Response of Ginsenoside Rb1 Production in Panax Ginseng Cells

Full Length Research Paper

Response of ginsenoside Rb 1 production in Panax ginseng cells to Amaranthus tricolor cells

Cai-Jun Yue*, Yan-Ping He, Zhong-Jing Zang and Yu-Dong Cui

College of Life Science and Biotechnology, Heilongjiang August First Land Reclamation University, Daqing 163319, P. R. China.

Accepted 31 March, 2010

Ginsenoside Rb 1 is one of the principal pharmacological ingredients of ginseng. The effects of Amaranthus tricolor suspension cells, containing high level of squalene, on ginsenoside Rb 1 production by Panax ginseng suspension cells were investigated. The maximum Rb1 content (51.5 ± 3.1 mg/L) reached during the next 4 d after A. tricolor cells (16 g fresh weight /L) were added into P. ginseng suspension cell cultures on day 16 of cultivation, which was around 2-fold that in control. The activity of ginsenoside Rb1 synthase was increased by 1.6 fold at this point. At the same time, addition of A. tricolor cells increased the content of squalene, a precursor of ginsenoside Rb 1, in the culture medium of P. ginseng suspension cell cultures. Feeding squalene (10 mg/L) led to a 16 ± 0.9% increase in ginsenoside Rb 1 production. The observation suggested that the enhanced Rb 1 production resulted from that the A. tricolor cells invoked the ginsenoside Rb 1 synthase and supplied the precursor squalene. Rational utilization of suspension cell co-culture could be a novel tactic for improving ginsenoside production by cell cultures.

Key words: Ginsenoside Rb 1, Panax ginseng , ginsenoside Rb 1 synthase, Amaranthus tricolor , plant cell suspension culture.

INTRODUCTION

Ginsenoside Rb 1 is one of the main active ingredients of in Panax notoginseng suspension cells and P. ginseng Panax ginseng which is considered as one of the most adventitious roots were investigated (Yue and Zhong, valuable medicinal herbs in traditional Asian medicine. 2005; Hu and Zhong, 2007; Yu et al., 2005), but no Ginsenoside Rb 1 has many pharmacological effects information about effects of any plant suspension cells on including immunostimulatory, antidiabetic effects, as well production of ginsenoside Rb 1 by P. ginseng suspension as effects on the central nervous system (Christensen, cells and its synthase activity was available yet. This work 2009). It is biosynthesized via the isoprenoid pathway. In is to investigate the influence of Amaranthus tricolor the pathway some important steps are included: oxidation suspension cells on ginsenoside Rb 1 production by P. of squalene to 2,3-oxidosqualene, cyclization of 2,3- ginseng suspension cells and ginsenoside Rb 1 synthase oxidosqualene to dammarenediol II, oxidation and activity. glycosylation of dammarenediol II to ginsenoside Rb 1 Interactions between natural plants have been (Han et al., 2006). The last step of ginsenoside Rb1 investigated for many years. Chemicals, especially biosynthesis is catalyzed by ginsenoside Rb 1 synthase secondary metabolites, are introduced by the plant into that is UDPG (uridine diphosphate glucose): ginsenoside the environment, which influence the growth and Rd glucosyltransferase (UGRdGT) (Yue and Zhong, development of neighboring plants (Gniazdowska and 2005). In recent year, effects of some incitants, e.g. Bogatek, 2005). Recently interactions between cultured synthetic jasmonate derivatives, external calcium signals organs of plants in vitro have been becoming a focal point and organic germanium, on ginsenoside Rb 1 biosynthesis of attention (Sidwa-Gorycka et al., 2003; Mohagheghzadeh et al., 2008; Wu et al., 2008). The investigations showed that the growth rates and the secondary metabolite contents were affected when a root *Corresponding author. E-mail: [email protected]. Tel: 086- was cultured with shoots or other roots. However, to our 459-6819299. Fax: 086-459-6819290. best knowledge, no report about effects of plant 898 J. Med. Plant. Res.

