Saikosaponin accumulation and antioxidative protection in drought-stressed Bupleurum chinense DC. plants

Environmental and Experimental Botany 66 (2009) 326–333

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Saikosaponin accumulation and antioxidative protection in drought-stressed Bupleurum chinense DC. plants Zaibiao Zhu a,b,c , Zongsuo Liang a,b,∗ , Ruilian Han a a

Research Center of Soil and Water Conservation and Ecological Environment, Chinese Academy of Sciences and Ministry of Education, No. 26 Xinong Road, Yangling, Shaanxi Province 712100, PR China College of Life Sciences, Northwest A&F University, Yangling, Shaanxi Province 712100, PR China c Graduate School of the Chinese Academy of Sciences, Beijing 100049, PR China b

a r t i c l e

i n f o

Article history: Received 16 September 2008 Received in revised form 23 March 2009 Accepted 31 March 2009 Keywords: Bupleurum chinense DC. Water deficit Lipid peroxidation Antioxidant system Saikosaponin

a b s t r a c t Dried root of Bupleurum spp. is one of the most popular ingredients in many oriental medicinal preparations. Potted Bupleurum chinense DC. seedlings were subjected to progressive drought stress by withholding irrigation followed by a rewatering phase. The changes in antioxidant system, hydrogen peroxide (H2 O2 ), superoxide radicals (O2 − ), and malondialdehyde (MDA) contents as well as saikosaponin a (SSa) and saikosaponin d (SSd) content in B. chinense roots were investigated. Additionally, the antioxidant activity of the roots extract was evaluated. The results showed that B. chinense root appeared highly resistant to water deficit. Both SSa and SSd content increased with the progressive water deficit, however, decreased under severe drought conditions or after water recovery. Moderate drought treatment resulted in 83% increase in SSa content and 22% increase in SSd content compared to the well-hydrated treatment. And increased SSa and SSd content during drought were accompanied by enhanced O2 − content and superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) activity until severe drought stress. Notably, in vitra antioxidant tests demonstrated that the lipid peroxidation inhibition capacity was positively correlated with the content of SSa and SSd, particularly significant at p = 0.05 with SSd content. These results suggest that B. chinense roots exhibit effective antioxidative protection mechanism to withstand drought stress. And it could be speculated that drought-induced SSa and SSd accumulation in B. chinense roots may be stimulated via active oxygen species, and consequently involve in mitigating the oxidative damage due to its high anti-lipid peroxidation capacity. © 2009 Published by Elsevier B.V.

1. Introduction Bupleuri Radix (Bupleurum spp. root) is one of the most important crude drugs used in many traditional Chinese medicines (TCM). It is believed that saikosaponins are responsible for part of the pharmaceutical properties of Bupleuri Radix (Zhu et al., 2006). Among saikosaponins, saikosaponin a (SSa) and saikosaponin d (SSd) are especially known for their pharmacological activity. They have antiallergic activity, analgesic action, anti-inflammatory action, plasma cholesterol-lowering action, action for hepatic injuries, etc. (Aoyagi et al., 2001). Due to its medicinal importance, the demand for Bupleuri Radix has increased steadily in recent years. About eight million kilograms of Bupleuri Radix are required for prescriptions or

∗ Corresponding author at: Research Center of Soil and Water Conservation and Ecological Environment, Chinese Academy of Sciences and Ministry of Education, No. 26 Xinong Road, Yangling, Shaanxi Province 712100, PR China. Tel.: +86 29 87014582; fax: +86 29 87092262. E-mail address: [email protected] (Z. Liang). 0098-8472/$ – see front matter © 2009 Published by Elsevier B.V. doi:10.1016/j.envexpbot.2009.03.017

exported from China each year. In Japan, the gross sales of manufactured prescription medicines containing Bupleuri Radix amounted to 27 billion yen in 2002 (Pan, 2006). Great attention had been paid to develop a high-speed saikosaponin production system using plant tissue culture technology, however, the production rate and the saikosaponin content in the root are generally low (Aoyagi et al., 2001). Although the basic outlines of saikosaponin production are fairly well understood, the enzymes, genes and biochemical pathway involved in saikosaponin biosynthesis are largely uncharacterized (Chen et al., 2007). Understanding the physiological mechanism of saikosaponin accumulation in response to environmental stress is critical for scale-up saikosaponin production. For the last few decades, several scales of physiological work have been conducted under drought stress in crop plants, but it is not so with response to medicinal plants (Jaleel et al., 2007a). A significant increase in the SSa, SSc and SSd content of the cork layer and surrounding tissues in Bupleurum falcutum after subjected to one month of water-stress (Minami et al., 1995), indicating the possible role of saikosaponins in response to stress conditions. However, little

