- Email: [email protected]
Flavonoid accumulation during florescence in three Chrysanthemum morifolium Ramat cv. ‘Hangju’ genotypes
Biochemical Systematics and Ecology 55 (2014) 79–83
Contents lists available at ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Flavonoid accumulation during florescence in three Chrysanthemum morifolium Ramat cv. ‘Hangju’ genotypes Tao Wang a, b, Qiao-sheng Guo a, *, Peng-fei Mao a a b
Institute of Chinese Medicinal Materials, Nanjing Agricultural University, Nanjing 210095, PR China Biology Post-doctoral Mobile Stations, College of Life Science, Nanjing Agricultural University, Nanjing 210095, PR China
a r t i c l e i n f o Article history: Received 25 December 2013 Accepted 21 February 2014 Available online Keywords: Chrysanthemum morifolium Flavonoids Quercetin Luteolin Optimum harvest time
1. Introduction Chrysanthemum morifolium Ramat. (Compositae) is famous as an ornamental plant, and C. morifolium Ramat. cv. ‘Hangju’ is taken as a functional drink (Van Der Ploeg and Heuvelink, 2006). Flos Chrysanthemi, the dried capitulum, is a popular herbal tea and food source in some Asian countries (Lin and Harnly, 2010; Wang et al., 2013); it is listed in the Chinese Pharmacopoeia as having the ability to “rid colds”, “remove fevers and toxins” and ‘‘brighten eyes” (Lai et al., 2007; Chinese Pharmacopoeia Committee, 2010; Jin et al., 2012). C. morifolium. cv. ‘Hangju’ originated in Tongxiang County, Zhejiang province. It has been cultivated for centuries, and several genotypes have been developed. It is rich in flavonoids, including quercetin, luteolin, apigenin and acacetin, in the form of free flavones and flavonoid glycosides. Flavonoids are used in the quality control of Flos Chrysanthemi (Chinese Pharmacopoeia Committee, 2010). The flavonoid contents of a species are determined by many factors, including the genotype and environment (Liu et al., 2010; Gao et al., 2011; Wang et al., 2013). Previous work has focused on the types of components in C. morifolium cv. ‘Hangju’ capitulum (Shao et al., 2010) and the genetic differences among the genotypes (Wang et al., 2009; Sun et al., 2010). Wang et al. (2013) found that the contents of the flavonoid glycosides and caffeic acid derivatives changed during florescence. However, few studies have monitored the levels of specific flavonoids during florescence. In this paper, a rapid and reliable HPLC method was developed for the simultaneous determination of four flavonoids (quercetin, luteolin, apigenin and acacetin) in C. morifolium cv. ‘Hangju’ capitulum. Furthermore, the flavonoid trend during florescence was compared and summarized based on their contents at different flowering stages.
* Corresponding author. Tel./fax: þ86 25 84395980. E-mail address: [email protected] (Q.-s. Guo). http://dx.doi.org/10.1016/j.bse.2014.02.023 0305-1978/Ó 2014 Elsevier Ltd. All rights reserved.
80
T. Wang et al. / Biochemical Systematics and Ecology 55 (2014) 79–83
Table 1 Sampling information for C. morifolium cv. ‘Hangju’. No.
Sample code
Origin
Location
1 2 3 4 5 6 7 8 9
TZ TW MH JZ JW JH SH RZ RW
Tong Xiang, Zhejiang province Tong Xiang, Zhejiang province Ma Cheng, Hubei province Ju Rong, Jiangsu province Ju Rong, Jiangsu province Ju Rong, Jiangsu province She Yang, Jiangsu province Rui Cheng, Shanxi province Rui Cheng, Shanxi province
N30 380 , N30 380 , N31 100 , N31 570 , N31 570 , N31 570 , N33 460 , N34 420 , N34 420 ,
Genotypes E120 320 E120 320 E115 000 E119 090 E119 090 E119 090 E120 150 E110 400 E110 400
Zao Yang Wan Yang Hong Xin Zao Yang Wang Yang Hong Xin Hong Xin Zao Yang Wan Yang
2. Materials and methods 2.1. Plant materials Three C. morifolium cv. ‘Hangju’ genotypes were collected from five areas in China in April 2010 (Table 1), and species identification was performed by Dr. Qiao-sheng Guo. These plants were re-cultivated under consistent and uniform field conditions and were then harvested at five flowering stages based on the openness of the tubular and ligulate flowers in October 2011. The following five stages were collected: stage I (tubiform flowers open 0%, ligulate flowers open 0%); II (tubiform flowers open 0%, ligulate flowers open 30%); III (tubiform flowers open 30%, ligulate flowers open 50%); IV (tubiform flowers open 50%, ligulate flowers open 70%); and V (tubiform flowers open 100%, ligulate flowers open 100%). Fresh capitulum enzymes were deactivated with 100 C steam for 3 min, and the samples were oven-dried at 60 C for 6 h and ground into a powder that could pass through a 0.5 mm sieve. 2.2. Reagents and solvents Quercetin, luteolin, acacetin and apigenin were purchased from China Pharmaceutical Biological Products (Nanjing, China). HPLC-grade methanol was purchased from Merck (Darmstadt, Germany). 2.3. Sample solution preparation Dried powder of C. morifolium (0.25 g) was mixed with 25 mL 70% methanol and extracted by sonication (300 W; 45 kHz) for 40 min. Then, 9 mL extraction was mixed with 1 mL 1 M HCl and hydrolyzed at 80 C for 1 h (Maksoud and El Hadidi, 1988). The hydrolysis solution was prepared for HPLC analysis.
