Journal of Chemistry
Volume 2020 (2020), Article ID 4301891, 14 pages
https://doi.org/10.1155/2020/4301891

Research Article

Active Compound Identification in Extracts of N. lappaceum Peel and Evaluation of Antioxidant Capacity

Mariel Monrroy,1,2 Onix Araúz,1,2 and José Renán García1,2

¹Centro de Investigación en Bioquímica y Química Aplicada, Facultad de Ciencias Naturales y Exactas, Universidad Autónoma de Chiriquí, 0426 David, Panama
²Departamento de Química, Facultad de Ciencias Naturales y Exactas, Universidad Autónoma de Chiriquí, 0426 David, Panama

Correspondence should be addressed to José Renán García; joregam@gmail.com

Received 18 October 2019; Revised 8 December 2019; Accepted 31 December 2019; Published 28 January 2020

Academic Editor: Maurizio Quinto

Copyright © 2020 Mariel Monrroy et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Nephelium lappaceum and its by-products have great potential in the agricultural, pharmaceutical, and food industries. Some studies have shown that N. lappaceum by-products exhibit antimicrobial, antioxidant, antidiabetic, and anticancer properties. However, studies focused on identifying these compounds are rare. The availability of polyphenolic compounds can vary according to environmental conditions, soil, plant variety, and agronomic management. Therefore, in this study, the active compounds in extracts of the N. lappaceum peel were identified, and their antioxidant properties were evaluated using various extraction solvents and both ultrasonic and boiling extraction techniques. The chemical characterization of the N. lappaceum peel exhibited carbohydrate and reducing sugar contents of 12 and 2%, respectively. Phytochemical analysis indicated the presence of flavonoids, tannins, terpenes, and steroids. The total phenolic and flavonoid contents and total antioxidant capacity were the highest in the hydroethanolic extract obtained by ultrasound, with values of 340 mg gallic acid equivalents g⁻¹, 76 mg quercetin equivalents g⁻¹, and 2.9 mmol of Trolox equivalents g⁻¹, respectively. Contrarily, the total anthocyanin content was higher in the acid extract obtained by ultrasound, with a value of 0.7 mg cyanidin-3-O-glucoside equivalents g⁻¹. A total of 18 compounds—including hydroxybenzene, phenolic acid, flavonoids, fatty acids (saturated, unsaturated, and ester), vitamin, arenecarbaldehyde, and phthalate—were identified for the first time in the N. lappaceum peel using gas chromatography-mass spectrometry. The identified compounds have been previously isolated from other plants and reportedly exhibit anticancer, anti-inflammatory, antimicrobial, and antioxidant activities. Thus, the N. lappaceum peel was shown to be a potential source of bioactive compounds of immense importance in the pharmacological and food industries.

1. Introduction

Nephelium lappaceum, also known as rambutan, is a tropical fruit with excellent nutraceutical properties. In recent years, several studies in Southeast Asia and Australia have revealed that different parts of the fruit, particularly the peel, have powerful antioxidant, antibacterial, and antidiabetic activities, as summarized in Table 1, which may be attributed to the presence of phenolic compounds.

Table 1: Biological properties in the extract from the seed, pulp, and peel of N. lappaceum.

Material
Extract or solvent
Property
Reference

Peel
Methanolic
Antioxidant including DPPH, lipid peroxidation, ABTS, FRAP, reducing power, b-carotene bleaching, linoleic peroxidation, and free radical scavenging
[1–3]

Peel
Ethanolic
Antidiabetic: significant decrease of 62% in blood glucose levels
Antioxidant including ABTS and DPPH assays
[4–6]

Peel
Aqueous
Antioxidant including ABTS and FRAP
Antimicrobial activity against Vibrio cholerae, Enterococcus faecalis, Staphylococcus aureus, and Staphylococcus epidermidis
[1, 7]

Peel
Ethyl acetate
Antioxidant including ABTS and FRAP
[8]

Peel
Hydroethanolic
Antioxidant including DPPH, ABTS, FRAP, lipid peroxidation, and nitrite and hydroxyl radical scavenging
Antiglycation activities
[9, 10]

Peel
Ether
Antimicrobial activity against V. cholerae, E. faecalis, S. aureus, and S. epidermidis
[1]

Pericarp
Ethanolic and aqueous
Inhibitory effects on the infection of herpes simplex virus type 1
[11]

Pulp
Ethanolic
Anti-inflammatory: decreasing secretion of tumor necrosis factor alpha (TNF-α)
Antioxidant: suppressive effect on reactive oxygen species (ROS) formation
[12]

Seed
Aqueous
Antibacterial: moderate inhibition against S. aureus, S. pyogenes, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa
Antiviral: inhibitory effect on HIV-1 reverse transcriptase (HIV-1 RT)
Antitumoral: trypsin/chymotrypsin inhibitory activity
[13, 14]

Seed
Methanolic
Antibacterial: inhibition against S. aureus, S. epidermidis, E. coli, and P. aeruginosa
Anti-inflammatory, antinociceptive, CNS, and antifungal
Antioxidant including DPPH, reducing power, b-carotene bleaching, linoleic peroxidation, and free radical scavenging
[1, 15, 16]

Seed
Ethyl acetate and ethanolic
Antioxidant using DPPH and ABTS assays
[17]

Seed
Hexane, ethyl acetate, butanol, and water
Antidiabetic: α-glucosidase inhibitor activity
[18]

Phenolic compounds are secondary metabolites that exist in all plants as a defense mechanism against stress caused by pathogens or adverse environmental conditions [19]. They act as reducing agents, eliminators of free radicals, and metal chelators [20]. Several studies carried out on phenolic extracts or isolated compounds have shown that they protect against disorders caused by oxidative damage, such as cancer, arteriosclerosis, cardiovascular disorders, and diabetes, by delaying and inhibiting the oxidation of lipids and other molecules [9, 21–25]. These compounds are of interest not only for their antioxidant properties but also for their antiallergic, anti-inflammatory, and antimicrobial properties [26–28].

Presently, about eight thousand phenolic compounds are known to exist in nature [29]. Although phenolic compounds are very diverse, they can be divided into six major classes according to their structure: simple phenols, phenolic acids, flavonoids, tannins, stilbenes, and lignans [26, 29–32].

George et al. [33] and Pande and Akoh [34] have reported that phenolic compounds mainly accumulate in the skin or peel, rather than the pulp of fruits and vegetables, making these by-products an attractive source for the isolation of natural active compounds. For example, studies of N. lappaceum have revealed the presence of numerous phenolic compounds with important properties in the peel, in even greater quantities than in the seed and pulp [18, 35]. Sun et al. [9] reported the presence of phenolic acids, hydroxycinnamic acids, and flavan-3-ols such as gallic acid, p-coumaric acid, catechin, and rutin in the peel. Palanisamy et al. [36] and Thitilertdecha et al. [2] identified a hydrolysable tannin called geraniin in the N. lappaceum peel with antihyperglycemic and antioxidant properties. Thitilertdecha et al. [2] further showed that this compound had greater antioxidant capacity than the synthetic antioxidant butylated hydroxytoluene. Such phenolic compounds can also act as antiviral agents that inhibit the replication of human immunodeficiency virus (HIV) [37]. These studies illustrate the promising potential of N. lappaceum peels. However, the chemical and biological diversity of these plants depends on factors such as location, variety, management, and climatic conditions [38, 39].

Although the largest commercial production of N. lappaceum is concentrated in Thailand, Malaysia, Indonesia, and Philippines [40], there are several countries in the Central American Region, such as Costa Rica, Guatemala, Honduras, Mexico, and Panamá, which are also dedicated to its cultivation. According to the Food and Agriculture Organization of the United Nations (FAO) [41], the global production of N. lappaceum reached an estimated 1.4 million tons in 2017, which generated 756 thousand tons of by-products such as peels and seeds [3]. While these by-products have found little or no use so far, they could represent a renewable resource from which many useful biological and chemical products could be obtained, providing an avenue to meet the demand of the food industry for functional foods and reutilization of agricultural by-products.

