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Meri-Rastilantie 3 B, FI-00980 Journal of Food, Agriculture & Environment Vol.9 (1): 360-363. 2011 www.world-food.net Helsinki, Finland e-mail: [email protected]

Effects of light intensity on growth and accumulation of triterpenoids in three accessions of Asiatic pennywort (Centella asiatica (L.) Urb.)

Jirapan Srithongkul 1, Sirichai Kanlayanarat 1*, Varit Srilaong 1, Apiradee Uthairatanakij 1 and Piya Chalermglin 2 1 Division of Postharvest Technology, School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi, 126 Pracha-Uthid Road, Bangmod,Thungkru, 10140, . 2 Thailand Institute of Scientific and Technological Research, Pathum Thani, 10120, Thailand. *e-mail: [email protected]

Received 20 October 2010, accepted 4 January 2011.

Abstract This study deals with the effects of light and accessions on leaf area, petiole length and accumulation of asiaticoside, madecassoside, asiatic acid and madecassic acid that were studied in three accessions of Asiatic pennywort (Centella asiatica (L.) Urb.). The accessions of C. asiatica, Nakon Si Thammarat, and Ubon Ratchathani, were collected from three different locations in Thailand and planted at the Thailand Institute of Scientific and Technological Research in Pathum Thani . They were grown for 90 days in replicated organic culture system under different light intensities expressed as photosynthesis photon flux densities (PPFD) (933.1 (full sun light), 362.5 and 93.3 µmol m-2s-1, respectively). Light intensities and accessions affected leaf area, petiole length, fresh and dry weight, asiaticoside, madecassoside, asiatic acid and madecassic acid content. At high light intensity, leaf area and petiole length were smaller than at low light intensity in each accession. The accession had the largest leaf area followed by the Ubon Ratchathani and Rayong accessions, respectively, while the longest petioles were found in Rayong, and Ubon Ratchathani had the highest fresh weight per unit area. Dry weight per kg fresh weight did not differ with genotype. With decreasing light intensity, there was increase in leaf area and petiole length, which differed with genotype, but decrease in fresh and dry weights, which did not vary much with genotype. Triterpenoid analysis by High Performance Liquid Chromatography (HPLC) revealed varying genotypic responses to light intensity. Asiaticoside and madecassoside contents were highest in Ubon Ratchathani and comparably lower in Nakhon Si Thammarat and Rayong. Low light intensity reduced the content of both triterpenoids in all genotypes, with Ubon Ratchathani exhibiting more pronounced decreases. Asiatic acid content did not differ with genotype while madecassic acid was highest in Nakhon Si Thammarat followed by that of Ubon Ratchathani and Rayong, respectively. With decreasing light incidence, asiatic acid content decreased in Nakhon Si Thammarat only under full sunlight (PPFD value 93.3 µmol m-2s-1) while in Ubon Ratchathani, under both 50% and 80% shading (PPFD value 362.5 and 933.1 µmol m-2s-1). Madecassic acid contents showed almost similar response to low light intensities. Contrastingly in Rayong, asiatic and madecassic acid contents remarkably increased under 50% shading (PPFD value 362.5 µmol m-2s-1). The results demonstrate genotypic variations in growth characteristics and triterpenoid contents and the detrimental effect of low light intensity on contents of asiaticoside and madecassoside, the main pharmacologically active triterpenoids of C. asiatica.

Key words: Centella asiatica, accessions, light intensity, leaf area, triterpenoids asiaticoside, madecassoside, medicinal compounds.

