Hort. Environ. Biotechnol. 54(5):441-449. 2013. DOI 10.

1007/s13580-013-0171-2

ISSN (p rin t) : 2211-3452 ISSN (online) : 2211-3460

Research Report

Accumulation of Flavonoids and Antioxidant Activity of Stellera chamaejasme by an Efficient Callus Culture
Junli Wang*, Xuan Xiao, Qian Wang, Xiaoxu Li, Lu Zhang, and Jianfei Li
College of Life and Environmental Sciences, Minzu University of China, Beijing, 100081, Peoples Republic of China
*Corresponding author: wangjunli2008@yahoo.com.cn, wangjunli1698@163.com

Received November 25, 2012 / Revised September 6, 2013 / Accepted September 17, 2013 GKorean Society for Horticultural Science and Springer 2013

Abstract. An efficient callus proliferation system of Stellera chamaejasme was developed. The calli were initially induced by cultivating the leaf explants on the MS medium containing 1.0 mgL-1 2,4-dichlorophenoxy acetic acid (2,4-D). The culture had its fresh and dry weights increased by about 29 and 25 times, respectively, through further cultivation on the MS medium containing 0.5 mgL-1 -naphthalene acetic acid (NAA) and 1.0 mgL-1 n-phenyl-n -1,2,3-thiadiazol-5-ylurea (TDZ). The concentrations of NAA and 6-benzylaminopurine (BA) for an efficient accumulation -1 -1 of the total flavonoids in the callus were found to be 1.0 mgL and 0.25 mgL , respectively. With this combination, -1 the content of the total flavonoids slightly increased to 10.8 mgg dry weight (DW) in comparison to 10.1 mgg-1 DW obtained in the root of wild-type plant. The antioxidant activities of all flavonoid extracts were evaluated by -1 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The flavonoid extracts from the callus as induced by 1.0 mgL NAA -1 and 0.25 or 0.5 mgL BA was very active in radical scavenging, and their IC50 values were 11.94 and 19.17 gmL-1, respectively. Compared to the ascorbic acid (IC50 21.21 gmL-1), the antioxidant activity of callus from S. chamaejasme was even stronger, suggesting that be another potential source of new natural antioxidants. Additional key words: callus proliferation, plant growth regulators, radical scavenging activity

Introduction
Free radicals are known to make oxidative damages to biomolecules and cells, accelerating aging and increasing the susceptibilities to cardiovascular diseases, cancers, impairment of immune system, and inflammatory diseases (Finkel and Holbrook, 2000; Hsouna et al., 2011; Wu and Hansen, 2008). Antioxidants, which could be produced as natural derivatives by some medicinal plants (Huo et al., 2010), are molecules capable of scavenging the free radicals that potentially attack the biomolecules and the cells (Erkana et al., 2011). Stellera chamaejasme L., known also as Gelsemium elegans, is a perennial plant that belongs to the Thymelaeaceae family (Flora of China Editorial Committee of Chinese Academy of Sciences, 1999). The root of Stellera chamaejasme has long been used as a traditional medicinal herb in Mongolia and Tibet of China with the following pharmacological functions: anti-bacterial, anti-inflammatory, relieving toxicity, purging fire, removing the necrotic tissue or ulcers and promoting granulation (Cao and Feng, 2004).

So far, some medicinal properties of S. chamaejasme have been studied, and a variety of flavonoids have been isolated from S. chamaejasm root, including chamaejasmins A, B, C and D (Yang et al., 2005), 7-methoxylchamaejasmine, neochamaejasmins A and B, isochamaejasmins A and B, chamaejasmine(dl-), isochamaejasmin(meso-), euchamaejasmins A, B and C, 3,14-dimethyl-4,11-dimethoxy-5,7-dihydroxybenzoflavanone, dihydrokaempferol, dihydrokaempferol-7-O-D-glucopyranoside, mohsenone (Cao and Feng, 2004), 7-methoxyneochamaejasmin A, isomohsenone (Feng et al., 2002), epiafzelechin, wikstrol B (Feng et al., 2001), chamaejasmenin B, isoneochamaejasmin A, and (+) -chamaejasmin (Feng et al., 2004). These studies found that 24.3% of total flavonoids can be extracted from S. chamaejasme by using ultrasonic technology and colorimetric determination, which show remarkable capacities of removing superoxide anion radical and hydroxyl radical in vitro (Huo et al., 2010). The increased demand for S. chamaejasme necessitates new approaches for sustainable development and utilization. Plant tissue culture techniques have provided such powerful ways for germplasm conservation and mass-multiplication