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information is available on the actual physiological role of saikosaponin in protecting Bupleurum spp. plants from drought stress. On the other hand, the antioxidant capacity of saikosaponin such as scavenging against reactive oxygen species and inhibiting lipid peroxidation was observed in pharmacological experiments (Fang and Zheng, 2002; Wang et al., 2004; Liu et al., 2005). To counteract the injurious effects of AOS, plants evolved complex antioxidant system which includes enzymatic antioxidants (superoxide dismutase (SOD, EC 1.15.1.1), peroxidase (POD, EC 1.11.1.7) and catalase (CAT, EC 1.11.1.6), etc.) and non-enzymatic antioxidants (ascorbate, glutathione, tocopherol, etc.). On the other hand, plants can interact with biotic and abiotic stress by physiological adaptation and altering the biochemical profile of the plant tissues and producing diverse array of secondary metabolites (Nacif de Abreu and Mazzafera, 2005; Zobayed et al., 2005), and it is well-known that accumulation of secondary metabolites is generally a defense mechanism of plants. Increasing evidences showed that cascade of events including lipid peroxidation, accumulation of H2 O2 content may be involved in the initiation of secondary metabolites production (Yuan et al., 2002; Shohael et al., 2006). Principally animals can produce the same AOS as plants and the mechanisms of oxygen activation are identical or analogous (Grassmann et al., 2002). Numerous studies showed that oxidative stress play an important role in the etiology of a wide variety of diseases in people such as Alzheimer’s disease, aging, cancer, inflammation, rheumatoid arthritis, and atherosclerosis (Jung et al., 2008). On the other hand, the antioxidant potency of some medicinally important compounds could be the basis for its medicinal potential, i.e. terpenoids represent the basis of numerous herbal drugs used in the treatments of pain, cold, bronchitis and gastrointestinal disease in which AOS may be involved. And it is also of importance for humans using phenolics as vitamins and/or protectants against “oxidative stress” (Grassmann et al., 2002). Together, it could be speculated that the accumulation of some secondary metabolites with antioxidant potential may be a need for scavenging AOS and protecting biomembrance from oxidative stress in plant cells subjected to environmental stresses, and AOS may be served as a mediator in initiating biosynthesis of certain secondary metabolites. Several previous works detected the increased antioxidant potential as well as an enhancement in certain secondary metabolites production under abiotic or biotic stress. Munné-Bosch et al. (2001) postulated that the antioxidant activity of carnosic acid, a membrane-associated antioxidant found in chloroplasts, provides protection for drought-stressed Salvia officinalis leaves, and according to the work of Misra and Gupta (2006) on Catharanthus roseus, increased peroxidase activity concomitant with the accumulation of alkaloid was found, suggesting the close correlation between the peroxidase activity and indole alkaloid. In addition, significant accumulation of ajmalicine (Jaleel et al., 2007a) or total indole alkaloid (Jaleel et al., 2007b) in roots of C. roseus was found under the oxidative stress resulted from drought stress. Meanwhile, enhancement in non-enzymatic antioxidants and antioxidant enzyme activities were found. Thus the authors suggested that plants under drought stress are highly regulated by components of antioxidative system and secondary metabolite contents. Moreover, volatile isoprenoids have been suggested to be involved in scavenging AOS and potentially protecting plant against photo-oxidative stress (Chen and Cao, 2008). Considering the above, the hypothesis of current study is that the biosynthesis and accumulation of SSa and SSd under water-stress conditions may be an important part of the complex antioxidant system. And the aim of current study was (1) to investigate the antioxidant potential of Bupleurumchinense DC. under drought stress; (2) to clarify the relationship among AOS, antioxidant system, and SSa, SSd content change in response to drought stress, and the possible mechanism of saikosaponin accumulation. For

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these purposes, the enzymatic- and non-enzymatic-antioxidants (i.e. SOD, CAT, APX, GSH, and AsA), stress marker (i.e. H2 O2 , O2 − , and MDA), and SSa, SSd content were measured in B. chinense roots under increasing water deficit and subsequent rewatering. In addition, the antioxidant activity of methanolic extract of the roots was assessed using anti-DPPH free radical method and anti-FeCl2 ascorbic acid-stimulated lipid peroxidation assay. 2. Materials and methods 2.1. Plant materials Seeds of B. chinense were obtained from Shaanxi TASLY Plant Pharmaceutical Co. Ltd., Shaanxi, China, and surface-sterilized with 0.1% HgCl2 for 3 min followed by repeated washings with distilled water, then sown in plastic pots (30 cm length, 20 cm width and 10 cm depth) with a 3 kg mixture of soil, sand, and vermiculite (1:5:2, v/v/v). Initial soil was collected from experimental field in Research Center of Soil and Water Conservation and Ecological Environment, Chinese Academy of Sciences and Ministry of Education, Shaanxi Province, China. Fresh soil was air dried, and passed through a sieve of 1.0 mm. After germination, seedlings were grown under controlled conditions (light/dark regime of 13/11 h at 25/18 ◦ C, relative humidity of 45–50%, photosynthetic photon flux density of 100 ␮mol m−2 s−1 ). All the pots were watered regularly to the field capacity with 1/4 strength Hoagland and Arnon solution. Thirty days after germination, ten seedlings of uniform size were left to grow in each pot. Half strength Hoagland and Arnon solution was added with 2-d intervals to provide the nutritional requirements of seedlings prior to the onset of the experiment, and the photon flux density increased up to 200 ␮mol m−2 s−1 . There were altogether 15 pots comprising 5 treatments with three replications for each. The pots were arranged following the completely randomized block design. Drought treatment was applied to 150-d-old seedlings, at vegetable and high-speed saikosaponin accumulation stage, by withholding water for 0 d (control treatment, C), 3 d (S1 treatment), 6 d (S2 treatment) and 9 d (S3 treatment), and recovery was performed by rehydrating S2 plants to field capacity for 3 d (RH treatment). Fresh leaves were used for relative water content (RWC) assay. Part of roots were frozen in liquid nitrogen and kept at −80 ◦ C for subsequent analysis. For measurements of SSa and SSd content, DPPH radical-scavenging ability, and lipid peroxidation inhibition ability, the rest of roots were dried at 60 ◦ C for 72 h, then were ground to pass through a 0.5 mm sieve. 2.2. Determination of relative water content (RWC) The RWC of leaves was calculated according to Smart and Bingham (1974). 2.3. Antioxidant enzymes and O2 − content assay One gram frozen root was homogenized with 8 mL 0.05 M Na phosphate buffer solution (pH 7.8) including 1% PVP, and centrifuged at 12,000 × g at 4 ◦ C for 15 min. The supernatant was used for enzyme analysis and O2 − assay. SOD activity was determined according to the method of Giannopolitis and Ries (1977), which measure the inhibition in the photochemical reduction of nitroblue tetrazolium (NBT) spectrophotometrically at 560 nm. One unit of enzyme activity was defined as the quantity of SOD required to produce a 50% inhibition of NBT and the specific enzyme activity was expressed as units g−1 fresh weight h−1 . The reaction mixture contained 50 mM Na phosphate buffer solution (pH 7.8), 750 ␮M NBT, 130 mM lmethionine, 100 mM EDTA and 20 ␮M riboflavin. Reaction was

