HPLC lavonoid proiles of Bupleurum L. species PHARMACIA, vol. 61, No. 2/2014 17 HPLC FLAVONOID PROFILES OF BUPLEURUM L. SPECIES R. Gevrenova*, N. Denkov, D. Zheleva-Dimitrova * Department of Pharmacognosy, Faculty of Pharmacy, Medical University - Soia, 2, Dunav street, 1000 Soia, Bulgaria Abstract: A reliable solid-phase extraction – high-performance liquid chromatography (SPE-HPLC) method for the determination of lavonoids in European Bupleurum species B. baldense Turra and B. afine Sadler was developed. Extraction of aerial parts by ultrasound with 80% aqueous methanol and fractionation of lavonoids by means of SPE allowed a good recovery of analytes between 99 and 104%. The subsequent HPLC separation of the lavonoids was performed on a Luna C18 column using gradient elution and UV detection. The optimized SPE-HPLC method was validated for precision, linearity, accuracy, recovery, limit of detection and limit of quantiication. The precision of the entire analytical procedure was < 2%. The total amount of assayed lavonoids was 11.53 mg/g dry weight (B. afine) and 39.16 ± 0.92 mg/g (B. baldense). Rutin was the dominant lavonol glycoside in B. baldense being present at 28.63 ± 1.57 mg/g, whilst B.afine demonstrated lower levels of lavonoids. Key Words: Bupleurum afine Sadler, Bupleurum baldense Turra, lavonoids, rutin, narcissin, SPE-HPLC. Introduction Bupleuri radix [roots of Bupleurum L. spp. (Apiaceae)] is one of the most frequently used herbs in Chinese herbal medicine [1]. Ashour and Wink (2011) reviewed the chemistry and pharmacology of the genus Bupleurum [2]. Most of the secondary metabolites isolated from Bupleurum species belong to the classes of phenolics, lignans, terpenoids (triterpenoids and sterols), mono- and sesquiterpenes (essential oils) and polyacetylenes [3-7]. Triterpene saponins (the so called saikosaponins) are considered as the major bioactive compounds of the drugs, mainly used for their anti-inlammatory [8], anti-tumor [9], hepatoprotective [10] and antiviral [11] activities. Flavonoids, including the minor compounds, are widely used as chemotaxonomical markers to distinguish between different Bupleurum species and different geographical sources [12]. Bupleurum species are important ingredients in many multi-herb remedies in traditional Chinese medicine [2]. Recently, few quantitative HPLC-UV and UPLC-PDA methods have been established for simultaneous determination of lavonoids in the aerial parts of several Bupleurum species for quality assessment of the raw materials of traditional Chinese medicines [13, 14, 15]. However, there are only a few studies with respect to the lavonoid content of annual European Bupleurum species and the antioxidant activity of these taxa [16, 17]. Previous investigation of the aerial parts from Bupleurum lavum Forsk. led to the isolation of ive lupane-type triterpenoids, one lignan and eight lavonoids (kaempferol, isokaempferide, gossipetin, quercetin, luteolin, isorhamnetin 3-O-βD-glucopyranoside, isorhamnetin 3-rutinoside, and rutin) [4]. Based on all these studies, we aimed at investigating the lavonoids and their variability in two European Bupleurum species, including B. baldense Turra and B. afine Sadler using a solid-phase extraction – high-performance liquid chromatography (SPE-HPLC) method. Materials and methods Plant material and solid-phase extraction procedure Aerial parts from B. baldense and B. afine were collected in August 2006, respectively, from Topolovgrad region (42°5'4" N – 26°20'12" E) and Radomir region (42°32'20" N – 22°57'31" E) in Bulgaria. These plants were identiied by one of us (R.G.) Air-dried powdered parts of each plant (0.1 g) were extracted with 10 ml 80% methanol (v/v) (× 2) by sonication for 15 min at room temperature. The combined extracts were iltered and the inal volume was made up to 10 ml. SPE was accomplished on VARIAN Vac Elut 10 vacuum manifold using cartridges Bond Elut C18, 500 mg, 3 ml and Bond Elut CN, 500 mg, 3 ml (Varian, CA, USA). The optimized SPE procedure was carried out as follows: 3 ml of the plant sample was diluted with 18 PHARMACIA, vol. 61, No. 2/2014 5 ml phosphate buffer (20 mM KH2PO4, pH 3.2) and was passed through a C18 SPE cartridge, previously conditioned with 2 ml of