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of many plant species (Guo et al., 2007; Jain et al., 2011; Wang et al., 2009, 2010; Yu et al., 2005), and therefore, it may also be capable of giving alternative opportunities in manufacturing economically important secondary metabolites such as flavonoids, flavors and pharmaceuticals in controlled laboratory environments (Bourgaud et al., 2001; Brent and Steven, 2008; Rao and Ravishankar, 2002; Sangwan et al., 2007; Vanisree et al., 2004; Verpoorte et al., 2002; Wang et al., 2003, 2011). It has been documented that in vitro culture techniques can rapidly increase biomass that accumulates secondary metabolites. To our knowledge, the effect of different plant growth regulators on the proliferation of S. chamaejasme callus and the accumulation of the secondary metabolites, such as flavonoids, during the proliferation have not been investigated. Herein, we report a research on the development of an effective protocol for in vitro callus proliferation of S. chamaejasme in terms of accumulation of total flavonoids, potential antioxidants, in the callus cultures.

harvested after 35 days of culture and their fresh and dry weights were measured. For the growth curve experiments,

Materials and Methods

3ODQW 0DWHULDOV Wild plants of Stellera chamaejasme, collected from Lingshan Mountain at a 1800 m above sea level in Beijing, China, were used as starting materials in this study. 6DPSOH 3UHSDUDWLRQ Leaves of Stellera chamaejasme were processed by thoroughly washing with tap water for 1-2 h before being sterilized with 75% alcohol for 30 s followed by 0.1% (w/v) mercuric chloride solution (HgCl2) for 8 min. The leaves were then rinsed 4-5 times with sterilized deionized water, and sectioned into explants of approximately 0.5 cm 0.5 cm in size. Six to eight explants were aseptically cultured in a 100 mL Erlenmeyer flask containing 30 mL of the MS medium supplemented with different concentrations of plant growth regulators (Murashige and Skoog, 1962). 0HGLD DQG &XOWXUH &RQGLWLRQV Leaf explants were incubated on the MS medium -1 supplemented with 0.5, 1.0, 2.0, or 4.0 mgL 2,4-D, each with 60 explants. The frequencies of callus induction were recorded after 35 days of culture. For the callus proliferation, effect of various concentrations of NAA, 2,4-D, kinetin -1 (KT), BA, and TDZ (0.5, 1.0, 2.0, and 4.0 mgL ), alone and in combinations of NAA either with BA or with TDZ (see Table 2) were studied. A callus with 0.5 g fresh weight (FW) was incubated per Erlenmeyer flask with a total of 8 replicates per treatment. Calli from all treatments were

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callus with 0.5 or 1.5 g FW was incubated per Erlenmeyer flask, and calli were collected every 7 days to measure their fresh and dry weights. All MS media used were adjusted to pH 5.8 using 1 M -1 -1 NaOH, and 30 gL sucrose and 7 gL agar (Sigma-Aldrich, St. Louis, MO, USA) were added before autoclaving at 121C for 20 min. Explants were cultured at 25-26C under -2 -1 illumination of 30-40 molm s PPF. All experiments were repeated three times. 4XDQWLWDWLYH $QDO\VLV For quantitative analysis of the total flavonoids, powders of the 5 g cultured calli and root of wild Stellera chamaejasme plants were used. Total flavonoids were extracted from the powders as following: the powder was mixed up with 100 mL 95% ethanol for 48 h at room temperature, and then extracted using an ultrasonic technology for 2 h (power at 500 W, temperature at 45C) for three repeated times. The solutions obtained were further partitioned by adding petroleum ether (1:1, v/v) for three times, and the petroleum ether layer was discarded. The remaining layer was concentrated to 25 mL by decompression using a rotary evaporator (RE-52B, Yarong, Shanghai, China). The total flavonoids obtained by extractions were measured by the absorbance at wavelength 490 nm by an UV spectrophotometer (U-2800, Hitachi, Tokyo, Japan). The calibration curve of rutin, obtained by the absorbance measurements versus the concentrations of the standard rutin, was adjusted to a linear equation: A = 12.872 c + 0.083 with a coefficient of correlation of R2 = 0.997. The linear range was within 0.0111-0.0888 mgmL-1. Each point in the calibration curve was the average of three measurements of absorbance. Flavonoid contents were determined by using this calibration curve. $QWLR[LGDQW $VVD\V Radical scavenging activities of the samples were determined by using a method described by Kirby and Schmidt (1997). The DPPH was purchased from Sigma-Aldrich, St. Louis, MO, USA and the radical generated by DPPH was used as a reagent. Ascorbic acid (purchased from Beijing Chemical Works) was used as a standard substance in measuring the scavenging effect. The percentage of the scavenging effect of DPPH was calculated using the following equation: Scavenging effect of DPPH (%) = [(Ac-At) /Ac] 100, where Ac is the absorbance of the control reaction and At is absorbance of the sample. The antioxidizing capacities of the fraction were expressed as the amount of IC50, which is defined as the concentration in g of dry material per mL when the generation of the

