Functional constituents (micronutrients and phytochemicals) and antioxidant activity of Centella asiatica (L.) Urban leaves

Functional constituents (micronutrients and phytochemicals) and antioxidant activity of Centella asiatica (L.) Urban leaves

Industrial Crops and Products 61 (2014) 115–119 Contents lists available at ScienceDirect Industrial Crops and Products journal homepage: www.elsevi...

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Industrial Crops and Products 61 (2014) 115–119

Contents lists available at ScienceDirect

Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop

Functional constituents (micronutrients and phytochemicals) and antioxidant activity of Centella asiatica (L.) Urban leaves Shrawan Singh ∗ , D.R. Singh, V. Shajeeda Banu, Avinash N Division of Horticulture & Forestry, Central Island Agricultural Research Institute (CIARI), Port Blair 744101, Andaman & Nicobar Islands, India

a r t i c l e

i n f o

Article history: Received 19 February 2014 Received in revised form 21 June 2014 Accepted 23 June 2014 Keywords: Micronutrients Phytochemicals DPPH activity Herbal industry RP-HPLC Andaman Islands

a b s t r a c t The Centella asiatica is a surface growing plant and has great potential in herbal industry. Its leaves are also used as vegetable or flavoring agents. The present study investigated the genetic influence on phytochemistry of C. asiatica using 11 collections from Andaman Islands. The study showed significant (p < 0.05) variations in dietary micronutrients, potential antioxidants, and antioxidant activity. The analysis of leaves (100 g) showed wide range for micronutrients like Mn (25.2–90.1 mg), Cu (2.9–46.8 mg), Na (23.6–697.7 mg), Zn (16.6–122.9 mg), Ca (1354.9–2870.8 mg), Fe (112.4–247.2 mg), and Mg (398.0–757.4 mg). Phytochemical analysis of 100 g fresh leaves of C. asiatica genotypes showed wide range for phenolics (120.0–318.0 mg), flavonoids (111.8–260.6 mg), tannin (206.6–856.6 mg), anthocyanin (176.2–315.2 mg), carotenoids (12.5–72.6 mg) and ascorbic acid (36.6–96.6 mg). Carotenoids and phenolics compounds were validated in composite leaf samples of tested genotypes with RP-HPLC analysis. Antioxidant activity of methanol extracts ranged from 72.0 to 85.7% which showed significantly positive correlation with anthocyanin (r = 0.75; p < 0.05), total phenol (r = 0.53; p < 0.05), flavonoids (r = 0.56, p < 0.05), and tannin (r = 0.42; p < 0.05). The study revealed the genotype affect on functional compounds and identified genotypes for use herbal or breeding purpose. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Centella asiatica (L.) Urban is a small herbaceous annual plant of the family Apiaceae, native to Asia. It is also known as gotu kola, Indian Pennywort, and Mandookaparni and has been referred for centuries as a medicinal herb in French pharmacopoeia, Chinese Shennong Herbal, and Indian Ayurveda (Jayshree et al., 2003; Singh et al., 2010). Leaves are in common use as source of herbal extracts (Gohil et al., 2010), vegetable item in form of chutney, taste or texture agent in pulse items, tubers, and other vegetables (Singh et al., 2012). Its sweet and flavoured herbal drinks are common in market (Vasantharuba et al., 2012). A number of reviews on traditional health uses of C. asiatica and their supportive evidences (Arora et al., 2002; Gohil et al., 2010; Singh et al., 2012; Chong and Aziz, 2013; Mutua et al., 2013) clearly indicate its strong potential in health sector. Further, various active ingredients were reported in C. asiatica such as asiaticoside, brahmoside, brahminoside, madecassoside, madecassic acid, thiamine, riboflavin, pyridoxine, vitamin K,

asparate, glutamate, serine, threonine, alanine, lysine, and histidine (Jayshree et al., 2003; Vasantharuba et al., 2012; Chong and Aziz, 2013). Besides, a number of macromolecules and dietary micronutrients are present in C. asiatica which contribute for ‘health effects’ but their concentration varies with genotypes, environment, and analysis method (Singh et al., 2012). Though, knowledge about such factors was needed for higher recovery of the active ingredient during industrial processes which was still scarce. Further, the underexplored regions like Andaman and Nicobar Islands, India required special attentions due to their rich diversity of plant species (Abraham et al., 2008; Singh and Singh, 2012) and strong traditional health system of indigenous tribals and settlers (Gupta et al., 2004). The Centella is one of the prominent herbal medicines for island people (Singh et al., 2012) and easy dietary item for poor households (Singh et al., 2012; Singh and Singh, 2012). Therefore, present study was conducted to study the dietary nutrients and phytochemicals in local genotypes of C. asiatica from Andaman Islands. 2. Materials and methods 2.1. Sample collection and preparation