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Z. Zhu et al. / Environmental and Experimental Botany 66 (2009) 326–333

carried out at 25 ◦ C, under light intensity of about 300 ␮mol m−2 s−1 through 25 min. Activity of CAT was measured using the method of Abassi et al. (1998) which measured the decline of the extinction of H2 O2 at the maximum absorption at 240 nm. The reaction mixture contained 50 mM Tris–HCl buffer solution (pH 7.0) and 50 mM H2 O2 . One unit of CAT was defined as decreasing 0.1 in absorbance at 240 nm in 1 min. APX activity was measured according to Dalton et al. (1987). The assay depends on the decrease in absorbance at 290 nm as ascorbate was oxidized. The reaction mixture contained Na-phosphate buffer solution (pH 7.0), 0.9 mM ascorbate, 0.3 mM EDTA-Na2 and 0.25 mM H2 O2 . The enzyme activity was expressed in terms of ␮M of ascorbate oxidized g−1 FW h−1 . Superoxide anion was assayed following the method of Yuan et al. (2002) with a slight modification. Briefly, 1 mL of supernatant was mixed with 1 mL of 10 mM hydroxylammonium chloride and 0.5 mL of 65 mM phosphate buffer (pH 7.8), and the mixture was incubated at 25 ◦ C for 20 min. Then 1 mL of sulphanilamide (17 mM) and 1 mL of ␣-aminonaphthalene (7 mM) were added and the mixture was incubated at 30 ◦ C for 30 min. The absorbance was measured at 530 nm. Calibration curve of OD530 against NO2 − concentration was established (r2 = 0.9977). The concentration of O2 − was calculated as twice of that of NO2 − based on the following reaction: 2O2 − + H+ + NH2 OH → H2 O2 + H2 O + NO2 −

2.4. Lipid peroxidation assay The level of lipid peroxidation was measured in terms of malondialdehyde (MDA) content by the thiobarbituric acid (TBA) reaction method (Chen and Cao, 2008). 2.5. Non-enzymatic antioxidants assays A modified method of Wu et al. (2006) was employed for the assay of AsA and GSH. One gram of root tissue was extracted with 5 mL of 5% (w/v) metaphosphoric acid and centrifuged at 14,000 × g for 10 min at 4 ◦ C. The supernatant was used for AsA and GSH assays. The AsA assay mixture contained 0.4 mL supernatant, 75 mM phosphate buffer (pH 7.7), 10% (w/v) metaphosphoric acid, 44% (v/v) H3 PO4 , 4% (w/v) 2,2-bipyridyl and 3% (w/v) FeCl3 . The final mixture was incubated in 37 ◦ C for 60 min and cooled to room temperature. The absorbance was recorded at 525 nm. The GSH assay mixture contained 0.3 mL supernatant, 150 mM Na phosphate buffer solution (pH 7.7) and 0.3 mM 5,5 -dithiobis (2-itrobenzoic acid) (DTNB). The absorbance was measured at 412 nm.

2.7. DPPH radical scavenging activity and anti-lipid peroxidation assays For preparation of methanolic extract, 0.5 g of powdered dried sample was extracted with 25 mL 5% ammonia–methanol solution for 30 min by sonication. After filtering, the extract was dried under vacuum at 60 ◦ C and dissolved in methanol, filled up to the final volume of 25 mL with methanol. 2.7.1. Scavenging DPPH radical assay The scavenging of DPPH radical was assayed following the method of Öztürk et al. (2007). Briefly, 0.1 mM solution of DPPH in methanol was prepared and 4 mL of this solution was added to 1 mL of sample solutions. Thirty minutes later, the absorbance was measured at 517 nm. The capacity to scavenge the DPPH radical was calculated using the following equation: DPPH scavenging effect (%) =

Acontrol − Asample Acontrol

× 100

2.7.2. Anti-lipid peroxidation assay The preparation of liposome samples followed the method of Yi et al. (1997) with a slight modification. Lecithin was suspended in 0.01 M Na phosphate buffer solution (pH 7.4) at a concentration of 10 mg mL−1 by sonication and stirring with a glass rod in a bathtype sonicator. A modified method of Yen et al. (2008) was employed for the assay of lipid peroxidation inhibition capacity. 50 ␮L of liposome sample, 30 ␮L of test sample, 10 ␮L of 4 mM FeCl2 , and 10 ␮L of 0.2 mM ascorbic acid were pipetted into a 2 mL microcentrifuge tube and then incubated at 37 ◦ C for 1 h. Subsequently, 100 ␮L of 0.1N HCl, 40 ␮L of 9.8% SDS, 180 ␮L of de-ionized water, and 400 ␮L of 0.6% TBA were successively pipetted into each tube and vigorously shaken. These tubes were then heated to 95 ◦ C for 30 min. After cooling, tubes were treated with 1000 ␮L of n-butanol and centrifuged at 1000 × g for 25 min, and the supernatant was subsequently measured at 532 nm. All determinations were performed in triplicate. The percentage of lipid peroxidation inhibition was calculated as: Lipid peroxidation inhibition (%)



= 1−

induced532 nm − sample532 nm induced532 nm − control532 nm



× 100%

2.8. Saikosaponin a and d content assay SSa and SSd content was determined using a Waters HPLC system (Milford, MA, USA). The methods and conditions for determination have been reported previously (Zhu et al., 2008). 2.9. Experimental design and statistical analysis

2.6. H2 O2 content assay Hydrogen peroxide was estimated by forming titanium-hydro peroxide complex (Rao et al., 1997). One and a half gram root material was ground with liquid nitrogen and the fine powdered material was mixed with 3 mL cooled acetone. Mixture was centrifuged at 3000 × g at 4 ◦ C for 10 min. And 1.0 mL of supernatant was mixed with 0.1 mL of 5% titanium reagent and 0.2 mL of ammonium solution to precipitate the titanium-hydro peroxide complex. Reaction mixture was centrifuged at 3000 × g for 10 min. Precipitate was dissolved in 5 mL of 2 M H2 SO4 and then recentrifuged. Supernatant was read at 415 nm against reagent blank. Hydrogen peroxide content was calculated based on a standard curve.

