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Plant Biotechnology Reports (2020) 14:381–386 https://doi.org/10.1007/s11816-019-00592-7

SHORT COMMUNICATION

Effect of nutritional factors on the accretion of secondary metabolites in Malaysian ginseng adventitious root cultures

Xi‑Hua Cui1 · Hosakatte Niranjana Murthy2 · Ji‑De Zhang1 · Hang‑Lin Song1 · Yin‑Ji Jiang1 · Wen‑Wen Qi1 · Yong Yi Li1 · Kee‑Yoeup Paek3 · So‑Young Park1

Received: 22 November 2019 / Accepted: 14 December 2019 / Published online: 21 December 2019 © Korean Society for Plant Biotechnology 2019

AbstractIn this study, we aimed to verify the effect of nutritional factors on the accretion of secondary metabolites in the adventitious root (AR) cultures of Malaysian ginseng (Eurycoma longifolia Jack) grown in small-scale bioreactors. AR were induced from leaf explants and cultured in different types of media including Murashige and Skoog (MS) medium, Driver Kuniyuki Walnut (DKW) medium, Gamborg’s B5 medium, Woody Plant Medium (WPM), and ¾ MS medium. Among these media, the MS and Gamborg’s B5 media induced lateral root development from initial inoculum, which accounted for the increase in AR biomass accretion. By contrast, the DKW and WPM media did not induce lateral root formation from the cultured explants. The ¾ MS medium was optimal for the growth of AR and accretion of secondary metabolites, after 7 weeks of culture, the biomass of AR increased by 8.6-fold in ¾ MS medium, and the total phenolic and flavonoid contents reached 5.23 and 2 mg g−1 of tissue dry weight, respectively. Analysis of mineral elements in the spent medium revealed that ¾ MS medium was most suitable for nutrient supply to developing AR. LC–MS analysis showed the accretion of eurycomanone, a therapeutically useful metabolite, in the AR of Malaysian ginseng.

Keywords Adventitious root · Bioreactor cultures · Eurycoma longifolia · Eurycomanone · Malaysian ginseng

Introduction

Malaysian ginseng (MG, Eurycoma longifolia Jack; Sima-roubaceae) commonly called as ‘Tongkat Ali’ is an impor-tant traditional plant in South East Asia. MG exhibits antimi-crobial, antidiabetic, antipyretic, and aphrodisiac properties (Bhat and Karim 2010). MG is used to cure malaria and treat syphilis and gonorrhea (Bhat and Karim 2010). The extract of MG is available in powder form as health supplement,

which contain alkaloids, quissinoid diterpenoids, and poly-phenols as the major bioactive compounds (Bhat and Karim 2010). MG is a highly overexploited plant species in South East Asia and has been declared as protected plant; there-fore, harvesting of MG plants from their natural habitats is highly restricted (Bhat and Karim 2010).

AR cultures serve as an attractive alternative for the large-scale production of valuable secondary metabolites (Murthy et al.; 2008, 2014a,2016). AR cultures have been used for the production of ginsenosides from Ginseng (Paek et al. 2009), caffeic acid derivatives from Echinacea (Wu et al. 2007), and hypericins from St. Jhon’s wort (Cui et al. 2014). Although production of secondary metabolites from MG-AR cultures has been attempted previously (Lulu et al. 2015), the set-ting-up of AR cultures requires optimization of chemical and physical parameters (Murthy et al. 2008, 2014a; 2016). Current study investigates the effect of growth medium and mineral elements on the accretion of secondary metabolites in the AR cultures of MG.

Online ISSN 1863-5474Print ISSN 1863-5466

* Hosakatte Niranjana Murthy nmurthy60@yahoo.co.in

* So-Young Park soypark7@cbnu.ac.kr

1 Yanbian Academy of Agriculture Sciences, Longyan Road 359, Yanji 133400, Kilin, China

2 Department of Botany, Karnatak University, Dharwad 580003, India

3 Research Center for the Development of Advanced Horticultural Technology, Department of Horticultural Sciences, Chungbuk National University, Cheongju 28644, Republic of Korea

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Materials and methods

Establishment of AR suspension cultures

AR of MG were induced from leaf explants, maintained in ¾ MS (Murashige and Skoog 1962) liquid medium, supple-mented with 3.0 mg L−1 indole-3-burtyric acid (IBA), 30 g L−1 sucrose (Lulu et al. 2015), and subcultured into fresh medium regularly at 4-week intervals. AR were cultured in a 3-L balloon-type bubble bioreactor (BTBB) containing 2-L of MS medium, supplemented with 3.0 mg L−1 IBA, 30 g L−1 sucrose. Cultures were incubated in the dark at 22 ± 1 °C for 7 weeks by aerating with sterile compressed air at 0.1 vvm (vvm is defined as the air volume per unit culture volume per min).

