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Evid Based Complement Alternat MedecamEvidence-based Complementary and Alternative Medicine : eCAM1741-427X1741-4288Oxford University Press 10.1093/ecam/nem009ReviewsGoniothalamus Species: A Source of Drugs for the Treatment of Cancers and Bacterial Infections? Wiart Christophe School of Pharmacy, The University of Nottingham (Malaysia Campus), Jalan Broga, 43500 Semenyih, Selangor, MalaysiaFor reprints and all correspondence: Dr C. Wiart, School of Pharmacy The University of Nottingham (Malaysia Campus), Jalan Broga, 43500 Semenyih, Selangor, Malaysia. Tel: +60-3-79675749; Fax: +60-3-79674964; E-mail: christophe_wiart@yahoo.com9 2007 12 3 2007 4 3 299 311 15 3 2005 16 1 2007 © 2007 The Author(s).2007This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Irrespective of the presence of cytotoxic acetogenins and styryl-lactones in the genus Goniothalamus, only 22 species in the genus Goniothalamus, out of 160 species (13.7%) have so far been investigated. In an effort to promote further research on the genus Goniothalamus which could represent a source of drugs for the treatment of cancers and bacterial infections, this work offers a broad analysis of current knowledge on Goniothalamus species. Therefore, it includes (i) taxonomy (ii) botanical description (iii) traditional medicinal uses and (iv) phytochemical and pharmacological studies. We discuss the molecular mechanisms of actions of acetogenins and styryl-lactones, with some emphasis on the possible involvement of protein kinase, Bax and TRAIL receptors in the cytotoxic effects of styryl-lactones. We also report (v) the growth inhibition of several nosocomial bacteria by Goniothalamus. scortechinii. The crude methanol extract of G. scortechinii showed a good and broad spectrum of antibacterial activity against both Gram-negative and Gram-positive bacteria.

acetogeninsantibacterialantifungalapoptosiscytotoxicfoodborn bacteriaGoniothalamusGoniothalamus scortechiniinosocomialstyryl-lactones.
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Introduction
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.

Botanical Description
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.



Traditional Medicinal Uses
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.

Phytochemical and Pharmacological Studies
General Concept
Twenty-two species (13.7%) in the genus Goniothalamus, out of 160 species, have so far been phytochemically investigated namely: G. amuyon, G. andersonii J.Sincl., G. arvensis Scheff, G. borneensis Mat-Salleh, G. cardiopetalus, G. cheliensis. Hu, G. donnaiensis Finet & Gagnep., G. gardneri Hook. f. & Thoms., G. giganteus Hook. f. & Thoms., G. griffithii Hook. f. & Thoms., G. howii Merr., G. leiocarpus (W.T.) Wang P.T. Li, G. malayanus Hook. f. & Thoms., G. marcanii Craib, G. montanus J. Sincl., G. scortechinii, G. sesquipedalis Hook. f. & Thoms., G. tapis Miq., G. thwaitesii Hook. f. & Thoms., G. umbrosus J. Sincl., G. uvaroides King and G. velutinus Airy Shaw. These phytochemical studies have resulted so far in the isolation of two very distinct classes of lipophilic secondary metabolites: acetogenins and styryl-lactones, both of them possessing complex stereochemistry and existing in different stereoisomeric forms (13). Testing of these chemicals for cytotoxicity showed that both acetogenins and styryl-lactones are toxic for several human tumors cell lines. Note that both acetogenins and styryl-lactones are cytotoxic for mammalian cells as the result of distinct biochemical pathways which however take both their molecular origin near or in the mitochondrial membrane and or mitochondrial respiratory system (14). To date some evidence clearly demonstrate that acetogenins have beneficial effects against the growth of tumors, including ovarian tumors (15), gastric tumors (16) and multidrug-resistant cancerous xenografts (17) via the activation of caspases enzymatic cascades (18). Most phytochemical reports found so far on Goniothalamus species deal with the chemical constituents of a medicinal species: G. giganteus which abounds with cytotoxic acetogenins (Table 1) as mentioned further.
Table 1. Antitumor activity of Goniothalamus species