Fig. 1. The typical retention times of four flavonoids: quercetin, luteolin, acacetin and apigenin.
T. Wang et al. / Biochemical Systematics and Ecology 55 (2014) 79–83
81
Table 2 Quercetin content in the genotypes of C. morifolium cv. ‘Hangju’ during florescence (n ¼ 3). Samples
I/mg g1
TZ TW MH JZ JW JH SH RZ RW
8.3 5.0 7.9 5.8 3.9 3.8 8.2 6.1 9.9
0.24 0.08 0.24 0.17 0.14 0.12 0.18 0.11 0.49
b dd eb dc ee de db ec ea e
II/mg g1
III/mg g1
13.9 0.34 db 16.2 0.53 ca 11.7 0.36 ccd 7.3 0.20 cf 11.1 0.47 cd 6.6 0.17 cg 9.5 0.27 ce 15.8 0.45 da 11.9 0.38 cc
20.9 18.8 12.8 12.7 15.0 7.7 12.0 18.5 16.0
0.13 0.51 0.17 0.36 0.60 0.19 0.48 0.63 0.67
IV/mg g1 a bb be be bd bf be cb bc b
28.7 23.6 18.6 13.8 17.5 9.0 17.2 22.6 25.0
0.51 0.60 0.56 0.60 0.64 0.25 0.55 0.71 0.82
V/mg g1 a ac ad af ae ag ae ac ab a
17.4 18.1 9.1 7.9 9.5 6.3 8.7 21.4 13.0
0.59 cc 0.63 bb 0.37 def 0.29cg 0.24 de 0.14 ch 0.20 df 0.67 ba 0.21 dd
Note: A superscript within a row with the same lowercase superscript letter indicates no significant difference; a subscript within a column with the same lowercase subscript letter indicates no significant difference (P < 0.05).
2.4. Determination of quercetin, luteolin, apigenin and acacetin All analyses were performed on an Agilent 1120 Compact LC (G4288A) system (Agilent Technologies, USA) equipped with a Shim–pack C18 column (4.6 mm 250 mm, 5 mm) at 25 C and was controlled by the Agilent 1120 software. The gradient elution system consisted of (A) methanol and (B) 0.2% H3PO4 as the mobile phase, and the ratio (A:B) was 60:40 (v:v) with a flow rate of 1 mL min1. The analytes were simultaneously monitored at 253 nm. A mixed stock standard solution of quercetin, luteolin, apigenin and acacetin was prepared in methanol with concentrations of 51.36, 102.40, 26.88, and 101.12 mg L1, respectively, and every injection volume was 20 mL. Calibration curves were generated for each of the flavonoid standards using linear regression, and these curves were used to calculate the amount of each flavonoid in the extracts. The retention times of the four flavonoids are shown in Fig. 1. 2.5. Statistical analysis All results are expressed as the mean values standard deviation (n ¼ 3). Significant difference was calculated using a oneway ANOVA, and values of p < 0.05 were considered significant. Statistical analyses were conducted using the SPSS 18.0 software package for Windows. 3. Results and discussion 3.1. A comparison of flavonoids in the genotypes from different areas 3.1.1. Comparison of quercetin content in genotypes from different areas The quercetin content varied among the genotypes, but the content increased from stage I to stage IV flower development in all genotypes then declined at stage V (Table 2). TZ had the greatest content of quercetin at stage IV (28.7 mg g1), whereas genotype JH contained the lowest amount of quercetin at stage IV (9.0 mg g1). 3.1.2. Comparison of luteolin content in genotypes from different areas Luteolin content in the capitulum varied over the course of flowering in all genotypes (Table 3). The content was greatest at either stage III or IV and then declined. At stage I, TW had the highest luteolin content (17.1 mg g1), which was over three times greater than that found in the SH (5.4 mg g1). Overall, SH had the lowest luteolin content, RW had the greatest luteolin content at stage III (26.4 mg g1), whereas TZ had the greatest luteolin at stage IV (26.5 mg g1).
Table 3 Luteolin content in the genotypes of C. morifolium cv. ‘Hangju’ during florescence (n ¼ 3). Samples
I/mg g1
TZ TW MH JZ JW JH SH RZ RW
12.7 17.1 10.2 12.5 9.0 7.4 5.4 11.2 16.5
0.45 0.32 0.11 0.34 0.23 0.09 0.20 0.38 0.55
II/mg g1 c a ee dc cf dg eh ed cb d c
17.4 20.9 14.7 16.3 12.9 9.2 7.9 17.2 23.8
0.69 0.28 0.24 0.38 0.37 0.31 0.18 0.44 0.28
III/mg g1 c b ce bd bf cg ch bc ba c
b
21.3 24.2 16.0 18.4 17.4 12.2 8.8 20.0 26.4
0.92 0.93 0.46 0.41 0.75 0.26 0.15 0.15 0.73
IV/mg g1 c b bf ae ae ag bh ad aa b a
26.5 14.5 18.0 13.9 12.5 10.5 9.6 15.7 17.3
0.57 0.39 0.75 0.12 0.43 0.48 0.28 0.53 0.62
V/mg g1 a d ab cd be bf ag cc cb a
d
14.3 13.1 13.0 10.1 9.2 8.6 5.9 12.9 14.1
0.38 0.37 0.35 0.15 0.16 0.36 0.13 0.49 0.57
a b db ec cd cd de db da e e
Note: A superscript within a row with the same lowercase superscript letter indicates no significant difference; a subscript within a column with the same lowercase subscript letter indicates no significant difference (P < 0.05).
