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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Solvent-based extraction optimisation for efficient ultrasonication-assisted ginsenoside recovery from Panax quinquefolius and P. sikkimensis cell suspension lines a
b
a
Tanya Biswas , P.V. Ajayakumar , Ajay Kumar Mathur & Archana Mathur
a
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Division of Plant Biotechnology, CSIR-Central Institute of Medicinal & Aromatic Plants, Council of Scientific & Industrial Research, PO CIMAP, Lucknow 226015, India b
Analytical Chemistry Division, CSIR-Central Institute of Medicinal & Aromatic Plants, Council of Scientific & Industrial Research, PO CIMAP, Lucknow 226015, India Published online: 27 Mar 2015.
To cite this article: Tanya Biswas, P.V. Ajayakumar, Ajay Kumar Mathur & Archana Mathur (2015): Solvent-based extraction optimisation for efficient ultrasonication-assisted ginsenoside recovery from Panax quinquefolius and P. sikkimensis cell suspension lines, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2015.1024119 To link to this article: http://dx.doi.org/10.1080/14786419.2015.1024119
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Natural Product Research, 2015 http://dx.doi.org/10.1080/14786419.2015.1024119
Solvent-based extraction optimisation for efficient ultrasonication-assisted ginsenoside recovery from Panax quinquefolius and P. sikkimensis cell suspension lines
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Tanya Biswasa, P.V. Ajayakumarb, Ajay Kumar Mathura and Archana Mathura* a Division of Plant Biotechnology, CSIR-Central Institute of Medicinal & Aromatic Plants, Council of Scientific & Industrial Research, PO CIMAP, Lucknow 226015, India; bAnalytical Chemistry Division, CSIR-Central Institute of Medicinal & Aromatic Plants, Council of Scientific & Industrial Research, PO CIMAP, Lucknow 226015, India
(Received 9 February 2015; final version received 20 February 2015)
The present study aims at developing an extraction protocol for efficient ginsenoside recovery from cell suspensions of Panax quinquefolius and P. sikkimensis. Methanol (100%, 70% and 30%), water (408C, 908C), water-saturated butanol and butanolsaturated water were compared for their ultrasonication-assisted ginsenoside retrieval efficacy. HPLC and HP-TLC analysis revealed 100% methanol as the best solvent for maximum retrieval of Rb (diol) and Rg (triol) ginsenosides (P. quinquefolius: Rb: 0.189, Rg: 3.163 mg/g DW; P. sikkimensis: Rb: 0.245, Rg: 4.073 mg/g DW), followed by water (908C). Methanolic solutions, especially 70%, proved to be significant retrievers of Rg1 (1.812 and 1.327 mg/g DW in P. quinquefolius and P. sikkimensis), with poor Re recovery (0.328 and 0.342 mg/g DW). Water-saturated butanol also led to significant ginsenoside extraction (72.4% of content extracted by methanol), selectively in P. quinquefolius, with a less than 50% of total content extracted by methanol, in P. sikkimensis. Keywords: Panax quinquefolius; Panax sikkimensis; ginsenosides; ultrasonicationassisted extraction; HPLC; HPTLC
1. Introduction Collectively known as ‘ginseng’, members of the Araliaceae family, under the genera Panax (P. ginseng or Korean ginseng, P. quinquefolius or American ginseng), are valued medicinal herbs in the global drug scenario, known for their general health promotory and protective effects on the cardiovascular, immune and central nervous systems (Yuan et al. 2010). Five- to
*Corresponding author. Email: [email protected], [email protected] q 2015 Taylor & Francis
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six-year-old mature roots which harbour the major bioactives, ginsenosides, are a part of triterpenoid saponins, falling into two broad classes: 20-S protopanaxadiol (Rb1, Rb2, Rc, Rd) and 20-S protopanaxatriol (Re, Rg1, Rg2, Rh1), the former having two and the latter having three sugar groups (Fournier et al. 2003). The pharmacological activity of these ginsenosides largely depend on their structural aspects, not just individually but even in combinations, thus enlarging the range of their bioactivities. Ginseng easily qualifies as one eligible candidate for plant tissue culture-based production platforms for faster and sustained metabolite procurement as compared to its traditional cultivation. Mature ginseng roots should be about 5 – 6 years of age before their ginsenoside quality and quantity can be put to use. Moreover, Ginseng cultivation is restricted to narrow geographic belts due to its extremely specific soil and moisture requirement. However, an important aspect in obtaining these bioactives from in vitro cultures is their safe recovery with minimum loss and post harvest processing. Ginsenosides present a complicated picture due to their structural similarity, often making their identification difficult and sample preparation tedious, leading to significant losses in ginsenoside recovery. Many techniques involving their extraction, purification and analysis have been employed from a variety of host tissues such as roots (Lin et al. 2013; Sunwoo et al. 2014), leaves (Lee et al. 2012) and even berries (Kim et al. 2013). The preferred solvent under use involves methanol often in aqueous solutions, in conjunction with heat, refluxing or sonication-based extraction methodologies (Corbit et al. 2005; Engelberth et al. 2010). Ultrasonication-assisted extraction has been widely utilised for extraction of herbal oils, anthocyanins and polyphenolics among other bioactives from plant sources with an increased yield (Vilkhu et al. 2008). Factors such as the nature of the tissue (fresh or dry), localisation of the bioactive within the tissue, the requirements of any pretreatments and the most crucial is the consideration of the disruptive effect of ultrasonics, heavily