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Chinese Journal of Natural Medicines 2014, 12(5): 0367−0372

Chinese Journal of Natural Medicines

Effects of feeding tyrosine and phenylalanine on the accumulation of phenylethanoid glycosides to Cistanche deserticola cell suspension culture HU Gao-Sheng 1, 2, JIA Jing-Ming 1, 2, Doh Hoon Kim 2, 3* 1

School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China;

2

Joint Laboratory of Shenyang Pharmaceutical University & Dong-A University, Shenyang 110016, China;.

3

College of Life Science and Natural Resources, Dong-A University, Busan 604-714, Republic of Korea Available online 20 May 2014

[ABSTRACT] AIM: To investigate the effects of feeding phenylalanine (Phe) and tyrosine (Tyr) on the accumulation of total phenolic compounds and four phenylethanoid glycosides (PeGs) to a cell suspension culture of the parasitic plant Cistanche deserticola. METHOD: A cell suspension culture of C. deserticola was established and precursors of different concentrations were fed. In each group, the cell was sampled at the 24th day after inoculation. The content of total phenolic compounds and four PeGs compounds were determined using the Folin-Ciocalteu method and an HPLC method, respectively. RESULTS: In the Phe fed cells, the maximum PeGs yield was achieved when Phe was fed at 1.5 mmol·L−1 and the yield reached 1.13 times the control cell concentration. In the Tyr fed cells, the maximum yield of PeGs was 1.60 times of control when 0.75

mmol·L−1 Tyr was fed to the cells. Furthermore, it was found that the salidroside yield was 4.01 times of control group when 5 mmol·L−1 Tyr was fed. CONCLUSION: Tyr is a better precursor for PeGs accumulation compared with Phe, and the rate limiting enzymes might be involved in the Tyr branch. [KEY WORDS] Cistanche deserticola; Cell suspension culture; Phenylalanine; Tyrosine; Precursor feeding; RP-HPLC; Salidroside; Biosynthesis; Phenylethanoid glycosides

[CLC Number] R965

[Document code] A

[Article ID] 2095-6975(2014)05-0367-06

Introduction Cistanche deserticola Ma, is a traditional Chinese medicinal plant that has been used for centuries in China for its nourishing effects. It is a perennial, parasitic medicinal herb belonging in the Orobanchaceae family. The plant grows in the desert areas of West China and is a parasite on the roots of Haloxylon ammodendron (C.A. Mey) Bunge (Amaranthaceae). Isolation, structural elucidation, and pharmacological studies on the constituents of C. deserticola were started in the 1980s. Based on these studies, the polyethanoid gly[Received on] 05-Feb.-2013 [*Corresponding author] Doh Hoon KIM: Tel: 82-51-2007507; Fax: 82-51-2007505, E-mail: [email protected] These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved

cosides (PeGs) were believed to be the main active compounds in C. deserticola. The demonstration of PeGs as effective compounds in protecting neuro cells from damage [1-4] caused by chemicals and aging, drove an increase in the market demand. Due to over-collection, corruption of habitats, and complex parasitism, the natural resources of C. deserticola were on the edge of extinction and it was listed in CITES (The Convention on International Trade in Endangered Species of Wild Fauna and Flora) in 2003. To solve the limited-resources related issues, cell and tissue culture studies of this medicinal plant were conducted in China in recent decades. In those studies, the effects of various factors including osmotic stress [5], precursor feeding [6] , light of different wavelength [7], fungal elicitors [8-10], and abiotic elicitors [11] on the cell growth and accumulation of PeGs were investigated. These studies provided valuable information for the large scale culture of C. deserticola. In the

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HU Gao-Sheng, et al. / Chin J Nat Med, 2014, 12(5): 367−372

reported precursor feeding experiments conducted in C. deserticola cells, the effects of phenylalanine, tyrosine, sodium acetate, and phenylacetic acid feeding on the accumulation of PeGs were investigated [6], and phenylalanine was found to be the best precursor to improve total PeGs accumulation. With the development of cultivation skills, C. deserticola can now be produced under cultivation in Ningxia Province in recent years. Therefore, the production of C. deserticola materials using plant cell and tissue culture techniques has become less attractive. However, it is still a powerful and convenient tool for the study of the biosynthetic pathway of PeGs in C. deserticola. A pioneer study on acteoside biosynthesis using isotope labeled precursor feeding [12] in Syringa vulgaris cell suspension cultures indicated that the caffeoyl group of acteoside was derived from L-phenylalanine, while the 3, 4-dihydroxyphenylethanol unit was derived from L-tyrosine. Another isotope labeled precursor study in an Olea europaea cell line also determined that the caffeoyl group was derived from phenylalanine, and the hydroxyl-phenylethanol group was derived from tyrosine through dopamine [13]. However, the effects of precursor feeding on the biosynthesis of specific compounds, especially salidroside, which was believed to be a potential intermediate [12-13] in the biosynthesis of PeGs, has not been investigated in Cistanche plant. In the present study, the effects of phenylalanine and tyrosine feeding on phenylalanine ammonia lyase (PAL) activity, cell growth, accumulation of total phenolic compounds, salidroside and three other phenylethanoid glycosides, including echinacoside, acteoside, and cistanoside A, were investigated. The results are discussed from a biosynthetic perspective.