82
T. Wang et al. / Biochemical Systematics and Ecology 55 (2014) 79–83
Table 4 Apigenin content in the genotypes of C. morifolium cv. ‘Hangju’ during florescence (n ¼ 3). Samples
I/mg g1
TZ TW MH JZ JW JH SH RZ RW
7.0 5.9 4.4 9.6 4.0 16.4 2.3 7.0 3.6
0.21 ec 0.16 dd 0.12de 0.47 db 0.20 ee 0.23 da 0.06 bg 0.15 cc 0.15 df
II/mg g1 11.9 8.5 5.1 12.5 10.1 18.7 3.4 9.0 11.5
0.16 0.25 0.18 0.33 0.31 0.15 0.13 0.30 0.38
III/mg g1 c cf cg bb bd ba ch be cc b
13.8 15.1 7.4 14.9 11.1 23.9 3.5 11.0 17.7
0.31 0.46 0.15 0.40 0.23 0.19 0.06 0.37 0.33
IV/mg g1 d bc bf ac ae aa cg ae ab a
9.8 23.8 8.6 11.2 6.5 19.1 3.7 6.0 12.3
0.29 0.44 0.21 0.22 0.09 0.25 0.11 0.19 0.42
V/mg g1 e aa af cd cg bb ai dh bc c
8.4 7.6 4.7 9.5 5.4 17.3 3.3 4.0 3.3
0.18 0.35 0.10 0.06 0.21 0.32 0.11 0.10 0.12
c d df db de ca ch eg dh d e
Note: A superscript within a row with the same lowercase superscript letter indicates no significant difference; a subscript within a column with the same lowercase subscript letter indicates no significant difference (P < 0.05).
3.1.3. Comparison of apigenin content in genotypes from different areas During stages I to V (Table 4), apigenin content exhibited significant variations in the different genotypes, with the highest content at either stage III or IV, followed by a decline. SH had the lowest values of 3.5 and 3.7 mg g1 apigenin at stages III and IV, respectively. JW possessed the highest apigenin content at stage III of 23.9 mg g1, followed by TW (23.8 mg g1) at stage IV. 3.1.4. Comparison of acacetin content in genotypes from different areas Acacetin (Table 5) variation among the genotypes over the course of flowering was similar to that of the previous three flavonoids. The greatest concentration appeared at either stage III or IV then declined. The lowest acacetin content at stage III was found in SH (1.2 mg g1), and RW exhibited the lowest concentration of 0.6 mg g1 at stage IV. In contrast, at stages I and IV, JH had the greatest concentration of 10.1 and 8.4 mg g1 acacetin, respectively. At the same stage, the contents of the mentioned flavonoids varied by genotype and cultivated area. Previous studies in other medicinal plants, such as Glechoma longituba, found that the flavonoid species were similar among the genotypes, but their contents also varied with genotypes and areas (Liu et al., 2012). 3.2. The trend of flavonoid accumulation during C. morifolium cv. ‘Hangju’ florescence Some studies indicated that flavonoid accumulation occurred with floral organ development (Dong et al., 1998; Weiss and Halevy, 1991). Flavonoid contents in C. morifolium cv. ‘Hangju’ constantly changed over the course of flowering with the degree of flower openness. At stage III or stage IV, the flavonoid contents were highest and then declined; the general trend was a “peak shape”. It was reported that the contents of two flavonoid glycosides in C. morifolium exhibited a dynamic process throughout flowering (Wang et al., 2013), and our results agreed with this study. The “peak shape” course may relate to the variation in chalcone isomerase (CHI), which is the key enzyme in flavonoid biosynthesis. During early florescence, CHI activity was enhanced, and the flavonoids gradually accumulated; at the end of florescence, CHI activity was inhibited, followed by a decrease in flavonoid contents (Nishihara et al., 2005). 3.3. Discussion of optimal cultivated area Tongxiang County is the traditional origin of C. morifolium cv. ‘Hangju’ (Shao et al., 2010; Sun et al., 2010). Wang et al. (2013) suggested that C. morifolium cv. ‘Hangju’ from Tongxiang County had the advantage in contents of flavonoid glycosides and caffeoylquinic acid derivatives than the other areas. Comparing the peaks of the four flavonoids, TZ (genotype ZaoYang from Tongxiang County) possessed the highest peak value of quercetin and luteolin, while the peak of apigenin occurred in TW
Table 5 Acacetin content in the genotypes of C. morifolium cv. ‘Hangju’ during florescence (n ¼ 3). Samples
I/mg g1
TZ TW MH JZ JW JH SH RZ RW
1.0 1.2 1.0 1.6 0.9 6.9 0.6 0.9 0.9
0.06 dd 0.06 dc 0.06 dd 0.07 cb 0.03cd 0.19 da 0.09 ee 0.02 cd 0.05 cd
II/mg g1 1.6 2.0 1.3 1.8 1.4 9.2 0.9 1.6 1.3
0.05 cde 0.08 bb 0.05 cg 0.06 bc 0.06bef 0.17 ba 0.08 ch 0.07bd 0.11 bfg
III/mg g1 2.7 2.6 1.4 2.3 1.8 10.1 1.2 2.8 1.8
0.08 abc 0.10 ac 0.06 bf 0.05 ad 0.09ae 0.23 aa 0.06 bg 0.10 ab 0.10 ae
IV/mg g1 1.9 1.9 2.0 1.7 0.9 8.4 1.9 1.5 0.6
0.07 bbc 0.11 bbc 0.11 ab 0.09 bc 0.06ce 0.11 ca 0.08 abc 0.05 bd 0.06 df
V/mg g1 1.6 1.4 0.9 1.4 0.8 8.1 0.8 0.6 0.6
0.06 cb 0.07 cc 0.05 dd 0.08dc 0.11 cd 0.19 ca 0.03 dde 0.06 def 0.04 df
Note: A superscript within a row with the same lowercase superscript letter indicates no significant difference; a subscript within a column with the same lowercase subscript letter indicates no significant difference (P < 0.05).
