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Improvement of phenylethanoid glycosides production by a fungal elicitor in cell suspension culture of Cistanche deserticola

Cui-tao Lu¹,∗ & Xing-guo Mei²

Keywords Cistanche deserticola, fungal elicitor, phenylethanoid glycoside, plant cell culture

Abstract
When, on the 15th day of growth, an elicitor from Fusarium solani was added at 40 mg l^-1 to Cistanche deserticola cell suspension cultures, the contents of echinacoside, acteoside, and total phenylethanoid glycosides (PeGs) in cultured cells all increased over the next 27 d by over 100% to 15 mg g^-1 dry wt, 9 mg g-1 dry wt and 57 mg g^-1 dry wt, respectively. The final biomass (1.3 mg dry wt ml^-1) was not affected.

Cistanche deserticola

Introduction

Cistanche deserticola Ma. (Orobanchaceae), a native herb of China is widely used in traditional medicine for various treatments including use as a sedative or an immune response intensifier (Hsu et al. 1995, Chang et al. 1995). Phenylethanoid glycosides (PeGs) are the major bioactive metabolites in Cistanche deserticola (Xiong et al. 2000) and are composed of many components such as echinacoside, acteoside, cisacteoside, isoacteoside, 2-acetylacteoside, cistanoside A, and osmanthuside B (Wang et al. 2000). With the increasing demand for these products and the rapid decline in Cistanche deserticola plants, cell culture would be an attractive alternative means of production. Many elicitors (especially fungal elicitors) can enhance metabolite production in plant cell culture (Wang et al. 2001). Li et al. (1998) found that a fungal elicitor of Fusarium solani isolated from root systems of both Cistanche deserticola and its host, Halaxylon ammodendron, could improve the amino acid contents in calli of Cistanche deserticola But the changes of PeGs content in this callus line were not mentioned in their study. In this paper, the effects of an elicitor from F. solani on the biosynthesis of total PeGs and two main bioactive PeGs (echinacoside and acteoside), and on the changes of cell growth were studied in the suspension culture of Cistanche deserticola.

Materials and methods

Plant cell cultures
Callus was induced from scales of Cistanche deserticola and maintained on Gamborg's B5 medium (1968) supplemented with 0.5 mg indole-3-acetic acid l^-1, 2 mg kinetin l^-1, and 1 g casein hydrolysate l^-1. The pH was adjusted to 6 before sterilization. All experiments were performed in 250 ml shake flasks, each containing 100 ml medium on a rotary shaker (120 rpm and 25 ℃) under darkness. The inoculum was about 100 g fresh wt l^-1.

effective contents of cistanche deserticola-- phenylethanoid glycoside

Preparation of fungal elicitor

Separation of fungus strain, Fusarium solani, and preparation of fungal elicitor were performed as previously described (Li et al. 1998, Yu et al. 2001). Various amounts (carbohydrate equivalents) of fungal elicitor were added to the shake flask cultures on the 15th d (total culture period was 42 d).

Biomass accumulation and viability assay
Cells from a shake flask were washed with distilled water and freeze-dried to a constant weight. The dry wt was thus determined. Cell viability (percentage of viable cells in a cell population) was determined by the reduction of 2,3,5-triphenyl tetrazolium chloride (Towill & Mazur 1975).

phenylethanoid glycoside of cistanche deserticola

Determination of the contents of echinacoside, acteoside, and total phenylethanoid glycosides (PeGs)
Extraction of PeGs was performed according to the method of Du & Liu (1993). Freeze-dried Cistanche deserticola cells were extracted with 70% (v/v) ethanol. The ethanol fraction was concentrated, degreased with water-saturated ethyl acetate, and then leached with water-saturated n-butanol. The n-butanol fraction was concentrated, dissolved in water, loaded into a macropore resin column (AB-8, chemical product of Nankai University, Tianjin, China), and eluted with water and methanol, successively. Methanol fraction was collected and the total PeGs content was determined by using echinacoside as standard at 334 nm. Echinacoside and acteoside were determined by using the Nova Pak C18 column (250 × 4.6 mm) in an HPLC system (waters) with a UV detector (334 nm).

Results

Effect of fungal elicitor on biomass accumulation and total PeGs biosynthesis Effects of fungal elicitor on cell biomass and total PeGs accumulation depended on the dosage added (carbohydrate equivalents). As shown in Figure 1, lower doses of elicitor were favorable to the dry wt of Cistanche deserticola cells. The peak was with the elicitor at 20 mg l^-1 (giving 1.76 mg dry wt ml^-1).
Total PeGs accumulation was significantly enhanced by the fungal elicitor at between 20 mg l^-1 and 80 mg l^-1. Elicitor at 40 mg l-1 obtained the highest yield (57 mg g^-1), which was over twice that obtained in the non-elicitor control culture. The further experiment used the fungal elicitor at 40 mg1^-1.

Fig. 1. Dry cell weight and total PeGs accumulation in suspension cell culture of Cistanche deserticola in response to various doses of fungal elicitor. A fungal elicitor was added to the medium on the 15th d. Samples were taken after a further 27 d. ▲, Final dry cell weight; ◆, the total content of PeG. Values are means of triplicate results and error bars represent standard deviations.

