The genus Goniothalamus Hk. f. et Thoms. (Family Annonaceae A.L. de Jussieu 1789 nom. conserv., the Custard-Apple Family) consists of 160 species of archaic shrubs and treelets which grow in the shady primary rainforest of tropical Asia. These plants can be quickly spotted in field collection by their aromatic bark and fusiform leathery flowers (1,2). A number of Goniothalamus species have been used for timber, as fiber sources (2), for ornamental and medicinal purposes, especially in relation with post-partum and abortion (3,4). The genus Goniothalamus belongs to a primitive taxon of flowering plants: the Annonaceae (Family Annonaceae A.L. de Jussieu 1789 nom. conserv., the Custard-Apple Family) (5). The Annonaceae form a large, generally recognizable family of about 122 genera and 2000 plant species which are widespread chiefly in tropics and subtropics (6–8). In regards to the pharmacological potentials of Goniothalamus species, there is a massive body of evidence to suggest that this taxon has the ability to elaborate series of acetogenins and styryl-lactones which are cytotoxic against a broad array of cancer cells including breast, colon, kidney and pancreatic carcinoma cells. Interestingly, both acetogenins and styryl-lactones are completely different in terms of chemical structure but their cellular activities are involving the same organelles in mammals: the mitochondria. An exciting fact about the mode of action of styryl-lactones, which is still an enigma, is their possible action via protein kinase and TRAIL receptors. In an effort to promote further research on the genus Goniothalamus which could be a promising source for chemotherapeutic agents, this work offers a broad analysis of current knowledge on Goniothalamus species. We also highlight the antibacterial activity of Goniothalamus scortechinii. This is the first antibacterial study report on Goniothalamus species.

The botanical characteristics of Goniothalamus species are homogenous and simple. When searching for Goniothalamus species in the rainforest, one is advised to look for few-leaved slender treelets or shrubs with smooth, thin and fibrous and strongly aromatic bark and upright blackish cylindrical trunk. The leaves are few, simple, alternate and exstipulate. The blade is glossy, oblong—lanceolate to obovate and thick. The secondary nerves are oblique, conspicuous, straight and parallel with scalariform reticulations (9). The flowers are axillary and characteristically woody, often dark green and fusiform (Fig. 1). The calyx consists of three sepals which are valvate, membranous, veined, free or connate. The corolla consists of two series of three petals which are veined and coriaceous, the inner smaller and fused in a vault above the androecium. The outer petals are marked with a prominent midrib. The androecium comprises several stamens which are linear and oblong. The gynaecium consists of several free carpels grooved at the anterior side. The fruits are stalked or sessile one to two seeded ripe carpels (6). The geographical pattern of distribution of this genus suggests the genus Goniothalamus to have been among the flowering plants to have colonized hearth during the post-Permian early Cretaceous time.

Figure 1. Acetogenins from Goniothalamus species.

Out of 160 species, five Goniothalamus species are medicinal. These have been used in traditional medicinal Asian system, and since a long period of time most of these in connection with abortion, childbirth and fever (10). The leaves of G. macrophyllus Hook.f & Thoms. are used to allay fever and a decoction of the roots is given as a post-partum remedy and to cause abortion (3). In Malaysia, a decoction of leaves is used externally to allay fever (3). The roots of G. giganteus Hook.f & Thoms. are used to abort and treat colds and the heated leaves are applied onto swellings (10). A decoction of G. scortechinii is given as a post-partum protective remedy (3). The roots of G. tapis Miq. are used as abortifacient during early months of pregnancy (3). In Java, Indonesia, an infusion of the roots is used to treat typhoid fever (11). In Taiwan, the seeds of G. amuyon Merr. are used to treat scabies (12). In the Philippines, the seeds are used to treat rheumatism and tympanites, and the fruit is stomachic (4). None of the traditional uses previously mentioned has been substantiated yet via strict pharmacological experimentation. However, these species have been studied for their chemical constituents.

