Several factors affecting hypericin production of Hypericum perforatum during adventitious root culture in airlift bioreactors
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Hypericum perforatum L. is a traditional medicinal plant for the treatment of depression and wound healing, and hypericin is one of the main effective active substances. To optimize the culture system for producing hypericin in adventitious root, this study used balloon-type airlift bioreactors to investigate the effect of air volume, inoculation density, indole-3-butyric acid (IBA) concentration and methyl jasmonate (MeJA) concentration on hypericin content and productivity during adventitious root culture. Hypericin content and productivity were improved with increasing air volume, and 0.1 vvm (air volume/culture volume/min) was optimal for hypericin production. Inoculation density also had a great effect on hypericin accumulation. Hypericin content and productivity were favorable in an inoculation density of 5.0 g l−1 and decreased when inoculation densities were lower or higher than 5.0 g l−1. Furthermore, 1.25 mg l−1 IBA enhanced hypericin content and productivity, but too low (≤0.50 mg l−1) or too high (≥1.50 mg l−1) IBA concentrations decreased hypericin accumulation. MeJA concentration significantly affected biomass accumulation and hypericin production. The biomass decreased and hypericin production increased with increasing MeJA concentration. Optimum hypericin content (1.61 mg g−1 DW) and productivity (15.57 mg l−1) were obtained at 350 μM MeJA. The hypericin content in bioreactor-grown adventitious roots was lower than in 3-year field-grown plants, but significantly higher than that in in vitro-grown plantlets and 1-year field-grown plants. Thus, the bioreactor culture of adventitious roots can realize rapid and mass production of hypericin in H. perforatum.
KeywordsAdventitious root Air volume Bioreactor Inoculation density Hypericin MeJA
Air volume/culture volume/min
High-performance liquid chromatography
Human immunodeficiency virus type 1
Murashige and Skoog
Revolutions per minute
Hypericum perforatum L. (St. John’s wort) is a perennial herb native to Europe and also a traditional medicinal plant which is gaining popularity mainly for the treatment of depression and wound healing (Lawvere and Mahoney 2005). The most characteristic constituents of H. perforatum are naphtodianthrones (hypericin and pseudohypericin) and phloroglucinols (hyperforin and adhyperforin) (Butterweck 2003). H. perforatum has potential in the treatment of mild to moderate form of depression due to the active constituents ‘hypericin and hyperforin’ and has become a popular medicinal plant due to its antiviral, anticancer, bactericidal, antiinflammatory and sedative properties (Linde 2009). Moreover, photodynamic hypericin activities displayed under the influence of light are used for therapy in various diseases. These properties allow hypericin to act as an antiviral agent. Attention has been focused on its use against human immunodeficiency virus type 1 (HIV-1) (Meruelo et al. 1988) and to enhance radiolytic sensitivity of tumor cells (Hadjur et al. 2008).
However, the level of hypericin in H. perforatum plants often varies with growth under different field environments (Southwell and Campbell 1991; Büter et al. 1998). The limited area of occurrence of this plant, seasonal harvesting, loss of biodiversity, variability in quality and contamination issues trigger the search for alternative methods of hypericin production (Kirakosyan et al. 2000; Sirvent and Gibson 2002; Walker et al. 2002; Kornfeld et al. 2007). In recent years, plant cell culture technology has been successfully applied to the production of many useful secondary metabolites, including pharmaceuticals, pigments and other fine chemicals (Verpoorte et al. 2002). The culturing of adventitious root tissues is an efficient means of biomass production because of fast growth rates and stable metabolite productivity (Murthy et al. 2008). Bioreactors are often used for large-scale production of plant metabolites as these automated or semi-automated processes save labor and production costs (Chakrabarty et al. 2003). For H. perforatum, active compounds such as phenolics, flavonoids, chlorogenic acid, quercetin and hyperoside also have been produced by culturing adventitious roots with bioreactors (Cui et al. 2010; Cui et al. 2011). During bioreactor culture, there have been many factors affecting adventitious root growth and accumulation of active compounds in H. perforatum. Auxin and auxin/cytokinin combinations, inoculation sizes and Murashige and Skoog (MS) medium dilutions affect adventitious root biomass and accumulation of phenols and flavonoids (Cui et al. 2011). Chlorogenic acid, polysaccharides, as well as phenolics and flavonoids are also affected by inoculation density, aeration volume and culture period (Cui et al. 2010). However, the information on the factors affecting hypericin production of H. perforatum by bioreactor culture of adventitious root has been limited. Cui et al. (2010) indicated that hypericin could be produced by adventitious root culture in a bioreactor, but a detailed analysis and research of the factors affecting hypericin production and increase of hypericin content have not been made.