DPPH radical was inhibited by 50%. Each data point was determined from the regression equation. Analysis was carried

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out for three times. 'DWD $QDO\VLV All experimental data were statistically analyzed by using one-way analysis of variance (ANOVA) using the protected least-significant-difference (LSD) test (P G0.05), and were presented as the average standard error (SE).

Results and Discussion

&DOOXV ,QGXFWLRQ Leaf explants were incubated on the MS medium containing 0.5, 1.0, 2.0, or 4.0 mgL-1 2,4-D for callus induction. It was observed that the explants became swollen after 7 days of culture (Fig. 1A), and calli developed systematically from the leaf explants by further culture for 14 days (Fig. 1B). Light green calli were harvested from the leaf explants and were subcultured at every 35 days. The response of the leaf explants to the concentration of 2,4-D on the MS medium -1 began at 1.0 mgL corresponding to an induction rate of 98.3%, which is much higher than the induction rates observed in other treatments. However, the frequency decreased to 18.3% on the MS medium supplemented with 4.0 mgL-1 2,4-D. &DOOXV 3UROLIHUDWLRQ In the callus proliferation experiments, effect of different concentrations of NAA, 2,4-D, KT, BA, and TDZ at 0.5, 1.0, 2.0, and 4.0 mgL-1 and NAA (at 0.25, 0.5, 1.0, and 2.0 -1 mgL ) in combination with BA or TDZ (at 0.25, 0.5, 1.0, -1 and 2.0 mgL ) on callus proliferation were investigated on the MS medium (Fig. 2). As can be seen in Figs. 2A and 2B, effect of 2,4-D and KT was less significant than NAA,BA and TDZ. The calli turned brown in two weeks of culture (Figs. 2A and 2B) and the maximum fresh and dry weights -1 of calli were 2.91 and 0.21g (1.0 mgL 2,4-D), and 2.23 -1 and 0.21 g (2.0 mgL KT), respectively. The effect of NAA or BA was much greater when compared to that of 2,4-D or KT, and the calli looked greenish (Figs. 2C and 2D) with maximum fresh and dry weights of 7.24 and 0.45 g (2.0 -1 -1 mgL NAA), 9.46 and 0.49 g (1.0 mgL BA), respectively. Compared with effect of 2,4-D, KT, NAA and BA, effect of TDZ was much more pronounced. When the -1 concentration of TDZ was at 1.0 mgL the maximum fresh and dry weights of calli were 12.75 and 0.53 g, re-1 spectively, increase of 25.5 and 22.1 folds (1.0 mgL TDZ), respectively (Fig. 2E and Table 1).

supplemented with 0.5 mgL NAA in combination with -1 0.25 mgL BA, fresh and dry weights of calli were 12.59 and 0.58 g, increase of 25.2 and 21.2 times, respectively. The callus cultured under this condition was yellow and friable (Fig. 2F). The MS medium supplemented with NAA in combination with TDZ was found to be very suitable for callus proliferation. Fresh and dry weights of calli cultured on the MS -1 -1 medium containing 0.5 mgL NAA and 1.0 mgL TDZ were 14.49 and 0.61 g, respectively, showing 29.0 and 25.4 fold increase, respectively (Fig. 2G and Table 2). Our observations were consistent with the report that the fresh and dry weights of callus of Rheum franzenbachii cultured on the MS medium containing 0.5 mgL-1 TDZ and 0.2
-1