suspension cell co-culture on the secondary metabolite suspension cells on ginsenoside Rb 1 production. production and the related synthases has been published. For increasing production of secondary Establishment of A. tricolor suspension cell cultures metabolites by means of plant-plant interactions, it is worthy to investigate the effect of a species plant A. tricolor young leaves from Guangdong province of China were suspension cells on the secondary metabolite surface-sterilized by a 10 second immersion in 70% (v/v) ethanol biosynthesis in another species plant suspension cells. and for 5 min in aqueous solution of 0.1% (w/v) HgCl 2 and induced A. tricolor P. ginseng to produce calli. The medium, conditions and procedure of callus In this work, and suspension cells induction and the establishment process of suspension cell cultures were used to investigate the effects of suspension cell co- were the same as callus induction and the suspension cell cultures culture on the secondary metabolite ginsenoside Rb 1 establishment of P. ginseng above. The suspension cells production. A. tricolor is a vegetable amaranth being subcultured 10 times were used to investigate the effects of A. cultivated since centuries in many parts of the world, its tricolor suspension cells on ginsenoside Rb 1 production by P. leaves and seeds contain high level squalene (0.027- ginseng cells.

0.080 mg/g dry leaf; 1.67-3.08 mg/g dry seed) (He and Corke, 2003). Squalene is an unsaponifiable lipid and Co-cultures of P. ginseng and A. tricolor suspension cells acts as a biosynthetic precursor to some saponins and steroids in plants. Squalene can be oxidized to 2,3- All the experiments were performed in 250 ml Erlenmeyer flasks oxidosqualene mediated by the squalene epoxidase in containing the liquid medium of 50 ml described above and under darkness and 24 ± 1°C, and the initial inoculum size was 2 g fresh ginseng cells, and is a precursor of ginsenoside Rb 1 in cell of P. ginseng and A. tricolor harvested by vacuum filtration, the ginsenoside biosynthesis pathway (Zhong and Yue, respectively. On day 6 and 16 of cultivation that is at the end of lag 2005). Addition of precursors would enhance the phase and log phase of the P. ginseng suspension cell the A. production of plant secondary metabolites (Smetanska, tricolor cells were harvested by vacuum filtration and then added 2008), so it is interesting whether A. tricolor suspension into the P. ginseng suspension cell cultures in different inoculum P. sizes, respectively. The mono-culture of P. ginseng suspension cells would boost the production of ginsenoside Rb 1 by cells and the mono-culture of A. tricolor suspension cells were used ginseng suspension cells or not, via providing squalene as controls. For all cultures, multiple flasks were run under each and other pathways when they were co-cultured together. condition and the data represent the mean values with standard This work aims to understand how A. tricolor suspension deviations from three independent samples. cells affected ginsenoside Rb 1 biosynthesis in P. ginseng cells and how to manipulate ginsenoside Rb 1 production Addition of squalene to P. ginseng suspension cell cultures by co-culturing the suspension cells of two different species. In order to investigate the effects of squalene on the ginsenoside Rb 1 production, different concentration of squalene were added into the P. ginseng