Species	Chemical component	Cells	Dose	Cell cycle/apoptosis	
G. giganteus	4-Deoxyanomontacin (27)	A-549a	6.45 × 10−7 μg/ml		
		MCF-7b	5.77 × 10−7 μg/ml		
		HT-29c	1.41 × 10−1 μg/ml		
		A-498d	1.50 × 10−1 μg/ml		
		PC-3e	1.73 × 10−1 μg/ml		
		PACA-2f	1.×10−5 μg/ml		
	(2,4-cis and trans)- annomontacinone (27)	HT-29	2.55 × 10−1 μg/ml		
		PACA-2	6.78 × 10−1 μg/ml		
	cis-Gigantrionenin (32)	A-549	5.99 × 10−2 μg/ml		
		MCF-7	2.68 × 10−1 μg/ml		
		HT-29	6.94 × 10−6 μg/ml		
		A-498	1.39 × 10−2 μg/ml		
		PC-3	1.11 × 10−1 μg/ml		
		PACA-2	1.15 × 10−1 μg/ml		
	4-Acetylgigantetrocin (25)	A-549	<10−2 μg/ml		
		MCF-7	8.5 × 10−1 μg/ml		
		HT-29	<10−2 μg/ml		
		A-498	1.55 × 10−1 μg/ml		
		PACA-2	<10−2 μg/ml		
	Annonacin (19)	PA1g	0.452 μg/ml	G1	
		SKOV3h	0.411 μg/ml		
		HeLai	0.219 μg/ml		
		HeLa S3j	0.426 μg/ml		
		MCF-7	0.433 μg/ml		
		T-24k	0.324 μg/ml		
		BCC-1l	0.427 μg/ml		
	Gigantransenin A (26)	A-549	0.16 μg/ml		
	Gigantransenin B (26)	A-549	0.21 μg/ml		
		MCF-7	2.1 × 10−1 μg/ml		
	Gigantrasenin C (26)	A-549	0.18 μg/ml		
	Goniotetrocin (29)	A-549	3.9 10−1 μg/ml		
		PC-3	2.1 10−1 μg/ml		
		PACA-2	2.6 10−2 μg/ml		
	(2,4-cis and trans)				
	Gonioneninone (29)	PACA-2	4.5 10−2 μg/ml		
	Goniothalamicin (24)	A-549	2.8010−1 μg/ml		
	Gonionenine (24)	PACA-2	4.5 10−1 μg/ml		
	Pyranicin (30)	A-549	2.8 10−1 μg/ml		
		MCF-7	3.6 × 10−1 μg/ml		
		A-498	1.8 × 10−1 μg/ml		
		PACA-2	1.3 10−3 μg/ml		
	Deoxyannomontacin (27)	A-549	6.45 10−7 μg/ml		
		MCF-7	1.41 × 10−1 μg/ml		
		A-498	1.50 × 10−1 μg/ml		
		PACA-2	1.5 10−5 μg/ml		
	Annomontacinone (27)	HT-29	2.55 × 10−1 μg/ml		
G. giganteus	Annomontacinone (27)	PACA-2	6.78 × 10−1 μg/ml		
	Pyragonicin (30)	A-549	<10−2 μg/ml		
		HT-29	3.4 10−1 μg/ml		
		A-498	1.55 × 10−1 μg/ml		
		PACA-2	5.8 10−3 μg/ml		
	Goniotrionin (30)	A-549	7.7 10−3 μg/ml		
		MCF-7	8.5 × 10−1 μg/ml		
		MCF-7	5.3 10−6 μg/ml		
		A-498	2.10−3 μg/ml		
		PC-3	3.6 10−1 μg/ml		
		PACA-2	5.4 10−3 μg/ml		
G. donnaiensis	Goniodonin (34,35)	HCT-8m	<10 μg/ml		
	Donhexocin (34,35)	HCT-8	0.82 μg/ml		
	Donbutocin (34,35)	L1210	0.81 μg/ml		
G. gardneri	Gardnerilin A (37)	Bel7402n	3.6 μg/ml		
	Gardnerilin B (37)	Bel7402n	8.5 μg/ml		
G. andersonii	Goniothalamin (47)	HL-60o		apoptosis	
		Jurkat Tp		apoptosis	
G. griffithii	Goniothalamin (82)	HepG2	8.83 μM	G2/apoptosis	
		HepG2R	8 μM		
	Altholactone (82)	HepG2	0.7 μM	apoptosis	
		HepG2R	6.17 μM		
	Goniodiol (82)	HepG2	10 μM	G2	
		HepG2R	8.33 μM		
G. malayanus	Altholactone (74,76)	HL60		apoptosis	
G. borneensis	Goniothalamin (49)	P388	0.75 μg/ml		
		WEHI164	1.70 μg/ml		
		MOLT-4	<1 μg/ml		
G. howii	Howiinol (79)	L1210	6.85 μg/ml	G1	
G. cheliensis	Goniolactone B (54)	A2780	7.40 μM		
		HCT-8	4.43 μM		
		KB	7.23 μM		
Synthesized	Goniothalamin (66)	MCF-7	10.5 μM		
		HT-29	11.2 μM		
ahuman lung carcinoma; bhuman breast carcinoma; chuman colon adenocarcinoma; dhuman kidney carcinoma, ehuman prostate adenocarcinoma, fhuman pancreatic carcinoma, g,hovarian cancer cells; I,jcervical cancer; kbladder cancer; lskin cancer, mhuman colon adenocarcinoma, nhepatoma cell-line, oleukemia cells, ppromyelocytic leukemia cells; qEhrlich ascites tumor cells.