The economic prospects and market demand of this fruit have seen rapid growth in recent years because studies have shown that it contains polyphenolic bioactive compounds [7]. Although the N. lappaceum peel has been documented in previous studies as a material that is rich in bioactive compounds with antioxidant and antihyperglycemic activities, studies identifying the molecules present in its extracts that could be associated with other bioactivities are rare. This is particularly true in Central America. Considering that the availability of polyphenolic compounds can vary according to environmental conditions, soil, plant variety, and agronomic management, studies on these by-products in different locations are necessary. In addition, the ability to obtain polyphenolic compounds may depend on the extraction technique and nature of the extraction solvent used. Polyphenolic compounds range from very polar to very nonpolar compounds, so the extraction solvent plays an important role in their extraction efficiency [42]. Therefore, in this study, we identified the active compounds present in the extract of the N. lappaceum peel and evaluated their antioxidant capacities using different extraction solvents (acid, alkaline, aqueous, ethanolic, and hydroethanolic) and both ultrasonic and boiling extraction techniques. This is one of the first studies utilizing acidic and alkaline media, as well as ultrasound in the extraction of active compounds from N. lappaceum. To the best of our knowledge, only Prakash Maran et al. [43] have used ultrasound in the extraction of bioactive compounds (total anthocyanin, phenolic, and flavonoid contents) from N. lappaceum L. peels. Our research, focusing on finding such bioactive compounds from renewable resources often considered waste, constitutes an important step towards sustainability within food, pharmacological, and other industries.

2. Materials and Methods

2.1. Raw Materials

A local farmer provided samples of N. lappaceum (red variety). The peels were removed from the fruit, washed, and dried for 48 h at 60°C and then milled and stored in dark plastic bags under dry conditions until experimental use for experiments. Sample analysis began immediately after collection.

2.2. Chemical Characterization

The samples were analyzed for the total carbohydrate, reducing sugar, and mineral contents, and phytochemical screening was conducted. All analyses were performed in triplicate. The total carbohydrate content was determined by the phenol-sulfuric acid method. A test tube containing the sample (0.1 g) and HCl (5 mL, 2.5 N) was heated in a water bath (Thomas Scientific, USA) at 95°C for 3 h. The resulting suspension was neutralized with Na₂CO₃, diluted with 5 mL water, and centrifuged (Digicen 21 R centrifuge, Ortoalresa, Madrid, Spain) at 3500 rpm for 5 min. The dilution/centrifugation process was repeated five times. The supernatant volume was adjusted to 100 mL with water, and a 0.1 mL aliquot of this solution was withdrawn. The aliquot was treated with 50 g·L⁻¹ aqueous phenol (1 mL) and concentrated 96% H₂SO₄ (5 mL), vortexed (MX-S vortex mixer, Scilogex, Thomas Scientific, USA) for a minute, and heated at 30°C for 20 min in a water bath. Finally, the total carbohydrate content was determined by visible-light spectrophotometry (Genesys 10S, Thermo Scientific, USA) at a wavelength of 490 nm, using a calibration curve prepared with glucose as a standard. The reducing sugars were quantified by visible-light spectrophotometry at a wavelength of 540 nm following the dinitrosalicylic acid (DNS) method [44]. Typically, a suspension of the sample (0.1 g) in hot ethanol (8 mL, 800 mL L⁻¹) in a test tube was heated in a water bath at 95°C for 10 min and then centrifuged at 2500 rpm for 5 min. The supernatant was collected, ethanol was evaporated in a water bath at 80°C, and the obtained residue was treated with water (10 mL). A 0.7 mL aliquot of this solution was transferred into a test tube and treated with 1.5 mL of the DNS reagent. The resulting mixture was heated in a boiling water bath for 5 min and then brought to room temperature in a 25°C water bath. Finally, the content of reducing sugars was determined from the absorbance of the cooled mixture at 540 nm, using a calibration curve prepared with glucose as a standard. The DNS reagent was prepared by dissolving 0.748 g of DNS, 0.564 g of crystalline phenol, 21.612 g of Rochelle salt, 1.412 g of NaOH, and 0.564 g of sodium sulfite in 100 mL water. The total nitrogen concentration was determined by the Kjeldahl method (K415-K350-F308 Kjeldahl system, Buchi, Switzerland) according to AOAC Method 988.05 [45]. The phosphorus content was determined by the vanadomolybdophosphoric method described by Kitson and Mellon [46]. For the determination of the mineral content, ashes of a sample were dissolved in hydrochloric acid and the resulting solution was analyzed by atomic absorption spectroscopy (AA-7000 flame atomic absorption spectrophotometer, Shimadzu, Kyoto, Japan) according to AOAC Method 985.35 [45].

Infrared spectra of the samples were measured by attenuated total reflectance (ATR). The spectra were recorded between 4000 and 650 cm⁻¹ using a Cary 630 Fourier-transform infrared spectrophotometer (FT-IR) (Agilent Technologies, USA) equipped with a deuterated triglycine sulfate detector. The diamond ATR sensor was cleaned with ethyl alcohol before each sample measurement. The spectral resolution was 4 cm⁻¹, and 64 scans were made for each spectrum.

The sample was subjected to a phytochemical screening by conducting the Salkowski test for sterols, Shinoda and sodium hydroxide tests for flavonoids, Dragendorff, Wagner, and Mayer tests for alkaloids, ferric chloride test for tannins, Rosenthaler test for saponins, and Dimroth test for 5-hydroxy flavones.

2.3. Extraction Method

The extracts were obtained by ultrasound and by boiling from alkaline, acidic, ethanolic, hydroethanolic, and aqueous solutions. For the extractions in acidic, alkaline, aqueous, and hydroethanolic solutions, 1 g of the dry sample was sonicated in 10 mL of the corresponding solvent of 10 g·L⁻¹ HCl, 10 g·L⁻¹ NaOH, water, 96% ethanol, or 600 g·L⁻¹ hydroethanol, respectively, for 20 min using an ultrasonic bath (Elmasonic P60H, Singen, Germany). The extractions were also carried out by boiling for 10 min with the same sample/solvent ratio. Subsequently, the extracts were cooled and filtered [47].

2.4. Phenolic, Flavonoid, and Anthocyanin Content Evaluation

The total phenolic content (TPC) was determined by the Folin–Ciocalteu assay, as described by Miliauskas et al. [39]. A volume of 500 μL of different diluted extracts was mixed with 2500 μL of the Folin–Ciocalteu reagent (0.2 mol·L⁻¹) and allowed to stand for 5 min. Next, 2 mL of 75 g·kg⁻¹ sodium carbonate solution was added to the mixtures, which then stood for 2 h at room temperature in the dark. The absorbances of the solutions were then measured at 754 nm using a visible-light spectrophotometer (Genesys 10S, Thermo Scientific, USA). To set up the calibration curve, gallic acid solutions of concentrations ranging from 0 to 12 μg mL⁻¹ were prepared. The results were expressed as mg of gallic acid equivalents (GAE) per g of dry peel mass.

The total flavonoid content (TFC) was determined by spectrophotometry using the AlCl₃ colorimetric method [48], with some modifications. In brief, 50 μL of the extract was mixed with distilled water to prepare a 5 mL solution, followed by the addition of 0.3 mL of 50 g·L⁻¹ NaNO₂. After 5 min, 0.3 mL of 100 g·L⁻¹ AlCl₃ was added to the mixture. Shortly thereafter, 2 mL of 1 mol·L⁻¹ NaOH was added and the total volume was increased to 10 mL by adding distilled water. After 15 min, the absorbance of the solution was measured at 374 nm using an ultraviolet spectrophotometer (Genesys 10S, Thermo Scientific, USA). The calibration curve was set up using quercetin solutions with concentrations ranging from 0 to 15 μg·mL⁻¹. The flavonoid content in the extract was expressed as mg of quercetin equivalents (QE) per gram of dry peel mass.

The total anthocyanin content (TAC) was estimated by a pH differential method described by Prakash Maran et al. [43] Aliquots of the extracts were adjusted to pH values of 1.0 and 4.5 using 25 mM KCl and 0.4 M CH₃COONa as buffer solution, respectively, and allowed to equilibrate for 20 min. Subsequently, their absorbances were measured at 520 and 700 nm. The TAC of the extracts was calculated using the following equation:

where A is the difference in the absorbances at a pH of 1.0 (A520 nm − A700 nm) and a pH of 4.5 (A520 nm − A700 nm), MW is the molecular weight of cyanidin-3-glucoside (449.2 g·mol⁻¹), DF is the dilution factor, and ε is the molar extinction coefficient of cyanidin-3-glucoside (26,900 L·mol⁻¹  ×  cm⁻¹). Therefore, the TAC was expressed as mg cyanidin-3-O-glucoside equivalents (Cy-3GE) per gram of dry peel mass.

