Industrial Crops and Products 35 (2012) 309312

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Industrial Crops and Products

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Growth and centelloside production in hydroponically established medicinal plant-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 Scientic & Industrial Research, PO CIMAP, Lucknow, Uttar Pradesh 226015, India Division of Analytical Chemistry, Central Institute of Medicinal & Aromatic Plants, Council of Scientic & Industrial Research, PO CIMAP, Lucknow 226015, India c Division of Plant Physiology, Central Institute of Medicinal & Aromatic Plants, Council of Scientic & Industrial Research, PO CIMAP, Lucknow 226015, India
b

a r t i c l e

i n f o

a b s t r a c t
Conditions to cultivate medicinally important herb Centella asiatica in hydroponic system are reported here for the rst 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 11 mg, 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.

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

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, varicose veins, ulcer, lupus and certain eczemas (Mathur et al., 2007). The plant contains many bioactive constituents, such as triterpenes glycosides and their respective genins, avonoids and phenols. Amongst these, the triterpene centellosides such as asiaticoside, madecassoside, asiatic acid and madecassic acid, are already in clinical 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 bioactive constituents. In India, Centella is harvested from the ditches that are generally contaminated with heavy metals, unacceptable microbial loads (including moulds and yeasts), excess dirt and 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

growth of the International market for C. asiatica has created a need for establishing more sustainable and economically viable production strategies for this herb and its pure bioactive molecules (McCaleb et al., 2000). The present study has got its genesis in the backdrop of these considerations and specically 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 dened 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 heterotrophically grown in vitro cultures, hydroponically cultivated plants are autotrophic and do not require external sugar or growth hormone supplements that are known to down-regulate plant secondary metabolism (Huttner and Dudy, 2003).

2. Materials and methods 2.1. Plant material

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). 0926-6690/$ see front matter 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2011.06.020

Plants of C. asiatica (L.) were obtained from Bhowali (Uttarakhand), India and established in soil under glass house (25 5 C) and eld environment at CIMAP, Lucknow. Rooted cuttings with

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A. Prasad et al. / Industrial Crops and Products 35 (2012) 309312

12 nodes from these stock plants were then grown in acid-washed silica sand for 2 weeks (Agarwala and Sharma, 1961). After 2 weeks of growth the uniform rooted cuttings (on the basis of leaf number and leaf size) were selected and transferred into 2 L hydroponic jar containing half-strength modied Hogland and Arnons nutrient 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 ow rate of 1.29 105 m3 s1 (46.44 L/h). The nutrient solution during cultivation cycle of 70 days was replaced with fresh solution every 10th day to avoid algal growth. For growth measurement, the rooted plants were periodically harvested every 7th day and their fresh and dry matter accumulation was measured. The biomass increment was calculated in term of growth index (GI = % increase over the initial weight). Mean performance of 10 plants per treatment was tabulated and the experiment was repeated thrice. The data is expressed as mean performance of replicates along with their standard deviation using a two-way ANOVA method of multiple comparison based on LSD test (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 separated 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. 200 mg of dried leaf powder of each treatment was soaked overnight in 80% methanol (3 20 mL) for 36 h and ltered 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 analysis. Quantitative analysis of centellosides was performed using a Waters modular HPLC system (Waters, Milford, USA) consisting of 2996 photo diode array detector, 600 E pump, 717 autosampler and C18 column (150 mm 4.6 mm i.d.; 3.5 m). 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 carried 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 ow rate was kept constant at 1 mL/min up to 25 min. After 30 min, ow rate was increased to 1.2 mL/min while the solvent composition was maintained at 10% A. After 35 min the initial conditions were restored. 10 L of sample was injected for each analysis and detection was done at 206 nm. The peaks were identied by co-injecting respective standards, i.e. madecassoside, asiaticoside, asiatic acid and madecassic acid. Reference compounds of asiaticoside, asiatic acid and madecassoside, madecassic acid were purchased from Fluka Analytical, France, SigmaAldrich, USA and ChromaDex Ltd., USA, respectively. 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 rst 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 evident from 56th day onward. The accumulation patterns of 4 major bioactive constituents of C. asiatica namely madecassoside, asiaticoside, 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 significant rise on 42nd day (11.0 mg g1 dry weight) and an equally steep fall in following weeks. Asiaticoside level in the leaves varied from 0.2 to 0.7 mg g1 dry weight till 28th day of growth, followed by its highest accumulation between 35th and 42nd day of culture (1.7 mg g1 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 consistent fall from 46.2 to 17.0 mg g1 dry weight during rst 4 weeks of growth, followed by an increase during 3542 days (28.8 and 36.6 mg g1 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 (3.36.3 mg g1 dry weight) followed by a decline in aging cultures. Cummulative data of these experiments have suggested that a cultivation period of 67 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% madecassoside, 0.17% asiaticoside, 3.6% madecassic acid and 0.63% asiatic acid). The concentration of various centellosides in 3542 days old hydroponically grown plants was also found comparable with those of eld-grown plants of C. asiatica under Lucknow conditions (data not shown). Interestingly, while eld cultivated plants are known to show high seasonal uctuations in their centelloside 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 inuences on triterpenoids content and composition in C. asiatica, Randriamampionona et al. (2007) have shown that plants samples collected from different locations in Madagascar signicantly differed in their growth and metabolite production under both in vivo and in vitro environments. The highest asiaticoside and madecassoside levels (6.42 and 5.89% dry weight, respectively) with a total triterpenoids content of 12.69% dry weight were detected in eld collections made from Mangoro region of Madagascar. This genotype maintained its highest metabolite production capacity (3.83% dry weight total triterpenoids with 1.78 and 1.40% dry weight asiaticoside 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. asiatica, 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 content on dry weight basis, therefore, provides an alternate mechanism to fulll this quality criteria with further renements 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 congeners. 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 suspensions and plantlet cultures in C. asiatica (Kim et al., 2004, 2007; Kiong et al., 2005; Mangas et al., 2006, 2008; Bonll et al., 2011). Though did not constitute a part of present study it will be interesting to test if the recovery of centellosides from hydroponically grown plants can be further improved by employing the recently reported modied extraction procedure of Kim et al. (2009) using