methanol and with 2 ml of phosphate buffer (20 mM KH2PO4, pH 3.2). The cartridge was washed with 3 ml of phosphate buffer and 3 ml of methanol-phosphate buffer (20:80, v/v) and then the lavonoids were eluted with 1.5 ml of pure methanol. The inal eluates were evaporated under gentle nitrogen stream and the residue was dissolved in 1 ml methanol. Ten μl aliquots of samples were injected into the chromatographic system. Chemicals and reagents The standards of rutin (quercetin 3-rutinoside), narcissin (isorhamnetin 3-rutinoside), isoquercitrin (quercetin 3-glucoside), astragalin (kaempferol 3-glucoside), isorhamnetin 3-glucoside, luteolin, quercetin, kaempferol and isorhamnetin were purchased from Extrasynthese (Genay, France); kaempferid was provided by Fluka (Buchs, Germany). HPLC-grade solvents and analytical-grade chemicals were provided by Merck (Darmstadt, Germany) and Sigma-Aldrich (Germany). Chromatographic equipment and conditions The chromatographic analyses were performed on a Varian (Walnut Creek, CA, USA) chromatographic system, which consisted of a tertiary pump model 9012, a Rheodyne injector with a 10 μl sample loop and a UV-VIS detector model 9050. The chromatograms were recorded at 360 nm. All data were acquired and processed with Varian Star Chromatography software (version 4.5). The separation was carried out with a Luna C18 column (150 × 4.6 mm i. d.; 5 μm) (Phenomenex, USA), itted with a pre-column (30 × 4.6 mm i.d.) dry packed with Perisorb RP-18 (30 - 40 μm) (Merck, Germany) and periodically changed. The binary solvent system consisted of solvent A (aqueous phosphoric acid, 0.1%) and solvent B (acetonitrile with 0.1% ortho-phosphoric acid). Gradient program was performed as follows: 0 min-10% B; 10 min-20% B; 25 min-30% B; 40 min-40% B; 45 min-40% B; 60 min-60% B and then return to the initial conditions in 5 min. The solvents were iltered through Millipore (Watford, Ireland) 0.45 μm ilters and degassed in an ultrasonic bath prior to use. The low rate was 1 ml/min. The oven temperature was set at 35°C. Quantitative HPLC analysis and analytical performance The quantiication of lavonoids was carried out using the external standard method. Because of the similar molecular structure, the responses of narcis- R. Gevrenova, N. Denkov, D. Zheleva-Dimitrova sin were related to rutin, assuming the responses at 360 nm to be equal. The concentrations of astragalin and isorhamnetin 3-glucoside were estimated using the isoquercitrin calibration curve; concentrations of luteolin, kaempferol and isorhamnetin were estimated using the quercetin calibration curve. The amounts of astragalin, isorhamnetin 3-glucoside, luteolin, kaempferol and isorhamnetin are thus relative and not absolute. External standard calibrations were established on seven data points covering the concentration range 0.001-1 mg/ml for rutin, 0.004-0.4 mg/ ml for isoquercitrin, and 0.005-0.5 mg/ml for quercetin. The stock standard solutions of appropriate concentration were prepared in methanol and were stored at 4°C in the dark. The working standard solutions of appropriate concentration were prepared by diluting the stock standard solutions with methanol. Triplicate HPLC analyses were performed for each concentration and the peak area was detected at 360 nm. Calibration curve was constructed from peak areas versus analyte concentrations. Slope, intercept, and other statistics of calibration lines were calculated with linear regression program using the Analytik-Software STL statistics programme (Leer, Germany). The regression equations were: y = 1.34 × 107 + 8604, r2 = 0.9999 (rutin); y = 1.57 × 107 + 44071, r2 = 0.9993 (isoquercitrin), and y = 2.27 × 107 + 48900, r2 = 0.9995 (quercetin). The examined compounds were assigned in the HPLC chromatograms by comparing individual peak retention times with these of authentic references standards, as well as by spiking techniques. For each sample, the complete assay procedure was carried out in triplicate and standard deviation was calculated. The repeatability was established by injecting the standard solution of rutin, isoquercetrin and quercetin (0.1 mg/ml) six times. The reproducibility was determined over 10 days by three injections per day of the same solution. The