suspension cell cultures on day 16 of cultivation time. Squalene solutions (concentration: 1 g/L and 10 g/L) were MATERIALS AND METHODS made by dissolving squalene in dimethyl sulfoxide (DMSO) and sterilized by passing through 0.22 µm polyvinylidenedifluoride Establishment of P. ginseng suspension cell cultures syringe filter (Millipore). The P. ginseng cell cultures were added with the squalene solutions (50 µL /50 mL culture), or DMSO (50 µ The kernels of P. ginseng seeds from Changbai Mountain in Jilin L /50 mL culture) without squalene as control, respectively. province of China were surface-sterilized by a 10 s immersion in 70% (v/v) ethanol and for 15 min in aqueous solution of 0.1% (w/v) HgCl 2 and induced to produce calli. The induction medium was Measurement of biomass and ginsenoside Rb 1 content Murashige and Skoog (MS) medium with 13.6 µM 2,4- dichlorophenoxyacetic acid, 10.7 µM naphthaleneacetic acid, 2.2 The analytical procedures for cell dry weight and ginsenoside Rb 1 µM 6-Benzylaminopurine, 3% sucrose(W/V) and 0.6% agar(W/V). content were similar as described previously (Yue and Zhong, After two months the induced calli under darkness and 24 ± 1°C 2005). For sampling, three identical shake flasks were used for were subcultured at an interval of 30 days on the same culture each data point. The mono-cultured P. ginseng suspension cells, medium as above described. For establishment of suspension cell the mono-cultured A. tricolor suspension cells and their co-culture cultures, ten gram of the loose calli subcultured 8 times were cells were harvested, respectively. The samples from flasks were inoculated into a 250 ml Erlenmeyer flask containing 50 ml liquid filtered under vacuum and dried at 45ºC, and weighed. For medium where contained the same ingredients as in the above ginsenoside Rb 1 extraction, 100 mg of powdered dry cells was medium except for agar, and cultured on a rotary shaker (110 rpm) soaked in 4 ml of n-butanol for 1 day, and then ultrasonicated for 30 under darkness and 24 ± 1°C. On day 3 of cultivation, the small min. After centrifugation at 4,000 g for 10 min, the extract was suspension cell masses (diameter < 2 mm) were collected by separated and the cell debris was extracted once more with 2 ml of filtration with a sieve and vacuum filtration with a Buchner funnel n-butanol. The combined extracts were finally evaporated to and Whatman filter papers, and the cell masses of 2 g fresh weight dryness at 45°C under vacuum. The residue was dissolved in 1 mL (FW) were subcultured in a 250 ml flask containing 50 ml liquid of methanol and filtered through a 0.22 µm PVDF syringe filter medium under the same culture conditions as above described. The (Millipore) and 20 µL were taken and analyzed by reversephase following subculture were performed at an interval of 20 days by HPLC, using a Shimadzu LC-2010 HPLC apparatus equipped with transferring 2 g fresh cells harvested by vacuum filtration to a 250 a variable-wavelength UV detector. A VP-ODS column (250 mm ml Erlenmeyer flask containing 50 ml medium under the same long, 4 mm i.d., 5 µm particles; Shimadzu) was used at 25°C. The conditions as above described. The suspension cells subcultured mobile phase consisted of acetonitrile and water and the following 10 times were used to investigate the effects of A. tricolor gradient procedure was used: 0 - 5 min, 30 - 32% acetonitrile, Yue et al. 899

