Tongkat Ali (Eurycoma Longifolia Jack): a Review on Its Ethnobotany and Pharmacological Importance

Fitoterapia 81 (2010) 669–679

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Review Tongkat Ali (Eurycoma longifolia Jack): A review on its ethnobotany and pharmacological importance

Rajeev Bhat ⁎, A.A. Karim

Food Technology Division, School of Industrial Technology, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia article info abstract

Article history: Eurycoma longifolia Jack is an herbal medicinal plant of South-East Asian origin, popularly Received 22 January 2010 recognized as ‘Tongkat Ali.’ The plant parts have been traditionally used for its antimalarial, Accepted in revised form 11 April 2010 aphrodisiac, anti-diabetic, antimicrobial and anti-pyretic activities, which have also been Available online 29 April 2010 proved scientifically. The plant parts are rich in various bioactive compounds (like eurycomaoside, eurycolactone, eurycomalactone, eurycomanone, and pasakbumin-B) among Keywords: which the alkaloids and quassinoids form a major portion. Even though toxicity and safety Antimalaria evaluation studies have been pursued, still a major gap exists in providing scientific base for Anticancer commercial utilization and clearance of the Tongkat Ali products with regard to consumer's Aphrodisiac safety. The present review aims at reviewing the research works undertaken till date, on this Ethnobotany fi Toxicity plant in order to provide suf cient baseline information for future works and for commercial Traditional medicine exploitation. © 2010 Elsevier B.V. All rights reserved.

Contents

1. Introduction ...... 669 2. Taxonomy, cultivation and ethnobotanical uses ...... 670 3. Chemical composition ...... 670 3.1. Volatiles ...... 672 4. Pharmacological properties ...... 672 4.1. Antimalarial and anticancer activity ...... 673 4.2. Anti-diabetic properties ...... 675 4.3. Aphrodisiac activities ...... 675 4.4. Antimicrobial activities ...... 676 5. Quality and safety (toxicology, contaminants and side effects) ...... 676 6. Conclusions ...... 677 References ...... 678

1. Introduction recently have commanded major attention worldwide due to their potential nutraceutical values [1–4]. Traditionally employed, indigenous plant based herbal Eurycoma longifolia Jack (Tongkat Ali, Genus: Eurycoma; medicines have been popular from time immemorial, and Family, Simaroubaceae) is one of the most popular tropical herbal plants, indigenous to South-East Asian countries like Malaysia, Indonesia, and Vietnam. Some of the plant species ⁎ Corresponding author. Tel.: +60 4653 5212; fax: +60 4657 3678. E-mail addresses: [email protected], [email protected] are also found in certain patches in regions of Cambodia, (R. Bhat). Myanmar and in Thailand. This plant is locally known as

