Improved production of withanolides in adventitious

root cultures of

Withania somnifera

by suspension

culture method

Sindhu Rangaraju*, A.N. Lokesha and Chenna Reddy Aswath

Division of Floriculture and Medicinal Crops, ICAR-Indian Institute of Horticultural Research, Hesaraghatta

Lake post, Bangalore-89

ABSTRACT

Jawahar Ashwagandha-20 (JA-20) and Arka Ashwagandha (AA) seeds were raised in in vitro conditions and phenotypic

differences between the plants was recorded. The adventitious roots were derived from leaves of the two varieties in in

vitro conditions. The effects of the strength of media and concentration and the combination of auxins (IAA and IBA)

for adventitious roots multiplication using suspension culture were studied. After 30 days of suspension culture, root

biomass was measured and HPLC analysis of major withanolides in leaves and adventitious roots was conducted. Arka

Ashwagandha variety had higher total withanolide content of 1.621 mg/g, Withaferin A content of 1.362 mg/g and root

yield of 4.066 g from 0.1g inoculum in 30 days compared to JA-20 which had total withanolide content of 1.156 mg/g,

Withaferin A content of 0.930 mg/g on dry weight basis and root mass of 3.71g from 0.1g of inoculum in 30 days.

The present study thus helps in the identi cation of an elite cultivar of Ashwagandha and development of a standard

protocol for mass multiplication of adventitious root in hormone-free media. This is bene cial in the preparation of

health supplements in terms of human health issues due to negligible residual effects of hormones in the  nal product.

KEY WORDS: ADVENTITIOUS ROOT CULTURE, ASHWAGANDHA, JA 20, IAA- INDOLE ACETIC ACID, IBA- INDOLE-3-BUTYRIC ACID

Pharmaceutical

Communication

Biosci. Biotech. Res. Comm. 12(1): 73-79 (2019)

ARTICLE INFORMATION:

Corresponding Author: sindhurangaraju63@gmail.co.in

Received 9

Feb, 2019

Accepted after revision 12

March, 2019

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007 CODEN: USA BBRCBA

Thomson Reuters ISI ESC / Clarivate Analytics USA

NAAS Journal Score 2019: 4.31 SJIF: 4.196

Online Contents Available at: http//www.bbrc.in/

DOI: 10.21786/bbrc/12.1/10

Sindhu Rangaraju, Lokesha and Chenna Reddy Aswath

74 COMPARATIVE ANALYSIS OF PHENOTYPIC TRAITS, ROOT YIELD, AND TOTAL WITHANOLIDES BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

INTRODUCTION

Ashwagandha (Withania somnifera Dunal) is a medici-

nal crop of commercial importance, belongs to family

Solanaceae, and is considered as an alternate to Panax

ginseng in its therapeutic values (CSIR.,1976). It is used

for curing a wide range of diseases in Ayurveda and

other indigenous systems of medicine for over 5000

years (Akram et al., 2011). The herb has the highest value

for its pharmacological activity in preparation of vari-

ous Ayurvedic formulations. It is anti-stress, anti aging,

and aids in recovering from neurodegenerative disorders

(Bhattacharya et al., 2002). It is a small, erect, branched,

woody shrub that grows up to 1.50m tall. It is cultivated

under rainfed condition in marginal soils by small and

marginal farmers of Madhya Pradesh, Rajasthan, Guja-

rat, Andhra Pradesh, Karnataka, and other states. Apart

from chemical constituents like alkaloids and witha-

nolides, it also contains a variety of amino acids includ-

ing aspartic acid, proline, tryptophan, tyrosine, cysteine,

alanine, glycine, and a high amount of iron.

Withanolides are C-28 steroidal lactones (Alfonso et al.,

1993). Major withanolides identi ed include Withanolide

A, Withanoside IV and VI (Tohda et al., 2005), Withaferin

A (Oh et al., 2008), Withanosides IV and V (Matsuda et

al., 2001) and Withanoside B (Pramanick et al., 2008).

