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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, People’s 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 mg·L-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 mg·L-1 Į-naphthalene acetic acid (NAA) and 1.0 mg·L-1 n-phenyl-nƍ -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 mg·L and 0.25 mg·L , respectively. With this combination, -1 the content of the total flavonoids slightly increased to 10.8 mg·g dry weight (DW) in comparison to 10.1 mg·g-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 mg·L NAA -1 and 0.25 or 0.5 mg·L BA was very active in radical scavenging, and their IC50 values were 11.94 and 19.17 ȝg·mL-1, respectively. Compared to the ascorbic acid (IC50 21.21 ȝg·mL-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, 3ƍ,14-dimethyl-4ƍ,11-dimethoxy-5,7-dihydroxybenzoflavanone, dihydrokaempferol, dihydrokaempferol-7-Oȕ-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

The frequencies of callus induction were recorded after 35 days of culture. 1. effect of various concentrations of NAA. 2007.0. 2011).. such as flavonoids.5 cm × 0..0 mg·L ). 2003. 2009. 2001. 54(5):441-449.. For the callus proliferation. For the growth curve experiments. 2002.0 mg·L 2. the effect of different plant growth regulators on the proliferation of S. 1. To our knowledge. were used as starting materials in this study. kinetin -1 (KT). and sectioned into explants of approximately 0. A callus with 0. Xuan Environ. Hort. during the proliferation have not been investigated. each with 60 explants.5 cm in size. flavors and pharmaceuticals in controlled laboratory environments (Bourgaud et al. chamaejasme in terms of accumulation of total flavonoids. Wang. 2007.5.0. potential antioxidants. harvested after 35 days of culture and their fresh and dry weights were measured. it may also be capable of giving alternative opportunities in manufacturing economically important secondary metabolites such as flavonoids.5 g fresh weight (FW) was incubated per Erlenmeyer flask with a total of 8 replicates per treatment.. 1962). BA.. Zhang.0. 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.4-D.. collected from Lingshan Mountain at a 1800 m above sea level in Beijing. we report a research on the development of an effective protocol for in vitro callus proliferation of S. and TDZ (0.442 442 Junli Wang. Brent and Steven. 2005). chamaejasme callus and the accumulation of the secondary metabolites. Wang et al. Qian Biotechnol. The leaves were then rinsed 4-5 times with sterilized deionized water. Xiaoxu Li. and 4.5. 2011. and Jianfei Li 442 442 of many plant species (Guo et al. Yu et al. or 4. in the callus cultures. Wang et al. Calli from all treatments were . 2. 0HGLD DQG &XOWXUH &RQGLWLRQV Leaf explants were incubated on the MS medium -1 supplemented with 0. Vanisree et al.4-D.0. Jain et al. It has been documented that in vitro culture techniques can rapidly increase biomass that accumulates secondary metabolites..1% (w/v) mercuric chloride solution (HgCl2) for 8 min. alone and in combinations of NAA either with BA or with TDZ (see Table 2) were studied. Materials and Methods 3ODQW 0DWHULDOV Wild plants of Stellera chamaejasme. Lu 2013. 2. and therefore. Verpoorte et al. Rao and Ravishankar.. China. 2008. Herein. 2. Xiao. Sangwan et al. 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. 2010.. 2002. 2004.

