ALKIMIA : Jurnal Ilmu Kimia dan Terapan View Article Online
ARTICLES
OPEN
ACCESS
View Journal | View Issue Effect of Temperature on Antioxidant Activity of Phaleria macrocarpa Fruits Via Green Hydrothermal Extraction Karinda Septiyaningruma.
Fahmi Hidayata*.
Aulia Dewi Rosantia.
Muhammad Alwi Syaharaa Abstract.
Phaleria macrocarpa (God's Crow.
is a medicinal plant rich in bioactive compounds, including minerals, vitamins, alkaloids .
uch as vincristin.
, and flavonoids.
These compounds have shown potential in treating various ailments.
This study aimed to optimize the extraction of bioactive compounds, particularly flavonoids and phenols, from P.
macrocarpa fruit.
Various extraction methods were employed, including hydrothermal extraction at different temperatures and decoction.
The resulting extracts were analysed for total flavonoid and phenol content and antioxidant activity.
Colorimetric methods, using AlClCE and Folin-Ciocalteu reagents, were used to determine the respective contents.
Antioxidant activity was assessed using the DPPH assay.
Hydrothermal extraction at 175AC provided the highest levels of flavonoids .
04 mg QE/1% extrac.
total phenols .
12 mg GAE/1% extrac.
and antioxidant activity .
73%).
However, further increases in temperature led to a decline in these parameters, indicating thermal degradation of heat-sensitive bioactive Keywords : Antioxidant activity, phenols, flavonoids, phaleria macrocarpa.
Program Studi Kimia Ae Universitas Islam Kadiri.
Kediri-64128.
Indonesia Correspondence and requests for materials should be addressed to Fahmi Hidayat .
mail: fahmihidayat@uniska-kediri.
ARTICLES | No.
2 Vol.
9 : 74-80 .
| DOI : https://doi.
org/10.
19109/ktzvbv78 ALKIMIA
ARTICLES
Introduction Phaleria macrocarpa is a plant with promising health benefits.
It exhibits antioxidant properties, protecting cells from damage, and may also have anti-inflammatory and immuneboosting effects .
, .
The potent bioactivity shown by P.
macrocarpa is due to abundant secondary metabolites, notably flavonoids and phenols.
These compounds have been welldocumented for their potent antioxidant properties, enabling them to neutralize free radicals and protect cells from oxidative damage .
Recent analyses have confirmed exceptionally high levels of these compounds in P.
macrocarpa fruit.
For example, one study found that oven-dried P.
macrocarpa fruits contained approximately 55.
mg gallic acid equivalent (GAE)/g total phenolics 5 mg quercetin equivalent (QE)/g flavonoids, yielding about 84.
5% inhibition in a DPPH antioxidant assay .
Based on Pratiwi's research .
, the flavonoid extract from P.
fruit has shown significant potential in reducing IL-17A levels, comparable to the commonly used endometriosis drug, dienogest .
Likewise, aqueous soxhlet extracts of the fruit were reported to have very high flavonoid content .
9 mg QE per 100 mL extrac.
In the context of phenolic compounds, the extraction method significantly influences their yield.
These findings indicate that P.
macrocarpa fruit is a potent source of natural antioxidants .
henolic and flavonoid compound.
that can scavenge free radicals and support cellular health.
The use of organic solvents in extraction, while effective in isolating various compounds, is frequently associated with toxicity and environmental hazards.
Organic solvents like methanol, ethanol, and hexane can be detrimental to human health and pollute the environment if not handled appropriately.
On the other hand, water provides a safer and more sustainable option for Water-based extraction methods significantly reduce contamination risks and are simpler to manage .
, .
, .
In particular, hydrothermal .
ubcritical wate.
extraction has several significant advantages over other extraction One of its primary advantages is its ability to extract bioactive compounds at high temperatures and pressures, which can increase extraction efficiency, accelerate the process and solubilize a wide range of phytochemicals .
, .
, .
Additionally, this method is often used Jurnal Ilmu Kimia dan Terapan due to water's ability to have selective polarity, allowing water to act as a polar or non-polar solvent depending on the applied conditions.
Under certain conditions, water can behave similarly to ethanol or methanol and can extract various phytochemicals from plant biomass, including medicinal plants .
, .
, .
Indeed, multiple studies report that subcritical water extraction yields higher total phenolic and flavonoid contents than conventional methods .
Such green extraction approaches improve efficiency and selectivity while avoiding toxic solvent use.
To date.
macrocarpa fruit extracts have largely been prepared with organic solvents or fixed drying conditions, and no studies have systematically optimized hydrothermal extraction conditions for this material .
, .
