INTRODUCTION:

Phytochemicals are secondary metabolites of plants. Their functions are to protect environmental stress and give the beneficial effect on consuming animals. These secondary metabolites are reported to be present in various edible herbs, medicinal plants and their pharmacological activities are evaluated. Especially, secondary metabolites like flavonoids which has antioxidant activity is widely distributed in edible vegetables, fruits and many herbs¹. Now-a-days many sophisticated instruments including HPTLC, HPLC, LC-MS, XRD etc., have been developed to analyse various phytochemical by simple methods². Using these instruments numerous phytochemicals have been identified isolated and their biological effects includes antioxidant, anti-inflammation and cytotoxic activities have been reported³. Some phytochemicals may exhibits toxic effects and interfere with the absorption of nutrients⁴.

Cocos nucifera belongs to the family Arecaceae is commonly referred as “coconut tree” and this plant are wide spread fruit tree on earth⁵. The origin of this plant was South East Asia⁶. India is the third largest producer of coconut and the yield was calculated as 10.56 million tonnes per year⁷. The tender coconut of this tree contains flesh and water called endosperm which is protected by three different sections that are exocarp, mesocarp, and endocarp⁸. The middle hard and fibrous part of tender coconut is mesocarp, which is used in many coir industries⁹.

The study on phytochemical analysis of ethanolic extract from coconut shell shows the presence of phenols, tannins, flavanoids, steroids and alkaloids¹⁰. The ethyl acetate extract of coconut shell is a rich source of polyphenolic compounds like catechins, epicatechins, tannins, and flavonoids¹¹.

Coconut fibre is a natural fibre extracted from the husk of tender coconut¹² and used in products such as floor mats, doormats, brushes and mattresses. Coir is the fibrous material found between the hard, internal shell and the outer coat of a tender coconut. Other uses of brown coir are in upholstery padding, sacking and horticulture. White coir, harvested from unripe coconuts, is used for making finer brushes, string, rope and fishing nets¹³.

Coconut water almost cures associated symptoms of Crohn’s disease, and illness of infected intestines¹⁴, ulcerative colitis and stomach ulcers. In addition to that coconut water is a rich source of quick energy. Coconut water is used in place of dextrose/ glucose in medical emergencies. It is also used as an antidote for poisons¹⁵. It is used to remove the intestinal parasites like tapeworms and Helicobacter pylori, which are responsible for causing indigestion and ulcer¹⁶.

Though various pharmacological and phytochemical evaluation of tender coconut has been reported earlier, scientific evaluation of phytoconstituents and pharmacological study on tender coconut mesocarp has not been reported earlier. Aim of the present study is to analyse the presence of various pharmacologically active phytoconstiuents in acetone extract of tender coconut mesocarp.

MATERIALS AND METHODS:

Collection and preparation of acetone extract:

Tender coconut mesocarp (TCM) was collected from Chennai, Tamilnadu, India. It was identified and authenticated at Institute of herbal botany and plant anatomy research centre, Chennai, Tamilnadu, India (No PARC/2019/3980). It was washed with distilled water and dried for 15 days at room temperature under shade. The dried plant material was coarsely powdered. 100.0 gm of coarsely powdered TCM was soaked in 1 litre of acetone for 72 hours at 4°C. The extract was filtered and concentrated under reduced pressure. The yield of acetone extract of TCM was calculated as 10.785±0.9 g/100g. The dried concentrated sample was stored under refrigerator and used for further phytochemical analysis.

Estimation of Phenol:

10.0mg of acetone extract was dissolved in 100.0ml of 5% ethanol. To 0.1ml extract, 2.5ml of 10.0% folin phenol was added. The mixture was incubated at room temperature for 5 minutes. To the mixture 2.5ml of 20.0 % sodium carbonate was added. Vortex well and incubate at room temperature for 2 hours. Absorbance was read spectrophotometrically at 720nm. Different concentration of Gallic acid was used as standard¹⁷.

Estimation of Tannin:

10.0mg of acetone extract was dissolved in 100.0ml of 5% ethanol. To 0.1ml of extract, add 0.5ml of Folin Dannis reagent. The tubes were allowed to stand for 5 minutes in room temperature and 2.0ml of 20% sodium carbonate was added. The tubes were then, kept in boiling water for 5 minutes. Formation of blue colour indicates the presence of tannins¹⁷. Optiical density was read spectrophotometrically at 620nm.