Louis.5 or 1. Xiao. The calibration curve of rutin.997. USA) were added before autoclaving at 121°C for 20 min. Explants were cultured at 25-26°C under -2 -1 illumination of 30-40 ȝmol·m ·s PPF. Each point in the calibration curve was the average of three measurements of absorbance. Analysis was carried . and calli were collected every 7 days to measure their fresh and dry weights. Zhang. The total flavonoids obtained by extractions were measured by the absorbance at wavelength 490 nm by an UV spectrophotometer (U-2800. The linear range was within 0. All MS media used were adjusted to pH 5. and then extracted using an ultrasonic technology for 2 h (power at 500 W. Each data point was determined from the regression equation. Xiaoxu Li.0888 mg·mL-1. 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. MO.8 using 1 M -1 -1 NaOH. where Ac is the absorbance of the control reaction and At is absorbance of the sample. and Jianfei Li 443 443 callus with 0. Hort. USA and the radical generated by DPPH was used as a reagent. 54(5):441-449. v/v) for three times. Qian Biotechnol. Yarong. Louis. temperature at 45°C) for three repeated times. St.443 443 Junli Wang. MO. Lu 2013.5 g FW was incubated per Erlenmeyer flask. The antioxidizing capacities of the fraction were expressed as the amount of IC50. Wang. The remaining layer was concentrated to 25 mL by decompression using a rotary evaporator (RE-52B.083 with a coefficient of correlation of R2 = 0. Ascorbic acid (purchased from Beijing Chemical Works) was used as a standard substance in measuring the scavenging effect. Japan). was adjusted to a linear equation: A = 12. Xuan Environ. China). 4XDQWLWDWLYH $QDO\VLV For quantitative analysis of the total flavonoids. Hitachi.872 c + 0. St. The percentage of the scavenging effect of DPPH was calculated using the following equation: Scavenging effect of DPPH (%) = [(Ac-At) /Ac] ×100. All experiments were repeated three times. $QWLR[LGDQW $VVD\V Radical scavenging activities of the samples were determined by using a method described by Kirby and Schmidt (1997). Tokyo. obtained by the absorbance measurements versus the concentrations of the standard rutin.0111-0. and the petroleum ether layer was discarded. Flavonoid contents were determined by using this calibration curve. and 30 g·L sucrose and 7 g·L agar (Sigma-Aldrich. which is defined as the concentration in ȝg of dry material per mL when the generation of the DPPH radical was inhibited by 50%. The solutions obtained were further partitioned by adding petroleum ether (1:1. Shanghai. powders of the 5 g cultured calli and root of wild Stellera chamaejasme plants were used. The DPPH was purchased from Sigma-Aldrich.

0 mg·L TDZ). The effect of NAA or BA was much greater when compared to that of 2.0 mg·L-1 and NAA (at 0. respectively. and calli developed systematically from the leaf explants by further culture for 14 days (Fig.49 and 0.75 and 0. Compared with effect of 2.5. and 2. When the -1 concentration of TDZ was at 1. KT. respectively. which is much higher than the induction rates observed in other treatments.0 mg·L TDZ were 14.0 -1 mg·L ) in combination with BA or TDZ (at 0. 1.5 mg·L NAA and 1.20-12. Wang.0. -1 and 2.59 and 0.24 and 0. 1B). 1A).QGXFWLRQ Leaf explants were incubated on the MS medium containing 0. 2F). 2E and Table 1). effect of different concentrations of NAA.5 and 22. 1. It was observed that the explants became swollen after 7 days of culture (Fig.65 g.53 g. respectively (Fig.4-D and KT was less significant than NAA.25. fresh and dry weights of calli were 12. 0. Hort.0 mg·L-1 2. Xiao.4 fold increase.4-D or KT. However.46 and 0.4-D.0 mg·L-1 2.21 g (2. and 2. respectively. Xuan Environ. The effect of the treatment of NAA in combination with BA on callus proliferation was very significant.0 times. 2G and Table 2).3% on the MS medium supplemented with 4.444 444 Junli Wang.0 mg·L KT). 2.49 g (1. showing 29.61 g. Fresh and dry weights of calli cultured on the MS -1 -1 medium containing 0. KT. 2A and 2B. 2).1 folds (1. BA.21g (1. respectively. or 4. supplemented with 0.25.58 g.0 mg·L ) on callus proliferation were investigated on the MS medium (Fig.5 mg·L NAA in combination with -1 0. re-1 spectively. 9.5 mg·L-1 TDZ and 0.0.0. '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. 2C and 2D) with maximum fresh and dry weights of 7.0 mg·L the maximum fresh and dry weights of calli were 12.35-0. 2.BA and TDZ. Zhang.0 -1 -1 mg·L NAA). The response of the leaf explants to the concentration of 2.0 mg·L 2. 54(5):441-449. the frequency decreased to 18. When callus was cultured on the MS medium .4-D.0. The MS medium supplemented with NAA in combination with TDZ was found to be very suitable for callus proliferation. and fresh and dry weights of calli were 5.4-D.5. 1. Results and Discussion &DOOXV .45 g (2.5. Xiaoxu Li. 0.23 -1 and 0. 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. respectively. 2. Light green calli were harvested from the leaf explants and were subcultured at every 35 days.0 and 25. increase of 25. effect of TDZ was much more pronounced.59 and 0. and 4.4-D on the MS medium -1 began at 1. 2A and 2B) and the maximum fresh and dry weights -1 of calli were 2. The calli turned brown in two weeks of culture (Figs. and the calli looked greenish (Figs. and Jianfei Li 444 444 out for three times. Qian Biotechnol.2 times.0. effect of 2.3%.05).3 and 15. 1.2 -1 -1 mg·L NAA increased 26.2 and 21.4-D for callus induction.4-D). As can be seen in Figs. The callus cultured under this condition was yellow and friable (Fig.5. and TDZ at 0.25 mg·L BA. increase of 25. &DOOXV 3UROLIHUDWLRQ In the callus proliferation experiments. respectively.0 mg·L BA). and were presented as the average ± standard error (SE).0. respectively (Fig. Lu 2013. NAA and BA.91 and 0.0 mg·L corresponding to an induction rate of 98.

