Effects of biotic and abiotic elicitors on cell growth and tanshinone accumulation in Salvia miltiorrhiza cell cultures
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This study examined the effects of biotic and abiotic elicitors on the production of diterpenoid tanshinones in Salvia miltiorrhiza cell culture. Four classes of elicitors were tested, heavy metal ions (Co2+, Ag+, Cd2+), polysaccharides (yeast extract and chitosan), plant response-signaling compounds (salicylic acid and methyl jasmonate), and hyperosmotic stress (with sorbitol). Of these, Ag (silver nitrate), Cd (cadmium chloride), and polysaccharide from yeast extract (YE) were most effective to stimulate the tanshinone production, increasing the total tanshinone content of cell by more than ten-fold (2.3 mg g-1 versus 0.2 mg g-1 in control). The stimulating effect was concentration-dependent, most significant at 25 μM of Ag and Cd and 100 mg l-1 (carbohydrate content) of YE. Of the three tanshinones detected, cryptotanshinone was stimulated most dramatically by about 30-fold and tanshinones I and IIA by no more than 5-fold. Meanwhile, most of the elicitors suppressed cell growth, decreasing the biomass yield by about 50% (5.1–5.5 g l-1 versus 8.9 g l-1 in control). The elicitors also stimulated the phenylalanine ammonia lyase activity of cells and transient increases in the medium pH and conductivity. The results suggest that the elicitor-stimulated tanshinone accumulation was a stress response of the cells.
KeywordsSalvia miltiorrhiza Cell culture Tanshinones Elicitors Stress response
Salvia miltiorrhiza Bunge (Lamiaceae), called Danshen in Chinese, is a well-known and important medicinal plant because its root is an effective herb for treatment of menstrual disorders and cardiovascular diseases and for the prevention of inflammation (Tang and Eisenbrand 1992). As its Chinese name refers, Danshen root is characterized by the abundance of red pigments which are mainly ascribed to numerous diterpene quinones generally known as tanshinones, e.g., tanshinone I (T-I), tanshinone-IIA (T-IIA), and T-IIB, isotanshinone I and II, and cryptotanshinone (CT). Tanshinones constitute a major class of bioactive compounds in S. miltiorrhiza roots with proven therapeutic effects and pharmacological activities (Wang et al. 2007). Danshen in combination with a few other Chinese herbs is an effective medicine widely used for the treatment of cardiovascular diseases and used as an emergency remedy for coronary artery disease and acute ischemic stroke. According to WHO statistics, cardiovascular diseases are and will continue to be the number one cause of death in the world (www.who.int/cardiovascular_diseases). It is of significance to develop more efficient means for the production of Danshen and its active constituents.
Although field cultivation is currently the major production means for Danshen and most other plant herbs, plant tissue cultures provide more well-controlled and sustainable systems for efficient production of desired bioactive compounds of the herb. Plant tissue cultures are the most useful and convenient experimental systems for examining various factors on the biosynthesis of desired products and for exploring effective measures to enhance their production. The importance of Danshen for traditional and modern medicines has promoted the long-lasting research interest in the development of S. miltiorrhiza tissue cultures for production of bioactive compounds for more than two decades. In an early study, Nakanishi et al. (1983) induced several cell lines from plant seedlings and screened out a cell line capable of producing significant amounts of CT and another diterpene, ferruginol. In later studies, the group performed a fuller evaluation and optimization of the medium for cell growth and CT production and, eventually, derived an effective production medium with a simpler composition (ten components) than the original Murashige and Skoog (MS) medium (about 20 components), achieving a high CT yield of 110 mg l-1 (Miyasaka et al. 1987). Many recent studies have been focused on hairy root cultures of S. miltiorrhiza transformed by Agrobacterium rhizogenes (Hu and Alfermann 1993; Chen et al. 2001) and by our group (Zhang et al. 2004; Ge and Wu 2005; Shi et al. 2007).
Most of the bioactive compounds in medicinal plants belong to secondary metabolites which are usually less abundant than primary metabolites in the plants. Since the accumulation of secondary metabolites in plants is a common response of plants to biotic and abiotic stresses, their accumulation can be stimulated by biotic and abiotic elicitors. Therefore, elicitation, treatment of plant tissue cultures with elicitors, is one of the most effective strategies for improving secondary metabolite production in plant tissue cultures (Chong et al. 2005; Smetanska 2008). The most common and effective elicitors used in previous studies include the components of microbial cells especially poly- and oligosaccharides (biotic) and heavy metal ions, hyperosmotic stress, and UV radiation (abiotic), and the signaling compounds in plant defense responses such as salicylic acid (SA) and methyl jasmonate (MJ; Zhou and Wu 2006; Smetanska 2008). Some of these elicitors, yeast extract (mainly the polysaccharide fraction), silver ion Ag+, and hyperosmotic stress (by an osmoticum) have also been applied and shown effective to enhance the production of tanshinones in S. miltiorrhiza hairy root cultures (Chen et al. 2001; Zhang et al. 2004; Shi et al. 2007).