Time course of cell growth and PeGs biosynthesis
Figure 2 shows the time course of cell viability and PeGs (echinacoside and acteoside) biosynthesis of suspension cells after cultures were subjected to a 40 mg l^-1 fungal elicitor on the 15th d. In the subsequent 27 d of culture, echinacoside and acteoside, the two main bioactive PeGs in Cistanche deserticola, both increased considerably. On the 39th d maximum accumulations were both achieved, reaching 14.7 mg g^-1 dry wt and 8.9 mg g^-1 dry wt, respectively. A significant difference in cell viability between the elicitor-treated cultures and the control was not observed.

Fig. 2. Cell viability and echinacoside and acteoside biosynthesis in suspension cell culture of Cistanche deserticola after treatment by 40 mg l^-1 fungal elicitor. Fungal elicitor was added to the medium on the 15th d.◆, Echinacoside-treated with elicitor; ◇, echinacoside-control; ●, acteoside-treated with elicitor;○,

acteoside-control; ▲, cell viability-treated with elicitor;△ , cell viability-control. Values are means of triplicate results and error bars represent standard deviations.

Discussion

Many components derived from fungi (cell wall fragments, polysaccharides, oligosaccharides, glycoproteins, etc.) have been used as various fungal elicitors in secondary metabolite production with many plant cell species. In this paper, the homogenate (mainly composed of polysaccharides) from fungus strain Fusarium solani isolated from root systems of Cistanche deserticola and Halaxylon ammodendron was shown to be an effective elicitor for PeGs biosynthesis in the cell culture of Cistanche deserticola. The use of this elicitor to stimulate PeGs synthesis in cells was not only significant but was exhibited soon after elicitation. This conclusion agreed with some previous studies on plant cell culture (Wang et al. 2001).

In the previous study of Li et al. (1998), when a fungal elicitor from F. solani was added to the culture medium, callus induced from the seeds of Cistanche deserticola could remain alive in the subsequent long-term subculture, and the iridoids production could be enhanced. It indicated that cultured cells could synthesize secondary metabolites of disease resistance when attacked by microorganisms. Though iridoids were not determined in our experiment, we have shown that polysaccharides from F. solani could be used as effective fungal elicitors in the production of PeGs with Cistanche deserticola cells.

effective contents of cistanche deserticola-- phenylethanoid glycoside

From: ' Improvement of phenylethanoid glycosides production by a fungal elicitor in cell suspension culture of Cistanche deserticola ' by Cui-tao Lu¹,∗ & Xing-Guo Mei²

----Biotechnology Letters 25: 1437–1439, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands.

References

Chang Y, Wu H, Wang SN, Cheng HC (1995) Study of the strong effects of Cistanche deserticola Ma., Cistanche salsa (C.A. Mey) G. Beck. and Cistanche tubulosa (Schenk) R Wight. China J. Chin. Mater. Med. 26: 143–146.
Du NS, Liu JL (1993) Determination of phenylethanoid glycosides in Cistanche deserticola by macrovesicular resin spectrophotometry. Nat. Prod. Res. Develop. 5: 30–33.

Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50: 151–158.

Hsu WH, Chu SH, Sheng LC, Yuan KW, Mao CN (1995) Study of the chemical constituents and pharmacological effects of Cistanche deserticola Ma. and Cistanche salsa (C.A. Mey) G. Beck. Chin. Tradit. Herb. Drugs 26: 143–146.

Li S, Wu X, Dong XC, Zheng X, Zheng JH, Lou ZC (1998) Effect of elicitor of Fusarium solani on the amino acid contents of Cistanche deserticola callus. China J. Northwest Pharm. 13: 247–248.
Towill LE, Mazur P (1975) Studies of the reduction of 2,3,5-triphenyltrazolium chloride as a viability assay for plant tissue cultures. Can. J. Bot. 53: 1097–1102.
Wang CG, Wu JY, Mei XG (2001) Enhancement of Taxol production and excretion in Taxus Chinensis cell culture by fungal elicitation and medium renewal. Appl. Microbiol. Biotechnol. 55: 404–410.
Wang YM, Zhang SJ, Luo GA, Hu YN, Hu JP, Liu L, Zhu Y, Wang HJ (2000) Analysis of phenylethanoid glycoside in the extract of herbs Cistanchis by LC/ESI- MS/MS. Acta Pharm. Sin. 35: 839–842.
Xiong QB, Yasuhiro Tezukay, Takuji Kaneko, Li HY, Le Quan Tran, Koji Hase, Tsuneo Namba, Shigetoshi Kadota (2000) Inhibition of nitric oxide by phenylethanoids in activated macrophages. Eur. J. Pharm. 400: 137–144.
Yu LJ, Lan WZ, Qin WM, Xu HB (2001) Effects of salicylic acid on fungal elicitor-induced membrane-lipid peroxidation and Taxol production in cell suspension cultures of Taxus Chinensis. Proc. Biochem. 37: 477–482.