4quercetin-toralba.pmd RP-HPLC Analysis of Quercetin in the Extract of Sambong 48 SCIENCE DILIMAN (JANUARY-JUNE) 27:1, 48-63 RP-HPLC Analysis of Quercetin in the Extract of Sambong (Bl umea bal samifera (L) DC) Leaves Joanna V. Toralba* University of the Philippines Manila Noel S. Quiming University of the Philippines Manila Jocelyn SB Palacpac University of the Philippines Manila _______________ *Corresponding Author ISSN 0115-7809 Print / ISSN 2012-0818 Online ABSTRACT Blumea balsamifera (L) DC, known in the Philippines as sambong, is an h e r b v a l u ed fo r i t s h e a l t h b e n e f i t s e s p ec i a l l y i n t h e m a n a g e m e n t of u r o l i t h i a s i s . Va r i o u s p h y t o c h e m i c a l s , i n c l u d i n g f l a v o n o i d s s u c h a s quercetin, have been determined in sambong leaves. A reversed-phase h i g h - p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h i c m e t h o d ( R P - H P LC ) w a s developed for the quantitative determination of quercetin in the methanol ex t r a c t of s a m b o n g l e a ve s o b t a i n ed f r o m Ley te, Co t a b a to, a n d N u ev a Ecija, Philippines. The methanol extracts of sambong were prepared by maceration followed by rotary evaporation. The solid phase extraction (SPE) for the sample cleanup involved the use of a C18 SPE packing, a 0.5-mL sample load (50 mg/mL solution), and elution with 4-mL of 80:20 M e t h a n o l : 0 . 5 % H 3 PO 4 . T h e H P LC c o n d i t i o n s f o r t h e d e te r m i n a t i o n of q u e r c e t i n i n v o l v e d t h e u s e o f a C 1 8 4 . 6 - m m x 2 5 0 - m m c o l u m n m a i n t a i n e d a t 3 0 ° C , 2 5 4 - n m U V d e t e c t i o n , a n d a m o b i l e p h a s e composition of 25 parts methanol and 75 parts mixture of 0.5% H 3 PO 4 and 0.2% triethylamine with a 1 mL/min flow rate in gradient elution. A g o o d l i n e a r i t y a t t h e c o n c e n t r a t i o n r a n g e o f 3 . 7 2 – 1 2 4 µ g / m L o f quercetin standard (r2=0.9989) was observed with the limits of detection (LOD) and quantitation (LOQ) at 0.68 ng/mL and 2.28 ng/mL, respectively. T h e i n t r a - d a y ( n = 5 - ) a n d i n t e r - d a y ( n = 3 ) p r e c i s i o n v a l u e s w e r e s a t i s f a c t o r y ( % R S D < 2 % ) . T h e r ecov e r y e f f i c i e n cy of t h e S P E s a m p l e c l e a n u p s t e p , w h i c h w a s c h e c k e d b y s p i k i n g s a m b o n g s o l u t i o n w i t h JV Toralba and others 49 quercetin standard, was 102.41%. The quercetin contents are 0.2337mg, 0 . 1 3 5 0 m g , a n d 0 . 2 9 4 0 m g p e r g r a m o f t h e p o w d e r e d d r i e d l e a v e s o f sambong from Nueva Ecija, Cotabato, and Leyte, respectively. This is the f i r s t r e p o r t of q u e r ce t i n co n te n t i n t h e l e a ve s of s a m b o n g co l l ected from the Philippines. Ke y w o r d s : S a m b o n g , Bl u m e a b a l samifera , Q u e r ce t i n , H P LC, S o l i d P h a s e Extraction LAYMAN’S ABSTRACT Blumea balsamifera (L) DC, known in the Philippines as sambong, is an h e r b v a l u ed fo r i t s h e a l t h b e n e f i t s e s p ec i a l l y i n t h e m a n a g e m e n t of u r o l i t h i a s i s . Va r i o u s p h y t o c h e m i c a l s , i n c l u d i n g f l a v o n o i d s s u c h a s quercetin, have been determined in sambong leaves collected from other Asian countries using instrumental techniques such as high performance liquid chromatography (HPLC). An HPLC method can be used to generate a c h e m i c a l f i n g e r p r i n t u s e f u l i n t h e a c c u r a t e a u t h e n t i c a t i o n a n d identif ication of herbal medicines, in the comparison of plant materials g r o w n i n d i f f e r e n t r e g i o n s , a n d d e t e r m i n a t i o n o f a m o u n t o f t h e chemically characteristic or pharmacologically active components. This s t u d y f o c u s e d o n t h e d e v e l o p m e n t o f a n H P LC m e t h o d f o r t h e d e t e r m i n a t i o n o f q u e r c e t i n i n s a m b o n g l e a v e s c o l l e c t e d f r o m N u e v a E c i j a , Co t a b a to, a n d Ley te. T h e m e t h a n o l ex t r a c t s of s a m b o n g l e a ves were prepared by maceration and by a solid phase extraction technique developed in this study. A reversed phase HPLC method was optimized and used in the determination of quercetin in the prepared extracts. The quercetin contents determined using the developed methods are 0 . 