‘Tongkat Ali’ wherein ‘Ali’ means “walking stick”—assigned plant is dioecious with male and female flowers borne on due to the presence of long twisted roots. The plant extract, different trees. especially roots, are exclusively used (traditionally) for Generally, harvesting of the root is carried out manually enhancing testosterone levels in men. The plant extract and requires lot of serenity and time, as for uprooting a single (particularly roots) have been used in indigenous traditional plant (of around 70–100 mm diameter size) possibly requires medicines for its unique antimalarial, anti-pyretic, antiulcer, between 8 and 10 h. The plant grows profusely in sandy, well cytotoxic and aphrodisiac properties [5–8]. Root extracts have drained soil in the presence of partial shade and with also been used traditionally to reduce blood pressure, fevers adequate quantity of water. Tongkat Ali plant has been (mainly due to the presence of quassinoids) and fatigue. As declared as a protected plant in most of the cultivated areas regular intake of root extracts is believed to enhance the (including Malaysia) and harvesting is highly restricted. This testosterone levels, increased interest has also been created plant grows naturally in the wild jungle slopes and is widely among the individuals involved in body building to enhance sought ‘singly’ or as an ‘essential component’ in herbal the muscle mass and strength. However, most of the times, the remedies for a variety of illnesses and as health supplements. plant extracts forms an essential component in herbal Almost all the parts of the Tongkat Ali plant have been remedies for a wide range of illnesses and as health supple- used traditionally for therapeutic purposes. The plant extracts ments. Though the exact statistic demand in the international have been traditionally employed for its antimalarial, aphro- market is obscure, the dried Tongkat Ali roots are known to disiac and anti-pyretic activities [13]. The roots are the most fetch somewhere between 20 and 25 US dollars/kg. However, valuable component and are used for the treatment of aches, the water extracts has been reported to have a better market persistent fever, malaria, sexual insufficiency, dysentery, value of 26 US dollars per bottle of 60 capsules [9]. glandular swelling, and as health supplements [14–16]. Of late, there has been a tremendous increase in demand Root extract of the plant have been administered for restoring for this plant and nearly 200 Tongkat Ali products, mainly energy and vitality, enhancing blood flow and functioning as highlighting the aphrodisiac properties, are available in the a herbal ingredient for women after child birth [7]. The health-food market [8]. Tongkat Ali products are available process of consuming the root also follows the traditional either in the form of raw crude powder (of root), as capsule way, wherein the roots are cut into smaller portions and mixed with other aphrodisiac herbs, as an additive mixed boiled to blood-warm temperature and is consumed as a tea. with coffee (and ginseng) or in certain health products as a However, due to extensive bitterness in the root, intense replacement for ginseng. boiling (in fresh water for several times) is also suggested to To our knowledge, till date, no meticulous review is remove bitterness in the decoction. Some traditional healers available on Tongkat Ali plant or on its products. Hence, the also advise mixing of extract (chipped roots) with honey, present review is intended to highlight some of the significant sugar syrup or dates for reducing the bitterness. In certain published scientific reports along with providing details cases the root powder is mixed with alcohol and consumed of the traditional knowledge of this wonder-plant (mainly after maintaining for specified time of incubation (extending E. longifolia Jack). It is envisaged that the baseline information up to 6–8 weeks). However, it is also opined by a few provided in this review will be useful and applicable for experienced traditional healers that ‘the more the bitterness’ future research works aiming towards exploiting the plants – the ‘better is the efficacy.’ nutraceutical potentials. The leaves of the plant have also been used by traditional healers to cure malaria, ulcers, prevent gum diseases and for 2. Taxonomy, cultivation and ethnobotanical uses the treatment of sexually transmitted diseases like syphilis and gonorrhea. Scientific names: there are four different species of Tongkat Ali plant reported till now, which includes: E. longifolia, 3. Chemical composition Entomophthora apiculata, Polyathia bullata and Goniothalamus sp. [10–12].However,E. longifolia is used more routinely for A wide range of chemical compounds have been isolated traditional medicine purposes. and characterized from E. longifolia, particularly from the Popular names: some of the common and popular names roots. Some of them include: canthin-6-one alkaloids,-β- of this plant includes: Long Jack, Malaysian Ginseng, Local carboline alkaloids, quassinoids, quassinoid diterpenoids, Ginseng, Natural Viagra, Pasak Bumi, Payung Ali, Penawar Eurycomaoside, tirucallane-type triterpenes, squalene deri- Pahit, Setunjang Bumi, Bedara Pahit, Tongkat Baginda, Pokok vatives, biphenylneolignans, eurycolactone, laurycolactone, Syurga, Tongkat Ali Hitam, Pokok Jelas, Cay ba binh, Ian-don, and eurycomalactone [17–22]. Fig. 2 depicts the structure of and Jelaih. some of the quassinoids isolated form E. longifolia with E. longifolia (Tongkat Ali) is an evergreen slow growing potential anti-tumor promoting and anti-parasitic activities. herbal plant (Fig. 1), attaining a maximum height of 15–18 m Kuo et al. [23] have reported the isolation of nearly sixty-five and bearing fruits after nearly 2–3 years of cultivation. compounds from the roots of E. longifolia, and characterized However, it is generally believed that for complete matura- them by 1D and 2D NMR and by mass spectral data. Among tion of the plant, it might take up to 25 years. But, for these isolates, for the first time they identified four quassinoids commercial uses, most of the times, roots are harvested after diterpenoids obtained from natural sources, viz., eurycomalide 4 years of cultivation. The fruits are green in color, 2–3cm A, eurycomalide B, 13β, 21-dihydroxyeurycomanol, and 5α, long and turn to dark red after ripening. The leaves are 14β,15β-trihydroxyklaineanone. Chan et al. [24] isolated a new pinnate, spirally arranged, long (10–15 inches) with 10–30 quassinoid glycoside from E. longifolia, eurycomanol-2-O-β-D- leaflets. The flowers are produced in large panicles and the glycopyranoside along with eurycomanol which was found R. Bhat, A.A. Karim / Fitoterapia 81 (2010) 669–679 671

Fig. 1. Eurycoma longifolia in a natural habitat at the Univeristi Sains Malaysia Campus, Malaysia.

to possess antimalarial activity. Tada et al. [25] separated hydroxy-9-methoxycanthin-6-one, 11-hydroxy-10-methoxy- four quassinoids, pasak bumin-A, -B, -C, and -D from Pasak bumi canthin-6-one, 5,9-dimethoxycanthin-6-one and 9-methoxy- (E. longifolia) and found that pasakbumin-A (= eurycoma- 3-methylcanthin-5,6-dione) from the bark and wood of none) and pasakbumin-B to exhibit potent antiulcer activity. E. longifolia. Morita et al. [26] isolated two novel isomeric 2,2′- Some of the quassinoids (seven types) was isolated from the dimethoxy-4-(3-hydroxy-1-propenyl)-4′-(1,2,3-trihy- leaves of E. longifolia, which includes: lonilactone, 6-dehydro droxypropyl) diphenyl ethers, and two novel biphenyls, lonilactone,11-dehydroklaineanone, 12-epi-dehydroklainea- 2-hydroxy-3,2′,6′-trimethoxy-4′-(2,3-epoxy-1-hydroxy- none, 15β-hydroxyklaineanone, 14,15 β-dihydroxyklainea- propyl)-5-(3-hydroxy-1-ropenyl)-biphenyl and 2-hydroxy- none, and 15-β-O-acetyl-14-hydroxyklaineanone [28].These 3,2′-dimethoxy-4′-(2,3-epoxy-1-hydroxypropyl)-5-(3-hy- compounds have also been reported to possess anti-tumor droxy-1-propenyl)-biphenyl, from the wood of E. longifolia. promoting and anti-parasitic activities. Further, Mitsunaga et al. [27] reported the isolation of 5 new However, E. longifolia extracts containing eurycomanone- canthin-6-one alkaloids (9,10-dimethoxycanthin-6-one, 10- type (bioactive quassinoid) compounds showed poor oral 672 R. Bhat, A.A. Karim / Fitoterapia 81 (2010) 669–679