∗ Corresponding author. Tel.: +913192250234; fax: +913192251068. E-mail addresses: [email protected], [email protected] (S. Singh). http://dx.doi.org/10.1016/j.indcrop.2014.06.045 0926-6690/© 2014 Elsevier B.V. All rights reserved.

Fresh young leaves of 11 local collections (LCCA 1 to 11; LCCA denotes to Local Collection of C. asiatica) of C. asiatica (Fig. 1)

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radical (DPPH) method (Blois, 1958) with appropriate modifications as described by Singh et al. (2012). Briefly, the sample extract was prepared as described for phytochemical estimation in subheading 2.1 and 0.1 ml of extract was added to 3 ml of 0.001 M DPPH methanol solution. The absorbance was taken at 517 nm in UV–visible spectrophotometer (Elico Pvt., Hyderabad, India). Similarly, absorbance were also taken of blank sample (1 ml of DPPH· and 5 ml of 80% methanol) and reference standards of rutin and ascorbic acid. Antioxidant activity was calculated using formula: [(Ao − Ae)/Ao] × 100 (Ao = absorbance without extract; Ae = absorbance with extract). Percentage increase in antioxidant activity of extracts with incubation period was calculated by the formula: [(Ae120min − Ae10min )/Ae10min × 100], where, Ae120min denotes antioxidant activity of extracts at 120 min of incubation and Ae10min represents antioxidant activity at 10 min of incubation. 2.5. HPLC analysis

Fig. 1. C. asiatica (L.) Urban leaves.

from Andaman Islands were taken from Indigenous Vegetables Germplasm Block, Central Agricultural Research Institute (CARI), Port Blair, Andaman and Nicobar Islands, India. The leaves were washed with millipore water from Heal Force® Water Purification System (Sanghai Canrex Analytic Instruments Co. Ltd, Shangai, China) and 2 g leaf sample ground with methanol (10 ml) and centrifuged (8000 × g × 10 min). It was filtered through Whatman No. 1 filter paper and concentrated by rotary evaporator (Amkette Analytics Ltd., Chennai, India). The samples were kept at −20 ◦ C for further analysis. 2.2. Micronutrient estimation The samples for micronutrient estimation were prepared according to the procedures described by Singh et al. (2012) using muffle furnace and Mg, Fe, Ca, Co, Cu, Mn, Na, and Zn were analysed through Atomic Absorption Spectrophotometer (AAS; Shimandzu AA 6200, Scientific Instruments Inc. Columbia, USA). 2.3. Estimation of antioxidant Total polyphenol was analysed by spectrophotometer at 700 nm using Folin–Ciocalteau reagent by the method described by Singleton et al. (1999) (10%, v/v) with some modifications and expressed as gallic acid equivalent using the equation based on the calibration curve: y = 0.147x, R2 = 0.974, where x is the gallic acid equivalent (mg/100 g) and y is the absorbance. Total tannin content was estimated using the Folin–Danis reagent (AOAC, 1995) and results were expressed as tannic acid equivalent using the equation based on the calibration curve: y = 0.045x, R2 = 0.961, where x is the tannic acid equivalent (mg/100 g) and y is the absorbance at 760 nm wavelength. Flavonoid content was determined by spectrophotometer using its standard protocol (Sadasivam and Manikam, 1996) with slight modification as suggested by Singh et al. (2012). Similarly, standard methods from Sadasivam and Manikam (1996) were employed for estimation of ascorbic acid, chlorophyll, and carotenoids content in the leaf samples. The pH differential method (Fuleki and Francis, 1968) was used for anthocyanin determination and cynidine-3-glucoside was used as reference standard. 2.4. DPPH antioxidant activity The electron-donating capacity of methanol extract of leaves of C. asiatica was determined using 1,1-diphenyl-2-picrylhydrazyl

