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Simultaneous quantification and peroxynitrite-scavenging activities of flavonoids in Polygonum aviculare L. herb
Journal of Pharmaceutical and Biomedical Analysis 89 (2014) 93–98
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Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Short communication
Simultaneous quantification and peroxynitrite-scavenging activities of flavonoids in Polygonum aviculare L. herb Agung Nugroho a,b , Eon Ji Kim c , Jae Sue Choi c , Hee-Juhn Park d,∗ a
Department of Applied Plant Sciences, Graduate School, Sangji University, Wonju 220-702, Republic of Korea Department of Agro-industrial Technology, Faculty of Agriculture, Lambung Mangkurat University, Banjarbaru 70712, Indonesia Department of Food Science and Nutrition, Pukyong National University, Busan 607-737, Republic of Korea d Department of Pharmaceutical Engineering, Sangji University, Wonju 220-702, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 23 September 2013 Received in revised form 23 October 2013 Accepted 25 October 2013 Available online 7 November 2013 Keywords: Polygonaceae Polygonum aviculare Flavonoids Desmanthin-1 Peroxynitrite HPLC
a b s t r a c t The plant Polygonum aviculare L. (Polygonaceae) is an annual herbaceous plant which is known to be beneficial for treating gastroduodenal ulcer, hypertension, diarrhea, hemorrhage, and hemorrhoids. Ten phenolic compounds, including nine flavonoids (myricetin, quercetin, kaempferol, myricitrin, desmanthin-1, isoquercitrin, quercitrin, avicularin, juglanin), and gallic acid were used for simultaneous HPLC quantification and peroxynitrite-scavenging assay. Simultaneous quantification of these substances were performed on five extracts (EtOH-, MeOH-, 70% MeOH-, 30% MeOH-, and H2 O extracts) as well as on the three fractions (Et2 O-, EtOAc-, and BuOH fractions), under the condition of a Capcell Pak C18 column (5 m, 250 mm × 4.6 mm i.d.) and a gradient elution of 0.05% trifluoroacetic acid (TFA) and MeOH CH3 CN (60:40). Of the three fractions, the EtOAc fraction displayed the highest content of flavonoids (sum, 208.9 mg/g) with the strongest peroxynitrite-scavenging activity (IC50 , 2.68 g/mL). The activities of the eight compounds (myricitrin, isoquercitrin, avicularin, quercitrin, myricetin, desmanthin-1, quercetin, and kaempferol) were comparable to that of the positive control (l-penicillamine; IC50 : 1.03 g/mL). These results suggest that folkloric medicinal uses of P. aviculare are mainly attributed to flavonoids, such as particularly highly contained myricetin, myricitrin, and desmanthin-1. © 2013 Published by Elsevier B.V.
1. Introduction The plant Polygonum aviculare L. (common knotgrass) belongs to the family Polygonaceae and is an annual herbaceous plant which is usually found in fields or roadsides. Traditionally, it has been used to treat several diseases, such as gastric ulcer, duodenal ulcer, hemorrhage, diarrhea, and hemorrhoids [1]. Scientifically, the alcohol extracts of P. aviculare have been reported to possess pharmacological activities, such as antioxidant activities [2], antiobesity effects [1], antimicrobial activities [3], anti-inflammatory effects [4], anti-gingivitis effect [5], antifibrotic effect [6], and also anti-cancer effects [7]. Several flavonoids, quercetin, kaempferol, myricetin, isorhamnetin, luteolin, and their glycosides, have been identified from P. aviculare [8–12]. Gallic acid [5], protocatechoic acid, and chlorogenic acid [13], have also been reported to be present in this species. Recently, Granica et al. [14] identified many flavonoids in P. aviculare using an UHPLC-DAD with two mass detectors, confirming the previous reports on the same constituents. However,
∗ Corresponding author. Tel.: +82 33 730 0564; fax: +82 33 730 0564. E-mail address: [email protected] (H.-J. Park). 0731-7085/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.jpba.2013.10.037
there has been no report on the quantification of flavonoids and peroxynitrite-scavenging activity in P. aviculare. A simultaneous quantification is required because several flavonoids or phenolic acids will individually scavenge peroxynitrite. Therefore, this study was aimed to evaluate the peroxynitrite-scavenging activity of P. aviculare and to analyze the content of the active principles in the extracts. 2. Materials and methods 2.1. Plant material, reagents and instruments P. aviculare was collected on August, 2012 at a certain roadside in Wonju City, Gangwon Province, Korea, and was verified by Prof. S. C. Lim (Department of Horticulture and Landscape Architecture, Sangji University). The voucher specimen (natchem-#43) was deposited to the Laboratory of Natural Products Chemistry, Department of Pharmaceutical Engineering, Sangji University. Chromatographic isolation was performed on the open columns using normal or reverse stationary phases. Spectra of the isolated compounds were measured using a NMR spectrometer (Bruker Co., Germany), an FT/IR spectrometer (Jasco Co., Japan), and an UV–vis spectrophotometer (Shimadzu Co., Japan). A quantitative analysis
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Fig. 1. Chemical structures of compounds identified in P. aviculare.
was performed on the HPLC system (Varian Co., CA, USA) consisting of two Prostar 210 pumps, a Prostar 325 UV–vis detector and a Shiseido Capcell PAK C18 column (5 m, 4.6 mm × 250 mm, Japan). The reagents used for the peroxynitrite-scavenging assay were diethylenetriaminepentaacetic acid (Sigma Co., St. Louis, MO, USA), dihydrorhodamine 123 (Molecular Probes, Eugene, OR, USA), and peroxynitrite (Cayman Chemicals Co., Ann Arbor, MI, USA).
to produce compound 4. EA-4 was also subjected to the same column chromatography with that of EA-2 to give 72 fractions. These fractions were grouped into three fractions, #9–13, #39–45, and #50–72, which were concentrated and recrystallized to produce compounds 2, 6, and 8, consecutively.