All treatments were arranged in a completely randomized block design with three replicates. Statistical analysis was performed using one-way analysis of variance (ANOVA) and followed by Duncan’s multiple range tests (DWRT) with SAS for Windows 8e (SAS Institute Inc., Cary, NC, USA). And SAS for Windows 8e was used to determine correlation coefficients. 3. Results 3.1. RWC, H2 O2 , O2 − content and lipid peroxidation level RWC in leaves is known as an alternative measure of plant water status, reflecting the metabolic activity in tissues, therefore we

Z. Zhu et al. / Environmental and Experimental Botany 66 (2009) 326–333

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Table 1 SOD, CAT, APX activities and AsA, GSH contents in B. chinense roots subjected to water deficit and subsequent rewatering. RWC of leaves were also provided. B. chinense seedlings were subjected to water deficit for 0 d (control, C), 3 d (S1), 6 d (S2), 9 d (S3), and rehydrated S2 plants for 3 d (RH). Treatment C S1 S2 S3 RH

SOD activity (U g−1 FW h−1 ) 48.88 33.33 210.14 241.22 112.80

± ± ± ± ±

2.37 d 3.77 d 6.17 b 7.21 a 7.93 c

CAT activity (U g−1 FW min−1 ) 1.40 1.44 2.60 2.70 2.01

± ± ± ± ±

0.07 c 0.08 c 0.13 a 0.11 a 0.10 b

APX activity (␮mol Vc g−1 FW h−1 ) 19257 24513 24669 33262 15629

± ± ± ± ±

724 b 3550 ab 1035 ab 1206 a 582 b

AsA content (␮g g−1 FW) 19.43 91.90 40.25 43.32 99.21

± ± ± ± ±

GSH content (␮g g−1 FW)

0.69 b 9.96 a 3.41 b 0.91 b 12.32 a

63.92 64.30 63.79 61.46 70.68

± ± ± ± ±

4.81 ab 4.80 ab 4.65 ab 6.33 b 2.56 a

RWC (%) 93.02 69.54 63.50 45.78 94.68

± ± ± ± ±

2.78 a 3.86 b 3.04 b 6.68 c 2.65 a

Different letters indicate significant difference at p = 0.05. Means ± standard deviation (S.D.) (n = 3) are shown.

monitored the RWC of B. chinense leaves to understand how water status was affected by drought stress. Following dehydration by withholding water for 9 d, the RWC decreased from 93.02% in the well-watered plants to the value of 45.78% in the S3 plants (Table 1), indicating that 9 d of drought exposure caused a marked reduction in water availability. After 3 d of rewatering, the RWC of B. chinense leaves was 94.68%, indicating the plants recovered quickly their hydration state. Drought-induced oxidative stress was measured in terms of lipid peroxidation level (expressed in MDA content), H2 O2 , and O2 − content. As shown in Fig. 1, MDA content remained relatively stable even up to the 6th of the drought (S2, RWC of leaves 63.50%). After withholding water for 9 d (S3, RWC of leaves 45.78%), a pronounced lipid peroxidation was detected, with a nearly double MDA content compared to the well-watered control treatment (C), indicating the inadequacy of antioxidant defenses in combating AOS mediated damage. After rewatering, the MDA content returned to the control level. The changes in H2 O2 content were in a similar pattern with that of MDA, with the exception that the H2 O2 content in rehydrated plant was higher significantly than that in well-watered control (C) though lower than that in S3 plants. O2 − formed even at well water availability conditions with a low level (0.186 ␮mol g−1 FW) (Fig. 1). Once exposed to water deficit, a significant and continuous increase of O2 − content was observed. The O2 − level in root of S1 treatment increased significantly up to 14.86-fold over control treatment (C), while the corresponding values enhanced to 54.67- and 82.89-fold in S2 and S3 plants, respectively. After 3 d of rewatering, the O2 − content remained unchanged in comparison with that in S2 plants, though significantly lower than the value in S3 plants.

3.2. Antioxidant enzymes activity When B. chinense seedlings were exposed to slight drought stress (S1, RWC of leaves 69.54%), non-significant difference in SOD activity was obtained compared to well-watered control (C) (Table 1). However, the further severity of water deficit caused significant enhancement in SOD activity, being 3.30-fold and 3.93-fold higher in the S2 and S2 treatment over well-watered control (C). After recovery from moderate drought stress, a marked decrease by 46.32% was observed compared to that in the S2 treatment. CAT activity, which decomposes the H2 O2 produced by SOD, changed in parallel with that of SOD activity, the highest value being observed in S3 plants (Table 1). However, there was no significant difference between S2 and S3 treatments. As water deficit progressed, another scavenger of H2 O2 , the APX activity of root followed a similar trend (Table 1). The highest APX activity was detected in S3 plants which had been withheld water for 9 d, with an increase of 73% compared to the well-watered control. After water availability was recovered to initial level, the APX activity decreased to 81.16% of that in S2 plants. 3.3. Non-enzymatic antioxidants contents A significant increase (3.73-fold) in AsA content was observed in S1 plants in comparison with that in well-watered plants (C) (Table 1). However, no significant difference was detected in S2, S3, and well-hydrated plants. AsA content enhanced after rewatering, being 4.11-fold higher than that in well-hydrated plants. It could be related to different rates of de novo AsA biosynthesis, AsA depletion by AOS scavenging and its recycling from oxidized forms of AsA, such as dehydroascorbate and semidehydroascorbate during progressive drought stress and subsequent rewatering phase. In case of GSH content, no significant difference was found during the progressive drought (Table 1). The maximum GSH content was recorded in rehydrated roots, which was significantly higher than that in S3 plants. 3.4. Saikosaponin a and d content

Fig. 1. H2 O2 , MDA, and O2 − contents in B. chinense roots subjected to water deficit and subsequent rewatering. B. chinense seedlings were subjected to water deficit for 0 d (control, C), 3 d (S1), 6 d (S2), 9 d (S3), and rehydrated S2 plants for 3 d (RH). Different letters indicate significant difference at p = 0.05. Means ± standard deviation (S.D.) (n = 3) are shown.

SSa content changed in parallel with that of SSd (Fig. 2). Both SSa and SSd content showed a linear increase when drought progressed from well-watered control (C) to moderate drought stress (S2). Relative to well-watered control (C), S2 treatment resulted in 82.61% increase in SSa content and 22.39% in SSd content. Endogenous SSa and SSd levels increased in roots of drought-stressed B. chinense, suggesting a role for SSa and SSd in response to drought. However, SSa and SSd content showed a significant decrease compared to that in the S2 treatment when exposed to severe water deficit, i.e. withholding water for 9 d (S3). And after enduring moderate water deficit (S2), the recovery of water availability to initial well-watered situation resulted in marked decrease in SSa and SSd content. The SSa and SSd content in roots of rehydrated plants were 7.14% and 88.01% of those in S2 plants.