Estimation of Flavanoids:

10.0mg of acetone extract was dissolved in 100.0ml of 5% ethanol. To 0.1ml of extract, 0.15ml of 5% Sodium nitrite was added and incubated at room temperature for 5 minutes. To this mixture 10% aluminium chloride was added. Vortex well and incubated at room temperature for six minutes. 1.0ml of 1N sodium hydroxide was added and absorbance was read at 520nm. Dfferent concentrations of epicatechin was used as standard¹⁷.

Estimation of Vitamin C:

10.0mg of acetone extract was dissolved in 100.0ml of 5% ethanol. To 0.1ml of extract, 0.1ml of DTC reagent (3g of DNPH, 0.4g of thiourea, 0.05g of copper sulphate, dissolved in 100ml of 9 N sulphuric acid) was added. All the tubes were incubated at 37°C for 3 hrs. 0.75ml of 65% sulphuric acid was added and all the tubes were incubated at room temperature for 30min. The reaction was arrested by adding 0.5ml of Trichloro acetic acid and absorbance was measured at 520nm. Different concentration of Vitamin C was used as standard¹⁸.

Liquid Chromatography-Mass Spectroscopy (LC-MS):

LC-MS analysis of the acetone extract of Tender coconut mesocarp (TCM) was carried out using Thermo/Finnigan Surveyor System consisting of a degasser, binary pump, auto sampler, and column heater. The column outlet was coupled to a Thermo fleet (LCQ-Fleet) Ion Trap mass spectrometer equipped with an ESI ion source. Data acquisition and mass spectrometric evaluation were carried out in a personal computer with Data Analysis software (Qual Browser; Thermo Electron, San Jose, CA). For the chromatographic separation, a phenomenexluna 5-µm C8 column (250 × 4.6 mm) was used. The column was held at 95% Solvent A (0.1% acetic acid in water) and 5% Solvent B (0.1% acetic acid in acetonitrile) for 1 min, followed by an 11 min step gradient from 5% B to 100% B, then 4 min with 100% B. Finally, elution was achieved with a linear gradient from 100% B to 5% B for 2 min. The flow rate was 200 µl/min and injection volume was 5µl. The following parameters were used throughout the MS experiment: for electro spray ionization with positive ion polarity the capillary voltage was set to 20V, the capillary temperature to 300°C, the nebulizer pressure to 40 psi, and the drying gas flow rate to 15L/ min.

Statistical Analysis:

Values are represented as Mean ± SD of triplicate.

RESULTS:

Table-1 Quantity of Phytoconstituents in Acetone extract of Tender coconut mesocarp

S. No	Phyto constituents	mg/g Mean ± SD
1	Phenolic compounds	6.3±1.9
2	Flavonoids	3.75±0.02
3	Tannins	0
4	Vitamin C	301.1±1.41

Values are Mean ±SD of triplicate

Values in Table 1 depicts the quantity of secondary metabolites in acetone extract of TCM. Phenolic compounds and flavanoids were found to be present as 6.3±1.9 and 3.75±0.02 mg/g of extract, respectively. Tannin was found to be absent in acetone extract of TCM.

69 different phyto-compounds were found to be present in acetone extract of TCM (Table 2, Fig 1). Among them 13 compounds were found to be present in ppm >24.88. Sulfinpyrazone, Didesmethylimipramine, Glucosylsphingosine were found to be present in the concentration of 24.88, 21.67, 13.32 ppm, respectively.

Figure – 1 Liquid Chromatogram spectra of Acetone extract Of Tender coconut mesocarp

Table 2: Phytochemicals identified from the Acetone extract of Tender coconut mesocarp by Liquid Chromatography – Mass Spectrometry