Introduction The Asiatic pennywort (Centella asiatica (L.) Urban) belongs to their biological activity 14. The sugars in the side chains vary both the family Umbelliferae that is commonly found in tropical and in type and number. The substances of therapeutic interest are the subtropical countries. The plant is creeping, perennial and saponins that contain triterpene acids and their sugar esters 16. flourishes in damp, moist and shady habitats. It is propagated Terpenoids in C. asiatica include asiaticoside, centelloside, sexually and vegetatively by seeds and stolons, respectively. madecassoside, brahmoside, brahminoside, thankuniside, Roots are produced at nodes, leaves are reniform-cordate and the sceffoleoside, cetellose, and asiatic, brahmic, cetellic and flowers are simple umbels and sessile 4. The C. asiatica is used as madecassic acids 1,12. The main active principals of C. asiatica a vegetable and is also an important herb. It has been used as a are two glycosides; asiaticoside and madecassoside 6, 10 (Fig. 1). medicinal herb since ancient times. Both the leaves and the entire These compounds have antibacterial, fungicidal and cell plants can be used therapeutically. It has been utilized for centuries proliferative activities which have been shown to aid in the treatment in Ayurvedic medicine to alleviate symptoms of anxiety 17 and to of wounds 13, ulcers, various skin diseases, vein insufficiency, promote fibroblast proliferation and collagen synthesis 3. tuberculosis and in the treatment of mental disorders 8. Analytical studies have shown that C. asiatica contains The pharmaceutical industry requires C. asiatica with a high triterpenoids 7. The triterpenoid structure (aglycone) of saponins content of the active compounds. The content of bio-active is hydrophobic but these compounds are normally present in plants compounds in medicinal species is influenced by both genetic with a hydrophilic sugar side chain (glycone) that contributes to and environmental factors. Thus, the environmental growing

360 Journal of Food, Agriculture & Environment, Vol.9 (1), January 2011 a b c

Asiaticoside Asiatic acid Figure 2. Three accessions of C. asiatica: Rayong (a), Nakhon Si Thammarat (b), and Ubon Ratchathani (c).

Determination of leaf area, petiole length, fresh and dry weight: The leaves and petioles were harvested from 1 square metre to determine the fresh weight. The length of the petioles was measured and the leaf area (cm2) of plants from each plot with a Li- Cor meter (LI-3100, LI-COR Inc., USA). One kg fresh weight from Madecassoside Madecassic acid each treatment (accession and light intensity) was dried in oven Figure 1. Structure of asiaticoside, asiatic acid, at 50°C. madecassoside and madecassic acid 6. Determination of asiaticoside, madecassoside, asiatic acid and conditions should be optimized to maximize synthesis of madecassic acid: The quantitative determination of asiaticoside, pharmacologically active constituents. However, the limits of such madecassoside, asiatic acid and madecassic acid in C. asiatica biosynthesis will always be determined by the genetic capacity of was done by high performance liquid chromatography (HPLC) 15. the plant 11. There are some relevant reports that studied factor of Dry tissue was ground with a milling machine to a powder and 5 g accumulation of asiaticoside, madecassoside, asiatic acid and of powder were extracted with 90 ml of 80% methanol in water at madecassic acid in C. asiatica. The asiaticoside, madecassoside, 60°C for 1.5 hours. The extracts were cooled to room temperature, asiatic acid and madecassic acid content are higher in plants grown filtered by vacuum suction through a filter paper and adjusted to in the field than in vitro 1,10, but the fringed leaf phenotype of C. 100 ml with deionized water. An aliquot (5 ml) of each extract was asiatica grown in a glasshouse in Malaysia had higher asiaticoside mixed with 1.0 ml of internal standard solution (10.0 mg and madecassoside concentrations than plants grown in the field 1. prednisolone was dissolved in methanol and adjusted volume to In C. asiatica of Korean origin, the asiaticoside content in micro- 25.0 ml) and adjusted to 10.0 ml with the mobile phase used for propagated shoots was only 50% of that of field grown material 2. HPLC (acetonitrile: phosphate buffer, 10 mM, pH 7.1(29:71)). Light intensity is an important environmental factor that affects Samples (2 ml) of these mixtures were loaded onto a 500 mg C18 plant growth, its development and synthesis of secondary SPE cartridge. Impurities in the extracts were removed by washing metabolites 8. C. asiatica growth was optimum in sandy loam the cartridge with 3 ml acetonitrile:water (1:9). Material retained soils and at high soil moisture levels 17. Both vegetative growth on the cartridges was eluted with six cycles of 0.5 ml of the mixture and flower production were favored under intense light, while of acetonitrile: phosphate buffer, pH 7.1 (45:55). The combined growth of the mother rosette was relatively greater when the plants eluates were injected into the HPLC, which was fitted with an were grown under shade 17. Alltech Alltima column (C 18, 5 mm diameter particle size; 4.6 × 150 The objective of this research was to study the effects of light mm). The mobile phase was acetonitrile-phosphate buffer (29:71) intensity and accessions of C. asiatica on yields and the content with a flow rate of 1 ml min-1. The detector was photo-diode array of asiaticoside, madecassoside, asiatic acid and madecassic acid at 210 nm, and the peak area of triterpenes was also calculated. in the leaves with petioles. % purity = (Au × Ws × %purity of reference standard)/(As ×Wu) Materials and Methods Plant material: Plant cuttings of three accessions of C. asiatica where Au = peak area of sample solution; As = peak area of (L.) Urban were obtained from different locations in Thailand and standard solution; Wu = weight of sample (mg); Ws = weight of grown at the Thailand Institute of Scientific and Technological standard (mg). Research (TISTR), in Pathum Thani Province. The accessions were Nakhon Si Thammarat, Rayong and Ubon Ratchathani. The Statistics: A 3 × 3 factorial completely randomized design with phenotypes were as follows: Nakhon Si Thammarat had thick and three replications was used. All data points represent the means slightly green leaves, green stolons and white flowers, Rayong and ± (SE) of three replications. Analysis of variance and mean Ubon Ratchathni had purple-red stolons and purple-red flowers comparison by Duncan’s Multiple Range Test (DMRT) were (Fig. 2). The plants were transplanted in Rungsit Series soil, in full performed using SAS software sunlight and under black shade cloth with 50 and 20% light transmission; photosynthesis photon flux densities (PPFD) were Results and Discussion 933.1, 362.5 and 93.3 µmol m-2s-1, respectively. For each treatment, Growth characteristics: Genotype and light intensity significantly 25 cuttings per square metre with three replications of each influenced growth of C. asiatica but their interaction effects were not accession were grown. Leaves and petioles were harvested after 90 significant (Table 1). Under PPFD value 933.1 µmol m-2s-1 (full sun days at temperatures of 24-27°C. Relative humidity ranged 70-85%. light), Nakhon Si Thammarat had the largest leaf area while Ubon