Table 1. Antitumor activity of Goniothalamus species

Jiang et al. isolated donhepocin (xx), goniodin (xxi), donhexocin (xxii) and donbutocin (xxiii), from G. donnaiensis Finet & Gagnep. collected from Guangxi Province, China (34–36). Gardnerilins A (xxiv) and B (xxv) from G. gardneri Hook.f. & Thoms collected from DiaoLo mount, Hainan Province, China, gave cytotoxic IC₅₀ values against Bel 7402 human tumor cell lines of 3.6 and 8.5μg/ml, respectively (37,38) (Fig. 1). The mode of action of acetogenins is discussed next.

Figure 2. Hypothetical molecular mode of action of bullatacin against Complex I. MM: Mitochondrial Matrix, MIN: Mitochondrial Inner Membrane; IS: Intermembrane Space.

The cytotoxic activity of acetogenin has prompted further work in an effort to discover synthetic acetogenins (44–46). Oberlies et al. (41) studied the cytotoxicity of acetogenins toward cancerous and normal cells and showed that they are selectively cytotoxic against cancerous cells and also effective for drug-resistant cancer cells, while exhibiting only minimal toxicity to ‘normal’ non-cancerous cells. However, further work is needed to render acetogenins more specific to cancerous cells and very much less active against normal cells or significantly heavy side-effects will preclude clinical trials. A possible approach would perhaps be to use antigen-guided or receptor-guided forms of administrations by associating acetogenins to specific carriers, hemisynthesis could be of value in this instance. More specific cytotoxic principles from Goniothalamus species are styryl-lactones reviewed in the next section.

Figure 3. Styryl-lactones from Goniothalamus species.

Jewers et al. (47) first reported goniothalamin as the active constituent of the bark of G. andersonii, G. macrophyllus Miq. and G. malayanus collected in the peat-swamp of Sarawak. Altholactone was characterized from G. arvensis Scheff. collected in the National Park of Varirata in the Central Province of Papua New Guinea and from the G. borneensis Mat-Salleh collected in Malaysia (48,49). Cardiopetalolactone was characterized from the stem bark of G. cardiopetalus Hook.f. & Thoms. collected from Palaruvi forest in Kerala in India, with altholactone, goniopypyrone, goniothalamin, goniodiol (iv), goniofufurone (v) and goniofupyrone (vi) (50,51). Goniofufurone, goniopypyrone, goniothalamin, goniodiol, goniotriol (vii) and 8-acetylgoniotriol (viii) were isolated from the roots of G. griffithii (52,53). An isomer of altholactone, (+)-isoaltholactone (ix), was isolated from stem bark of G. malayanus, and from the leaves of G. montanus J. Sincl. and the roots of G. tapis Miq. (54). Goniolactones A–F were identified from the roots of G. cheliensis (55). Digoniodiol, deoxygoniopypyrone A, goniofupyrone, goniothalamin, deoxygoniopypyrone A, gonodiol-8-monoacetate and gonotriol (x) and were characterized from the aerial parts of G. amuyon collected in the southern part of Taiwan near the coastal regions (56–59). The petroleum ether extract of the stem bark of G. sesquipedalis collected in Bangladesh yielded 5-isogoniothalamin oxide (60). 5-Acetyl goniothalamin (xii) was characterized from G. uvaroides King collected in Bangladesh (61). Chen et al. (62) isolated howiinol A from G. howii Merr. (xii). The mode of cytotoxic action of styryl-lactone is described subsequently.

Indicating that not all Goniothalamus styryl-lactones are exclusively apoptogenic, Zhong et al. (83) investigated the apoptosis-inducing effect of styryl-lactones from G. cheliensis, on human promyelocytic leukemia HL-60 and showed the activation of caspase-3, reduced the expression of the anti-apoptotic gene Bcl-2, and increased the expression of the pro-apoptotic gene Box via cAMP-dependent protein kinase mechanism. Taking into consideration the available evidence, one might propose the hypothesis that goniothalamin and congeners induce apoptosis at the TRAIL-BAX system level via protein kinase modulation. Protein kinase has long been known to be involved in cell growth and proliferation. Wang et al. showed that protein kinase is involved in apoptosis mediated by TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) (84). An example of styryl-lactone which inhibits kinase is flavokavain A from kava, or Piper methysticum in the closely related family Piperaceae (85). The Fig. 4 shows the similitude of chemical structure between flavokavain and goniothalamin.