The Reverse phase-High Performance Liquid Chromatography [RP-HPLC; (DIONEX, Ultimate 3000 series)] analysis of composite sample from 11 genotypes was done for carotenoids and phenolics as per procedure described by Singh et al. (2013a). The internal standard for carotenoids was ␤-carotene while for phenolics it was constituted by mixing of catechin, caffeic acid, sinapic acid, ellegic acid, and naringin. The RP-HPLC used in present study had C18 Silica column, a solvent rack (SRD-3200), a pump (HPG-3200SD), a column oven (TCC-3000SD), and a diode array detector (variable wavelength detectors VWD-3100 and VWD-3400). Mobile phase had methanol (solvent A) and acetonitrile (solvent B) in 90:10 ratio and flow rate was 1.0 ml/min for carotenoids. For phenolics, solvent A (dilute acetic acid, 0.9%; pH, 2.7) and solvent B (100% acetonitrile) were used at a flow rate of 0.8 ml/min with a gradient of 9% (0–5 min), 11% (5–15 min), 18% (15–22 min), 23% (22–38 min), 90% (38–43 min), 80% (43–44 min), 80% (44–45 min), and 5% (45–60 min). The column temperature was 22 ◦ C and 38 ◦ C and the absorbance was read at 450 nm and at 280 nm for carotenoids and phenolics, respectively. 2.6. Potential recovery of antioxidants The potential recovery of functional elements i.e. antioxidants and micronutrients from C. asiatica were estimated by the formula: potential recovery of functional element = [(FErg − FEpm )/EFpm] × 100; where, FErg and FEpm denote the value of functional element in richest genotype and population mean, respectively. 2.7. Statistical analysis Data generated were presented as mean and standard deviation of triplicate results using Microsoft EXCEL 2007 software. Analysis of variance and Pearson’s correlation were done using OPSTAT online software (http://hau.ernet.in/opstat.html). 3. Results and discussion 3.1. Micronutrients The results of micronutrient analysis of C. asiatica collections from islands are presented in Table 1 along with analysis of variance in Table 1. The study showed that the Mn content was ranged from 25.2 mg/100 g to 90.1 mg/100 g, highest in ‘LCCA 1’ and lowest in ‘LCCA 8’. Copper is essential for functioning and development of brain and C. asiatica is good accumulator of it (Mokhtar et al., 2011) and present study showed ‘LCCA 9’ (46.8 mg/100 g) as rich genotype in Cu while ‘LCCA 6’ had lowest value of 3.0 mg/100 g (Table 1). The

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Table 1 Dietary micronutrients and antioxidant in Centella asiatica genotypes from Andaman Islands. Genotypes

LCCA 1 LCCA 2 LCCA 3 LCCA 4 LCCA 5 LCCA 6 LCCA 7 LCCA 8 LCCA 9 LCCA 10 LCCA 11 CD (p = 0.05)

Micronutrient content (mg/100 g DW)

Phytochemical (mg/100 g FW)

Mn

Cu

Na

Zn

Ca

Fe

Mg

Phenol

Flavonoids

Tannin

Anthocyanin

Chlorophyll (␮g/100 g)

Carotenoids (mg/100 ml)

Vitamin C

90.1 55.4 83.9 47.8 83.9 41.9 34.1 25.2 28.6 35.4 32.2 16.3

5.0 6.0 15.3 13.0 15.7 3.0 19.4 34.9 46.8 26.1 28.8 9.1

697.8 23.6 109.1 55.2 136.6 203.7 195.2 301.0 429.4 140.4 152.2 130.2

20.7 38.3 68.9 46.6 123.0 48.9 23.5 16.7 20.9 38.5 37.6 20.4

1354.9 2791.5 2058.1 2870.8 2281.7 1398.4 1568.7 1371.2 1405.8 1384.5 1428.1 39.7

