Detection and Quantification of Eurycomanone Levels in Tongkat Ali Herbal Products

MATERIALS AND METHODS

Sample sourcing: A total of 41 Tongkat Ali products, in various pharmaceutical dosage forms, both registered and unregistered with NPCB Malaysia, were sourced from Malaysia and international market. Any products bearing the scientific (Eurycoma longifolia) and common name (Tongkat Ali) were chosen randomly from the market. Those products were categorized into; Malaysian Registered Products (MRP), Malaysian Unregistered Products (MUP) and International Products (IP). Two herbal products without Tongkat Ali but were promoted as having aphrodisiac effect, were bought as negative controls. Eight samples of premixed coffee, allegedly containing Tongkat Ali were sourced from various herbal outlets in Kuantan, Pahang. Tongkat Ali root sample was sourced from Bentong Pahang. The morphological comparison of the plant was done and found to be similar with the previous deposit at Forest Research Institute of Malaysia (FRIM) having voucher number of MTA 0001.

Water extraction of Tongkat Ali: Fifty gram of root chips were put in 600 mL deionized water. The sample then was boiled under reflux for 5 h, followed by filtration with Whatman No 1 filter paper and freeze drying process.

HPLC analysis: HPLC (Waters) with photodiode array was utilized and XBridge column (Supelcosil 5 μm, 250×4.6 mm) was used. The mobile phase consisted of isocratic mixture of water and acetonitrile (86:14) with a flow rate of 0.8 mL min^-1. Five milligram of each sample were dissolved in 1.5 mL water and filtered through 0.45 μm nylon membrane filter. Ten microliter of each sample was injected into the HPLC system. Analysis of each samples were done in triplicate. Limit of Detection (LOD) and Limit Of Quantification (LOQ) for eurycomanone standard were calculated from eurycomanone standard calibration curve in Microsoft Excel.

Eurycomanone standard preparation: Five milligram eurycomanone standard (ChromaDex@, USA) was dissolved in 2 mL deionized water. The stock solution then diluted serially to produce 0.5, 0.25, 0.125, 0.0625 and 0.01325 mg mL^-1 solution, respectively. Contents of eurycomanone were determined from calibration curve generated from the eurycomanone standard compounds.

RESULTS

A total of 41 products were analyzed for eurycomanone content using HPLC. Eurycomanone level in all products were calculated based on calibration curve of eurycomanone standard in Fig. 1. Figure 2, 3 and 4 show the chromatograms of eurycomanone standard, freeze dried Tongkat Ali extract and product without any Tongkat Ali, respectively. LOD and LOQ for eurycomanone in this analysis are 0.0227 and 0.0690, respectively. HPLC analysis of all 41 products in this study showed data that is well above the LOD and LOQ of eurycomanone. Table 1 shows that 24 of these products contain eurycomanone while 17 without. From Table 2, it can be observed that freeze dried Tongkat Ali extracts in this study has the highest content of eurycomanone than the rest of the products. Malaysian Standard (2011) stated that the level of eurycomanone should be from 0.8-1.5 w/v (%). Out of the 24 products that contain eurycomanone, 11 of the products have eurycomanone level well above the criteria set by Malaysian Standard.

Fig. 1:	Calibration curve of eurycomanone standard

Fig. 2:	HPLC chromatogram of eurycomanone standard

Fig. 3:	HPLC chromatogram of freeze dried Tongkat Ali extracts

Table 1:	Eurycomanone level in Tongkat Ali products
C*: Product code, MRPMalaysian Registered Product, MUPMalaysian Unregistered Product, IPInternational product, nd: not detected

Fig. 4:	HPLC chromatogram of a product without any eurycomanone.

Table 2:	Summary of products with (+) and without (-) eurycomanone

No eurycomanone were detected in nine samples of the premixed coffees (data not shown).

