ANTIOXIDANT ACTIVITY OF NIPA (NYPA FRUTICANS) SAP

The nipa palm, sometimes known as the mangrove palm, is Nypa fruticans. The nipa palm is
quite popular in industries as it had a lot of benefits. Every part of the nipa palm will be used
fully in this worldwide. The distinctive nipa tree sap is employed extensively in a variety of
fields including health, food, biofuels, and many more. It is because the Nipa saps include
sugars, minerals, yeasts, and nutrients that can be utilized as raw materials for bioethanol
production. Furthermore, it is owing to the extremely high sugar content of nipa sap. Since it
was genuinely high in sugar, the sap of the nipa palm was utilized to produce sugar and was
very famous in the food industry (Hossain,2020). Nipa saps have a high sucrose, glucose, and
fructose content, allowing them to constantly generate rich sugar saps in large yields. The Nipa
sap was usually gathered frequently by tapping the stem to extract the sugar sap. Tapping
results produce little leftover trash and have no negative impact on nipa palm development.
Because it produces a larger output of sap, Nipa palm sap is tapped from unopened mature
palm inflorescences by chopping off the blooming head. Nipa palm sap is gathered from intact
fully developed palm inflorescences by slicing off the blooming head, pounding the new
inflorescence, and stomping the stalk's base, which stimulates a greater sap release.

As we already know, the nipa palm contained higher sugar. Sugar is a fundamental dietary
carbohydrate derived from Nypa fruticans, which are natural sweeteners, and it has been widely
employed in various food businesses for a long time to produce sugar. Thus, the sap of the nipa
palm was famously used as a localized to make sugar especially “Gula Apong”. Gula Apung
was made from the sap extracted from palm trees that are at least five years old. The sap is
collected by cutting the blossom and allowing the sap to run into bamboo containers. The
process of making “Gula Apong” localized only includes heating fresh sap in an open pan
above a wood-fired stove, stirring until a golden-brown tint resembling treacle was obtained.
Numerous chemical events, notably non-enzymatic browning reactions, occur during
prolonged heating and impact the nutritional values and sensory features of the sap. Heating
and non-enzymatic browning processes (Maillard reaction and caramelization) have been
shown to have antioxidative activity due to their radical scavenging and reducing power.

Antioxidants have become a hot issue in the world of health. Antioxidant activity is defined as
the restriction or prevention of food oxidation (particularly of lipids and proteins) via oxidative
chain reactions. A high-antioxidant diet may lower the risk of several illnesses (including heart
disease and certain cancers). Antioxidants scavenge free radicals from bodily cells and prevent
or minimize oxidative damage. Antioxidants' protective function is still being explored all over
the world. Antioxidant research in common food items has revealed that disease-fighting
qualities may be found in practically all fruits and vegetables, as well as processed foods.
Maillard reaction has played an important part in providing food a nice look and taste for as
long as it has been cooked, and it has also been demonstrated to create antioxidant components.
Just know, The Maillard reaction is also involved in the production of nipa palm. The Maillard
reaction is responsible for the creation of brown colors from the condensation of the carbonyl
groups of reducing sugars, aldehydes, and ketones, the free amino groups of lysine or other
free amino acids (such as amino acids, peptides, and proteins), or any nitrogenous substance.
Thus, this study, it was aimed to investigate the antioxidant activities of Nipa Sap in other to
provide a healthy sugar. This study was conducted by analysis of DPPH radical scavenging
activity, FRAP assay, total phenolic content (TPC), and total flavonoid content (TFC)

OBJECTIVE

To study the activities of the antioxidant nipa sap

SAMPLE EXTRACTION

CHEMICALS:

1. Methanol (400 ml)

MATERIALS AND APPARATUS:

1. Sample
2. Beaker
3. Magnetic stirrer
4. Filter funnel
5. Filter paper
6. Volumetric flask
7. Pipette and tips
8. Freezer/refrigerator (-20°C)
DPPH RADICAL SCAVENGING ACTIVITY

CHEMICALS:

1. Methanol
2. DPPH solution

MATERIAL AND APPARATUS:

1. Sample (extract solutions)

2. Distilled water (blank)
3. Weighing balance (mg unit)
4. Volumetric flask (100 ml)
5. Aluminum foil
6. Vortex
7. Test tube
8. Measuring cylinder (100 ml)
9. Micropipette and tips
10. Cuvettes
11. UV-Vis spectrophotometer
TOTAL PHENOLIC CONTENT

CHEMICALS:

1. Folin-Ciocalteu reagent
2. 2% Sodium carbonate solution
3. Gallic acid standard solution (0.1 – 2.5 mg/ml)
MATERIALS AND APPARATUS:

1. Extract solution
2. Distilled water
3. Test tube
4. Pipette and tips
5. Aluminum foil
6. Dark cabinet
7. Cuvettes
8. UV-Vis spectrophotometer
FRAP ASSAY

CHEMICALS:

1. 10 mM TPTZ (2,4,6-tripridyl-s-triazine) solution in 40 mM HCl

2. 300 mM Acetate buffer (pH 3.6)
3. 20 mM ferric chloride (FeCl3.6H2O)
4. 2 mM Trolox solution (standard curve)

MATERIALS AND APPARATUS:

1. Sample extract solutions

2. Distilled water (blank)
3. Aluminum foil
4. Micropipette and tips
5. Test tube/volumetric flask (10 ml)
6. Cuvettes
7. UV-Vis Spectrophotometer

TOTAL FLAVONOID CONTENT

CHEMICALS:

1. Methanol
2. 10% AlCl3 in distilled water solution
3. 1 M potassium acetate in distilled water solution
4. Rutin (reagent grade)
5. Ethanol (blank)

MATERIALS AND APPARATUS

1. Extract solution
2. 10 ml volumetric flask or test tube
3. Micropipette and tips
 4. Pipette and tips
5. Vortex
6. Cuvettes
7. UV-Vis Spectrophotometer

PROCEDURE

This experiment is divided into three parts: total phenolic content, DPPH radical scavenging
activity, FRAP assay and TFC. To begin with the DPPH radical scavenging activity are by To
begin, the DPPH radical scavenging activity experiment was carried out by weighing 4.728 mg
DPPH and adding 100 ml of ethanol to the powder to prepare a DPPH solution. The solution
was then kept in a cool, dark place. Following that, 400 ml of extract and 2 ml of DPPH solution
were poured into a test tube. The mixture was then vigorously shaken. Then after, the mixture
was left in the dark at room temperature for 30 minutes. The absorbance was measured four
times using UV-VIS at 517nm. Afterwards, a blank solution was prepared in the same manner,
but with distilled water instead of sample. The DPPH activity was then calculated using the
following formula: % = [(A-A1/a0)] × 100.For the next procedure which is the Frap Assay. A
FRAP reagent was freshly prepared containing 10 mM TPTZ solution in 40 Mm HCI ,300mM
acetate buffer and 20mM FeCl3. Next, a 150 ml of the extract were mixed with 2859 ml of
FRAP reagent. The mixture was then measured at 593 nm using UV-VIS.A standard curve was
prepared by using Trolox ranging from 50 to 400 mM.The result was then expressed as mmol
TE/ dry weight. Finally, an experiment procedure of total phenolic content was conducted by
diluting 0.5 ml of the extract with 0.5 ml of distilled water. A 0.5 ml of Folin-Ciocalteau reagent
and 2.5ml of 2% sodium carbonate solution were added. The mixture were mix thoroughly
and was placed in the dark for 40 minutes. Next, the absorbance was measured at 725nm with
a spectrophotometer. A blank was prepared in the same manner except the sample is substituted
with distilled water. Gallic acid was used as the standard. The standard curve of gallic acid was
used to calculate the TPC.Lastly, the total flavonoid content were conducted by diluting
500microlitre of extract with 2.5 ml of water. Then 150 microliter of 5% NANO2 solution were
added into the solution. The mixture was left to stand for 6 min at room temperature.300
microliter of 10% ALCL3.6H20 were also added and left to stand for another 5 minutes.1 ml
of 1M NAOH was added together with 550 microliter of water into the mixture and were vortex
thoroughly. The absorbance was then measure with 510 nm with rutin as reference standard.
DATA AND RESULTS

i. DPPH Activity

ABSORBANCE DPPH ACTIVITY (%)

SAMPLE & TRIAL TRIAL TRIAL MEAN
GROUP 1 2 3
NYPA SAP 0.8572 0.6383 0.7552 0.7502 7.4855

ii. FRAP

ABSORBANCE FRAP value (mg FeSO4/g sample)

SAMPLE & GROUP TRIAL 1 TRIAL 2 TRIAL 3 MEAN 0.0572
NYPA SAP 0.074 0.0734 0.073 0.074

FeSO4.7H2O Standard
concentration (mM) Absorbance
0 0
0.2 0.262
0.4 0.514
0.6 0.819
0.8 1.078
1 1.315

Frap FeSo4.7H2O Standard

1.4
1.3
1.2
1.1
1
0.9
Absorbance

0.8
0.7
0.6
0.5
0.4
0.3
0.2 0.074
0.1
0
0 0.2 0.4 0.6 0.8 1 1.2
Concentration (mM)
iii. TFC