Ashwagandha is available in the Western world as a die-

tary supplement. Its also known as “Indian Ginseng” and

“Winter Cherry”. Withanolide A has strong neuro phar-

macological properties of promoting outgrowth, synap-

tic reconstruction, and a potential to reconstruct neural

networks (Kuboyama et al., 2005; Tohda et al., 2005 a,b).

Withaferin A inhibits angiogenesis (Mohan et al., 2004),

metastasis (Misico et al., 2002). The major pharmacologi-

cal activity of Withania somnifera is contributed to two

major withanolides, Withaferin and Withaferin D (Gupta

et al., 2007, Sindhu et al., 2018).

Arka Ashwagandha is a variety identi ed at ICAR-

Indian Institute of Horticulture Research, for high dry

root yield and high total withanolide content. The vari-

ety has double the dry root yield (10 q/ha) than JA-20

(5.27q/ha). The other signi cant features are early vigor,

 eld tolerance to bacterial wilt, late blight, leaf spot dis-

eases and pests (Epilachna beetle, mites and aphids). It

matures in 180 days and is characterized by desired root

thickness and depth. The distinguishing features of the

variety are lengthy tertiary branch, thick stem which

has dense curved pubescence, lanceolate leaves with

obtuse leaf tip, bigger fruit capsules and fruits. JA-20

is a released variety from MPKVV, Mandsaur, Madhya

Pradesh used as a check in AICRP National trials and it

yields about 5 q/ha.

Recent advances in tissue culture methodologies have

improved secondary metabolite production across vari-

ous medicinal plants. Selection of high bioactive produc-

ing lines, optimization of culture conditions, metabolic

engineering, elicitation strategies and use of bioreac-

tor culture systems has made the production of useful

metabolites in vitro at a shorter duration of time (Sarin

et al., 2005). Plant-speci c metabolites can be effec-

tively obtained from organ and plant culture systems

(Verpoorte et al., 2002). Plant cell and organ culture sys-

tems are promising methodologies as they aid in rapid

proliferation of cells/organs, condensed biosynthetic

cycles in comparison to  eld grown plants (Ramachan-

dra Rao and Ravishankar, 2002, Thanh Tam et al., 2019).

Adventitious roots suspension cultures are found to be

ideal for biomass accumulation in Echinacea purpurea (Wu

et al., 2007) and P. notoginseng (Gao et al., 2005). Recent

studies indicate that explant type and genotype affect the

accumulation of bioactive compounds in adventitious

root cultures of Polygonum multi orum (Ho et al.,2019)

Lack of post-harvest storage technology for roots (Govil

et al., 1993; Singh and Kumar., 1998), excessive exploita-

tion of natural resources, problems in  eld cultivation as

it is dependent on monsoon, time consuming and labori-

ous are reasons enough to multiply adventitious roots of

Withania somnifera in suspension culture which meet the

global market requirement of Ashwagandha.

MATERIALS AND METHODS

Seeds of Withania somnifera like JA-20 and Arka Ash-

wagandha were selected. Seed pretreatment was con-

ducted as per our earlier reports (Sindhu et al., 2018),

morphological and phenotypic examination of 30-day

old plants was done. The adventitious roots were induced

in these two varieties, which were then harvested from

10-day old leaf culture bottles supplemented with aux-

ins. These roots were washed with sterile distilled water

two times to remove the small traces of agar followed by

treatment for 1 minute with 3% sodium hypochloride,

then the traces of sodium hypochlorite from roots were

removed by washing again with sterile distilled water.

The roots were dried by blotting with sterile tissue paper.

The known quantity of roots (100 mg) were weighed and

transferred into 100 ml of full strength MS liquid media

supplemented with different concentration and a combi-

nation of auxins in conical  asks with 3% sucrose con-

centration under 16 hours of photoperiod and placed in

an orbital shaker at 90 rpm at 25°C. The mass of mul-

tiplied roots was observed after 30 days of inoculation

and was compared with the control.

