A Simple Isocratic HPLC Method for the Simultaneous Determination of Sinensetin, Eupatorin, and 3′-hydroxy-5,6,7,4′-tetramethoxyflavone in Orthosiphon stamineus Extracts

Abstract

Orthosiphon stamineus extracts contain three flavonoids (3'-hydroxy-5,6,7,4'-tetramethoxyflavone, sinensetin, and eupatorin) as bioactive substances. Previous reported high performance liquid chromatography- ultraviolet (HPLC-UV) methods for the determination of these flavonoids have several disadvantages, including unsatisfactory separation times and not being well validated according to International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) standard guidelines. A rapid, specific reversed-phase HPLC method with isocratic elution of acetonitrile: isopropyl alcohol: 20mM phosphate buffer (NaH(2)PO(4)) (30:15:55, v/v) (pH 3.5) at a flow-rate of 1ml/minute, a column temperature of 25°C, and ultraviolet (UV) detection at 340 nm was developed. The method was validated and applied for quantification of different types of O stamineus extracts and fractions. The method allowed simultaneous determination of 3'-hydroxy-5,6,7,4'-tetramethoxyflavone, sinensetin, and eupatorin in the concentration range of 0.03052-250 μg/ml. The limits of detection and quantification, respectively, were 0.0076 and 0.061 μg/ml for 3'-hydroxy-5,6,7,4'-tetramethoxyflavone, 0.0153 and 0.122 μg/ml for sinensetin and 0.0305 and 0.122 μg/ml for eupatorin. The percent relative standard deviation (% RSD) values for intraday were 0.048-0.368, 0.025-0.135, and 0.05-0.476 for 3'-hydroxy-5,6,7,4'-tetramethoxyflavone, sinensetin, and eupatorin, respectively, and those for intraday precision were 0.333-1.688, 0.722-1.055, and 0.548-1.819, respectively. The accuracy for intraday were 91.25%-103.38%, 94.32%-109.56%, and 92.85%-109.70% for 3'-hydroxy-5,6,7,4'-tetramethoxyflavone, sinensetin, and eupatorin, respectively, and those for interday accuracy were 97.49%-103.92%, 103.58%-104.57%, and 103.9%-105.33%, respectively. The method was found to be simple, accurate and precise and is recommended for routine quality control analysis of O stamineus extract containing the three flavonoids as the principle components in the extract.

Orthosiphon stamineus protects Caenorhabditis elegans against Staphylococcus aureus infection through immunomodulation

“…Several active chemical constituents that are prominently present in this plant are flavonoids (sinensetin and eupatorin) (Laavola et al, 2012; Yam et al, 2012) and a caffeic acid derivative (rosmarinic acid) (Olah et al, 2003; Pan et al, 2011). The chemical structures of sinensetin, eupatorin and rosmarinic acid are presented in supplementary material Fig.…”

“…Interference with bacterial quorum sensing and biofilm production also appears to represent a promising intervention strategy (Wu et al, 2004; Balaban et al, 2007). At this juncture, we cannot rule out the possibility that UE-12 extract may also affect bacterial virulence as the plant extract contains numerous active constituents (Yam et al, 2012). It has been reported that plants possess compounds that can attenuate the virulence of a pathogen by inhibiting virulence factors including quorum sensing and biofilm formation (Adonizio et al, 2008; Rudrappa and Bais, 2008).…”

Discovery of potential anti-infectives against Staphylococcus aureus using a Caenorhabditis elegans infection model

“…On the other hand, O. stamineus leaf extract is widely used as an herbal remedy for kidney and urinary tract disorders. Previous studies revealed the anti-oxidant [51,52], anti-apoptotic [51], hepatoprotective [52] and gastroprotective [53] properties and also identified the major active fractions include eupatorin, sinensetin, 3′-hydroxy-5, 6, 7,4′-tetramethoxyflavone [54] and rosmarinic acid [55]. N. fruticans and O. stamineus extracts enabled the host to survive both antibiotic-sensitive and antibiotic-resistant S. aureus infection without interfering with bacterial viability, suggesting that these extracts possess compounds capable of targeting the host defense mechanism or attenuating bacterial virulence traits.…”

Potent α-glucosidase and α-amylase inhibitory activities of standardized 50% ethanolic extracts and sinensetin from Orthosiphon stamineus Benth as anti-diabetic mechanism

“…The total amounts of 3′hydroxy-5,6,7,4′-tetramethoxyflavone, sinensetin and eupatorin (purchase from Indofine Chemical Company, NJ, USA) in the 50% ethanol extract of O. stamineus were quantified using developed HPLC method (n = 3). The contents of these three compounds were expressed as percentages of the dried extract [18]. …”