Fig. 2. Some of the quassinoids isolated form Eurycoma longifolia with potential anti-tumor promoting and anti-parasitic activities. (Source: Ang e t al. 2002a; Jiwajinda et al. 2002; license number: 2352940174049 and 2351811481183, respectively obtained from Rights link). bioavailability in rat models, probably due to poor membrane Shafiqul Islam et al. [30] analyzed E. longifolia extracts permeability in view of their low P values and/or high first- (water and methanol extract-freeze or spray dried) by pass metabolism [29]. employing GC–MS using solid-phase micro-extraction sam- Even though various types of chemical compounds have pling technique and correlated the 12 identified compounds been isolated and characterized from E. longifolia, research with a QCM array sensor (see Table 1). Further, the major reports on the bioactivity and the mechanism of action of the volatile compounds identified included: curcumene, massoi- isolated compounds under in vivo conditions are limited. lactone, 3-Phenoxy-1-propanol, octanoic acid, benzoic and Additionally the effects of these compounds on other acetic acids, menthol, and 2-Phenoxyethanol, 4-Ethynyl-4- ailments like cancer, HIV, blood pressure, cardio-vascular hydroxy-3,5,5-trimethyl-2-cyclohex-1-enone. From their disease and others, need to be investigated in detail. results, a correlation was established between the headspace volatiles and the quartz crystal sensors for the classification of 3.1. Volatiles plant extracts, indicating sensitivity of the technique. The authors concluded by highlighting the ability of the electronic The volatiles (aroma compounds) of each plant species nose to detect small changes in volatiles (smell/aroma) not possess their own characteristic smell, which might be useful only in Tongat Ali extracts, but also extendable as a rapid from differentiating the plant from other closely related sub- detection method for other herbal volatiles. species. Characterizing the volatile compounds is highly However, additional works are warranted to look for the essential with regard to Tongkat Ali plant as similar plants possible biological activities of these volatile compounds mimicking the original E. longifolia are available in wild (including oils) and also the possibilities for their commercial (forest region) and can be used as a low-cost substitute, exploitation. leading to serious ailments and side effects on long term consumption. Additionally, characterizing and identification 4. Pharmacological properties of volatile compounds is essential as some of the manufac- turer's mix the genuine crude extract/ powder with low- Even though several traditional uses of Tongkat Ali are priced chemicals like sildenafil (a known active ingredient in recognized, a scientific validity and supporting evidence is a Viagra) to highlight better efficacy of their products. pre-requisite for commercial exploitation. In the preceding R. Bhat, A.A. Karim / Fitoterapia 81 (2010) 669–679 673

Table 1 Some of the major compounds identiﬁed in Eurycoma longifolia extracts using SPME coupled with GC–MS. (Source: A.K.M. Shaﬁqul Islam [30]; reproducing license no. 2351811080811 obtained from Rights click copyright clearance centre).

Retention Compound Relative peak area (%)* time 123456789101112

11.66 Acetic acid 1.64 2.07 53.35 21.75 26.88 2.53 51.09 17.01 27.57 19.46 27.78 17.49 19.14 Curcumene 9.37 14.15 1.03 2.40 4.87 0.49 0.64 1.04 1.41 1.23 0.94 0.85 23.76 (R)-(−)-Massoilactone 20.68 18.62 2.07 6.49 10.45 0.35 0.76 3.34 4.68 4.17 3.95 2.63 24.80 3-Phenoxy-1-propanol 4.32 4.42 0.64 0.93 3.56 0.72 0.56 3.28 2.13 3.36 1.05 0.38 25.22 Octanoic acid 2.47 2.26 0.43 0.67 2.00 0.86 2.34 1.00 0.76 0.82 0.42 0.27 26.04 Menthol, 1_-[butyn-3-one-1-yl]-, [1R,2 S,5R]- 7.47 6.91 0.77 0.81 6.33 - 0.18 0.74 0.66 0.73 0.67 0.19 26.60 2-Phenoxyethanol 1.41 1.46 0.23 0.43 1.19 0.06 0.20 1.47 0.95 0.95 0.47 0.16 27.71 4-Ethynyl-4-hydroxy-3,5,5- trimethyl-2-cyclohex-1- 8.04 7.50 0.13 0.88 2.03 0.19 0.31 0.70 0.84 0.77 0.61 0.35 enone 31.63 Benzoic acid 13.34 11.78 5.41 3.53 14.41 0.91 1.56 25.66 15.68 11.16 7.44 1.97

* (Samples 1–6 were freeze dried and samples 7–12 were spray dried: samples 2 and 5–12 were commercial samples: 1, methanol extract; 2, Eu. Long batch 1202– 8002; 3, TA Stem (without bark) MeOH; 4, TA (Sabah) [(Ac) 2O Ext.] (FD); 5, MMBPP206 (34)-84-TAFD; 6, MMBPP206 (34)-84-TAET; 7, MMBPP206 (34)-84- TASD; 8, TA Powder (01)-Nusantara; 9, TA Powder (02)-Nusantara; 10, TA Ext. TAE 2003/7–10 (1); 11, TA Ext. TAE 2003/7–10 (2); and 12, 02A/TA-PE/06/02).