influence any extraction procedure involving ultrasonication. The resultant acoustic cavitations, inter-particle collisions and turbulence, elicited by ultrasonic stress, lead to cellular membrane erosion and disintegration and release of cell contents. Ultrasonication is a convenient and efficient laboratory bench level tool for extraction of ginsenosides from P. quinquefolius and P. sikkimensis cell suspension lines used in the present study. It also offers an attractive option for extraction of thermally unstable ginsenosides at low temperatures as compared to refluxing methods. Different solvents [methanol, 70% MeOH, 30% MeOH, water {408 C(NW) and 908 C(HW)}, water-saturated butanol (WSB) and butanolsaturated water (BSW)] widely used for extraction of eight major ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rf, Rg1 and Rg2), present in in vivo ginseng tissues constructing its ginsenoside fingerprint for quality assessment of ginseng samples, were compared with an extraction methodology in order to devise a protocol for their efficient recovery from cell suspensions. 2. Results and discussion The samples were analysed for their ginsenoside content using HP-TLC densitometry and HPLC – UV quantification (see Methodology, Supplementary material). Out of the eight ginsenoside markers, HP-TLC led to successful resolution of six authentics with the exception of Rb2 and Rc (Figure 1(A)). These ginsenosides could not be resolved due to their similar Rf. For further resolution and confirmation of ginsenoside identity, HPLC – UV analysis was performed which resulted in the resolution of all eight ginsenosides within a run time of 70 min [retention time: Rb1: 50.8, Rb2: 53.6, Rc: 52.4, Rd: 55.01, Re: 42.3, Rf: 49.8, Rg1: 41.8, Rg2: 53.9 min, Figure 1(B)]. All the solvents were found to retrieve ginsenosides from both the cell lines of Panax, however to markedly different extents. Solvents affect the dissolving capacity of the ginsenosides due to their differential polarities. Methanol (100%) registered a maximum
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Figure 1. (A) and (B) HPTLC densitogram and HPLC chromatogram of the eight ginsenoside authentics.
retrieval of both triol (Rg group) and diol groups (Rb group), in both P. quinquefolius and P. sikkimensis cell suspension lines. 2.1. Effect of different solvents on UAE-assisted ginsenoside retrieval from cell suspensions of P. quinquefolius In P. quinquefolius cell suspensions, production of four different ginsenosides out of the eight standard markers, Rb1, Rb2, Re, Rg1 (Rg2 present only in traces), was observed. Maximum ginsenoside recovery was registered with the use of 100% methanol (total saponin – 3.352 mg/g
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DW) as the extracting solvent, coupled with the fixed schedule of ultrasonication (Figure 2(A), (B); Tables 1 and 2). The efficacy of the solvents as far as total saponin retrieval is concerned, followed the order: 100% MeOH . HW . WSB . 70% MeOH . NW . BSW . 30% MeOH. However, the pattern of extraction of individual ginsenosides did not necessarily mimic the above trend. Although 100% methanol was capable of extracting all the ginsenosides maximal (Rb1: 0.139 mg/g DW; Rb2: 0.050 mg/g DW; Re: 1.191 mg/g DW; Rg1:1. 972 mg/g DW), 70% methanol proved much better than the rest of the solvents used, for extraction of Rb class of ginsenosides (Rb1: 0.109 mg/g DW; Rb2: 0.028). Among the triols, significant ginsenoside Re retrieval was observed with the use of hot water as the extractant (0.737 mg/g DW). However, not much variability was observed within the solvents in terms of ginsenoside Rg1 recovery (Tables 1 and 2). The extraction efficiencies were found to be differing in the recovery of ginsenosides Rb1 and Re. Rb2 recovery was observed to be 50% less than 100% MeOH with the use of hot water and 70% MeOH. Among the latter two solvents, 70% MeOH was observed to be a better Rb1 retriever and HW, a better Re retriever. Water-saturated butanol, also demonstrated Rg1, Re and Rb1 retrieval, when compared to 100% methanol, however, with a more than 50% loss in Re and Rb1 recovery. 2.2. Effect of different solvents on UAE-assisted ginsenoside retrieval from cell suspensions of P. sikkimensis In P. sikkimensis cell suspensions, in vitro production of Rb1, Rb2, Re, Rg1 and Rg2 were observed (Figure 3(A),(B); Tables 1 and 2) with maximum saponin retrieval (total saponin: 4.442 mg/g DW) registered with the use of 100% methanol as the extracting solvent (0. 215 mg/g DW; 0.030 mg/g DW; 1.568 mg/g DW; 2.505 mg/g DW; 0.124 mg/g DW, respectively). With respect to the total saponin content, the efficacy of the different solvents towards ginsenoside retrieval from cell suspensions followed the order: 100%MeOH . HW . BSW . 70% MeOH . WSB , 30% MeOH . NW (Tables 1 and 2). However, as with P. quinquefolius suspensions, different ginsenosides were recovered at different levels with the solvents used, not necessarily in the order elucidated above. Ginsenoside Rb1 was found to be recovered from P. sikkimensis cell suspensions significantly, only with the use of 100% methanol as the extractant. Ginsenoside Re was recovered appreciably with the use of hot water as the extractant (0.960 mg/g DW). However, in contrast to P. quinquefolius cells, ginsenoside Rg1 recovery varied with different solvent combinations, with BSW registering comparable recovery with 100% MeOH (1.757 mg/g DW) along with appreciable Rg2 recovery (0.108 mg/g DW). However, its use led to the reduced ginsenoside Re recovery which was 19 and 11.5 fold less, when compared with 100% MeOH and hot water, respectively. The total saponin content with
Figure 2. (A) and (B) HPTLC densitogram and HPLC chromatogram of P. quinquefolius cell suspension cells extracted with 100% methanol.