2.2. Extraction, fractionation, and isolation
Through this study, ten constituents were identified from P. aviculare. Compound numbers were assigned depending on the retention time of the HPLC chromatogram. Four compounds (2, 4, 6, and 8) were identified by chromatographic isolation and comparison of their spectroscopic data (1 H-and 13 C-NMR) with reported values of the literatures as mentioned below:
The air-dried and crushed leaves of P. aviculare (800 g) were extracted under reflux with 7 L of EtOH for 6 h and repeated three times. Fractionation was carried out by suspending the EtOH extract (190 g) in the H2 O and partitioned three times with 800 mL diethyl ether (Et2 O). In the same way, the water layer was successively fractionated with EtOAc and BuOH in order to give EtOAc fraction (14.56 g) and BuOH fraction (14.88 g). The EtOAc fraction was chromatographed on the silica gel column (SiO2 330 g, ø 55 mm × 35 cm) with CHCl3 MeOH H2 O (75:25:10, lower layer) by collecting 50 mL for each fraction. The collected fractions were grouped into EA-1 (#01–24), EA-2 (#25–30), EA-3 (#31–62), and EA-4 (#62–95). EA-2 was subjected to ODS column chromatography (150 g, ø 25 mm × 35 cm) with MeOH H2 O (50:50), collected by 20 mL (each). Fraction #12–30 was combined and concentrated
2.3. Identification of the constituents
Compound 2 (myricitrin) Yellowish powder, mp 205–207 ◦ C, 1 H and 13 C-NMR: literature [15]. Compound 4 (avicularin) Yellowish powder, mp 172–175 ◦ C, 1 H and 13 C-NMR: literature [16]. Compound 6 (myricetin) Yellowish powder, mp 352–355 ◦ C, 1 H and 13 C-NMR: literature [17]. Compound 8 (desmanthin-1) Brownish powder, mp 196–199 ◦ C, 1 H and 13 C-NMR: literature [18].
Referring to the literature, six standard compounds, gallic acid (1), isoquercitrin (3), quercitrin (5), juglanin (7), quercetin (9), and kaempferol (10), were used to detect the compounds in P. aviculare. The existence of the six compounds was confirmed by the
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Table 1 Linearity of standard curves and detection/quantification limits for the standard compounds. Standard compounds
Regression equation (linear model)a
Linear range (g/mL)
R2 b
LODc (g/mL)
LOQd (g/mL)
Gallic acid Myricitrin Isoquercitrin Avicularin Quercitrin Myricetin Juglanin Desmanthin-1 Quercetin Kaempferol
y = 145.02x + 33.59 y = 101.10x + 66.65 y = 472.75x + 75.62 y = 247.29x + 65.75 y = 458.11x + 62.66 y = 106.79x + 54.78 y = 182.69x + 58.73 y = 90.40x + 52.08 y = 660.71x + 57.15 y = 456.75x + 55.57
1.56–50.00 3.13–100.00 1.56–50.00 1.56–50.00 1.56–50.00 3.13–100.00 1.56–50.00 3.13–100.00 0.50–30.00 0.50–50.00
0.9997 0.9998 0.9998 0.9999 0.9998 0.9999 0.9998 0.9998 0.9999 0.9998
0.46 0.33 0.05 0.14 0.08 0.42 0.23 0.53 0.06 0.10
1.52 1.10 0.17 0.46 0.27 1.41 0.75 1.77 0.21 0.32
a b c d
y, peak area at 254 nm; x, concentration of the standard (g/mL). R2 , correlation coefficient (n = 4). LOD, limit of detection (S/N = 3). LOQ, limit of quantification (S/N = 10).
HPLC recovery- and co-TLC tests. Chemical structures of those ten compounds are shown in Fig. 1.
detection wavelength was fixed at 254 nm and monitored during 40 min for each sample. 2.5. Preparation of standard and sample solutions
2.4. HPLC analytical method Two solvents, solvent A (H2 O with 0.05% trifluoroacetic acid, v/v) and solvent B (MeOH:CH3 CN = 60:40 with 0.05% TFA, v/v) were used in this method. The linear gradient elution of the solvents was programmed as follows: 0–35 min (15 → 65% B), 35–40 min (65% B), 40–42 min (65 → 100% B), 42–46 min (100% B), 46–49 min (100 → 15% B), and 49–55 min (15% B). The flow rate and column temperature was set constantly at 1.0 mL/min and 40 ◦ C, respectively. The
Ten compounds were used for the quantitative analysis of the constituent in P. aviculare. Four compounds were obtained by isolation (purity ≥ 98% by HPLC), five compounds were purchased from Sigma–Aldrich Co. (St. Louis, MO, USA), i.e., isoquercitrin (lot no. BCBC1548 V), quercitrin (059K1201), quercetin (14H0957), kaempferol (075K1574), and gallic acid (066K1432). Juglanin (purity ≥ 98% HPLC) was provided by Prof. Jae Sue Choi (Dept. of Food Science and Nutrition, Pukyong National University). Standard
Table 2 Precision and recovery data of each analyte. Analyte
Precision test tR (min)
Gallic acid Myricitrin Isoquercitrin Avicularin Quercitrin Myricetin Juglanin Desmanthin-1 Quercetin Kaempferol
4.162 16.26 17.15 19.19 19.64 20.40 21.80 23.56 25.06 29.08
Recovery test
Intra-day variability RSD (%)
Inter-day variability RSD (%)
tR
Area
tR
Area
0.45 0.22 0.23 0.21 0.17 0.17 0.30 0.13 0.12 0.08
1.49 0.53 2.66 0.77 1.30 0.55 1.08 1.46 1.21 2.90
0.67 0.51 0.42 0.43 0.36 0.36 0.77 0.29 0.30 0.24
3.96 2.59 5.15 1.99 2.13 2.09 3.18 2.48 5.00 3.99
Initial conc. (g/mL)
Amount added (g)
7.837 30.67 2.087 11.82 5.898 31.09 6.641 15.90 0.769 0.743
6.250 25.00 1.563 12.50 6.250 25.00 6.250 12.50 1.875 1.563
Concentration after addition (g/mL)
Expected
Measured
14.09 55.67 3.650 24.32 12.15 56.09 12.89 28.40 2.644 2.305
13.79 54.24 3.475 23.98 11.75 55.22 12.33 27.40 2.586 2.208
Recovery (%)
RSD (%)
97.90 97.42 95.20 98.61 96.74 98.45 95.68 96.48 97.80 95.78
0.68 0.48 0.61 0.59 0.67 0.59 0.67 0.69 0.44 0.81
Relative standard deviation (RSD) values were calculated for both retention time (tR ) and peak area of five experiments. Recovery tests were performed in the 7% MeOH extract spiked with each standard compound.