Journal of Food, Agriculture & Environment, Vol.9 (1), January 2011 361 Table 1. Effects of PPFD on leaf area, petiole length, fresh and dry weight of three and 93.3 µmol m-2s-1) on these two triterpenoids accessions of C. asiatica. depended on the genotype which accounted PPFD Leaf area Petiole Fresh weight Dry weight for the significant interaction effect of Accession (µmol m-2s-1) (cm2) length (cm) (kg m-2) (g m-2) genotype and light intensity. In Rayong, both Nakhon Si 933.1 11.21 bc 3.94 a 2.78 d 430.70 g low light intensities had comparably reducing Thammarat 362.6 13.87 d 5.75 b 1.45 b 213.88 f effect on asiaticoside and madecassoside 93.3 14.73 d 11.15 c 0.83 a 100.32 ab Rayong 933.1 6.79 a 7.33 b 2.02 c 305.94 e contents while in Ubon Ratchathani and 362.6 9.90 b 9.80 c 1.22 ab 168.13 b Nakhon Si Thammarat, asiaticoside and 93.3 12.87 cd 14.7 d 0.69 a 82.36 a madecassoside contents correspondingly Ubon Ratchathani 933.1 7.45 a 5.30 a 3.50 e 526.96 g decreased with decreasing PPFD. Ubon 362.6 10.73 b 9.56 c 1.66 bc 223.14 d Ratchathani exhibited more pronounced 93.3 14.36 d 10.16 bc 0.85 a 104.15 b Accessions * * * * decreases than Nakhon Si Thammarat as the PPFD * * * * asiaticoside and madecassoside contents at the Accessions ×x PPFD ns ns ns * lowest light intensity were comparable in both % C.V. 17.75 23.19 23.87 4.76 genotypes. For the two other triterpenoids, Values followed by the same letter are not significantly different based on DMRT (P≤0.05). asiatic acid and madecassic acid, genotype, PPFD and interaction effects were similarly Ratchathani and Rayong had comparably lower leaf area. The significant (Table 2). Under unshaded conditions, asiatic acid did petioles were comparably shorter in Nakhon Si Thammarat and Ubon not differ with genotype while madecassic acid was highest in Ratchathani than in Rayong. Fresh weight per unit area was highest Nakhon Si Thammarat followed by that of Ubon Ratchathani and in Ubon Ratchathani followed by that of Nakhon Si Thammarat and Rayong, respectively. With decreasing light, asiatic acid content Rayong, respectively. However, dry weights per kg fresh weight significantly decreased in Nakhon Si Thammarat only at PPFD did not significantly differ with genotype. With decreasing light value 93.3 µmol m-2s-1 while in Ubon Ratchathani, both PPFD value intensity, leaf area and petiole length increased. The increase in leaf 362.5 and 93.3 µmol m-2s-1 had comparable reducing effect. area was more dramatic in Ubon Ratchathani and Rayong than in Madecassic acid contents showed similar trend except that in Nakhon Si Thammarat while the increase in petiole length was about Nakhon Si Thammarat, it decreased with decreasing PPFD. A three-fold in Nakhon Si Thammarat and two-fold in Ubon different response of Rayong genotype was obtained as reduced Ratchathani and Rayong at the lowest PPFD 93.3 µmol m-2s-1. Leaf PPFD value of 362.5 µmol m-2s-1 remarkably increased the asiatic expansion and petiole elongation are natural adaptation responses and madecassic acid contents. When PPFD was further decreased of plants to shading in order to receive more light for to 93.3 µmol m-2s-1, asiatic acid content decreased but was still photosynthesis9, 17. In contrast, fresh and dry weights decreased significantly higher than that under full sun, while madecassic with decreasing light intensity which did not vary much with acid was significantly lower than that under full sun. genotype. This was expected as low light incidence decreases The results show that shading had detrimental effect on the photosynthetic rates which in turn decrease growth processes 9. main pharmacologically active triterpenoids of C. asiatica, Similarly, decreases in fresh weight in response to shading in asiaticoside and madecassoside 10, regardless of genotype. Both different C. asiatica genotypes in India were obtained 8. compounds markedly decreased in the three genotypes used in this study at lower light intensities (50-80% shading) compared to Triterpenoid contents: Asiaticoside and madecassoside contents that under unshaded conditions. The report earlier found that differed with genotype and generally decreased with decreasing light intensity lower than 15% of full sunlight reduced the light intensity (Table 2). Under PPFD value 933.1 µmol m-2s-1 (full asiaticoside content of C. asiatica in India 8. Interestingly, partial sun light), or unshaded conditions, they were highest in Ubon shading (50% or 362.5 µmol m-2s-1 light intensity) promoted asiatic Ratchathani and comparably lower in Nakhon Si Thammarat and and madecassic acid accumulation in Rayong unlike that in the Rayong. The decreasing effect of low light intensities (i.e. 362.5 two other genotypes. This genotypic response could be elucidated