T. Wang et al. / Biochemical Systematics and Ecology 55 (2014) 79–83
83
(genotype Wanyang from Tongxiang County). Overall, based on the standard of flavonoid quality, we agree that Tongxiang County is a favorable area for C. morifolium cv. ‘Hangju’ cultivation. 3.4. Optimum harvest time depends on quercetin, luteolin, apigenin and acacetin The greatest quercetin concentration occurred at florescence Stage IV, so this stage may be the reasonable harvest time for quercetin. Similarly, TW, JZ, JW, JH, RZ and RW should be harvested at stage III due to the high luteolin concentrations; for TZ, MH and SH, this opportunity was at stage IV. Considering the optimum apigenin level, TZ, JZ, JW, JH, RZ and RW should be harvested at stage III, while TW, MH and SH should be harvested at stage IV. Stage III would be the appropriate time to harvest TZ, TW, JW, JH, RZ and RW based on the highest acacetin level; stage IV would suit MH, JZ and SH. Kang and Lee (2011) considered that determining the optimal harvest time was the key to obtaining phenolic phytochemicals (including flavonoids) in Perilla frutescens. Wang et al. (2013) noted that more flavonoid glycosides could be obtained if C. morifolium cv. ‘Hangju’ was picked at a suitable time. For maximum flavonoid bioavailability, choosing a reasonable harvest time seems to be particularly meaningful for C. morifolium cv. ‘Hangju’. Acknowledgments This study was financially supported by the National Major Scientific and Technological Special Project for ‘Significant New Drug Development’ (2009ZX09308-002). References Chinese Pharmacopoeia Committee, 2010. In: Pharmacopoeia of the People’s Republic of China, Chinese ed. 2010. China Medico-Pharmaceutical Science and Technology Press, Beijing, p. 292. Part I. Dong, Y.H., Beuning, L., Davies, K., Mitra, D., Morris, B., Kootstra, A., 1998. Aust. J. Plant Physiol. 25, 245. Gao, C.Y., Lu, Y.H., Tian, C.R., Xu, J.G., Guo, X.P., Zhou, R., Hao, G., 2011. Food Chem. 127, 615. Jin, M., Zhu, Z.B., Guo, Q.S., Shen, H.J., Wang, Y.R., 2012. J. Med. Plants Res. 6, 398. Kang, N.S., Lee, J.H., 2011. Food Chem. 124, 556. Lai, J.P., Lim, Y.H., Su, J., Shen, H.M., Choon, N.O., 2007. J. Chramatogr. B. 848 (2), 215. Lin, L.Z., Harnly, J.M., 2010. Food Chem. 120, 319. Liu, L., Zhu, Z.B., Guo, Q.S., Zhang, L.X., He, Q., Liu, Z., 2012. J. Med. Plants Res. 6, 122. Liu, W., Zu, Y.G., Fu, Y.J., Kong, Y., Ma, W., Yang, M., Li, J., Wu, N., 2010. Variation in contents of phenolic compounds during growth and post-harvest storage of pigeon pea seedlings. Food Chem. 121, 732–739. Maksoud, S.A., El Hadidi, M.N., 1988. Plant Syst. Evol. 160, 153. Nishihara, M., Nakatsuka, T., Yamamura, S., 2005. FEBS Lett. 579, 6074. Shao, Q.S., Guo, Q.S., Deng, Y.M., Guo, H.P., 2010. Biochem. Syst. Ecol. 38, 1160. Sun, Q.L., Hua, S., Ye, J.H., Zheng, X.Q., Liang, Y.R., 2010. Afr. J. Biotechnol. 25, 3817. Van Der Ploeg, A., Heuvelink, E., 2006. J. Hortic. Sci. Biotechnol. 81, 174. Wang, T., Zhu, Z.B., Guo, Q.S., Mao, P.F., 2013. Biochem. Syst. Ecol. 47, 74. Wang, Y.J., Yang, X.W., Guo, Q.S., Liu, H.T., 2009. Chromatographia 70, 109. Weiss, D., Halevy, A.H., 1991. Physiol. Plantarum 81, 127.