135.8 112.2 154.3 157 169.7 176.4 221.4 247.2 235.5 174.1 166.5 27.9

663.7 647.6 745.8 547.1 757.4 502.3 466.8 482.4 398.1 412.5 432.4 88.5

316.7 312.6 261.6 157.5 318.0 216.0 214.6 158.8 120.1 243.2 295.6 47.4

178.1 111.8 165.7 166.3 260.7 111.8 196.1 114.0 164.6 233.7 220.8 33.8

856.7 263.3 228.9 433.3 234.4 368.9 287.8 308.9 206.7 367.8 337.8 121.3

315.2 187.6 245.7 218.8 309.1 233.6 176.3 201.5 180.6 280.5 323.0 37.5

451.0 469.8 400.0 420.7 421.7 459.0 491.0 486.9 424.4 387.2 425.8 23.0

28.6 31.8 55.6 72.7 48.5 52.3 12.5 31.0 31.3 54.7 23.8 11.9

36.7 50.0 56.8 76.7 70.0 96.7 56.6 60.0 76.7 46.4 86.7 12.2

DW—dry weight; FW—fresh weight.

LCCA11

LCCA10

LCCA9

LCCA8

LCCA7

LCCA6

LCCA5

LCCA4

100 95 90 85 80 75 70 65 60 55 50

LCCA3

The C. asiatica genotypes showed significant (p < 0.05) difference for antioxidant activity at 30 min of incubation and it was highest in ‘LCCA 8’ (85.7%) while lowest in ‘LCCA 10’ (72.3%) (Fig. 2). The antioxidant activity was increased with incubation period and mean value of DPPH activity from test genotypes was increased from 64.3% (10 min) to 96.7% (120 min) (Fig. 3). The highest percentage increase in antioxidant activity (41.2%) was observed in ‘LCCA 8’ (from 68.2% at 10 min to 96.2% at 120 min) and lowest (28.1%) in ‘LCCA 6’ (74.5% at 10 min to 95.7% at 120 min) (Fig. 3). Similar observations were made by Singh et al. (2012) in traditional vegetables of Andaman and Nicobar Islands and (Singh et al. 2013b) in Hibiscus sabdariffa.

LCCA2

The data pertaining to antioxidants in the C. asiatica genotypes from different parts of the islands are presented in Table 1. Total phenol content was ranged 120.1 to 328.0 mg/100 g, highest in ‘LCCA 5’ and lowest ‘LCCA 9’. The values found in the study are in agreement with the earlier reports of 286.0 mg/100 g by Pittella et al. (2009) and in the range of 323.0 to 117.0 mg/100 g as reported by Zainol et al. (2003). The genotypes showed significant difference for flavonoid content and it was highest in ‘LCCA 5’ (260.7 mg/100 g) followed by ‘LCCA 10’ (233.7 mg/100 g). The flavonoid content in C. asiatica genotypes was higher than earlier reported content of 36.1 mg/100 g by Pittella et al. (2009) which might be due variation in genotypes, climate, soil and growing conditions. The maximum amount of tannin was determined in ‘LCCA 1’ (856.7 mg/100 g) while ‘LCCA 9’ had its lowest value of

3.3. DPPH antioxidant activity

LCCA1

3.2. Antioxidants

206.7 mg/100 g. Anthocyanin content also had significant (p < 0.05) difference among genotypes with maximum content in ‘LCCA 7’ genotype. Maximum amount of carotenoid was estimated in ‘LCCA 4’ (72.7 mg/100 ml) while lowest in ‘LCCA 7’ (12.5 mg/100 ml) (Table 1). C. asiatica is consumed as crushed form or chutney therefore, the ascorbic acid may be readily available to body. Ascorbic acid rich genotypes were identified as ‘LCCA 6’ (96.7 mg/100 g) followed by ‘LCCA 11’ (86.7 mg/100 g). Ascorbic contents were in the range of earlier reports of Norhayati et al. (2011) while difference in carotenoids might be due to genotype, environment, and method of estimations. Similarly, chlorophyll content also showed significant (p < 0.05) difference among genotypes and it was highest in ‘LCCA 7’ (490.9 ␮g/100 g) while lowest in ‘LCCA 10’ (387.2 ␮g/100 g) (Table 1).