Acetogenins: Unusual Polyketides
Acetogenins are unusual series of polyketides which have so far only been characterized from members of the family Annonaceae including in the genus Goniothalamus particularly G. giganteus, G. donnaiensis and G. gardneri (9,10). In the genus Goniothalamus, acetogenins were first characterized as the active principles responsible for shrimp lethality from the bark of G. giganteus collected from Thailand. Extract of the bark showed toxicity in the brine shrimp test and showed murine cytotoxicity in the 3PS (P388) leukemia bioassay. The cytotoxicity of this extract compelled a series of phytochemical studies which resulted in the identification of a series of cytotoxic acetogenins including notably: (2,4-cis and trans-)-annomontacinones (i), annonacin (ii), giganenin (iii), gigantecin (iv), 4-deoxygigantecin, (2,4-cis and trans)-gigantecinones, 4-acetylgigantetrocin A (v), goniotrionin (vi), gigantransenin A (vii) and C (viii), gigantrionenin (ix), gigantetrocin (x), goniotetrocine (xi), (2,4-cis and trans)-gigantetrocinones, gonionenin (xii), (2,4-cis and trans)-gonioneninones (xiii), 4-deoxygigantenin (xiii), 4-deoxyannomontacin (xiv), goniothalamicin (xv), pyranicin (xvi), gigantriocin (xvii), goniotriocin, (2,4-cis and trans)-isoannonacins, longicoricin, longifolicicin, longimicin C, cis-gigantrionenin (xviii), pyragoniocin (xix), xylomaticin, and (2,4-cis and trans)-xylomaticinones (Fig. 1) (19–33). Gigantransenins A, and C showed selective inhibitory effects on the human breast tumor cell-line (MCF-7) comparable with the potency of adriamycin (26). Both goniotetracin, and 2,4-cis- and trans-gonioneninone are selectively and significantly cytotoxic to the human pancreatic tumour cell line (PACA-2) (29). Pyranicin exhibited a selective cytotoxic against the pancreatic cell line (PACA-2) in a panel of six human solid tumor cell lines, with pyranicin showing 10 times the potency of adriamycin (30).

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 8.5 μg/ml, respectively (37,38) (Fig. 1). The mode of action of acetogenins is discussed next.

Mode of Action of Acetogenins: Inhibition of NADH-ubiquinone Oxidoreductase
Acetogenins have very potent and diverse biological effects owing to the fact that they inhibit enzymatic activity of a key enzyme in Eukaryotic cells: mitochondrial NADH-ubiquinone oxidoreductase (complex I). To date the most potent existing inhibitor of this enzyme is an acetogenin known as bullatacin (39–41). In regards to the precise molecular mode of action of acetogenins against the enzyme, there is an expanding body of evidences to suggest that the most lipophilic moieties are embedded in the mitochondrial membrane allowing suitable position of the pharmacophore. One might set the hypothesis that the tetrahydrofuran (THF) or tetrahydropyran rings as well as the free alkyl substituent fix the molecule, whereas the lactones maintained by an alkyl spacer acts on the active site of the enzyme as illustrated in Fig. 2 (42,43). The work of Motoyuki et al. (13) lends strong support to that hypothesis. They synthesized series of acetogenins and assessed their activity against bovine heart mitochondrial complex I and showed that the length of the alkyl spacer and the polarity of THF surroundings were very important structural factor and that the γ-lactone and THF ring moieties act in a cooperative manner on complex I with the support of some specific conformation of the alkyl spacers as illustrated in Fig. 2.
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.

Styryl-lactones: Phenolic Compounds
Styryl-lactones are low molecular weight phenolic compounds, which, like acetogenins are essentially found in members of the Annonaceae family and present a lactonic pharmacophore (9). Examples of styryl-lactones from Goniothalamus species are goniothalamin (i), altholactone (ii) and cardiopetalolactone (iii) (Fig. 3).
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.

Mode of Cytotoxic Action of Styryl-lactones: Apoptosis
The evidence currently available clearly indicate that goniothalamin and congeners are toxic for several sorts of cancer cells cultured in vitro including HL-60 leukemia cells, breast cancer cell line MCF-7, liver cancer cell line HepG2, PANC-1, HeLa cell lines (63–80) (Table 1). Current paradigms of apoptosis suggest that styryl-lactones from Goniothalamus activate in mammalian cells the caspases enzymatic cascades via a loss of mitochondrial transmembrane which results in the release of mitochondrial cytochrome c (72). To date, the very precise premitochondrial mechanism involved in this activation remains an enigma, and an exciting fact is that the activation of caspases, 3, 6, 7 and 9 is a sign of TRAIL receptors/Bax activation (65). Other examples of goniothalameous styryl-lactones of possible chemotherapeutic value are altholactone, goniolactone B and howiinol. Altholactone is apoptogenic in HL-60 promyelocytic leukemia cells via oxidative stress and mitochondrial respiratory abrogation (75,76). Goniolactone B exhibited significant cytotoxicity against A2780, HCT-8 and KB cells with IC50 values of 7.40, 4.43 and 7.23 μM, respectively (55). Howiinol A showed significant antitumor activities toward human tumor cell in vitro and in vivo (77–81). A remarkable advance in the pharmacological knowledge of howiinol A has been provided by the work of He et al. Using techniques of cell growth curve determination, MTT test, soft agar colony assay and experimental therapy of transplantable tumors in mice, they showed that howiinol exerts potent inhibitory effect on cancer cells including drug-resistant cell line, KB/VCR 2000, whereas normal cells are less affected. Howiinol is active in rodents infected with H22 hepatoma and Lewis lung cancer and ascetic sarcoma 180. In addition to flow cytometry technique, they showed that the cycle of howiinol A block is used to analyze the cell cycle of L1210 cells from G1 phase to S phase with structural damage on DNA molecules. Tian et al. (82) showed that Goniothalamus styryl-lactones which are cytotoxic against both HepG2 and HepG2-R cell lines show less toxicity on normal mice hepatocytes as the IC50 values of them on normal mouse hepatocytes were about 3 times of that on HepG2. They demonstrated that cells treated with goniothalamin and altholactone stopped to multiply at G(2)/M and were apoptotic, whereas cells with chromosomes gathered at the equator were easily found in gonodiol-treated cultures.

