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Industrial Crops and Products 35 (2012) 309–312

Contents lists available at ScienceDirect

Industrial Crops and Products

 journal homepage: www.elsevier .com/ locate / indcrop

Short communication

Growth and centelloside production in hydroponically established medicinalplant-Centella asiatica (L.)

Archana Prasad a, V.S. Pragadheesh b, Archana Mathur a,∗, N.K. Srivastava c, Manju Singh b, A.K. Mathur a

a Division of Plant Biotechnology, Central Institute of Medicinal & Aromatic Plants, Council of Scientific & Industrial Research,PO CIMAP,Lucknow, Uttar Pradesh 226015, Indiab Division of Analytical Chemistry, Central Institute of Medicinal & Aromatic Plants, Council of Scientific & Industrial Research,PO CIMAP,Lucknow 226015, Indiac Division of Plant Physiology, Central Institute of Medicinal & Aromatic Plants, Council of Scientific & Industrial Research,PO CIMAP, Lucknow 226015, India

a r t i c l e i n f o

 Article history:Received 28 February 2011

Received in revised form 15 June 2011

Accepted 16 June 2011

Available online 12 July 2011

Keywords:

C. asiatica

Centelloside

Growth kinetics

Hydroponic cultures

a b s t r a c t

Conditions to cultivate medicinally important herb Centella asiatica in hydroponic system are reported

here for the first time. Growth kinetics of hydroponically grown plants was monitored over a period of 70

days. The maximum growth and dry matter accumulation (156.3% increment over the initial inoculum

weight) in the cultured plants occurred around 42nd day. High Performance Liquid Chromatography

(HPLC) analysis of the bioactive centellosides in the crude triterpenoids extract of the harvested leaves

showed the presence of  11mg, 1.7 mg, 36.6 mg and 6.3 mg of  madecassoside, asiaticoside, madecassic

acid and asiatic acid on per gram dry weight basis, respectively. The results of this study suggest that the

cultivation of C. asiatica in hydroponic systems can be an effective platform for the production of clean

and good quality C. asiatica herb for the pharmaceutical companies.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Plants are valuable source of phytochemicals, many of which

are used as additives in functional foods or bioactive components

in pharmaceutical preparations. Centella asiatica (L.) Urban, is a

prostrate, faintly aromatic, stoloniferous perennial medicinal herb

belonging to the Family Apiaceae. It is valued in the traditional

systems of medicine for the treatment of Alzheimer, leprosy, vari-

cose veins, ulcer, lupus and certain eczemas (Mathur et al., 2007).

The plant contains manybioactive constituents,such as triterpenes

glycosides and their respective genins, flavonoids and phenols.

Amongst these, the triterpene centellosides such as asiaticoside,

madecassoside, asiatic acidand madecassicacid, are already in clin-

ical usage (Skopinska-Rozewska et al., 2002). At present most of 

the C. asiatica material collected from the wild for pharmaceutical

companies is of very poor quality in terms of its purity and bioac-tive constituents. In India, Centella is harvested from the ditches

that are generally contaminated with heavy metals, unacceptable

microbialloads (including moulds andyeasts),excess dirtand other

harmful chemicals due to which the raw plant material frequently

fail to obtain quality clearance as per raw herb purity guidelines of 

WHO (http://www.greenbush.net/gotukolanotes.html). The rapid

∗ Corresponding author. Tel.: +91 522 235 9623/7134;

fax: +91 522 234 2666; mobile: +91 9793196005.

E-mail addresses: a.mathur@cimap.res.in,

archnacimap@yahoo.co.in (A. Mathur).

growth of the International market for C. asiatica has created a

need for establishing more sustainable and economically viableproduction strategies forthis herb andits pure bioactive molecules

(McCaleb et al., 2000). The present study has got its genesis in

the backdrop of these considerations and specifically aimed to

standardise and exploit hydroponic production technology for the

cultivation of C. asiatica. So far no report on hydroponic culture of 

this plant exists in literature. Hydroponic culture-based cultivation

technology can provide many advantages, such as a more defined

and reproducible production system under control conditions as

compared to the plants grown in soil, the improved quality of the

raw material for industrial processing and, predictable metabolites

yield. The hydroponics-based cultivation besides saving upon time

can also provide ease in applying biotic and abiotic elicitors for

hyper-expression of a targeted metabolite pathway. Unlike het-

erotrophically grown in vitro cultures, hydroponically cultivatedplants are autotrophic and do not require external sugar or growth

hormone supplements that are known to down-regulate plant sec-

ondary metabolism (Huttner and Dudy, 2003).

