Phytochemical Profiling and Antioxidant Potential of Coconut Inflorescence Sap

Background: The coconut palm is so much more than just a tree; because all of this is essential with medicinal benefits. Coconut inflorescence sap (CIS) is the sweet, oyster-white colored, non-fermented juice tapped from the immature coconut spadix. Coconut products are widely used in Indian folk medicine for their effects on hemorrhaging, bronchitis, antimicrobial, radical scavenging, analgesics, anti-inflammation, anthelmintics, and immunotherapies. Many studies reported the fantastic health benefit of the coconut palm tree. The coconut inflorescence has cytoprotective and antihyperglycemic properties [Renjith, R.S. et al ., 2013]. But not many systematic studies have been conducted on Coconut Inflorescence Sap. The objective of the present study is to analyze the nutritional components, detect the mineral composition, perform preliminary phytochemical screening, and find out biologically active compounds using FTIR Spectrum analysis and LCMS analysis. Materials and Methods: CIS’s nutritional components and mineral content analysis were done following the standard procedures. The phytochemical screening, FTIR Spectrum analysis, and LCMS analysis are also done to confirm the health benefits of CIS. Results: CIS exhibited a significant amount of micro and macronutrients. The Phytochemical analysis also showed the presence of flavonoids, alkaloids, terpenes, steroids, phenols, and glycosides. The LCMS Spectrum analysis of CIS shows the presence of biologically significant compounds, namely 4-HydroxyCoumarin, P-Coumaric acid, Mellein, Leucopelargonidin, Coumarin, 3 Caffeoylquinic acid, Ferulic acid 4-O-Glucosidase and also three essential aminoacids L-Phenylalanine, L-isoleucine and D-Tryptophan. Conclusion: The nutritional composition, active phytochemical constituents, and polyphenolic compounds of CIS showed that CIS has a significant beneficial effect in upgrading the antioxidant status. CIS is a natural drink with effective antioxidant potential. Concerning the nutrient composition, mineral and amino acid composition, and scavenging assays, it would be possible to exploit this natural drink in clinical therapy.


Nutritional Analysis
The nutritional analysis was analyzed based on the A.O.A.C. lnternational 21 st Edition 2019 and quantified the total fat, total ash, total protein, total energy, and total carbohydrates present in 100ml of the sample.

Mineral Content Analysis
The minerals detection was done using an inductively coupled mass spectrometry system (Thermo Scientific ICAP Qc).

Qualitative Phytochemical Analysis
The naturally occurring active secondary metabolites in plants constitute phytochemicals.(The word Phyto in Greek indicates "plant"), and are responsible for color and biological properties.The phytochemical means those chemicals that may have biological significance but are not established as essential nutrients.The CIS was analyzed for the presence of Alkaloids, Phenols, Flavonoids, Terpenes, Steroids, and Glycosides.

DPPH Radical Scavenging Assay
DPPH radical scavenging activity was analyzed following the procedure of Blois, 1958. 12Ascorbic acid was used as a standard and the IC 50 of the extracts was compared with the standard.The lower IC 50 value indicates higher free radical scavenging activity.

Hydrogen Peroxide Radical Scavenging Activity
The H 2 O 2 scavenging ability of the CIS was determined by the method of Ruch et al., 1989. 13Phosphate buffer devoid of H 2 O 2 was used as blank.Quercetin was used as the standard and the IC 50 of the extract was compared with the standard.

FTIR Spectrum of CIS
Fourier transform infrared spectroscopy (FTIR) is the method used to identify the chemical bond or functional group.CIS was loaded in the FTIR spectrometer (Perkin-Elmer LS-5S-Luminescence Spectrometer).The measurement was taken at ATRA mode at room temperature.(Range from 400 cm −1 -4000 cm −1 ), and changes in the spectral pattern were analyzed and noted.

Determination of Phenolic Compounds of CIS using LCMS Analysis
LCMS analysis was performed using SHIMADZU LC MS-8045.To identify the active components, the particulate matters are to be separated by centrifugation for 20 min at 4000rpm, followed by sterilization using a filter of 0.22 μm.The filtrate thus obtained was dried by lyophilization.The final powdered material (1 mg) was dissolved in 1 ml of ultrapure water and prepared in a stock solution (1 mg ml −1 ) to detect the active constituents.

Nutritional Composition of CIS
The nutritional composition of CIS is depicted in Table 1 and it showed that CIS possesses an excellent macronutrient composition with a total energy of 57.4Kcal /100ml.

Mineral Composition of CIS
CIS showed the presence of micronutrients Iron and Zinc in significant quantity and also showed the presence of other essential minerals as shown in Table 2.

Preliminary Phytochemical Analysis
The phytochemical characteristics of Coconut Inflorescence Sap (CIS) were summarized in Table 3.The results showed the presence of bioactive molecules such as flavonoids, alkaloids, glycosides, phenols, and tannins were present in CIS.

Antioxidant Assays
Antioxidant potential of CIS was determined by DPPH assay, Hydroxyl radical scavenging assay and Superoxide radical scavenging assay.Figure 1 shows the DPPH radical scavenging activity of CIS and the standard.The IC 50 of CIS was 2.92 μg/mL, and ascorbic acid (standard) was 4.35 μg/mL Figure 2 shows that CIS's hydroxyl radical scavenging activity was dose-dependent.The IC 50 of CIS was 35.29 µg/ ml, and GA was 14.69 µg/ ml, indicates that the scavenging activity of CIS was adequate and comparable to that of the standard GA Figure 3 represents the abilities of the CIS and the standard quercetin to scavenge superoxide radicals from the reaction medium.As depicted

FTIR Spectrum Analysis of CIS
The FTIR Spectrum of CIS is depicted in Figure 4, and the prominent functional group present in CIS is illustrated in Table 4.