0.050 0.028 0.014 0.024 0.010 nd nd 0.005 0.007
0.030 0.013 nd 0.015 0.012 nd nd 0.005 0.006
0.215 (0.191 ^ 0.06) 0.048 (nd) 0.056 (nd) 0.035 (nd) 0.058 (nd) Nd (nd) 0.091 (nd) 0.014 0.020
Rb2
0.139 (0.175 ^ 0.03) 0.109 (0.101 ^ 0.05) 0.062 (0.066 ^ 0.06) 0.086 (0.075 ^ 0.03) 0.040 (0.054 ^ 0.08) 0.072 (0.089 ^ 0.04) 0.093 (0.088 ^ 0.05) 0.019 0.026
Rb1
nd nd nd nd nd nd nd 0 0
nd nd nd nd nd nd nd 0 0
Rc
nd nd nd nd nd nd nd 0 0
nd nd nd nd nd nd nd 0 0
Rd
1.568 0.342 0.200 0.960 0.472 0.083 0.057
(1.366 ^ 0.10) (0.333 ^ 0.15) (0.169 ^ 0.07) (0.924 ^ 0.11) (0.340 ^ 0.08) (0.096 ^ 0.03) (0.063 ^ 0.02) 0.078 0.108
(1.106 ^ 0.14) (0.233 ^ 0.05) (0.134 ^ 0.11) (0.624 ^ 0.09) (0.163 ^ 0.12) Nd (Tr) 0.502 (0.424 ^ 0.18) 0.04 0.05
1.191 0.328 0.149 0.737 0.171
Re
nd nd nd nd nd nd nd 0 0
nd nd nd nd nd nd nd 0 0
Rf
2.505 1.327 1.063 0.921 0.569 1.757 1.155
1.972 1.812 1.306 1.747 1.451 1.585 1.834
(1.938 ^ 0.11) (1.161 ^ 0.11) (1.058 ^ 0.12) (0.784 ^ 0.07) (0.466 ^ 0.16) (1.703 ^ 0.04) (1.082 ^ 0.10) 0.282 0.391
(1.938 ^ 0.07) (1.861 ^ 0.11) (1.379 ^ 0.13) (1.704 ^ 0.06) (1.563 ^ 0.11) (1.549 ^ 0.19) (1.762 ^ 0.14) 0.102 0.142
Rg1
Notes: Tr, traces; nd, not detected; values in parentheses (mean ^ SD) is content calculated via HP-TLC densitometry.
P. quinquefolius 100% Methanol 70% Methanol 30% Methanol HW NW BSW WSB CD (5%) CD (1%) P.sikkimensis 100% Methanol 70% Methanol 30% Methanol HW NW BSW WSB CD (5%) CD (1%)
Sample ID
Ginsenoside content (mg/g DW)
0.124 0.088 0.092 0.086 0.098 0.108 0.120
(Tr) (Tr) (Tr) (Tr) (Tr) (Tr) (Tr) 0 0 (0.133 ^ 0.04) (0.095 ^ 0.06) (0.096 ^ 0.08) (0.092 ^ 0.07) (0.097 ^ 0.10) (0.107 ^ 0.03) (0.112 ^ 0.07) 0.013 0.018
Tr Tr Tr Tr Tr Tr Tr
Rg2
4.442 1.818 1.411 2.018 1.211 1.948 1.423 0.003 0.004
3.352 2.277 1.531 2.594 1.672 1.657 2.429 0.002 0.003
Total saponin content (mg/g DW)
Table 1. Comparative ginsenoside content in P. quinquefolius and P. sikkimensis cell suspension lines extracted with different solvents assisted with ultrasonication (120 US power for 30 min £ 4).
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Figure 3. (A) and (B) HPTLC densitogram and HPLC chromatogram of P. sikkimensis cell suspension cells extracted with 100% methanol.