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.

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 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?.


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 translocation of Bax to mitochondria, 4, 5: release of cytochrome C and activation of caspases, SMAC/Diablo, EndoG, 6: chromatin condensation and cellular death.



Other Phytochemicals
The aerial parts of G. gardneri have yielded the known flavonoids 2′-hydroxy-4,4′,6′-trimethoxychalcone (flavokavain A), 2′,4′-dihydroxy-4,6′-dimethoxydihydrochalcone, 4,2′,4′-trihydroxy-6′-methoxydihydrochalcone, 5,7,4′-trimethoxyflavanone (naringenin trimethyl ether) and 7-hydroxy-5,4′-dimethoxyflavanone (tsugafolin) together with three novel compounds, the dimer characterized as (rel)-1β,2α-di-(2,4-dihydroxy-6-methoxybenzoyl)-3β,4α-di-(4-methoxyphenyl)-cyclobutane, 2′,4′-dihydroxy-4,6′-dimethoxychalcone and 2′-hydroxy-4,4′,6′-trimethoxydihydrochalcone (86). A similar study of the aerial parts of G. thwaitesii led only to the isolation of the known flavonoids myricetin 4′-O-methyl ether-3-O-α-l-rhamnopyranoside (mearnsitrin) and myricetin-3-O-methyl ether (annulatin), together with a series of triterpenes friedelinol, friedelin and betulinic acid (86).

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.

Antibacterial Activity of G. Scortechinii
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, 300 m 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 (800 g) were extracted with methanol (2 l) using a soxhlet apparatus. Hexane (250 ml), dichloromethane (250 ml), and water fractions (250 ml) were obtained by the partitioning of liquid methanol extract (250 ml) (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 20 ml 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 5 min. Sterile filter paper disks (6 mm diameter) soaked with 50 μl of extract (100 mg/ml) were placed onto the agar with flamed forceps and gently pressed down to ensure contact. Streptomycin (10 μg/disc) and nystatin (100 IU) 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 24 h. 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: 23 mm, S. aureus ATCC 29213: 27 mm, E. faecalis ATCC 24922: 20 mm, Escherichia coli ATCC 25922: 19 mm, Bacillus sp.: 20 mm, K. pneumoniae: 13 mm (Fig. 6), S. sonnei: 28 mm, S. flexneri: 13 mm, and Proteus sp. 17 mm. The extract inhibited the growth of C. albicans ATCC 90028. It was inactive against P. aeruginosa ATCC 27853.
Figure 6. Antibacterial activity of G. scortechinii against Klebsiella pneumoniae.


Table 2. Antibacterial activity of extractives from G. scortechinii

	H	D	A	M	S	N	
Gram-positive bacteria							
Bacillus sp.	13	20	–	20	18		
Staphylococcus aureus ATCC 25923	14	27	–	21	16		
Staphylococcus aureus ATCC 29213	14	23	–	20	16		
Enterococcus faecalis ATCC 24922	–	20	–	14	14		
Streptococcus pneumoniae ATCC 49619	–	21	–	13	10		
Gram-negative bacteria							
Pseudomonas aeruginosa ATCC 27853	–	–	–	–	17		
Escherichia coli ATCC 25922	–	19	10	13	20		
Klebsiella pneumoniae	–	11	–	–	20		
Shigella sonnei	16	28	–	18	20		
Shigella flexneri	–	13	7	–	13		
Proteus sp.	–	17	12	10	20		
Yeast							
Candida albicans ATCC 90028	13	23	–	9		17	
Average zone of inhibition (in mm) of triplicate including the diameter of the filter paper disc (6 mm). H = hexane fraction (5 mg/disc) D = dichloromethane fraction (5 mg/disc) A = aqueous fraction (5 mg/disc) M = methanol fraction (5 mg/disc) S = streptomycin (10 μg/disc) N = Nystatin (100 IU).



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.

Conclusion
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.

