Heliyon 6 (2020) e03411

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Heliyon
journal homepage: www.cell.com/heliyon

Research article

Phytochemical composition and antioxidant activity of coconut cotyledon

Udaya Prakash Nyayiru Kannaian a, *, Jasmine Brighty Edwin b, Vidhya Rajagopal c,
Sripriya Nannu Shankar b, Bhuvaneswari Srinivasan d
a
Department of Biotechnology, Vels Institute of Science, Technology and Advanced Studies, Pallavaram, Chennai 600117, India
b
R and D, Marina Labs, 14, Kavya Gardens, N.T. Patel Road, Nerkundram, Chennai 600107, India
c
Department of Biotechnology, Valliammal College for Women, Anna Nagar, Chennai 600040, India
d
Department of Botany, Bharathi Women's College (Autonomous), Broadway, Chennai 600108, India

A R T I C L E I N F O A B S T R A C T

Keywords: Coconut tree (Cocos nucifera L.), a perennial, monocot tree, belonging to the family Arecaceae, is distributed
Coconut through the tropics. Bioactivities of coconut water, husk fiber, oil, flowers, spadix and mesocarp of coconut fruit
Cotyledon are widely reported. However, there is no study on cotyledon of coconut. In this study, carbohydrates, proteins,
Cold percolation
lipids, phenols, flavonoids, tannins, alkaloids and antioxidants were quantified in hot and cold percolated extracts
Cardiac glycosides
Antioxidant
of coconut cotyledon. Further, the antioxidant activity was studied using 2,2-diphenyl-1-picrylhydrazyl (DPPH);
Biochemical composition of food ferric reducing antioxidant power (FRAP); ferric thiocyanate (FTC); thiobarbituric acid (TBA); nitric oxide (NO)
Plant products radical scavenging and β-carotene bleaching assays. Among the secondary metabolites, only cardiac glycosides
Phytochemistry were detected. Methanolic extraction by cold percolation extracted high content of secondary metabolites and
Bioactive plant product exhibited significant antioxidant activity in DPPH, FRAP, NO and β-carotene bleaching assays, with EC50 of 0.12,
Secondary metabolite 6.43, 16.21 and 8.09 mg/ml respectively. The chloroform extracts recorded high lipid content and scavenged the
Food science radicals in FTC (EC50 13.31 mg/ml) and TBA (EC50 9.21 mg/ml) assays. The study recommends extraction of
compounds using methanol through cold percolation. The cotyledon of coconut is found to be a potent nutritive
source equivalent to the endosperm.

1. Introduction such as coconut water, husk fiber, oil, flowers, spadix and mesocarp of
coconut fruit are documented. However, the bioactivity of C. nucifera
Cocos nucifera L., popularly called as coconut tree, is a perennial, cotyledon has not been studied so far.
monocot tree, belonging to the family Arecaceae. The tree, native to The cotyledon of coconut, also known as coconut apple, sprout and
Southeast Asia and Melanesia, is distributed throughout the tropics and pearl, is a white, off-white or creamy, spongy structure, formed during
sub-tropics of the world (Chan and Elevitch, 2006). Nearly 60.77 million the germination of zygotic embryo. They form the basis of nutrition for
metric tons of coconuts were produced during the year 2017 globally the developing plant. Cotyledons are found to possess parenchyma cells
(Shahbandeh, 2019). C. nucifera has wide range of uses, which include with few vascular tissues. Though the cotyledons of coconut are being
making of brooms and thatched roofs using leaves; ropes and mats using consumed by people at large, they have been explored only for their role
coir; furniture using lumber; fuel using shell and husk; use of coconut oil & in clonal propagation (Nguyen et al., 2015). The present study is focused
milk for cooking, and coconut water as a refreshment in sports drink & on the quantification of the primary and secondary metabolites present in
alcoholic beverages (Toddy). The importance of different parts of cotyledons, and evaluation of their free radical scavenging activity.
C. nucifera in different industries has been documented (Roopan and
Elango, 2015). Nutritionists claim that coconuts contain vitamins, elec- 2. Materials and methods
trolytes, fibre and few minerals like potassium, phosphorus & manganese.
The bioactivities viz., antiulcerogenic (Nneli and Woyike, 2008), 2.1. Plant source and solvent extraction
wound healing (Srivastava and Durgaprasad, 2008), antimicrobial,
anti-inflammatory, anti-diabetic, anti-neoplastic, anti-parasitic, insecti- Germinated coconut (Cocos nucifera) (Figure 1a) was procured from
cidal, leishmanicidal (Roopan, 2016), antioxidant (Muritala et al., 2018) local markets of Chennai, Tamil Nadu, India. The cotyledons were
and cell proliferation (Dhanyakrishnan et al., 2018) of one or more parts, separated from the shell (Figure 1b), sliced into pieces (0.5 cm3) and
* Corresponding author.
E-mail address: [email protected] (U.P. Nyayiru Kannaian).