5 – 32 min, 32 - 46% acetonitrile, at a constant flow rate of 1 established and cultivated as described in materials and ml/min, at 25°C; detection wavelength: 203 nm. Ginsenoside Rb 1 methods. On day 6 and 16 of cultivation, respectively, the was identified by comparison with its authentic sample (Sigma). The A. tricolor cells were harvested and then added into the P. quantification was done according to the external calibration graph obtained from the peak areas vs. different concentrations of ginseng suspension cell cultures to co-culture. ginsenoside Rb 1 standard. The standard curve equation: A = Ginsenoside Rb 1 in these suspension cell cultures was 226.51, G (ng) + 722, where A is the peak area of ginsenoside Rb 1, determined. Figure 1 showed a set of HPLC chromato- and G is the content of the assay solution. grams of ginsenoside Rb 1 determination in different

suspension cell cultures. Ginsenoside Rb 1 was not detected in A. tricolor suspension cells. Analyses of squalene contents The suspension cell biomasses and ginsenoside Rb 1 The cellular squalene content was analyzed according to Yue and production by P. ginseng mono-cultures and co-cultures Jiang (2009) with some modifications. Dry cells were ground to were shown in Figure 2. The growth rate of the A. tricolor powder. The powder (100 mg) was saponified by 5 ml of 10% (w/v) cells was higher than that of the P. ginseng cells. On day KOH-75% (v/v) ethanol solution at 50°C for 15 min. The mixture 20 of cultivation, the biomasses and the ginsenoside Rb 1 was extracted with 5 mL of hexane for three times. The hexane production of the co-cultures, composed of P. ginseng cell layer was collected and evaporated to dryness under vacuum at 45°C. The residue was dissolved in 1.0 ml of acetonitrile for HPLC cultures and the A. tricolor cells (4 g FW/L) introduced on analysis. Shimadzu LC-2010AHT HPLC with a UV detector (SPD- day 6, were not obviously different from those of P. 10S) and a VP-ODS column (150 mm length, 4.6 mm i.d., 5 µm ginseng cell mono-cultures. The biomasses of the co- particles; Shimadzu) were used. The separation of squalene was cultures, made up of P. ginseng cell cultures and the achieved by using a mobile phase of 100% acetonitrile at a introduced A. tricolor cells (8 and 12 g FW /L) on day 6, constant flow rate of 1.5 ml/min. The squalene was monitored at were higher than that of the P. ginseng cell cultures wavelength of 195 nm. The squalene released into culture media was analyzed as (Figure 2A). However, Figure 2 C showed that the follow: medium of 20 ml was extracted by hexane of 20 ml for three ginsenoside Rb 1 production in the P. ginseng cell cultures times. The hexane layer was collected and evaporated to dryness added with A. tricolor cells (4, 8 and 12 g FW /L) on day 6 under vacuum at 45°C. The residue was dissolved in 0.2 ml of was lower than that of the P. ginseng cell mono-cultures. acetonitrile for subsequent HPLC analysis as described above. The Ginsenoside Rb 1 production was enhanced by addition of peak of squalene was identified based on its retention time obtained A. tricolor from squalene standard (Sigma Chemical Co.). The quantification cells on day 16, and reached the maximum was done according to the external calibration graph obtained from (51.5 ± 3.1 mg/L) on day 20 in the co-cultures added A. the peak areas vs. different concentrations of squalene standard. tricolor cells (16 g FW /L), which was about 2.0-fold that The standard curve equation: A = 5012.9 · S (ng) + 1024, where A is of the mono-cultures of P. ginseng suspension cells the peak area of squalene, and S is the content of the assay (Figure 2D). The increment of ginsenoside Rb 1 solution. production in the P. ginseng cell cultures added with A. tricolor cells (8 and 24 g FW /L) on day 16 was lower than that of the P. ginseng cell cultures added with A. tricolor Extraction and assay of UGRdGT activity cells (16 g FW /L) on the same day. The observation UGRdGT of cultured cells was extracted and detected similarly as above suggested that effects A. tricolor cells on described previously in Yue and Zhong (2005), the only difference ginsenoside Rb 1 production by P. ginseng suspension was that the cells were P. ginseng , A. tricolor and co-cultured cells, cells were addition time and dosage dependent. not P. notoginseng cells. Fresh cells (0.5 g) were frozen in liquid nitrogen and homogenized in an ice bath with 1.5 mL extraction buffer consisting of 50 mM phosphate buffer (pH 7.5) and 1 mM Effects of squalene addition on growth and PMSF (phenylmethyl sulfonylfluoride). The homogenate was centrifuged at 15,000g for 30 min at 4°C. The supernatant was ginsenoside Rb 1 production of P. ginseng cells used as a crude enzyme extract for the assay. The reaction mixture (0.2 ml) for UGRdGT contained 50 mM phosphate buffer (pH 7.5), 5 Squalene is a precursor of ginsenoside Rb 1 biosynthesis. mM UDPG, and 0.5 mM ginsenoside Rd. For accuracy, the reaction A. tricolor is a high-squalene-content plant. In order to mixture without ginsenoside Rd was used as control. The reaction investigate possible enhancement mechanism of was started by an addition of 0.05 ml enzyme extract. After 20 min ginsenoside Rb 1 production when A. tricolor cells were at 30°C, the reaction was stopped by an addition of 0.25 ml n- added into the P. ginseng cell cultures on day 16, butanol. The n-butanol was extracted and volatilized and then the squalene contents of the cultured cells and the media residue was redissolved in methanol for ginsenoside Rb 1 assay by HPLC. were measured on day 20. Figure 3 showed a set of HPLC chromatogram of squalene determination in different culture media. The results were shown in Table RESULTS 1. The squalene content of the cultured A. tricolor cells was about 5 fold that of the P. ginseng suspension cells, Growth and ginsenoside Rb 1 production of different and the squalene content of the A. tricolor cell culture cell cultures medium was also markedly higher than that of the P. ginseng cell culture media. After the A. tricolor cells were P. ginseng and A. tricolor suspension cell cultures were added into the P. ginseng suspension cell cultures, the 900 J. Med. Plant. Res.

