Contamination with heavy metals in other tongkat ali is associated with risks of severe nerve and brain damage     . Our tongkat ali may be a little more expensive, but it is harvested in jungles far from any civilisation and certified free of heavy metals. Read more here
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5 Metal toxicity in the central nervous system., Environ Health Perspect. 1987 Nov; 75: 59?64.
HPLC coupled on-line to ESI-MS and a DPPH-based assay for the rapid identification of anti-oxidants in Butea superba
A reversed-phase HPLC coupled on-line to a radical scavenging detection system and MS/MS was developed in order to combine separation, activity determination and structural identification of anti-oxidants in complex mixtures in one run. The sample was separated by HPLC and the eluate split into two flows. The major portion was fed into an electrospray ionisation MS/MS system, while the minor part was mixed with a free radical, 2,2′-diphenyl-1-picrylhydrazyl (DPPH), and the reaction determined spectrophotometrically. The negative peaks, which indicated the presence of anti-oxidant activity, were monitored by measuring the decrease in absorbance at 517 nm. The developed method was successfully applied to the identification of anti-oxidant compounds in a fraction, obtained by solid-phase extraction, of an extract of a Thai medicinal plant, Butea superba Roxb. The anti-oxidant compounds were separated and identified as procyanidin B2, (−)-epicatechin and procyanidin B5. Copyright © 2005 John Wiley & Sons, Ltd.
A number of plants are reported to have anti-oxidantproperties (Rajalakshmi and Narasimhan, 1995). Severalstudies have suggested that anti-oxidant compounds canprevent or be used against chronic diseases associatedwith ageing such as cancer, cardiovascular diseases,cataracts, immune system decline and brain dysfunction(Block et al., 1992; Ames et al., 1993; Vinson et al., 1995).For this reason, the isolation and determination ofthe chemical structures of the anti-oxidants is veryimportant in order to apply them as therapeutic com-pounds. Following the recent advances in hyphenatedtechniques, HPLC-MS/MS rapidly provides structuralinformation leading to the partial, or even complete,on-line structure determination of a compound ofinterest (Wolfender et al., 1998). As a complement tothis approach, bioassays performed on-line can permitefﬁcient determination of the bioactivity associatedwith the peaks in the chromatograms.In the investigation of bioactive compounds from plantmaterial, the presence of hundreds of compounds withdifferent types of structures makes their separation andfast identiﬁcation, even on a tentative basis, almost im-possible. Several attempts have been made to acceleratethe isolation, identiﬁcation and bioactivity evaluationprocesses. In recent years, on-line coupling of separationand activity determination techniques has been achievedby mixing a solution of a free radical with the eluate of an HPLC column (Dapkevicious et al., 1999; Kolevaet al., 2000, 2001; Bandoniene and Murkovic, 2002a,b;Pukalskas et al., 2002; Kosar et al., 2003). Such techniquesallow for a rapid and selective detection of radicalscavenging substances in the presence of many inactiveconstituents with a minimum of sample preparation.Koleva et al. (2000) presented an on-line HPLC methodwhereby analytes, separated by HPLC, were reactedpost-column with the free radical, 2,2′-diphenyl-1-picrylhydrazyl (DPPH). The removal of the radicalresults in bleaching of the colour of the reagent andthis can be detected as a negative signal at 515–517 nm.This method has been applied to evaluate anti-oxidantcompounds in fruit and vegetables (Bandoniene andMurkovic, 2002a,b; Pukalskas et al., 2002; Kosar et al.,2003).In the present study, the aim was to couple the HPLC-DPPH method on-line to MS/MS for the analysis of theanti-oxidant compounds in plant extracts. This techniquewould permit the rapid determination of anti-oxidantactivity and provide structural identiﬁcation of the anti-oxidant compounds involved. The Thai medicinal plant,Butea superba Roxb. or ‘Kwao Kreu Daeng’ (Smitinand,1989) was used as the test material as it showed highactivity in off-line anti-oxidant activity screening. Thetubers of this plant have been used traditionally as atonic and rejuvenating agent (Loung-Anusarnsoontorn,1931). Flavonoids and ﬂavonoid glycosides isolated fromthe tuber of this plant were shown to inhibit cAMPphosphodiesterase activity (Roengsumran et al., 2000).The stem was also reported to contain ﬂavone glycosides(Yadava and Reddy, 1998a) and ﬂavonol glycosidesexhibiting anti-microbial activities (Yadava and Reddy,1998b). However, the free radical scavenging activity ofthe plant has not been reported before.