https://doi.org/10.1016/j.heliyon.2020.e03411
Received 4 September 2019; Received in revised form 5 November 2019; Accepted 28 January 2020
2405-8440/© 2020 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
U.P. Nyayiru Kannaian et al. Heliyon 6 (2020) e03411

Tannins: A few drops of 0.1% ferric chloride were added to 5 ml of the

extract. Brownish green or blue black colour indicated the presence of
tannins.
Phlobatannins: Upon boiling the extract (10 ml) with 1% HCl,
development of red precipitate indicated the presence of phlobatannins.
Saponins: Water (3 ml) was added to 10 ml of the extract and shaken
well. A few drops of olive oil were then added. The presence of a stable
emulsion denotes saponins.
Flavonoids: Yellow coloration of the solution, upon addition of a few
drops of 1% liquor ammonia to the extract confirms the presence of
flavonoids.
Terpenoids: To 5 ml of the extract, 2 ml of chloroform and 3 ml of
concentrated sulphuric acid were added. The presence of terpenoids was
indicated by a reddish brown interface.
Cardiac glycosides: Two ml of glacial acetic acid containing a drop of
ferric chloride and concentrated sulphuric acid (1 ml) were added
consecutively to 5 ml of the extract. Development of a brown ring indi-
Figure 1. a. Germinated coconut. b. Cotyledon of C. nucifera. cated the presence of cardiac glycosides.
Steroids: To the extract (2 ml), acetic anhydride (2 ml) and a few
drops of concentrated sulphuric acid were added. The presence of ste-
subjected to extraction. Solvent extraction of the cotyledons was per-
roids was indicated by a blue green ring.
formed by hot and cold percolation methods, using the solvents methanol
Alkaloids: The presence of reddish brown precipitate, upon addition
and chloroform. Soxhlet apparatus was used for the hot percolation
of Wagner's reagent (2 ml) to the plant extract (1 ml) indicated alkaloids.
method, with the cotyledon-solvent ratio being 1:10 (w/v). The tem-
Quinones: Sodium hydroxide (10%) was added in drops to 1 ml of the
perature was set at 70
C and the apparatus was operated for 5 cycles.
extract. The presence of quinones was indicated by blue green or red
Extraction by cold percolation was carried out by the addition of 100 ml
color in the solution.
of solvent to 10 g of the sliced cotyledon. The extracts were maintained at
Coumarins: To the extract, NaOH (10%) and chloroform were added
30  2
C in a temperature controlled shaker for 48 h and then filtered.
at equal proportions. An yellow colored solution indicated the presence
The extracts thus obtained through hot and cold percolation were
of Coumarins.
concentrated to obtain the crude. The crude was diluted with respective
solvents for further analysis. The methanol extract obtained through hot
percolation is abbreviated as HPME and that of chloroform extract as 2.2.2.2. Quantitative analysis. Total phenolic content (TPC): According
HPCE. The respective solvent extracts obtained by cold percolation are to Folin-Ciocalteu method, 500 μl of distilled water and 100 μl of Folin-
presented as CPME and CPCE. Ciocalteu reagent were added to 100 μl of the extract and incubated for 6
min at room temperature. The volume was made to 3 ml using water,
after adding 1.25 ml of 7% sodium carbonate. After incubation for 90
2.2. Analysis of plant metabolites min, the absorbance was recorded at 760 nm, and the result expressed as