2. Materials and methods

 2.1. Plant material

Plants of C. asiatica (L.) were obtained from Bhowali (Uttarak-

hand), India and established in soil under glass house (25±5 ◦C)

and field environment at CIMAP, Lucknow. Rooted cuttings with

0926-6690/$ – seefrontmatter © 2011 Elsevier B.V. All rights reserved.

doi:10.1016/j.indcrop.2011.06.020

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310 A. Prasadet al./ Industrial Crops and Products 35 (2012) 309–312

1–2 nodesfrom thesestockplants were then grownin acid-washed

silicasandfor 2 weeks (Agarwala and Sharma, 1961). After 2 weeks

of growth the uniform rooted cuttings (on the basis of leaf num-

berand leaf size) were selected andtransferred into 2 L hydroponic

 jar containing half-strength modified Hogland and Arnon’s nutri-

ent solution (Hogland and Arnon, 1938). The pH of the nutrient

solution was adjusted to 6.2±0.4. Each jar contained three rooted

plants. After 2 weeks of acclimatization, the nutrient solution was

replaced with full-strength Hogland and Arnon salt solution and

intermittently aerated at an air flow rate of 1.29×10−5 m3 s−1

(46.44 L/h). The nutrientsolutionduringcultivationcycle of 70 days

was replaced with fresh solution every 10th day to avoid algal

growth. For growth measurement, the rooted plants were peri-

odically harvested every 7th day and their fresh and dry matter

accumulation was measured. The biomass increment was calcu-

lated in term of growth index (GI = % increase over the initial

weight). Mean performance of 10 plants per treatment was tabu-

latedand theexperiment wasrepeatedthrice.The datais expressed

as mean performance of replicates along withtheir standard devia-

tionusing a two-wayANOVA method of multiplecomparison based

on LSDtest (P < 0.01).

 2.2. Extraction and analysis of centellosides

For qualitative and quantitative analysis of the centellosides in

the hydroponically grown plants, the leaves from all the replicated

plant samples of different day harvests were individually sepa-

rated and lyophilized (Labconco, Free Zone 2.5, USA). After the

determination of dry biomass yield of each sample, the lyophilized

leaves of each treatment were pooled and powdered. 200mg of 

dried leaf powder of each treatment was soaked overnight in 80%

methanol (3× 20mL) for 36h and filtered through a Whatman

paper Grade-1. The methanolic fraction was defatted with equal

amount of hexane and the samples were concentrated in vacuum

(BUCHI, vacuum controller V-850, Switzerland) for further analy-

sis. Quantitative analysis of centellosides was performed using a

Waters modular HPLC system (Waters, Milford, USA) consistingof 2996 photo diode array detector, 600 E pump, 717 autosam-

pler and C18 column (150 mm×4.6 mm i.d.; 3.5m). The gradient

elution was performed by using solvent system-A comprising

water:acetonitrile:methanol:acetic acid (70:10:20:0.15, v/v/v/v),

and solvent-B comprising water:acetonitrile:methanol:acetic acid

(10:50:40:0.15, v/v/v/v). A linear gradient programming was car-

ried out at 27 ◦C with initial composition of 100% A, changing

to 80% A at 5.0 min, changing to 10% A at 25.0 min, while

flow rate was kept constant at 1 mL/min up to 25 min. After

30 m in, flow rate was increased to 1.2 mL/min while the sol-

vent composition was maintained at 10% A. After 35min the

initial conditions were restored. 10L of sample was injected

for each analysis and detection was done at 206 nm. The

peaks were identified by co-injecting respective standards, i.e.

madecassoside, asiaticoside, asiatic acid and madecassic acid.