Figure 4. Note the proximity of chemical structure between flavokavain A, an inhibitor of protein kinase, and goniothalamin. What is the precise activity of styryl lactones against protein kinases?.

A possible mechanism of action for Goniothalamus styryl-lactones would be a cAMP-dependent protein kinase-mediated TRAIL-induced apoptosis, by stimulating TRAIL-induced translocation of Bax from cytosol to mitochondria, loss of mitochondrial transmembrane potential, and subsequent release of cytochrome c from mitochondria and activation of caspases, SMAC/Diablo, endo G and finally chromatin deterioration (Fig. 5). Protein kinase modulators are of immense therapeutic usefulness. Note that flavokavains are present in the genus Goniothalamus, as discussed next.

Figure 5. Putative mechanism of action of Goniothalamus styryl-lactones in apoptosis. TRAIL R: TRAIL receptor, Cyt. c: Cytochrome c; C: caspases. 1: styryl-lactone interacts with cellular kinase, 2: kinase mediation of TRAIL induction of apoptosis, 3: TRAIL induced (more ...)

Isoquinoline alkaloids were characterized from G. amuyon (87). Other miscellaneous secondary metabolites isolated from members of this genus include goniopedaline, a phenanthrene lactam, aristololactam A-II and its N,O-diacetyl derivative, taliscanine, aurantiamide acetate and β-sitosterol and its β-d-glucoside were isolated from the leaves and twigs of G. sesquipedalis Hook.f. & Thoms. (88). 3-Amino naphthoquinones were characterized from the stem bark of G. marcanii (89). Alkaloids were characterized from G. griffithii and essential oils were distilled from G. malayanus, G. uvarioides, G. macrophyllus and G. andersonii (90,91). In the genus Goniothalamus, 138 species still await to be phytochemically investigated, including G. scortechinii, the antibacterial property of which is reported in the next section.

G. scortechinii or in Malay akar gajah beranak (climber of the elephant bringing forth) is a small tree found from Penang to Selangor and Pahang used by Malays apparently freely, either alone or with other substances after childbirth, and taken internally to prevent bacterial infection (3). The plant is known to exhibit potent schizonticidal activity in vitro (92). We report the first evaluation of the antibacterial activity of hexane, dichloromethane and aqueous fractions of G. scortechinii. The plant was collected from 6° North and 98° East, near Kuala Kangsar, State of Perak, Malaysia in August 2004, 300m above sea level. The plant material was identified on comparison with specimens available at the Herbarium of the ‘Forest Research Institute of Malaysia’, Kepong, Malaysia. A voucher specimen (number W1332) has been deposited in our Herbarium collection for future reference. Finely powdered, air-dried leaves of G. scortechinii (800g) were extracted with methanol (2l) using a soxhlet apparatus. Hexane (250ml), dichloromethane (250ml), and water fractions (250ml) were obtained by the partitioning of liquid methanol extract (250ml) (yield: 5.52, 8.43, 64.5). The different fractions obtained were concentrated with a rotary evaporator and brought to complete dryness over water bath to yield the crude extracts. Hexane fraction (yield: 5.52) gave a positive chemical test for steroids, dichloromethane fraction (yield: 8.43) gave a positive chemical test for steroids and terpenes, and aqueous fraction (yield: 64.5) gave a positive chemical test for tannins (93). These extracts were screened for antibacterial activity using the following antibacterial assay.