2.5. 2,2′-Azino-bis-(3-ethylbenzothiazoline-6-sulphonic Acid) Radical Cation-Scavenging Activity Determination

The 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assay was performed using the method described by Re et al. [49]. The ABTS radical cation was prepared by reacting equal portions of 7 mM ABTS solution and 2.45 mM potassium persulfate solution. The mixture was incubated at room temperature for 16 h in the dark at 25°C and then diluted with 96% ethanol until its absorbance of 754 nm reached 0.7 ± 0.02. The prepared ABTS•+ solution (1900 μL) was mixed with 100 μL of the extract that was diluted and then allowed to stand for 10 min at room temperature in the dark. The absorbance of the sample was measured at 754 nm using a visible-light spectrophotometer (Genesys 10S, Thermo Scientific, USA). The ABTS•+ scavenging activity was calculated by comparison with a standard curve of Trolox (40 and 320 mmol·L⁻¹ Trolox), and the results were expressed as mmol of Trolox equivalents (TE) per gram of dry peel mass (mmol TE·g⁻¹).

2.6. 1,1-Diphenyl-2-picrylhydrazine Radical-Scavenging Activity Determination

The 1,1-diphenyl-2-picrylhydrazine (DPPH) assay was done as described by Thaipong et al. [50], with some modifications. The DPPH radical solution was prepared by dissolving 13 mg DPPH with 100 mL methanol, and then 10 mL of the solution was diluted with 45 mL of methanol to obtain an absorbance of 0.70 ± 0.02 units at 515 nm using the spectrophotometer (Genesys 10S, Thermo Scientific, USA). The extracts (100 μL) were reacted with 2900 μL of the DPPH solution for 30 min at room temperature in the dark. The absorbance of the sample was measured at 515 nm. The DPPH scavenging activity was calculated by comparison with a standard curve of Trolox (40 and 600 mmol·L⁻¹ Trolox). Results are expressed as mmol of Trolox equivalents (TE) per gram of dry peel mass (mmol TE·g⁻¹).

2.7. Compound Isolation

The hydroethanolic extract was reextracted with ethyl acetate and hexane. In this procedure, the extract was evaporated and the residue was resuspended in water. The aqueous extract (3 mL) was then acidified to a pH of 2 with HCl and extracted using liquid-liquid extraction (LLE) with 3 mL ethyl acetate three times. The ethyl acetate phase was treated with activated charcoal to adsorb the pigments. After filtration, the organic phase was dried over anhydrous Na₂SO₄ and evaporated. The residue was resuspended in ethanol for the GC-MS analysis. The LLE process was repeated using hexane.

2.8. GC-MS Analysis

The identification of bioactive compounds from the crude hydroethanol extract and ethanol extract obtained by LLE with ethyl acetate and hexane was performed on a 7890A GC system coupled with a 5975C MS system (Agilent Technologies, USA), fitted with an HP-5MS column (30 m in length × 250 μm in diameter × 0.25 μm in thickness of the film). Pure He was used as the carrier gas at a constant flow rate of 1 mL·min⁻¹. One microliter of the extract was injected in a splitless mode. The ionization of the sample components was carried out at 70 eV. The injector and ion source temperatures were 260°C and 230°C, respectively. The oven temperature ramp was programmed as 60°C at 0 min, 10°C min⁻¹, and 280°C for 30 min. Mass spectra were scanned from m/z 45–550 at a rate of 2 scans/s. The total running time of the GC was 52 min. The compounds were identified by comparing their mass spectra with those from the NIST05 Mass Spectral Library.

2.9. Statistical Analysis

Each assay was performed three times per extract to determine their reproducibility. Data were expressed as the mean ± standard deviation values. Two-way analysis of variance (ANOVA) and multiple-comparison test (the Tukey procedure) were used to determine significant differences. The results were deemed significant at p < 0.05. Correlations between the total phenolic, flavonoid, and antioxidant capacities were made using Pearson’s procedure (). Statistical analyses were conducted using the Statgraphics Centurion XVII software (Statgraphics Technologies, USA).

3. Results and Discussion

3.1. Chemical Composition of the N. lappaceum Peel

The total carbohydrate content in the N. lappaceum peel was 120 g·kg⁻¹, which is less than the value of 238 g·kg⁻¹ reported by Fila et al. [51] in Nigeria. In addition, the N. lappaceum peel is an excellent source of essential elements such as nitrogen, calcium, potassium, phosphorus, magnesium, copper, zinc, manganese, sodium, and iron, as summarized in Table 2. These elements are of considerable interest to the nutraceutical and food industries because they are crucial for the healthy functioning of the human body due to their roles in various vital processes such as cellular functioning, enzymatic activation, genetic expression, and bone and hemoglobin formation [52, 53]. Phosphorus is an important constituent of adenosine triphosphate and nucleic acid and is also essential for acid-base balance and bone and tooth formation. Nitrogen is the basic component of macromolecules such as proteins. Iron is an important component of the cytochromes that function in cellular respiration, and red blood cells cannot function properly without iron in hemoglobin. Magnesium, copper, zinc, iron, manganese, and molybdenum are important cofactors found in the structure of certain enzymes and are indispensable in numerous biochemical pathways [52, 53].

Table 2: Carbohydrate and mineral content of the N. lappaceum peel.

Total carbohydrate
Reducing sugar
N
P
K
Ca
Mg
Fe
Cu
Mn
Zn
Na

g·kg⁻¹
g·kg⁻¹
g·kg⁻¹
mg·kg⁻¹

120 ± 10
24 ± 2
9.1 ± 0.2
5.7 ± 0.2
7.6 ± 0.1
0.9 ± 0.1
0.66 ± 0.08
41 ± 2
10 ± 1
160 ± 5
8.7 ± 0.1
45 ± 3

The FT-IR spectrum of the N. lappaceum peel is shown in Figure 1. The functional group bands observed in the spectrum are characteristic of the lignocellulosic material. The bands at 3275 and 2925 cm⁻¹ correspond to the O–H and C–H stretching vibrations, respectively, of the hydroxyl and carboxylic functional groups in phenols and pyranose. The band at 1707 cm⁻¹ is attributed to the –C=O stretching vibrations of the ester carbonyl or carboxylic acid functional group [54], which are characteristic of fatty acids. The –C=O absorptions might correspond to either conjugated or nonconjugated, saturated or unsaturated, amides, acids, or other systems [55]. The bands at 1506 and 1440 cm⁻¹ correspond to aromatic C–C stretching, while the peak at 1317 cm⁻¹ may be attributed to C–H asymmetric deformation and COO⁻ anion stretching. The bands observed at ∼1440–1201 cm⁻¹ can be assigned to C–O stretching and O–H deformation vibrations [56]. The absorption band at 1024 cm⁻¹ corresponds to the C–O stretching of either an ester or an ether [54], while that at 752 cm⁻¹ is attributed to =CH bending.

Figure 1: FT-IR spectrum of the N. lappaceum peel.

The phytochemical analysis shown in Table 3 reveals the presence of terpenes/steroids, flavonoids, and tannins. Although alkaloids were not detected in this study, Nethaji et al. [57] have reported the presence of alkaloids, steroids and sterols, glycosides, flavonoids, triterpenoids, and tannins in the methanolic extracts of the N. lappaceum epicarp. Lestari et al. [58] have reported that the ethanolic, aqueous, and n-butanol extracts of the N. lappaceum peel contain compounds such as terpenoids, steroids, flavonoids, sugars, amino acids, and peptides. Thus, the chemical composition of N. lappaceum is clearly quite variable, depending on several factors such as the chemical characteristics of the soil, geographic location, environmental conditions, age, and species.

Table 3: Phytochemical analysis of the N. lappaceum peel.