Acknowledgements
The staffs of the Forest Institute of Malaysia are gratefully acknowledged.
==== Refs
References
1 Sinclair J  A revision of the Malayan Annonaceae The garden's Bulletin Singapore 1955 2 149 516 
2 Watt G  Dictionary of the Economic Products of India 1890 III London Allen 533 
3 Burkill IH  A Dictionary of the Economic Products of the Malay Peninsula 1953 1 London Crown Agent 1097 
4 Quisumbing E  Medicinal Plants of the Philippines 1951 Manila Bureau of Printing 324 
5 Cronquist A  An Integrated System of Classification of Flowering Plants 1981 New York Columbia University Press 
6 Saunders RMK  A synopsis of Goniothalamus species(Annonaceae) in Peninsular Malaysia, with a description of a new species Bot J Linn Soc 2003 142 321 39 
7 Takhtajan A  Diversity and Classification of Flowering Plants 1997 New York Columbia University Press 
8 Koek-Noorman J Westra LY Th Maas PJM  Studies in Annonaceae. XIII. The role of morphological characters in subsequent classifications of Annonaceae: a comparative survey Taxon 1990 39 16 32 
9 Wiart C  Medicinal Plants of Asia-Pacific: Drugs for the Future? 2006 Singapore World Scientific Publishing 
10 Wiart C  Ethnopharmacology of Medicinal Plants: Asia and the Pacific 2007 USA Humana Press 
11 Greshoff M  Beschriving der giftige en bedwelmende planten bij de vischvangt in gebruik 1900 29 1 253 II. Ibid 
12 Heyne K  De Nuttige Planten van Indonesie 3rd ed. 1950; Part I: 1–1450; Lui TS List of Economic Plants in Taiwan 1952 92 Taipei, Taiwan 
13 Motoyuki T Kaoru K Hironori N Akira T Hajime I Hideto M  Definition of crucial structural factors of acetogenins, potent inhibitors of mitochondrial complex I Biochim Biophys Acta 2000 1460 302 10 11106771 
14 Yang S Yu J Xu L  Chemical constituents of Annonaceae plants and their antitumour activities Zhongguo Yi Xue Ke XueYuan Xue Bao 2000 22 376 82 
15 Nakanishi Y Chang FR Liaw CC Wu YC Bastow KF Lee KH  Acetogenins as selective inhibitors of the human ovarian 1A9 tumor cell line J Med Chem 2003 46 3185 8 12852747 
16 Huang GR Jiang S Wu YL Jin Y Yao ZJ Wu JR  Induction of cell death of gastric cancer cells by a modified compound of the annonaceous acetogenin family Chem Biochem 2003 4 1216 21 
17 Fu LW He LR Liang YJ Chen LM Xiong HY Yang XP  Experimental chemotherapy against xenografts derived from multidrug resistant KBv200 cells and parental drug-sensitive KB cells in nude mice by annonaceous acetogenin 89-2 Yao Xue Xue Bao 2003 38 565 70 14628443 
18 Zhu XF Xie BF Li ZM Feng GK Zeng YX Liu ZC  Mechanism of apoptosis induced by squamocin in leukemia cells Yao Xue Xue Bao 2001 36 498 501 12585079 
19 Alkofahi A Rupprecht J Smith DL Chang CJ Mclaughlin JL  Goniothalamin and annonacin: bioactive acetogenins from Goniothamalus giganteus (Annonaceae) Experientia 1988 44 83 5 3350126 
20 Alkofahi A Ma WW McKenzie AT Byrn SR McLaughlin JL  Goniotriol from G. giganteus J Nat Prod 1989 52 1371 3 2614426 
21 Alkofahi A Rupprecht J Liu YM Chang CJ Smith DL Mclaughlin JL  Gigantecin: a novel antimitotic and cytotoxic acetogenin, with non adjacent tetrahydrofurane rings, from G. giganteus (Annonaceae) Experientia 1990 46 539 41 2347408 
22 Fang XP Anderson JE Smith DL Mc Laughlin JL Wood KV  Gigantetronenin and gigantrionenin: novel cytotoxic acetogenins from G. giganteus J Nat Prod 1992 55 1655 63 1479382 
23 Xin-ping F Rong S Zhe-ming G Rieser MJ Miesbauer LR Smith DL  A new type of cytotoxic annonaceous acetogenin: giganin from G. giganteus. Bioorg Med Chem Lett 1993 3 1153 6 
24 Gu ZM Fang XP Zeng L Song R Ng JH Wood KV  Gonionenin: a new cytotoxic annonaceous acetogenin from G. giganteus and the conversion of mono-THF acetogenins to bis –THF acetogenins J Org Chem 1994 59 3472 9 
25 Zeng L Zhang Y Ye Q Shi G He K McLaughlin JL  Cis-gigantrionenin and 4-acetyl gigantetrocin A, two new bioactive annonaceous acetogenins from G. giganteus, and the stereochemistries of acetogenin 1,2,5-triols Bioorg Med Chem 1996 4 1271 9 8879548 
26 Zeng L Yan Z McLaughlin JL  Gigantransenins A, B, and C, novel mono-THF acetogenins bearing trans double bonds, from G. giganteus (Annonaceae) Tetrahedr Lett 1996 37 5449 52 
27 Feras QA Zeng L Zhang Y Ye Q Hopp DC Schwedler JT  4-deoxyannomontacin and (2,4-cis and trans)-annomontacinone, new bioactive mono-tetrahydrofuran annonaceous acetogenins from G. giganteus Bioorg Med Chem 1997 5 549 55 9113333 