use of HW and BSW was almost similar; however, differences lay in their abilities towards Re recovery from cells. Almost all the solvents employed in the present study were able to recover ginsenoside Rg2, from P. sikkimensis cells, to a lesser degree of variability. Water-saturated butanol exhibited good potential towards its recovery (similar to 100% methanol; 0.120 mg/g DW). Both the aqueous solutions of methanol (70% and 30%), although effective in recovery of ginsenoside Rg1 and Rg2, were observed to be poor extractants for ginsenoside Re (Table 1) in P. sikkimensis cells. Ginsenoside Rb2 recovery was best when extracted with 100% MeOH. Use of other solvents led to minute or no recovery of this particular ginsenoside. Dong and co-workers have previously reported the maximum ginsenoside retrieving capacity of methanol as did Rodin and co-workers in 2013 (Dong et al. 2005; Rodin et al. 2013). Ginsenosides are also reported to have been recovered in water solvents in significant yields (Engelberth et al. 2010; Liu et al. 2012). Our results also support the fact, as HW (908 C) proved better than NW(408C) towards extraction of the triol ginsenosides (Re and Rg1) from P. quinquefolius and P. sikkimensis suspension cells. In both the cell lines, hot water extraction was observed to be the second best (P. quinquefolius) and third best extractant (P. sikkimensis) for saponins. Although the heat lability of ginsenosides, with the use of hot water cannot be completely ruled out, it has been observed by Corbitt et al. (2005)) to be significantly less than their degradation following refluxing. Corbitt and team also found 100% methanol as a better extractant than 70% methanol, which further corroborates the results obtained in our study. Butanolic solutions have also been reported to yield ginsenosides from tissues (Engelberth et al. 2010). In the present study, butanol-saturated water (P. sikkimensis) and water-saturated butanol (P. quinquefolius) were found to be effective extractants working in reverse order for these two Panax spp., with one solvent, successful for cell suspension of one species and unsuccessful for the other. The extraction differences may probably exist because of the differential membrane structure and ultrasonic disruption in the cell suspensions of the two Panax species, affecting the dissolution of the ginsenosides. Even though being an excellent solvent for ginsenosides, use of butanol might not lead to higher ginsenoside yield primarily due the heat degradation of the ginsenosides due to its high boiling point. Thirty per cent methanolic solution was not found to be as capable as the other solvent systems towards efficient recovery of ginsenosides. This particular solvent system was observed to have low triol (Re and Rg1) recovery. Similar trend of poor ginsenoside recovery with 30% methanol has been previously reported by Li and Fitzloff (2002).
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Table 2. Analysis of variance using CRD. Sources of variation P. quinquefolius Treatments Error Total P. sikkimensis Treatments Error Total
Degree of freedom
Mean sum of squares Rb1
Rb2
Re
Rg1
Rg2
Total content
6 14 20
0.003** 0.0001
0.0009** 0.00001
0.509** 0.0005
0.165** 0.003
0 0
0.0021** 0.0002
6 14 20
0.014** 0.00006
0.0003** 0.000008
0.916** 0.002
1.205** 0.026
0.0006** 0.00005
0.0014** 0.004
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Note: **p , 0.01; three replicates employed per treatment.
3. Conclusion An effective extraction procedure is of critical importance towards the outcome of metabolomic experiments. Various factors, ranging from sample collection to storage, the selection of an ideal extraction solvent and methodology heavily influence the recovery of metabolites, discrepancies in which, often lead to incomplete information. This report combines extractants with ultrasonication as a strategy for recovery of ginsenosides from cell suspensions in significant yields at the bench level. Ultrasonication has immense potential as an extraction tool as it might prove to be a cost-effective strategy, even if used as a pre-treatment, for ginsenoside extraction even at the industrial level from bioreactor-harvested cell biomass. It may even be applied in a combinatorial way with other extraction techniques for superior yields of saponins. Ultrasonication-based extraction techniques may eradicate the dependence of a particular solvent for bioactive extraction and permit use of safe solvents such as water, with minimum losses, and inculcate safe, economical and environment-friendly industrial practices. Supplementary material Experimental details relating to this article are available online as supplementary data. Acknowledgements The authors are grateful to the director, CIMAP, for the infrastructure and the lab facilities required to carry out the work.
Disclosure statement No potential conflict of interest was reported by the authors.
Funding Tanya Biswas also acknowledges the University Grants Commision for grant of a UGC-JRF fellowship.
References Corbit RM, Ferreira JF, Ebbs SD, Murphy LL. 2005. Simplified extraction of ginsenosides from American ginseng (Panax quinquefolius L.) For high-performance liquid chromatography 2 ultraviolet analysis. J Agric Food Chem. 53:9867–9873. doi:10.1021/jf051504p.