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Z. Zhu et al. / Environmental and Experimental Botany 66 (2009) 326–333 Table 2 Correlation analysis of SSa, SSd content, the scavenging DPPH radical activity and anti-FeCl2 -ascorbic acid-stimulated lipid peroxidation activity in 5% ammonia– methanol extract of B. chinense roots. Scavenging DPPH radical activity (%) Scavenging DPPH radical activity (%) Anti-lipid peroxidation activity (%) SSd content (%) SSa content (%) *

Fig. 2. SSa and SSd content in roots of B. chinense subjected to drought and subsequent rewatering. B. chinense seedlings were subjected to water deficit for 0 d (control, C), 3 d (S1), 6 d (S2), 9 d (S3), and rehydrated S2 plants for 3 d (RH). Different letters indicate significant difference at p = 0.05. Means ± standard deviation (S.D.) (n = 3) are shown.

3.5. DPPH radical scavenging activity and anti-lipid peroxidation activity Absorbance at 517 nm decreases when DPPH radical accepts an electron or hydrogen radical; thus DPPH radical is used as a substrate to evaluate the scavenging activity of free radicals. And the liposome peroxidation induced by reactive metal ions and reducing agents is widely used to simulate the vivo lipid peroxidation. Hence, in this study the antioxidant potential of methanolic extracts of B. chinense roots was evaluated using two antioxidative tests, namely DPPH radical-scavenging activity and inhibition activity of ferrous ions-induced lipid peroxidation. When exposed to slight drought stress (S1), the methanolic extract of the roots exhibited lower DPPH radical-scavenging capacity than that in well-hydrated control (C) (Fig. 3). Relative to well-watered control (C), methanolic extract of the root of S3 plants exerted the highest capacity to detoxify oxygen radicals, followed by that of S2 plants, being 44.83% increase in S3 plants and 27.81%

Anti-lipid peroxidation activity (%)

SSd content (%)

SSa content (%)

1 0.59

1

1

0.75

1

0.82* 0.47

0.38 −0.16

The level of significance is indicated as follows: 0.05 > p >0.01.

increase in S2 plants. After 3 d of rewatering, 9.29% decrease was observed in comparison with that of S2 plants. In case of lipid peroxidation inhibition activity of the methanolic extract of different treatments, it increased with the progressive drought from C to S2, and decreased after further dehydration or rehydration. As shown in Table 2, there was no significant linear correlation between SSa (or SSd) content and DPPH radical-scavenging activity. However, a significant linear correlation between SSd content and lipid peroxidation inhibition activity was established (r2 = 0.83, p < 0.05). 3.6. Correlation analysis There were significant positive correlations among SOD, CAT activities and O2 − content (Table 3). This result can be interpreted by that SOD is known to be a substrate-inducible antioxidant enzyme, and that CAT and SOD shows close connection in the substrate and product. The higher CAT activity indicates that plants have greater capacity to decompose H2 O2 generated by SOD. The positive correlation between APX activity and GSH content suggested the important role of AsA–GSH cycle in protecting B. chinense from oxidative stress. The level of lipid peroxidation (as measured in MDA content) was directly related to H2 O2 content, and H2 O2 content was positively related with O2 − content, indicating the close relationship among these parameters. The correlation coefficient between SSa (or SSd) and any other parameter was not significant (p > 0.05) (Table 3). This mainly resulted from the different response of these parameters to severe drought stress (S3). SOD, CAT, and APX activities as well as O2 − , MDA, and H2 O2 content reached their maximum values under severe drought stress conditions, while SSa and SSd content in S3 plants was significantly lower than that in S2 plants. However, the Table 3 Correlation analysis of SSa, SSd contents, antioxidant system and AOS, MDA contents in roots of B. chinense subjected to drought and subsequent rewatering. CAT

Fig. 3. DPPH radical-scavenging activity and anti-FeCl2 -ascorbic acid-stimulated lipid peroxidation activity of the methanolic extract of B. chinense subjected to drought and subsequent rewatering. B. chinense seedlings were subjected to water deficit for 0 d (control, C), 3 d (S1), 6 d (S2), 9 d (S3), and rehydrated S2 plants for 3 d (RH). Different letters indicate significant difference at p = 0.05. Means ± standard deviation (S.D.) (n = 3) are shown. The test samples were prepared at the concentration of 20 mg mL−1 .

CAT APX SOD AsA GSH H2 O2 O2 − MDA SSd SSa * **

APX

SOD

AsA

1 0.58 1 1 0.99** 0.63 −0.19 −0.31 −0.3 1 0.68 −0.25 −0.83* −0.34 0.63 0.59 0.65 0.12 0.94** 0.58 0.93** 0.03 0.73 −0.2 0.68 0.85* 0.69 0.41 0.63 0.07 0.07 0.3 0.07 −0.4

O2 −

GSH

H2 O2

1 −0.14 −0.13 −0.54 −0.19 −0.58

1 0.83* 1 0.91* 0.79 0.07 0.55 −0.55 −0.2

MDA

SSd

SSa

1 0.17 −0.2

1 0.59

1

The level of significance is indicated as follows: 0.05 > p >0.01. The level of significance is indicated as follows: 0.01 > p > 0.001.

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parallel changes in SSa (or SSd) content and SOD, CAT, and APX activities as well as O2 − content in C, S1, S2, and RH plants indicated the possible intimate relationship between the accumulation of SSa, SSd and antioxidant potential, as well as O2 − accumulation.