mg Tannic acid equivalents (TAE) per gram dry weight (DW) of the plant
2.2.1. Primary metabolites material (Udayaprakash et al., 2015).
The carbohydrate content in the extracts of C. nucifera cotyledons was Total flavonoid content (TFC): The solvent in 200 μl of the extract was
quantified by DNSA (Dinitrosalicyic acid) method (Saqib and Whitney, evaporated and 5 ml of aluminium chloride (0.1 M) was added to the
2011). To 1 ml of the extract, 1 ml of DNSA reagent was added and residue. After incubation for 40 min, the absorbance was measured at
incubated in a boiling water bath at 100
C for 10 min. The volume was 415 nm. The TFC was expressed with reference to quercetin equivalents
made upto 10 ml using distilled water and the absorbance was measured (QE) per gram dry weight (DW) of the plant material (Udayaprakash
at 540 nm, with glucose as the standard. et al., 2015).
Lowry's method was employed for estimating the protein content of Total tannin content (TTC): The extract (100 μl), water (8.4 ml),
C. nucifera cotyledons (Thangaraj, 2016). To 100 μl of the extract, 100 μl Folin-Ciocalteau reagent (500 μl) and sodium bicarbonate (1 ml) were
of 2N NaOH was added and maintained at 100
C for 10 min in a boiling mixed together and incubated for 30 min. The results are expressed as mg
water bath. Upon cooling to room temperature, 1 ml of Lowry's reagent Tannic acid equivalents (TAE) per gram dry weight (DW) of the plant
was added and incubated at room temperature for 10 min. To this, 100 μl material (Tambe and Bhambar, 2014).
of Folin reagent was added and maintained at room temperature for 30 Total alkaloid content (TAlC): One ml of 2 N HCl was added to the
min, after which the absorbance was measured at 680 nm in UV-Vis extract and filtered. Boromocresol green (5 ml) and phosphate buffer (5
spectrophotometer. Standard plot of bovine serum albumin was used to ml) were added to the filtrate. To this, chloroform was added in suc-
quantify the protein content. cessive volumes (1–4 ml) in a separating funnel and mixed vigorously.
The total lipid content was estimated according to Bligh & dyer The chloroform layer was then separated and the volume made up to 10
method (Thangaraj, 2016). The extract (10 ml) was added to a pre- ml. The absorbance was measured at 470 nm and expressed as caffeine
weighed petridish and the solvent was allowed to evaporate in an oven at equivalents (CE) per gram dry weight (DW) of the plant material (John
100  2
C. The total lipid content was measured from the difference of et al., 2014).
weight between the petridish prior to heating and after heating. Total antioxidant content (TAC): The TAC was studied by phospho-
molybdenum method. To the extract (0.5 ml), 4.5 ml of reagent solution
2.2.2. Secondary metabolites containing 0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM
ammonium molybdate were added. The solution was maintained at 95
C
2.2.2.1. Qualitative analysis. The presence of secondary metabolites such for 90 min in a boiling water bath. Subsequently, the absorbance was
as tannins, phlobatannins, saponins, flavonoids, terpenoids, cardiac gly- measured at 695 nm and the TAC was expressed as mg Tannic acid
cosides, steroids, alkaloids, quinones and coumarins was studied ac- equivalents (TAE) per gram dry weight (DW) of the plant material
cording to Evans (2009) & Udayaprakash et al. (2013). (Udayaprakash et al., 2015).