Antioxidant activity (%)

observations are in agreement with reports of Mokhtar et al. (2011) where they reported Cu content in shoots of C. asiatica was in the range of 19.0 to 122.5 mg/kg and the differences were due to genotypes or growing conditions. Though, Na is essential element for proper functioning of body but excess intake affect the physiology of organs like kidney and heart and it ranges from 23.6 mg/100 g (‘LCCA 2’) to 697.8 mg/100 g (‘LCCA 1’). Zinc is one of the essential micronutrients for plants and its concentration in vegetable foodstuffs was found to be in the range of 25.2–50.0 mg/kg (Broadley et al., 2007). The present study identified Zn rich genotypes as ‘LCCA 5’ (122.9 mg/100 g) and ‘LCCA 3’ (68.9 mg/100 g) while ‘LCCA 8’ was poor source with lowest content of 16.7 mg/100 g. Zinc is cofactor in many of the physiological enzyme of body system and also acts as cofactor in free radical scavenging capacity of various enzymes (Evans and Helliwell, 2001). C. asiatica was reported as rich source of Ca (Baruah and Borah, 2009; Singh et al., 2012) and present study observed wide variation in Ca content in Centella genotypes which ranged from 1354.9 mg/100 g in ‘LCCA 1’ to 2870.8 mg/100 g in ‘LCCA 4’. Thus, daily intake of small quantity of mature raw leaves of C. asiatica can contribute the major part of Ca requirement of human body. Similarly, the C. asiatica leaves were rich in Fe (Singh et al., 2012) and it was supported by findings of the present study. The ‘LCCA 8’ (247.2 mg/100 g) was richest among the tested genotypes while ‘LCCA 2’ (112.2 mg/100 g) had minimum amount of Fe content (Table 1). Magnesium play crucial role in chlorophyll synthesis in plants and the present study identified the Mg rich genotypes as ‘LCCA 5’ (757.4 mg/100 g) and ‘LCCA 3’ (745.8 mg/100 g) while it was minimum in ‘LCCA 9’ (398.2 mg/100 g). The observations for micronutrient contents in C. asiatica are in agreements with the findings of Singh et al. (2012) and Baruah and Borah (2009).

Genotype Fig. 2. Antioxidant activity of C. asiatica genotypes at 30 min of incubation (LCCA denotes for Local Collection of C. asiatica 1 to 11).

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S. Singh et al. / Industrial Crops and Products 61 (2014) 115–119 Table 2 Correlation between micronutrients and antioxidant activity C. asiatica genotypes. Micronutrient

Antioxidant activity

Mn Cu Na Zn Ca Fe Mg

0.49 −0.55 −0.09 0.50 0.07 −0.46 0.35

acid, and naringin were important constituents in phenolics group of compounds. 3.5. Correlation studies Fig. 3. Change in antioxidant activity of C. asiatica genotypes with incubation period (LCCA denotes for Local Collection of C. asiatica 1 to 11).

3.4. HPLC analysis The HPLC analysis of composite leaf sample of 11 genotypes for carotenoids (Fig. 4a) and phenolics (Fig. 4b) showed presence of ␣-carotene, zeaxanthin, ␤-cryptoxanthin, and ␤-carotene in carotenoids group while catechin, caffeic acid, sinapic acid, ellegic

Pearson’s correlation analysis showed that Zn (r = 0.50; p < 0.05), Mn (r = 0.49; p < 0.05), Mg (r = 0.35; p < 0.05), and Ca (r = 0.07; p > 0.05) had positive correlation with antioxidant activity in C. asiatica leaves while Cu (r = −0.55; p < 0.05), Fe (r = 0.46; p < 0.05), and Na (r = −0.09; p > 0.05) had negative correlation (Table 2). Antioxidants had positive and strong correlation with anthocyanin (r = 0.75; p < 0.05), flavonoids (r = 0.56; p < 0.05), and phenolics (r = 0.53; p < 0.05). Tannin and carotenoids also showed strong correlation with antioxidant capacity of C. asiatica extracts (Table 3). This kind of correlation between different antioxidants and their activity is rarely observed in plant extracts (Singh et al., 2012). 3.6. Antioxidant recovery Higher recovery of functional elements in herbal products add to their health benefits and present showed great influence of genotypes on recovery of antioxidants and micronutrients in C. asiatica which ranged from 11.5% of chlorophyll to 214.0% of Na (Fig. 5). The recovery from best genotypes was high for Zn (179.7%), tannin (141.9%), Cu (140.5%), and carotenoids (80.6%) (Fig. 5). The strong antiradical activity of genotype ‘LCCA 8’ (85.7%) could be due to presence of high amount of tannin, chlorophyll, ascorbic acid, and supported by copper and iron. While it might be phenol and carotenoids which had played key role with Mn and Mg for higher free radical scavenging property of ‘LCCA 5’. However, genotype ‘LCCA 1’ had tannin, phenolics, anthocyanin and zinc for strong activity. The study suggests that this was the complex of more than one factor which contributed in antioxidant activity of methanol extract of C. asiatica genotypes. Thus, a mixture of extract from rich genotypes may more helpful for higher recovery of functional elements in herbal products and health supplements.