Metabolite
Test
Evidence

Alkaloids
Meyer
(−)

Dragendorff
(−)

Wagner
(−)

Tannins
FeCl₃
(+)

Terpenes/steroids
Salkowski
(+)

Flavonoids
Reaction with NaOH
(+)

3.2. Total Phenolic, Flavonoid, and Anthocyanin Contents in the Extracts

N. lappaceum by-products are rich in phenolic acids, flavonoids, and anthocyanins, as mentioned above. The phenolic acid, flavonoid, and anthocyanins contents in the extracts obtained by ultrasound ranged from 208 to 340 mg·g⁻¹, 44 to 76 mg·g⁻¹, and 0.53 to 0.7 mg·g⁻¹, respectively, while in the extracts obtained by boiling, these same contents ranged from 189 to 315 mg·g⁻¹, 41 to 72 mg·g⁻¹, and 0.15 to 0.68 mg·g⁻¹, respectively (Table 4).

Table 4: Total phenolic, flavonoid, and anthocyanin contents based on the extraction method and solvent.

Extraction method
Nature of the solvent
Properties

TPC (mg GAE g⁻¹)
TFC (mg QE g⁻¹)
TAC (mg Cy-3GE g⁻¹)
ABTS (mmol TE g⁻¹)
DPPH (mmol TE g⁻¹)

Ultrasound
Acidic 1%
231 ± 2d
43.7 ± 0.3e
0.70 ± 0.01a
1.91 ± 0.06c
1.65 ± 0.04d

Alkaline 1%
262 ± 6c
72 ± 2b
ND
2.12 ± 0.03b
1.34 ± 0.02e

Aqueous
280 ± 2b
54 ± 1c
0.533 ± 0.004b
1.76 ± 0.02d
2.4 ± 0.1b

Ethanolic
208 ± 1e
48.1 ± 0.3d
0.69 ± 0.01a
1.76 ± 0.02d
1.78 ± 0.02c

Hydroethanolic 60%
340 ± 4a
76 ± 2a
0.57 ± 0.03b
2.9 ± 0.1a
3.26 ± 0.02a

Boiling
Acidic 1%
189 ± 2d
40.7 ± 0.3e
0.68 ± 0.02a
1.79 ± 0.01c
0.86 ± 0.03c

Alkaline 1%
258 ± 3b
62.2 ± 0.4b
ND
1.96 ± 0.02b
0.78 ± 0.04c

Aqueous
235 ± 2c
44 ± 1d
0.18 ± 0.05c
1.54 ± 0.02d
1.38 ± 0.04b

Ethanolic
233 ± 5c
51 ± 1c
0.15 ± 0.03c
1.73 ± 0.03c
2.3 ± 0.1a

Hydroethanolic 60%
315 ± 7a
72 ± 1a
0.35 ± 0.04b
2.6 ± 0.1a
1.53 ± 0.02b

p value
Extraction method
0.0026
0.0001
0.0001
0.0001
0.0001

Nature of the solvent
0.0001
0.0001
0.0003
0.0001
0.0001

ND: not determined; TPC: total phenolic content; TFC: total flavonoid content; TAC: total anthocyanin content; ABTS: 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid); DPPH: 1,1-diphenyl-2-picrylhydrazine. All values are expressed as mean ± SD (n = 3). values <0.05 indicate significant differences by two-way ANOVA. Different letters in the same column within each extraction method indicate significantly different values at the 95% confidence level, using the Tukey test.

Active compound extraction is a critical process that depends on the solvent, temperature, time, and chemical nature of the sample [48], as well as the extraction technique. In this investigation, we evaluated the solvent and extraction process. To determine the effect of the extraction process and the nature of the extraction solvent, a two-way ANOVA was performed. It was found that there were significant differences at a confidence level of 95.0% () between the TPC, the TFC, and the TAC for the extracts obtained through different extraction processes and solvents (Table 4). To determine the significantly different mean among the solvents at a confidence level of 95.0%, we applied the Tukey test for each extraction method.

For ultrasound, the TPC values for all solvent extractions varied significantly. Meanwhile, the pair of ethanolic and aqueous extracts did not present any significant differences for ebullition. The results obtained by ultrasound were significantly different from those obtained by ebullition. The highest TPC was obtained with the extract produced by ultrasound in hydroethanol, as this solvent allowed for the extraction of both polar and semipolar compounds. These results are similar to the findings in previous studies about extraction of the TPC. Ngo et al. [59] and Dailey and Vuong [42] indicated that extraction solvents play a key role in the extraction of the TPC and that the combined organic solvents and water had a higher recovery yield in comparison with the absolute solvent. The TPC values obtained in this work were similar to, and even higher than, those reported in the literature for the N. lappaceum peel extract. While Thitilertdecha et al. [1] reported a TPC of 293.3 mg GAE g⁻¹ by extraction with ether, Prakash Maran et al. [43] obtained 5.5 mg GAE g⁻¹ of the phenolic material in the aqueous extract by ultrasound. The TPC value reported by Nguyen et al. [3] from the methanolic extract was 128 mg GAE g⁻¹.

For the TFC, the results showed that solvents also significantly affected the extraction yields of flavonoids. The acidic, alkaline, aqueous, ethanolic, and hydroethanolic extracts showed significant differences in all cases, for both extraction methods. The highest TFC for both extraction methods was that of the hydroethanolic extracts, though it was higher for the extracts obtained by ultrasound. The TFC in our study was higher than the values reported previously for the N. lappaceum peel extract. Gusman and Tsai [60] showed that, under the two extraction methods of conventional extraction (12 h) and ultrasonic extraction (2 min), the TFC values were 7.7 mg QE g⁻¹ and 6.4 mg QE g⁻¹, respectively, with hydroethanol as a solvent (40% ethanol). Prakash Maran et al. [43] have reported 1000 μg rutin g⁻¹ in the aqueous extract obtained by ultrasound.

The impact of the solvent nature and extraction technique on the extraction of the TAC was also studied. Anthocyanins are the natural plant pigments categorized as flavonoids. Interestingly, the pair of ethanolic and acidic extracts, and hydroethanolic and aqueous extracts, did not exhibit any significant differences for the TAC when ultrasound was used. In contrast, the acid extract exhibits significant differences from the other extracts obtained by ebullition. Although the TAC obtained by boiling is different from that obtained by ultrasound, the acid extracts presented similar values in both extraction methods. In general, the highest TAC was obtained by the acid extracts. This is reasonable because, typically, anthocyanins are more stable under acidic conditions [61]. For all extracts, these TAC values were intermediate to those reported in the literature. Sun et al. [62] and Maran et al. [43] reported 1.8 mg Cy-3GE g⁻¹ and 1 mg Cy-3GE g⁻¹ TAC of the ethanolic/aqueous/acidic and aqueous extracts, respectively. These compounds, which are mainly responsible for the red color of the ripe N. lappaceum fruit, exhibit antioxidant, anti-inflammatory, antioedema, and antitumor activities [62, 63].

In this investigation, besides traditional extraction solvents such as water and ethanol, acidic and alkaline aqueous media were also used to compare the extraction efficiencies of the active compounds. While the alkaline media make the acidic compounds more soluble in an aqueous medium, the acidic media similarly increase the water solubility of organic bases. Our work is among the few studies that report the extraction of secondary metabolites in N. lappaceum using acidic and alkaline media. The results indicated that the compounds in the extracts obtained from N. lappaceum could be significantly affected by the extraction solvents, and the variation can be explained by the difference in solubility of different compounds, highlighting the relevance of identifying the optimal extraction solvent for each case. Diverse reports in the literature have shown that extraction of active compounds varies with different solvents used [64–66]. Koffi et al. [66] reported that ethanolic extracts of Ivorian plants yielded a higher amount of phenolic compounds compared to acetone, water, and methanol. Do et al. [65] reported that the Limnophila aromatica extract obtained by 100% ethanol showed the highest TPC, TFC, and Trolox equivalent antioxidant capacity (TEAC) extracts compared with pure water, other organic solvents, and aqueous organic mixtures. Contrarily, we found that the extraction with pure solvent (water and ethanol) was less effective than that of aqueous organic mixtures, as reported by Ngo et al. [59] and Dailey and Vuong [42]. In our case, the highest TFC and TPC were those of the hydroethanolic extracts in both extraction methods. In contrast, the TAC was higher in the acid extract.