Table 3 Content of analytes in the lyophilized extracts and fractions of P. aviculare (mg/g). Analyte
Extracts
Fractions
EtOH
MeOH
70% MeOH
30% MeOH
H2 O
Et2 O
EtOAc
BuOH
Gallic acid Myricitrin Isoquercitrin Avicularin Quercitrin Myricetin Juglanin Desmanthin-1 Quercetin Kaempferol
0.63 11.73 0.13 5.79 2.44 11.81 3.21 7.73 0.84 0.59
1.59 13.58 0.37 5.72 2.31 12.29 3.05 5.90 0.52 0.43
2.85 11.15 0.76 4.30 2.15 11.30 2.42 5.78 0.28 0.27
5.03 7.67 0.55 2.36 1.71 6.82 2.02 3.04 LOQ LOQ
7.34 3.94 0.34 0.95 0.74 0.58 0.50 1.50 LOQ LOQ
3.32 2.41 LOQ 8.46 0.80 6.68 4.32 5.59 0.74 0.94
9.67 84.30 2.98 26.34 11.44 64.04 6.74 12.45 0.62 LOQ
1.17 9.78 0.78 LOQ 0.84 LOQ LOQ LOQ LOQ LOQ
Total of flavonoids
44.27
45.17
38.41
24.17
8.55
29.94
209.1
11.40
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Table 4 Peroxynitrite-scavenging activity of the ten compounds, extracts and fractions of P. aviculare. Sample
Peroxynitrite-scavenging (%) 0.4 g/mL
Gallic acid Myricitrin Isoquercitrin Avicularin Quercitrin Myricetin Juglanin Desmanthin-1 Quercetin Kaempferol L-Penicillaminec EtOH extract MeOH extract Et2 O fraction EtOAc fraction BuOH fraction a b c
30.53 ± 27.22 ± 25.17 ± 20.99 ± 32.87 ± 35.00 ± 19.76 ± 27.52 ± 47.44 ± 28.95 ± 39.95 ± – – – – –
4.80a 3.85 4.11 2.61 0.69 1.85 1.00 0.69 8.90 6.18 0.69
IC50 (g/mL) 2 g/mL 68.01 51.47 76.71 62.07 70.61 77.09 35.04 62.70 82.68 92.50 65.51 23.97 10.09 23.41 46.39 24.71
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
10 g/mL 0.97 0.36 3.55 0.67 0.50 0.08 2.21 1.26 3.79 1.45 0.82 3.49 1.16 2.28 1.99 2.26
96.77 86.06 94.58 88.95 90.41 95.20 51.39 89.99 94.82 99.41 84.72 48.57 50.92 65.42 88.50 62.13
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.37 0.19 1.49 0.99 0.06 0.23 2.86 0.57 1.49 0.11 0.44 0.70 0.38 3.98 0.24 1.99
50 g/mL – – – – – – – – – – – 80.58 ± 84.26 ± 94.24 ± 98.59 ± 92.10 ±
0.21 0.38 0.13 0.13 0.42
1.23 (7.23)b 1.90 (4.09) 1.17 (2.52) 1.53 (3.52) 1.10 (2.45) 0.97 (3.04) 9.32 (22.3) 1.42 (2.30) 0.51 (1.69) 0.92 (3.21) 1.03 (6.90) 11.78 9.82 7.08 2.68 7.40
Value represents mean ± S.D. (n = 2). Unit of the value in the parenthesis is M. Positive control.
stock solution (1000 g/mL) was prepared by dissolving each standard compound in MeOH. Working standard solutions were prepared by a serial dilution of the standard stock solution. Five extracts were prepared using EtOH, MeOH, 70% MeOH, 30% MeOH, and H2 O in order to compare which solvents more efficiently extract flavonoids from P. aviculare. The extraction method was designed by extracting a crushed and lyophilized plant material (3.0 g) with a 100 mL solvent in a 250 mL Erlenmeyer flask on an ultrasonicator for 5 h at 45 ◦ C. The extracted solution was filtered through a disposable syringe filter (0.50 m, Dismic-25JP Advantec, Japan) prior to the injection into the HPLC system.
2.6. Validation of the HPLC analytical method The HPLC analysis method used in this study has been validated through several tests. Linearity was evaluated by the value of R2 (correlation coefficient) in the calibration curve of serial concentrations. Sensitivity was determined by the value of limit of detection (LOD) and limit of quantification (LOQ). A Signal-to-noise ratio (S/N) method was used to determine the LOD and LOQ value. The S/N ratio was 3 for LOD and 10 for LOQ. The precision and stability of the analysis method was assessed by measurements of the intra- and inter-day variability of both retention time and peak area values. To evaluate the accuracy, the recovery test was performed, spiking each standard compound to the sample solution.
2.7. Peroxynitrite-scavenging activity An assay method described by Kooy et al. [19] was used to measure the peroxynitrite-scavenging activity of the compounds, extracts, and fractions from P. aviculare. The principle of this method is to monitor the intensity of highly fluorescent rhodamine formed from non-fluorescent DHR 123 under the presence of ONOO− . The concentration of DHR 123 was 5 M. The samples were dissolved in 10% DMSO (concentration: 5 g/mL). The final fluorescent intensity was measured with or without the treatment of 10 M ONOO− in 0.3 N NaOH. The fluorescence intensity of the oxidized DHR 123 was measured at the excitation and emission of 480 nm and 530 nm using a microplate fluorescence reader FL 500 (Bio-Tek Instruments Inc., Winooski, VT, USA).