2 3 3 7 m g , 0 . 1 3 5 0 m g , a n d 0 . 2 9 4 0 m g p e r g r a m o f t h e p o w d e r e d d r i e d leaves of sambong from Nueva Ecija, Cotabato, and Leyte, respectively. This is the f irst repor t of quercetin content in the leaves of sambong collected from the Philippines. INTRODUCTION Blumea balsamifera (L) DC, commonly known in the Philippines as sambong, is an herb that can grow anywhere in the Philippines and is also abundant in other Asian countries like India, China, and Malaysia (Quisumbing 1978). The leaves of sambong are extensively used in folk medicine to address various conditions including arthritis, rheumatism, headache, chest pains, diarrhea, dysentery, stomach pain, cough, and fever relief. The decoction is used as a diuretic in edema, in expelling kidney RP-HPLC Analysis of Quercetin in the Extract of Sambong 50 stones, and in the management of urolithiasis (Quisumbing 1978; DOH BFAD 2005). The Philippine National Drug Formulary (PNDF) Essential Drugs List (DOH-NFC 2005) includes sambong in its list of drugs under diuretics, taken orally as a 250 mg or as a 500 mg tablet. Alternative preparations include capsules and bags for infusion. The leaf infusions and dosage forms were used to provide evidence of the pharmacologic activities of B balsamifera including intraocular pressure lowering effect (Arroyo and others 1990), antimutagenic activity (Lim-Sylianco and others 1987), and dissolution of calcium/urinary tract stones (Rico 1992). Phytochemical investigation of sambong leaves reveals a diverse list of chemical constituents present which includes the flavonoids. The high amount of flavonoid content of the crude methanol extracts was attributed for the xanthine oxidase inhibitory and antioxidant activities of B. balsamifera (Fazilutan and others 2003; Fazilutan and others 2004). Among the flavonoids found in the leaves of sambong are quercetin, velutin, luteolin, luteolin-7-methyl ether, rhamnetin, tamarixetin, ombuin, 3,3’-dimethyoxyquercetin, 3,7-dimethylquercetin, quercetin-3,7,3.-trimethyl e t h e r, 3 , 7, 4 ’- t r i m e t h y l q u e r ce t i n , p e r s i co g e n i n , a n d 3 ’, 4 ’, 5 - t r i h yd r oxy - 3 , 6 , 7- trimethoxyflavone (Barua and Sharma 1992; Fazilutan and others 2004; Ali and others 2005). The use of HPLC has been documented for the determination of flavonoids in extracts of plant materials and food products (Hertog and others 1993; Crozier and others 1997; Zu and others 2006; Andres-Lacueva and others 2008; Lin and others 2008). The research of Fazilutan and others (2005) focused on the HPLC quantitative determination of f ive major flavonoids in the methanol extract of sambong leaves from f ive different sources in Malaysia. The flavonoids included in their study are q u e r ce t i n , d i h yd r o q u e r ce t i n - 7, 4 '- d i m e t h y l e t h e r, b l u m e a t i n , 5 , 7, 3 ', 5 '- te t r a - hydroxyflavanone, and dihydroquercetin-4'- methyl ether. An HPLC method can be used to generate a chemical f ingerprint useful in the accurate authentication and identif ication of herbal medicines, in the comparison of plant materials grown in different regions, and the determination of amount of the chemically characteristic or pharmacologically active components. In this study, HPLC was used in the determination of quercetin (Figure 1) in the extracts of sambong leaves collected from Leyte, Cotabato, and Nueva Ecija. At the time of the writing of this manuscript, these are the f irst repor ted values of quercetin in sambong samples cultivated in the Philippines. JV Toralba and others 51 MATERIALS AND METHODS Chemicals, Reagents, and Materials Methanol (RCI Labscan® Limited) and acetonitrile (JT Baker®) solvents used