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2.3. Free radical scavenging assays of the free radical scavenging assays. Correlation was performed at 95%
confidence interval and the Pearson correlation coefficient (R2) was
2.3.1. 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay recorded. Regression, correlation and PCA were carried out using Mini-
Varying concentrations of C. nucifera extracts (2, 4, 6, 8 & 10 mg/ml) tab, LLC (2019).
were made upto 1 ml using the respective solvents. To this, 1 ml of 0.01
mM DPPH was added and incubated in the dark for 30 min, after which 3. Results
the absorbance was measured at 517 nm (Udayaprakash et al., 2014).
3.1. Primary metabolites
2.3.2. Ferric reducing antioxidant power (FRAP) assay
To different concentrations of the extract (2, 4, 6, 8 & 10 mg/ml), 2.5 The total protein content was the highest in HPME (1.93  0.04 mg/
ml of 0.2 M phosphate buffer (pH 7) and 2.5 ml of 1% potassium ferri- g) followed by that in cold percolation method, i.e., CPME (1.62  0.15
cyanide were added and maintained at 50
C for 30 min. Further, 10% mg/g). The chloroform extract possessed protein content of 1.27  0.10
trichloroacetic acid (2.5 ml) was added and centrifuged for 10 min at mg/g and 1.14  0.05 mg/g in hot and cold percolation methods
6500 rpm. To the supernatant, 2.5 ml of distilled water and 0.5 ml of respectively. Similarly, the total carbohydrate content was highest in
0.1% ferric chloride were added and the absorbance was measured at HPME (12.72  0.15 mg/g), followed by CPME (10.39  0.16 mg/g).
700 nm (Udayaprakash et al., 2015). However, the total lipid content was found to differ, with higher quantity
in the chloroform extracts when compared to methanol. The total content
2.3.3. Thiobarbituric acid (TBA) assay of proteins, carbohydrates and lipids of coconut cotyledons extracted
One ml of 2.51% linoleic acid, 20% trichloroacetic acid (200 μl) and using hot and cold percolation method with respective solvents is pre-
0.67% thiobarbituric acid (200 μl) were added to varying concentrations sented in Table 1.
of C. nucifera extracts (2, 4, 6, 8 & 10 mg/ml) and maintained for 10 min
in boiling water bath. After centrifugation at 3000 rpm, the absorbance of 3.2. Secondary metabolites
the supernatant was measured at 532 nm (Udayaprakash et al., 2015).
Preliminary screening for the detection of different secondary me-
2.3.4. Ferric thiocyanate (FTC) tabolites in the cotyledon of C. nucifera revealed the presence of cardiac
To the extracts of C. nucifera (2, 4, 6, 8 & 10 mg/ml), 120 μl of 98% glycosides alone in the chloroform extracts obtained through hot and
ethanol, 100 μl of 2.51% linoleic acid and 9 ml of 40 mM phosphate cold percolation methods. However, cardiac glycosides were not detec-
buffer (pH 7) were added. Further, the solution was maintained at 40
C ted in methanol extract either through hot or through cold percolation
in dark for 40 min. To this solution (100 μl), 75% ethanol (9.7 ml), 30% method. All other secondary metabolites analysed were not detected in
ammonium thiocyanate (100 μl) and 20 mM FeCl3 in 3.5% HCl (100 μl) the preliminary screening.
were added successively. The absorbance was measured at 500 nm The yield of total phenolic content was found to be high (30.35 
(Udayaprakash et al., 2015). 0.94 mg TAE/g dw) in CPME, followed by HPME (29.58  0.82 mg TAE/
g dw). The total flavonoids were in trace amounts, in both the extraction
2.3.5. Nitric oxide (NO) radical scavenging assay methods. There was no difference in the total tannins content irrespective
Two ml of sodium nitroprusside (5 mM) was added to different of the extraction method or solvent, in the range 1.85  0.03 mg TAE/g
concentrations of C. nucifera extracts (2, 4, 6, 8 & 10 mg/ml). The solu- dw to 1.94  0.04 mg TAE/g dw.
tions were incubated at room temperature for 1 h, to which 5 ml of Griess The total alkaloid content of coconut cotyledon was high in chloroform
reagent was added. The absorbance was then measured at 546 nm extracts, with the values 19.7  0.32 and 16.68  0.42 mg CE/g dw
(Jagetia et al., 2004). respectively, in cold and hot percolation methods. The TAlC of the
methanol extracts was recorded as 5.74  0.37 mg CE/g dw through cold
2.3.6. β-carotene bleaching assay percolation and 5.01  0.57 mg CE/g dw through hot percolation. Per-
An emulsion was prepared by mixing 11 μl of β-carotene (8.2 μM), 4.4 taining to total antioxidant content, the yield of CPME was the highest
μl of linoleic acid (628 μM) and 22 μl of Tween 40 (0.2 g/ml). On removal (155.87  0.39 mg TAE/g dw), followed by HPME (143.28  0.79 mg
of the solvent from the emulsion, 2.4 ml of phosphate buffer (0.02 M, pH TAE/g dw). However, the solvent chloroform extracted nearly 20 fold
7) and varying concentrations of C. nucifera extracts (2, 4, 6, 8 & 10 mg/ lower quantities in both, hot and cold percolation methods. The total
ml) were added. The solutions were maintained at 50
C for 10 min and phenolics, flavonoids, tannins, alkaloids and antioxidant contents extrac-
the absorbance was measured at 460 nm (Lelono et al., 2009). ted from cotyledons of coconut using hot and cold percolation methods
The percent of inhibition exhibited by different concentrations of the using methanol and chloroform as the solvents are presented in Table 1.
extracts in all the free radical scavenging assays was calculated as:

ðAbsorbance of control  Absorbance of sampleÞ 3.3. Free radical scavenging activity

Percent inhibition ¼  100
Absorbance of control
C. nucifera extracts had high scavenging activity against DPPH radi-
cals. The methanol extract of coconut cotyledon showed nearly 88% of
where control refers to the solution containing all the reagents as
inhibition at the concentration of 10 mg/ml. There was no difference in
applicable for each assay, and does not possess the sample (extract). The
percent inhibition of DPPH at higher concentration of the extracts.
concentration at which 50% of the radicals were scavenged i.e., EC50 was
However, significant difference was noticed at lower concentration (2
calculated from the percent inhibition values, by regression analysis.
mg/ml) of the extract. Nearly 75% of inhibition was recorded for the
CPME and only 55% for HPCE. The EC50 values were recorded as 0.44,
2.4. Statistical analysis 1.52, 0.12 and 1.47 mg/ml, respectively for HPME, HPCE, CPME and
CPCE. The inhibition percent of DPPH radicals at different concentra-
The EC50 values for the free radical scavenging assays were calculated tions of the solvent extracts is presented in Figure 2a.
by the best fit as applicable for each assay (logarithmic regression for Similar trend was observed in the ferric reducing antioxidant power
DPPH assay and linear trend for all other radical scavenging assays). (FRAP) assay. The methanolic extract by cold percolation scavenged the
Correlation and Principal Component analysis (PCA) were performed to radicals by 66.67%, while hot percolation resulted in 51.9%. The chlo-
correlate the mean values of the phytochemicals quantified with the EC50 roform extracts of cold and hot percolation showed 48.67 and 16.67%

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U.P. Nyayiru Kannaian et al. Heliyon 6 (2020) e03411

Table 1. Quantification of primary and secondary metabolites in C. nucifera cotyledons.

Hot percolation Cold percolation

Methanol Chloroform Methanol Chloroform

Total protein content (mg/g) 1.93  0.04 1.27  0.1 1.62  0.15 1.14  0.05
Total carbohydrate content (mg/g) 12.72  0.15 1.84  0.14 10.39  0.16 1.33  0.12
Total lipid content (mg/g) 14  0.05 15.4  0.04 13.8  0.04 16  0.03
TPC (mg TAE/g) 29.58  0.82 6.44  0.72 30.35  0.94 15.82  0.81
TFC (μg QE/g) 23.67  2.61 11.97  5.17 24.3  2.88 16.3  4.94
TTC (mg TAE/g) 1.94  0.04 1.85  0.03 1.94  0.01 1.85  0.05
TAlC (mg CE/g) 5.01  0.57 16.68  0.42 5.74  0.37 19.7  0.32
TAC (mg TAE/g) 143.28  0.79 7.05  0.66 155.87  0.39 8.17  0.88

TAE: Tannic acid equivalent; QE: Quercetin equivalent; CE: Caffeic acid equivalent.