-1

mgL NAA increased 26.3 and 15.0 times, respectively, The effect of the treatment of NAA in combination with BA on callus proliferation was very significant, and fresh and dry weights of calli were 5.20-12.59 and 0.35-0.65 g, respectively. When callus was cultured on the MS medium

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within 35 days of culture (Wang et al., 2011). *URZWK &XUYH The growth curve of callus cultured on the MS medium -1 -1 containing 1.0 mgL NAA and 0.5 mgL BA was presented in Fig. 3. As can be seen in Fig. 3, the growth rates of the cultures were slow in the first two weeks, corresponding to

A B

Fig. 1. The induction of calli: Explants became swollen after one week (A); and compact and light greenish calli formed on the -1 MS + 2,4-D 1.0 mgL (B).

Fig. 2. Effects of plant growth regulator on callus proliferation: MS -1 -1 + 1.0 mgL 2,4-D (A); MS + 2.0 mgL KT (B); MS + 2.0 mgL
1

NAA (C); MS + 1.0 mgL BA (D); MS + 0.5 mgL TDZ (E); -1 -1 MS + 0.5 mgL NAA + 0.25 mgL BA (F); and MS + 0.5 -1 -1 mgL NAA + 1.0 mgL TDZ (G).
-1 -1

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Table 1. Effect of NAA, 2,4-D, KT, BA and TDZ on callus proliferation of S. chamaejasme . 3ODQW JURZWK UHJXODWRU 1$$ &RQFHQWUDWLRQ PJ/ &DOOXV IUHVK ZHLJKW J s s s s s ' s s s s s .7 s s s s s %$ s s s s s 7'= s s s s s
]

&DOOXV GU\ ZHLJ KW J s s s s s s s s s s s s s s s s s s s s s s s s s D E F G D D E D F F D E D D E D F G E D D E F E E

D E E F D D F E G G D G E E F D E F E D D FG G F E

7KH 06 PHGLXP ZDV XVHG ,QLWLDO LQRFXODWHG IUHVK ZHLJKW RI FDOOXV ZDV J &DOOXV ZDV KDUYHVWHG IURP GLIIHUHQW PHGLD DIWHU GD\V RI FXOWXUH 7KH H[SHULPHQWV ZHUH UHSHDWHG WKUHH WLPHV 9DOXHV UHSUHVHQW PHDQV s 6( 7KH YDOXHV ZLWK GLIIHUHQW OHWWHUV DUH VLJQLILFDQWO\ GLIIHUHQW 3 XVLQJ WKH /6' WHVW

the increase of the fresh weight of the calli from initial incubated weight of 0.5 or 1.5 g to 1.5 or 4.0 g when callus cultured on the MS medium containing 1.0 mgL-1 NAA and 0.5 mgL-1 BA. However, the growth rates increased rapidly in the subsequent two weeks of culture, while fresh and dry weights reached the maximum at the end of fifth week of culture to 10.7 and 10.4 g and 0.58 and 0.55 g respectively. This observation is similar to the growth of Eucommia ulmoides observed in a suspension culture (Wang et al., 2003) and the growth of Rheum franzenbachii on the MS medium supplemented with 0.5 mgL-1 TDZ in combination with 0.2 mgL-1 NAA (Wang et al., 2011). In this work, we also found lack of significant effects of the weight (0.5 and 1.5 g) of the initial culture material on the growth curve.