were of HPLC grade. The phosphoric acid 85% (RCI Labscan® Limited) and triethylamine (JT Baker®) used were of analytical reagent (AR) grade. Distilled water was used in the aqueous solutions that were prepared. The quercetin standard was from Sigma- Aldrich®. Solvents used as mobile phase were f iltered using a 0.45-µm 47-mm (diameter) nylon membrane f ilter discs (Whatman®) while solutions injected to the HPLC system were f iltered using a 0.45-µm 25-mm (diameter) PuradiscTM 25 NYL nylon membrane with polypropylene housing f ilter (Whatman®). The solid phase extraction (SPE) was done using a 6-mL capacity SPE tube packed with 500-mg octadecylsilane (C-18) packing (Varian® Bond Elut-C18). Instruments The spectrophotometer used in the method developed is a Genesys 10UV Scanning ThermoSpectronic® Spectrophotometer. The HPLC analysis was done using a Perkin- Elmer® S200 HPLC System equipped with a quaternary pump, manual injector, column oven, UV detector, and TotalChrom Workstation® Software. The HPLC column used was a 4.6-mm internal diameter, 250-mm length Brownlee Analytical Perkin- Elmer® column with an octadecylsilane (C1-8) 5-µm particle size packing. Figure 1. Structure of the flavonoid quercetin determined in Blumea balsamifera (L) D C RP-HPLC Analysis of Quercetin in the Extract of Sambong 52 Plant Materials The healthy and mature leaves of sambong were bought from farms located in three provinces of the Philippines: Nueva Ecija, Leyte, and Cotabato. The drying and comminution of the sambong leaves were done in the processing facility of each area. A Certif icate of Analysis for the sambong samples bought from Leyte and Cotabato was provided by Philippine Institute of Traditional and Alternative Heath Care (PITAHC). A voucher specimen for the authentication of the sample from Nueva Ecija has been deposited in the Botany Division of the National Museum. Sample Preparation Crude extraction of 50 grams of powdered dried leaves was done by maceration for 18 hours using 500 mL methanol as solvent, with prior heating to 40°C in a water bath for 6 hours. The extract was collected and f iltered. The f iltrate was dried at 45°C in vacuo using a rotary evaporator. Sample Extraction and Cleanup Different solvent volumes and the sonication times for the preparation of the sample solutions were investigated in this study. The optimized conditions of 50 mg/mL sambong solution in methanol, prepared with the aid of sonication for 15 minutes, were used. Different volumes of methanol extract to be loaded, composition and volume of eluting solvent, and total volume of eluate to be collected were examined. The optimized conditions, described in the succeeding sentences, were used in the sample cleanup. The SPE tube was preconditioned by sequentially passing 6 mL of methanol and 6 mL of 0.5% H 3 PO 4 . A 0.5 mL portion of the sambong solution was loaded to the same SPE tube and eluted with 4.00 mL of 80:20 methanol:0.5% H 3 PO 4 . The eluate was collected. Another 0.5 mL portion of the sambong solution was loaded to another separate SPE tube and treated in the same manner. The eluates of each of the two SPE tubes (about 8 mL) were pooled and diluted to 10.0-mL with methanol. The sambong solution was f iltered through a 0.45-µm nylon membrane f ilter and used in the HPLC analysis. The recovery eff iciency of the SPE as a cleanup method was determined by adding 124 µL of 1.26 mg/mL quercetin standard solution to a 1.00 mL aliquot of a 50.12 JV Toralba and others 53 mg/mL sambong solution. The spiked sambong solution was equally divided and delivered to two SPE tubes as described previously, to achieve a f inal concentration of 15.62 µg/mL of quercetin standard in the pooled eluate. The quercetin standard solution and unspiked sambong solution were prepared in the same manner. The solutions were f iltered and injected to the HPLC system. Quercetin Standard Solution Preparation Standard stock solution (1.24 mg/mL) of quercetin was prepared in methanol. Aliquots of the stock solution were diluted with methanol to prepare six