Although the highest TPC and TFC were obtained in the hydroethanolic ultrasound extracts, and TAC in the acid extract, the TPC, TFC, and TAC from all extracts obtained by both extraction methods were comparable to those reported in the literature as mentioned above [3, 43, 60, 62].

On the contrary, the active compound extraction from N. lappaceum also depends on the extraction technique, and generally, the ultrasound technique shows a greater extraction capacity. This technique improves the efficiency of the extraction solvent and has excellent destructive effects on a matrix because it alters the fluid around the solid particles, via cavitation processes, and the internal structure, by disrupting cell walls and/or enlarging its pores, thus allowing the solvent to better penetrate into the cell and release the target molecules [67, 68]. Additionally, this technique is economical and environmentally friendly and presents the ability to produce concentrated extracts in a time-efficient manner [68]. According to Quintero Quiroz et al. [69], the ultrasound technique increased the yield of bioactive compounds and preserved the metabolites with a greater efficiency and thus promoted their functional activities.

3.3. Antioxidant Activity of N. lappaceum

Antioxidant activity plays a crucial role in mediating free radicals and reactive oxygen species (ROS), which are considered harmful to human health [35]. Because multiple oxidation mechanisms exist, no single assay will accurately reflect the entire antioxidant capacity of N. lappaceum peel extracts. Thus, to elucidate a complete profile of antioxidant capacity, multiple antioxidant capacity assays are required. However, in this study, only assays based on the single electron transfer (SET) mechanism, ABTS and DPPH assays, were evaluated. The ABTS assay has been widely used to evaluate the antioxidant activity of aqueous and lipophilic systems in vitro, while the DPPH assay has been used for evaluating the scavenging activities of antioxidants in lipophilic systems [70, 71].

Although the antioxidant properties of the extract can vary depending on the antioxidant assays used, the nature of solvents and the extraction techniques also have a great impact on the antioxidant properties of extracts. The antioxidant activity, determined using the ABTS and DPPH assays, was expressed as Trolox equivalent antioxidant capacity (TEAC), which indicates the free radical-scavenging activity. The extracts evaluated exhibited good antioxidant capacity with both high inhibitory activity against ABTS∙+ radical cations and strong scavenging activity against DPPH radicals. The values obtained by both tests were comparable. The TEAC for the ABTS assay ranged from 1.76 mmol TE g⁻¹ to 2.90 mmol TE g⁻¹ and 1.54 mmol TE g⁻¹ to 2.6 mmol TE g⁻¹, in the extracts obtained by ultrasound and boiling, respectively. Additionally, the TEAC for the DPPH assay ranged from 1.65 mmol TE g⁻¹ to 3.26 mmol TE g⁻¹ and 0.78 mmol TE g⁻¹ to 2.3 mmol TE g⁻¹, in the extracts obtained by ultrasound and boiling, respectively (Table 4). The ANOVA indicated that there was a significant difference, at a confidence level of 95.0%, between the different extraction processes and the solvents used for both assays. The Tukey test showed that there is a significant difference in the TEAC values between the hydroethanolic extract and the rest of the extracts. Meanwhile, the pairs of ethanolic and acidic, and ethanolic and aqueous, extracts did not present any significant differences when ebullition and ultrasound methods, respectively, were used. The differences in antioxidant capacity are attributed to the variation of the types of compounds extracted by the different solvents. Hosseinian et al. [72] indicated that the effect of antioxidant activity varies if antioxidants are water- or lipid-soluble. The highest antioxidant capacity was that of the hydroethanolic extract obtained by ultrasound for both assays. The values obtained are similar to those recently reported by Nguyen et al. [3], 2.16 mmol TE g⁻¹ for the ABTS assay and 1.85 mmol TE g⁻¹ for the DPPH assay in the methanolic extract of the N. lappaceum peel.

Compared with some other fruits and vegetables, the antioxidant capacity of N. lappaceum determined through the radical DPPH method is higher than those reported for apples (14 μmol TE g⁻¹), bananas (5.2–11 μmol TE g⁻¹), grapes (19–55 μmol TE g⁻¹), kiwis (10 μmol TE g⁻¹), nectarines (12 μmol TE g⁻¹), plums (17–22 μmol TE g⁻¹), broccoli (6–1.8 μmol TE g⁻¹), garlic (0.6–13 μmol TE g⁻¹), onions (0.6 μmol TE g⁻¹), potatoes (3.5–4 μmol TE g⁻¹), peppers (11–12 μmol TE g⁻¹), and spinach (1.8 μmol TE g⁻¹) [73–75]. Similarly, the antioxidant capacity of the N. lappaceum peel, determined through the radical ABTS method, is much higher than those reported for apples (10–14 μmol TE g⁻¹), berries (16–31 μmol TE g⁻¹), bananas (9.2 μmol TE g⁻¹), grapes (8.7 μmol TE g⁻¹), kiwis (7.8 μmol TE g⁻¹), nectarines (8 μmol TE g⁻¹), plums (9.8 μmol TE g⁻¹), broccoli (6.8 μmol TE g⁻¹), onions (4–7.2 μmol TE g⁻¹), peppers (3.6–19.8 μmol TE g⁻¹), and spinach (22.4 μmol TE g⁻¹ [71, 76].

Antioxidant capacity has an intrinsic relationship with phenolic compounds. Different studies have shown that these compounds can act as powerful antioxidants because of their activity against free radicals and their ability to chelate metals [2, 4, 7]. The Pearson correlation coefficients showed that the TFC and TPC have a strong correlation with antioxidant activity found using an ABTS assay, with r = 0.84 and r = 0.83, respectively, at a confidence level of 95.0% (). However, the correlation with antioxidant activity found using a DPPH assay was lower, with r = 0.68 and r = 0.77 for the TFC and TPC, respectively. The TAC was found to have a weak correlation with antioxidant capacity, with r = 0.15 and r = 0.10 found using ABTS and DPPH, respectively. These findings reveal that the antioxidant capacity of the N. lappaceum peel was mainly caused by phenolic acids and flavonoids. These results are consistent with the studies on the Salacia chinensis root extract, as reported by Ngo et al. [59], which concluded that phenolic compounds and flavonoids are mainly responsible for antioxidant activity, while anthocyanidins have a weak correlation with antioxidant capacity. Although anthocyanins have been reported in some works to have antioxidant properties, it is important to emphasize that these properties depend on their specific chemical structure.

In summary, these results show that extraction solvents and extraction technique significantly affected the active compound yield and antioxidant properties of the extracts.

3.4. GC-MS Analysis

GC-MS was used for analyzing volatile organic compounds present in N. lappaceum. The bioactive compounds present in the extracts obtained from the N. lappaceum peel are summarized in Figure 2 and Table 5. The total ion chromatograms of the three extracts presented in Figure 2 show the retention time and signals that correspond to the active compounds present in the extract. The elution time, molecular formula, molecular weight, and fragment ions are given in Table 5.

(a)

(b)

(c)

Figure 2: Total ion chromatograms obtained by GC-MS of N. lappaceum peel extracts. (a) Ethanolic extract obtained by LLE with ethyl acetate. (b) Ethanolic extract obtained by LLE with hexane. (c) Hydroethanolic crude extract.

Table 5: Active compounds identified in the mass spectra of N. lappaceum peel extracts.