ABSORBANCE
SAMPLE & GROUP TRIAL 1 TRIAL 2 TRIAL 3 MEAN TFC value (mg RE/g DW)
NYPA SAP 0.669 0.679 0.669 0.672 57.205

Rutin standard solution

concentration (mg/ml) absorbance
0 0
10 0.123
20 0.235
30 0.361
40 0.496
50 0.579
60 0.693

TFC Rutin Standard solution

0.8
0.672
0.7
0.6
Absorbance

0.5
0.4
0.3
0.2
0.1
0
0 10 20 30 40 50 60 70
Concentration (mg/ml)
 iv. TPC

ABSORBANCE
SAMPLE & GROUP TRIAL 1 TRIAL 2 TRIAL 3 MEAN TPC value (mg GAE/100 g sample)
NYPA SAP 0.194 0.194 0.194 0.194 17.565

Gallic acid standard solution:

concentration (mg/ml) Absorbance
0 0
10 0.119
20 0.235
30 0.354
40 0.414
50 0.491
60 0.602

TPC Gallic acid standard curve

0.7

0.6

0.5
Absorbance

0.4

0.3
0.194
0.2

0.1

0
0 10 20 30 40 50 60 70
Concentration (mg/ml)
Nipa palm sap, also known as nipa sap, is a watery fluid that is phloem sap generated by the
nipa plant and is used to transfer nutrients throughout the plant. Furthermore, once the floral or
fruit heads have been removed, nipa releases an abundance of sap from the chopped stalks of
fully developed young inflorescences. This Nipa sap is commonly used to create sugar and
vinegar. It can also be drunk fresh. Because it is utilized as food, it is worthwhile to investigate
the antioxidant activity of nipa sap. It is because antioxidant-rich foods are beneficial to our
health. Analysis of antioxidant activities always be used to assuring the quality of functional
meals, and, more crucially, research the effectiveness of dietary antioxidants in preventing and
treating oxidative stress-related disorders. Before starting the analysis of the antioxidant
activity, the sample was prepared first by extraction. This sample will be used until the end of
this experiment. The sample was mixed with methanol solvent using magnetic stirring and was
filtered with Whatman No. 4 filter paper. The extracted sample was stored at -20℃ in the dark
until the end of the analysis. This extracted sample needs to store in dark to avoid the possibility
it will occur the photosynthesis reaction since it is sensitive to light.

The first analysis of antioxidant activities is DPPH radical scavenging activity. The DPPH free
radical scavenging mechanism is a well-established method for testing the antioxidant activity
of plant extracts. In this analysis, a slightly modified approach published by Azlim et al. (2010)
was used to determine the free radical-scavenging activity of samples on DPPH radical. The
DPPH solution was made at first by mixing the 4.728 mg powder of DPPH with 100 mL
methanol and stored in the dark, cool solution. The DPPH solution needs to be stored in a dark
condition because it is sensitive to light which may interfere with the results as the light would
oxidize the DPPH radical in the solution to be tested. There are two samples were prepared
which are the blank solution (DPPH + methanol) and the mixture of the extracted sample with
DPPH solution. The sample was left in the dark at room temperature for 30 minutes. The blank
solution was prepared as a negative control in this experiment. Thus, when calculating the
DPPH activity, the normal result should get is positive. A UV-VIS spectrophotometer was used
to measure the absorbance of the resultant solution at 517 nm. Due to its odd electron, DPPH
exhibits a significant absorption band at 517 nm, and the solution appears a deep violet color;
however, the absorption fades when the electron pairs off. We calculate the mean which we
get is 0.7502. The absorbance read for the blank solution is 0.8109. By using the formula below,
we can obtain the percent of DPPH activity for the Nipa Sap which is 7.4855%.

𝐴0 − 𝐴1
𝐷𝑃𝑃𝐻 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 % = × 100
𝐴0
DPPH is one of the chemicals that contains a proton-free radical with a distinct absorbance that
lowers dramatically when exposed to proton radical scavenging. The production of the non-
radical form, DPPH-H, is responsible for the decrease of alcoholic DPPH solution in the
presence of a hydrogen-donating antioxidant. When antioxidants scavenge the DPPH-radical
by donating hydrogen to produce a stable DPPH-H molecule, the color changes from purple to
yellow. When compared to glucose, the Maillard reaction products and caramelization products
from fructose had the strongest radical-scavenging activity. Sugar, on the other hand, is mostly
composed of sucrose, which contains glucose and fructose. As a result, the higher the reduced
sugar is, the higher the percentage of scavenging activity. Generally, when comparing the type
of sugar that extracts from plants such as cane and nipa. Sugar cane has a high percentage of
DPPH which is in the range of 20-35% while sugar from nipa has a range of DPPH which is
5-10%. Thus, for our data, we obtained the DPPH activities is 7.4855% which is in the range
of nipa palm sugar. Then, the sap of the nipa palm can act as an antioxidant through the
donation of a hydrogen atom. In addition, when compared with other sugar from other plants,
the nipa palm has the second higher activity of DPPH.