Material and Methods Cell suspension culture of C. deserticola A cell suspension culture of C. deserticola was maintained in B5 [14] liquid media supplemented with 30 g·L−1 sucrose, 0.5 mg·L−1 6-BA, and 2.0 mg·L−1 NAA at (25 ± 1) °C with shaking (180 r·min−1). The light period was 16 h (L)/8 h (D). The cell suspension culture was sub-cultured every 24 days. During subculture, cell suspension culture was filtered first using an autoclaved miracloth, the fresh cell pellet was weighed and inoculated into new media at a concentration of 2 g FW/50 mL. Growth investigation Cell growth was mainly measured by cell fresh weight (FW) and dry weight (DW). Cell cultures were sampled at the 24th day and filtered with vacuum filtration using miracloth until there was no liquid from the funnel. The cell pellet was weighed and recorded as FW. Half of the fresh cells were frozen in liquid N2 and stored in −78 °C for further PAL activity assay. The other half of the fresh cells were dried at 60 °C for 24 h until constant weight was observed. The dried cells were cooled in a desiccator to room temperature, weighed, and designated as DW. The dried cell samples were

then ground to a fine powder (100% pass through 80 mesh sieve), put in brown tubes, sealed, and stored at −20 °C for future extraction. Content determination of total phenolic compounds A sample (30 mg) of dried cell powder was extracted using 80% methanol (400 μL) under sonication for 30 min, followed by centrifugation. The supernatant was transferred to a new tube, and the debris was extracted twice more using the same procedure. The supernatants were combined, filtered, and used for content determination of the total phenolic compounds and for RP-HPLC analysis. The total phenolic compound content was analyzed using the modified Folin-Ciocalteu method [15]. The Folin-Ciocalteu reagent was diluted with 1 volume of DW, and the extract obtained was diluted 10 times, and 10 μL was used in each assay. Ten μL of each diluted extract was added in freshly prepared 2% sodium carbonate solution (200 μL). The preparation was mixed vigorously and briefly spun down. The mixture was incubated at room temperature for 5 min, and subsequently mixed with diluted Folin reagent (10 μL). The final preparation was mixed vigorously and incubated at room temperature for 30 min. The absorbance A750 was measured in a UV/Vis spectrophotometer and recorded for the calculation of total phenolic compounds. Linear regression analysis was carried out between the amount of chlorogenic acid added (Y) and A750 (X). The standard formula was as follows: Y = 0.821 X – 0.089 8 (R2 = 0.992 6). Precursor feeding Different amounts of phenylalanine and tyrosine were dissolved in the media individually, and the final concentrations of phenylalanine and tyrosine were 0.75, 1.5, 3.0, and 5.0 mmol·L−1. The media was subjected to autoclave and the cells were inoculated into the media with precursors at 2 g FW/50 mL. Cells were sampled at the 24th day. Triplicate flasks inoculated with cells collected from the same bottle were used in all groups to minimize systematic errors. HPLC analysis The extract obtained in 2.3 was filtered through a 0.45 μm filter before applied to HPLC. HPLC analysis was carried out using Hitachi HPLC (L-2130 pump, L-2420 UV-Vis detector, and L-2200 autosampler) with C18 column (250 mm × 4.6 mm, 5 μm, Waters), mobile phase (MeOH/H2O gradient elution), with detection wavelength at 330 nm and 220 nm for the detection of PeGs and salidroside, respectively. Each standard compound dilution and sample extract (10 μL) was subjected to analysis. Content determination of echinacoside, acteoside, cistanoside A, and salidroside was carried out using the standard curve of each reference compound. Salidroside was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The rest of the reference compounds were provided by Yomeisu Co., Nagano, Japan. The purity of all standard compounds was higher than 90%. The sum of four PeGs yield is defined as the total PeGs yield.