250 Potential recovery (% over mean value)

200 150 100

Fig. 4. (a) HPLC chromatogram of carotenoids in composite sample of C. asiatica. (b) HPLC chromatogram of phenolics in composite sample of C. asiatica.

Vitamin C

Carotenoid

Chlorophyll

Anthocyanin

Tannin

Flavonoid

Mg

Fe

Ca

Zn

Na

Phenol

-50

Cu

0

Mn

50

Fig. 5. Potential recovery (%) of functional elements from respective genotype over the mean value.

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Table 3 Correlation between antioxidants and antioxidant activity C. asiatica genotypes.

Phenol Flavonoids Tannin Anthocyanin Carotenoids Ascorbic acid Antioxidant activity

Phenol

Flavonoids

Tannin

Anthocyanin

Carotenoids

Ascorbic acid

Antioxidant activity

1.00 0.36 0.28 0.66 −0.15 −0.34 0.53

1.00 −0.01 0.63 0.02 −0.09 0.56

1.00 0.45 −0.04 −0.38 0.42

1.00 0.14 −0.04 0.75

1.00 0.21 0.33

1.00 0.10

1.00

4. Conclusion The present study attempted to investigate the genetic influence on phytochemistry of C. asiatica using 11 genotypes from Andaman Islands. The significant variations in dietary micronutrients, potential antioxidants and antioxidant activity suggest that phytochemical composition affected by the genotypes. The C. asiatica genotypes of Andaman Islands showed high contents of dietary micronutrients and antioxidants. Information conveys positive indication in support of traditional health perception of the Centella leaves and its potential in herbal industry. However, identified genotypes can be used for breeding of desirable varieties for higher recovery of functional elements in herbal products from this underutilized plant. Acknowledgement The authors express sincere thanks for the Director, Central Island Agricultural Research Institute, Port Blair for financial and research facilities. The contribution of plant materials by the local farmers/tribes to the germplasm was also acknowledged by the authors. References Abraham, Z., Senthilkumar, R., Joseph, K.J., Sharma, T.V.R.S., et al., 2008. Collection of plant genetic resources from Andaman and Nicobar Islands. Genet. Resour. Crop Evol. 55, 1279–1289. AOAC, 1995. Official Methods of Analysis. Association of Official Analytical Chemists, Washington, DC. Arora, D., Kumar, M., Dubey, S.D., 2002. Centella asiatica—a review of its medicinal uses and pharmacological effects. J. Nat. Remedies 2 (2), 143–149. Baruah, A.M., Borah, S., 2009. An investigation on sources of potential minerals found in traditional vegetables of north-eastern India. Int. J. Food Sci. Nutr. 60 (S4), 111–115. Blois, M.S., 1958. Antioxidant determinations by the use of a stable free radical. Nature 181, 1199–1200. Broadley, M.R., White, P.J., Hammond, J.P., Zelko, I., Lux, A., 2007. Zinc in plants. New Phytol. 173 (4), 677–702. Chong, N.J., Aziz, Z., 2013. A systematic review of the efficacy of Centella asiatica for improvement of the signs and symptoms of chronic venous insufficiency. Evidence Based Complement. Altern. Med., 1–10, http://dx.doi.org/ 10.1155/2013/627182.