Characterization of Phenolic Compounds of CIS using LCMS Analysis
The LCMS Spectrum analysis of CIS in Figure 5 showed the presence of biologically significant compounds, as depicted in Table 5.       product, diphenylpicryl hydrazine, by adding the extract concentrationdependent.The method requires a very short period for analysis and is thus extensively used for antioxidant activity assays.The degrees of discoloration of DPPH by its reduction indicated the radical scavenging activity of CIS.This study revealed that the CIS had a similar free radical scavenging activity to ascorbic acid.The IC 50 value of CIS was 4.35 µg/ml and standard ascorbic acid 2.92 µg/ ml, demonstrating that the inhibitory activity of CIS was higher than the standard.The results obtained in this study indicate that CIS possess radical scavenging activity due to their hydrogen donating or electron transfer or ability.In the present study, CIS showed significant antioxidant activity and inhibition percentage.The present study suggests that CIS contains phytochemical constituents capable of donating hydrogen atoms to free radicals and thus alleviating the potential damage.The ability of CIS and the standard to scavenge superoxide radicals from the reaction mixture are indicated by the decrease in absorbance at 560 nm.The IC 50 values of CIS and the standard on SO 2-scavenging activity were 45.785 μg/mL and 32.20 μg/mL, respectively.At the pathogenesis, biomolecules are damaged directly or indirectly by superoxide radicals due to the formation of peroxyl nitrite, H 2 O 2 , singlet oxygen, orˉˉOH.Superoxide anions now initiate lipid peroxidation.The PMS-NADH system assayed the superoxide anion radical scavenging activity of CIS.SO 2-scavenging activity of CIS was increased significantly with the increase in concentrations.Thus, the increased inhibitory effects of the CIS on radical formation suggest the potential of CIS as an effective antioxidant.The FTIR spectrum shows a broad spectrum 3261cm -1 corresponds to X-H stretch (X is C, O, N), indicating the presence of alkanes, hydroxyl group, or imides.The C= C stretch at 1636cm -1 is due to the presence of alkenes.The C-N stretch observed at 1054cm -1 indicates the presence of aliphatic amines.The peaks 996 and 572 indicate the presence of ethers and alkyl halides.The LCMS Spectrum analysis of CIS shows the presence of biologically significant compounds, namely 4-Hydroxy Coumarin, P-Coumaric acid, Mellein, Leucopelargonidin, Coumarin, 3 Caffeoylquinic acid, Ferulic acid 4-O-Glucosidase and also three essential aminoacids L-Phenylalanine, L-isoleucine and D-Tryptophan.The studies confirmed the presence of biologically active compounds with protective efficacy.Coumarin and its derivative, 4-hydroxycoumarin, p-coumaric acid was reported to have significant roles in alleviating inflammation.Geranylnaringenin is also reported to have potential antioxidant and anti-inflammatory effects.
In short, the results of this study scientifically validated the nutritional composition, phytochemical constitution, and antioxidant potential of the natural drink Coconut Inflorescence Sap.The regular consumption of CIS may protect the body from various chronic diseases, especially where oxidative stress plays a key role.

CONCLUSION
The findings of the present study confirm the nutritional importance and health benefits of CIS.The phytochemical analysis showed the presence of flavonoids, alkaloids, terpenes, steroids, phenols, and glycosides.The antioxidant assays, mineral content analysis, FTIR spectrum and LCMS analysis suggest that CIS is an effective nutritional drink to be exploited in clinical therapy.So further in vivo and in vitro studies are essential to elucidate the biochemical effects of the natural drink in alleviating oxidative stress and inflammation.

DISCUSSION
Phytochemicals are secondary metabolites obtained from plants having a protective role in preventing diseases.Preliminary phytochemical screening of CIS indicates the presence of significant phytochemicals such as flavonoids, alkaloids, phenolics, terpenes, steroids, and glycosides.The antioxidant assays also indicate that CIS possesses high antioxidant potential comparable to standard antioxidants.The biochemical analysis of CIS elevates the importance of CIS as a health drink.CIS was subjected to analysis of mineral contents and identified the presence of 15 elements.Magnesium, an essential element, is present in ample quantity in CIS.The presence of Cobalt, a vital trace element, Iron, Potassium, and Sodium in a significantly better portion.The present investigation observed that CIS has significant amounts of alkaloids, phenolic compounds, glycosides, and flavonoids.Antioxidants have the property of scavenging free radicals, offering resistance to oxidative stress and inhibiting lipid peroxidation. 14Antioxidant activity is expressed in IC 50 , and a lower IC 50 value corresponds to a more significant scavenging activity. 15The beneficial effects of antioxidants are due to their ability to provide hydrogen atoms to DPPH.The scavenging activities of natural antioxidants can prevent the toxic effects of free radicals.The antioxidant activity of CIS was analyzed by DPPH free radical scavenging assays.In the assay, the violet color DPPH solution was reduced to a yellow-colored

Figure 5 :
Figure 5: Characterisation of Phenolic compounds of CIS using LCMS analysis.
O2-scavenging activity of the CIS was analyzed by Fontana et al., 2001.Phosphate buffer devoid of the CIS served as control.IC 50 of the extracts was compared with quercetin.

Table 4 : List of functional groups in FTIR Spectrum of CIS. FTIR Peak Values Functional group 3261
X-H stretch (X is C, O, or N) 1636 C=C stretch(alkene) 1054 C-N stretch(aliphatic Amines) 996 C-O(ethers) 572 C-Br(alkyl halides)