28 Feras QA Zhang Y Rogers L McLaughlin JL  (2,4-cis and trans)-gigantecinone and 4-deoxygigantecin, bioactive nonadjacent bis- tetrahydrofuran annonaceous acetogenins, from G giganteus J Nat Prod 1997 60 929 33 9322367 
29 Feras QA Yan Z Jingling R McLaughlin JL  Mono-tetrahydrofuran acetogenins from G. giganteus Phytochemistry 1998 49 761 8 9779595 
30 Feras QA Lingling R Yan Z McLaughlin JL  Unusual bioactive annonaceous acetogenins from G. giganteus Tetrahedron 1998 54 5833 44 
31 Feras QA Rogers L Zhang Y McLaughlin JL  Goniotriocin and (2,4-cis- and -trans)-xylomaticinones, bioactive annonaceous acetogenins from G. giganteus J Nat Prod 1999 62 31 4 9917277 
32 Zeng L Zhang Y Qing Y Gouen S Kan H McLaughlin JL  cis-Gigantrionenin and 4-acetyl gigantetrocin A, two new bioactive annonaceous acetogenins from G. giganteus, and the stereochemistries of acetogenin 1,2,5-triols Bioorg Med Chem 1996 4 1271 9 8879548 
33 Feras QA  Novel anti-cancer and pesticidal components from G. giganteus (Annonaceae) Dissertation 1998 Purdue Libraries 
34 Jiang Z Chen Y Ruo-Yun Ch De-Quan Y  Mono-tetrahydrofuran ring acetogenins from G. donnaiensis Phytochemistry 1997 46 327 31 
35 Jiang Z Chen Y Chen RY Yu DQ  Linear acetogenins from G. donnaiensis Phytochemistry 1998 49 769 75 9779596 
36 Jiang Z Chen RY Chen Y Yu DQ  Donnaienin, a new acetogenin bearing a hydroxylated tetrahydrofuran ring J Nat Prod 1998 61 86 8 9548832 
37 Chen Y Jiang Z Chen RR Yu DQ  Two linear acetogenins from G. gardneri Phytochemistry 1998 49 1317 21 9842730 
38 Seidel V Bailleul F Waterman PG  A linear acetogenin from G. gardneri Phytochemistry 1999 52 1101 3 
39 Morre DJ de Cabo R Farley C Oberlies NH McLaughlin JL  Mode of action of bullatacin, a potent antitumor acetogenin: inhibition of NADH oxidase activity of HeLa and HL-60, but not liver, plasma membranes Life Sci 1995 56 343 8 7837933 
40 Feras AQ Kaakeh W Bennett GW McLaughlin JL  Annonaceous acetogenins as natural pesticides: potent toxicity against insecticide-susceptible and -resistant German cockroaches (Dictyoptera: Blattellidae) J Econ Entomol 1998 J91 641 9 
41 Oberlies NH Croy VL Harrison ML McLaughlin JL  The Annonaceous acetogenin bullatacin is cytotoxic against multidrug-resistant human mammary adenocarcinoma cells Cancer Lett 1997 115 73 9 9097981 
42 Shimada H Kozlowski JF Mclaughlin JL  The localizations in liposomal membranes of the tetrahydrofuran ring moieties of the annonaceous acetogenins, annonacin and sylvaticin, as determined by 1H NMR spectroscopy Pharmacol Res 1998 37 357 64 9642031 
43 Zeng BB Wu Y Jiang S Yu Q Yao ZJ Liu ZH  Studies on mimicry of naturally occurring annonaceous acetogenins: non-THF analogues leading to remarkable selective cytotoxicity against human tumor cells Chemistry 2003 9 282 90 12506384 
44 Kojima N  Systematic synthesis of antitumor annonaceous acetogenins Yakugaku Zasshi 2004 124 673 81 15467275 
45 Rodier S Le Huerou Y Renoux B Doyon J Renard P Pierre A  New cytotoxic analogues of annonaceous acetogenins Anticancer Drug Des 2001 16 109 17 11962508 
46 Jiang S Liu ZH Sheng G Zeng BB Cheng XG Wu YL  Mimicry of annonaceous acetogenins: enantioselective synthesis of a (4R)-hydroxy analogue having potent antitumor activity J Org Chem 2002 17;67 3404 8 12003552 
47 Jewers K Davis JB Dougan J Manchanda AH Blunden G Aye K  Goniothalamin and its distribution in four Goniothalamus species Phytochemistry 1972 11 2025 30 
48 Bermejo A Blazquez MA Rao KS Cortes D  Styryl-lactones from G. arvensis Phytochemistry 1998 47 1375 80 9611830 
49 Shu-Geng C Xiao-Hua W Keng-Yeow S Tan BHK Pereira JT Swee-Hock G  Styryl-lactone derivatives and alkaloids from G. borneensis (Annonaceae) Tetrahedron 1998 54 2143 8 
50 Hisham A Harassi A Shuaily W Shizue E Fujimoto Y  Cardiopetalolactone: a novel styryl-lactone from G. cardiopetalus Tetrahedron 2000 56 9985 89 
51 Hisham A Toubi M Shuaily W Ajitha MDB Fujimoto Y  Cardiobutanolide, a styryl-lactone from G. cardiopetalus Phytochemistry 2003 62 597 600 12560032 
52 Mu Q Tang WD Liu RY Li CM Lou LG Sun HD  Constituents from the stems of G. griffithii Planta Med 2003 69 826 30 14598208 
53 Zhang YJ Zhou GX Chen RY Yu DQ  Styryl-lactones from the rhizomes of G. griffithii J Asian Nat Prod Res 1999 1 189 97 11254031 