To optimize the bioreactor culture for mass production of hypricin, the present study used airlift bioreactors to explore the effects of air volume, inoculation density, IBA concentration, culture period and MeJA on hypericin content and productivity during adventitious root culture. Finally, the hypericin contents from different source extracts were also compared.
Materials and methods
Plant material and adventitious root culture
Adventitious roots provided by Chungbuk National University (South Korea) were cultured in 250 ml conical flasks containing 70 ml MS (Murashige and Skoog 1962) medium supplemented with 30 g l−1 sucrose and IBA, and the pH was adjusted to 5.8. The cultures were maintained in the dark at 25 ± 2 °C on a gyratory shaker at 100 rpm (revolutions per minute), then subcultured to fresh medium once in 30 days.
Effects of air volume, inoculation density and IBA concentration on hypericin content and productivity
All the experiments were carried out in 5-l balloon-type airlift bioreactors, and the bioreactors were maintained in the dark at 25 ± 2 °C. Firstly, the 30-day adventitious roots cultured in conical flasks were cut to 1 cm long and transferred to bioreactors with 4-l working volume. To study the effect of air volume on hypericin production, the medium was aerated at 0.025, 0.05, 0.075 and 0.1 vvm (air volume/culture volume/min). 5 g l−1 adventitious roots (fresh weight, FW) were inoculated to each bioreactor. The culture medium was MS supplemented with 30 g l−1 sucrose and 1.0 mg l−1 IBA; the pH was adjusted to 5.8. Next, the inoculation density was tested, the initial density of adventitious roots was adjusted to 2.5, 5.0, 7.5, 10.0 and 12.5 g l−1 FW, air volume was adjusted at 0.1 vvm and other culture conditions were in keeping with the air volume experiment. Then, the IBA concentration in the culture medium was also studied and the MS medium supplemented with different concentrations of IBA (0, 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75 and 2.00 mg l−1) and 30 g l−1 sucrose (pH 5.8). 5 g l−1 of inoculation density and 0.1 vvm of air volume were applied. The hypericin content and productivity of adventitious root in the above three experiments were determined after 30 days of bioreactor culture.
To determine the changes of hypericin content during the culture period, adventitious roots were inoculated in the bioreactor with MS medium containing 1.25 mg l−1 IBA and 30 g l−1 sucrose (pH 5.8) at an inoculation density of 5.0 g l−1 and an air volume of 0.1 vvm for 50 days. The bioreactors were maintained in the dark at 25 ± 2 °C. The adventitious roots were sampled at 5-day intervals from bioreactors and the fresh weight, dry weight (DW) and hypericin content were determined.
Effect of MeJA on hypericin production
MeJA (MUST Biotechnolog co. Ltd, Chengdu, China) was used as an elicitor for improving hypericin accumulation during bioreactor culture of adventitious root. Five-liter balloon-type airlift bioreactors were used to select a suitable MeJA concentration. Each bioreactor was inoculated with 5 g l−1 FW of adventitious roots, aerated at 0.1 vvm and filled with 4-l MS medium supplemented with 30 g l−1 sucrose and 1.25 mg l−1 IBA (pH 5.8). The different amounts of MeJA (0, 50, 100, 150, 200, 250, 300, 350 and 400 μM) were supplied to the bioreactors from the initial culture. The bioreactors were maintained in the dark at 25 ± 2 °C. After 40 days of culture, the adventitious roots were harvested and used for measuring the biomass and the hypericin content.