Optimization of culture medium for the growth of AR

AR cultures of MG were initiated from 6 g L−1 inoculum in various growth media including the MS medium, DKW medium (Driver and Kuniyuki 1984), B5 medium (Gamborg et al. 1968), WPM (Lloyd and McCrown 1980), and ¾ MS medium containing 3.0 mg L−1 IBA, 30 g L−1 sucrose. The cultures were grown in 3 or 10-L BTBB under dark condi-tions for 7 weeks, and the effect of medium composition on the growth, biomass and secondary metabolites content of AR cultures were determined.

Analysis of the growth pattern and AR weight

To determine the growth pattern of AR, the medium was sampled at 0, 2, 4, 6, and 7 weeks from two bioreactors, and each experiment was terminated at the time of sampling. To determine the root weight of the cultures, root were sam-pled from the medium, and the fresh weight (FW) and dry weight (DW) of the roots were measured as described previ-ously (Cui et al. 2014).

Measurement of secondary metabolites in AR extracts

Dried AR (0.5  g) were refluxed with 10  mL of 80% methanol using a side-mounted float liquid sensor (LS-2050-S10; LSTECH, Korea) at 80 °C for 1 h. The samples were centrifuged (Kubota 5930, Tokyo, Japan) at 4000xg for 10 min, the supernatant was filtered through Advantec 110 mm filter paper (Toyo Rosishi Kaisha Ltd., Japan). The residue remaining on the filter paper was re-extracted as described above, and the final volume of the sample was

adjusted to 25 mL with 80% methanol. The total phenolic and flavonoids contents of AR were analyzed spectropho-tometrically, as described previously (Cui et al. 2014).

Determination of electrical conductivity (EC), pH, water potential and residual ion and sugar content of the growth media

The electrical conductivity (EC) of the medium was meas-ured using a conductivity meter (Model LF-54, WTW GmbH, Germany) and the hydrogen ion concentration (pH) of the culture medium was measured using a pH meter (Model Inolab, WTW GmbH, Germany). Water potential (WP) of the residual media were determined using a dew-point potentiometer (Model WP4, Decagon Devices Inc., Washington, USA). The residual ions and sugar in the medium were quantified by HPLC method (Water Co., Mildford, USA).

Identification of eurycomanone in AR extract

To identify eurycomanone, about 2 g of the ground-dried roots were extracted in 10  mL of 80% methanol and sonicated for 1 h. Crude extract was filtered through fil-ter paper (Advantec 110 mm, Toyo Rosihi Kaisha Ltd., Japan) and concentrated using a vacuum rotary evapora-tor (Tokyo Rikakikai Co., Japan) in a water bath at 50 °C. The residues were dissolved with 50% acetonitrile up to 0.5 mg mL−1 and filtered through a 0.2 µm membrane filter (Gelman, USA) for analysis. Eurycomanone (ChromaDex; Laguna Hills, CA, USA) was identified on an ESI–MS/MS system consisting of an AB SCIEX 3200 QTRAP MS/MS (Applied Biosystems, Foster City, CA, USA) with an ESI source (Turbo Ionspray). Data acquisition and processing were achieved with AB SCIEX Analyst 1.5 software. All samples were quantified by product ion (MS2) analysis in positive mode. The syringe pump method properties (tune control) employed were as follows: diameter of 4.6 mm and flow rate of 10 μL min−1.

Statistical analysis

Data are presented as the averages of three independent experiments. Statistically significant differences among means were identified using analysis of variance using SAS program (SAS Institute Inc., USA).

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Results and discussion

Influence of medium composition on biomass and secondary metabolite accretion in AR cultures

Optimization of medium constituents is essential for maxi-mizing the accretion of biomass and secondary metabolites

in AR cultures (Murthy et al. 2014a, 2016). In this study, AR of MG grown for 7 weeks in different media, including MS, Gamborg’s B5, WPM, DKS and ¾ MS, and growth and development of AR were assessed. AR cultures grown in MS and B5 media showed profuse lateral root develop-ment from the main root, which accounted for an increase in biomass accretion, whereas cultures maintained in DKW and WPM showed only root elongation without the

Fig. 1 Effect of medium composition on the growth of AR cultures of Malaysian ginseng (Eurycoma longifolia) in bioreactors for 7 weeks. a–o Images of adventitious root cultures grown in Murashige and

Skoog medium (MS) medium (a, f, k), MS medium (b, g, i), Driver Kuniyuki Walnut (DKW) medium (c, h, m), Woody Plant Medium (WPM) (d, i, n), and 3/4 MS medium (e, j, o)

Table1 Effect of medium formulation on adventitious root growth of Malaysian ginseng and amount of phenolics and flavonoids after 7 weeks of cultures

a Mean separation within columns by Duncan’s multiple range test at 5% level

Medium formu-lation

Fresh wt. (g L−1) Dry wt. (g L−1) Total phenolics (mg g−1 DW)

Total flavo-noids (mg g−1 DW)