Figure 1. HPLC analysis of ginsenoside Rb 1 in suspension cells on day 20 of cultivation. The red line represents standard ginsenoside Rb 1; the blue line represents the co-culture cells added A. tricolor cells (16 g FW/L) on day 16 of cultivation; the green line represents P. ginseng mono-culture cells; the black line represents A. tricolor mono-culture cells.

13 13 A B 11 11

9 9

7 7 Cell DW (g/L) DW Cell 5 5 Addition of Addition of A tricolor A tricolor 3 3 30 60 C D

(mg/L) 20 40 1

10 20

Addition of Addition of

Ginsenoside Rb Ginsenoside A tricolor A tricolor 0 0 0 5 10 15 20 25 0 5 10 15 20 25 Cultivation time (day) Cultivation time (day)

Figure 2. Growth of co-cultured cells (A, B) and ginsenoside Rb 1 production (C, D) after A. tricolor cells were introduced into P. ginseng cell cultures on day 6 and 16 of cultivation, respectively. Symbols for addition of A. tricolor cells on day 6 (A, C): ♦ 0 g (FW /L); ▲4 g; ■ 8 g; ● 12 g. Symbols for addition of A. tricolor cells on day 16 (B, D): ♦ 0 g (FW /L); ▲8 g; ■ 16 g; ● 24 g . Symbol ◊: A. tricolor cell mono-cultures (A, B). Yue et al. 901

Figure 3. HPLC analysis of squalene in culture media on day 20 of cultivation. The red line represents standard squalene; the blue line represents the co-culture media added A. tricolor cells (16 g FW/L) on day 16 of cultivation; the green line represents P. ginseng mono-culture media; the black line represents A. tricolor mono-culture media.

Table 1. Squalene content of the cultured cells and the media on day 20 of cultivation.

Cultured cells Squalene content (µg /g DW) Culture media Squalene content (µg /L) P. ginseng cells 49 ± 3.7 P. ginseng cell culture medium 10 ± 0.8 A. tricolor cells 245 ± 20 A. tricolor cell culture medium 50 ± 4.2

Co-culture cells* Co-culture media* (1) 77 ± 4.3 (1) 12 ± 0.7 (2) 92 ± 5.5 (2) 18 ± 1.2 (3) 105 ± 5.4 (3) 23 ± 1.4

* (1), (2), (3) represent addition of 8 g, 16 g, 24 g (FW) of A. tricolor cells into P. ginseng cell cultures of one liter on day 16 of cultivation, respectively.

squalene content of co-culture media increased A. tricolor cells in the co-cultures was one of driving force significantly. This suggested that squalene in A. tricolor to promote ginsenoside Rb 1 production. cells could be secreted into the co-culture media to supply the precursor for production of ginsenoside Rb 1. To evaluate the contribution of external squalene to UGRdGT activities of cultured cells enhancement of ginsenoside Rb 1 production, squalene feeding into P. ginseng suspension cell cultures on day For further understanding the influence of A. tricolor cells 16 was done. No effect of DMSO (0.05 mL in every 50 on ginsenoside Rb 1 production, it is necessary to mL culture media) on the growth and ginsenoside Rb 1 investigate the effect of the cells on UGRdGT its activities contents in P. ginseng cells was observed (data not in P. ginseng suspension cells. Here, the UGRdGT shown), so DMSO was selected as the solvent to activity in A. tricolor cells was not observed, UGRdGT dissolve squalene in this study. The results were shown activities of P. ginseng suspension cells were detected in Table 2. In this work the ginsenoside Rb 1 production and effects of A. tricolor cells on the enzyme activities increased 16 ± 0.9% by squalene feeding at the were examined (Figure 4). The UGRdGT activities were concentration of 10 mg/L. The results were possible to inhibited by A. tricolor cell addition (8 g FW /L and 12 g suggest that supplying more precursor squalene by FW /L) on day 6. The results were consistent with the 902 J. Med. Plant. Res.