Comparison of hypericin content from the different sources
To define the hypericin production level in adventitious roots, the hypericin content of adventitious roots was compared with plantlets and 1-year or 3-year plants. The bioreactor-grown adventitious roots (treated with 350 μm MeJA) and in vitro-grown plantlets were obtained after 40 days of culture. The 1-year (plant without flowers) and 3-year (plant with flowers) plants derived from in vitro cultured-plantlets, i. e., the 40-day in vitro-grown plantlets were transplanted to the pots and cultivated in a green house, then harvested in September after 1 or 3 years.
Determination of biomass and hypericin content
The adventitious roots were separated from the medium after 40 days of bioreactor culture, then the fresh weight was measured after rinsing three times with sterile water and stripping the surface moisture with a dehydrator for 2 min. The fresh roots were dried at 40 °C with a drying oven for 2 days and the dry weight was recorded.
The hypericin content was determined using the method described by Tolonen et al. (2003) with modifications. The dry adventitious roots were ground into fine powder in a grinder. The powdered material (0.4 g) was extracted with 8 ml of high-performance liquid chromatography (HPLC)-grade methanol for 60 min in an ultrasonic bath at 35 °C. The extracting solution was filtered with a 0.45-μm filter (PVDF syringe filter, Beijing, China) before injection for HPLC analysis. Hypericin was quantified by an HPLC system (Agilent 1100, Agilent Technologies, USA) equipped with a 5 C18-AR-II column (particle size 5 μm, 250 × 4.6 mm) maintained at 35 °C. Fractions were eluted with 80 % HPLC-grade acetonitrile in 5 mM 20 % ammonium acetate, the flow rate of eluent was 1 ml min−1 and the total run time was 15 min. The injection volume was 10 μl. Hypericin was detected at 284 nm. The hypericin standards were obtained from MUST Biotechnology Co., Ltd. (Chengdu, China). Measurements of all the sample materials were integrated by comparison with an external standard calibration curve. The retention time (tR) for hypericin was 4.15 min.
The hypericin productivity of adventitious roots was calculated as [harvested DW (g) × hypericin content (mg g−1 DW)]/medium volume (l).
Experimental design and data analysis
All experiments were independently replicated three times and the data expressed as the mean value for each experiment. The bars indicate the standard error (SE) of the mean for each replicate.
Results and discussion
The effect of air volume, inoculation density and IBA concentration on hypericin content and productivity.
Inoculation density significantly affected hypericin accumulation; the maximum hypericin content and productivity were obtained when 5 g l−1 FW of adventitious roots was used as inoculum (Fig. 1b). When inoculation density was lower than 5 g l−1 FW (2.5 g l−1 inoculation density), low hypericin content (0.25 mg g−1 DW) and productivity (0.99 mg l−1) was measured. Inoculation densities higher than 5 g l−1 FW inhibited hypericin production. At 12.5 g l−1 inoculation density, only 0.19 mg g−1 DW of hypericin content and 0.88 g l−1 of hypericin productivity were determined. Low inoculation densities wasted space, extended culture time and reduced hypericin production. High inoculation densities resulted in poor growth because the adventitious roots rapidly reached their stationary phase, which limited the availability of nutrients and oxygen as well as affected the production of secondary metabolites (Min et al. 2007; Cui et al. 2010). This finding was also observed in other plant species during adventitious root culture (Jeong et al. 2009; Lee et al. 2006). In addition, the effect of inoculation density on H. perforatum was also verified by Cui et al. (2011); the greatest increment of chlorogenic acid, phenolics, flavonoids and polysaccharides content occurred at an inoculation density of 3 g l−1. This result differed with our study in which 5 g l−1 inoculation density favored hypericin production. Consequently, during bioreactor culture of adventitious roots, a suitable inoculation density should be selected according to plant species, bioreactor size, working volume, target compounds, etc. 5 g l−1 of inoculation density was considered optimal for hypericin production of H. perforatum in a 5-l balloon-type airlift bioreactor with 4-l working volume.