4XDQWLWDWLYH $QDO\VLV RI 7RWDO )ODYRQRLGV The contents of total flavonoids in different calli reached

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to about 5.3-10.8 mgg-1 DW, as compared to 10.1 mgg-1 DW in the root of the wild plant (Table 3). Supplements of BA or NAA showed a significant accumulation of flavonoids in callus as compared with the supplement of TDZ (Fig. 4). Different analyses on contents of total flavonoids showed that contents in callus in the treatments with different concentrations of NAA or BA alone were higher than that in the control group (P 0.05). When the concentration of NAA or BA was at 0.5 mgg-1, total flavonoid content was as high as 8.67 and 9.11 mgg-1 DW, which was similar to the effect of BA on the accumulation of total flavonoids and content of flavonoids in callus of Hylotelephium tatarinowii cultured on the MS medium (Wang et al., 2010). The combination of NAA and BA in the culture was ideal for accumulating total flavonoids in callus. As can be seen in Fig. 5, the combination of 1.0 mgL-1 NAA and 0.25 mgL-1 BA in the MS medium enhanced the total flavonoid

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Table 2. Effect of NAA in combination with BA or TDZ on callus proliferation of S. chamaejasme . 1$$ PJ/ %$ PJ/ )UHVK ZHLJKW J s s s s s s s s s s s s s s s s s s s s
]

'U\ ZHLJKW J s s s s s s s s s s s s s s s s s s s s D F EF G E D H G F E D G F EF E D E EF F E

1$$ PJ/

7'= PJ/

)UHVK ZHLJKW J s s s s s s s s s s s s s s s s s s s s F G H E D E E E F D F F E D D E F F F D

'U\ ZHLJKW J s s s s s s s s s s s s s s s s s s s s D E E F G D F G H E D E EF EF F D F E E D

D EF EF F E D G F E E D F F F E D E F F E

7KH 06 PHGLXP ZDV XVHG ,QLWLDO LQFXEDWHG IUHVK ZHLJKW RI FDOOXV ZDV J &DOOXV ZDV KDUYHVWHG IURP GLIIHUHQW PHGLD DIWHU G 7KH H[SHULPHQWV ZHUH UHSHDWHG WKUHH WLPHV 9DOXHV UHSUHVHQW PHDQV s 6( 7KH YDOXHV ZLWK GLIIHUHQW OHWWHUV DUH VLJQLILFDQWO\ GLIIHUHQW 3 XVLQJ WKH /6' WHVW
-1

content in the callus to as high as 10.8 mgg DW, which is higher than total flavonoid content in the root of the wild plant (Fig. 5). '33+ )UHH 5DGLFDO VFDYHQJLQJ $FWLYLWLHV The DPPH is a stable organic free radical with an absorption peak at 517 nm. This absorption disappears at acception of an electron or a free radical species, and can be reflected by changing color from purple to yellow (Hseu et al., 2008). This activity can also be measured by the IC50 value, which is defined as the amount of extracts needed to scavenge 50% of the DPPH radicals in a reaction. High IC50 values correspond to low antioxidant activities. The DPPH radical scavenging activities of different calli and the root of the wild plant are presented in Fig. 6. The values of IC50 of -1 samples were in a range of 11.94-35.25 gmL , showing strong antioxidant activities. And also as can be seen in Table 4, the most potent radical scavenger (IC50 = 11.94 -1 gmL ) was the calli cultured on the MS medium containing -1 -1 1.0 mgL NAA and 0.25 mgL BA, followed by the callus -1 cultured on the MS medium supplemented with 1.0 mgL

NAA in combination with 0.5 mgL-1 BA (IC50 = 19.17 -1 -1 gmL ) and the root of the wild plant (IC50 = 20.34 gmL ).

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The scavenging activities of these three samples were significant as compared to that made by the positive -1 control reagent of ascorbic acid (IC50 = 21.21 gmL ) The total flavonoids in the S. chamaejasme root was also extracted by using an ultrasonic technology, and the radical- scavenging activity of the total flavonoids against superoxide anion radical and hydroxyl radical was determined. The result showed that the flavonoids extracted from the root had remarkable activity in removing superoxide anion radical and hydroxyl radical in vitro, which is more efficient than that of ascorbic acid (Huo et al., 2010). The stronger antioxidant activities seen in the calli and the wild plant root as compared to that of ascorbic acid in the DPPH reaction was likely be due to the higher accumulations of the total flavonoid contents (Fig. 7). As shown in Fig. 7, a direct correlation can be seen between the total flavonoid contents and the 2 antioxidant activities, and the correlation coefficient was R = 0.719 (Fig. 7). Our findings are consistent with the findings reported by Kim et al. (2011). In summary, an efficient callus proliferation system of S. chamaejasme has been developed. It was found that the -1 MS medium supplemented with 1.0 mgL NAA and -1 0.25 mgL BA was ideal for both the proliferation and the