4. Discussion Bupleuri Radix, dried roots of Bupleurum spp. (Apiaceae), has been used as medicine in China for over 2000 years, and it is one of the most common components of Chinese traditional medicine prescriptions for the treatment of chronic hepatitis, kidney syndrome, inflammatory diseases, and ulcers of the digestive system (Abe et al., 1982; Katakura et al., 1991; Pistelli et al., 1996; Guo et al., 2000; Sánchez-Contreras et al., 2000; Pan, 2006). And it is believed that saikosaponin content is one of the most important criteria for determining Bupleuri Radix quality (Pan, 2006; Zhu et al., 2007). The concentration of saikosaponins depends on the growth location, the time of harvest and the part of the root (Zhu et al., 2006), and is also affected by plant condition, characteristics of individual plant, fertilization, and cultivation methods (Pan, 2006). Large body evidence indicated that the extracts from Bupleurum spp. possess antioxidant activity. Wang et al. (2004) observed that fraction R and F1 have the most saikosaponins and most effectively protect against CCl4 -induced hepatotoxicity. Similarly, Liu et al. (2005) found that as the highest saikosaponin as well as baicalin and baicalein (flavonoid compounds) contents were detected in the 95% ethanol extract, the best inhibitory effect of lipid peroxidation and the highest DPPH and superoxide anion radical scavenging activity were observed. Additionally, pharmacological experiment showed that saikosaponin, a kind of triterpenoid compounds, possessed scavenging activity against reactive oxygen species and inhibited peroxidation of rat liver homogenate (Fang and Zheng, 2002; Wang et al., 2004; Liu et al., 2005). Abe et al. (1982) found that pretreatment with SSd produced a remarkable inhibitory action on acute hepatic injury by CC14 , and a significant inhibition of lipid peroxidation induced by an acute dose of CC14 in the liver of rats pre-treated with SSd was also noted. The authors suggested that SSd brings about its protective effect by the alteration of plasma membranes or a direct membrane stabilizing effect in addition to the change of drug-metabolizing enzyme activities or inhibitory action against lipid peroxidation. Nishiura et al. (1994) suggested that since saikosaponins have been known to have various pharmacological effects, including stabilization of cell membranes, the protective action of saikosaponin against halothane-induced hepatitis may be partly due to stabilization of the cell membrane of hepatocytes. In present study, the significant correlation between SSd content and lipid peroxidation inhibition capacity demonstrated the dominant role of SSd in protecting cell membrane against oxidative stress. The biochemistry of oxygen-activation and -detoxification analyzed in the past, the identification of similarities between plants and animals opened a new field of research (Grassmann et al., 2002). Lipid peroxidation is associated with some diseases such as carcinogenesis, atherosclerosis, and coronary heart disease by interfering in metabolic reactions (Wang et al., 2005). Furthermore, terpenoids have been shown to possess antioxidative properties in different situations, particularly against lipid peroxidation as a result of their high lipophilicity (Grassmann et al., 2002). Based on information mentioned above, the membrane-stabilizing capacity of SSa and SSd under drought stress is assumed for their physiological and pharmacological function in human disease. SSa and SSd are a pair of stereoisomers (Shyu et al., 2004), and just differ in the configuration of the hydroxyl function at C16, i.e. SSa has a ␤-hydroxyl

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function and SSd has an a-hydroxyl function (Dobashi et al., 1995). The pharmacological experiments demonstrated that the activity of SSd was more than that of SSa. Therefore it was suggested that a-OH of C-16 had more pharmacological activity than ␤-OH (Pan, 2006). Predicted by Growth/Differentiation Balance Hypothesis (GDBH), rapidly growing plants have low secondary metabolite concentration as a result of a resource-based trade-off between primary and secondary metabolic pathways. However, moderate water limitation slows growth more than carbon assimilation, which can result in the accumulation of carbohydrates in source leaves. And this may increase the substrate available for secondary metabolism. However, when resource limitation is severe enough to depress carbon assimilation, secondary metabolism is predicted to fall because of energy and substrate concentration on biosynthesis (Hale et al., 2005). Coinciding with this prediction, SSa and SSd content increased as the drought progressed, while it decreased under severe drought conditions or when rewatered from moderate drought. It is well known that drought stress induces formation of AOS and consequently activates expression of genes encoding antioxidant enzymes (Torres-Franklin et al., 2008). Our results showed that B. chinense exhibited strong protection against AOS reflected by the low MDA and H2 O2 contents when exposed to slight or moderate drought stress. This may be explained at least partly by that there was a significant increase in SOD, CAT, and APX activities of roots as drought stress progressed, which suggested the stimulation of antioxidant mechanism for effective removal of AOS. Since a higher activity of SOD under drought stress was accompanied by increase in APX and CAT activities as well as O2 − content in the root of B. chinense, it may be suggested that SOD, CAT, and APX are working more efficiently to decompose oxidants such as O2 − and H2 O2 . This is in agreement with numerous previous reports in the literature that underline the intimate relationship between enhanced or constitutive antioxidant enzyme activities and increased resistance to environmental stress (Türkan et al., 2005; Misra and Gupta, 2006; Chen and Cao, 2008). After withholding water for 9 d, the physiological balance was considerably disturbed and MDA, H2 O2 , and O2 − contents increased drastically, which revealed the increased lipid peroxidation resulted from elevated oxidative stress. The production of AOS in plants, known as the oxidative burst, is an early event of plant defense response to different stress and acts as a secondary massager to trigger subsequent defense reaction in plants (Shohael et al., 2006). Hence the drought-induced biosynthesis and accumulation patterns of SSa and SSd, as well as the parallel increase of SSa, SSd and O2 − content in C, S1, S2, RH plants demonstrated that the accumulation of SSa and SSd, especially SSd is assumed to be the result of physiological adaptation and to protect B. chinense from drought-induced oxidative stress. The AOS possibly represent a stress signal that triggers the activation of antioxidant enzymes and accumulation of SSa and SSd. These results were in close conformity with previous works on other medicinal plants (Munné-Bosch et al., 2001; Misra and Gupta, 2006; Jaleel et al., 2007a,b). Relatively stable organic radical DPPH has been widely used to evaluate the antioxidant activity of various samples (Jung et al., 2008). The method is based on the reduction of DPPH solution in the presence of hydrogen-donating antioxidant due to the formation of non-radical-donating DPPH-H (Öztürk et al., 2007; Jung et al., 2008). Nevertheless, the non-significant relationship between SSa (or SSd) content and DPPH radical-scavenging ability suggested that the radical-scavenging capacity may be not the main mechanism for membrane protection of SSa and SSd. And according to the result of Wang et al. (2004), saikosaponins suppress the activities of P450 and cytochrome b5 (consequently lower