Contents lists available at ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Flavonoid accumulation during florescence in three Chrysanthemum morifolium Ramat cv. ‘Hangju’ genotypes Tao Wang a, b, Qiao-sheng Guo a, *, Peng-fei Mao a a b
Institute of Chinese Medicinal Materials, Nanjing Agricultural University, Nanjing 210095, PR China Biology Post-doctoral Mobile Stations, College of Life Science, Nanjing Agricultural University, Nanjing 210095, PR China
a r t i c l e i n f o Article history: Received 25 December 2013 Accepted 21 February 2014 Available online Keywords: Chrysanthemum morifolium Flavonoids Quercetin Luteolin Optimum harvest time
1. Introduction Chrysanthemum morifolium Ramat. (Compositae) is famous as an ornamental plant, and C. morifolium Ramat. cv. ‘Hangju’ is taken as a functional drink (Van Der Ploeg and Heuvelink, 2006). Flos Chrysanthemi, the dried capitulum, is a popular herbal tea and food source in some Asian countries (Lin and Harnly, 2010; Wang et al., 2013); it is listed in the Chinese Pharmacopoeia as having the ability to “rid colds”, “remove fevers and toxins” and ‘‘brighten eyes” (Lai et al., 2007; Chinese Pharmacopoeia Committee, 2010; Jin et al., 2012). C. morifolium. cv. ‘Hangju’ originated in Tongxiang County, Zhejiang province. It has been cultivated for centuries, and several genotypes have been developed. It is rich in flavonoids, including quercetin, luteolin, apigenin and acacetin, in the form of free flavones and flavonoid glycosides. Flavonoids are used in the quality control of Flos Chrysanthemi (Chinese Pharmacopoeia Committee, 2010). The flavonoid contents of a species are determined by many factors, including the genotype and environment (Liu et al., 2010; Gao et al., 2011; Wang et al., 2013). Previous work has focused on the types of components in C. morifolium cv. ‘Hangju’ capitulum (Shao et al., 2010) and the genetic differences among the genotypes (Wang et al., 2009; Sun et al., 2010). Wang et al. (2013) found that the contents of the flavonoid glycosides and caffeic acid derivatives changed during florescence. However, few studies have monitored the levels of specific flavonoids during florescence. In this paper, a rapid and reliable HPLC method was developed for the simultaneous determination of four flavonoids (quercetin, luteolin, apigenin and acacetin) in C. morifolium cv. ‘Hangju’ capitulum. Furthermore, the flavonoid trend during florescence was compared and summarized based on their contents at different flowering stages.
* Corresponding author. Tel./fax: þ86 25 84395980. E-mail address: [email protected] (Q.-s. Guo). http://dx.doi.org/10.1016/j.bse.2014.02.023 0305-1978/Ó 2014 Elsevier Ltd. All rights reserved.
80
T. Wang et al. / Biochemical Systematics and Ecology 55 (2014) 79–83
Table 1 Sampling information for C. morifolium cv. ‘Hangju’. No.
Sample code
Origin
Location
1 2 3 4 5 6 7 8 9
TZ TW MH JZ JW JH SH RZ RW
Tong Xiang, Zhejiang province Tong Xiang, Zhejiang province Ma Cheng, Hubei province Ju Rong, Jiangsu province Ju Rong, Jiangsu province Ju Rong, Jiangsu province She Yang, Jiangsu province Rui Cheng, Shanxi province Rui Cheng, Shanxi province
N30 380 , N30 380 , N31 100 , N31 570 , N31 570 , N31 570 , N33 460 , N34 420 , N34 420 ,
Genotypes E120 320 E120 320 E115 000 E119 090 E119 090 E119 090 E120 150 E110 400 E110 400
Zao Yang Wan Yang Hong Xin Zao Yang Wang Yang Hong Xin Hong Xin Zao Yang Wan Yang
2. Materials and methods 2.1. Plant materials Three C. morifolium cv. ‘Hangju’ genotypes were collected from five areas in China in April 2010 (Table 1), and species identification was performed by Dr. Qiao-sheng Guo. These plants were re-cultivated under consistent and uniform field conditions and were then harvested at five flowering stages based on the openness of the tubular and ligulate flowers in October 2011. The following five stages were collected: stage I (tubiform flowers open 0%, ligulate flowers open 0%); II (tubiform flowers open 0%, ligulate flowers open 30%); III (tubiform flowers open 30%, ligulate flowers open 50%); IV (tubiform flowers open 50%, ligulate flowers open 70%); and V (tubiform flowers open 100%, ligulate flowers open 100%). Fresh capitulum enzymes were deactivated with 100 C steam for 3 min, and the samples were oven-dried at 60 C for 6 h and ground into a powder that could pass through a 0.5 mm sieve. 2.2. Reagents and solvents Quercetin, luteolin, acacetin and apigenin were purchased from China Pharmaceutical Biological Products (Nanjing, China). HPLC-grade methanol was purchased from Merck (Darmstadt, Germany). 2.3. Sample solution preparation Dried powder of C. morifolium (0.25 g) was mixed with 25 mL 70% methanol and extracted by sonication (300 W; 45 kHz) for 40 min. Then, 9 mL extraction was mixed with 1 mL 1 M HCl and hydrolyzed at 80 C for 1 h (Maksoud and El Hadidi, 1988). The hydrolysis solution was prepared for HPLC analysis.