limits of detection (LOD) and limits of quantiication (LOQ) were calculated according to ICH recommendation [18]. They were based on standard deviation of the regression line of speciic calibration curve and its slope, using analyte concentrations in the range of LOD and LOQ solutions. The recovery of the analyte was evaluated by applying the entire SPE procedure to a control plant matrix (B. baldense) that had been spiked with a standard solution of rutin, isoquercitrin and quercetin, and measured in triplicate. The percentage recovery was determined by subtracting the values obtained for the control matrix preparation from those samples that had been prepared with the add- HPLC lavonoid proiles of Bupleurum L. species PHARMACIA, vol. 61, No. 2/2014 ed standard, divided by the amounts added of standards and multiplied by 100. The accuracy of the overall method was assessed analysing analytes (rutin, isoquercitrin and quercetin) added to the control plant matrix and tested in triplicate. The obtained peak areas were corrected using the values recorded for the control matrix, and the amounts of standards were determined from the corresponding calibration curves. The accuracy of the method (expressed as a percentage) was calculated by dividing the deviation of the mean concentrations found from the nominal value by the nominal value of analyte [19]. Results A method for puriication and isolation of lavonoids from aerial parts of the studied Bupleurum species was developed by SPE. In general, the pKa of lavonoids with different positions of hydroxyl group are ranged from 7-12 [20]. Based on this information, the lavonol glycosides were retained on the SPE cartridges as neutral solutes by using an acidic phosphate buffer (20 mM KH2PO4, pH 3.2) before the SPE procedure. Four different procedures using two separate sorbents were tested for the determination of rutin, narcissin, isoquercitrin and astragalin using standard solutions (Table 1). The percentage extraction eficiency of tested lavonoids obtained on Bond Elut C18 cartridges gave satisfactory results (recovery up to 99%) in comparison with sorbent cyanopropyl (CN). After neutral phenolics were loaded, an additional washing step with water was necessary in order to eliminate the most polar compounds. The washing step was controlled to avoid an insuficient recovery using the acidic phosphate buffer. It was examined the effect of three eluents, methanol, 75% methanol or tetrahydrofuran (THF), on analytical recovery. The optimum elution of the lavonol glycosides was achieved with pure methanol and it was used in all of the following experiments (Table 1). The quantiication of the main compounds in the lavonoid mixture isolated by SPE was performed by an improved HPLC-UV method. Based on the com- 19 mon approach, HPLC analysis of lavonoids from two Bupleurum species were performed with mobile phase composed of acetonitrile and water (0.1% phosphoric acid), and with UV detection at 360 nm [20]. Flavonols have their λmax in the 240-280 nm and 350-385 nm ranges. Therefore, to enhance selectivity, the longer λmax was chosen for detection of the lavonoids and data were collected at 360 nm. In respect to the analytical performance, for triplicate analysis of both standards and plant samples, RSDs of the retention times were ≤ 0.34 % for the determined compounds (n = 6). The proposed method was found to be linear in the studied concentration ranges of rutin, isoquercitrin, quercetin. The instrument precision was composed of repeatability and reproducibility studies of the assayed compounds. The RSDs of the repeatability and the reproducibility were estimated to be ≤ 2.47% and ≤ 2.87%, respectively. The detection limits (LODs) and quantiication limits (LOQs) were 1.2 μg/ml and 3.5 μg/ml (rutin), 0.7 μg/ml and 2.2 μg/ml (isoquercitrin), 0.5 μg/ml and 1.5 μg/ml (quercetin), respectively. On the basis of the above results, the accuracy and recovery of the overall method were assessed employing the selected SPE conditions (Table 2). A good agreement between the spiked and determined concentrations indicating acceptable accuracy was found. In the recovery assessment the percentage ratios are within the acceptance range of 90 - 110% [21]. The average precision