respectively at the concentration of 10 mg/ml (Figure 2b). The EC50 secondary stage. The percent inhibition of the CPME ranged between 6
values of HPME, HPCE, CPME and CPCE were calculated as 9.36, 37.99, and 24.47% in FTC assay, while it was 22.47–40.26% in TBA assay
6.43 and 10.41 mg/ml respectively. (Figure 2c-d). Consequently, the EC50 values were lower in TBA assay
Scavenging of radicals generated in lipid peroxidation is studied when compared to FTC assay. The respective EC50 values of HPME,
through FTC and TBA assays. In both the assays, the chloroform extract of HPCE, CPME and CPCE in FTC assay were 22.2, 16.86, 21.78 and 13.31
cold percolation yielded higher inhibition percent, followed by that mg/ml; which further decreased to 16.27, 10.25, 14.53 and 9.21 mg/ml
through hot percolation. In the first stage of lipid peroxidation as studied in TBA assay.
through FTC assay, upto 37% radicals were inhibited by the chloroform Analogous to DPPH and FRAP assays, the methanolic extract of the
extract (cold percolation), which further increased to 51.31% in the coconut cotyledon extracted through cold percolation exhibited high
percentage in scavenging NO radicals, with EC50 value of 16.21 mg/ml.
About 45.74 and 41.4% of NO radicals were scavenged respectively by
CPME and HPME (EC50 ¼ 18.98 mg/ml). Not much difference in percent
inhibition was observed between the chloroforms extracts obtained by
hot (34.91%) and cold percolation (35.11%). However, the corre-
sponding EC50 were 23.58 and 33.99 mg/ml.
Similarly, CPME and HPME exhibited 65.66% and 59.6% respectively
in β-carotene bleaching assay, with respective EC50 of 8.1 and 8.92 mg/
ml. The chloroform extracts obtained by cold and hot percolation
revealed 43.43% and 41.41% of radical inhibition and EC50 of 11.45 and
11.65 mg/ml. The percent inhibition recorded in NO free radical scav-
enging assay and β-carotene bleaching assay are presented in Figure 2e
and 2f respectively.
The loading plot (Figure 3.) and the correlation matrix (Table 2)
represent the relation between the quantification of metabolites and the
EC50 values of the free radical scavenging assays. TPC, TFC, TTC and TAC
are found to reside in the same cluster, with high positive value in the
first principal component. Lipids and alkaloids were the only metabolites
which correlated positively with DPPH, NO radical scavenging and
β-Carotene bleaching assays, while all other metabolites exhibited strong
negative correlation. Similarly, total carbohydrates, proteins, tannins and
total antioxidant contents positively correlated with FTC and TBA assays.
However, no significant positive correlation was observed for FRAP assay
with any of the metabolites, while TPC (R2 ¼ -0.859) and TFC (R2 ¼
-0.841) showed strong negative correlation.

4. Discussion

Coconut tree is an important subsistence crop in the tropics and

almost every part of the plant is explored for application in different
fields. Although many studies are available on the application of different
plant parts (Lima et al., 2015), the same related to cotyledons of the tree
has not been reported so far. Thus, the present study was conducted to
know the presence of primary & secondary metabolites in the cotyledons
of coconut and evaluate their free radical scavenging potency.
The cotyledon of coconut is described as a haustorial like tissue
developed during embryogenesis (Buffard-Morel et al., 1995). As the
embryo develops, the cotyledon becomes the dominant tissue, over-
whelming the growth of root and shoot primordium (Haccius and Philip,
Figure 2. Percent inhibition of radicals recorded by C. nucifera cotyledon in a)
1979). Cotyledon is a significant part of the embryo and is hypogeal in
DPPH radical scavenging assay, b) FRAP assay, c) FTC assay, d) TBA assay, e)
NO radical scavenging assay, f) β-carotene bleaching assay. germination, which remains below the ground. The cotyledons are not

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Figure 3. Loading plot recorded in principal component analysis.

Table 2. Correlation of primary and secondary metabolites in C. nucifera cotyledons with EC50 recorded in free radical scavenging assays.