text some of the available reports pertaining towards the tone, eurycomanone and eurycomanol from the plant, which pharmacological potential of the plant extracts are being showed antimalarial activities. discussed. Tables 2 and 3 provide an overview of some Kardono et al. [17] isolated and characterized five important works on the pharmacological properties, the cytotoxic constituents from the roots of E. longifolia obtained isolated chemical compounds and their activity undertaken from Kalimantan, Indonesia. Nearly four of the alkaloids were on the Tongkat Ali plant. found to possess cytotoxic effects against a panel of cell lines like: human cancer cell types [(breast, colon, fibrosarcoma, lung, melanoma, KB, and KB-V1 (a multi-drug resistant 4.1. Antimalarial and anticancer activity cell line derived from KB)] and murine lymphocytic leukemia (P-388). The isolated alkaloids were canthin-6-one alkaloids, Malaria being one of the deadliest diseases the world has namely, 9-methoxycanthin-6-one, 9-methoxycanthin-6-one- ever witnessed, is estimated for nearly 2–3 million deaths Noxide, 9-hydroxycanthin-6-one, and 9-hydroxycanthin-6- annually, and still has not been eradicated due to the one-N-oxide and one quassinoid, eurycomanone. Two resistance developed by the infesting Plasmodium sp. (mainly compounds viz., Eurycomanone and 7-methoxy-P-carboline- Plasmodium falciparum malaria) against chloroquine treat- 1-propionic acid showed significant antimalarial activity ment [31,32]. Hence, a necessity to initiate research and against P. falciparum strains. development of new antimalarial drug involving the current In one of their experiments, Ang et al. [35] evaluated the and new potential drug assumes high importance [33]. effects of daily replacement of culture medium containing Interestingly, several studies have been reported to provide E. longifolia root extracts (rich in quassinoids) against six scientific base for the traditionally popular E. longifolia Malaysian P. falciparum isolates. Their results were encouraging, extracts against malaria or the intermittent fever. wherein, complete inhibitions were observed at 1.25– Chan et al. [34] tested the extracts of E. longifolia for 5.00 µg ml−1 extract after 3 days post-treatment and 0.62 and antiplasmodial activity against a multi-drug resistant 0.31 µg ml−1 after 4 and 6 days of post-treatments, respectively. Thailand strain (K-1) of P. falciparum under in vitro condi- Hout et al. [36] in one of their in vitro antiplasmodial activity tions. They isolated 10-Hydroxycanthin-6-one, eurycomalac- screening studies, found that aqueous extracts E. longifolia (from

Cambodian origin) with IC50 values of ≤3 µg/ml were as active as dichloromethane extracts and the extracts displayed a Table 2 marked cytotoxicity with low selectivity (SI≤10). Root extracts

An overview of some important works on the pharmacological properties of in water revealed highest IC50 (against P. falciparum strain W2 Tongkat Ali (Eurycoma longifolia Jack) plant. strain) (21.2 µg/ml) and IC50 (THP1-cytotoxicity) (39.0 µg/ml) Plant part used Activity Reference values. Recently, Mohd Ridzuan et al. [37] investigated the antimalaria properties of the root extracts of E. longifolia Roots, stem, bark Antiplasmodial [13,17,23,24,35–38] standardized extract (TA164) alone and in combination with activity (antimalarial) Leaves [39] artemisinin in vivo and found that the combination treatment Herb sample Antihyperglycaemic [47] suppressed Plasmodium yoelii infection in the experimental (not clear as to whether activity mice. root or aerial part used) Bioactivity-guided fractionation of the ethanolic extract of Roots Cytotoxic [13,17,23,41,42,45] Roots Sexual/aphrodisiac [5,21,48– the roots of E. longifolia was studied by Chan et al. [38] for the activity 50,53,54,68] possible antiplasmodial activities using the lactate dehydro- Leaves Anti-tumor [39] genase activity assay, and were further compared with their Roots Antiulcer [25] known in vitro cytotoxicity against KB cells. Their results Leaves, stem, and roots Antimicrobial [55] revealed that some of the quassinoids isolated like: 674 R. Bhat, A.A. Karim / Fitoterapia 81 (2010) 669–679

Table 3 Isolated chemical compounds and their activity of different parts of Tongkat Ali plant.