Reference compounds of asiaticoside, asiatic acid and madecas-

soside, madecassic acid were purchased from Fluka Analytical,

France, Sigma–Aldrich, USA and ChromaDex Ltd., USA, respec-

tively.

3. Results and discussion

Growth and metabolite production kinetics data collected at

7 day intervals through a 70 day long cycle of hydroponically

grown plants of C. asiatica (Table 1, Fig. 1) revealed that after an

initial lag during first 2 weeks of culture, the plants entered in

exponential growth phase from 21st day (GI= 74.94) onwards and

acquired highest biomass (GI = 156.3) around 42nd day of culture.

Fall in biomass and subsequent sign of senescence became evi-

dent from 56th day onward. The accumulation patterns of 4 major

bioactive constituents of C. asiatica namely madecassoside, asiati-

coside, madecassic acid and asiatic acid were also found to vary

in a age-dependent manner in these hydroponically grown plants.

Concentration of madecassoside in the harvested leaves showed

a steady decline up to 35th day of growth followed by a signif-

icant rise on 42nd day (11.0mg g−1 dry weight) and an equally

steep fall in following weeks. Asiaticoside level in the leaves varied

from 0.2 to 0.7 mgg−1 dry weight till 28th day of growth, followed

by its highest accumulation between 35th and 42nd day of cul-

ture (1.7mg g−1 dry weight). Madecassic acid which constituted

the major component of the crude saponin+ sapogenic mixture

through out the 70 days long growth cycle, registered a consis-

tent fall from 46.2 to 17.0mgg−1 dry weight during first 4 weeks

of growth, followed by an increase during 35–42 days (28.8 and

36.6mg g−1 dry weight) and a subsequent decline again. Asiatic

acid concentration in the leaves was also steadily maintained in

the hydroponically cultivated plants during the initial 7 weeks

of growth (3.3–6.3mg g−1 dry weight) followed by a decline in

aging cultures. Cummulative data of these experiments have sug-

gested that a cultivation period of 6–7 weeks under hydroponic

conditions can be adopted for the production of C. asiatica herb

with a consistent yield of 5.5% total triterpenoids (1.1% madecas-soside, 0.17% asiaticoside, 3.6% madecassic acid and 0.63% asiatic

acid). The concentration of various centellosides in 35–42 days

old hydroponically grown plants was also found comparable with

those of field-grown plants of C. asiatica under Lucknow condi-

tions (data not shown). Interestingly, while field cultivated plants

are known to show high seasonal fluctuations in their centel-

loside content, the hydroponically grown plants showed a steady

quantitative and qualitative yields of these bioactives. Earlier in a

detailed study to assess the genotypic and environmental influ-

ences on triterpenoids content and composition in C. asiatica,

Randriamampionona et al. (2007) have shown that plants samples

collected from different locations in Madagascar significantly dif-

fered in their growthand metabolite production under both in vivo

and in vitro environments. The highest asiaticoside and madecas-soside levels (6.42 and 5.89% dry weight, respectively) with a total

triterpenoids content of 12.69% dry weight were detected in field

collections made from Mangoro region of Madagascar. This geno-

type maintained its highest metabolite production capacity (3.83%

dry weight total triterpenoids with 1.78 and 1.40% dry weight asi-

aticoside and madecassoside, respectively) over the samples of 

six other regions when grown as rooted plantlets under in vitro

conditions. Though the total triterpenoids levels detected in this

Mangoro collection are still the highest reported values in C. asi-

atica, the European Pharmacopoeia considers a total triterpenoids

level of around 6.0% dry weight to be a good quality parameter

for this herb. The nutrient and growth cycle parameters employed

for the hydroponic cultivation of C. asiatica in our study allowed

the biomass production with a total of 5.5% triterpenoids contenton dry weight basis, therefore, provides an alternate mechanism

to fulfill this quality criteria with further refinements in growth

conditions. It is pertinent to mention here that Das and Mallick

(1991) have earlier reported that Indian cultivars generally had

three to seven times less centellosides than their Madagascar con-

geners. The triterpenoids yield obtained in the present study are

either comparable or better than reported values in other in vivo

(Schaneberg et al., 2003) or in vitro studies on callus, cell suspen-

sions and plantlet cultures in C. asiatica (Kim et al., 2004, 2007;

Kiong et al., 2005; Mangas et al., 2006, 2008; Bonfill et al., 2011).