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the production of AOS) may be involved in the reduction in the amount of CCl4 metabolite and hepatoprotective effects. Therefore, further studies are required to confirm the elaborate function of SSa and SSd in antioxidant system, such as scavenging and/or detoxifying free radicals, blocking their production, or enhancing the activities of antioxidant enzymes, which is underway in our laboratory. Despite a few decades of research, the enzymes, genes, and biochemical pathways involved in saikosaponin biosynthesis are largely uncharacterized. The results of current investigation suggest the potential of using suitable water stress as strategy for increasing the content of SSa and SSd. To meet the strong market demand, regulation of the biosynthesis of SSa and SSd in terms of their possible function in plant cell may be feasible to bypass the low product yield bottle neck. In summary, B. chinense showed strong drought resistance and protected themselves from oxidative stress with various physiological and biochemical changes such as the activation of antioxidant system. Additionally, this study strongly supports the hypothesis that SSa and SSd play significant physiological role in B. chinense drought resistance against oxidative damage and subsequent lipid peroxidation. AOS may be serve as signal massager to trigger the activation of antioxidant system and the accumulation of SSa and SSd. The elaborate physiological and biochemical mechanism that SSa and SSd play a crucial role under drought or other environmental stress remains unclear and needs to be addressed in the further. In addition, to take advantage of the physiological function of saikosaponins for increasing the content and total yield of in the cultivation of B. chinense, a production protocol consisting of the applied drought stress at suitable phonological stages and degree is needed to investigate. Acknowledgement This work was supported by the Knowledge Innovation Project of Chinese Academy of Science (KZCX2-XB2-05-01). References Abassi, N.A., Kushad, M.M., Endress, A.G., 1998. Active oxygen-scavenging enzymes activities in developing apple flowers and fruits. Sci. Horticult. 74, 183– 184. Abe, H., Sakaguchi, M., Odashima, S., Arichi, S., 1982. Protective effect of saikosaponin-d isolated from Bupleurum falcatum L. on CCl4 -induced liver injury in the rat. Naunyn-Schmiedeberg’s Arch Pharmacol. 320, 266–271. Aoyagi, H., Kobayashi, Y., Yamada, K., Yokoyama, M., Kusakari, K., Tanaka, H., 2001. Efficient production of saikosaponins in Bupleurum falcatum root fragments combined with signal transducers. Appl. Microbiol. Biotechnol. 57, 482– 488. Chen, J.W., Cao, K.F., 2008. Changes in activities of antioxidative system and monoterpene and photochemical efficiency during seasonal leaf senescence in Hevea brasiliensis trees. Acta Physiol. Plant 30, 1–9. Chen, L.R., Chen, Y.J., Lee, C.Y., Lin, T.Y., 2007. MeJA-induced transcriptional changes in adventitious roots of Bupleurum kaoi. Plant Sci. 173, 12–24. Dalton, D.A., Hanus, F.J., Russell, S.A., Evans, H.J., 1987. Purication, properties and distribution of ascorbate peroxidase in legume root nodules. Plant Physiol. 83, 789–794. Dobashi, I., Tozawa, F., Horiba, N., Sakai, Y., Sakai, K., Suda, T., 1995. Central administration of saikosaponin-d increases corticotropin-releasing factor mRNA levels in the rat hypothalamus. Neurosci. Lett. 197, 235–238. Fang, Y.Z., Zheng, R.L., 2002. Theory and Application of Free Radical Biology. Science Press, Beijing (in Chinese). Giannopolitis, C.N., Ries, S.K., 1977. Superoxide dismutase, occurrence in higher plants. Plant Physiol. 59, 309–314. Grassmann, J., Hippeli, S., Elstner, E.F., 2002. Plant’s defence and its benefits for animals and medicine: role of phenolics and terpenoids in avoiding oxygen stress. Plant Physiol. Biochem. 40, 471–478. Guo, Y.J., Matsumoto, T., Kikuchi, Y., Ikejima, T., Wang, B.X., Yamada, H., 2000. Effects of a pectic polysaccharide from a medicinal herb, the roots of Bupleurum falcatum L. on interleukin 6 production of murine B cells and B cell lines. Immunopharmacology 49, 307–316. Hale, B.K., Herms, D.A., Hansen, R.C., Clausen, T.P., Arnold, D., 2005. Effects of drought stress and nutrient availability on dry matter allocation, phenolic glycosides, and