Chemical compounds Plant Activity Reference isolated parts

Three new alkaloids: Roots Compounds showed signiﬁcant cytotoxicity against human lung cancer Kuo et al. n-pentyl β-carboline-1-propionate, (A-549) and human breast cancer (MCF-7) cell lines [13] 5-hydroxymethyl-9-methoxycanthin-6-one, and 1-hydroxy-9-methoxycanthin–6-one Canthin-6-one alkaloids, namely, Roots Isolated compounds were found to possess cytotoxicity effects against: Kardono 9-methoxycanthin-6-one, 9-methoxycanthin-6-one-Noxide, human cancer cell types [(breast, colon, ﬁbrosarcoma, lung, melanoma, et al. [17] 9-hydroxycanthin-6-one, and 9-hydroxycanthin- KB, and KB-V1 (a multi-drug resistant cell line derived from KB)] and 6-one-N-oxide (alkaloids), and murine lymphocytic leukemia Eurycomanone (a quassinoid) Eurycomanone and 7-methoxy-P- Roots Compounds showed antimalarial activity against Plasmodium Kardono carboline-1-propionic acid falciparum strains et al. [17] Quassinoids diterpenoids: Roots Compounds exhibited strong cytotoxicity toward human lung cancer Kuo et al. eurycomalide A, eurycomalide B, 13β, (A-549) cell lines and human breast cancer (MCF-7) cell lines [23] 21-dihydroxyeurycomanol, and 5α,14β,15β-trihydroxyklaineanone Quassinoid glycoside Roots Antimalarial activity Chan et al. [24] 10-Hydroxycanthin-6-one, Plant Antiplasmodial activity against a multi-drug resistant Thailand strain Chan et al. eurycomalactone, eurycomanone and (Root) (K-1) of Plasmodium falciparum [34] eurycomanol Eurycomanone and 13,21- Roots Antiplasmodial activities Chan et al. dihydroeurycomanone [38] Quassinoids: Roots Compounds possess antiulcer activity Tada et al. Pasakbumin-A, -B, -C, and -D [25] 9-methoxycanthin-6-one (an alkaloid) Roots Compounds exhibited cytotoxic effects Nurhanan et al. [45] Squalene-type triterpene, named Roots Compounds showed cytotoxic effects Itokawa eurylene et al. [41] 2 novel isomeric compounds: Wood -NR- Morita 2,2′-dimethoxy-4-(3-hydroxy-1- et al. [26] propenyl)-4′-(1,2,3-trihydroxypropyl) diphenyl ethers and 2 novel biphenyls: 2-hydroxy-3,2′,6′-trimethoxy-4′-(2,3- epoxy-1-hydroxypropyl)-5-(3- hydroxy-1-ropenyl)-biphenyl and 2- hydroxy-3,2′-dimethoxy-4′-(2,3- epoxy-1-hydroxypropyl)-5-(3- hydroxy-1-propenyl)-biphenyl 5 new canthin-6-one alkaloids (9,10- Bark -NR- Mitsunaga dimethoxycanthin-6-one, 10-hydroxy- and et al. [27] 9-methoxycanthin-6-one, 11-hydroxy- wood 10-methoxycanthin-6-one, 5,9- dimethoxycanthin-6-one and 9-methoxy- 3-methylcanthin-5,6-dione) Seven quassinoids: lonilactone, 6- Leaves Anti-tumor and anti-parasitic activities Jiwajinda dehydro lonilactone,11- et al. [28] dehydroklaineanone, 12-epi- dehydroklaineanone, 15β- hydroxyklaineanone, 14,15 β- dihydroxyklaineanone, and 15- β -O- acetyl-14-hydroxyklaineanone 6 quassinoid compounds: Leaves Compounds posses anti-tumor promoting, antischistosomal and Jiwajinda (longilactone, dehydro longilactone, plasmodicidal activities et al. [39] 11-dehydroklaineanone, 15β- hydroxyklaineanone, 14,15 dihydroxyklaineanone, and 15β-O- acetyl-14 -hydroxyklaineanone)

NR, indicates — not reported. Plant extracts (methanol, aqueous, and chloroform). Alkaloids (canthin-6-one alkaloids, namely, 9-methoxycanthin-6-one, 9-methoxycanthin-6-one-Noxide, longilactone. 10-Hydroxycanthin-6-one, eurycomalactone, eurycomanone, eurycomanol, 7-methoxy-P-carboline-1-propionic acid, and 13,21-dihydroeurycomanone.

eurycomanone and 13,21- dihydroeurycomanone possessed However, as not much information's is available on the potential antimalarial properties against in vitro culture of possible antimalarial activities of E. longifolia in vivo or in chloroquine-resistant P. falciparum and showed higher human studies, further detailed studies are warranted on selectivity indices for cytotoxicity against KB cells. these aspects. R. Bhat, A.A. Karim / Fitoterapia 81 (2010) 669–679 675

Jiwajinda et al. [39] isolated six quassinoids (long- Nurhanan et al. [45] evaluated the methanol, n-butanol, ilactone, dehydro longilactone, 11-dehydroklaineanone, chloroform and water extracts obtained from the root of 15β-hydroxyklaineanone, 14,15 dihydroxyklaineanone, E. longifolia for its possible cytotoxic effect against KB, DU- and 15β-O-acetyl-14 -hydroxyklaineanone) from the leaves 145, RD, MCF-7, CaOV-3, and MDBK cell lines. Their results of E. longifolia and studied their possible effects on anti-tumor indicated that except for the water extract, all the other promoting, antischistosomal and plasmodicidal activities by in extracts produced signiﬁcant cytotoxic effect on these cell vitro methods. Their results revealed that 14, 15β-dihydrox- lines with no signiﬁcant cytotoxic effect on MDBK (kidney) yklaineanone to be more potent active compound for inhibition normal cell line. An alkaloid 9-methoxycanthin-6-one was of tumor promoter-induced Epstein–Barr-virus activation detected in each extract with different intensities, which was