To the best of our knowledge, only a few studies have been documented on the effects of elicitors, YE, SA, and MJ, on the secondary metabolite production in Agrobacterium tumefaciens transformed S. miltiorrhiza cell cultures from one research group (Chen and Chen 1999, 2000) but not any study in normal cell cultures. The present study focuses on the effects of common biotic and abiotic elicitors including polysaccharides, heavy metal ions, SA and MJ, and osmotic stress (with sorbitol) on the growth and accumulation of three major tanshinones T-I, T-IIA, and CT in suspension culture of normal S. miltiorrhiza cells. In addition to the effects of various elicitors on the total tanshinone content of cells, the study will examine the effects on different tanshinone species and the potential relationship to plant stress response.
Material and methods
Callus induction and cell suspension culture
Young stem explants of S. miltiorrhiza Bunge were collected from the botanical garden at the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Beijing, China, in May 2005. The fresh explants were washed with tap water, surface-sterilized with 75% ethanol for 1 min, and then soaked in 0.1% mercuric chloride for 10 min and rinsed thoroughly with sterilized water. The clean and sterilized explants were cut into ∼0.5-cm segments and placed on solid MS medium (Murashige and Skoog 1962) supplemented with sucrose (30 g l-1), 2,4-D (2 mg l-1) and 6-BA (2 mg l-1) to induce callus formation. The callus culture of S. miltiorrhiza was maintained on a solid, hormone-free MS medium with 8 g l-1 agar and 30 g l-1 sucrose at 25°C in the dark and subcultured every 4 weeks. The culture was deposited in Lab Y1210 at The Hong Kong Polytechnic University with a collection number of Danshen cell-1. All experiments in this study were performed in suspension culture of S. miltiorrhiza cells in a liquid medium of the same composition as for the solid culture but excluding agar. The cell suspension culture was maintained in shake-flasks, i.e., 125-ml Erlenmeyer flasks on an orbital shaker operated at 110–120 rpm, at 25°C in the dark. Each of the flasks was filled with 25 ml medium and inoculated with 0.3 g fresh cells from 18–21-day-old shake–flask culture.
Elicitor preparation and administration
Elicitors and concentrations tested in the initial experiments
Cobalt chloride (Co)
Silver nitrate (Ag)
Cadmium chloride (Cd)
Salicylic acid (SA)
Methyl jasmonate (MJ)
Yeast elicitor (YE)
Elicitor treatment was administered to the shake–flask culture of S. miltiorrhiza cell on day 18, which was about 2–3 days before reaching the stationary phase. This time point is usually favorable for elicitation when the biomass concentration is high (compared with earlier days of growth), and the cell metabolism is still active (compared with that during or after stationary phase; Buitelaar et al. 1992; Cheng et al. 2006). Each of the elicitor solutions was added into the culture medium with a micropipette at the desired concentration. After the elicitor addition, the shake–flask culture of cells was maintained for another 7 days and then harvested for analysis. All treatments were performed in triplicate, and the results were averaged. After the initial experiments on the eight elicitors, the three most effective ones, Ag (25 µM), Cd (25 µM), and YE (100 mg l-1) were applied in the following experiments on the time courses of elicitor-treated cell growth and tanshinone accumulation in the S. miltiorrhiza cell culture.
Measurement of cell weight, sucrose concentration, medium pH, and conductivity
The cells were separated from the liquid medium by filtration. The cell mass on the filter paper was rinsed thoroughly with water and filtered again, and blotted dry by paper towels and then dried at 50°C in an oven to attain the dry weight. Sucrose concentration in the liquid medium was determined by the Anthrone test using sucrose as a reference (Ebell 1969), and the medium pH and conductivity were measured with the respective electrodes on an Orion 720A+ pH meter (Thermo Fisher Scientific, Inc., Beverly, MA, USA) and a CD-4303 conductivity meter (Lutron, Taiwan), respectively.
Measurement of PAL activity
Phenylalanine ammonia lyase (PAL) was extracted from fresh S. miltiorrhiza cells with borate buffer (pH 8.8). The cells were ground in the buffer (0.15 g ml-1) for 2 min with a pestle and mortar on ice, and then centrifuged at 10,000 rpm and 4°C for 20 min to obtain a solid-free extract. The PAL activity was determined based on the conversion of l-phenylalanine to cinnamic acid as described by Wu and Lin (2002).