Table 2. Effect of PPFD on triterpenoid contents of three accessions of C. asiatica. PPFD Asiaticoside Madecassoside Asiatic acid Madecassic Triterpenoids Accessions (µmol m-2 s-1) (%w/w) (%w/w) (%w/w) acid (%w/w) (g m-2) Nakhon Si 933.1 2.77 d 2.73 d 0.343 c 0.656 f 24.77 f Thammarat 362.5 2.36 c 2.04 c 0.340 c 0.581 e 12.28 d 93.3 1.83 b 1.71 b 0.154 a 0.505 c 4.56 b Rayong 933.1 2.80 d 2.82 d 0.260 bc 0.488 a 19.47 e 362.5 1.26 a 1.07 a 0.735 d 0.772 d 6.47 c 93.3 1.21 a 1.21 a 0.295 c 0.447 b 2.55 a Ubon 933.1 3.50 e 3.30 e 0.343 c 0.547 d 38.82 g Ratchathani 362.5 2.82 d 2.26 c 0.172 ab 0.435 ab 12.69 d 93.3 2.04 b 1.63 b 0.126 a 0.427 ab 4.75 b Accessions * * * * * PPFD * * * * * Accessions ×x PPFD * * * * * % C.V. 5.65 6.5 18.02 3.17 6.51 Values followed by the same letter are not significantly different based on DMRT (P≤0.05).

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