3. Results and discussion 3.1. Optimization and validation of the HPLC analytical method To establish a reliable HPLC analytical method, four parameters of mobile phase composition, gradient elution, UV wavelength, and column temperature were optimized through repetitive experimentations. An optimum formula consisting of solvent A and solvent B was selected. Solvent A was H2 O and solvent B was the mixture of MeOH and CH3 CN with a ratio of 60:40 (v/v). This composition was selected because it showed a better separation and the solvents were environment-friendly. A gradient elution method, as described in the Materials and methods section, was employed for better chromatographic separation on a wide range of polarity in a shorter time. That method worked very well when the temperature of the column was maintained at 40 ◦ C. The optimized HPLC method was validated by examining the linearity, sensitivity, precision, and accuracy. Every R2 value was more than 0.999, verifying the linearities of the calibration equations (Table 1). In the inter-day variability test, the RSDs were observed over 1.99–5.15% (Table 2), indicating that the analysis is precise and stable under the present method. Recovery rates were observed from 95.20 to 98.61% (Table 2), which indicate a sufficient accuracy of this experiment.
3.2. Peroxynitrite-scavenging effect of the constituents The peroxynitrite-scavenging activities of the ten compounds were determined (Table 4). The M unit was used for making comparisons on the structure–activity relationship. The activities of the eight flavonoids, except for juglanin, were higher than that of the positive control (l-penicillamine, 6.90 M). Comparing the activities of the three flavonols, kaempferol, quercetin, and myricetin, the order was quercetin (1.69 M) > myricetin (3.04 M) > kaempferol (3.21 M), indicating that the catechol ring in the B-ring of flavonol contributes to the activity of flavonol rather than to the pyrogallol in myricetin and phenol in the kaempferol. In the flavonol glycosides, the activities ranged over 2.45 M (quercitrin)–4.09 M (myricitrin). These results suggest that the activities of flavonol glycosides are primarily due to the aglycone rather than to the sugar moiety. Comparing the activities of myricetin, myricitrin and desmanthin-1 (2 -O-galloylmyricitrin), the order was desmanthin1 (2.30 M) > myricetin (3.04 M) > myricitrin (4.09 M). These
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Fig. 2. HPLC chromatograms of mixed standards, extract and fractions of P. aviculare.
results suggest that galloylation to myricitrin increases the peroxynitrite-scavenging activity. 3.3. Content of substances in the extract and fractions: correlation to their peroxynitrite-scavenging activities In the evaluation of peroxynitrite-scavenging activity (Table 4), the EtOAc fraction exhibited a potent activity (IC50 : 2.68 g/mL), significantly stronger than other fractions or the extract (IC50 more than 7.08 g/mL). As presented in Table 3 and Fig. 2, the flavonoids were mainly distributed in the EtOAc fraction (208.9 mg/g) rather than in the BuOH (11.90 mg/g) or Et2 O (29.92 mg/g) fractions. Therefore, it is considered that the peroxynitrite-scavenging activity of P. aviculare could be influenced by the composition or contents of the flavonoids. Contents of ten compounds in the extracts and
fractions were shown in Table 3. The sum of the flavonoid contents were shown in the order of MeOH ext. (45.16 mg/g) > 70% MeOH ext. (38.40 mg/g) > 30% MeOH (24.17 mg/g) > 0% MeOH (8.55 mg/g), indicating that a high ratio of MeOH efficiently extracts flavonoids in P. aviculare. The effects of MeOH and EtOH solvents on the extraction of flavonoids were similar. 4. Conclusion Three flavonols (myricetin, quercetin, kaempferol), their six glycosides (myricitrin, desmanthin-1, isoquercitrin, quercitrin, avicularin, and juglanin), and one phenolic acid (gallic acid) were identified in P. aviculare. Except for juglanin, the eight flavonoids showed potent peroxynitrite-scavenging activities which were stronger than l-penicillamine (IC50 : 6.90 M). It was demonstrated
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that the EtOAc fraction with the highest content of flavonoids (208.9 mg/g) exhibits the strongest peroxynitrite-scavenging activity (IC50 , 2.68 g/mL). Thus, it is suggested that flavonoids should act as the active principles of P. aviculare for its peroxynitritescavenging activity. In addition, extraction using a high ratio of MeOH efficiently extracts the flavonoids in P. aviculare. Acknowledgement This research was supported by the Sangji University Research Fund, 2013. References [1] Y.Y. Sung, T. Yoon, W.K. Yang, S.J. Kim, D.S. Kim, H.K. Kim, The antiobesity effect of Polygonum aviculare L. ethanol extract in high-fat diet-induced obese mice, Evid. Based Complement. Alternat. Med. (2013) 1–11. [2] C.Y. Hsu, Antioxidant activity of extract from Polygonum aviculare L, Biol. Res. 39 (2006) 281–288. [3] H.M.H. Salama, N. Marraiki, Antimicrobial activity and phytochemical analysis of Polygonum aviculare L. (Polygonaceae), naturally growing in Egypt, Saudi J. Biol. Sci. 17 (2010) 57–63. [4] H. Tunón, C. Olavsdotter, L. Bohlin, Evaluation of anti-inflammatory activity of some Swedish medicinal plants. Inhibition of prostaglandin biosynthesis and PAF-induced exocytosis, J. Ethnopharmacol. 48 (1995) 61–76. [5] M. González Begné, N. Yslas, E. Reyes, V. Quiroz, J. Santana, G. Jimenez, Clinical effect of a Mexican Sanguinaria extract (Polygonum aviculare L.) on gingivitis, J. Ethnopharmacol. 74 (2001) 45–51. [6] J.X. Nan, E.J. Park, H.J. Kim, G. Ko, D.H. Sohn, Antifibrotic effects of the methanol extract of Polygonum aviculare in fibrotic rats induced by bile duct ligation and scission, Biol. Pharm. Bull. 23 (2000) 240–243.