MS 21.57ba 2.37b 7.51bc 2.42d

B5 27.46ab 3.28a 7.87c 2.70c

DKW 7.42d 1.15c 8.29a 2.94a

WPM 10.97c 1.64c 8.12ab 2.82b

3/4MS 31.07a 3.22a 8.04ab 2.95a

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development of lateral roots (Fig. 1). Biomass accretion was the highest in ¾ MS medium (31.07 g L−1 FW and 3.22 g L−1 DW), which represented an 8.6-fold increment. Additionally, high levels of phenolics (9.04 mg g−1 DW) and flavonoids (2.90 mg g−1 DW) were obtained in ¾ MS medium (Table 1). Consistent with our results, previous studies showed that MS medium favored anthocyanin and withanolide-A production in AR cultures of radish and aswagandha, respectively (Betsui et al. 2004; Praveen and Murthy 2010). In Scopolia parviflora AR cultures, Gam-borg’s B5 medium improved the accretion of alkaloids (Min et al. 2007). In Korean ginseng AR cultures, ¾ MS medium was satisfactory for biomass accretion, whereas, ½ MS medium was good for ginsenoside accumulation (Murthy et al. 2014b).

Time‑course analysis of AR growth

Next, we established AR cultures of MG in bioreactors to monitor the growth over duration of 7 weeks. The AR cul-ture of MG grew well in bioreactor system (Fig. 1). The cultures showed a typical growth pattern characterized by a lag phase from 0 to 2 weeks, an exponential phase from 3 to 7 weeks, and a stationary phase thereafter (Fig. 2a). At

7 weeks of culture, the biomass of MG-AR cultures reached a peak of 3.22 g L−1 DW (Table 1). These results are concur-rent with earlier reported results of Jeong et al. (2009) with AR cultures of Echinacea.

Effect of minerals on the growth of AR

To assess the role of minerals of culture medium on the growth of AR of MG, we continuously monitored changes in anion and cation concentration in the growth medium during the 7-week culture period (Fig. 2b, c). The results showed a rapid depletion of ammonium (NH4

+), potassium (K+), nitrate (NO3

−) and hydrogen phosphate (HPO42−) ion

in the growth medium. Preferential utilization of these ions has been reported previously in AR cultures of Echinacea (Jeong et al. 2009), beetroot (Shin et al. 2002), and soapwort (Morad et al. 1998). The results of this study suggest that the bioreactor culture adequately meet the nutritional require-ment of AR cultures of MG.

Figure  2d presents the EC and pH data of ¾ MS medium during the 7-week period. The EC of the culture medium decreases with the lapse of time, especially from 4 to 7 weeks (Fig. 2d); this reflects the accretion of bio-mass in the suspension. These results are consistent with

Fig. 2 a Changes in fresh weight (FW) and dry weight (DW) of Malaysian ginseng AR during 7  weeks of culture. Data represent mean ± standard error (SE) of three replicates. b Cation concen-trations in the medium, as affected by the medium formulation, at 0 and 7  weeks of culture of Malaysian ginseng AR. Data represent mean ± SE of three replicates. c Anion concentrations in the medium,

as affected by the medium formulation, at 0 and 7 weeks of culture of Malaysian ginseng AR. Data represent mean ± SE of three replicates. d Electrical conductivity (EC) and pH of the medium, as affected by the medium formulation, at 7 weeks of culture of Malaysian ginseng AR. Data represent mean ± SE of three replicates

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those of AR cultures of Echinacea (Jeong et al. 2009). The concentration of H+ ions in the medium decreased considerably over time. It has been shown previously that H+ ion concentration increased during NO3

− uptake and decreases during ammonia (NH3) consumption in suspen-sion cultures (McDonold and Jackman 1989).

ESI–MS/MS analysis of eurycomanone in AR extracts

Eurycomanone is the major secondary metabolite in MG; therefore, we investigated whether eurycomanone was present in the AR extracts by ESI–MS/MS (Fig. 3). Ion isolation data revealed presence of eurycomanone in the AR extracts of MG (Fig. 3). These results suggest that AR cultures of MG could be efficiently used for the produc-tion of secondary metabolites.

Conclusion

Among the five different media tested in this study, ¾ MS medium was most suitable for cultivation of AR of MG, as it resulted in the highest AR biomass as well as excellent amount of secondary metabolites. Our results revealed a rapid decline in NH4

+, K+, NO3−, and

HPO42−concentrations in the medium and ¾ MS medium

is useful for production of MG secondary metabolites by AR root suspension cultures.

Acknowledgements This work was supported by Yanbin Academy of Agricultural Sciences, China.

Author contribution J-DZ, H-LS, Y-JJ, Y-YL, K-L and W-WQ con-ducted experiments. HNM and S-YP planned the experimental work, interpreted the data and helped in manuscript preparation.

Fig. 3 Electrospray ionization tandem mass spectrometry (ESI–MS/MS) chromatogram of eurycomanone standard and Malaysian ginseng AR extract. ESI–MS/MS was performed using the negative ion mode

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Compliance with ethical standards

Conflict of interest Authors do not have any conflict of interest.

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