FRAP assays are ferric reducing antioxidant power. In comparison to other assays that evaluate
free radical inhibition, the FRAP test is the only one that directly analyses antioxidants (or
reductants) in a sample. The principle of the FRAP is the test was based on a compound's
reducing power (antioxidant). A potential antioxidant converts the ferric ion (Fe3+) to the
ferrous ion (Fe2+), which produces a blue complex (Fe2+/TPTZ) and boosts absorbance at 593
nm. The FRAP reagent was prepared freshly before starting the experiment. The fresh FRAP
reagent needs to be put in the incubate at 37℃ for 30 minutes. The FRAP reagent needs to be
put in the incubate for 30 minutes because at 37℃ electron transport from the sample to the ion
is favored. The extracted sample was mixed with the FRAP reagent and the absorbance was
recorded by using UV-VIS spectroscopy. The absorbance reading, we obtained for this FRAP
data is 0.074, the second is 0.0734 and the third is 0.073. The mean for the data we get is 0.074.
The FRAP value we obtain is 0.0572. This value was obtained by preparing a standard curve
using Trolox ranging from 50 to 400 µM.

Total phenolic content (TPC) is the next step in the analysis of antioxidant activity. To
determine the presence and amount of phenolic compounds in each sample, the total phenolic
content (TPC) test utilizing the Folin-Ciocalteu technique was utilized. The Folin-Ciocalteu
technique is an electron transfer-based test that provides reducing capability as phenolic
content. In general, the solubility of phenolic compounds is determined by the polarity of the
solvent. This approach works based on the creation of complex blue chemicals that may be
detected at a wavelength of 725 nm. The diluted extracted sample with distilled water was
added with Folin-Ciocalteau reagent and sodium carbonate solution. For 40 minutes, the
mixture was put in a dark place. By using UV-VIS spectroscopy, the absorbance of the TPC
was recorded. For this data, all data we obtained is 0.194 with a mean of 0.914. The TPC value
is 17.565. The value of this TPC got from calculated from the standard curve of gallic acid.
Because DPPH radical scavenging activity was highly connected to total phenol content, total
phenol content rose as DPPH radical scavenging activity increased. It shows that the phenolic
does exist in the nipa sap.

The last analysis of antioxidant activities is total flavonoid content, TFC. The aluminum
chloride colorimetric test was used to determine the total flavonoid concentration. Because of
their propensity to donate hydrogen atoms to free radicals, flavonoid molecules are key
antioxidant components that are responsible for deactivating free radicals. The essential
premise of the aluminum chloride colorimetric approach is that aluminum chloride forms acid
stable complexes with flavone and flavonol C-4 keto groups and either the C-3 or C-5 hydroxyl
groups. The extracted sample was diluted with water and mixed with sodium nitrate,
AlCl3.6H2O, and sodium hydroxide, water. The absorbance was measured at 510 nm with rutin
as the reference standard. The reading of the absorbance we get for the first trial is 0.669, the
second is 0.679 and the third is 0.669. The mean data we get is 0.672. By plotting the data, the
value TFC we get is 57.205. The result of TFC was believed to be influenced by both
endogenous and exogenous polyphenols. Endogenous polyphenols and flavonoids collected in
the nipa palm inflorescences can be released into the sap after harvesting. Exogenous
polyphenols can be obtained from wood pieces that were purposefully added as a natural
preservative for fresh sap. This indicates that the nipa sap contains a flavonoid which is a
chemical antioxidant. From the data, nipa sap had a higher flavonoid than phenolic.
CONCLUSION

In conclusion, the nipa sap of the palm tree shows a positive reaction to antioxidant activity.
Sugar from the nipa palm can be used to replace other sugar in our diet. The DPPH activity of
the nipa sap is 7.4855%. For FRAP, it is was 0.0572. TPC and TFC got 17.565 and 57.205
respectively. It is shown that the nipa sap does have antioxidant activities and contains a
compound of antioxidants. Antioxidants are compounds that can prevent or reduce cell damage
caused by free radicals, which are unstable molecules produced by the body in response to
environmental and other stressors. Antioxidants are considered to assist our bodies to neutralize
free radicals, which is thought to improve general health. Thus, Sugar, particularly nipa palm
sugar derived from sap, might be employed as an antioxidant-rich sweetener in food
preparation.