of the extract was dissolved in 3 ml of methanol and heated. A small magnesium metal was added to the mixture followed by the addition of a few drops of concentrated HCl. The occurrence of a red or orange colouration was indicative of the presence of flavonoids or any other phenolic compounds.

About 0.5 g of the plant extract was dissolved in 5 ml of 1% HCl and warmed on steam bath. The filtrate (1 ml) was mixed with drops of Dragendorff’s reagent. Reddish orange precipitation was considered as indicative of the presence of alkaloids.

The extract (1 g) was dissolved in 20 ml of distilled water and filtered. Three drops of 10% of FeCl₃ were added to 2 ml of the filtrate. The appearance of blackish-blue or blackish-green colouration was indicative of tannins. Some 2 ml of the filtrate was added 1 ml of bromine water and a precipitate was taken as positive for tannins.

The 7% blood agar medium was used. The extract in methanol was applied with distilled water and methanol used as negative control while commercial saponin (BDH) solution was used as positive control. The plates were incubated at 35^oC for 6 hours. A total haemolysis of the blood around the extract was indicative of saponins. About 0.5 g of the extract was dissolved in 3 ml of CHCl₃ and filtered. Concentrated H₂SO₄ was added to the filtrate. A reddish brown colour was taken as positive for steroid ring.

Results

Sixteen plant extracts were tested in an HIV-1 viral replication assay using MT-4 cells infected with the NL4−3 strain, which is a T-cell adapted X4 wild type HIV-1 virus. Among the species showing significant activity were the ethanol extracts of the fruits of G. livingstoneii and G. semseii that indicated anti-HIV-1 replication effects with EC₅₀ values of 2.25 and 0.93 µg/ml, respectively.

Table 2:  Anti-HIV replication activity of fruit extracts of G. livingstoneii and G. semseii

*Results are expressed as EC₅₀ and CC₅₀ values (µg/ml) ±SD
N = # of independent experiments;
CC₅₀, = the concentration of test sample that was toxic to 50% of the infected cells;
EC₅₀, = the concentration of the test sample that was able to suppress HIV replication by 50%
AZT ( +ve control),  EC₅₀ = 0.0062± 0.0007µg/ml and CC₅₀ value > 40 µg/ml

The cytotoxicity of the G. livingstoneii extract on the T cells was CC₅₀ = 6.1 µg/ml with a very narrow selectivity index (SI) of 2.7 while the CC₅₀ value of G. semseii was 3.12 µg/ml with a very narrow selectivity index of 3.4. The comparisons of EC₅₀ values of the fruits of G. livingstoneii and G. semseii with that of standard drug revealed 362.9 and 150.0 fold far lower anti-HIV activity as compared to the standard drug, AZT (Table 2) respectively. The rest of the crude extracts were not active (EC₅₀ > 20 µg/ml) on this assay and having very narrow SI < 2 (Table 2).

Table 3: Cytotoxicity results of Tanzanian Garcinia species against four human cancer cell lines

* CC₅₀ values >20 µg/ml ; not considered to be significant and not calculated.
A549 = lung adenocarcinoma; DU145 = prostate carcinoma; KB = nasopharyngeal carcinoma; Kbvin = vincristine resistant nasopharyngeal

In the cytotoxicity assay, eighteen Garcinia plant extracts were screened for cytotoxicity against A549, DU145, KB and Kbvin cell lines (Table 3). Among the tested extracts, only the fruit extracts of G. livingstoneii and G. semseii showed moderate to mild cytotoxic activities against four human cell lines with CC₅₀ values ranging from 5.7-12.0 µg/ml. The extracts from fruit hulls, seed and stem bark of G. semseii showed marginal activity to all four cancer cells with CC₅₀ values ranging from 7.8-20 µg/ml.

Table 4: Comparative cytotoxicity results of Garcinia extracts against cancer cell lines

The comparison of the bioactivity of the crude extracts to that of a standard showed to be far lower cytotoxic than that of a standard drug (Table 4). The rest of the crude extracts were not active on this assay showing CC₅₀ values > 20 µg/ml. In this test, taxol was used as a positive control. All Garcinia extracts were subjected to phytochemical screening methods that revealed the presence of mainly phenolics and some steroidal compounds (Table 5).

Table 5: Phytochemical screening of the extracts from some Garcinia species

*   +: present;        -: absent

Discussion

The present study has shown that some crude extracts from the Garcinia plant species growing in Tanzania have both cytotoxic and anti-HIV-1 activities. Out of sixteen extracts tested in an HIV-1 viral replication assay, two extracts (fruit extracts of G. semseii and G. livingstoneii) showed moderate to mild anti-HIV activities, respectively. The literature indicated that some polyisoprenylated benzophenone derivatives isolated from Clusia (Piccinelli et al., 2005) and Garcinia (Gustafson et al., 1992) genera exhibited potent anti-HIV-1 activities. Similar findings have been reported by Gustafson, et al., 1992 of the isolation of guttiferones, benzophenone compounds with anti-HIV activities from Garcinia plants. Recent study on the stem bark of G. semseii reported the isolation of three novel prenylated benzophenones (Magadula et al, 2008), which were not tested due to the small amount of pure compounds obtained. Therefore, this class of compounds may be responsible for the activity indicated by extracts of G. livingstoneii and G. semseii hence phytochemical work on these two plants have to be  undertaken.

Our results also show that, four extracts indicated moderate to mild cytotoxic activities with CC₅₀ in between 5.7-20.0 µg/ml on four human cancer cell lines. The phytochemical screening indicated the presence of phenolic and steroidal compounds. These compounds are widely reported in the genus Garcinia (Nyemba et al., 1990; Oliveira et al., 1999) displaying significant cytotoxic activities. For instance, three polyisoprenylated benzophenones, isogarcinol, garcinol and xanthochymol were isolated from the pericarps of G. purpurea and evaluated to show growth inhibition in four human leukemia cell lines (NB4, HL60, U937 and K562) (Matsumoto et al., 2003). Furthermore, two benzophenones, gutifferone H and gambogenone isolated from fruits of G. xanthochymus indicated cytotoxicity activity in the SW-480 colon cancer cell line with CC₅₀ values of 12 and 188 µM respectively (Baggett et al., 2005). Hence, there is a need to investigate phytochemically, the active fractions from these plants in order to establish chemical constituents responsible for the cytotoxic and HIV-1 activities.

Acknowledgements

We are grateful to Prof. K.H. Lee and his research group at the Natural Products Research Laboratories, University of North Carolina, USA for HIV-1 and cytotoxicity assays. This study was supported by the International Foundation for Sciences, Stockholm, Sweden and part by the Organisation for the Prohibition of Chemical Weapons, the Hague through a grant number F/4572-1 extended to JJM.

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