Extraction of bioactive principles from W. somnifera: The

adventitious roots were extracted from the liquid medium

and the in vitro leaves were taken washed using distilled

water to remove the traces of medium, dried and pow-

dered using pestle and mortar. Adventitious roots were

Sindhu Rangaraju, Lokesha and Chenna Reddy Aswath

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS COMPARATIVE ANALYSIS OF PHENOTYPIC TRAITS, ROOT YIELD, AND TOTAL WITHANOLIDES 75

assessed for total withanolides and also different com-

ponents that contribute to total withanolides whereas the

leaves were assessed only for Withaferin A content. The

analysis of bio actives was done using High Performance

Liquid Chromatography method. One gram of dry root

powder was extracted with methanol at 80

C on a water

bath and the residue was re-extracted twice with metha-

nol, till the extract was colorless. The extracts were pooled

and  ltered through sample clari cation kit and were

subjected to analysis by HPLC with Photo Diode Array.

Seven standards such as Withanoside IV, Withanoside

V, Withaferin A, Withanolide A, Withanolide B, Witha-

none and Withanostramolide from Natural Remedies Pvt.

Ltd, Bangalore were used to quantify the total amount of

withaferin A present in leaf samples and withanolides in

root samples. The chromatogram was recorded at 227 nm

and later the contents of various withanolides were added

to estimate the total withanolide and expressed as mg/g

on a dry weight basis.

Data Analysis:

All experiments were repeated thrice. Mean values of

treatments were subjected to ANOVA and signi cant

differences were separated by Duncan’s Multiple Range

Test. To determine signi cance at P<0.05SPSS (Win-

dows version 75.1, SPSS Inc., Chicago) was used.

RESULTS AND DISCUSSION

1. Morphological comparison between two varieties of

Ashwagandha.

In vitro plants were assessed for plant height using 1

mm ruler from base of the stem to apical meristem. Leaf

shape was also observed.

Table 1. Phenotypic differences between Arka

Ashwagandha and JA-20 in in vitro conditions.

Traits Arka Ashwagandha JA -20

Plant height (cm) 6.30 5.50

Number of roots 17 16

Leaf shape Lanceolate Ovate

Leaf base Concave Concave

Leaf tip Obtuse Acute

Table 2. Data represent mean ± standard error of  ve replications in three independent experiments, each

with one explant per treatment. Values followed by the different letters are signi cant P<0.05 according to

Duncan’s Range Multiple Test. Data were scored after 30 days of culture. Growth Conditions: Media- Full

MS liquid medium supplement with different hormone combinations, 3% sucrose, 16 hours photoperiod at

25±2°C.

Treatment

Combination of auxins

mg/l

The quantity of root obtained in gram/100

ml of media after 30 days of culture

Remarks

IAA IBA AA JA-20

Control 0.0 0.0 4.066±0.118f 3.718±0.150f Normal roots

T1 0.25 0.75 8.340±0.098b 7.254±0.135b Fluffy roots

T2 0.50 0.50 6.400±0.139c 6.248±0.072c Fluffy roots

T3 0.75 0.25 5.640±0.083d 5.364±0.068d Fluffy roots

T4 1.0 0.0 4.682±0.086e 4.098±0.114e Fluffy roots

T5 0.0 1.0 9.612±0.088a 8.314±0.138a Fluffy roots

2. In uence of hormone supplementation on the

proliferation of

Withania somnifera

adventitious roots

in suspension culture

For large scale production of useful bioactive metabo-

lites, the use of cell suspension cultures is favored due

to its rapid growth cycles over other kinds of cell culture

methods. Qualitative and quantitative analysis requires

a considerable quantity of cells to determine growth

responses and metabolism of phytochemicals, for these

studies cell suspension cultures are found to be best

suited (Vanishree et al., 2004).