concentrations of quercetin standard solution (3.72-124.00 µg/mL). Solutions were f iltered through a 0.45-µm nylon membrane f ilter and used in the HPLC analysis. Chromatographic Cond itions The conditions that were optimized for the HPLC determination of quercetin were wavelength of detection, and the composition and gradient program of the mobile phase. The optimized procedure, described in the succeeding sentences, were used in the analysis of the sambong solution. Chromatographic separation was carried out using an octadecylsilane (C18) 5 µm particle size stationary phase packed in a 4.6-mm x 250-mm column maintained at 30°C. The UV detector was operated at 254 nm. The volume of solutions injected to the HPLC was 20 µL. The mobile phase was run with a flow rate of 1 mL/min in a gradient elution (Table 1). The mobile phase was f iltered through a 0.45-µm nylon membrane f ilter and sonicated prior to use. The chromatographic peak of quercetin in the sambong solution was conf irmed by comparing the retention time with those of the quercetin standard solutions. Quantif ication was made according to the linear calibration curves of the quercetin standard solutions. Using the data generated by the injection of the six (6) quercetin standard solutions and methanol as blank solution (n=5), an evaluation of the peak areas as a function of the concentration of the solutions was done by calculating the parameters slope, intercept, and coeff icient of correlation. The limit of detection (LOD) and limit of quantitation (LOQ) were computed from the same set of data. The intra-day precision RP-HPLC Analysis of Quercetin in the Extract of Sambong 54 (n=3) for the peak area and retention time of quercetin were reported as % relative standard deviation (%RSD). The intra-day (n=5) and inter-day (n=3) precision of the methods were also tested using the sambong solution and expressed as %RSD. Statistical Analysis The quercetin content of the extracts was expressed as mean ± %RSD. A t-test (p< 0.05) was performed to determine the signif icance of the difference between means of the quercetin content of sambong from the different sources. RESULTS AND DISCUSSIONS Optimization of the Sample Extraction and Cleanup Effect of Solvent and Sonication Different solvent ratios were tested for the preparation of the sample solution from the sambong extracts prepared from maceration and rotary evaporation (Table 2). Although the quercetin standard dissolved in solvents containing 80% or 20% methanol, an incomplete dissolution was observed when these solvents were applied to the sambong extract. Even with the aid of sonication, the amount of insoluble components did not appreciably decrease (Table 3). The sambong solution prepared in pure methanol that was subjected to sonication for 15 minutes was used in this study. *0.5% H 3 PO 4 , 0.2%TEA: This was prepared by adding 1.75 mL of 85% phosphoric acid to water, swirling thoroughly, then adding 1.38 mL of triethylamine (TEA), and diluting to 500.0-mL with water Table 1. Mobile phase composition and grad ient program for the HPLC analysis of solutions T ime (minutes) Mobile Phase Composition Elution Type 0.0 25:75 methanol:0.5% H 3 PO 4 , 0.2%TEA* Gradient 0.0 – 10.0 45:55 methanol:0.5% H 3 PO 4 , 0.2%TEA Gradient 10.0 – 30.0 55:45 methanol:0.5% H 3 PO 4 , 0.2%TEA Gradient 30.0 – 40.0 100% methanol Gradient 40.0 – 50.0 100% methanol Isocratic JV Toralba and others 55 Effect of Vol ume Load ing and El uting Solvent in SPE The SPE cleanup procedure in this study was modif ied from the work of Chen and others (2001) wherein flavonoids and other phenolic compounds in cranberry juice were extracted in a C18 SPE car tridge and analyzed by HPLC. The aim of the SPE in this study was to remove most of the unwanted components including the pigments in the methanol extract while allowing the quercetin to be collected completely. When 0.3 mL and 0.5 mL volumes of 50 mg/mL sambong solution were loaded to separate SPE tubes, no peak corresponding to quercetin retention time was observed in the HPLC analysis