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Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Short communication
Simultaneous quantification and peroxynitrite-scavenging activities of flavonoids in Polygonum aviculare L. herb Agung Nugroho a,b , Eon Ji Kim c , Jae Sue Choi c , Hee-Juhn Park d,∗ a
Department of Applied Plant Sciences, Graduate School, Sangji University, Wonju 220-702, Republic of Korea Department of Agro-industrial Technology, Faculty of Agriculture, Lambung Mangkurat University, Banjarbaru 70712, Indonesia Department of Food Science and Nutrition, Pukyong National University, Busan 607-737, Republic of Korea d Department of Pharmaceutical Engineering, Sangji University, Wonju 220-702, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 23 September 2013 Received in revised form 23 October 2013 Accepted 25 October 2013 Available online 7 November 2013 Keywords: Polygonaceae Polygonum aviculare Flavonoids Desmanthin-1 Peroxynitrite HPLC
a b s t r a c t The plant Polygonum aviculare L. (Polygonaceae) is an annual herbaceous plant which is known to be beneficial for treating gastroduodenal ulcer, hypertension, diarrhea, hemorrhage, and hemorrhoids. Ten phenolic compounds, including nine flavonoids (myricetin, quercetin, kaempferol, myricitrin, desmanthin-1, isoquercitrin, quercitrin, avicularin, juglanin), and gallic acid were used for simultaneous HPLC quantification and peroxynitrite-scavenging assay. Simultaneous quantification of these substances were performed on five extracts (EtOH-, MeOH-, 70% MeOH-, 30% MeOH-, and H2 O extracts) as well as on the three fractions (Et2 O-, EtOAc-, and BuOH fractions), under the condition of a Capcell Pak C18 column (5 m, 250 mm × 4.6 mm i.d.) and a gradient elution of 0.05% trifluoroacetic acid (TFA) and MeOH CH3 CN (60:40). Of the three fractions, the EtOAc fraction displayed the highest content of flavonoids (sum, 208.9 mg/g) with the strongest peroxynitrite-scavenging activity (IC50 , 2.68 g/mL). The activities of the eight compounds (myricitrin, isoquercitrin, avicularin, quercitrin, myricetin, desmanthin-1, quercetin, and kaempferol) were comparable to that of the positive control (l-penicillamine; IC50 : 1.03 g/mL). These results suggest that folkloric medicinal uses of P. aviculare are mainly attributed to flavonoids, such as particularly highly contained myricetin, myricitrin, and desmanthin-1. © 2013 Published by Elsevier B.V.
1. Introduction The plant Polygonum aviculare L. (common knotgrass) belongs to the family Polygonaceae and is an annual herbaceous plant which is usually found in fields or roadsides. Traditionally, it has been used to treat several diseases, such as gastric ulcer, duodenal ulcer, hemorrhage, diarrhea, and hemorrhoids [1]. Scientifically, the alcohol extracts of P. aviculare have been reported to possess pharmacological activities, such as antioxidant activities [2], antiobesity effects [1], antimicrobial activities [3], anti-inflammatory effects [4], anti-gingivitis effect [5], antifibrotic effect [6], and also anti-cancer effects [7]. Several flavonoids, quercetin, kaempferol, myricetin, isorhamnetin, luteolin, and their glycosides, have been identified from P. aviculare [8–12]. Gallic acid [5], protocatechoic acid, and chlorogenic acid [13], have also been reported to be present in this species. Recently, Granica et al. [14] identified many flavonoids in P. aviculare using an UHPLC-DAD with two mass detectors, confirming the previous reports on the same constituents. However,
∗ Corresponding author. Tel.: +82 33 730 0564; fax: +82 33 730 0564. E-mail address: [email protected] (H.-J. Park). 0731-7085/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.jpba.2013.10.037
there has been no report on the quantification of flavonoids and peroxynitrite-scavenging activity in P. aviculare. A simultaneous quantification is required because several flavonoids or phenolic acids will individually scavenge peroxynitrite. Therefore, this study was aimed to evaluate the peroxynitrite-scavenging activity of P. aviculare and to analyze the content of the active principles in the extracts. 2. Materials and methods 2.1. Plant material, reagents and instruments P. aviculare was collected on August, 2012 at a certain roadside in Wonju City, Gangwon Province, Korea, and was verified by Prof. S. C. Lim (Department of Horticulture and Landscape Architecture, Sangji University). The voucher specimen (natchem-#43) was deposited to the Laboratory of Natural Products Chemistry, Department of Pharmaceutical Engineering, Sangji University. Chromatographic isolation was performed on the open columns using normal or reverse stationary phases. Spectra of the isolated compounds were measured using a NMR spectrometer (Bruker Co., Germany), an FT/IR spectrometer (Jasco Co., Japan), and an UV–vis spectrophotometer (Shimadzu Co., Japan). A quantitative analysis
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Fig. 1. Chemical structures of compounds identified in P. aviculare.
was performed on the HPLC system (Varian Co., CA, USA) consisting of two Prostar 210 pumps, a Prostar 325 UV–vis detector and a Shiseido Capcell PAK C18 column (5 m, 4.6 mm × 250 mm, Japan). The reagents used for the peroxynitrite-scavenging assay were diethylenetriaminepentaacetic acid (Sigma Co., St. Louis, MO, USA), dihydrorhodamine 123 (Molecular Probes, Eugene, OR, USA), and peroxynitrite (Cayman Chemicals Co., Ann Arbor, MI, USA).
to produce compound 4. EA-4 was also subjected to the same column chromatography with that of EA-2 to give 72 fractions. These fractions were grouped into three fractions, #9–13, #39–45, and #50–72, which were concentrated and recrystallized to produce compounds 2, 6, and 8, consecutively.