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Dong TTX, Zhao KJ, Huang WZ, Leung KW, Tsim KWK. 2005. Orthogonal array design in optimizing the extraction efficiency of active constituents from roots of Panax notoginseng. Phytother Res. 19:684–688. doi:10.1002/ptr. 1728. Engelberth AS, Clausen EC, Carrier DJ. 2010. Comparing extraction methods to recover ginseng saponins from American ginseng (Panax quinquefolium), followed by purification using fast centrifugal partition chromatography with HPLC verification. Sep Purif Technol. 72:1–6. doi:10.1016/j.seppur.2009.12.002. Fournier AR, Proctor JT, Gauthier L, Khanizadeh S, Be´langer A, Gosselin A, Dorais M. 2003. Understory light and root ginsenosides in forest-grown Panax quinquefolius. Phytochemistry. 63:777–782. doi:10.1016/S0031-9422(03) 00346-7. Kim SJ, Kim JD, Ko SK. 2013. Changes in ginsenoside composition of Ginseng berry extracts after a microwave and vinegar process. J Ginseng Res. 37:269–272. doi:10.5142/jgr.2013.37.269. Lee HJ, Lee HS, Cho HJ, Kim SY, Suh HJ. 2012. Utilization of hydrolytic enzymes for the extraction of ginsenosides from Korean ginseng leaves. Process Biochem. 47:538–543. doi:10.1016/j.procbio.2011.12.004. Li W, Fitzloff JF. 2002. HPLC determination of ginsenosides content in ginseng dietary supplements using ultraviolet detection. J Liq Chromatogr Relat Tech. 25:2485–2500. doi:10.1081/JLC-120014269. Lin H, Zhang Y, Han M, Yang L. 2013. Aqueous ionic liquid based ultrasonic assisted extraction of eight ginsenosides from ginseng root. Ultrason Sonochem. 20:680–684. doi:10.1016/j.ultsonch.2012.10.003. Liu Z, Li Y, Li X, Ruan CC, Wang LJ, Sun GZ. 2012. The effects of dynamic changes of malonyl ginsenosides on evaluation and quality control of Panax ginseng C.A. Meyer. J Pharm Biomed Anal. 64 –65:56– 63. doi:10.1016/ j.jpba.2012.02.005. Rodin IA, Stavrianidi AN, Braun AV, Shpigun OA. 2013. Modern methods of identifying and determining ginsenosides. Moscow Univ Chem Bull. 68:127–142. doi:10.3103/S0027131413030024. Sunwoo HH, Gujral N, Huebl AC, Kim CT. 2014. Application of high hydrostatic pressure and enzymatic hydrolysis for the extraction of ginsenosides from fresh ginseng root (Panax ginseng C.A. Myer). Food Bioprod Technol. 7:1246–1254. doi:10.1007/s11947-013-1234-1. Vilkhu K, Mawson R, Simons L, Bates D. 2008. Applications and opportunities for ultrasound assisted extraction in the food industry – a review. Innovative Food Sci Emerg Technol. 9:161–169. doi:10.1016/j.ifset.2007.04.014. Yuan CS, Wang CZ, Wicks SM, Qi LW. 2010. Chemical and pharmacological studies of saponins with a focus on American ginseng. J Ginseng Res. 34:160–167. doi:10.5142/jgr.2010.34.3.160.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Solvent-based extraction optimisation for efficient ultrasonication-assisted ginsenoside recovery from Panax quinquefolius and P. sikkimensis cell suspension lines a
b
a
Tanya Biswas , P.V. Ajayakumar , Ajay Kumar Mathur & Archana Mathur
a
a
Click for updates
Division of Plant Biotechnology, CSIR-Central Institute of Medicinal & Aromatic Plants, Council of Scientific & Industrial Research, PO CIMAP, Lucknow 226015, India b
Analytical Chemistry Division, CSIR-Central Institute of Medicinal & Aromatic Plants, Council of Scientific & Industrial Research, PO CIMAP, Lucknow 226015, India Published online: 27 Mar 2015.
To cite this article: Tanya Biswas, P.V. Ajayakumar, Ajay Kumar Mathur & Archana Mathur (2015): Solvent-based extraction optimisation for efficient ultrasonication-assisted ginsenoside recovery from Panax quinquefolius and P. sikkimensis cell suspension lines, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2015.1024119 To link to this article: http://dx.doi.org/10.1080/14786419.2015.1024119
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or
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This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2015 http://dx.doi.org/10.1080/14786419.2015.1024119
Solvent-based extraction optimisation for efficient ultrasonication-assisted ginsenoside recovery from Panax quinquefolius and P. sikkimensis cell suspension lines
Downloaded by [University of Connecticut] at 18:00 01 April 2015
Tanya Biswasa, P.V. Ajayakumarb, Ajay Kumar Mathura and Archana Mathura* a Division of Plant Biotechnology, CSIR-Central Institute of Medicinal & Aromatic Plants, Council of Scientific & Industrial Research, PO CIMAP, Lucknow 226015, India; bAnalytical Chemistry Division, CSIR-Central Institute of Medicinal & Aromatic Plants, Council of Scientific & Industrial Research, PO CIMAP, Lucknow 226015, India
(Received 9 February 2015; final version received 20 February 2015)
The present study aims at developing an extraction protocol for efficient ginsenoside recovery from cell suspensions of Panax quinquefolius and P. sikkimensis. Methanol (100%, 70% and 30%), water (408C, 908C), water-saturated butanol and butanolsaturated water were compared for their ultrasonication-assisted ginsenoside retrieval efficacy. HPLC and HP-TLC analysis revealed 100% methanol as the best solvent for maximum retrieval of Rb (diol) and Rg (triol) ginsenosides (P. quinquefolius: Rb: 0.189, Rg: 3.163 mg/g DW; P. sikkimensis: Rb: 0.245, Rg: 4.073 mg/g DW), followed by water (908C). Methanolic solutions, especially 70%, proved to be significant retrievers of Rg1 (1.812 and 1.327 mg/g DW in P. quinquefolius and P. sikkimensis), with poor Re recovery (0.328 and 0.342 mg/g DW). Water-saturated butanol also led to significant ginsenoside extraction (72.4% of content extracted by methanol), selectively in P. quinquefolius, with a less than 50% of total content extracted by methanol, in P. sikkimensis. Keywords: Panax quinquefolius; Panax sikkimensis; ginsenosides; ultrasonicationassisted extraction; HPLC; HPTLC