Fig. 1. The typical retention times of four flavonoids: quercetin, luteolin, acacetin and apigenin.
T. Wang et al. / Biochemical Systematics and Ecology 55 (2014) 79–83
81
Table 2 Quercetin content in the genotypes of C. morifolium cv. ‘Hangju’ during florescence (n ¼ 3). Samples
I/mg g1
TZ TW MH JZ JW JH SH RZ RW
8.3 5.0 7.9 5.8 3.9 3.8 8.2 6.1 9.9
0.24 0.08 0.24 0.17 0.14 0.12 0.18 0.11 0.49
b dd eb dc ee de db ec ea e
II/mg g1
III/mg g1
13.9 0.34 db 16.2 0.53 ca 11.7 0.36 ccd 7.3 0.20 cf 11.1 0.47 cd 6.6 0.17 cg 9.5 0.27 ce 15.8 0.45 da 11.9 0.38 cc
20.9 18.8 12.8 12.7 15.0 7.7 12.0 18.5 16.0
0.13 0.51 0.17 0.36 0.60 0.19 0.48 0.63 0.67
IV/mg g1 a bb be be bd bf be cb bc b
28.7 23.6 18.6 13.8 17.5 9.0 17.2 22.6 25.0
0.51 0.60 0.56 0.60 0.64 0.25 0.55 0.71 0.82
V/mg g1 a ac ad af ae ag ae ac ab a
17.4 18.1 9.1 7.9 9.5 6.3 8.7 21.4 13.0
0.59 cc 0.63 bb 0.37 def 0.29cg 0.24 de 0.14 ch 0.20 df 0.67 ba 0.21 dd
Note: A superscript within a row with the same lowercase superscript letter indicates no significant difference; a subscript within a column with the same lowercase subscript letter indicates no significant difference (P < 0.05).
2.4. Determination of quercetin, luteolin, apigenin and acacetin All analyses were performed on an Agilent 1120 Compact LC (G4288A) system (Agilent Technologies, USA) equipped with a Shim–pack C18 column (4.6 mm 250 mm, 5 mm) at 25 C and was controlled by the Agilent 1120 software. The gradient elution system consisted of (A) methanol and (B) 0.2% H3PO4 as the mobile phase, and the ratio (A:B) was 60:40 (v:v) with a flow rate of 1 mL min1. The analytes were simultaneously monitored at 253 nm. A mixed stock standard solution of quercetin, luteolin, apigenin and acacetin was prepared in methanol with concentrations of 51.36, 102.40, 26.88, and 101.12 mg L1, respectively, and every injection volume was 20 mL. Calibration curves were generated for each of the flavonoid standards using linear regression, and these curves were used to calculate the amount of each flavonoid in the extracts. The retention times of the four flavonoids are shown in Fig. 1. 2.5. Statistical analysis All results are expressed as the mean values standard deviation (n ¼ 3). Significant difference was calculated using a oneway ANOVA, and values of p < 0.05 were considered significant. Statistical analyses were conducted using the SPSS 18.0 software package for Windows. 3. Results and discussion 3.1. A comparison of flavonoids in the genotypes from different areas 3.1.1. Comparison of quercetin content in genotypes from different areas The quercetin content varied among the genotypes, but the content increased from stage I to stage IV flower development in all genotypes then declined at stage V (Table 2). TZ had the greatest content of quercetin at stage IV (28.7 mg g1), whereas genotype JH contained the lowest amount of quercetin at stage IV (9.0 mg g1). 3.1.2. Comparison of luteolin content in genotypes from different areas Luteolin content in the capitulum varied over the course of flowering in all genotypes (Table 3). The content was greatest at either stage III or IV and then declined. At stage I, TW had the highest luteolin content (17.1 mg g1), which was over three times greater than that found in the SH (5.4 mg g1). Overall, SH had the lowest luteolin content, RW had the greatest luteolin content at stage III (26.4 mg g1), whereas TZ had the greatest luteolin at stage IV (26.5 mg g1).
Table 3 Luteolin content in the genotypes of C. morifolium cv. ‘Hangju’ during florescence (n ¼ 3). Samples
I/mg g1
TZ TW MH JZ JW JH SH RZ RW
12.7 17.1 10.2 12.5 9.0 7.4 5.4 11.2 16.5
0.45 0.32 0.11 0.34 0.23 0.09 0.20 0.38 0.55
II/mg g1 c a ee dc cf dg eh ed cb d c
17.4 20.9 14.7 16.3 12.9 9.2 7.9 17.2 23.8
0.69 0.28 0.24 0.38 0.37 0.31 0.18 0.44 0.28
III/mg g1 c b ce bd bf cg ch bc ba c
b
21.3 24.2 16.0 18.4 17.4 12.2 8.8 20.0 26.4
0.92 0.93 0.46 0.41 0.75 0.26 0.15 0.15 0.73
IV/mg g1 c b bf ae ae ag bh ad aa b a
26.5 14.5 18.0 13.9 12.5 10.5 9.6 15.7 17.3
0.57 0.39 0.75 0.12 0.43 0.48 0.28 0.53 0.62
V/mg g1 a d ab cd be bf ag cc cb a
d
14.3 13.1 13.0 10.1 9.2 8.6 5.9 12.9 14.1
0.38 0.37 0.35 0.15 0.16 0.36 0.13 0.49 0.57
a b db ec cd cd de db da e e
Note: A superscript within a row with the same lowercase superscript letter indicates no significant difference; a subscript within a column with the same lowercase subscript letter indicates no significant difference (P < 0.05).