(anti-tumor promotion) wherein the IC50 value was 5 µM. envisaged to be responsible for the possessed activities. Significant antischistosomal effect (200 mg/ml) was exhib- The cytotoxic effects of root extracts of E. longifolia have ited by longilactone, while 11-dehydroklaineanone and also been worked out by Abd Razak et al. [46]. They reported 15β-O-acetyl-14-hydroxyklaineanone showed potent plas- that the extract of E. longifolia is found to be cytotoxic with modicidal activity (IC50 = 2 mg/ml). IC50 of 11 μg/ml and 13 μg/ml on Hep2 and HFL1 cell lines Kuo et al. [23] isolated and identified nearly 65 compounds respectively and that the combined extracts of E. longifolia from the roots of E. longifolia and screened them for the and Hunteria zeylanica are more cytotoxic than the single potential cytotoxicity, anti-HIV and antimalarial activities by extract on Hep2 cell lines. in vitro assays. Among the compounds evaluated, nearly eight of them demonstrated strong cytotoxicity toward human lung 4.2. Anti-diabetic properties cancer (A-549) cell lines, while seven of them exhibited strong cytoxicity towards human breast cancer (MCF-7) cell Traditionally, daily consumption of Tongkat Ali leaves and lines. Two of the compounds displayed potent antimalarial roots has been belied to control blood sugar levels. However, activity against the resistant P. falciparum. However, none of the scientific information available on this aspect relevant to the tested compounds showed significant anti-HIV effects. human model is very scarce, indicating the need for The results of these observations have definitely opened elaborative research works to be undertaken in the near up the chances for researchers to evaluate the effects of future. Tongkat Ali root extract to look for the potential anti- Husen et al. [47] have reported the possible hyperglycae- cancerous activity against other common cancers. mic activity of E. longifolia in the rat model system. They Several reports are available on the possible inhibitory of screened the aqueous extracts (50, 100 and 150 mg/kg BW) cancer by the extracts of E. longifolia. The quassinoids isolated of freeze dried and spray dried samples of E. longifolia to from E. longifolia have been studied for their in vitro determine the blood glucose lowering effect in normo- cytotoxicities against KB cells derived from human epider- glycaemic and Streptozotocin-induced hyperglycaemic rats. moid carcinoma of the nasopharynx [40]. Itokawa et al. [41] From the results obtained, the administration of freeze dried isolated a new squalene-type triterpene, named eurylene, and spray dried aqueous extracts revealed positive results in from E. longifolia which were found to be cytotoxic. Chan et al. hyperglyacaemic rats and was high at a concentration of