Compound (type)
Molecular formula
Tr (min)
Peak area (%)
Molecular weight (g·mol⁻¹)
Top peak
Fragment ions (m/z), 2nd and 3rd highest
Extract

5-Methylfuran-2-carbaldehyde (arenecarbaldehyde)
C₆H₆O₂
4.1
0.20
110.11
110
109 and 53
Crude

1,2-Benzenediol (hydroxybenzene)
C₆H₆O₂
7.4
2.97
110.11
110
64 and 63
Crude

1,2,3-Benzenetriol or pyrogallol (catechin)
C₆H₆O₃
9.7
29.17
126.11
126
52 and 80
Crude

3-Hydroxybenzoic acid (phenolic acid)
C₇H₆O₃
11.3
7.86
138.12
138
121 and 60
Crude

Hexadecanoic acid or palmitic acid (phenolic acid)
C₁₆H₃₂O₂
16.0
1.11
256.43
73
60 and 55
Crude

Hexadecanoic acid, ethyl ester (fatty acid ethyl ester)
C₁₈H₃₆O₂
16.4
0.18
284.50
88
101 and 55
Crude

Oleic acid methyl ester (fatty acid methyl ester)
C₁₉H₃₆O₂
17.4
0.18
296.5
55
182 and 73
Crude

(9Z,12Z)-Octadeca-9,12-dienoic acid or linoleic acid (fatty acid)
C₁₈H₃₂O₂
17.7
0.29
280.4
67
81 and 55
Crude

9-Octadecenoic acid or elaidic acid (phenolic acid)
C₁₈H₃₄O₂
17.8
1.30
282.5
55
73 and 60
Crude

Beta-tocopherol or vitamin E (vitamin)
C₂₈H₄₈O₂
26.0
0.40
416.7
416
151 and 207
Crude

2,4-Di-tert-butylphenol (alkylbenzene)
C₁₄H₂₂O
11.24
7.78 and 9.21
206.32
191
206 and 57
LLE with hexane and ethyl acetate

Isobutyl octyl phthalate
C₂₀H₃₀O₄
15.22
15.66 and 16.46
334.45
149
57 and 223
LLE with hexane and ethyl acetate

Hexadecanoic acid, methyl ester (fatty acid methyl ester)
C₁₇H₃₄O₂
15.75
4.02 and 2.44
270.5
74
87 and 57
LLE with ethyl acetate

Hexadecanoic acid, ethyl ester (fatty acid ethyl ester)
C₁₈H₃₆O₂
16.38
16.54 and 17.79
284.5
88
101 and 55
LLE with hexane and ethyl acetate

Octadecanoic acid, methyl ester or methyl stearate (fatty acid methyl ester)
C₁₉H₃₈O₂
17.6
3.55 and 4.17
298.50
74
87 and 57
LLE with hexane and ethyl acetate

Oleic acid, ethyl ester (fatty acid ethyl ester)
C₂₀H₃₈O₂
18.0
4.37 and 3.07
310.51
69
55 and 83
LLE with hexane and ethyl acetate

Octadecanoic acid, ethyl ester (fatty acid ethyl ester)
C₂₀H₄₀O₂
18.2
24.30 and 25.2
312.5
88
101 and 55
LLE with hexane and ethyl acetate

Diisooctyl phthalate (phthalate ester)
C₂₄H₃₈O₄
21.2
8.88 and 5.33
390.6
149
167 and 57
LLE with ethyl acetate

Tr: retention time.

A total of 18 compounds were identified, based on their fragmentation patterns, and have been reported for the first time in the N. lappaceum peel. These compounds were hydroxybenzenes, phenolic acids, flavonoids, fatty acids (saturated, unsaturated, and ester), vitamins, arenecarbaldehydes, and phthalates, with the specific species including 5-methylfuran-2-carbaldehyde, 1,2-benzenediol, 1,2,3-benzenetriol (pyrogallol), 3-hydroxybenzoic acid, hexadecanoic acid (palmitic acid), hexadecanoic acid, ethyl ester, oleic acid methyl ester, (9Z,12Z)-octadeca-9,12-dienoic acid (linoleic acid), 9-octadecenoic acid (elaidic acid), beta-tocopherol (vitamin E), 2,4-di-tert-butylphenol, isobutyl octyl phthalate, hexadecanoic acid, methyl ester, hexadecanoic acid, ethyl ester, octadecanoic acid, methyl ester (methyl stearate), oleic acid, ethyl ester, octadecanoic acid, ethyl ester, and diisooctyl phthalate. These identified compounds have been previously isolated from other plants and are known to exhibit important activities (Table 6). Notably, 1,2,3-benzenetriol exhibits activity against lung cancer cell lines, hexadecanoic acid attacks multiple myeloma cells and inhibits HIV-1 infection, and (9Z,12Z)-octadeca-9,12-dienoic acid reduces the risk for type 2 diabetes. Among the compounds with antioxidant properties, we identified beta-tocopherol and 2,4-di-tert-butylphenol, which likely contribute to the antioxidant capacity observed in the N. lappaceum peel.

Table 6: Activities and uses reported for the compounds identified in N. lappaceum peel extracts.

Compounds
Activity or uses

5-Methylfuran-2-carbaldehyde
Food additive (flavoring agent) [77]

1,2-Benzenediol
Precursor to pesticides, flavors, and fragrance [77]

1,2,3-Benzenetriol (pyrogallol)
Cytotoxic effect on human lung cancer cell lines [78], inhibition or retardation of oxidation reactions, effective antiseptic and antipsoriatic agent [79, 80]

3-Hydroxybenzoic acid
Production of glycol benzoates used as plasticizers [77]

Hexadecanoic acid (palmitic acid)
Novel therapeutic agents that specifically attack multiple myeloma cells and inhibit HIV-1 infection [55, 81]

Hexadecanoic acid, ethyl ester
Food additive (flavoring agent) [77]

Oleic acid methyl ester
Additive for detergents, emulsifiers, wetting agents, stabilizers, textile treatment, plasticizers for duplicating inks, rubbers, waxes, chromatographic reference standard [77]

(9Z,12Z)-Octadeca-9,12-dienoic acid (linoleic acid)
Improving insulin sensitivity and reducing the risk for type 2 diabetes. Anti-inflammatory effects that may alleviate migraine [82, 83]

9-Octadecenoic acid (elaidic acid)
Food additive [77]

Beta-tocopherol (vitamin E)
Antioxidant [77]

2,4-Di-tert-butylphenol isobutyl octyl phthalate
Antifungal, antioxidant, and anticancer [84–86]

Hexadecanoic acid, methyl ester
Anti-inflammatory [87], antifibrotic [88], biodiesel [89], neuroprotector [90], additive for detergents, emulsifiers, wetting agents, stabilizers, resins, lubricants, plasticizers, animal feed, and medical research [91]

Hexadecanoic acid, ethyl ester
Anti-inflammatory [87], antimicrobial [92], acaricidal [93], and flavoring agent or adjuvant [94]

Octadecanoic acid, methyl ester (methyl stearate)
Neuroprotector [90], anti-inflammatory [95], additive for detergents, emulsifiers, wetting agents, stabilizers, resins, lubricants, plasticizers [91]

Oleic acid, ethyl ester
Solvent, plasticizer, lubricant, hydrophobic agent, flavoring [96]

Octadecanoic acid, ethyl ester
Flavoring agent or adjuvant

Diisooctyl phthalate
Plasticizer for vinyl, cellulosic, and acrylate resins and synthetic rubber [97]

To the best of our knowledge, there are no studies that identify the compounds reported here. Researchers from China, Malaysia, and Thailand have previously reported the presence of other phenolic compounds such as ellagic acid, gallic acid, corilagin, p-coumaric acid, catechin, rutin, and geraniin in N. lappaceum peels, found by HPLC-MS, HPLC-UV, and NMR [2, 3, 9, 36]. Recently, researchers in Vietnam have also identified other compounds, such as kaempferol and quercetin [3]. Additionally, researchers in Mexico have been the first to identify ellagitannins, such as brevifolin carboxylic acid, ellagic acid pentoside, castalagin/vescalagin, and galloyl-bis-HHDP-hexoside (casuarinin), in N. lappaceum peels [7]. This shows that the chemical diversity of N. lappaceum plants varies depending on their geographical location and climate. The chemical pattern of our sample is noticeably different in composition from that of other N. lappaceum peels reported in other countries [2, 3, 7, 9, 36].

4. Conclusions

The results presented here indicate that the compounds in the extracts obtained from the N. lappaceum peel could be significantly affected by the extraction solvents and extraction technique used. The highest TPC and TFC were obtained in the hydroethanolic ultrasound extracts, and the highest TAC was obtained in the acid extract. The extracts evaluated exhibited good antioxidant capacity with both high inhibitory activity against ABTS∙+ radical cations and strong scavenging activity against DPPH radicals. In this work, we identified 18 compounds that had not previously been isolated from N. lappaceum and found that their peels are rich in phenolic acids, flavonoids, and fatty acids. They contain compounds that have excellent antioxidant capacity and are being studied intensively in other plants for their ability to improve human health and counteract chronic diseases. The new compounds can also be used for industrial and food applications, including detergent additives, biodiesel, emulsifiers, wetting agents, lubricants, stabilizers, food additives, and flavoring agents. Additional studies will be carried out to evaluate the biological activities and to identify more specific phytochemicals responsible for these activities.