82
T. Wang et al. / Biochemical Systematics and Ecology 55 (2014) 79–83
Table 4 Apigenin content in the genotypes of C. morifolium cv. ‘Hangju’ during florescence (n ¼ 3). Samples
I/mg g1
TZ TW MH JZ JW JH SH RZ RW
7.0 5.9 4.4 9.6 4.0 16.4 2.3 7.0 3.6
0.21 ec 0.16 dd 0.12de 0.47 db 0.20 ee 0.23 da 0.06 bg 0.15 cc 0.15 df
II/mg g1 11.9 8.5 5.1 12.5 10.1 18.7 3.4 9.0 11.5
0.16 0.25 0.18 0.33 0.31 0.15 0.13 0.30 0.38
III/mg g1 c cf cg bb bd ba ch be cc b
13.8 15.1 7.4 14.9 11.1 23.9 3.5 11.0 17.7
0.31 0.46 0.15 0.40 0.23 0.19 0.06 0.37 0.33
IV/mg g1 d bc bf ac ae aa cg ae ab a
9.8 23.8 8.6 11.2 6.5 19.1 3.7 6.0 12.3
0.29 0.44 0.21 0.22 0.09 0.25 0.11 0.19 0.42
V/mg g1 e aa af cd cg bb ai dh bc c
8.4 7.6 4.7 9.5 5.4 17.3 3.3 4.0 3.3
0.18 0.35 0.10 0.06 0.21 0.32 0.11 0.10 0.12
c d df db de ca ch eg dh d e
Note: A superscript within a row with the same lowercase superscript letter indicates no significant difference; a subscript within a column with the same lowercase subscript letter indicates no significant difference (P < 0.05).
3.1.3. Comparison of apigenin content in genotypes from different areas During stages I to V (Table 4), apigenin content exhibited significant variations in the different genotypes, with the highest content at either stage III or IV, followed by a decline. SH had the lowest values of 3.5 and 3.7 mg g1 apigenin at stages III and IV, respectively. JW possessed the highest apigenin content at stage III of 23.9 mg g1, followed by TW (23.8 mg g1) at stage IV. 3.1.4. Comparison of acacetin content in genotypes from different areas Acacetin (Table 5) variation among the genotypes over the course of flowering was similar to that of the previous three flavonoids. The greatest concentration appeared at either stage III or IV then declined. The lowest acacetin content at stage III was found in SH (1.2 mg g1), and RW exhibited the lowest concentration of 0.6 mg g1 at stage IV. In contrast, at stages I and IV, JH had the greatest concentration of 10.1 and 8.4 mg g1 acacetin, respectively. At the same stage, the contents of the mentioned flavonoids varied by genotype and cultivated area. Previous studies in other medicinal plants, such as Glechoma longituba, found that the flavonoid species were similar among the genotypes, but their contents also varied with genotypes and areas (Liu et al., 2012). 3.2. The trend of flavonoid accumulation during C. morifolium cv. ‘Hangju’ florescence Some studies indicated that flavonoid accumulation occurred with floral organ development (Dong et al., 1998; Weiss and Halevy, 1991). Flavonoid contents in C. morifolium cv. ‘Hangju’ constantly changed over the course of flowering with the degree of flower openness. At stage III or stage IV, the flavonoid contents were highest and then declined; the general trend was a “peak shape”. It was reported that the contents of two flavonoid glycosides in C. morifolium exhibited a dynamic process throughout flowering (Wang et al., 2013), and our results agreed with this study. The “peak shape” course may relate to the variation in chalcone isomerase (CHI), which is the key enzyme in flavonoid biosynthesis. During early florescence, CHI activity was enhanced, and the flavonoids gradually accumulated; at the end of florescence, CHI activity was inhibited, followed by a decrease in flavonoid contents (Nishihara et al., 2005). 3.3. Discussion of optimal cultivated area Tongxiang County is the traditional origin of C. morifolium cv. ‘Hangju’ (Shao et al., 2010; Sun et al., 2010). Wang et al. (2013) suggested that C. morifolium cv. ‘Hangju’ from Tongxiang County had the advantage in contents of flavonoid glycosides and caffeoylquinic acid derivatives than the other areas. Comparing the peaks of the four flavonoids, TZ (genotype ZaoYang from Tongxiang County) possessed the highest peak value of quercetin and luteolin, while the peak of apigenin occurred in TW
Table 5 Acacetin content in the genotypes of C. morifolium cv. ‘Hangju’ during florescence (n ¼ 3). Samples
I/mg g1
TZ TW MH JZ JW JH SH RZ RW
1.0 1.2 1.0 1.6 0.9 6.9 0.6 0.9 0.9
0.06 dd 0.06 dc 0.06 dd 0.07 cb 0.03cd 0.19 da 0.09 ee 0.02 cd 0.05 cd
II/mg g1 1.6 2.0 1.3 1.8 1.4 9.2 0.9 1.6 1.3
0.05 cde 0.08 bb 0.05 cg 0.06 bc 0.06bef 0.17 ba 0.08 ch 0.07bd 0.11 bfg
III/mg g1 2.7 2.6 1.4 2.3 1.8 10.1 1.2 2.8 1.8
0.08 abc 0.10 ac 0.06 bf 0.05 ad 0.09ae 0.23 aa 0.06 bg 0.10 ab 0.10 ae
IV/mg g1 1.9 1.9 2.0 1.7 0.9 8.4 1.9 1.5 0.6
0.07 bbc 0.11 bbc 0.11 ab 0.09 bc 0.06ce 0.11 ca 0.08 abc 0.05 bd 0.06 df
V/mg g1 1.6 1.4 0.9 1.4 0.8 8.1 0.8 0.6 0.6
0.06 cb 0.07 cc 0.05 dd 0.08dc 0.11 cd 0.19 ca 0.03 dde 0.06 def 0.04 df
Note: A superscript within a row with the same lowercase superscript letter indicates no significant difference; a subscript within a column with the same lowercase subscript letter indicates no significant difference (P < 0.05).