Though did not constitute a part of present study it will be inter-

esting to test if the recovery of centellosides from hydroponically

grown plants can be further improved by employing the recently

reported modified extraction procedure of Kim et al. (2009) using

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 A. Prasad et al. / Industrial Crops and Products 35 (2012) 309–312 311

 Table 1

Growth kinetics and centelloside production in hydroponically grown plants of C. asiatica.

Culture age

(days)

Fresh wt. of whole

plant (g)a

Dry wt. of whole

plant (g)

Growth indexb Centelloside content in leaves (mgg−1 drywt.)

Madecassoside Asiaticoside Madecassic a cid Asiatic a cid

7 0.843 ± 0.254c 0.4327 ± 0.106 16.23 ± 3.09 3.5 0.3 46.2 4.4

14 1.025 ± 0.444 0.4627 ± 0.131 40.42 ± 13.89 0.7 0.2 30.8 3.4

21 1.319 ± 0.802 0.641 ± 0.159 74.94 ± 13.70 0.3 0.2 23 4.1

28 1.731 ± 1.077 0.6611 ± 0.192 131.541 ± 14.0 0.2 0.7 17 3.4

35 1.849 ± 1.021 0.7490 ± 0.178 132.51 ± 33.92 0.1 1.0 28.8 5.0

42 1.997 ± 0.828 0.7709 ± 0.206 156.30 ± 8.76 11.0 1.7 36.6 6.3

49 2.115 ± 1.328 0.3527 ± 0.124 122.54 ± 16.45 0.2 0.6 18 3.3

56 1.246 ± 1.23 0.3486 ± 0.122 106.08 ± 11.08 0.4 0.2 6.2 1.5

63 1.182 ± 0.381 0.2941 ± 0.103 98.48 ± 16.54 0.6 0.2 6.2 1.8

70 0.847 ± 0.521 0.1943 ± 0.007 51.64 ± 4.28 0.4 0.1 4.2 1.0

Analysis of variance (ANOVA) using RBD, replicates-10, treatments-10 for the three parameters studied

Sources of variation Degree of freedom Mean sum of squares

Fresh weight Dry weight Growth index

Replications 9 0.316 0.041 290.564

Treatments 9 0.653** 0.122** 6152.654**

Error 81 0.155 0.018 239.354

**P < 0.01.a

Initial inoculum fresh weight in various treatments varied from 600 to 800mg.b % incrementin fresh biomass accumulation over theinitial inoculum weight.c Valuesare mean±S.D. (n =10).

Fig. 1. Cultivation of C. asiatica in hydroponics culture. (A) 42 days old hydroponic culture. (B) HPLC chromatogram of leaf extract of a 42 days old culture. (C) HPLC

chromatogram of referencecompounds madecassoside (1), asiaticoside (2), madecassic acid (3)and asiatic acid(4).

the subcritical water as a extraction solvent in place of organic

solvents.

4. Conclusions

This study demonstrates for the first time the feasibil-

ity of cultivating C. asiatica through hydroponics approach to

address/eliminate the problem of heavy metal and microbial con-

tamination in the wild or field grown materials. Quality biomass

with high centellosides content could be obtained in such plants

within a short culture cycle of just 42 days. Further refinements in

this hydroponic culture approach will provide a novel production

platform for this important medicinal herb with better chemo-

profile of in-demand centellosides.

 Acknowledgements

The present work is being carried under a sponsored project

grant no. SR/SO/Ps-28/07 of the Department of Science and Tech-

nology, New Delhi (India). The authors are also grateful to Director

CIMAP, Lucknow and Council of Scientific and Industrial Research,

New Delhi for providing the necessary facilities.

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