of the overall analytical procedure, expressed by the relative standard deviations of the parallel results (n = 3), was estimated by measuring the within-day repeatability being lower than 2.09% (Table 2). It should be noted that the results of the overall method are acceptable [21]. Typical HPLC proiles of Bupleurum species and a standard mixture of the investigated compounds are presented in Figure 1 and Figure 2. The content of lavonoids in the assayed samples is shown in Table 3.With respect to the lavonol glycosides, rutin was present in the highest amount in B. baldense, while its content was considerably lower in B. afine (Table 3). The content of rutin Table 1. Mean recovery (n = 3) of selected lavonoids obtained by SPE using sorbents C18 and CN, and different eluents Sorbent (Eluent) C18 (MeOH) C18 (75% MeOH) C18 (THF) CN (MeOH) 1 Rutin 99.67 ± 1.811 87.68 ± 2.00 63.33 ± 3.61 49.45 ± 1.38 Mean recovery ± SD (standard deviation) Isoquercitrin 92.64 ± 3.10 82.35 ± 0.99 71.32 ± 5.06 72.73 ± 1.00 Narcissin 96.57 ± 5.29 80.26 ± 1.70 62.69 ± 1.58 94.46 ± 2.00 Astragalin 95.34 ± 5.92 74.13 ± 2.66 69.16 ± 6.04 89.47 ± 3.21 20 PHARMACIA, vol. 61, No. 2/2014 R. Gevrenova, N. Denkov, D. Zheleva-Dimitrova Table 2. Accuracy, mean recovery and precision of rutin, isoquercitrin and quercetin obtained from SPE of the spiked plant matrix (n = 3). Flavonoids Rutin Isoquercitrin Quercetin 1 2 Concentration Added Found±SD1 0.3828 0.1175 0.1410 (mg/ml) ± SD1 RSD2 (%) Accuracy (%) Recovery ± SD (%) RSD (%) 0.3902 ± 0.0145 0.1255 ± 0.0017 0.1489 ± 0.0022 3.72 1.39 1.48 + 1.93 + 6.75 +5.01 98.88 ± 2.07 104.22 ± 1.45 104.02 ± 1.54 2.09 1.39 1.48 SD – standard deviation RSD – relative standard deviation was from 73.1% (B. baldense) to 25.9% (B. afine) of the total amount of assayed compounds. The content of isoquercitrin was substantially higher in B. afine and reached 31% of the studied lavonoids. Astragalin and isorhamnetin 3-glucoside (as isoquercitrin equivalents) were presented in small concentrations or below the LOQ in B. baldense. However, in B. afine isorhamnetin 3-glucoside occurred in relatively higher concentration (Table 3, Fig. 1B). In particular, the highest content of aglycones (as quercetin equivalents) was found in B. afine. Kaempferol was not presented in B. baldense. The total amount of assayed lavonoids was estimated to be 11.53 ± 0.19 mg/g (B. afine) and 39.16 ± 0.92mg/g (B. baldense) (Table 3). Discussion In the present study, the content of lavonoids in two annual European Bupleurum species, including B. baldense and B. afine, is reported. HPLC lavonoid proiles of assayed species were characterized for the irst time. Table 3. Content of lavonoids in aerial parts of assayed Bupleurum species (mg/g dry weight) (n = 3). Flavonoids Rutin Isoquercitrin Narcissin Astragalin Isorhamnetin 3-glucoside Quercetin Kaempferol Isorhamnetin Luteolin Kaempferid Total B. baldense 28.63 ± 1.57 0.57 ± 0.04 9.90 ± 1.69 0.0001 B. afine 2.99 ± 0.55 3.58 ± 0.06 2.71 ± 0.37 0.04 below LQ 1.43 ± 0.07 below LQ 0.06 ± 0.03 below LQ below LQ 39.16 ± 0.92 0.33 ± 0.09 0.03 ± 0.008 0.16 ± 0.05 0.25 ± 0.07 below LQ 11.53 ± 0.19 The behavior of the identiied lavonol glycosides on SPE and HPLC supports was predicted by preliminary assessment of the lipophilicity parameter partition coeficient logP calculated for n-octanol – water system: -0.374 (rutin), 0.319 (isoquercitrin), 0.245 (narcissin), 0.904 (astragalin) and 0.930 (isorhamnetin 3-glucoside) [22]. In respect to SPE procedure, it has been reported for materials with pore sizes between 60 and100 Å, that a higher average pore size increases the retention capacity, due to the stronger interactions between the nonpolar surface and the analyte [23]. Also, a close relationship between carbon loading (17.4%) of C-18 cartridges might account for the greater retention of the analytes compared to the CN support (8.1% C) (Table 1). C-18 solid-phase support was tested with three eluents, tetrahydrofuran (THF), methanol or 75% methanol, searching for the optimal fractionation ability. THF was tested as the higher elutropic solvent. Moreover, the recovery improved when solvent polarity increased and methanol was selected as the eluent in the solid phase extraction procedure (Table 1). Due to the apolar interactions of the aglycons with modiied silica surface, the recovery decreased with increasing eluent polarity (75% methanol). In keeping with the reports of Zhang et al., 2010, our results indicated that B. baldense aerial parts contained signiicant quantities of rutin [13]. Several differences were observed in the qualitative and quantitative pattern of lavonoids in Chinese and European Bupleurum species. The contents of individual lavonoids reported here were generally higher than those reported by Zhang et al., 2010 [13]. Our SPE-HPLC analysis revealed the presence of higher amounts of rutin (Table 3) than those reported for Chinese species (up to 0.40 mg/g for B. chinense and B. longicaule) [13]. This inding agreed with the high content of rutin in B. lavum hydromethanolic extract (45.2 mg/g dry extract) HPLC lavonoid proiles of Bupleurum L. species PHARMACIA, vol. 61, No. 2/2014 Figure 1. HPLC chromatogram of a standard mixture Key to peaks identities: 1 – Rutin; 2 – Isoquercitrin; 3 – Narcissin; 4 – Astragalin; 5 – Isorhamnetin 3-glucoside; 6 – Luteolin; 7- Quercetin; 8 - Kaempferol; 9 – Isorhamnetin; 10 – Kaempferid. (For chromatographic protocol see Chromatographic equipment and conditions). Figure 2. HPLC chromatograms of the assayed Bupleurum species after SPE: (A) B. baldense (A1 – the same chromatogram from 20 to 60 min); (B) B. afine. (For key to peaks see Figure 1) 21 22 PHARMACIA, vol. 61, No. 2/2014 [17]. The contents of isorhamnetin and quercetin were in the same order of magnitude as that previously reported (up to 0.70 and 0.35 mg/g, respectively in B. yunnanense) [13]. The results obtained using Folin-Chiocalteu method for total polyphenols in other Bupleurum species varied from small level in B. chinense (2.63 mg gallic acid equivalent/g dry weight) [24] to higher levels in Turkish endemic Bupleurum species (between 31.48 and 61.48 mg gallic acid equivalent/g root extract) [25] and Bulgarian B. lavum aerial parts (350.59 ± 9.28 mg pyrogallol equivalent/g dry extract) [17]. In comparison with HPLC methods for the analysis of lavonol derivatives in Bupleurum species as reported above, the SPE-HPLC method optimized in this study allowed a good recovery of the compounds of interest. Formerly it was believed that most lavonoids in the genus Bupleurum are derivatives of the lavonol aglycones kaempferol, isorhamnetin or quercetin [26]. Recently, however, some other aglycones like apigenin, acacetin, chrysin, luteolin, tamarixetin, kaempferide, isokaempferide, and gossipetin have been characterized [3,4,12,13]. About 30 lavonoids have been isolated; rutin was found to be the most widespread [2]. The relative composition of the lavonoids in different Chinese Bupleurum species has been previously investigated [13], but the content of lavonoid glycosides in European species B. baldense and B. afine is reported for the irst time in this study. Acacetin 7-rutinoside (linarin) and apigenin 6, 8-di-C-β-D- glucopyranoside (vicenin-2), both lavon glycosides, have proved to be useful chemotaxonomic markers for B. chinense [12]. In contrast, our HPLC analysis revealed that B. baldense is different with a high yield of lavonol glycoside rutin. B. afine was noticeably separated by the highest content of isoquercitrin. Recently, a high content of narcissin was found in B. lavum aerial parts (64.10 ± 3.50 mg/g dry extract) [17]. The presence of narcissin and isoquercitrin seems to be characteristic of Bupleurum genus, which agrees with previous reports on species originating from Russia and Mediterranean area [17,26,27]. Conclusion A validated SPE-HPLC method for quantiication of lavonoids in the aerial parts of two European Bupleurum species was developed and showed excellent recovery. Here, lavonoid proiles of R. Gevrenova, N. Denkov, D. Zheleva-Dimitrova B. baldense and B. afine were characterized for the irst time. From the pharmaceutical point of view, B. baldense might be of interest, considering the high amount of lavonoids determined in its aerial parts. References 1. B a u e r R, Franz G. Modern European monographs for quality control of Chinese herbs (Review). Planta Med 2010; 76: 2004-2011. 2. 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