T. Wang et al. / Biochemical Systematics and Ecology 55 (2014) 79–83
83
(genotype Wanyang from Tongxiang County). Overall, based on the standard of flavonoid quality, we agree that Tongxiang County is a favorable area for C. morifolium cv. ‘Hangju’ cultivation. 3.4. Optimum harvest time depends on quercetin, luteolin, apigenin and acacetin The greatest quercetin concentration occurred at florescence Stage IV, so this stage may be the reasonable harvest time for quercetin. Similarly, TW, JZ, JW, JH, RZ and RW should be harvested at stage III due to the high luteolin concentrations; for TZ, MH and SH, this opportunity was at stage IV. Considering the optimum apigenin level, TZ, JZ, JW, JH, RZ and RW should be harvested at stage III, while TW, MH and SH should be harvested at stage IV. Stage III would be the appropriate time to harvest TZ, TW, JW, JH, RZ and RW based on the highest acacetin level; stage IV would suit MH, JZ and SH. Kang and Lee (2011) considered that determining the optimal harvest time was the key to obtaining phenolic phytochemicals (including flavonoids) in Perilla frutescens. Wang et al. (2013) noted that more flavonoid glycosides could be obtained if C. morifolium cv. ‘Hangju’ was picked at a suitable time. For maximum flavonoid bioavailability, choosing a reasonable harvest time seems to be particularly meaningful for C. morifolium cv. ‘Hangju’. Acknowledgments This study was financially supported by the National Major Scientific and Technological Special Project for ‘Significant New Drug Development’ (2009ZX09308-002). References Chinese Pharmacopoeia Committee, 2010. In: Pharmacopoeia of the People’s Republic of China, Chinese ed. 2010. China Medico-Pharmaceutical Science and Technology Press, Beijing, p. 292. Part I. Dong, Y.H., Beuning, L., Davies, K., Mitra, D., Morris, B., Kootstra, A., 1998. Aust. J. Plant Physiol. 25, 245. Gao, C.Y., Lu, Y.H., Tian, C.R., Xu, J.G., Guo, X.P., Zhou, R., Hao, G., 2011. Food Chem. 127, 615. Jin, M., Zhu, Z.B., Guo, Q.S., Shen, H.J., Wang, Y.R., 2012. J. Med. Plants Res. 6, 398. Kang, N.S., Lee, J.H., 2011. Food Chem. 124, 556. Lai, J.P., Lim, Y.H., Su, J., Shen, H.M., Choon, N.O., 2007. J. Chramatogr. B. 848 (2), 215. Lin, L.Z., Harnly, J.M., 2010. Food Chem. 120, 319. Liu, L., Zhu, Z.B., Guo, Q.S., Zhang, L.X., He, Q., Liu, Z., 2012. J. Med. Plants Res. 6, 122. Liu, W., Zu, Y.G., Fu, Y.J., Kong, Y., Ma, W., Yang, M., Li, J., Wu, N., 2010. Variation in contents of phenolic compounds during growth and post-harvest storage of pigeon pea seedlings. Food Chem. 121, 732–739. Maksoud, S.A., El Hadidi, M.N., 1988. Plant Syst. Evol. 160, 153. Nishihara, M., Nakatsuka, T., Yamamura, S., 2005. FEBS Lett. 579, 6074. Shao, Q.S., Guo, Q.S., Deng, Y.M., Guo, H.P., 2010. Biochem. Syst. Ecol. 38, 1160. Sun, Q.L., Hua, S., Ye, J.H., Zheng, X.Q., Liang, Y.R., 2010. Afr. J. Biotechnol. 25, 3817. Van Der Ploeg, A., Heuvelink, E., 2006. J. Hortic. Sci. Biotechnol. 81, 174. Wang, T., Zhu, Z.B., Guo, Q.S., Mao, P.F., 2013. Biochem. Syst. Ecol. 47, 74. Wang, Y.J., Yang, X.W., Guo, Q.S., Liu, H.T., 2009. Chromatographia 70, 109. Weiss, D., Halevy, A.H., 1991. Physiol. Plantarum 81, 127.