Hypericin content and productivity increased as IBA concentration increased from 0 to 1.25 mg l−1; the highest hypericin content and productivity appeared at 1.25 mg l−1 IBA (Fig. 1c). IBA concentrations higher than 1.25 mg l−1 adversely affected adventitious root growth and hypericin content. The optimum concentration of plant growth regulators is critical in controlling the accumulation of secondary metabolites. Auxin is a root-inducing agent critical to the formation of adventitious roots, whereas IBA affects metabolite accumulation. A previous study revealed that 7.0 mg l−1 IBA was the optimum concentration for promoting both cell growth and saponin production during cell suspension culture of Panax ginseng (Lian et al. 2002). Baque et al. (2010) found that the biomass and secondary metabolite accumulation of Morinda citrifolia adventitious roots could reach maximum values at 5.0 mg l−1 IBA. These results demonstrated that the optimum IBA concentration for cell and organ growth varied per plant species. In the present study, 1.25 mg l−1 IBA is favored for the hypericin production of adventitious roots in bioreactors.
Kinetic changes in hypericin content and productivity during culture period
Biomass accumulation and hypericin production in response to different MeJA concentrations
MeJA promoted the hypericin production of H. perforatum. The hypericin content and productivity increased with increasing MeJA concentrations (ranging from 0 μM to 350 μM). The maximum hypericin content (1.61 mg g−1 DW) and productivity (15.57 mg l−1) were achieved at 350 μM MeJA (Fig. 4b). However, excess MeJA (400 μM) slightly decreased hypericin production. MeJA is a plant hormone involved in regulating plant response to environmental stress through the modulation of gene expression. Exogenously applied MeJA induced biosynthesis of many secondary metabolites (Chen et al. 2007). Modern bioreactor culture systems provide a more advanced technology to produce higher secondary metabolites from plant cell, tissue or organ using artificial nutrients with MeJA. Yu et al. (2002) found that the ginsenoside content was obviously enhanced by the addition of 100 μM MeJA during adventitious root culture of Panax ginseng; Donnez et al. (2011) examined that 0.2 mM MeJA was optimal for the efficient production and high accumulation of resveratrol in grape cell; Shohael et al. (2008) indicated that the accumulation of eleutherosides and chlorogenic acid in the somatic embryo of Eleutherococcus sessiliflorus were promoted when 200 μM MeJA was supplied to the culture medium. Our study found that 350 μM MeJA efficiently elicited the hypericin synthesis of H. perforatum adventitious roots. Therefore, it can be assumed that the presence of MeJA might be beneficial in triggering the expression of biosynthetic genes in active compounds during plant cell, tissue or organ culture, which results in an increase of metabolite production.
Comparison of hypericin contents from different sources
Comparison of hypericin content in bioreactor-grown adventitious roots with in vitro-grown plantlets, 1- and 3-year field-grown plants
Hypericin content (mg g−1 DW)
Bioreactor-grown adventitious roots
1.68 ± 0.065
In vitro-grown plantlets
0.15 ± 0.033
1-year field-grown plants
0.27 ± 0.051
3-year field-grown plants
2.41 ± 0.062
MS medium supplemented with 1.25 mg l−1 IBA and 30 g l−1 sucrose at 0.1 vvm air volume and 5.0 g l−1 inoculation density is efficient for hypericin production in the bioreactor culture of H. perforatum adventitious roots. Moreover, a suitable MeJA concentration (350 μM) can enhance hypericin content and productivity. Some other chemical or biotic elicitors should be used in further studies that aim at increasing the hypericin content of adventitious roots.
All authors contributed extensively to the work presented in this paper (several factors affect hypericin production of Hypericum perforatum during adventitious root culture in airlift bioreactors). Song-Quan Wu, Xiao-Kun Yu, Mei-Lan Lian and So-Young Park designed the experiments and wrote the paper under the guidance of Xuan-Chun Piao.
This research was supported by the National Science Foundation of China (81160497 and 31260182). We thank Chungbuk National University for providing experimental materials.
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