Table 2. Effects of squalene addition on biomass and ginsenoside Rb 1 production*.

Addition concentration (mg/L) Biomass (g DW/L) Ginsenoside Rb 1 production (mg/L) 0 8.6 ± 0.39 25.8 ± 1.5 ab 1 8.7 ± 0.42 26.0 ± 2.0 bc 10 9.0 ± 0.50 30.1 ± 2.2 c

*Squalene addition was added into P. ginseng cell cultures on day 16 of cultivation. The biomass and ginsenoside Rb 1 content were determined on day 20 of cultivation. a,b,c The same letter noted in a single column indicates not significantly different according to Tukey’s Honestly Significant Difference multiple-comparison test with a family error rate of 0.05.

0.8 1 A B

0.6 0.8 Addition of A tricolor 0.4 /g Cell DW • min) • DW Cell /g

1 Addition of A tricolor 0.6

UGRdGT activities UGRdGT 0.2 (nmol Rb (nmol

0 0.4 0 5 10 15 20 25 0 5 10 15 20 25 Cultivation time (day) Cultivation time (day)

Figure 4. Responses of UGRdGT in P. ginseng cells to A. tricolor cell addition on day 6 (A) and 16 (B) of cultivation. The symbols are the same as in Figure 2 C and D, respectively.

above observation of inhibition of ginsenoside Rb 1 by A. P. ginseng and Echiancea purpurea was inhibited (Wu et tricolor cells. The UGRdGT activities could be induced by al., 2008). In this work it was observed that addition of A. A. tricolor cell addition on day 16, and the maximum tricolor suspension cells on day 6 of cultivation into P. activity was about 60% higher than that of control on day ginseng suspension cell cultures decreased ginsenoside 20. The results coincided with the above observation of Rb 1 production and the addition on day 16 of cultivation improvement of ginsenoside Rb 1 by A. tricolor cells. improved ginsenoside Rb 1 production (Figure 2C and D). However, addition of squalene (1 and 10 mg/L) into the P. This suggested that ginsenoside Rb 1 production could be ginseng cell cultures did not affect the UGRdGT activity affected by co-culture of P. ginseng and A. tricolor significantly (data not shown). These results suggested suspension cells. This might be the first observation that that influencing UGRdGT activity was another driving the influence of plant secondary metabolite occurred in force that A. tricolor cells affected ginsenoside Rb 1 the co-cultures of two plant suspension cells of different production. species. Precursor feeding has been an obvious and popular approach to increasing secondary metabolite production DISCUSSION in plant cell cultures (Smetanska, 2008; Namdeo, 2007). Lee et al. (2007) reported that the solasodine level in It was reported that the biosynthesis of secondary Solanum lyratum cells was about 10-fold higher than the metabolism products were affected when two different control after stigmasterol feeding. Veeresham et al. cultures were grown within the same culture device. The (2003) reported that addition of precursors production of phytoestrogen 5-hydroxyisoflavones was (phenylalanine, sodium benzoate, hippuric acid) improved when Genistia tinctoria shoots and their hairy improved the production of paclitaxel, deacetylbaccatin roots were co-cultured (Łuczkiewicz and Kokotkiewicz, III, and baccatin III in Taxus. wallichiana cell cultures. 2005). The xanthotoxin accumulation in the co-cultures of Phenylalanine is also one of the biosynthetic Ammi majus hairy roots and Ruta graveolens shoots was precursors of rosmarinic acid, and its addition to Salvia induced (Sidwa-Gorycka et al., 2003). The ginseng ofﬁcialis suspension cultures stimulated the production of saponin accumulation in adventitious root co-culture of rosmarinic acid (Hippolyte et al., 1992). Furthermore, Yue et al. 903

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