of the eluate. However, at 0.8 mL and 1 mL volumes of the same concentration of sambong solution, the eluates showed a peak corresponding to the retention time of quercetin. The eluates of two SPE tubes (about 4 mL each) loaded with 0.5 mL of the sambong solution were pooled to achieve a quantif iable amount in the HPLC analysis. Solvent Description of Solution Observed at 5-minute Time Intervals 0 min 5 min 10 min 15 min 20 min 25 min 100% Small portion Small portion Very small Complete Complete Complete Methanol undissolved undissolved portion solubility solubility solubility undissolved 80:20 Large portion Large portion Large portion Large portion Large portion Large portion Methanol: (lumps) (lumps) (lumps) (lumps) (dispersed) (dispersed) 0.5% H 3 PO 4 undissolved undissolved undissolved undissolved undissolved undissolved 50:50 Large portion Large portion Large portion Large portion Large portion Large portion Methanol: (lumps) (lumps) (lumps) (lumps) (lumps) (dispersed) 0.5% H 3 PO 4 undissolved undissolved undissolved undissolved undissolved undissolved Table 3. Effect of sonication on the solubil ity of sambong extracts in various solvents Table 2. Solubil ity of quercetin standard and sambong extract in d ifferent solvents Solvent Quercetin Standard Sambong Extract 100% Methanol Soluble Soluble 80:20 Methanol:0.5% H 3 PO 4 Soluble; Precipitation was Large portion remained observed after one week insoluble 50:50 Methanol:0.5% H 3 PO 4 Soluble; Precipitation was Large portion of remained observed after two days insoluble 20:80 Methanol:0.5% H 3 PO 4 Insoluble Large portion of remained insoluble 100% 0.5% H 3 PO 4 Insoluble Insoluble RP-HPLC Analysis of Quercetin in the Extract of Sambong 56 Methanol or acetonitrile can remove quercetin completely from the SPE packing. However, the use of pure organic solvents caused the elution of more undesired compounds. The addition of 0.5% H 3 PO 4 to methanol increased the polarity of the eluting solvent which enhanced the solubility of the polar quercetin to the eluting solvent. The sambong solution in the SPE tube was eluted with 4.00 mL of 80:20 methanol: 0.5% H 3 PO 4 . A second 4.00 mL of eluting solvent added to the same SPE tube did not result to additional elution of quercetin. Recovery Eff iciency The eff iciency of extraction of quercetin of the sambong solution treated by SPE was demonstrated by spiking a sambong extract solution with quercetin standard solution and allowing this to pass through the SPE tube (Figure 2). The solutions were analyzed using the optimized HPLC method. A mean recovery of 102.41% (± 1.15) for quercetin was achieved in this study. Figure 2. Overlay of chromatograms of solutions that were treated in the SPE tube in the same manner: (A) methanol; (B) quercetin standard solution (15.62 µg/mL); (C) sambong solution spiked with 15.62 µg/mL quercetin standard; and (D) unspiked sambong solution Optimization of the Chromatographic Cond itions Effect of Wavelength of Detection Using the UV spectrophotometer, a spectral scan from 200 nm to 350 nm of a quercetin standard solution was done. The three wavelengths that registered JV Toralba and others 57 maximum absorbance values were 254 nm, 263 nm, and 274 nm. The wavelength chosen for quercetin determination in this study was 254 nm because quercetin standard exhibited the highest value of area under the curve at this wavelength when tested using the HPLC-UV detector (Table 4). Table 4. Peak area of quercetin standard tested using an HPLC-UV detector Wavelength Mean Peak Area (n=3, ± %RSD) 255 nm 11,342,168.100 ± 2.56 270 nm 7,001,168.213 ± 1.62 360 nm 9,525,722.357 ± 5.27 Effect of Composition and Grad ient Program of the Mobile Phase The HPLC conditions used in this study were modif ied from the work of Fazilutan and others (2005) where f ive major flavonoids, including quercetin, were quantitatively determined in the methanol extract of sambong collected from the different areas in Malaysia. Their study used a C18 (250- x 4.6-mm, 5 ìm particle size) column as the stationary phase and