DPPH FRAP FTC TBA NO β CBA

TP -0.84 -0.447 0.932 0.988 -0.792 -0.832
TC -0.94 -0.59 0.945 0.995 -0.811 -0.935
TL 0.961 0.452 -0.986 -0.954 0.938 0.956
TPC -0.942 -0.859 0.771 0.866 -0.603 -0.945
TFC -0.954 -0.841 0.792 0.878 -0.633 -0.957
TTC -0.984 -0.639 0.939 0.969 -0.827 -0.981
TAlC 0.958 0.501 -0.983 -0.985 0.898 0.952
TAC -0.993 -0.647 0.933 0.954 -0.831 -0.991

TP: Total protein content.

TC: Total carbohydrate content.
TL: Total lipid content.
TPC: Total phenolic content.
TFC: Total flavonoid content.
TTC: Total tannin content.
TAlC: Total alkaloid content.
TAC: Total antioxidant content.
DPPH: 2,2-diphenyl-1-picrylhydrazyl free radical scavenging assay.
FRAP: Ferric reducing antioxidant power.
FTC: Ferric thiocyanate assay.
TBA: Thiobarbituric acid assay.
NO: Nitric oxide radical scavenging assay.
β CBA: β-carotene bleaching assay.

photosynthetic and simply act as an organ to store and supply food for a Screening for the presence of secondary phytoconstituents revealed
developing embryo. This favours our study on the primary and secondary the presence of only cardiac glycosides. The medical significance of
metabolites in highly nutritive tissues, i.e. cotyledons. Further, as they cardiac glycosides includes treatment of cardiac arrhythmias, conges-
are widely consumed, their antioxidant potency was examined. tive heart failure and as anticancer agents (Riganti et al., 2011). This
The study on the primary metabolites revealed the carbohydrate favours the utilization of cotyledon of coconut as a good nutritive
content of the cotyledon as 12.72  0.15 mg/g, the protein content as source to prevent heart ailments. Absence of other phytochemicals in
1.93  0.04 mg/g and lipid content as 16  0.03 mg/g. The protein preliminary screening may be due to their presence in trace quantities.
content of coconut water or the endosperm of the coconut with different The quantification of the total phenolics, flavonoids, tannins and al-
age groups of 7–10 months was reported to be in the range of 0.02%– kaloids revealed their presence and further confirms that they are at
0.13% and fat content as 0.5%–1.8% (Jackson et al., 2004). It is noticed meagre level. Further, certain phytochemicals, though present in the
that there is no significant difference between the nutritive content of plant extracts, may provide negative results in the qualitative
cotyledon and the liquid endosperm of coconut. However, the difference screening assays, owing to structural idiosyncrasies (Jones and King-
is only attributable to the solvents used in the study. horn, 2006).

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Phytoconstituents such as proteins, carbohydrates, polyphenols, fla- 5. Conclusion