0 mgᨿL (B). As can be seen in Fig. Xiaoxu Li.0 mg·L NAA and 0. *URZWK &XUYH The growth curve of callus cultured on the MS medium -1 -1 containing 1.0 mgᨿL KT (B).5 mgᨿL TDZ (E). Xiao. 2011). Fig.5 -1 -1 mgᨿL NAA + 1. MS + 2.0 mgᨿL 2. and MS + 0.4-D (A). and Jianfei Li 445 445 within 35 days of culture (Wang et al. Zhang. Effects of plant growth regulator on callus proliferation: MS -1 -1 + 1.5 mgᨿL NAA + 0.5 mg·L BA was presented in Fig..0 mgᨿL BA (D). MS + 1. MS + 2. the growth rates of the cultures were slow in the first two weeks. -1 -1 . 54(5):441-449. and compact and light greenish calli formed on the -1 MS + 2. Wang. The induction of calli: Explants became swollen after one week (A).0 mgᨿL 1 NAA (C). 3. -1 -1 MS + 0. 1.445 445 Junli Wang. MS + 0. Hort.4-D 1. Xuan Environ. Lu 2013. 3. Qian Biotechnol.0 mgᨿL TDZ (G). 2.25 mgᨿL BA (F). corresponding to A B Fig.

Effect of NAA.58 and 0. Xiaoxu Li. Qian Biotechnol.2 mg·L-1 NAA (Wang et al. 2.. chamaejasme .0 g when callus cultured on the MS medium containing 1.4 g and 0.5 mg·L-1 TDZ in combination with 0. Hort. 2011).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. while fresh and dry weights reached the maximum at the end of fifth week of culture to 10. Xiao.446 446 Junli Wang. Wang. In this work.5 g) of the initial culture material on the growth curve. we also found lack of significant effects of the weight (0. However. 3ODQW JURZWK UHJXODWRU 1$$ &RQFHQWUDWLRQ PJᨿ/ &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 ] z &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 . 54(5):441-449. Xuan Environ. Lu 2013. 4XDQWLWDWLYH $QDO\VLV RI 7RWDO )ODYRQRLGV The contents of total flavonoids in different calli reached ..5 mg·L-1 BA. KT.5 and 1.7 and 10. Zhang.5 or 1. the growth rates increased rapidly in the subsequent two weeks of culture. and Jianfei Li 446 446 Table 1.4-D.55 g respectively. BA and TDZ on callus proliferation of S. 2003) and the growth of Rheum franzenbachii on the MS medium supplemented with 0.5 or 4.5 g to 1.0 mg·L-1 NAA and 0. This observation is similar to the growth of Eucommia ulmoides observed in a suspension culture (Wang et al.