[42] isolated a new C19 quassinoid6α-hydroxyeurycomalac- 150 mg/kg BW of the aqueous extract was used. However, it tone from the roots of E. longifolia and have reported that the was observed that in the normo-glycaemic rats, no significant cytotoxic activity of the quassinoids from this plant was not reduction to occur after administering the extracts. mediated through DNA cleaving properties. Fractions of E. longifolia extract have also been reported 4.3. Aphrodisiac activities to induce apoptosis in breast cancer cells [43]. Further, Tee et al. [44] elucidated the mode of action of F16 (a plant- The root extracts of E. longifolia has gained wide derived pharmacologically active fraction, inhibited the recognition for its unique feature of enhancing the virility proliferation of MCF-7 human breast cancer cells by and sexual prowess [5], when the prepared decoction is taken inducing apoptotic cell death) and observed that the internally. Even though traditionally, this plant has gained intrinsic apoptotic pathway was invoked, with the reduction wide popularity for its aphrodisiac activities, when it comes of Bcl-2 protein and the apoptosis in the MCF-7 cells was to the consumers it is a necessity to validate the same and accompanied by the specific proteolytic cleavage of poly clear it “as safe for human consumption.” Though certain (ADP-ribose) polymerase-1 (PARP-1) and the caspase-9 and extensive research might be taking place worldwide, still p53 were unchanged with F16 treatment. From their study, there is a wide gap in the scientific information generated they concluded that the F16-induced apoptosis in MCF-7 with reference to the human model system. cells occur independently of caspase-9 and p53 indicating There is a wealth of literature available on the aphrodisiac that F16 from E. longifolia exerts anti-proliferative action activities of E. longifolia roots, wherein it has been adminis- and growth inhibition on MCF-7 cells through apoptosis tered to increase male virility and sexual prowess. Ang and induction and that it may have anticancer properties. Sim [48] evaluated the effects of E. longifolia roots extracts Kuo et al. [13] reported three new [n-pentyl β-carboline- (chloroform, methanol, water and butanol) on the libido of 1-propionate, 5-hydroxymethyl-9-methoxycanthin-6-one, sexually experienced male rats treated with various doses and 1-hydroxy-9-methoxycanthin-6-one along with a (200, 400 and 800 mg/kg BW, 2 times daily for 10 days). Their known β-carboline alkaloids from the roots of E. longifolia, results showed a dose-dependent increase in mounting which showed significant cytotoxicity against human lung frequency of the treated animals with 400 mg/kg of chloro- cancer (A-549) and human breast cancer (MCF-7) cell lines. form, methanol, water and butanol fractions resulting in 676 R. Bhat, A.A. Karim / Fitoterapia 81 (2010) 669–679 mounting frequencies of 5.3+/−1.2, 4.9+/−0.7, 4.8+/−0.7 basal release of testosterone, but increase the human and 5.2+/− 0.1, and 800 mg/kg respectively, with no chorionic gonadotropin (hcG)-induced production of testos- erections, intromissions, ejaculations or seminal emissions terone by rat Leydig cells. during the 20-min observation period. From the obtained As there is not much clinical evidence reported to support results they concluded that E. longifolia is a potent stimulator specific doses of E. longifolia plant extract, this is worth of sexual arousal in sexually vigorous male rats with the exploring under in vivo conditions. absence of feedback from genital sensation. Ang and Ngai [49] in their experiments, studied the aphrodisiac effect of 4.4. Antimicrobial activities fractions of E. longifolia roots (0.5 g/kg) in non-copulator male rats using an electrical cage and were able to support Exploring the possibilities of any herbal plant extract to be the use of the roots for aphrodisiac property. Later on, Ang a potential agent against pathogenic microbes assumes and Lee [50] were able to provide evidence pertaining to importance from a consumer's point of view. changes in sexual behavior (orientation activities) among Farouk and Benafri [55] evaluated various extracts the middle-aged male rats after administering different (methanol, ethanol, acetone and aqueous extracts) from fractions of the E. longifolia root extracts. They reported that different parts of E. longifolia leaves, stem, and roots for the male rats treated with 800 mg/kg of E. longifolia increased possible antibacterial activity against Gram-positive and orientation activities towards the receptive females (ano- Gram-negative bacteria. From their study, they reported that genital sniffing, licking and mounting) accompanied with the extracts of leaves and stem were active on both Gram- the increased genital grooming towards themselves and positive and Gram-negative bacteria, except for the 2 strains restricted movements to a particular area of the cage. of Gram-negative bacteria (Escherichia coli and Salmonella However, it was also noticed that there was a decrease typhi). Their studies also revealed that aqueous leaves interest in the external environment (climbing, raring, and extract to possess antibacterial activity against Staphylococcus exploration) compared with the normal rats. aureus and Serratia marscesens. However, surprisingly the root Sexual motivation activity in adult, middle-aged male extracts did not show any antibacterial activity. mice and in retired breeders, after administering E. longifolia However, based on the availability of the reports, still a root extract (500 mg/kg in chloroform, methanol, butanol, wide gap persists in looking for the antibacterial activities and water; for control, 3 ml/kg of normal saline+30 mg/kg of against other pathogenic microorganisms (such as Listeria yohimbine daily for 10 d) have been reported by Ang et al. monocytogenes or other pathogenic fungi). Hence, further [51]. Their results showed a momentary increase in the studies in this regard are warranted. percent of male mice responding after consumption of the fractions with 50% of the adult middle-aged male mice 5. Quality and safety (toxicology, contaminants and treated with E. longifolia. side effects) Ang et al. [52] investigated the effects of different fractions of E. longifolia roots on sexual qualities in middle-aged male The major drawback of employing traditional medicines is rats using an electrical copulation cage (after treating 0.5 g/kg the lack of adequate supporting scientific information on the of various fractions of extract wherein the control group levels of safety, quality and toxicity associated. To our received 3 ml/kg of normal saline daily for 12 weeks). Their knowledge, at present, there are no available reports on the results revealed that after treatment the sexual qualities of purity (in terms of quality and safety) and on the side effects the middle-aged male rats were enhanced accompanied by a of long term use of the products prepared from the Tongkat decrease in their hesitation time as compared to controls with Ali plant. Also, the plant extracts used for commercial various fractions. It was also noticed that there was a fleeting preparations might not meet the standard criteria regarding enhancement in the percent of the male rats responding the concentration of the bioactive constituents, as there to the right choice after chronic administration of 0.5 g/kg mightbewidevariationswithregardtoage,growth E. longifolia extract, with more than 50% of the male rats scoring conditions, and plant source. the right choice after 2 weeks post-treatment and the effect Today, with the increasing concern over the use of was more prominent at the dose of the observation period. chemical preservatives and the banned pesticides and Ang et al. [53] assessed sexual arousal in sexually sluggish fertilizers for cultivation of herbal plants, it is highly advisable old male rats, (24 months old, retired breeders) administered to grow Tongkat Ali plant in an organic way. This is of utmost with 200, 400, or 800 mg/kg of various fractions of E. longifolia importance as roots are the major ingredient used for extract (2 times daily, for 10 days) with control rats receiving therapeutic purposes, and are capable of