1. Introduction Collectively known as ‘ginseng’, members of the Araliaceae family, under the genera Panax (P. ginseng or Korean ginseng, P. quinquefolius or American ginseng), are valued medicinal herbs in the global drug scenario, known for their general health promotory and protective effects on the cardiovascular, immune and central nervous systems (Yuan et al. 2010). Five- to
*Corresponding author. Email: [email protected], [email protected] q 2015 Taylor & Francis
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six-year-old mature roots which harbour the major bioactives, ginsenosides, are a part of triterpenoid saponins, falling into two broad classes: 20-S protopanaxadiol (Rb1, Rb2, Rc, Rd) and 20-S protopanaxatriol (Re, Rg1, Rg2, Rh1), the former having two and the latter having three sugar groups (Fournier et al. 2003). The pharmacological activity of these ginsenosides largely depend on their structural aspects, not just individually but even in combinations, thus enlarging the range of their bioactivities. Ginseng easily qualifies as one eligible candidate for plant tissue culture-based production platforms for faster and sustained metabolite procurement as compared to its traditional cultivation. Mature ginseng roots should be about 5 – 6 years of age before their ginsenoside quality and quantity can be put to use. Moreover, Ginseng cultivation is restricted to narrow geographic belts due to its extremely specific soil and moisture requirement. However, an important aspect in obtaining these bioactives from in vitro cultures is their safe recovery with minimum loss and post harvest processing. Ginsenosides present a complicated picture due to their structural similarity, often making their identification difficult and sample preparation tedious, leading to significant losses in ginsenoside recovery. Many techniques involving their extraction, purification and analysis have been employed from a variety of host tissues such as roots (Lin et al. 2013; Sunwoo et al. 2014), leaves (Lee et al. 2012) and even berries (Kim et al. 2013). The preferred solvent under use involves methanol often in aqueous solutions, in conjunction with heat, refluxing or sonication-based extraction methodologies (Corbit et al. 2005; Engelberth et al. 2010). Ultrasonication-assisted extraction has been widely utilised for extraction of herbal oils, anthocyanins and polyphenolics among other bioactives from plant sources with an increased yield (Vilkhu et al. 2008). Factors such as the nature of the tissue (fresh or dry), localisation of the bioactive within the tissue, the requirements of any pretreatments and the most crucial is the consideration of the disruptive effect of ultrasonics, heavily influence any extraction procedure involving ultrasonication. The resultant acoustic cavitations, inter-particle collisions and turbulence, elicited by ultrasonic stress, lead to cellular membrane erosion and disintegration and release of cell contents. Ultrasonication is a convenient and efficient laboratory bench level tool for extraction of ginsenosides from P. quinquefolius and P. sikkimensis cell suspension lines used in the present study. It also offers an attractive option for extraction of thermally unstable ginsenosides at low temperatures as compared to refluxing methods. Different solvents [methanol, 70% MeOH, 30% MeOH, water {408 C(NW) and 908 C(HW)}, water-saturated butanol (WSB) and butanolsaturated water (BSW)] widely used for extraction of eight major ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rf, Rg1 and Rg2), present in in vivo ginseng tissues constructing its ginsenoside fingerprint for quality assessment of ginseng samples, were compared with an extraction methodology in order to devise a protocol for their efficient recovery from cell suspensions. 2. Results and discussion The samples were analysed for their ginsenoside content using HP-TLC densitometry and HPLC – UV quantification (see Methodology, Supplementary material). Out of the eight ginsenoside markers, HP-TLC led to successful resolution of six authentics with the exception of Rb2 and Rc (Figure 1(A)). These ginsenosides could not be resolved due to their similar Rf. For further resolution and confirmation of ginsenoside identity, HPLC – UV analysis was performed which resulted in the resolution of all eight ginsenosides within a run time of 70 min [retention time: Rb1: 50.8, Rb2: 53.6, Rc: 52.4, Rd: 55.01, Re: 42.3, Rf: 49.8, Rg1: 41.8, Rg2: 53.9 min, Figure 1(B)]. All the solvents were found to retrieve ginsenosides from both the cell lines of Panax, however to markedly different extents. Solvents affect the dissolving capacity of the ginsenosides due to their differential polarities. Methanol (100%) registered a maximum
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Figure 1. (A) and (B) HPTLC densitogram and HPLC chromatogram of the eight ginsenoside authentics.