Analysis of tanshinone contents
The cell mass from culture was dried and ground into powder and extracted with methanol/dichloromethane (4:1, v/v, 10 mg ml-1) under sonication for 60 min. After removal of the solid, the liquid extract was evaporated to dryness and redissolved in methanol/dichloromethane (9:1, v/v). Tanshinone content was determined by high performance liquid chromatography (HPLC) on a HP1100 system using C18 column, acetonitrile/water (55:45, v/v) as the mobile phase, and UV detection at 275 nm as described previously (Shi et al. 2007). Three tanshinone species CT, T-I, and T-IIA were detected and quantified with authentic standards obtained from the Institute for Identification of Pharmaceutical and Biological Products (Beijing, China). Total tanshinone content is the total content of the three tanshinones in the cells. Tanshinone content in the culture medium was negligible and not determined.
Cell growth and tanshinone accumulation in S. miltiorrhiza cell culture
Effects of various elicitors on cell growth and tanshinone production
Effects of elicitor treatments on different tanshinone species
Effects of various elicitors on the accumulation of three tanshinones in S. miltiorrhiza cells
Content, μg/g (fold of content control)
PAL activity, pH, and conductivity changes induced by elicitors
The effects of various elicitors on tanshinone accumulation found here in the normal S. miltiorrhiza cell cultures are in general agreement with those found in transformed cell and hairy root cultures of S. miltiorrhiza. In transformed cell cultures (Chen and Chen 1999), the CT accumulation was also stimulated significantly by YE but not by SA or MJ alone, and YE also inhibited the cell growth. The tanshinone (mainly CT) production in hairy root cultures was also enhanced significantly (3–4 fold) by Ag (Zhang et al. 2004) and YE (Shi et al. 2007). In all these culture systems, CT was the major tanshinone species stimulated by various elicitor treatments. CT has been identified as a phytoalexin in S. miltiorrhiza plant which plays a defense role against pathogen invasion of the plant (Chen and Chen 2000). In this connection, the stimulated CT accumulation by the elicitors may be a defense or stress response of the cells. CT was also the major diterpenoid produced by a normal S. miltiorrhiza cell line which was initially grown in the MS medium and then transferred to a production medium containing only about half of the nutrient components of the MS medium (Miyasaka et al. 1987). It is very possible that the improvement of CT yield in this production medium was also attributed, at least partially, to the stress imposed by the nutrient deficiency which suppressed growth but stimulated secondary metabolite accumulation.
MJ or its relative jasmonic acid has been shown effective for stimulating a variety of secondary metabolites in plant tissue cultures such as hypericin in Hypericum perforatum L. (St. John’s Wort) cell cultures (Walker et al. 2002), paclitaxol (diterpenoid) and related taxanes in various Taxus spp. and ginsenosides in Panax spp. (Zhong and Yue 2005), and bilobalide and ginkgolides in Ginkgo biloba cell cultures (Kang et al. 2006). However, MJ showed only a moderate or insignificant stimulating effect on tanshinone accumulation in normal and transformed S. miltiorrhiza cell cultures. The discrepancy suggests that the effects of various elicitors on secondary metabolite production in plant tissue cultures are dependent on the specific secondary metabolites. This argument is also supported by the much stronger stimulation of CT than T-I and T-IIA by most elicitors found in our S. miltiorrhiza cell cultures. In addition, the hairy roots appeared more tolerant to the elicitor stress, and the growth was less inhibited by the elicitors or even enhanced in some cases, e.g., by YE (Chen et al. 2001) and sorbitol (Shi et al. 2007). Moreover, sorbitol as an osmotic agent significantly stimulated the tanshinone accumulation (3–4 folds) in S. miltiorrhiza hairy root cultures, but not so significantly in the cell cultures. This shows that the elicitor activities for the same metabolites can vary with the tissue culture systems.
In conclusion, the polysaccharide fraction of yeast extract and two heavy metal ions Ag+ and Cd2+ were potent elicitors for stimulating the tanshinone production in S. miltiorrhiza cell culture. The stimulated tanshinone production by most elicitors was associated with notable growth suppression. CT was more responsive to the elicitors and enhanced more dramatically than another two tanshinones, T-I and IIA. The results from this study in the S. miltiorrhiza cell cultures and from previous studies in hairy root cultures suggest that the cell and hairy root cultures may be effective systems for CT production, provided with the elicitors. As most of the elicitor chemicals are commercially available or can be readily prepared in the laboratory and easily administered to the cell and root cultures, they are suitable for practical applications in the laboratory or large-scale production.
This work was supported by grants from The Hong Kong Polytechnic University (G-U502 and 1-BB80) and the China Hi-Tech Research and Development Program (2006AA10A209).
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