In the current study, the establishment of adventi-

tious root suspension culture of Withania somnifera was

done for two varieties Arka Ashwagandha and JA-20

with different hormone supplementation on media and

further the biomass accumulation and total withanolides

were estimated. Signi cant phenotypic differences were

recorded in the proliferation of root cultures in MS

medium, supplemented with hormones and without

hormones. In the hormone supplemented medium, the

root biomass was higher, but there was no elongation

of lateral roots. The suspension culture media with hor-

mones gave rise to  uffy roots, with callus like exudates

formed around the senescent root tissues and subse-

quently released into the medium which could not be

further multiplied in suspension cultures whereas media

without supplementation of hormones had normal roots

Sindhu Rangaraju, Lokesha and Chenna Reddy Aswath

76 COMPARATIVE ANALYSIS OF PHENOTYPIC TRAITS, ROOT YIELD, AND TOTAL WITHANOLIDES BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

with important factors of proliferation like lateral root

formation, elongation of lateral roots which favored

further multiplication of adventitious roots. Lateral root

formation was essential for rapid growth and higher

biomass production in Rauwol a serpentine L (Pandey

et al., 2010), thus indicating that phytohormone sup-

plementation does not always enhance the regeneration

frequency (Roychowdhury et al., 2013).

Genes involved in the synthesis of auxins are

expressed in roots, which contribute to normal root

growth and maintenance (Petersson et al., 2009). Auxin

biosynthesis genes are expressed at root stem cell nice

to increase the level of auxins (Stepanova et al., 2008).

Signaling pathways for other plant hormones also in u-

ence the auxin response in roots (Kuppusamy et al.,

2008). Though there was higher root mass in media

supplemented with hormones, considering lateral root

formation, proliferation and morphology of roots, MS

media without hormone supplementation was consid-

ered best.

3. In uence of media strength on proliferation of

Withania somnifera

adventitious roots in suspension

cultures

Both half strength MS medium and full strength MS

medium were found to be suitable for biomass produc-

tion of adventitious roots in Panax ginseng, but high-

est secondary metabolite content was induced in full

strength MS medium (Yu et al., 2000). Full strength MS

medium supplemented with 2.0mg/l IBA under contin-

uous agitation increased the biomass of root tissue in

Vernonia amygdalina (Khalaffala et al., 2009). Several

researchers have also reported cell suspension culture

studies in Withania somnifera (Sivanandan et al., 2012b

and Praveen et al., 2011), hence full strength media

without hormone supplementation was considered best

suited for adventitious root multiplication in suspension

culture as it provides more nutrients and reduces the

frequency of subculturing in both varieties of Ashwa-

gandha.

Different types of roots obtained by hormone

treatments in

Withania somnifera

Table 3. Data represent mean ± standard error of  ve replications in three independent experiments, each

with one explant per treatment. Values followed by the different letters are signi cant P<0.05 according to

Duncan’s Range Multiple Test. Data were scored after 30 days of culture. Growth Conditions- Media- Full

MS liquid medium supplement with different hormone combinations, 3% sucrose, 16 hours photoperiod at

25±2°C.

Treatment Arka Ashwagandha JA-20

Half MS 3.684±0.074b 3.520±0.073b

Full MS 4.066±0.066a 3.718±0.054a

FIGURE 1. Different types of roots obtained in

Arka Ashwagandha. A-Control, B-Hormone

treated roots, C-Normal roots obtained after

suspension culture in hormone-free medium,

D-Fluffy roots obtained after suspension culture

in hormone media.

4. Withanolides identi ed and total withanolide content

in leaf and adventitious roots by HPLC analysis

HPLC analysis of Withania somnifera using methanolic

extraction was reported by many researchers like Ganz-

era et al., (2003), Sangwan et al., (2004). The present

study indicated that the roots had higher total witha-

nolide content i.e., 1.621mg/g on a dry weight basis in

Arka ashwagandha, compared to the total withanolide

content in JA-20 root was 1.156 mg/g on a dry weight

basis. Withanolide A which has a biological activity of

sedative and hypnotic was 0.38mg/g on a dry weight

basis in Arka Ashwagandha in comparison to JA 20

Sindhu Rangaraju, Lokesha and Chenna Reddy Aswath

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS COMPARATIVE ANALYSIS OF PHENOTYPIC TRAITS, ROOT YIELD, AND TOTAL WITHANOLIDES 77

Withaferin A contributes to most of the pharmacologi-

cal activity of Withania somnifera with its antibacte-

rial, antifungal, antiarthritic, antitumor and antibiotic

properties. Dalavayi et al., (2006) identi ed Withaferin

A in roots and leaves of Ashwagandha. The Withaferin

A content in Arka Ashwagandha roots was identi ed to

be 1.316mg/g on a dry weight basis compared to JA- 20

with a content of 0.926mg/g on a dry weight basis. The

Withaferin A content in Arka Ashwagandha leaves was

0.1733mg/g on a dry weight basis compared to JA-20

leaves 0.116 mg/g on a dry weight basis.