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PAL activity assays Crude protein extracted from the samples was stored at −78 °C. A sample (0.1 g) was ground into a fine powder with liquid nitrogen, and 600 μL of protein extraction buffer was added to the cell powder, followed by vortexing for 5 min at 4 °C. The mixture was centrifuged at 13 000 r·min−1 at 4 °C for 10 min, and the supernatant was defined as the crude protein extract. Crude protein extract (10 μL) was added to Tris-HCl (pH 8.5) buffer containing 5 mmol·L−1 phenylalanine (10 μL) on ice. The mixture was incubated at 55 °C for 20 min, the reaction was quenched by the addition of 2 mol·L−1 HCl (50 µL), and the change in A270 was recorded to calculate the production of cinnamic acid, as based on a standard curve of trans-cinnamic acid.

Effects of precursor feeding on salidroside and three phenylethanoid glycosides accumulation in cell suspension culture of C. deserticola. To obtain the detailed effects of precursor feeding on the PeGs accumulation and biosynthesis, the sample extract was applied to RP-HPLC. The retention times (RT) of salidroside and three phenylethanoid glycosides are listed in Table 1. The four standard compounds in the sample extract were baseline separated (Fig. 2). Representative chromatograms of each sample are presented in Figs. 2b–2g). Table 1 Standard formula and retention time of four standard compounds Compound

Results and Discussion Effects of precursor feeding on cell growth and accumulation of total phenolic compounds in cell suspension culture of C. deserticola Phenylalanine and tyrosine are the major carbon sources of phenolic compounds, and in C. deserticola, PeGs are the major phenolic compounds. Therefore, the accumulation of total phenolic compounds can be a general index of the effects of added precurors. The results (Fig. 1a) showed that phenylalanine and tyrosine had no significant effects on the cell growth at most concentrations. Tyrosine at 5 mmol·L−1 concentration inhibited cell growth nearly 50%. Tyrosine feeding at a concentration of 3 mmol·L−1 increased the accumulation of total phenolic compounds by 59.3% (Fig. 1b). However, a higher concentration of tyrosine (5 mmol·L−1) resulted in a decrease of cell growth and total phenolic compound accumulation. For phenylalanine, the enhancing rate of total phenolic compounds was 21.2% when the concentration was 3 mmol·L−1, and as the concentration increased further, the accumulation of total phenolic compounds decreased (Fig. 1b).

Fig. 1 Effects of precursor feeding on the cell growth (a) and total phenolic compound yields (b) of C. deserticola. (DW: Dry weight; FW: Fresh weight; Phe: phenylalanine; Tyr: tyrosine)

Standard formula

R2

tR (min)

Echinacoside

y = 5E-07x + 0.058 7 0.999 5

49.26

Cistanoside A

y = 7E-07x + 0.009 6 0.999 8

56.77

Acteoside

y = 6E-07x + 0.008

0.999 1

50.45

Salidroside

y = 3E-07x – 0.001 2 0.999 1

20.92

To calculate the content of the four standard compounds, standard curves were prepared using series dilutions of each standard compound. Peak area (x) and compound concentration (y) were used for the linear regression analysis. The standard formulas of the four standard compounds are listed in Table 1. Using the standard formula of each compound, the yield of each compound was calculated and is indicated in Fig. 3. As demonstrated in Fig. 2b, in the Phe fed cells, the maximum yield of total PeGs was achieved when the Phe concentration was 1.5 mmol·L−1, and the yield is 1.14 times that of control cells. The yield of salidroside was not significantly affected under all concentrations. In the tyrosine fed cells, the yield of total PeGs was 1.60 times that of control when 0.75 mmol·L−1 Tyr was fed to the cultured cells. As the feeding concentration increased, the accumulation of total PeGs decreased, and when the cells were fed with 5 mmol·L−1 Tyr, the yield of total PeGs was only 23.7% of control group. However, the yield of salidroside, showed a quite different pattern compared with the three other PeGs. The yields under four Tyr feeding concentrations were 2.55, 2.25, 3.25, and 4.01 times the control group, as shown in Fig. 3. These results suggested that, compared with phenylalanine, tyrosine is a better precursor for the biosynthesis of salidroside, supporting the previous findings from a precursor feeding experiment using cell suspension cultures of Syringa vulgaris [12], Rhodiola sachalinensis Boriss. [16], and in hairy root cultures of R. sachalinensis [17]. As shown in Fig. 3, in the Tyr fed cells, there was a much stronger peak observed during HPLC analysis, with an RT of 3–4 min. A large scale culture and treatment is underway to isolate this compound(s). The structure may provide useful information for understanding the PeGs biosynthetic pathway in C. deserticola. As reported, salidroside is derived from tyrosine, and in the biosynthetic pathway from tyrosine to salidroside, tyrosine decarboxylase (TyrDc) played an important role. Besides,