vonoids and tannins are known to be responsible for antioxidant activity
(Hu et al., 2016; Kim et al., 2003). Mahayothee et al. (2016) reported the The study reports the quantification of primary metabolites, detection
TPC of coconut water in the range of 5.18–7.17 mg GAE/100 ml while and quantification of secondary metabolites and free radical scavenging
the coconut meat possessed 6.28–10.01 mg GAE/100 g. The results of the activity of the cotyledon of coconut for the first time. Among the primary
present study reveal that the cotyledons of coconut possess high phenolic metabolites, high carbohydrate and lipid contents were recorded in the
content when compared with coconut water and coconut meat. The TFC methanolic and chloroform extracts respectively. The detection of sec-
in the extracts varied between 12-24 μg QE/g dw. The TTC was almost ondary metabolites showed the presence of cardiac glycosides which
the same, irrespective of the solvent and the extraction method. The total favours the use of cotyledon as a nutritive source for people ailing with
alkaloid contents were high in the chloroform extracts when compared cancer and heart disease. The total phenolics, tannins and alkaloids from
with the methanolic extracts of C. nucifera cotyledons. the cotyledons proved that they are nutritionally equivalent to the
The studies on quantification of secondary metabolites, i.e. total endosperm of coconut. The free radical scavenging assays also proved
phenols, flavonoids, tannins, alkaloids and antioxidants from the coty- that they act as medium/weak antioxidants. The percent inhibition and
ledons of coconut revealed that their yield is higher in cold percolation EC50 values indicate that the antioxidant activity of the cotyledons is
method than hot percolation. Among the solvents studied, all the sec- equivalent to that of coconut water or the endosperm. Antioxidant ac-
ondary metabolites except for alkaloids were extracted highly in meth- tivity by ferric thiocyanate radical scavenging and β-carotene bleaching
anol when compared with chloroform. Yields of different phytochemicals assays is reported for the first time from coconut. Lipids and alkaloids
from plant extracts are found to vary depending on the method of were the only metabolites which positively correlated with DPPH, NO
extraction and the solvent used (Udayaprakash et al., 2019). radical scavenging and β-Carotene bleaching assays; while carbohy-
Different radicals generated during the metabolism of biomolecules drates, proteins and tannins correlated with FTC and TBA assays. Further,
in the biological system have the potency to hinder normal activities it is recommended to extract compounds through cold percolation, with
and lead to various diseases (Phaniendra et al., 2015). Hence, a balance methanol as the preferred solvent. The study concludes the cotyledon of
between oxidants and antioxidants is necessary. Thus, using cotyledons coconut to be a potent nutritive source equivalent to the endosperm, i.e.,
of coconut as a nutritive source balances the ratio between oxidants and either coconut meal or coconut water.
antioxidants. The EC50 value against the DPPH radicals was observed to
be less than 2 mg/g of the extract of coconut cotyledon. The EC50 was Declarations
recorded as >1 μg/μl for coconut water (Fonseca et al., 2009). Chak-
raborty and Mitra (2008) conducted a study on the potency of coconut Author contribution statement
meal in scavenging of radicals in DPPH, FRAP and TBA assays. They
reported the efficiency of scavenging DPPH radicals in concentration Udaya Prakash Nyayiru Kannaian: Conceived and designed the ex-
range of 22.7–90.2 μg/ml. The EC50 recorded in FRAP assay was 6.434 periments; Wrote the paper.
mg/ml by the cotyledons, whereas that of coconut meal ranged from Jasmine Brighty Edwin: Performed the experiments.
768 to 1292 mM. In TBA assay, inhibition percent of 51.31% was Vidhya Rajagopal: Analyzed and interpreted the data.
recorded at the concentration of 10 mg/g of coconut cotyledon. The Sripriya Nannu Shankar: Performed the experiments; Wrote the
same was reported as 40.6% in coconut meal. The NO scavenging ac- paper.
tivity recorded for the cotyledon was 45.74%. Singla et al. (2011) re- Bhuvaneswari Srinivasan: Conceived and designed the experiments;
ported the EC50 of coconut endosperm against NO radicals to be 538.44 Analyzed and interpreted the data.
μg/ml. The potency of radical scavenging by coconut through ferric
thiocyanate assay and β-carotene bleaching assay is reported here for Funding statement
the first time, among any of the coconut parts studied so far. Antioxi-
dants are classified as very strong (<50 μg/ml), strong (50–100 μg/ml), Research fund was supported by R & D, Marina Labs, Chennai (Grant
medium (101–150 μg/ml), weak (>150 μg/ml). Based on this, cotyle- Number: ML-2018-RD008).
dons of coconut are categorised as medium antioxidants against DPPH
radicals and weak antioxidants against radicals in FRAP, FTC, TBA, NO
Competing interest statement
& β-carotene bleaching assays.
In general, correlation coefficient values near 1 or -1 shows a strong
The authors declare no conflict of interest.
relationship between the variables (Schober and Schwarte, 2018). From
the correlation analysis of metabolite yield and EC50 of the free radical
scavenging assays, it is evident that all the metabolites correlated Additional information
strongly either positively or negatively with DPPH, FTC, TBA, NO radical
scavenging and β-Carotene bleaching assays. However, only TPC and TFC No additional information is available for this paper.
showed strong negative correlation to FRAP assay.
In the present study, radicals generated in the DPPH, FRAP, NO and References
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