Madhavi et al (2012) reported that Withaferin A was

not detected in 120 day old hairy root cultures, whereas

it was 136μg /g dry weight in 210 day old hairy root

cultures of Withania somnifera, Sivanandan et al (2012)

reported 0.85mg/g in 40 day old callus cultures of With-

ania somnifera, which is much lesser than the Withaf-

erin A content reported in the present study. Dewir et al

(2010) reported a Withaferin A content of 0.013 mg/g

dry weight in in vitro roots, the present study indicates

100 times increased Withaferin A content.

CONCLUSION

This current study helps in the identi cation of an elite

cultivar of Withania somnifera for mass production of

withaferin A and total withanolides. Field cultivation

of Withania somnifera is a laborious task, as the crop

is prone to diseases like seed rot and blight, harvest-

ing of roots is a tedious task. The provision for alterna-

tive sources of Ashwagandha through cell cultures and

micropropagation must be encouraged as it reduces the

heavy dependence on the wild population to ful ll the

global demand of Withania somnifera. Adventitious root

multiplication through suspension cultures in hormone

free medium provide an easy way for mass production

of useful withanolides and can be used by neutraceuti-

cal and pharmaceutical industries as there would not be

traces of hormones in the end product.

FIGURE 2. Withaferin A production in leaves and

roots of Withania somnifera analyzed by HPLC.

FIGURE 3. Standard HPLC chromatogram of W. som-

nifera root extracts. A. Withanoside IV, B. Withano-

side V, C. Withaferin A, D. 12-Deoxy Withastramon-

olide, E. Withanolide A and F. Withanolide B.

Table 4. Analysis of Withanolides in adventitious roots of Withania somnifera by HPLC Data represents mean ± standard

error of three replications. Values followed by the different letters are signi cant P<0.05 according to Duncan’s Range

Multiple Test. Data were scored after 30 days of culture.

Variety

Withanoside

Withaferin

12-Deoxy

withastromalide

Withanolide

Total

Withanolides

mg/g on dry weight basis

Arka

ashwagandha

root

0.013±0.000

0.025±0.001

1.362±0.032

0.181±0.004

0.038±0.001

0.003±0.000

1.621±0.069

JA-20 root 0.011±0.001

0.037±0.003

0.930±0.005

0.173±0.004

0.028±0.001

0.002±0.000

1.1558±0.013

which has 0.28mg/g on a dry weight basis. Madhavi et

al (2012) reported a Withanolide A content of 136 μg/g

dry weight in 120 days old hairy root culture and 13μg/g

dry weight in 210 days old hairy root culture of Witha-

nia somnifera, Dewin et al., (2010) reported Withanolide

A content of 0.019 mg/g in in vitro roots of Withania

somnifera which is much lesser than the Withanolide A

content reported in the present study. The methanolic

extract of Withania somnifera has GABA mimetic activ-

ity, anti-in ammatory and anti-stress properties (Mir

et al., 2012).

Comparison of Withaferin A content between adventitious roots and

in vitro

leaves in

Withania somnifera

Sindhu Rangaraju, Lokesha and Chenna Reddy Aswath

78 COMPARATIVE ANALYSIS OF PHENOTYPIC TRAITS, ROOT YIELD, AND TOTAL WITHANOLIDES BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

ACKNOWLEDGMENTS

This research was conducted at ICAR- Indian Institute of

Horticulture Research and was funded by the Department

of Science and Technology through DST Inspire fellowship.

The  rst author also acknowledges Centre for postgradu-

ate study, Jain University for conducting this research.

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