rapid induced resistance of poplar to two lymantriid defoliators. J. Chem. Ecol. 31, 2601–2620. Jaleel, C.A., Manivannan, P., Sankar, B., Kishorekumar, A., Gopi, R., Somasundaram, R., Panneerselvam, R., 2007a. Induction of drought stress tolerance by ketoconazole in Catharanthus roseus is mediated by enhanced antioxidant potentials and secondary metabolite accumulation. Colloids Surf. B: Biointerfaces 60, 201–206. Jaleel, C.A., Manivannan, P., Sankar, B., Kishorekumar, A., Gopi, R., Somasundaram, R., Panneerselvam, R., 2007b. Water deficit stress mitigation by calcium chloride in Catharanthus roseus: effects on oxidative stress, proline metabolism and indole alkaloid accumulation. Colloids Surf. B: Biointerfaces 60, 110–116. Jung, M.J., Heo, S.I., Wang, M.H., 2008. Free radical scavenging and total phenolic contents from methanolic extracts of Ulmus davidiana. Food Chem. 108, 482– 487. Katakura, M., Kimura, T., Endo, I., 1991. Production of saikosaponins by tissue culture of Bupleurum falcatum L. Bioproc. Biosyst. Eng. 7, 97–100. Liu, C.T., Wu, C.Y., Weng, Y.M., Tseng, C.Y., 2005. Ultrasound-assisted extraction methodology as a tool to improve the antioxidant properties of herbal drug Xiao-chia-hu-tang. J. Ethnopharmacol. 99, 293–300. Minami, M., Sugino, M., Sadaoka, M., Hasegawa, C., Ohe, C., Ashida, A., Ogaki, K., 1995. Physiological response and improvement of tolerance to environmental stress in Bupleurum falcatum L. (II) Comparison of sensitivities to dry stress between rosette and bolting plants. Nat. Med. 49, 401–408 (in Japanese with English abstract). Misra, N., Gupta, A.K., 2006. Effect of salinity and different nitrogen sources on the activity of antioxidant enzymes and indole alkaloid content in Catharanthus roseus seedlings. J. Plant Physiol. 163, 11–18. Munné-Bosch, S., Mueller, M., Schwarz, K., Alegre, L., 2001. Diterpenes and antioxidative protection in drought-stressed Salvia officinalis plants. J. Plant Physiol. 158, 1431–1437. Nacif de Abreu, I., Mazzafera, P., 2005. Effect of water and temperature stress on the content of active constituents of Hypericum brasiliense Choisy. Plant Physiol. Biochem. 43, 241–248. Nishiura, T., Marukawa, S., Ishida, H., Orita, M., Abe, H., 1994. Effects of saikosaponins on hepatic damage induced by halothane and hypoxia in phenobarbitalpretreated rats. J. Anesth. 8, 87–92. Öztürk, M., Aydo˘gmus¸-Öztürk, F., Duru, M.E., Topc¸u, G., 2007. Antioxidant activity of stem and root extracts of Rhubarb (Rheum ribes): an edible medicinal plant. Food Chem. 103, 623–630. Pan, S.L., 2006. Bupleurum Species: Scientific Evaluation and Clinical Applications. Taylor & Francis Group, CRC Press, New York. Pistelli, L., Bertoli, A., Rita, A., Morelli, I., 1996. Minor constituents from Bupleurum fruticosum. Phytochemistry 41, 1579–1582. Rao, M.V., Paliyath, G., Ormrod, D.P., Murr, D.P., Watkins, C.B., 1997. Influence of salicylic acid on H2 O2 production, oxidative stress and H2 O2 metabolizing enzymes. Plant Physiol. 115, 137–149. Sánchez-Contreras, S., Díaz-Lanza, A.M., Bartolomé, C., Bernabé, M., 2000. Minor sulfated saikosaponins from the aerial parts of Bupleurum rigidum L. Phytochemistry 54, 783–789. SAS, 2000. SAS Version 8e, SAS Systems for Windows. SAS Institute Inc., Cary, NC, USA. Shohael, A.M., Ali, M.B., Yu, K.W., Hahn, E.J., Islam, R., Paek, K.Y., 2006. Effect of light on oxidative stress, secondary metabolites and induction of antioxidant enzymes in Eleutherococcus senticosus somatic embryos in bioreactor. Process Biochem. 41, 1179–1185. Shyu, K.G., Tsai, S.C., Wang, B.W., Liu, Y.C., Lee, C.C., 2004. Saikosaponin C induces endothelial cells growth, migration and capillary tube formation. Life Sci. 76, 813–826. Smart, R.E., Bingham, G.E., 1974. Rapid estimates of relative water content. Plant Physiol. 53, 258–260. Torres-Franklin, M.L., Contour-Ansel, D., Zuily-Fodil, Y., Pham-Thi, A.T., 2008. Molecular cloning of glutathione reductase cDNAs and analysis of GR gene expression in cowpea and common bean leaves during recovery from moderate drought stress. J. Plant Physiol. 165, 514–521. Türkan, I˙ ., Bor, M., Özdemir, F., Koca, H., 2005. Differential responses of lipid peroxidation and antioxidants in the leaves of drought-tolerant P. acutifolius Gray and drought-sensitive P. vulgaris L. subjected to polyethylene glycol mediated water stress. Plant Sci. 168, 223–231. Wang, B.J., Liu, C.T., Tseng, C.Y., Wu, C.P., Yu, Z.R., 2004. Hepatoprotective and antioxidant effects of Bupleurum kaoi Liu (Chao et Chuang) extract and its fractions fractionated using supercritical CO2 on CCl4 -induced liver damage. Food Chem. Toxicol. 42, 609–617. Wang, B.J., Liu, C.T., Tseng, C.Y., Wu, C.P., Yu, Z.R., 2005. Antioxidant activity of Bupleurum kaoi Liu (Chao et Chuang) fractions fractionated by supercritical CO2 . LWT 38, 281–287. Wu, Q.S., Xia, R.X., Zou, Y.N., 2006. Reactive oxygen metabolism in mycorrhizal and non-mycorrhizal citrus (Poncirus trifoliata) seedlings subjected to water stress. J. Plant Physiol. 163, 1101–1110. Yen, F.L., Wu, T.H., Lin, L.T., Cham, T.M., Lin, C.C., 2008. Concordance between antioxidant activities and flavonol contents in different extracts and fractions of Cuscuta chinensis. Food Chem. 108, 455–462. Yi, O., Meyer, A.S., Frankel, E.N., 1997. Antioxidant activity of grape extracts in a lecithin liposome system. JAOCS 74, 1301–1307. Yuan, Y.J., Li, J.C., Ge, Z.Q., Wu, J.C., 2002. Superoxide anion burst and taxol production induced by Ce4+ in suspension cultures of Taxus cuspidata. J. Mol. Catal. B: Enzym. 18, 251–260.

Z. Zhu et al. / Environmental and Experimental Botany 66 (2009) 326–333 Zhu, S., Shimokawa, S., Shoyama, Y., Tanaka, H., 2006. A novel analytical ELISAbased methodology for pharmacologically active saikosaponins. Fitoterapia 77, 100–108. Zhu, S.H., Morinaga, O., Shimokawa, S., Shon, T.K., Lee, S.C., Shoyama, Y., Tanaka, H., 2007. Eastern blotting and use of anti-saikosaponin a monoclonal antibodies for detection of saikosaponins. J. Nat. Med. 61, 178–183.

333

Zhu, Z.B., Liang, Z.S., Han, R.L., Dong, J.E., 2008. Growth and saikosaponin production of the medicinal herb Bupleurum chinense DC. under different levels of nitrogen and phosphorus. Ind. Crops Prod., 010, doi:10.1016/j.indcrop.2008.04. Zobayed, S.M.A., Afreen, F., Kozai, T., 2005. Temperature stress can alter the photosynthetic efficiency and secondary metabolite concentrations in St. John’s wort. Plant Physiol. Biochem. 43, 977–984.