The crude methanol extract of G. scortechinii and fractions were subjected to antimicrobial assay using the disc diffusion method of Bauer et al. (94). Both Gram-positive and Gram-negative bacteria (Table 2) were obtained from the stock cultures of the Department of Medical Microbiology at the University of Malaya. The organisms were of the American Typed Culture Collections (ATCC) and some nosocomial isolates. The organisms included Bacillus sp., Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 24922, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Klebsiella pneumoniae, Shigella sonnei, Shigella flexneri and a yeast Candida albicans (ATCC 90028). The organisms selected for testing in this experiment are commonly responsible for foodborn and nosocomial bacterial infections (95). Mueller–Hinton agar was prepared according to the manufacturer's instruction. It was dispensed into sterile plates in 20ml aliquots. After gelling and drying, the plates were seeded with appropriate organisms by streaking evenly in three planes onto the surface of the medium with cotton swabs. The inoculum was dried for 5min. Sterile filter paper disks (6mm diameter) soaked with 50μl of extract (100mg/ml) were placed onto the agar with flamed forceps and gently pressed down to ensure contact. Streptomycin (10μg/disc) and nystatin (100IU) were used as a positive standard against bacteria and fungi as they are both inexpensive and broad spectrum antimicrobials. The plates were incubated at 37°C for 24h. The zones of inhibition were measured with a ruler. The experiment was carried out in triplicate. Results obtained for antibacterial activity of the crude methanol extract of G. scortechinii and fractions are reported in Table 2. Methanol, hexane, dichloromethane and water used for reconstitution of the extracts showed no activity. Analysis of the data revealed that among the tested fractions, the dichloromethane fraction exhibited the highest rates of antibacterial activity. It showed antibacterial activity against S. aureus ATCC 25923: 23mm, S. aureus ATCC 29213: 27mm, E. faecalis ATCC 24922: 20mm, Escherichia coli ATCC 25922: 19mm, Bacillus sp.: 20mm, K. pneumoniae: 13mm (Fig. 6), S. sonnei: 28mm, S. flexneri: 13mm, and Proteus sp. 17mm. The extract inhibited the growth of C. albicans ATCC 90028. It was inactive against P. aeruginosa ATCC 27853.

Table 2. Antibacterial activity of extractives from G. scortechinii

Figure 6. Antibacterial activity of G. scortechinii against Klebsiella pneumoniae.

This report is the first data available on the antibacterial activity of Goniothalamus species and lends support the traditional use of Goniothalamus species as post-partum remedy. An interesting development from these results would be first to identify the active constituents and next to study their precise molecular activity against bacteria. Note that mitochondria in eukaryotic cells take their origin in pro-bacterial ancestors from which they inherited NADH:ubiquinone oxidoreductase (96). One can perhaps envisage a new antibacterial pathway that would encompass a ‘bacterial apoptosis’. One wonders.

G. scortechinii was investigated as part of our study on the medicinal plants of Asia-Pacific (9,10,97–100) A critical factor for Goniothalamus’ use as a medicinal herb is its content of styryl-lactones, which promote apoptosis in mammalian cells. One might propose the hypothesis that the abortifacient and/or post-natal and anti-inflammatory reported traditional uses of Goniothalamus species might involve styryl-lactones since apoptosis is known to play a crucial role in trophoblasts of patients with recurrent spontaneous abortion of unidentified cause, and in T cells in the human decidua as defense mechanism against rejection of fetal allograft by the maternal immune system (101,102). In addition, goniothalamin induces apoptosis in vascular smooth muscle cells, the growth of which is required to allow embryo implantation and the development of the blood supply for fetal survival and inhibit the cell surface expression of intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 on the surface of murine endothelial cells (103,104)

In regards to the result obtained for antibacterial activity of the crude methanol extract of G. scortechinii and fractions, it can be concluded that the dichloromethane extract of G. scortechinii is very active against both Gram-positive and Gram-negative bacteria. and the results obtained tend to answer positively the question of Chinnok et al. (105). This work illustrates the fact that the careful study of the biochemical architecture of medicinal plants represents a fascinating and fruitful aspect of pharmaceutical research (106). In regards G. scortechinii, it will be interesting to know whether further studies on this plant disclose any molecules the treatment of nosocomial urinary, respiratory and wound nosocomial infections (S. aureus, E. coli and K. pneumoniae) which are developed by hospital patients.

In summary the evidence for the existence of anticancer, antibacterial and antiviral agents in the genus Goniothalamus is strong and it seems likely that further consistent and systematic research on this genus of flowering plants will lead to the discovery of antineoplastic and antimicrobial agents. If enough botanical, phytochemical and pharmacological work is dedicated to this discrete tropical genus of flowering plants, a couple of drugs for the treatment of tumors and/or bacterial and even viral infections should be developed in the relatively close future.

The staffs of the Forest Institute of Malaysia are gratefully acknowledged.