2.2. Extraction, fractionation, and isolation
Through this study, ten constituents were identified from P. aviculare. Compound numbers were assigned depending on the retention time of the HPLC chromatogram. Four compounds (2, 4, 6, and 8) were identified by chromatographic isolation and comparison of their spectroscopic data (1 H-and 13 C-NMR) with reported values of the literatures as mentioned below:
The air-dried and crushed leaves of P. aviculare (800 g) were extracted under reflux with 7 L of EtOH for 6 h and repeated three times. Fractionation was carried out by suspending the EtOH extract (190 g) in the H2 O and partitioned three times with 800 mL diethyl ether (Et2 O). In the same way, the water layer was successively fractionated with EtOAc and BuOH in order to give EtOAc fraction (14.56 g) and BuOH fraction (14.88 g). The EtOAc fraction was chromatographed on the silica gel column (SiO2 330 g, ø 55 mm × 35 cm) with CHCl3 MeOH H2 O (75:25:10, lower layer) by collecting 50 mL for each fraction. The collected fractions were grouped into EA-1 (#01–24), EA-2 (#25–30), EA-3 (#31–62), and EA-4 (#62–95). EA-2 was subjected to ODS column chromatography (150 g, ø 25 mm × 35 cm) with MeOH H2 O (50:50), collected by 20 mL (each). Fraction #12–30 was combined and concentrated
2.3. Identification of the constituents
Compound 2 (myricitrin) Yellowish powder, mp 205–207 ◦ C, 1 H and 13 C-NMR: literature [15]. Compound 4 (avicularin) Yellowish powder, mp 172–175 ◦ C, 1 H and 13 C-NMR: literature [16]. Compound 6 (myricetin) Yellowish powder, mp 352–355 ◦ C, 1 H and 13 C-NMR: literature [17]. Compound 8 (desmanthin-1) Brownish powder, mp 196–199 ◦ C, 1 H and 13 C-NMR: literature [18].
Referring to the literature, six standard compounds, gallic acid (1), isoquercitrin (3), quercitrin (5), juglanin (7), quercetin (9), and kaempferol (10), were used to detect the compounds in P. aviculare. The existence of the six compounds was confirmed by the
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Table 1 Linearity of standard curves and detection/quantification limits for the standard compounds. Standard compounds
Regression equation (linear model)a
Linear range (g/mL)
R2 b
LODc (g/mL)
LOQd (g/mL)
Gallic acid Myricitrin Isoquercitrin Avicularin Quercitrin Myricetin Juglanin Desmanthin-1 Quercetin Kaempferol
y = 145.02x + 33.59 y = 101.10x + 66.65 y = 472.75x + 75.62 y = 247.29x + 65.75 y = 458.11x + 62.66 y = 106.79x + 54.78 y = 182.69x + 58.73 y = 90.40x + 52.08 y = 660.71x + 57.15 y = 456.75x + 55.57
1.56–50.00 3.13–100.00 1.56–50.00 1.56–50.00 1.56–50.00 3.13–100.00 1.56–50.00 3.13–100.00 0.50–30.00 0.50–50.00
0.9997 0.9998 0.9998 0.9999 0.9998 0.9999 0.9998 0.9998 0.9999 0.9998
0.46 0.33 0.05 0.14 0.08 0.42 0.23 0.53 0.06 0.10
1.52 1.10 0.17 0.46 0.27 1.41 0.75 1.77 0.21 0.32
a b c d
y, peak area at 254 nm; x, concentration of the standard (g/mL). R2 , correlation coefficient (n = 4). LOD, limit of detection (S/N = 3). LOQ, limit of quantification (S/N = 10).
HPLC recovery- and co-TLC tests. Chemical structures of those ten compounds are shown in Fig. 1.
detection wavelength was fixed at 254 nm and monitored during 40 min for each sample. 2.5. Preparation of standard and sample solutions
2.4. HPLC analytical method Two solvents, solvent A (H2 O with 0.05% trifluoroacetic acid, v/v) and solvent B (MeOH:CH3 CN = 60:40 with 0.05% TFA, v/v) were used in this method. The linear gradient elution of the solvents was programmed as follows: 0–35 min (15 → 65% B), 35–40 min (65% B), 40–42 min (65 → 100% B), 42–46 min (100% B), 46–49 min (100 → 15% B), and 49–55 min (15% B). The flow rate and column temperature was set constantly at 1.0 mL/min and 40 ◦ C, respectively. The
Ten compounds were used for the quantitative analysis of the constituent in P. aviculare. Four compounds were obtained by isolation (purity ≥ 98% by HPLC), five compounds were purchased from Sigma–Aldrich Co. (St. Louis, MO, USA), i.e., isoquercitrin (lot no. BCBC1548 V), quercitrin (059K1201), quercetin (14H0957), kaempferol (075K1574), and gallic acid (066K1432). Juglanin (purity ≥ 98% HPLC) was provided by Prof. Jae Sue Choi (Dept. of Food Science and Nutrition, Pukyong National University). Standard
Table 2 Precision and recovery data of each analyte. Analyte
Precision test tR (min)
Gallic acid Myricitrin Isoquercitrin Avicularin Quercitrin Myricetin Juglanin Desmanthin-1 Quercetin Kaempferol
4.162 16.26 17.15 19.19 19.64 20.40 21.80 23.56 25.06 29.08
Recovery test
Intra-day variability RSD (%)
Inter-day variability RSD (%)
tR
Area
tR
Area
0.45 0.22 0.23 0.21 0.17 0.17 0.30 0.13 0.12 0.08
1.49 0.53 2.66 0.77 1.30 0.55 1.08 1.46 1.21 2.90
0.67 0.51 0.42 0.43 0.36 0.36 0.77 0.29 0.30 0.24
3.96 2.59 5.15 1.99 2.13 2.09 3.18 2.48 5.00 3.99
Initial conc. (g/mL)
Amount added (g)
7.837 30.67 2.087 11.82 5.898 31.09 6.641 15.90 0.769 0.743
6.250 25.00 1.563 12.50 6.250 25.00 6.250 12.50 1.875 1.563
Concentration after addition (g/mL)
Expected
Measured
14.09 55.67 3.650 24.32 12.15 56.09 12.89 28.40 2.644 2.305
13.79 54.24 3.475 23.98 11.75 55.22 12.33 27.40 2.586 2.208
Recovery (%)
RSD (%)
97.90 97.42 95.20 98.61 96.74 98.45 95.68 96.48 97.80 95.78
0.68 0.48 0.61 0.59 0.67 0.59 0.67 0.69 0.44 0.81
Relative standard deviation (RSD) values were calculated for both retention time (tR ) and peak area of five experiments. Recovery tests were performed in the 7% MeOH extract spiked with each standard compound.