DISCUSSION

Currently, regulatory control on herbal products ensures that herbal products are free from contaminant such as microbes and heavy metal (Poison Act 1952, Revised 1989), (http://www.bpfk.gov.my/newst.html) but does not take into account the quality of a product. Due to different geographical factor, extraction and processing method, quality of products may differ. People buy herbal products due to the assumed healing power and health enhancing property of particular herbs and quality of herbal products might influence its efficacy. This situation called for the quality of herbal products to be checked. It is also to prevent unscrupulous manufacturers from taking advantage of the demand towards Tongkat Ali by producing fake products. The production of herbal remedies is not controlled or regulated and quality assurance of testing for herbal adulteration is a novel task (Bogusz et al., 2006). There are several publications (Abdul Rahman et al., 2004; Ali et al., 2005; Ang and Lee, 2006; Islam et al., 2006; Razak and Aidoo, 2011; Venhuis et al., 2012) that highlighted the quality and safety of herbal products.

41 samples were analysed in this study. There were 16 Malaysian Registered Products (MRP), 13 Malaysian Unregistered Products (MUP) and 12 International Products (IP). Briefly, 70% of the samples in this study were acquired in Malaysia, while 30% were acquired internationally. The large portion of Malaysian products in this study is partly because Tongkat Ali is indigenous to Malaysia (Bhat and Karim, 2010) and it can be said that Malaysia is one of the major producer of Tongkat Ali products, so the sampling could signify the products that are consumed by people worldwide. It appears to be valid to adapt Malaysian Standard regulatory parameter due to similar reason.

Eurycomanone level was chosen as regulatory parameter in this study because it is the major compound in Tongkat Ali, thus it can signify the level of Tongkat Ali in a product. Furthermore, the analysis utilised High Performance Liquid Chromatography (HPLC), which is simple, highly sensitive and automated equipment.

It is interesting to note that only 4 (C14, C34, C40 and C41) out of 12 international products contain Tongkat Ali. These products were acquired from a reputable online selling website (amazon.com) and it can be said that most people worldwide acquire Tongkat Ali products from this website. It shows here that regulating the quality of Tongkat Ali product worldwide using eurycomanone can be a good indicator on the quality of Tongkat Ali products.

Some products (C11, C19 and C16) showed high level of eurycomanone even though the product did not register with Drug Control Authority (DCA), Ministry of Health (MOH) of Malaysia. This is partly because the processing steps of Tongkat Ali product are so simple, even the cottage industry can get involve easily and probably they do not aware of the necessities to register their product.

It can be observed that some of the products (C3, C8, C9, C20, C25 and C35) were devoid of eurycomanone even though the products have been registered with herbal regulatory body. Thus it can be generalized that product registration offers assurance on product safety but not the quality. It is a major concern when these products that have been registered with DCA, MOH, Malaysia but did not possess any eurycomanone. Although the products have passed the safety aspect set by MOH, it can be said that the product were low in quality as they did not contain any eurycomanone whereby eurycomanone can be indicative of the presence of Tongkat Ali altogether.

Table 2 shows that 6 (C7, C2, C29, C41, C19 and C14) out of 10 products with the highest eurycomanone content consist of products which contain solely Tongkat Ali. It can be generalized that single formulation has been found with higher amount of eurycomanone compared to those mixed herbal products.

CONCLUSION

The eurycomanone level in 41 products was quantified and 24 viz 58% of these products contain eurycomanone while 17 viz 42% of did not contain any eurycomanone. The 14 of these products complied with the eurycomanone level set by Malaysian Standard (0.8-1.5 w/v (%)) where the eurycomanone level was from 0.99-8.48 w/v (%) while 10 of these products did not comply with the level stated where the amount were below 0.8 w/v (%). It is recommended that regulatory method applying eurycomanone level in determining the quality of Tongkat to be utilized worldwide. MS 2409: 2011 states that the level of eurycomanone should be around 0.8-1.5% (w/v/). However, out of 24 products that contain eurycomanone, 11 of them had eurycomanone level higher than 1.5% (w/v). So to further increase the quality of Tongkat Ali products, it can be proposed that the standard may be raised as most of current products in the market have reached the current level easily.

ACKNOWLEDGMENT

The authors would like to acknowledge University Malaysia Pahang for providing financial support in the form of grants bearing internal numbers of RDU130396, RDU140305 and GRS120332.