In particular, the effect of extraction temperature on the yield of antioxidant phenolics and flavonoids from P.
macrocarpa fruit has not been examined.
Optimizing this parameter is scientifically important, because identifying the ideal hydrothermal temperature will maximize recovery of valuable antioxidants while minimizing degradation .
This study aims to optimize the extraction of flavonoids and phenols from P.
macrocarpa fruit.
Experimental Materials and Equipment.
The materials used in this study included powdered P.
fruits obtained from Balai Materia Medica.
Batu.
The plant material was authenticated at the UPT Lab.
Herbal BMM.
Batu.
The chemical used included ethanol (Merc.
, distilled water, concentrated hydrochloride acid (HCl, 37%.
Merc.
Magnesium powder (Merc.
NaOH (Merc.
Folin-Ciocalteu reagent.
AlCl3 (Merc.
CH3COONa (Merc.
, 1,1diphenyl-2-picrylhydrazyl (DPPH.
Smart La.
, gallic acid standard (Sigm.
, and quercetin standard (Sigm.
The equipment used included an analytical balance (US Solid Precision Balanc.
, hydrothermal reactor .
tainless-steel autoclave using inner teflon 100 mL capacit.
, whatman No.
1 filter paper, a set of laboratory glassware.
Quantitative analysis was performed using a UV-Vis Spectrophotometer (Shimadzu UV-T.
Hydrothermal Extraction.
Hydrothermal extraction was carried out following an adaptation of previous studies on subcritical water extraction of ARTICLES | No.
2 Vol.
9 : 74-80 .
| DOI : https://doi.
org/10.
19109/ktzvbv78 ALKIMIA
ARTICLES
medical plants .
, .
, with modifications to accommodate P.
macrocarpa samples.
This method has been widely applied for plant-based antioxidant extraction, although this is the first systematic optimization for P.
macrocarpa using this Four grams of powdered plant were mixed with 60 mL of distilled water in a hydrothermal reactor.
The mixture was heated at temperatures of 150AC, 175AC, and 200AC for 30 After cooling to room temperature, the resulting extracts were filtered through whatman No.
1 filter paper and stored at 4 AC for subsequent analysis.
Decoction Method.
A decoction method was employed for extraction, using distilled water.
Four grams of powdered P.
macrocarpa were boiled in 60 mL of distilled water at 100 AC in a glass beaker on hotplate for 30 minutes.
The mixture was cooled and filtered using Whatman No.
1 filter paper.
Phytochemical Assay Flavonoid.
A one-millilitre aliquot of P.
macrocarpa extract was introduced into a test Thereafter, three to four drops of concentrated hydrochloric acid and one gram of magnesium powder were added to the aliquot.
The formation of a reddish-black, yellow, or orange coloration indicates a positive outcome for the presence of flavonoid compounds .
Phenols.
One millilitre of P.
extract was transferred to a test tube.
Subsequently, three to four drops of ferric chloride were added to the extract.
The development of a dark blue or black hue signifies a positive result for the presence of phenolic compounds .
Determination of Total Flavonoid Content.
This method was adapted from Sembiring et al.
A stock solution of quercetin with a concentration of 100 ppm was prepared and subsequently diluted to generate a series of standard solutions.
Each standard solution was treated with aluminium chloride and sodium acetate, followed by a 30-minute incubation period.
The absorbance of each solution was measured spectrophotometrically at a wavelength of 425 nm.
calibration curve was constructed to correlate the concentration of quercetin with the corresponding absorbance values.
Jurnal Ilmu Kimia dan Terapan A 1% solution of P.
macrocarpa extract was prepared and subjected to the same experimental protocol.
The total flavonoid content of the extract was quantified by comparing its absorbance to the established calibration curve .
Determination of Total Phenol Content.
series of standard solutions containing gallic acid were prepared.
Each standard solution was reacted with Folin-Ciocalteu reagent, followed by the addition of sodium hydroxide.
After a 30-minute incubation period, the absorbance of each solution was measured at 735 nm using spectrophotometer.
calibration curve was constructed to correlate the concentration of gallic acid with the corresponding absorbance values.
A portion of each P.
macrocarpa extract was subjected to the same experimental protocol.
The total phenol content of the extracts was quantified by comparing their absorbance to the established calibration curve .
Evaluating Antioxidant Activity.
Antioxidant evaluation was performed using the DPPH method following a procedure adapted from Hidayat and Fatmawati .
A 6 x 10-5 M solution of DPPH radical was prepared, and its maximum absorbance wavelength was determined at 520 nm.
Each extract .
5 mL) was mixed with 2 mL of DPPH solution and incubated in the dark for 30 minutes at room temperature.
As a positive control, ascorbic acid .
was prepared and treated in the same way as the sample extracts.