Table 3 Content of analytes in the lyophilized extracts and fractions of P. aviculare (mg/g). Analyte
Extracts
Fractions
EtOH
MeOH
70% MeOH
30% MeOH
H2 O
Et2 O
EtOAc
BuOH
Gallic acid Myricitrin Isoquercitrin Avicularin Quercitrin Myricetin Juglanin Desmanthin-1 Quercetin Kaempferol
0.63 11.73 0.13 5.79 2.44 11.81 3.21 7.73 0.84 0.59
1.59 13.58 0.37 5.72 2.31 12.29 3.05 5.90 0.52 0.43
2.85 11.15 0.76 4.30 2.15 11.30 2.42 5.78 0.28 0.27
5.03 7.67 0.55 2.36 1.71 6.82 2.02 3.04 LOQ LOQ
7.34 3.94 0.34 0.95 0.74 0.58 0.50 1.50 LOQ LOQ
3.32 2.41 LOQ 8.46 0.80 6.68 4.32 5.59 0.74 0.94
9.67 84.30 2.98 26.34 11.44 64.04 6.74 12.45 0.62 LOQ
1.17 9.78 0.78 LOQ 0.84 LOQ LOQ LOQ LOQ LOQ
Total of flavonoids
44.27
45.17
38.41
24.17
8.55
29.94
209.1
11.40
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Table 4 Peroxynitrite-scavenging activity of the ten compounds, extracts and fractions of P. aviculare. Sample
Peroxynitrite-scavenging (%) 0.4 g/mL
Gallic acid Myricitrin Isoquercitrin Avicularin Quercitrin Myricetin Juglanin Desmanthin-1 Quercetin Kaempferol L-Penicillaminec EtOH extract MeOH extract Et2 O fraction EtOAc fraction BuOH fraction a b c
30.53 ± 27.22 ± 25.17 ± 20.99 ± 32.87 ± 35.00 ± 19.76 ± 27.52 ± 47.44 ± 28.95 ± 39.95 ± – – – – –
4.80a 3.85 4.11 2.61 0.69 1.85 1.00 0.69 8.90 6.18 0.69
IC50 (g/mL) 2 g/mL 68.01 51.47 76.71 62.07 70.61 77.09 35.04 62.70 82.68 92.50 65.51 23.97 10.09 23.41 46.39 24.71
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
10 g/mL 0.97 0.36 3.55 0.67 0.50 0.08 2.21 1.26 3.79 1.45 0.82 3.49 1.16 2.28 1.99 2.26
96.77 86.06 94.58 88.95 90.41 95.20 51.39 89.99 94.82 99.41 84.72 48.57 50.92 65.42 88.50 62.13
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.37 0.19 1.49 0.99 0.06 0.23 2.86 0.57 1.49 0.11 0.44 0.70 0.38 3.98 0.24 1.99
50 g/mL – – – – – – – – – – – 80.58 ± 84.26 ± 94.24 ± 98.59 ± 92.10 ±
0.21 0.38 0.13 0.13 0.42
1.23 (7.23)b 1.90 (4.09) 1.17 (2.52) 1.53 (3.52) 1.10 (2.45) 0.97 (3.04) 9.32 (22.3) 1.42 (2.30) 0.51 (1.69) 0.92 (3.21) 1.03 (6.90) 11.78 9.82 7.08 2.68 7.40
Value represents mean ± S.D. (n = 2). Unit of the value in the parenthesis is M. Positive control.
stock solution (1000 g/mL) was prepared by dissolving each standard compound in MeOH. Working standard solutions were prepared by a serial dilution of the standard stock solution. Five extracts were prepared using EtOH, MeOH, 70% MeOH, 30% MeOH, and H2 O in order to compare which solvents more efficiently extract flavonoids from P. aviculare. The extraction method was designed by extracting a crushed and lyophilized plant material (3.0 g) with a 100 mL solvent in a 250 mL Erlenmeyer flask on an ultrasonicator for 5 h at 45 ◦ C. The extracted solution was filtered through a disposable syringe filter (0.50 m, Dismic-25JP Advantec, Japan) prior to the injection into the HPLC system.
2.6. Validation of the HPLC analytical method The HPLC analysis method used in this study has been validated through several tests. Linearity was evaluated by the value of R2 (correlation coefficient) in the calibration curve of serial concentrations. Sensitivity was determined by the value of limit of detection (LOD) and limit of quantification (LOQ). A Signal-to-noise ratio (S/N) method was used to determine the LOD and LOQ value. The S/N ratio was 3 for LOD and 10 for LOQ. The precision and stability of the analysis method was assessed by measurements of the intra- and inter-day variability of both retention time and peak area values. To evaluate the accuracy, the recovery test was performed, spiking each standard compound to the sample solution.
2.7. Peroxynitrite-scavenging activity An assay method described by Kooy et al. [19] was used to measure the peroxynitrite-scavenging activity of the compounds, extracts, and fractions from P. aviculare. The principle of this method is to monitor the intensity of highly fluorescent rhodamine formed from non-fluorescent DHR 123 under the presence of ONOO− . The concentration of DHR 123 was 5 M. The samples were dissolved in 10% DMSO (concentration: 5 g/mL). The final fluorescent intensity was measured with or without the treatment of 10 M ONOO− in 0.3 N NaOH. The fluorescence intensity of the oxidized DHR 123 was measured at the excitation and emission of 480 nm and 530 nm using a microplate fluorescence reader FL 500 (Bio-Tek Instruments Inc., Winooski, VT, USA).