0 mg·L-1 NAA and 0.3-10. 54(5):441-449.1 mg·g-1 DW in the root of the wild plant (Table 3). Xiaoxu Li. 4). Supplements of BA or NAA showed a significant accumulation of flavonoids in callus as compared with the supplement of TDZ (Fig. The combination of NAA and BA in the culture was ideal for accumulating total flavonoids in callus. 5. When the concentration of NAA or BA was at 0. Hort.11 mg·g-1 DW.5 mg·g-1. As can be seen in Fig.. Zhang. the combination of 1.05).25 mg·L-1 BA in the MS medium enhanced the total flavonoid . 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. Qian Biotechnol. Xuan Environ.447 447 Junli Wang. total flavonoid content was as high as 8.8 mg·g-1 DW. Xiao.67 and 9. and Jianfei Li 447 447 to about 5. Wang. Lu 2013. as compared to 10. 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).

Effect of NAA in combination with BA or TDZ on callus proliferation of S.8 mg·g DW. which is higher than total flavonoid content in the root of the wild plant (Fig. Hort. Qian Biotechnol.448 448 Junli Wang. 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.. 5). Zhang.25 ȝg·mL . chamaejasme .17 -1 -1 ȝg·mL ) and the root of the wild plant (IC50 = 20. followed by the callus -1 cultured on the MS medium supplemented with 1. 1$$ PJᨿ/ %$ PJᨿ/ )UHVK ZHLJKW J s s s s s s s s s s s s s s s s s s s s ] z 'U\ ZHLJKW J s s s s s s s s s s s s s s s s s s s s D F EF G E D H G F E D G F EF E D E EF F E 1$$ PJᨿ/ 7'= PJᨿ/ )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 . This activity can also be measured by the IC50 value. And also as can be seen in Table 4. showing strong antioxidant activities.34 ȝg·mL ).94 -1 ȝg·mL ) was the calli cultured on the MS medium containing -1 -1 1.5 mg·L-1 BA (IC50 = 19.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. . which is defined as the amount of extracts needed to scavenge 50% of the DPPH radicals in a reaction. Wang. 54(5):441-449. and Jianfei Li 448 448 Table 2. Lu 2013.0 mg·L NAA and 0. '33+ )UHH 5DGLFDO VFDYHQJLQJ $FWLYLWLHV The DPPH is a stable organic free radical with an absorption peak at 517 nm. Xiaoxu Li. 6.0 mg·L NAA in combination with 0.94-35. Xiao. The DPPH radical scavenging activities of different calli and the root of the wild plant are presented in Fig.25 mg·L BA. the most potent radical scavenger (IC50 = 11. High IC50 values correspond to low antioxidant activities. 2008). The values of IC50 of -1 samples were in a range of 11. Xuan Environ.

As shown in Fig.21 ȝg·mL ) The total flavonoids in the S. 2010).25 mg·L BA was ideal for both the proliferation and the . The result showed that the flavonoids extracted from the root had remarkable activity in removing superoxide anion radical and hydroxyl radical in vitro.719 (Fig. a direct correlation can be seen between the total flavonoid contents and the 2 antioxidant activities. and the radical. 7). Lu 2013. In summary. which is more efficient than that of ascorbic acid (Huo et al. 7). 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.0 mg·L NAA and -1 0. chamaejasme has been developed.449 449 Junli Wang. It was found that the -1 MS medium supplemented with 1. Zhang. Xuan Environ. Hort. Xiaoxu Li. and the correlation coefficient was R = 0. Our findings are consistent with the findings reported by Kim et al. Wang. Xiao.. 54(5):441-449.scavenging activity of the total flavonoids against superoxide anion radical and hydroxyl radical was determined. Qian Biotechnol. (2011). 7. chamaejasme root was also extracted by using an ultrasonic technology. an efficient callus proliferation system of S. and Jianfei Li 449 449 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.

