Determination of Total Phenolic Content and Total Flavonoid Content of Nutmeg Flesh (Myristica Fragrans Houtt) Ethanol Extract from South Aceh Province

The nutmeg plant (Myristica fragrans Houtt) is often used as a medicinal ingredient and has medicinal efficacy. Nutmeg samples were obtained from South Aceh district, which is the main commodity obtained in the area. This study aims to measure the total phenolic content (TPC) and total flavonoid content (TFC) of the ethanol extract of nutmeg flesh. The dried nutmeg flesh was extracted (maceration) using an ethanol solvent, and the extract weight was 18.35%. The TPC and TFC analysis use the UV-visible spectrophotometer method and are measured at wavelengths of 759 nm and 495 nm, respectively. Gallic acid standards were used to measure total phenolic levels and quercetin standards to measure total flavonoid levels. Data of linearity, R square (R2), LoD, and LoQ were also calculated for each standard. The TPC and TFC contents in the ethanol extract of nutmeg flesh were found to be 76.972 ± 0.97 mg GAE/g and 15.625 ± 0.26 mg QE/g respectively. The presence of phenolic compounds and flavonoids shows that the ethanol extract of nutmeg flesh has high value metabolites that show good biological activity.


Introduction
The nutmeg plant (Myristica fragrans Houtt) is a traditional Indonesian spice plant which has medicinal efficacy.Today, nutmeg is still the dominant tree in Indonesia, grows well in the mountains on height 0-700 meters above sea level [1].South Aceh Regency is one of the largest producer nutmeg in Indonesia.In South Aceh Regency, nutmeg is easy to find and its become main agriculture commodity [2].Based on Department data Forestry and Aceh Plantations in 2020, area plant nutmeg in South Aceh Regency reached 16.898 hectare with results harvest up to 5.362,5 dry tons [3].
Nutmeg contains flavonoid and phenolic compounds [4].The phytochemical content of nutmeg extract contains secondary metabolites such as alkaloids, flavonoids, saponins, tannins, phenols and terpenoids [5].The flavonoid and phenolic content has pharmacological activity such as active substances as antibacterial [6]- [7], antimicrobial [8]- [9], antioxidant, antifungal and anti-inflammatory [10]- [11].Until now, complete data on the content of active compounds in nutmeg originating from the South Aceh region has not been found.Several researchers have carried out antioxidant and anticancer activities of nutmeg extract [9]- [12]- [13], antioxidant activity of nutmeg oil [14], phytochemical screening of n-hexane extract of nutmeg stems [15] and identification of flavonoid compounds from nutmeg leaves [16].Therefore, in this study we were interested in determining the total flavonoid content and total phenolic content using UV-visible spectrophotometer method of ethanol nutmeg flesh extract from South Aceh district that has never been carried out before.Data of total phenolic content that obtained is expressed as QE (quercetin equivalent), that is the equivalent number of standard milligrams of quercetin in gram samples.And data of total flavonoids content that obtained is expressed as GAE (galic acid equivalent), that is the equivalent number of standard milligrams of gallic acid in gram samples [17].

Nutmeg samples
Nutmeg samples were collected from plantations at Sawang Village of South Aceh Regency.The flesh part of the nutmeg weighed as much as ± 2 Kg, washed, thinly sliced and dried away from direct sunlight for 7 days.After dried, the nutmeg flash was ground into powder.Its called simplicia and ready for analysis.

Nutmeg Flesh Extraction
100 grams of simplicia were weighed and extracted (maceration) for 72 hours (3 days) using 1000 mL of 96% ethanol solvent.After the maceration process, the extract was filtered using a Buchner funnel and filter paper.The filtered extract is subjected to a rotary evaporator to obtain a thick extract and then used for analysis.The extract that obtained is calculated by using a formula.

Determination of total phenolic content (TPC)
Standard of gallic acid was reacted with reagent Folin-Ciocalteu (1:10 v/v water) and Na 2 CO 3 solution (7.5 % w/v water).Standard solution gallic acid was prepared with concentration 10.0; 20.0; 30.0; 40.0; and 50.0 µg/mL with a final volume of 100 mL.And than, its measured their absorbance on wavelength maximum 759 nm, created curve calibration connection between concentration of gallic acid (µg/ml) with absorbance [18].
Total phenolic content extract of nutmeg flesh determined based on Wulandari et.al. [19] method.Extract of nutmeg flesh weighed 20 mg and dissolved in ethanol to 10 mL, with tripplecate experiment.100 µL sample solution and 500 µL of Folin-Ciocalteu (1:10 v/v water) was added and incubated for 6 minutes, and than added 400 µL Na 2 CO 3 (7.5% w/v water) [20].Absorbance was measured on wavelength 759 nm using UV-visible spectrophotometer.Absorption sample calculated based on equality regression standard of gallic acid to produced total phenolic content as percentage equivalent of gallic acid with w/w (% w/w GAE) [19].