a mobile phase consisting of 50:50 methanol: 0.5% H 3 PO 4 in an isocratic mode and delivered at 0.9 mL/min. The flavonoids were detected at 285 nm. Quercetin, a polar molecule, can be eluted from a C18 stationary phase by using a polar mobile phase. This study explored gradient elution of various proportions of methanol and 0.5% H 3 PO 4 to increase the resolution of quercetin from adjacent peaks and to improve the peak shape of the compound. The flow rate for all tests was held constant at 1.00 mL/min. Eventually, a gradient that starts with 25:75 methanol: 0.5% H 3 PO 4 and the methanol content increasing to 100% in a 50-minute program yielded a satisfactory separation of quercetin (Figure 3A). The addition of triethylamine was also investigated to improve the peak shape of quercetin in the sambong sample. The concentration of triethylamine in the 25:75 methanol: 0.5% H 3 PO 4 mobile phase was varied from 0.1% to 1%. A 0.2% concentration of triethylamine resulted to an acceptable resolution of quercetin from adjacent peaks, as well as an improved peak shape (Figure 3B). Triethylamine, at a suitable concentration, acts as an ion pairing reagent for the stationary phase, which reduced the tailing of quercetin. When the concentration of triethylamine was increased, peak breaking and band broadening resulted (Figure 3C). RP-HPLC Analysis of Quercetin in the Extract of Sambong 58 Figure 3. Chromatograms of sambong solutions run in different mobile phase compositions. The mobile phase composition was changing to 100% methanol in a 50-minute gradient program starting with the following solvent combinations: (A) 25:75 methanol:0.5% H 3 PO 4 ; (B) 25:75 methanol:0.5% H 3 PO 4 with 0.2% triethylamine; and (C) 25:75 methanol:0.5% H 3 PO 4 with 0.5% triethylamine. The following HPLC conditions were then applied in the analysis of quercetin in the sambong solution: C18,5 µm particle size stationary packed in a 4.6-mm x 250-mm column and maintained at 30°C, 254-nm wavelength of detection, 1.00 mL/min mobile phase flow rate, and a mobile phase and gradient condition as outlined in Table 1. Linearity, Limit of Detection and Limit of Quantitation, Precision A series of six quercetin standard solutions (3.72-124.00 µg/mL) was tested to determine the linearity between the concentration and peak areas. A high correlation coeff icient of 0.9989 and regression equation of y=102789x-159435 was JV Toralba and others 59 generated from the HPLC analysis of the quercetin standard. The chromatograms of the six quercetin standard solutions are provided in Figure 4. The limits of detection and quantitation were evaluated on the basis of a signal-to-noise ratio of 3 and 10, respectively. The detection limit was determined as 0.68 ng/mL, while limit of quantitation was at 2.28 ng/mL. This indicates that the HPLC method is suff iciently sensitive for the determination of quercetin in sambong. Figure 4. Overlay of chromatograms of the quercetin standard solutions analyzed using the optimized HPLC method: (A) 124.00 µg/mL; (B) 62.00 µg/mL; (C) 31.00 µg/mL; (D) 15.50 µg/mL; (E) 7.44 µg/mL; and (F) 3.72 µg/mL RP-HPLC Analysis of Quercetin in the Extract of Sambong 60 The %RSD of the peak areas and retention times of the quercetin standards are both quite low which indicated that precision is good (Table 5). The precision was also measured using the peak areas of the quercetin in the sambong solutions. Both the intra-day (%RSD = 1.52%, n=5) and inter-day (%RSD = 1.73%, n=3) precision studies also showed that the method is both repeatable and reproducible. Quantitation of quercetin in sambong leaves The optimized SPE sample preparation and cleanup and HPLC method were applied to the sambong leaves collected from Leyte, Cotabato, and Nueva Ecija. The corresponding chromatograms of the sambong samples are shown in Figure 5. The summary of results on the quantitation is presented in Table 6. The quercetin content ranged from 0.135 to 0.294 mg per gram of powdered dried leaves. At the time of the writing of this article, these are the f irst reported