The blank was prepared by mixing 2 mL of DPPH solution with 0.
5 mL of ethanol.
The antioxidant activity of each sample was determined by calculating the percentage inhibition of DPPH radical absorption using the following formula .
% Absorbance = Where: A0 = Absorbance of control (DPPH solution without sampl.
As = Absorbance of sample or positive control.
Result and Discussion Extraction of P.
Macrocarpa.
Extraction was performed using a variety of methods, including hydrothermal extraction at 150AC, 175AC, and 200AC, as well as traditional methods such as boiling.
The results of the hydrothermal extraction demonstrated a correlation between increasing temperature ARTICLES | No.
2 Vol.
9 : 74-80 .
| DOI : https://doi.
org/10.
19109/ktzvbv78 ALKIMIA
ARTICLES
Jurnal Ilmu Kimia dan Terapan and increasing color intensity of the extract.
This phenomenon can be attributed to the accelerated extraction process at higher temperatures, which results in an increased concentration of extracted components.
Phytochemical Screening.
A qualitative phytochemical screening was performed to identify secondary metabolites, specifically flavonoids and phenols, present in P.
macrocarpa extracts.
The results, summarized in Table 1, demonstrate that all extracts, obtained through various hydrothermal extraction methods .
AC, 175AC, 200A C) as well as conventional methods .
, contain both classes of compounds.
The presence of flavonoids was confirmed by a positive colorimetric test, characterized by the formation of a red or orange flavylium salt upon the addition of HCl and Mg.
The qualitative test results for flavonoids are visually represented in Figure 1.
Figure 1.
Flavonoid qualitative test results of Macrocarpa: .
150AC extract, .
175AC extract, .
200AC extract, .
extract boiling Similarly, the presence of phenols was confirmed by a positive colorimetric test, evidenced by a dark colour change upon the addition of FeCl3.
The qualitative test results for phenols are visually represented in Figure 2.
This suggests the presence of catechol tannins, which are derivatives of phenolic compounds.
The reaction involves the complexation between Fe3 ions from FeCl3 and ortho-dihydroxy .
groups in phenol compounds during phytochemical screening with FeCl3.
The ferric ion coordinates with one or more catechol groups via their hydroxyl functional group, forming mono-, bis-, or tris-catecholate complexes.
These complexes exhibit intense color change ranging from green to black, associated with ligand-to-metal charge transfer transmitions (Figure .
These results agree with the findings of Kurang and Malaipada .
, who also observed a black colour change in the presence of phenols on P.
macrocarpa extract .
Total Flavonoid.
The total flavonoid content was quantified using a colorimetric method Figure 2.
Phenol qualitative test results of P.
Macrocarpa fruit: .
150AC extract, .
175AC extract, .
200AC extract, .
extract boiling Figure 3.
Mechanism of ferric ion (Fe3 ) complexation with catechol groups in phenolic compounds that relies on the formation of coloured complexes between flavonoids and aluminium chloride (AlClCE).
Hydrothermal extraction at 175AC proved to be the optimal method for extracting flavonoids from P.
macrocarpa, yielding a total flavonoid content of 04 mg quercetin equivalents per 1% extract.
higher temperatures, such as 200AC, a decline in flavonoid content was observed.
This reduction can be attributed to the thermal degradation of flavonoids, which are susceptible to high temperatures and oxidation.
This finding is in agreement with the observations reported by Prihandini and Purnama .
, which identified optimal total flavonoids content at temperatures between 140-170 AC in hydrothermal extraction.
Further increase to 200 AC led to a decrease in flavonoid content .
24 mg QE/1%), likely due to thermal degradation, as supported by general stability studies of plant extracts .
The test results of total flavonoid content of P.
macrocarpa fruit extract can be seen in Table 2.
Total Phenol Content.
The total phenol content was quantified using the Folin-Ciocalteu method, which relies on an oxidation-reduction reaction between polyphenols and phosphomolybdic acid/ phosphotungstic acid in the reagent.
This reaction ARTICLES | No.
2 Vol.
9 : 74-80 .
| DOI : https://doi.
org/10.
19109/ktzvbv78 ALKIMIA
ARTICLES
Jurnal Ilmu Kimia dan Terapan Table 1.
Phytochemical screening test results Secondary Hydrothermal 150AC Flavonoids Phenols Boiling Description:
Sign ( ): The sample contains the tested secondary metabolite compound Sign (-): Samples do not contain the tested secondary metabolite compounds results in a colorimetric change from yellow to Among the various extraction methods tested, hydrothermal extraction at 175AC yielded the highest total phenolic content .
12 mg GAE/1% extrac.