54 Steven MC Laily BD Abdul L Kamarudin MS Mohd WS Brian WS  (+)Isoaltholactone: a furanopyrone isolated from G. species Phytochemistry 1990 29 1701 4 
55 Wang S Zhang YJ Chen RY Yu DQ  Goniolactones A-F, six new styrylpyrone derivatives from the roots of G. cheliensis J Nat Prod 2002 65 835 41 12088424 
56 Lan YH Chang FR Liaw CC Wu CC Chiang MY Wu YC  Digoniodiol, deoxygoniopypyrone A, and goniofupyrone A: three new styryl-lactones from G. amuyon J Nat Prod 2002 40 835 41 
57 Lan YH Chang FR Yu JH Yang YL Chang YL Lee SJ  Cytotoxic styrylpyrones from G. amuyon J Nat Prod 2003 66 487 90 12713398 
58 Wu YC Duh CY Chang FR Chang GY Wang SK Chang JJ  The crystal structure and cytotoxicity of goniodiol-7-monoacetate from G. amuyon J Nat Prod 1991 54 1077 81 1791473 
59 Yang-Chang W Fang-Rong C Chang-Yih D Shang-Kwei W Tian-Shung W  Cytotoxic styrylpyrones of G. amuyon Phytochemistry 1992 31 2851 3 
60 Hasan CM Mia MY Rashid MA Connolly JD  5-Acetoxyisogoniothalamin oxide, an epoxystyryl lactone from G. sesquipedalis Phytochemistry 1994 37 1763 4 
61 Fasihuddin BA Wan AT Siraj O Atan MS  5-Acetyl goniothalamin, a styryl dihydropyrone from G. uvaroides Phytochemistry 1991 30 2430 1 
62 Chen R Yu D Ma L Wu F Song W  The chemical constituents of G. howii Merr Yao Xue Xue Bao 1998 33 453 6 12016916 
63 Inayat-Hussain SH Osman AB Din LB Ali AM Snowden RT MacFarlane M  Caspases-3 and -7 are activated in goniothalamin-induced apoptosis in human Jurkat T-cells FEBS Lett 1999 456 379 383 10462048 
64 Teck A Chien L Lope H Tan AN  Styrylpyrone Derivative (SPD) induces apoptosis in a caspase-7-dependent manner in the human breast cancer cell line MCF-7 Cancer Cell Int 2003 3 16 22 14580263 
65 Chien AL Pihie AH  Styrylpyrone derivative induces apoptosis through the up-regulation of Bax in the human breast cancer cell line MCF-7 J Biochem Mol Biol 2003 36 269 74 12787481 
66 de Fatima A Kohn LK Antonio MA de Carvalho JE Pilli RA  (R)-Goniothalamin: total syntheses and cytotoxic activity against cancer cell lines Bioorg Med Chem 2005 13 2927 33 15781402 
67 Pihie AHL Stanslas J Din LB  Non-steroid receptor-mediated anti-proliferative activity of styrylpyrone derivative in human breast cancer cell lines Anticancer Res 1998 18 1739 43 9673398 
68 Hawariah A Stanslas J  Antagonistic effects of styrylpyrone derivative (SPD) on 7,12-dimethylbenzanthracene-induced rat mammary tumors In Vivo 1998 12 403 10 9706492 
69 El-Sharkawi S Yusuf Z Pihie AHL Ali AM  Metabolism of goniothalamin in animal and microbial systems Bull Chim Farmaceutica 1996 135 35 40 
70 Ali AM MacKeen MM Hamidi M Aun QB Zauyah Y Azimahtol HLP  Cytotoxicity and electron death cell induced by goniothalamin Planta Med 1997 63 81 3 9063100 
71 Ali AM Umar-Tsafe N Mohamed SM Inayat-Hussein SH Oo KT Yusoff K  Apoptosis induction in CEM-SS T-lymphoblastic leukemic cell line by goniothalamin J Biochem Mol Biol Biophys 2001 5 253 61 
72 Inayat-Hussain SH Annuar BO Din LB Ali AM Ross D  Loss of mitochondrial transmembrane potential and caspase-9 activation during apoptosis induced by the novel styryl-lactone goniothalamin in HL-60 leukemia cells Toxicology in Vitro 2003 17 433 9 12849726 
73 Umar-Tsafe N Mohamed-Said MS Rosli R Din BD Lai LC  Genotoxicity of goniothalamin in CHO cell line Mutat Res 562 91 102 15279832 
74 Mereyala HB Joe M  Cytotoxic activity of styryl lactones and their derivatives Curr Med Chem Anti-Canc Agents 2001 1 293 300 
75 Peris E Estornell E Cabedo N Cortes D Bermejo A  3-Acetylaltholactone and related styryl-lactones, mitochondrial respiratory chain inhibitors Phytochemistry 2000 54 311 5 10870186 
76 Inayat-Hussain SH Osman AB Din LB Taniguchi N  Altholactone, a novel styryl-lactone induces apoptosis via oxidative stress in human HL-60 leukemia cells Toxicol Lett 2002 131 153 9 11992734 
77 He J Ye Y Xu C  Antitumor activity of howiinol (GHM-10) on L1210 cells in vitro Yao Xue Xue Bao 1998 33 566 70 12016894 
78 He J Ye Y Xu C  Studies on the anticancer effect of howiinol A, a new compound isolated from G. howii Yao Xue Xue Bao 1998 33 493 7 12016881 
79 He J Xu C  Inhibitory effect of Howiinol A(GHM-10) on the synthesis of biological macromolecules in L1210 cells Yao Xue Xue Bao 1998 33 886 90 12016851 