A. Prasad et al. / Industrial Crops and Products 35 (2012) 309312 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 (mg g1 dry wt.)

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Madecassoside 7 14 21 28 35 42 49 56 63 70 0.843 1.025 1.319 1.731 1.849 1.997 2.115 1.246 1.182 0.847 0.254c 0.444 0.802 1.077 1.021 0.828 1.328 1.23 0.381 0.521 0.4327 0.4627 0.641 0.6611 0.7490 0.7709 0.3527 0.3486 0.2941 0.1943 0.106 0.131 0.159 0.192 0.178 0.206 0.124 0.122 0.103 0.007 16.23 40.42 74.94 131.541 132.51 156.30 122.54 106.08 98.48 51.64 3.09 13.89 13.70 14.0 33.92 8.76 16.45 11.08 16.54 4.28 3.5 0.7 0.3 0.2 0.1 11.0 0.2 0.4 0.6 0.4

Asiaticoside 0.3 0.2 0.2 0.7 1.0 1.7 0.6 0.2 0.2 0.1

Madecassic acid 46.2 30.8 23 17 28.8 36.6 18 6.2 6.2 4.2

Asiatic acid 4.4 3.4 4.1 3.4 5.0 6.3 3.3 1.5 1.8 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 Replications Treatments Error
**

Dry weight 0.041 0.122** 0.018

Growth index 290.564 6152.654** 239.354

9 9 81

0.316 0.653** 0.155

P < 0.01. a Initial inoculum fresh weight in various treatments varied from 600 to 800 mg. b % increment in fresh biomass accumulation over the initial inoculum weight. c Values are 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 reference compounds 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 hydroponic culture approach will provide a novel production platform for this important medicinal herb with better chemoprole of in-demand centellosides.

Acknowledgements This study demonstrates for the rst time the feasibility of cultivating C. asiatica through hydroponics approach to address/eliminate the problem of heavy metal and microbial contamination in the wild or eld grown materials. Quality biomass with high centellosides content could be obtained in such plants within a short culture cycle of just 42 days. Further renements in The present work is being carried under a sponsored project grant no. SR/SO/Ps-28/07 of the Department of Science and Technology, New Delhi (India). The authors are also grateful to Director CIMAP, Lucknow and Council of Scientic and Industrial Research, New Delhi for providing the necessary facilities.

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A. Prasad et al. / Industrial Crops and Products 35 (2012) 309312 Mangas, S., Bonll, M., Osuna, L., Moyano, E., Tortoriello, J., Cusid, R.M., Pinol, M.T., Palazn, J., 2006. The effect of methyl jasmonate on triterpene and sterol metabolisms of Centella asiatica, Ruscus aculeatus and Galphimia glauca cultured plants. Phytochemistry 67, 20412049. Mangas, S., Moyano, E., Osuna, L., Cusid, R.M., Bonll, M., Palazon, J., 2008. Triterpenoid saponin content and the expression level of some related genes in calli of Centella asiatica. Biotechnol. Lett. 30, 1853 1859. Mathur, A., Mathur, A.K., Yadav, S., Verma, P., 2007. Centella asiatica (L.) Urbanstatus and scope for commercial cultivation. J. Med. Arom. Plant Sci. 129, 151162. McCaleb, R., Morien, K., Schott, T., 2000. Market Report on Herbs and Spices. Herb Research Foundation, Boulder, CO, USA. Randriamampionona, D., Diallo, B., Rakotoniriana, F., Rabemanantsoa, C., Cheuk, K., Corbisier, A.M., Mahillion, J., Ratsimamanga, S., Jaziri, M.E.J., 2007. Comparative analysis of active constituents in Centella asiatica samples from Madagascar: application for ex situ conservation and clonal propagation. Fitoterapia 78, 482489. Schaneberg, B.T., Mikell, J.R., Bedir, E., Khan, I.A., 2003. An improved HPLC method for quantitative determination of six triterpenes in Centella asiatica extracts and commercial products. Pharmazie 58 (6), 381384. Skopinska-Rozewska, E., Furmanowa, M., Guzewska, J., Sokolnicka, I., Sommer, E., Bany, J., 2002. The effect of Centella asiatica, Echinacea purpurea and Melaleuca alternifolia on cellular immunity in mice. Cent. Eur. J. Immunol. 27, 142 148.

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