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Fig. 2 RP-HPLC Chromatogram of standard compounds and treated cell extracts. a: chromatogram of four standard compounds; b: chromatogram of tyrosine fed cell T1-T4 at 330 nm; c: chromatogram of phenylalanine-fed cells P1-P4 at 330 nm; d: chromatogram of tyrosine-fed cells T1-T4 at 220 nm; e: chromatogram of phenylalanine-fed cells P1-P4 at 220 nm; f: chromatogram of control cells, tyrosine- (T2) and phenylalanine-(P3) fed cells at 330 nm; g: chromatogram of control, phenylalanine- (P2) and tyrosine- (T4) fed cells at 220 nm. Samples named T1, T2, T3, T4, P1, P2, P3, and P4 were cell samples fed with 0.75, 1.5, 3.0, and 5.0 mmol·L−1 tyrosine and phenylalanine, respectively. The sample named conc. is a sample with no precursor feeding

Fig. 3 Effects of precursor feeding on PeGs accumulation in cell suspension cultures of C. deserticola

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it was reported that, acteoside in an O. europaea cell line was biosynthesized through dopamine from tyrosine [13], which provided evidence that acteoside was not further modified from salidroside. As is known, dopamine is derived from tyrosine and catalyzed by tyrosinase (TYR), which is also the key enzyme in the browning of cell and tissues in plant physiology. However, how this enzyme regulates PeGs biosynthesis and the effects of precursor feeding on this enzyme activity have not been reported until now. Phenylalanine ammonia lyase (PAL), as the key enzyme in the phenylpropanoid biosynthesis pathway, attracted much more attention in PeGs biosynthesis and regulation research than the other enzymes involved in the tyrosine branch. PAL activity assay results showed (Fig. 4) that tyrosine had stronger inhibitory effects on PAL activity as the concentration of Tyr increased. Combined with the HPLC data, it was concluded that the reason why tyrosine feeding can increase the accumulation of salidroside continuously, but decrease the accumulation of PeGs as the concentration increased, might lie in the stimulation of the biosynthesis of salidroside by providing enough precursor, and inhibition of the production of caffeoyl groups, which is involved in the structure of echinacoside, acteoside, and cistanoside A. However, in Phe fed cells, the PAL activity was slightly increased from 0.75 mmol·L−1 to 3 mmol·L−1, and the same as control at 5 mmol·L−1 (Fig. 4). Combined with the HPLC analysis, it was found that the PAL activity is not inhibited and is even higher than control cells, although the yield of total PeGs was lower than the Tyr fed groups. Therefore, it was concluded that, the tyrosine branch might be more important than the phenylalanine branch in the biosynthesis of PeGs in C. deserticola cell cultures. Similarly, it was reported that TyrDc and tyramine hydroxycinnamoyl transferase are rate limiting enzymes in the biosynthesis of hydroxycinnamic acid amide in transgenic tobacco [18]. After all, the core structure of PeGs is derived from tyrosine, but not phenylalanine.

Fig. 4 Effects of precursor feeding on PAL activity in cell suspension cultures of C. deserticola

Acknowledgement This work was supported by the Dong-A University research fund. The authors also would like to express appreciation to Dr. Nobuhisa Ezaki at Yomeishu Seizo Company for providing the standard compounds echinacoside, acteoside, and cistanoside A.

Conclusions The data demonstrate that tyrosine is a better precursor for the production of salidroside and PeGs in cell suspension culture of C. deseticola than phenylalanine. In addition, the decrease of PAL activity in tyrosine fed cells did not affect the accumulation of three other PeGs, except at 5 mmol·L−1, which meant that the functional role of PAL needs to be reevaluated. The other enzymes involved in the tyrosine branch and the integration steps of the PeGs biosynthesis pathway should be studied further in the future. When fed 5 mmol·L−1 Tyr, the accumulation of PeGs decreased significantly, which might be due to the inhibition of cell growth and the enzyme activity involved in the tyrosine branch.

Acknowledgement This experiment was funded by Dong A University. The authors also would like to express appreciation to Dr. Nobuhisa Ezaki at Yomeishu Seizo Company for providing the standard compounds echinacoside, acteoside, and cistanoside A.

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Cite this article as: HU Gao-Sheng, JIA Jing-Ming, Doh Hoon Kim. Effects of feeding tyrosine and phenylalanine on the accumulation of phenylethanoid glycosides to Cistanche deserticola cell suspension culture [J]. Chinese Journal of Natural Medicines, 2014, 12 (5): 367-372

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