values of quercetin in sambong samples grown in the Philippines. These results are close to the values reported in the study of Fazilutan and others (2005) where quercetin was reported to be present in the range of 0.021 to 0.958 mg per gram of dried B. balsamifera leaves cultivated in the different regions of Malaysia. The quercetin content of sambong from Cotabato differed signif icantly (t-test, p<0.05) from that found in leaves from Nueva Ecija and Leyte. The quercetin content of samples from Leyte and Nueva Ecija did not differ signif icantly from each other. The variation in the quantity of quercetin may be due to various factors such as cultivation conditions (soil, temperature, moisture) and agricultural practices (use of fertilizers and pesticides). Table 5. %RSD of the retention time and peak area response of quercetin standard solutions 124.00 0.07 0.89 62.00 0.13 0.16 31.00 0.65 1.02 15.50 0.88 1.30 7.44 0.64 0.99 3.72 0.21 1.48 Quercetin Standard Concentration (µg/mL) Intra-day %RSD for Retention T ime Intra-day %RSD for Peak Area JV Toralba and others 61 Table 6. Quercetin retention time, peak area, and content as determined by HPLC analysis of the sambong leaves from Leyte, Cotabato, and Nueva Ecija Leyte 20.67 ± 0.65 1,345,645.36 ± 1.27 0.2940 Cotabato 20.59 ± 0.82 698,866.84 ± 1.05 0.1350 Nueva Ecija 20.57 ± 0.80 1,334,535.34 ± 1.24 0.2337 Source Retention T ime (minutes, n=3, mean ±%RSD) Peak Area (Au, n=3, mean ±%RSD Quercetin in mg per gram Powdered Leaves Figure 5. Overlay of chromatograms of the sambong from (A) Leyte, (B) Cotabato, and (C) Nueva Ecija analyzed using the optimized HPLC method ACKNOWLEDGMENTS The main author would like to thank the College of Pharmacy. Appreciation is also extended to Dr. Noel S. Quiming for generously providing the quercetin standard used in this study. RP-HPLC Analysis of Quercetin in the Extract of Sambong 62 REFERENCES Ali DMS, Wong KC, Lim PK. 2005. Flavonoids from Blumea balsamifera. Fitoterapia. 76:128-30. 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JV Toralba and others 63 Lin JK, Weng MS. 2006. Flavonoids as nutraceuticals. In: Grotewold E, editor. The Science of Flavonoids. New York: Springer Science, Inc. p. 213-238. Quisumbing E. 1978. Medicinal plants of the Philippines. Quezon City: Katha Publishing Co. Rico F. 1992. Sambong (Blumea balsamifera): Its effect on calcium stone. Philippine Journal of Urology. 2(1):9-13. Zu Y, Li C, Fu Y, Zhao C. 2006. Simultaneous determination of catechin, rutin, quercetin, kaempferol and isorhamnetin in the extract of sea buckthorn (Hippophae rhamnoides L.) leaves by RP-HPLC with DAD. Journal of Pharmaceutical and Biomedical Analysis. 41(3):714-19. _______________ Joanna V. Toralba is an Assistant Professor of the Department of Pharmaceutical Chemistry, College of Pharmacy, University of the Philippines Manila, where she is currently teaching and advising undergraduate research on pharmaceutical analysis and methods development. She graduated from the University of the Philippines Manila with degrees in BS Pharmacy and MS Pharmacy (Pharmaceutical Chemistry). Noel S. Quiming is an Associate Professor of the Department of Physical Sciences and Mathematics, College of Arts and Sciences, University of the Philippines Manila. He has published articles on chromatography and its application to analysis of natural products in international peer reviewed journals. He received his Doctor of Engineering degree from Toyohashi University of Technology in Japan and MS Chemistry degree from University of the Philippines Diliman. Jocelyn SB Palacpac is an Associate Professor of the Department of Industrial Pharmacy, College of Pharmacy, University of the Philippines Manila, where she served as chair of the Department and as Dean of the College. She is currently the Assistant Director of the Institute of Herbal Medicine, National Institutes of Health, University of the Philippines Manila. She is the project leader of the research project on the formulation of dosage forms of Philippine medicinal plant constituents.