3. Results and discussion 3.1. Optimization and validation of the HPLC analytical method To establish a reliable HPLC analytical method, four parameters of mobile phase composition, gradient elution, UV wavelength, and column temperature were optimized through repetitive experimentations. An optimum formula consisting of solvent A and solvent B was selected. Solvent A was H2 O and solvent B was the mixture of MeOH and CH3 CN with a ratio of 60:40 (v/v). This composition was selected because it showed a better separation and the solvents were environment-friendly. A gradient elution method, as described in the Materials and methods section, was employed for better chromatographic separation on a wide range of polarity in a shorter time. That method worked very well when the temperature of the column was maintained at 40 ◦ C. The optimized HPLC method was validated by examining the linearity, sensitivity, precision, and accuracy. Every R2 value was more than 0.999, verifying the linearities of the calibration equations (Table 1). In the inter-day variability test, the RSDs were observed over 1.99–5.15% (Table 2), indicating that the analysis is precise and stable under the present method. Recovery rates were observed from 95.20 to 98.61% (Table 2), which indicate a sufficient accuracy of this experiment.
3.2. Peroxynitrite-scavenging effect of the constituents The peroxynitrite-scavenging activities of the ten compounds were determined (Table 4). The M unit was used for making comparisons on the structure–activity relationship. The activities of the eight flavonoids, except for juglanin, were higher than that of the positive control (l-penicillamine, 6.90 M). Comparing the activities of the three flavonols, kaempferol, quercetin, and myricetin, the order was quercetin (1.69 M) > myricetin (3.04 M) > kaempferol (3.21 M), indicating that the catechol ring in the B-ring of flavonol contributes to the activity of flavonol rather than to the pyrogallol in myricetin and phenol in the kaempferol. In the flavonol glycosides, the activities ranged over 2.45 M (quercitrin)–4.09 M (myricitrin). These results suggest that the activities of flavonol glycosides are primarily due to the aglycone rather than to the sugar moiety. Comparing the activities of myricetin, myricitrin and desmanthin-1 (2 -O-galloylmyricitrin), the order was desmanthin1 (2.30 M) > myricetin (3.04 M) > myricitrin (4.09 M). These
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Fig. 2. HPLC chromatograms of mixed standards, extract and fractions of P. aviculare.
results suggest that galloylation to myricitrin increases the peroxynitrite-scavenging activity. 3.3. Content of substances in the extract and fractions: correlation to their peroxynitrite-scavenging activities In the evaluation of peroxynitrite-scavenging activity (Table 4), the EtOAc fraction exhibited a potent activity (IC50 : 2.68 g/mL), significantly stronger than other fractions or the extract (IC50 more than 7.08 g/mL). As presented in Table 3 and Fig. 2, the flavonoids were mainly distributed in the EtOAc fraction (208.9 mg/g) rather than in the BuOH (11.90 mg/g) or Et2 O (29.92 mg/g) fractions. Therefore, it is considered that the peroxynitrite-scavenging activity of P. aviculare could be influenced by the composition or contents of the flavonoids. Contents of ten compounds in the extracts and
fractions were shown in Table 3. The sum of the flavonoid contents were shown in the order of MeOH ext. (45.16 mg/g) > 70% MeOH ext. (38.40 mg/g) > 30% MeOH (24.17 mg/g) > 0% MeOH (8.55 mg/g), indicating that a high ratio of MeOH efficiently extracts flavonoids in P. aviculare. The effects of MeOH and EtOH solvents on the extraction of flavonoids were similar. 4. Conclusion Three flavonols (myricetin, quercetin, kaempferol), their six glycosides (myricitrin, desmanthin-1, isoquercitrin, quercitrin, avicularin, and juglanin), and one phenolic acid (gallic acid) were identified in P. aviculare. Except for juglanin, the eight flavonoids showed potent peroxynitrite-scavenging activities which were stronger than l-penicillamine (IC50 : 6.90 M). It was demonstrated
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that the EtOAc fraction with the highest content of flavonoids (208.9 mg/g) exhibits the strongest peroxynitrite-scavenging activity (IC50 , 2.68 g/mL). Thus, it is suggested that flavonoids should act as the active principles of P. aviculare for its peroxynitritescavenging activity. In addition, extraction using a high ratio of MeOH efficiently extracts the flavonoids in P. aviculare. Acknowledgement This research was supported by the Sangji University Research Fund, 2013. References [1] Y.Y. Sung, T. Yoon, W.K. Yang, S.J. Kim, D.S. Kim, H.K. Kim, The antiobesity effect of Polygonum aviculare L. ethanol extract in high-fat diet-induced obese mice, Evid. Based Complement. Alternat. Med. (2013) 1–11. [2] C.Y. Hsu, Antioxidant activity of extract from Polygonum aviculare L, Biol. Res. 39 (2006) 281–288. [3] H.M.H. Salama, N. Marraiki, Antimicrobial activity and phytochemical analysis of Polygonum aviculare L. (Polygonaceae), naturally growing in Egypt, Saudi J. Biol. Sci. 17 (2010) 57–63. [4] H. Tunón, C. Olavsdotter, L. Bohlin, Evaluation of anti-inflammatory activity of some Swedish medicinal plants. Inhibition of prostaglandin biosynthesis and PAF-induced exocytosis, J. Ethnopharmacol. 48 (1995) 61–76. [5] M. González Begné, N. Yslas, E. Reyes, V. Quiroz, J. Santana, G. Jimenez, Clinical effect of a Mexican Sanguinaria extract (Polygonum aviculare L.) on gingivitis, J. Ethnopharmacol. 74 (2001) 45–51. [6] J.X. Nan, E.J. Park, H.J. Kim, G. Ko, D.H. Sohn, Antifibrotic effects of the methanol extract of Polygonum aviculare in fibrotic rats induced by bile duct ligation and scission, Biol. Pharm. Bull. 23 (2000) 240–243.
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