S. No.	Compound Name	DB Diff (PPM)	Retention Time	Molecular Formula	Molecular Mass
1	Sulfinpyrazone	24.88	5.106	C23 H20 N2 O3 S	404.10
2	Glucosylsphingosine	13.32	15.53	C24 H47 N O7	461.32
3.	Didesmethylimipramine	21.67	26.99	C17 H20 N2	252.15
4.	Glutathione oxidized	8.15	5.081	C20 H32 N6 O12 S2	612.14
5.	Caffeic acid	2.16	4.947	C9 H8 O4	180.64
6.	Chlorogenic acid	1.74	4.937	C16 H18 O9	354.09
7.	Dihydro-Obliquin	4.79	4.403	C14 H14 O4	246.08
8.	6, 7, dihydroxy 4-oxo2-heptenoic acid	5.74	1.115	C7 H10 O5	174.05
9.	Quinic acid	4.07	1.115	C7 H12 O6	192.06
10.	D-Mannonate	3.00	1.082	C6 H12 O7	196.05
11.	2, 3, Dihydroxy benzoic acid	6.26	3.311	C7 H6 O4	154.02
12.	Epicatechin	0.95	4.982	C15 H14 O6	290.07
13.	Phenylacetic acid	1.94	5.088	C8 H8 O2	136.05

DISCUSSION:

Research on phytochemicals and evaluation of their pharmacological activity plays an important role in new drug development by pharmaceutical industry.

Acetone extract of TCM depicts the presence of phenolic compounds and flavonoids in higher concentration rather than tannins (Table 1). Presence of phenolic compounds and flavonoids may prone Acetone extract of TCM to serve as a cardio protective, anti-inflammatory or anticancer drug. Cardio protective effect of phenolic compound has been reported earlier. The phenolic compounds isolated from plant material were reported to exhibit anti-cancer activity and this effect might be due to its antioxidant potency¹⁹. Scientists evaluated anti-aging, anti-inflammatory, antioxidant, antiproliferative activity of isolated phenolic compounds ²⁰. Amylase inhibitory activity of isolated phenolic compounds in Invitro studies has been reported earlier²¹. The recent studies shows that the Intravenous administration of high – dose of vitamin C improved the quality of life for cancer patients²². In addition to that the meta-analysis on vitamin C and breast cancer shows the post diagnosis use of vitamin C supplementation which may associate in reducing the risk of mortality²³.

69 compounds were reported to be present in LC-MS analysis of acetone extract from TCM (Fig 1, Table 2), Sulfinpyrazone constitutes 24.88 ppm (Table 2) plays an important role in reducing the ischemic attacks, thrombombolism associated with vascular and cardiac prostheses²⁴. Sulfinpyrazone has been reported to treat gout²⁵.

Glucosylsphingosine (Table 2) is mainly used as Biomarker of Gaucher Disease²⁶. Oxidized Glutathione (Table 2), serves as an antioxidant and also prevent cellular damage from free radicals, lipid peroxides and heavy metals²⁷.

Caffeic acid and Chlorogenic acid (Table 2) both plays an important in inhibiting oxidation of LDL in vitro and thereforeit protect against cardiovascular disease ²⁸. Caffeic acid and chlorogenic acid possess inhibitory effects on important enzymes linked with hypertension and prevented preoxidant‐induced oxidative damage in rat's heart ²⁹

Quinic acid (Table 2) acts as anti-inflammatory agents ³⁰. 2, 3-Dihydroxy benzoic acid exhibits antioxidant and anti-radical activity ³¹.

Antioxidant activity of epicatechin ³²(Table 2) has been reported earlier. This epicatechin plays an important role in lowering blood glucose levels in diabetic patients. It is well known for its anticancer, antioxidant and antiproliferative effects against cancer cells ³³.

Phenyl acetic acid has been used as a precursor to manufacture penicillin-G³⁴. Pharmacological activity of Didesmethylimipramine, Dihydro-obliquin 6, 7-dihydroxy-4-oxo-2-heptenoic acid has not been reported earlier.

In conclusion acetone extract of TCM is a rich source of secondary metabolites like phenolic compounds, flavonoids. Various reported antioxidants like oxidized glutathione, caffeic acid, chlorogenic acid, epicatechin were found to be present in the extract selected for present study. Acetone extract of TCM may serve as a source for new drug development by pharmacological industry against various diseases.

ACKNOWLEDGEMENT:

We would like to convey our heartfelt thanks to Chancellor and Faculties, Department of Biochemistry, VISTAS, Chennai, for their support to complete this research article successfully.

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Received on 16.04.2019 Modified on 28.06.2019

Research J. Pharm. and Tech. 2019; 12(12): 5781-5785.

DOI: 10.5958/0974-360X.2019.01000.X