retrieval of both triol (Rg group) and diol groups (Rb group), in both P. quinquefolius and P. sikkimensis cell suspension lines. 2.1. Effect of different solvents on UAE-assisted ginsenoside retrieval from cell suspensions of P. quinquefolius In P. quinquefolius cell suspensions, production of four different ginsenosides out of the eight standard markers, Rb1, Rb2, Re, Rg1 (Rg2 present only in traces), was observed. Maximum ginsenoside recovery was registered with the use of 100% methanol (total saponin – 3.352 mg/g
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DW) as the extracting solvent, coupled with the fixed schedule of ultrasonication (Figure 2(A), (B); Tables 1 and 2). The efficacy of the solvents as far as total saponin retrieval is concerned, followed the order: 100% MeOH . HW . WSB . 70% MeOH . NW . BSW . 30% MeOH. However, the pattern of extraction of individual ginsenosides did not necessarily mimic the above trend. Although 100% methanol was capable of extracting all the ginsenosides maximal (Rb1: 0.139 mg/g DW; Rb2: 0.050 mg/g DW; Re: 1.191 mg/g DW; Rg1:1. 972 mg/g DW), 70% methanol proved much better than the rest of the solvents used, for extraction of Rb class of ginsenosides (Rb1: 0.109 mg/g DW; Rb2: 0.028). Among the triols, significant ginsenoside Re retrieval was observed with the use of hot water as the extractant (0.737 mg/g DW). However, not much variability was observed within the solvents in terms of ginsenoside Rg1 recovery (Tables 1 and 2). The extraction efficiencies were found to be differing in the recovery of ginsenosides Rb1 and Re. Rb2 recovery was observed to be 50% less than 100% MeOH with the use of hot water and 70% MeOH. Among the latter two solvents, 70% MeOH was observed to be a better Rb1 retriever and HW, a better Re retriever. Water-saturated butanol, also demonstrated Rg1, Re and Rb1 retrieval, when compared to 100% methanol, however, with a more than 50% loss in Re and Rb1 recovery. 2.2. Effect of different solvents on UAE-assisted ginsenoside retrieval from cell suspensions of P. sikkimensis In P. sikkimensis cell suspensions, in vitro production of Rb1, Rb2, Re, Rg1 and Rg2 were observed (Figure 3(A),(B); Tables 1 and 2) with maximum saponin retrieval (total saponin: 4.442 mg/g DW) registered with the use of 100% methanol as the extracting solvent (0. 215 mg/g DW; 0.030 mg/g DW; 1.568 mg/g DW; 2.505 mg/g DW; 0.124 mg/g DW, respectively). With respect to the total saponin content, the efficacy of the different solvents towards ginsenoside retrieval from cell suspensions followed the order: 100%MeOH . HW . BSW . 70% MeOH . WSB , 30% MeOH . NW (Tables 1 and 2). However, as with P. quinquefolius suspensions, different ginsenosides were recovered at different levels with the solvents used, not necessarily in the order elucidated above. Ginsenoside Rb1 was found to be recovered from P. sikkimensis cell suspensions significantly, only with the use of 100% methanol as the extractant. Ginsenoside Re was recovered appreciably with the use of hot water as the extractant (0.960 mg/g DW). However, in contrast to P. quinquefolius cells, ginsenoside Rg1 recovery varied with different solvent combinations, with BSW registering comparable recovery with 100% MeOH (1.757 mg/g DW) along with appreciable Rg2 recovery (0.108 mg/g DW). However, its use led to the reduced ginsenoside Re recovery which was 19 and 11.5 fold less, when compared with 100% MeOH and hot water, respectively. The total saponin content with
Figure 2. (A) and (B) HPTLC densitogram and HPLC chromatogram of P. quinquefolius cell suspension cells extracted with 100% methanol.
0.050 0.028 0.014 0.024 0.010 nd nd 0.005 0.007
0.030 0.013 nd 0.015 0.012 nd nd 0.005 0.006
0.215 (0.191 ^ 0.06) 0.048 (nd) 0.056 (nd) 0.035 (nd) 0.058 (nd) Nd (nd) 0.091 (nd) 0.014 0.020
Rb2
0.139 (0.175 ^ 0.03) 0.109 (0.101 ^ 0.05) 0.062 (0.066 ^ 0.06) 0.086 (0.075 ^ 0.03) 0.040 (0.054 ^ 0.08) 0.072 (0.089 ^ 0.04) 0.093 (0.088 ^ 0.05) 0.019 0.026
Rb1
nd nd nd nd nd nd nd 0 0
nd nd nd nd nd nd nd 0 0
Rc
nd nd nd nd nd nd nd 0 0
nd nd nd nd nd nd nd 0 0
Rd
1.568 0.342 0.200 0.960 0.472 0.083 0.057
(1.366 ^ 0.10) (0.333 ^ 0.15) (0.169 ^ 0.07) (0.924 ^ 0.11) (0.340 ^ 0.08) (0.096 ^ 0.03) (0.063 ^ 0.02) 0.078 0.108
(1.106 ^ 0.14) (0.233 ^ 0.05) (0.134 ^ 0.11) (0.624 ^ 0.09) (0.163 ^ 0.12) Nd (Tr) 0.502 (0.424 ^ 0.18) 0.04 0.05
1.191 0.328 0.149 0.737 0.171
Re
nd nd nd nd nd nd nd 0 0
nd nd nd nd nd nd nd 0 0
Rf
2.505 1.327 1.063 0.921 0.569 1.757 1.155
1.972 1.812 1.306 1.747 1.451 1.585 1.834
(1.938 ^ 0.11) (1.161 ^ 0.11) (1.058 ^ 0.12) (0.784 ^ 0.07) (0.466 ^ 0.16) (1.703 ^ 0.04) (1.082 ^ 0.10) 0.282 0.391
(1.938 ^ 0.07) (1.861 ^ 0.11) (1.379 ^ 0.13) (1.704 ^ 0.06) (1.563 ^ 0.11) (1.549 ^ 0.19) (1.762 ^ 0.14) 0.102 0.142
Rg1
Notes: Tr, traces; nd, not detected; values in parentheses (mean ^ SD) is content calculated via HP-TLC densitometry.