readily absorbing 3 ml/kg of normal saline. They studied the aphrodisiac effects and accumulating the toxic ingredients (e.g., heavy metals, by visualizing the act of yawning and stretching (alone or natural radionuclide, etc.) from the soil [4,56,57]. However, with stretching) which has been considered as identity for there is a limited research undertaken in this area with sexual arousal. Their results indicated that rats administered reference to Tongkat Ali plant species, which needs to be with the extract at various doses increased yawning and envisaged immediately, along with the practicing and stretching, which was highest at the administered dose of implementation of GAP, GMP and GSPs (Good Agricultural, 800 mg/kg of E. longifolia extract increased yawning by 50% Good Manufacturing, and Good Storage practices) with an and stretching by 16.7% in sexually sluggish old male rats HACCP approach to meet international standards. supporting the aphrodisiac activity. Chan and Choo [58] reported the toxicity of some However, Lin et al. [54] have reported that the crude quassinoids isolated from the roots of E. longifolia. In their ethanolic extract of the root of E. longifolia could decrease the study, 50% aqueous ethanol extract was partitioned with R. Bhat, A.A. Karim / Fitoterapia 81 (2010) 669–679 677 diethyl ether, n-butanol and water, and acute toxicity of each wherein myocotoxins have been encountered in herbal drugs fraction was evaluated on mice (administered orally) and during storage [63,64]. Hence, this aspect is highly essential with brine shrimps. Their results showed n-butanol fraction and needs to be attained for commercial exploitation of the to be the most toxic one and the responsible compound was plant and for safety reasons. identified to be as eurycomanone. Also, 13,21-Dihydroeur- ycomanone, eurycomanol, longilactone, 14,15 beta-dihydrox- 6. Conclusions yklaineanone and eurycomanol-2-O-beta-glucopyranoside showed toxicity which were found to be less toxic on brine It is evident from the available literatures that E. longifolia shrimps. Jack, possesses adequate therapeutic potential and could be Satayavivad et al. [59] in one of their communications, explored further for commercial purposes, and nevertheless have recommended that Tongkat Ali should be advocated could be designated as a “wonder drug plant.” However, only orally, as other means (direct administration by certain features like overcoming international barriers for injecting, etc.) could enhance the toxicity by approximately quality and safety needs to be addressed appropriately. Also, 100-fold. In one of their studies [59] they have reported the as most of the scientific reports are based on animal toxicity effects to be at 430 mg/kg daily and a daily dose level experiments, human trials might throw more insight onto of 270–350 mg/kg was found to be rather safe. The sub-acute the long term safety concerns of consuming the plant extract. signs of toxicity were correlated to the increased weight of As the plant E. longifolia is quite endangered, measures liver, kidneys, spleen, and testes. have to be taken to preserve the natural germplasm of this Contamination of herbal products by heavy metals is also plant. Application of tissue culture techniques might prove of grave concern owing to its side effects. There is a scarcity of to be beneficial for mass multiplication of this plant and for data pertaining to authentic contamination rates of Tongkat commercial exploitation. Development of suitable genetic Ali products by toxic heavy metals (e.g., Al, Cr, Pb, Hg, etc.). markers might prove to be beneficial for propagation and However, higher contamination above the permissible limits breeding programs to support conservation of various by lead has been reported by Ang and Lee [60]. From a total of species of ‘Tongkat Ali.’ In this regard, excellent works 100 products of Tongkat Ali analyzed, 17% of the products have been initiated by Osman et al. [65] wherein a genome contained 10.3–20.3 ppm of lead, and did not comply with complexity reduction strategy was applied to identify a the quality requirement for traditional medicines in Malaysia. series of single nucleotide polymorphisms (SNPs) within the Earlier, Ang et al. [61] evaluated a total of 100 products genomes of several E. longifolia accessions and were able to containing Tongkat Ali at different pharmaceutical doses for identify that the presence of these SNPs could reflect the mercury contamination. Accordingly they reported that 36% geographic origins of individual plants, and between of the products possessed 0.52 to 5.30 ppm of mercury and different natural populations. They also opined that the did not comply with the quality requirement for traditional SNP markers developed can be useful for identifying genetic medicines in Malaysia. Ang and Lee [62] also reported that the fingerprints, which can correlate with other properties of E. level of mercury contamination in some of the registered longifolia, like high regenerability or the appearance of Tongkat Ali hitam preparations available at various dosages bioactive metabolites. (a total of 100 products in the form of capsules, tablet, sachet Additionally, employing new modern techniques, which and pill) obtained in Malaysia revealed that 26% of the are more economical for better extraction of the required products possessed 0.53–2.35 ppm of mercury, and were phytochemicals, needs to be envisaged. In this regard, above the permissible level found in herbal medicines, and Athimulam et al. [66] have reported the possible use of a did not meet the quality criteria for traditional medicines in batch process simulator for modelling and optimizing the Malaysia. process for Tongkat Ali water extract, the production of which In general, studies have revealed that consumption of the was based on an existing pilot scale manufacturing setup. parts of E. longifolia is known to enhance the testosterone Three debottlenecking schemes were proposed by them levels. However, the safety of employing the extracts by wherein analysis was performed through simulation. They pregnant women, and men with cardio-vascular diseases still reported a final alternative scheme to achieve a product yield needs to be answered. Long term consumption of the root of 3%, with an annual production of 1137.72 kg of Tongkat Ali extract has been correlated with sleep disturbances or sleep extract with a reduced minimum batch cycle time (from apnoea, hot-headiness with facial flushing, pressure in the 24.44 h in the base case to 8.32 h). Their economic analysis testicles in men, and hyper-aggressiveness. However, there is determined that the proposed alternative production scheme a lack of scientific evidence for this, wherein research needs to have an annual revenue of $6.32 M, with an 86% gross to be pursued in the near future. It is also worthwhile margin and a 55% return on investment (ROI). investigating and standardizing the exact dosage and the time Development and application of new genetic based techni- duration for prescribing the plant extract, either directly or as ques for authentic identification of ‘Tongkat Ali’ plant is also one in capsule forms. of the essential areas that have to be explored. This might be Further, there are no authentic records on the microbial beneficial to minimize the mis-identification of the species, and contaminants in this plant or their products either after fresh also to verify the authentic presence of the active herbal harvest or during storage. Microbial toxins are one of the ingredient in the market samples. Abdul Rahman et al. [67] major reasons for the rapid outbreak of human illness, and have developed a microcontroller-based electronic taste some of the secondary metabolites are also considered to be sensing system capable of discriminating between liquid potential carcinogens (e.g., aflatoxins, ochartoxins, fumoni- samples containing E. longifolia and those that do not. sins, zearalenone, etc.). Of late, several reports are available Application of this technology at market level might be 678 R. Bhat, A.A. Karim / Fitoterapia 81 (2010) 669–679 beneficial to identify and authentisize the samples. 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