Phenolic content increased with temperature up to 175 AC, followed by a decrease at 200 AC due to thermal degradation.
In hydrothermal extraction, phenolic compounds can undergo thermal degradation, leading to a decrease in their concentration and antioxidant This degradation is often due to the breakdown of phenolic structures and the formation of new, less beneficial compounds .
The test results of total phenol content of crown fruit extract can be seen in Table 3.
Antioxidant Activity Test.
The P.
macrocarpa extract was added to a DPPH solution and Table 2.
Test results of total flavonoid content of P.
Macrocarpa fruit extracts Sample Flavonoid Content .
g QE/1% extrac.
Macrocarpa extract
150 AC
2,66
Macrocarpa extract
175 AC
4,04
Macrocarpa extract
200 AC
3,24
Boiling 2,10 Table 3.
Test results of total phenol content of Macrocarpa extracts Sample Phenol Content .
g GAE/1% extrac.
subsequently analyzed using UV-Vis spectrophotometry at a wavelength of 520 nm.
The percentage inhibition values obtained from this analysis quantify the extract's capacity to inhibit free radical activity.
The antioxidant potential of the fruit extracts, as measured by the DPPH assay, was found to be significant, with inhibition rates ranging from 66.
727% (Table .
The extract processed at 175A C demonstrated the highest antioxidant activity.
The improved extraction at 175 AC could be attributed to enhances cell wall disruption and increase water diffusivity, which facilitates the release of intracellular bio-actives like phenolic and flavonoids.
At this temperature, subcritical water reduces its dielectric constant, enhancing the solubility of moderately polar bio-actives, including flavonoid and phenolic compounds, thereby contributing to maximal antioxidant activity as observed in the DPPH assay.
These compounds can act as potent antioxidants by donating hydrogen atoms and reducing free radicals .
Similar trends were also reported by Prihandini and Purnama .
in their hydrothermal extraction study Moringa oleifera leaves, where antioxidant activity peaked between 140-170 AC, followed by a decline at higher temperatures .
A comparable pattern was described by Antony and Farid .
Who demonstrated that phenolic and flavonoid-rich extracts showed maximal DPPH radical scavenging activity at moderate subcritical temperatures, while activity decreased beyond 180 AC due to thermal degradation and the formation of degradation products.
This antioxidant activity decreased with increasing temperature, as observed in the 200AC extract.
This decline can be attributed to the thermal degradation of phenolic and flavonoids compounds, which are known to be sensitive to prolonged high-temperature exposure, as previously This temperature-dependent pattern is consistent with prior subcritical water extraction studies, where antioxidant capacities increased up to moderate temperatures .
Ae200 AC), but declined beyond due to thermal degradation and Maillard reaction product formation .
This study is the first to systematically optimize hydrothermal extraction conditions for P.
macrocarpa fruit and confirm 175 AC as the optimal temperature for maximizing antioxidant yield.
Macrocarpa
24,66
Macrocarpa
30,12
Conclusion Macrocarpa
27,49
This study demonstrated that hydrothermal extraction is an effective green extraction method for obtaining bioactive compounds from P.
Boiling 26,84 ARTICLES | No.
2 Vol.
9 : 74-80 .
| DOI : https://doi.
org/10.
19109/ktzvbv78 ALKIMIA
ARTICLES
Jurnal Ilmu Kimia dan Terapan Table 4.
%Inhibition values of P.
macrocarpa fruit extracts Sample Blank
(DPPH
Onl.
Ascorbic acid extract 150 AC extract 175 AC extract 200 AC Boiling Absorbance 1 Absorbance 2 Absorbance 3 Mean A SD 726 A 0.
117 A 0.
238 A 0.
162 A 0.
240 A 0.
205 A 0.
carpa fruit.
The quantitative analysis showed that increasing extraction temperature up to175 AC enhanced the yield of total flavonoids .
04 mg QE/1% extrac.
and total phenols .
12 mg GAE/1% extrac.
, along with the highest antioxidant activity .
73% DPPH inhibitio.
However, higher temperatures .
AC) led to a decrease in these parameters, suggesting that excessive heat can degrade bioactive compounds .
eat-sensitive Compared to boiling, hydrothermal extraction at 175 AC provided higher yields of antioxidant compounds and activity.
These findings highlight the importance of optimizing extraction conditions to maximize antioxidant yield and the recovery of valuable bioactive compounds from P.
macrocarpa fruit using a waterbased, organic solvent-free method.
Acknowledgements The authors are grateful for the laboratory support from Chemistry Laboratory.
Universitas Islam Kadiri-Kediri.
Author Contributions All the authors have contributed sufficiently to this research, from coming up with the idea, collecting the data, supervising, and producing the paper.
References