P. quinquefolius 100% Methanol 70% Methanol 30% Methanol HW NW BSW WSB CD (5%) CD (1%) P.sikkimensis 100% Methanol 70% Methanol 30% Methanol HW NW BSW WSB CD (5%) CD (1%)
Sample ID
Ginsenoside content (mg/g DW)
0.124 0.088 0.092 0.086 0.098 0.108 0.120
(Tr) (Tr) (Tr) (Tr) (Tr) (Tr) (Tr) 0 0 (0.133 ^ 0.04) (0.095 ^ 0.06) (0.096 ^ 0.08) (0.092 ^ 0.07) (0.097 ^ 0.10) (0.107 ^ 0.03) (0.112 ^ 0.07) 0.013 0.018
Tr Tr Tr Tr Tr Tr Tr
Rg2
4.442 1.818 1.411 2.018 1.211 1.948 1.423 0.003 0.004
3.352 2.277 1.531 2.594 1.672 1.657 2.429 0.002 0.003
Total saponin content (mg/g DW)
Table 1. Comparative ginsenoside content in P. quinquefolius and P. sikkimensis cell suspension lines extracted with different solvents assisted with ultrasonication (120 US power for 30 min £ 4).
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Figure 3. (A) and (B) HPTLC densitogram and HPLC chromatogram of P. sikkimensis cell suspension cells extracted with 100% methanol.
use of HW and BSW was almost similar; however, differences lay in their abilities towards Re recovery from cells. Almost all the solvents employed in the present study were able to recover ginsenoside Rg2, from P. sikkimensis cells, to a lesser degree of variability. Water-saturated butanol exhibited good potential towards its recovery (similar to 100% methanol; 0.120 mg/g DW). Both the aqueous solutions of methanol (70% and 30%), although effective in recovery of ginsenoside Rg1 and Rg2, were observed to be poor extractants for ginsenoside Re (Table 1) in P. sikkimensis cells. Ginsenoside Rb2 recovery was best when extracted with 100% MeOH. Use of other solvents led to minute or no recovery of this particular ginsenoside. Dong and co-workers have previously reported the maximum ginsenoside retrieving capacity of methanol as did Rodin and co-workers in 2013 (Dong et al. 2005; Rodin et al. 2013). Ginsenosides are also reported to have been recovered in water solvents in significant yields (Engelberth et al. 2010; Liu et al. 2012). Our results also support the fact, as HW (908 C) proved better than NW(408C) towards extraction of the triol ginsenosides (Re and Rg1) from P. quinquefolius and P. sikkimensis suspension cells. In both the cell lines, hot water extraction was observed to be the second best (P. quinquefolius) and third best extractant (P. sikkimensis) for saponins. Although the heat lability of ginsenosides, with the use of hot water cannot be completely ruled out, it has been observed by Corbitt et al. (2005)) to be significantly less than their degradation following refluxing. Corbitt and team also found 100% methanol as a better extractant than 70% methanol, which further corroborates the results obtained in our study. Butanolic solutions have also been reported to yield ginsenosides from tissues (Engelberth et al. 2010). In the present study, butanol-saturated water (P. sikkimensis) and water-saturated butanol (P. quinquefolius) were found to be effective extractants working in reverse order for these two Panax spp., with one solvent, successful for cell suspension of one species and unsuccessful for the other. The extraction differences may probably exist because of the differential membrane structure and ultrasonic disruption in the cell suspensions of the two Panax species, affecting the dissolution of the ginsenosides. Even though being an excellent solvent for ginsenosides, use of butanol might not lead to higher ginsenoside yield primarily due the heat degradation of the ginsenosides due to its high boiling point. Thirty per cent methanolic solution was not found to be as capable as the other solvent systems towards efficient recovery of ginsenosides. This particular solvent system was observed to have low triol (Re and Rg1) recovery. Similar trend of poor ginsenoside recovery with 30% methanol has been previously reported by Li and Fitzloff (2002).
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Table 2. Analysis of variance using CRD. Sources of variation P. quinquefolius Treatments Error Total P. sikkimensis Treatments Error Total
Degree of freedom
Mean sum of squares Rb1
Rb2
Re
Rg1
Rg2
Total content
6 14 20
0.003** 0.0001
0.0009** 0.00001
0.509** 0.0005
0.165** 0.003
0 0
0.0021** 0.0002
6 14 20
0.014** 0.00006
0.0003** 0.000008
0.916** 0.002
1.205** 0.026
0.0006** 0.00005
0.0014** 0.004
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Note: **p , 0.01; three replicates employed per treatment.
3. Conclusion An effective extraction procedure is of critical importance towards the outcome of metabolomic experiments. Various factors, ranging from sample collection to storage, the selection of an ideal extraction solvent and methodology heavily influence the recovery of metabolites, discrepancies in which, often lead to incomplete information. This report combines extractants with ultrasonication as a strategy for recovery of ginsenosides from cell suspensions in significant yields at the bench level. Ultrasonication has immense potential as an extraction tool as it might prove to be a cost-effective strategy, even if used as a pre-treatment, for ginsenoside extraction even at the industrial level from bioreactor-harvested cell biomass. It may even be applied in a combinatorial way with other extraction techniques for superior yields of saponins. Ultrasonication-based extraction techniques may eradicate the dependence of a particular solvent for bioactive extraction and permit use of safe solvents such as water, with minimum losses, and inculcate safe, economical and environment-friendly industrial practices. Supplementary material Experimental details relating to this article are available online as supplementary data. Acknowledgements The authors are grateful to the director, CIMAP, for the infrastructure and the lab facilities required to carry out the work.
Disclosure statement No potential conflict of interest was reported by the authors.
Funding Tanya Biswas also acknowledges the University Grants Commision for grant of a UGC-JRF fellowship.
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