Data Availability

The data used to support the findings of this study are included within the article.

Conflicts of Interest

The authors declare that there are no conflicts of interest regarding the publication of this paper.

Acknowledgments

Financial support was received from the National Secretariat of Science, Technology, and Innovation (SENACYT), as members of the Panamá Research National System (SNI). The authors thank the analysts Rosa Parada and Glenda Maldonado and Juanita Freer, Ph.D., of the Renewable Resources Laboratory, Biotechnology Center, Universidad de Concepcion-Chile, for the support provided in identifying compounds by GC-MS. The authors would also like to acknowledge the analytical technology company (Panamá) for providing the FT-IR equipment.

References

N. Thitilertdecha, A. Teerawutgulrag, and N. Rakariyatham, “Antioxidant and antibacterial activities of Nephelium lappaceum L. extracts,” LWT—Food Science and Technology, vol. 41, no. 10, pp. 2029–2035, 2008.

N. Thitilertdecha, A. Teerawutgulrag, J. D. Kilburn, and N. Rakariyatham, “Identification of major phenolic compounds from Nephelium lappaceum L. And their antioxidant activities,” Molecules, vol. 15, no. 3, pp. 1453–1465, 2010.

N. M. P. Nguyen, T. T. Le, H. Vissenaekens et al., “In vitroantioxidant activity and phenolic profiles of tropical fruit by-products,” International Journal of Food Science and Technology, vol. 54, no. 4, pp. 1169–1178, 2019.

U. Palanisamy, H. M. Cheng, T. Masilamani, T. Subramaniam, L. T. Ling, and A. K. Radhakrishnan, “Rind of the rambutan, Nephelium lappaceum, a potential source of natural antioxidants,” Food Chemistry, vol. 109, no. 1, pp. 54–63, 2008.

S. Okonogi, C. Duangrat, S. Anuchpreeda, S. Tachakittirungrod, and S. Chowwanapoonpohn, “Comparison of antioxidant capacities and cytotoxicities of certain fruit peels,” Food Chemistry, vol. 103, no. 3, pp. 839–846, 2007.

Muhtadi, A. U. Primarianti, and T. A. Sujono, “Antidiabetic activity of durian (Durio Zibethinus Murr.) and rambutan (Nephelium lappaceum L.) fruit peels in alloxan diabetic rats,” Procedia Food Science, vol. 3, pp. 255–261, 2015.

C. Hernández, J. Ascacio-Valdés, H. De la Garza et al., “Polyphenolic content, in vitro antioxidant activity and chemical composition of extract from Nephelium lappaceum L. (Mexican rambutan) husk,” Asian Pacific Journal of Tropical Medicine, vol. 10, no. 12, pp. 1201–1205, 2017.

R. Khonkarn, S. Okonogi, C. Ampasavate, and S. Anuchapreeda, “Investigation of fruit peel extracts as sources for compounds with antioxidant and antiproliferative activities against human cell lines,” Food and Chemical Toxicology, vol. 48, no. 8-9, pp. 2122–2129, 2010.

L. Sun, H. Zhang, and Y. Zhuang, “Preparation of free, soluble conjugate, and insoluble-bound phenolic compounds from peels of rambutan (Nephelium lappaceum) and evaluation of antioxidant activities in vitro,” Journal of Food Science, vol. 77, no. 2, pp. C198–C204, 2012.

Z. Yongliang, M. Qingyu, G. Yan, and S. Liping, “Purification and identification of rambutan (Nephelium lappaceum) peel phenolics with evaluation of antioxidant and antiglycation activities in vitro,” International Journal of Food Science and Technology, vol. 52, no. 8, pp. 1810–1819, 2017.

A. a. Nawawi, N. Nakamura, M. Hattori, M. Kurokawa, and K. Shiraki, “Inhibitory effects of Indonesian medicinal plants on the infection of herpes simplex virus type 1,” Phytotherapy Research, vol. 13, no. 1, pp. 37–41, 1999.

P. Chingsuwanrote, C. Muangnoi, K. Parengam, and S. Tuntipopipat, “Antioxidant and anti-inflammatory activities of durian and rambutan pulp extract,” International Food Research Journal, vol. 23, no. 3, p. 939, 2016.

R. S. Bhat and S. Al-daihan, “Antimicrobial activity of Litchi chinensis and Nephelium lappaceum aqueous seed extracts against some pathogenic bacterial strains,” Journal of King Saud University-Science, vol. 26, no. 1, pp. 79–82, 2014.

E. F. Fang and T. B. Ng, “A trypsin inhibitor from rambutan seeds with antitumor, anti-HIV-1 reverse transcriptase, and nitric oxide-inducing properties,” Applied Biochemistry and Biotechnology, vol. 175, no. 8, pp. 3828–3839, 2015.

A. Rajasekaran, S. Ganesan, N. Kamini, C. Lavanya, L. Lee Yoon, and H. Shian Oh, “Anti-nociceptive, CNS, antibacterial and antifungal activities of methanol seed extracts of Nephelium lappaceum L,” Oriental Pharmacy and Experimental Medicine, vol. 13, no. 2, pp. 149–157, 2013.

T. M. I. Morshed, P. R. Dash, F. A. Ripa, T. Foyzun, and S. Mohd Ali, “Evaluation of pharmacological activities of methanolic extract of Nephelium lappaceum L. seeds,” Int.J.Pharmacog, vol. 1, no. 10, pp. 632–639, 2014.

I. Fidrianny, L. Fikayuniar, and M. Insanu, “Antioxidant activities of various seed extracts from four varieties of rambutan (Nephelium lappaceum) using DPPH and ABTS assays,” Asian Journal of Pharmaceutical and Clinical Research, vol. 8, no. 5, pp. 215–219, 2015.

S. Soeng, “Antioxidant and hypoglycemic activities of extract and fractions of Rambutan seeds (Nephelium lappaceum L.),” Biomedical Engineering, vol. 1, no. 1, p. 6, 2015.

N. Thitilertdecha and N. Rakariyatham, “Phenolic content and free radical scavenging activities in rambutan during fruit maturation,” Scientia Horticulturae, vol. 129, no. 2, pp. 247–252, 2011.

O. A. Asimi, N. P. Sahu, and A. K. Pal, “Antioxidant capacity of crude water and ethylacetate extracts of some Indian species and their antimicrobial activity against Vibrio vulnificus and Micrococcus luteus,” Journal of Medicinal Plants Research, vol. 7, no. 26, pp. 1907–1915, 2013.

K. Kim, R. Tsao, R. Yang, and S. Cui, “Phenolic acid profiles and antioxidant activities of wheat bran extracts and the effect of hydrolysis conditions,” Food Chemistry, vol. 95, no. 3, pp. 466–473, 2006.

A. Y. Chen and Y. C. Chen, “A review of the dietary flavonoid, kaempferol on human health and cancer chemoprevention,” Food Chemistry, vol. 138, no. 4, pp. 2099–2107, 2013.

E. O. L. Santos, L. M. Kabeya, A. S. G. Figueiredo-Rinhel et al., “Flavonols modulate the effector functions of healthy individuals' immune complex-stimulated neutrophils: a therapeutic perspective for rheumatoid arthritis,” International Immunopharmacology, vol. 21, no. 1, pp. 102–111, 2014.

M. Singh, M. Kaur, and O. Silakari, “Flavones: an important scaffold for medicinal chemistry,” European Journal of Medicinal Chemistry, vol. 84, pp. 206–239, 2014.

A. Chanwitheesuk, A. Teerawutgulrag, and N. Rakariyatham, “Screening of antioxidant activity and antioxidant compounds of some edible plants of Thailand,” Food Chemistry, vol. 92, no. 3, pp. 491–497, 2005.

N. Balasundram, K. Sundram, and S. Samman, “Phenolic compounds in plants and agri-industrial by-products: antioxidant activity, occurrence, and potential uses,” Food Chemistry, vol. 99, no. 1, pp. 191–203, 2006.

M. d. S. Lima, I. d. S. V. Silani, I. M. Toaldo et al., “Phenolic compounds, organic acids and antioxidant activity of grape juices produced from new Brazilian varieties planted in the Northeast Region of Brazil,” Food Chemistry, vol. 161, pp. 94–103, 2014.