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Table 3. The content of total flavonoids in the different calli and the root of the wild plant . 1XPEHU RI &XOWXUH PHGLXP &RQWHQW RI WRWDO IODYRQRLGV VDPSOHV PJ/ PJJ ': 06 7'= s G 06 7'= s D 06 7'= s E 06 06 06 06 06 06 06 06 1$$ 1$$ 1$$ 1$$ 1$$ 1$$ 1$$ 1$$ 06 1$$ 1$$ 1$$ 1$$ 1$$ 1$$ 1$$ 1$$ 06 1$$ 1$$ 1$$ 1$$ 1$$ 1$$ 1$$ 1$$ 06 1$$ 1$$ 1$$ 1$$ 1$$ 1$$ 1$$ 7'= 7'= %$ %$ %$ %$ %$ 1$$ %$ %$ %$ %$ 7'= 7'= 7'= 7'= 1$$ %$ %$ %$ %$ 7'= 7'= 7'= 7'= 1$$ %$ %$ %$ %$ 7'= 7'= 7'= 7'= 1$$ %$ %$ %$ %$ 7'= 7'= 7'= s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s F H D G F E E F D E F G HI F I H FG F F H G E G D F E I H F G EF DE EF D E F D G G F E G I D

06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06

1$$ 7'= 5RRW RI ZLOG SODQW

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Junli Wang, Hort. Xuan Environ. Xiao, Qian Biotechnol. Wang, 54(5):441-449. Xiaoxu Li, Lu 2013. Zhang, and Jianfei Li

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7KH H[SHULPHQWV ZHUH UHSHDWHG WKUHH WLPHV 9DOXHV UHSUHVHQW PHDQ s 6( 7KH YDOXHV ZLWK GLIIHUHQW OHWWHUV DUH VLJQLILFDQWO\ GLIIHUHQW 3 E\ WKH /6' WHVW

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Table 4. The IC50 values of different samplesz. 1XPEHU RI VDPSOHV &RQWURO 06 06 06 06 0HGLXP FRPSRVLWLRQ RU VDPSOHV PJ/ DVFRUELF DFLG 5RRW RI ZLOG SODQW 1$$ 1$$ 1$$ 1$$ 06 06 06
]

,& wJP/ s s s s s s s s F EF D E H J I G I K

%$ %$ %$ %$ %$ %$ %$ %$

1$$ 06

s s

9DOXHV UHSUHVHQW PHDQ s 6( 7KH H[SHULPHQWV ZHUH UHSHDWHG WKUHH WLPHV 7KH YDOXHV ZLWK GLIIHUHQW OHWWHUV DUH VLJQLILFDQWO\ GLIIHUHQW 3 E\ WKH /6' WHVW

Fig. 3. The growth curve of calli with initial tissue fresh weight of either 0.5 g (A) or 1.5 g (B).

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Fig. 4. Influence of BA, NAA, and TDZ on total flavonoids accumulation. Bars represent mean SE. The columns with different letters are significantly different (P 0.05) by the LSD test.

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Fig. 5. The comparison of the total flavonoid contents. Bars represent mean SE. The columns with different letters are significantly different (P 0.05) by the LSD test.

Fig. 6. The DPPH radical scavenging capacities of different samples. Data represent the mean SE of three experiments.

accumulations of flavonoids in S. chamaejasme calli. The total flavonoids accumulated under this condition was as -1 high as 10.78 mgg DW, much higher than that accumulated

Fig. 7. Correlation between DPPH radical scavenging activity and total flavonoid content. Data represent the mean SE of three experiments.

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by the root of the wild plant. Moreover, the calli proliferated under this circumstances were also found to be the most -1 potent radical scavengers (IC50 = 11.94 gmL ). Acknowledgment: This work was financially supported by National Basic Research Program of China (973 Program, 2009CB522300), the 985 Project (MUC98504-14 and MUC 98507-08), 111 Project (B08044) and the Fundamental Research Funds for the Central Universities (0910KYZY46).

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