Evans, P., Helliwell, B., 2001. Micronutrients: oxidant/antioxidant status. Br. J. Nutr. 85 (2), S67–S74. Fuleki, T., Francis, F.J., 1968. Quantitative methods for anthocyanins extraction and determination of total anthocyanin in cranberries. J. Food Sci. 33, 72–78. Gohil, K.J., Patel, J.A., Gajjar, A.K., 2010. Pharmacological review on Centella asiatica: a potential herbal cure-all. Indian J. Pharm. Sci. 72 (5), 546–556. Gupta, S., Porwal, M.C., Roy, P.S., 2004. Indigenous knowledge on some medicinal plants among the Nicobari Tribe of Car Nicobar Island. Indian J. Tradit. Knowl. 3 (3), 287–293. Jayshree, G., Muraleedhara, G.K., Sudarslal, S., Jacob, V.B., 2003. Antioxidant activity of Centella asiatica on lymphoma-bearing mice. Fitoterapia 74, 431–434. Mokhtar, H., Morad, N., Fizani, F., Fizri, A., 2011. Phytoaccumulation of copper from aqueous solutions using Eichhornia crassipes and Centella asiatica. Int. J. Environ. Sci. Dev. 2 (3), 205–210. Mutua, P.M., Gicheru, M.M., Makanya, A.N., Kiama, S.G., 2013. Anti-proliferative activities of Centella asiatica extracts on human respiratory epithelial cells in vitro. Int. J. Morphol. 31 (4), 1322–1327. Norhayati, Y., Nor-Aini, M.F., Misri, K., Marziah, M., Azman, J., 2011. ␣-Tocopherol, ascorbic acid and carotenoid content in Centella asiatica leaf tissues and callus cultures. Pertanika J. Trop. Agric. Sci. 34 (2), 331–339. Pittella, F., Dutra, R.C., Dalton, D.J., Lopes, M.T.P., Barbosa, N.R., 2009. Antioxidant and cytotoxic activities of Centella asiatica (L) Urb. Int. J. Mol. Sci. 10 (9), 3713–3721. Sadasivam, S., Manikam, A., 1996. Biochemical Methods. Wiley Eastern Ltd., Madras, India. Singh, S., Gautam, A., Sharma, A., Batra, A., 2010. Centella asiatica (L.): a plant with immense medicinal potential but threatened. Int. J. Pharm. Sci. Rev. 4 (2), 9–17. Singh, S., Singh, D.R., Salim, K.M., Srivastava, A., Singh, L.B., Srivastava, R.C., 2011. Estimation of proximate composition, micronutrients and phytochemical compounds in traditional vegetables from Andaman and Nicobar Islands. Int. J. Food Sci. Nutr. 62 (7), 765–773. Singh, S., Singh, D.R., 2012. Species diversity of indigenous vegetables in Andaman and Nicobar islands: efforts and strategies for utilization. In: Singh, D.R., et al. (Eds.), Souvenir, National Seminar on Innovative Technologies for Conservation of Island Biodiversity. Central Agricultural Research Institute, Port Blair, India, pp. 130–133. Singh, S., Singh, D.R., Shajeeda-Banu, V., Salim, K.M., 2013a. Determination of bioactives and antioxidant activity in Eryngium foetidum L.: a traditional culinary and medicinal herb. Proc. Nat. Acad. Sci., India, Sect. B: Biol. Sci. 83 (3), 453–460. Singh, S., Singh, D.R., Avinash, N., Salim, K.M., 2013b. Estimation of phytochemicals and antioxidant activity in Hibiscus sabdariffa L. Progressive Hortic. 45 (1), 174–181. Singleton, V.L., Orthofer, R., Lamuela-Raventos, R.M., 1999. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin–Ciocalteu reagent. Methods Enzymol. 299, 152–178. Vasantharuba, S.V., Banumathi, P., Premalatha, M.R., Sundaram, S.P., Arumugam, T., 2012. Functional properties of Centella asiatica (L.): a review. Int. J. Pharm. Pharm. Sci. 4 (5), 8–14. Zainol, M.K., Abd-Hamid, A., Yusof, S., Muse, R., 2003. Antioxitative activity and total phenolic compounds of leaf, root, and petiole of four accessions of Centella asiatica (L.) Urban. Food Chem. 81, 575–581.