Determination of total flavonoid content (TFC)
Quercetin standard dissolved in ethanol.Quercetin standard solution was prepared with concentration 4.0; 6.0; 8.0; 10.0; and 12.0 µg/mL with a final volume of 10 mL.Standard solution was added AlCl 3 10% solution and 1 M Potassium acetate.And its measured their absorbance on wavelength maximum 495 nm, curve calibration made with connecting between concentration of quercetin (µg/mL) with absorbance [18].
Total flavonoid content extract of nutmeg flesh determined based on Antasionasti et.al. [21] method.The extract of nutmeg flesh weighed 20 mg and dissolved in ethanol to 10 mL, with tripplecate experiment.Added 1 mL of extract nutmeg flesh solution with 4 mL of distilled water, and than added 0.3 mL of 10% NaNO 2 and incubated for 6 minutes.After incubated, added 4 mL AlCl 3 10% up to a volume of 10 mL.Absorbance extract was measured on UV-visible spectrophotometer at wavelength 495 nm.The total flavonoid content was stated as mg equivalent quercetin mg/mL extract.The total of flavonoid content in sample is stated as gram quercetin equivalent (QE)/100 gram sample (%w/w QE) [21].

Data analysis
Data TPC and TFC are presented as mean concentration ± standard deviation (SD) with tripplecate experiment for every sample.The data was analysis using linear regression with y = ax + b, R square (R 2 ), limit of detection (LoD), and limit of quantitation (LoQ) was found in calculation using Microsoft Excel 2010 [22].

Results and discussion
In this study using 96% ethanol solvent for the maceration process.The choice of ethanol solvent used is due to its polarity which can extract polar components in samples such as phenolic and flavonoid compounds [17].50.33 grams dried sample of nutmeg flesh powder (simplicia) was used and its extracted using 500 mL of 96% ethanol solvent, obtains 9.24 grams of ethanol extract or an extraction yield percentage of 18.35%, as shown in table 1.Based on the results, ethanol solvent can dissolve polar compounds in the metabolites of nutmeg flesh [23].Analysis of total phenolic and flavonoid levels using UV-visible spectrophotometer method was measured with wavelengths of 759 nm and 495 nm respectively [17].Gallic acid standards were used to measure total phenolic levels and quarcetin standards to measure total flavonoid levels.Linearity data, R square (R 2 ), LoD and LoQ for each standard are shown in table 2 and the calibration curve is shown in figure 2. Based on the calibration data, TPC and TFC calculations were then carried out in the ethanol extract of nutmeg flesh.The TPC and TFC contents in the ethanol extract of nutmeg flesh are 76,972 ± 0.97 mg GAE/g and 15,625 ± 0.26 mg QE/g respectively, shown in table 3. The results of this analysis are calculated based on the linearity equation of the calibration standard that has been measured and expressed in grams of extract equivalent to milligrams of Gallic Acid Equivalent (mg GAE/g) and milligrams of Quercetin Equivalent (mg QE/g).Research by Gupta et.al. [24] showed that the total phenolic content of the ethanol extract of nutmeg flash reached 70.69 ± 2.06 mg GAE/g and a high total phenolic content was found in the methanol extract of nutmeg flesh base on studied by Assa et.al. [25] with a content reaching 388.36 ± 30.85 mg GAE/g.The total flavonoid content in the ethanol extract of nutmeg flesh was also reported by Makanaung et.al. [26] with a total concentration of 9.99 mg QE/g.The differences in phenolic and flavonoid content illustrate the diversity of nutmeg soil chemical constituents as a result of environmental conditions [27].Phenolic compounds and flavonoids are high-value plant metabolites that exhibit a wide range of biological activities, including antimicrobial, antifungal and anticancer [28].The high phenolic content is not harmful to human's health, the use of plants with high phenolic amounts in the food industry aiming to improve food quality [29].One of the utilization of nutmeg is often found in syrup products as herbal drinks [30]- [31].High phenol and flavonoid content shows a good correlation to antioxidant activity [32].Research by Antasionasti et.al. [21] nutmeg extract has antioxidant activity that is classified as strong with the antioxidant activity value obtained of 105.669 μg/mL.The presence of phenolic compounds and flavonoids in the ethanol extract of nutmeg flesh shows that there are many compounds that are polar in nature [33].The total phenolic and flavonoid content in the ethanol extract of the nutmeg flesh studied was different from previous research, this could be caused by geographical, climatic and genetic factors [34].

Conclusions
The TPC and TFC contents in the ethanol extract of nutmeg flesh were 76.972 ± 0.97 mg GAE/g and 15.625 ± 0.26 mg QE/g respectively.The presence of phenolic compounds and flavonoids compounds indicates that a plant has high-value metabolites that exhibit various biological activities.The amount of extract produced reached 18.35%, indicating that the compounds contained in the nutmeg flesh extract are relatively polar.

Figure 1 .
Figure 1.Nutmeg samples from plantation at South Aceh District.

Table 1 .
Extraction yield of nutmeg flesh ethanolic.

Table 2 .
Linearity data for gallic acid and quercetin standards.