The content of total flavonoids in the different calli and the root of the wild plant . Lu 2013. Hort. 1XPEHU RI &XOWXUH PHGLXP &RQWHQW RI WRWDO IODYRQRLGV VDPSOHV PJᨿ/ PJᨿJ ': 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$$ %$ %$ %$ %$ 7'= 7'= 7'= s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s F H D G F E E F D E F G HI F I H FG F F H G E G D F E I H F G EF DE EF D E F D G G F E G I D z 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 1$$ 7'= 5RRW RI ZLOG SODQW . Xuan Environ. Wang. Qian Biotechnol.446 446 Junli Wang. Xiaoxu Li. and Jianfei Li 446 446 Table 3. Zhang. Xiao. 54(5):441-449.

447 447 ] Junli Wang. 54(5):441-449. Lu 2013. Zhang. Hort. Xiaoxu Li. Qian Biotechnol. and Jianfei Li 447 447 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 . Xuan Environ. Xiao. Wang.

Qian Biotechnol.5 g (B). Hort. Wang. Xiao. . The IC50 values of different samplesz. The growth curve of calli with initial tissue fresh weight of either 0. 1XPEHU RI VDPSOHV &RQWURO 06 06 06 06 0HGLXP FRPSRVLWLRQ RU VDPSOHV PJᨿ/ DVFRUELF DFLG 5RRW RI ZLOG SODQW 1$$ 1$$ 1$$ 1$$ 06 06 06 ] . Lu 2013.5 g (A) or 1.& wJᨿP/ 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. Xiaoxu Li.448 448 Junli Wang. Xuan Environ. Zhang. and Jianfei Li 448 448 Table 4. 54(5):441-449. 3.

54(5):441-449. Hort.449 449 Junli Wang. NAA. Xuan Environ. Influence of BA. Xiao. Xiaoxu Li. and Jianfei Li 449 449 Fig. Zhang. Wang. 4. Lu 2013. The columns with different letters are significantly different (P ᧸ 0. and TDZ on total flavonoids accumulation. Qian Biotechnol. Bars represent mean ± SE. .05) by the LSD test.

05) by the LSD test. Zhang.78 mg·g DW. Data represent the mean ± SE of three experiments. accumulations of flavonoids in S. Hort. Lu 2013. The comparison of the total flavonoid contents. Xuan Environ. chamaejasme calli. The DPPH radical scavenging capacities of different samples. The columns with different letters are significantly different (P ᧸ 0. Wang. 5. 54(5):441-449. Correlation between DPPH radical scavenging activity and total flavonoid content. and Jianfei Li 450 450 Fig. Xiaoxu Li. Qian Biotechnol. Xiao. much higher than that accumulated Fig. Fig. .450 450 Junli Wang. 7. Bars represent mean ± SE. The total flavonoids accumulated under this condition was as -1 high as 10. 6. Data represent the mean ± SE of three experiments.

and F. plantlets in vitro. Feng. 161:839-851. A. Xuan Environ. the “985” Project (MUC98504-14 and MUC 98507-08). Qian Biotechnol. Milesi. J. Gontier. Zhang. 54(5):441-449.W. The latest progress in studies of chemical components of Euphorbiae Ebracteolatae. Lu 2013. and Jianfei Li 451 451 by the root of the wild plant. T. Cao. morphogenesis. the calli proliferated under this circumstances were also found to be the most -1 potent radical scavengers (IC50 = 11.. and E. F. X. 2009CB522300). Moreover. Chinese Med.-Plant 44:40-50.V. Gravot.451 451 Junli Wang. Hort. In Vitro Cell Dev.H. and essential oil production in Mentha spicata L. Wang. . Production of plant secondary metabolites: A historical perspective. S. Plant Sci. Growth. 19:81-84. Steven. Xiao. and W. Acknowledgment: This work was financially supported by National Basic Research Program of China (973 Program.94 ȝg·mL ). 2008. 2001. “111” Project (B08044) and the Fundamental Research Funds for the Central Universities (0910KYZY46). Literature Cited Bourgaud. Biol. Henan College Tradi. Xiaoxu Li. Brent. 2004.

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