80 He J Ye Y Xu C  Antitumor activity of howiinol (GHM-10) on L1210 cells in vitro Yao Xue Xue Bao 1998 33 566 70 12016894 
81 Sun S Yu D  [Studies on the synthesis and antitumour activities of Howiinol A and its analogies Yao Xue Xue Bao 1998 33 502 11 12016883 
82 Tian Z Chen S Zhang Y Huang M Shi L Huang F Fong C  The cytotoxicity of naturally occurring styryl lactones Phytomedicine 2006 13 181 6 16428026 
83 Zhong L Li CM Hao XJ Lou LG  Induction of leukemia cell apoptosis by cheliensisin A involves down-regulation of Bcl-2 expression Acta Pharmacol Sin 2005 26 623 8 15842784 
84 Wang G Ahmad KA Ahmed K  Modulation of death receptor-mediated apoptosis by CK2 Mol Cell Biochem 2005 274 201 5 16342415 
85 Folmer F Blasius R Morceau F Tabudravu J Dicato M Jaspars M  Inhibition of TNFα-induced activation of nuclear factor κB by kava (Piper methysticum) derivatives Biochem Pharmacol 71 1206 18 16464438 
86 Seidel S Bailleul F Waterman PG  (Rel)-1β,2α-di-(2,4-dihydroxy-6-methoxybenzoyl)-3β, 4α-di-(4-methoxyphenyl)-cyclobutane and other flavonoids from the aerial parts of G. gardneri and G. thwaitesii Phytochemistry 2000 55 439 6 11140605 
87 Sheng-Teh L Yang-Chang W Shiow-Piaw L  Isoquinoline alkaloids of formosan Fissistigma and G. species Phytochemistry 1985 24 1829 34 
88 Sunil KT Dipankar B Pratap C Bani T  Aristololactams of G. sesquipedalis Wall. Revised structures of the 2-oxygenated aristololactams Phytochemistry 1988 27 903 6 
89 Soonthornchareonnon N Suwanborirux K Bavovada R Patarapanich C Cassady JM  New cytotoxic 1-azaanthraquinones and 3-aminonaphthoquinone from the stem bark of G. marcanii J Nat Prod 1999 62 1390 4 10543899 
90 Zhang YJ Kong M Chen RY Yu DQ  Alkaloids from the roots of G. griffithii. J Nat Prod 1999 62 1050 2 10425141 
91 Jantan I Ahmad F Ahmad AS  A comparative study of the essential oils of four Goniothalamus species Bot J Linn Soc 2003 142 321 
92 Siti NMJ Noor RA Mohamad KAG Syed ZSI Khozirah S Lokman HS  The screening of extracts from G. scortechinii, Aralidium pinnatifidum and Andrographis paniculata for anti-malarial activity using the lactate dehydrogenase assay J Ethnopharmacol 2002 82 239 242 12242001 
93 Harborne JB  Phytochemical Methods 1984 London Chapman and Hall 
94 Bauer AW Kirby WMM Sherries JC  Antibiotic susceptibility testing by standard single disc diffusion method Amer J Clin Path 1966 45 426 33 
95 Greenwood D Slack RCB Peutherer JF  Medical Microbiology 1997 15th Edinburgh, UK: Churchill Livingstone 
96 Gabaldon T Rainey D Huynen MA  Tracing the evolution of a large protein complex in the eukaryotes, NADH:ubiquinone oxidoreductase (Complex I) J Mol Biol 2005 348 857 70 15843018 
97 Wiart C  Medicinal Plants of Asia and the Pacific 2006 USA CRC Press 
98 Wiart C Hannah A Yassim M Hamimah H Sulaiman M  Antimicrobial activity of Acalypha siamensis Oliv. ex Gage J Ethnopharmacol 2004 9 285 6 15507350 
99 Wiart C Mogana S Khalifah S Mahan M Ismail S Buckle M  Antimicrobial screening of plants used for traditional medicine in the state of Perak, Peninsular Malaysia Fitoterapia 2004 75 68 73 14693223 
100 Wiart C Hannah NA Yassim M Hamimah H Sulaiman M  Antimicrobial activity of tiger's betel (Piper porphyrophyllum N.E. Br., Piperaceae) Phytother Res 2004 18 783 4 15478188 
101 Vadillo OF Avila VMA Guerrero HC Arechavaleta VF Montoya BJ  Apoptosis in trophoblast of patients with recurrent spontaneous abortion of unidentified cause Ginecol Obstet Mex 2000 68 122 31 10808617 
102 Jerzak M Kasprzycka M Wierbicki P Kotarski J Gorski A  Apoptosis of T cells in the first trimester human deciduas C Am J Reprod Immunol 1998 40 130 5 
103 Chan KM Rajab NF Ishal MHA Ali AM Yusoff K Din LB Inayat-Hussain SH  Goniothalamin induces apoptosis in vascular smooth muscle cells Chem Biol Interact 2005 159 129 40 16297902 
104 Tanaka S Yoichi S Ao L Matumoto M Morimoto K Akimoto N  Potential immunosuppressive and anti-inflammatory activities of Malaysian medicinal plants characterized by reduced cell surface expression of cell adhesion molecules Phythoter Res 2001 15 681 6 
105 Chinnock P Siegfried N Clarke M  Is Evidence-Based Medicine Relevant to the Developing World? Evid-Based Complement Altern Med 2005 2 321 4 
106 Cooper EL  Drug Discovery, CAM and Natural Products Evid-Based Complement Altern Med 2004 1 215 7