E. W. Chan, V. P. Ng, V. V. Tan, and Y. Y. Low, “Antioxidant and antibacterial properties of alpinia galanga, curcuma longa, and etlingera elatior (zingiberaceae),” Pharmacognosy Journal, vol. 3, no. 22, pp. 54–61, 2011.

M. Oroian and I. Escriche, “Antioxidants: characterization, natural sources, extraction and analysis,” Food Research International, vol. 74, pp. 10–36, 2015.

R. Apak, K. Güçlü, B. Demirata et al., “Comparative evaluation of various total antioxidant capacity assays applied to phenolic compounds with the CUPRAC assay,” Molecules, vol. 12, no. 7, pp. 1496–1547, 2007.

C. D. Stalikas, “Extraction, separation, and detection methods for phenolic acids and flavonoids,” Journal of Separation Science, vol. 30, no. 18, pp. 3268–3295, 2007.

A. Pat, Dietary Phytochemicals and Microbes, Springer, London, UK, 2012.

B. George, C. Kaur, D. S. Khurdiya, and H. C. Kapoor, “Antioxidants in tomato (Lycopersium esculentum) as a function of genotype,” Food Chemistry, vol. 84, no. 1, pp. 45–51, 2004.

G. Pande and C. C. Akoh, “Antioxidant capacity and lipid characterization of six Georgia-grown pomegranate cultivars,” Journal of Agricultural and Food Chemistry, vol. 57, no. 20, pp. 9427–9436, 2009.

C. Hernández-Hernández, C. N. Aguilar, R. Rodríguez-Herrera et al., “Rambutan (Nephelium lappaceum L.):Nutritional and functional properties,” Trends in Food Science and Technology, vol. 85, pp. 201–210, 2019.

U. D. Palanisamy, L. T. Ling, T. Manaharan, and D. Appleton, “Rapid isolation of geraniin from Nephelium lappaceum rind waste and its anti-hyperglycemic activity,” Food Chemistry, vol. 127, no. 1, pp. 21–27, 2011.

F. Notka, G. Meier, and R. Wagner, “Concerted inhibitory activities of on HIV replication in vitro and ex vivo,” Antiviral Research, vol. 64, no. 2, pp. 93–102, 2004.

Z.-F. Wei, S. Jin, M. Luo et al., “Variation in contents of main active components and antioxidant activity in leaves of different pigeon pea cultivars during growth,” Journal of Agricultural and Food Chemistry, vol. 61, no. 42, pp. 10002–10009, 2013.

G. Miliauskas, P. R. Venskutonis, and T. A. van Beek, “Screening of radical scavenging activity of some medicinal and aromatic plant extracts,” Food Chemistry, vol. 85, no. 2, pp. 231–237, 2004.

S. Mehdizadeh, O. Lasekan, K. Muhammad, and B. Baharin, “Variability in the fermentation index, polyphenols and amino acids of seeds of rambutan (Nephelium lappaceum L.) during fermentation,” Journal of Food Composition and Analysis, vol. 37, no. 0, pp. 128–135, 2015.

FAO, Minor Tropical Fruits. Mainstreaming a Niche Market. Food Outlook Julio 2018, Food and Agriculture Organization of the United Nations, Rome, Italy, 2018.

A. Dailey and Q. V. Vuong, “Effect of extraction solvents on recovery of bioactive compounds and antioxidant properties from macadamia (Macadamia tetraphylla) skin waste,” Cogent Food and Agriculture, vol. 1, no. 1, p. 1115646, 2015.

J. Prakash Maran, S. Manikandan, C. Vigna Nivetha, and R. Dinesh, “Ultrasound assisted extraction of bioactive compounds from Nephelium lappaceum L. fruit peel using central composite face centered response surface design,” Arabian Journal of Chemistry, vol. 10, pp. S1145–S1157, 2017.

G. L. Miller, “Use of dinitrosalicylic acid reagent for determination of reducing sugar,” Analytical Chemistry, vol. 31, no. 3, pp. 426–428, 1959.

AOAC, Official Methods of Analysis of the Association of Official Analytical Chemists, AOAC, Rockville, MD, USA, 20th edition, 2016.

R. E. Kitson and M. G. Mellon, “Colorimetric determination of phosphorus as molybdivanadophosphoric acid,” Industrial & Engineering Chemistry Analytical Edition, vol. 16, no. 6, pp. 379–383, 1944.

R. M. Selvam, G. Athinarayanan, A. U. R. Nanthini, A. J. A. R. Singh, K. Kalirajan, and P. M. Selvakumar, “Extraction of natural dyes from Curcuma longa, Trigonella foenum graecum and Nerium oleander, plants and their application in antimicrobial fabric,” Industrial Crops and Products, vol. 70, pp. 84–90, 2015.

J.-H. Xie, C.-j. Dong, S.-P. Nie et al., “Extraction, chemical composition and antioxidant activity of flavonoids from Cyclocarya paliurus (Batal.) Iljinskaja leaves,” Food Chemistry, vol. 186, pp. 97–105, 2015.

R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang, and C. Rice-Evans, “Antioxidant activity applying an improved ABTS radical cation decolorization assay,” Free Radical Biology and Medicine, vol. 26, no. 9-10, pp. 1231–1237, 1999.

K. Thaipong, U. Boonprakob, K. Crosby, L. Cisneros-Zevallos, and D. Hawkins Byrne, “Comparison of ABTS, DPPH, FRAP, and ORAC assays for estimating antioxidant activity from guava fruit extracts,” Journal of Food Composition and Analysis, vol. 19, no. 6-7, pp. 669–675, 2006.

W. A. Fila, E. H. Itam, J. T. Johnson et al., “Comparative proximate compositions of watermelon citrullus lanatus, squash cucurbita pepo'l and rambutan Nephelium lappaceum,” International Journal of Science and Technology, vol. 2, no. 1, pp. 81–88, 2013.

K. Soetan, C. Olaiya, and O. Oyewole, “The importance of mineral elements for humans, domestic animals and plants: a review,” African Journal of Food Science, vol. 4, 2009.

L. Prashanth, K. Kattapagari, R. Chitturi, V. Baddam, and L. Prasad, “A review on role of essential trace elements in health and disease,” Journal of Dr. NTR University of Health Sciences, vol. 4, no. 2, pp. 75–85, 2015.

J. R. Memon, S. Q. Memon, M. I. Bhanger, G. Z. Memon, A. El-Turki, and G. C. Allen, “Characterization of banana peel by scanning electron microscopy and FT-IR spectroscopy and its use for cadmium removal,” Colloids and Surfaces B: Biointerfaces, vol. 66, no. 2, pp. 260–265, 2008.

M. Thirumavalavan, Y.-L. Lai, and J.-F. Lee, “Fourier transform infrared spectroscopic analysis of fruit peels before and after the adsorption of heavy metal ions from aqueous solution,” Journal of Chemical and Engineering Data, vol. 56, no. 5, pp. 2249–2255, 2011.

Y.-L. Han, J. Gao, Y.-Y. Yin, Z.-Y. Jin, X.-M. Xu, and H.-Q. Chen, “Extraction optimization by response surface methodology of mucilage polysaccharide from the peel of Opuntia dillenii haw. fruits and their physicochemical properties,” Carbohydrate Polymers, vol. 151, pp. 381–391, 2016.

R. Nethaji, G. Thooyavan, K. Mullai Nilla, and K. Ashok, “Phytochemical profiling, antioxidant and antimicrobial activity of methanol extract in rambutan fruit (Nephelium lappaceum) epicarp against the human pathogens,” International Journal of Current Innovation Research, vol. 1, no. 9, pp. 201–206, 2015.

S. Lestari, M. Djati, A. Rudijanto, and F. Fatchiyah, “Production and potency of local rambutan at East Java as a candidate phytopharmaca,” Agrivita, vol. 35, no. 3, pp. 270–276, 2013.

T. V. Ngo, C. J. Scarlett, M. C. Bowyer, P. D. Ngo, and Q. V. Vuong, “Impact of different extraction solvents on bioactive compounds and antioxidant capacity from the root of Salacia chinensis L.,” Journal of Food Quality, vol. 2017, Article ID 9305047, 8 pages, 2017.

J. Gusman and P.-J. Tsai, “Extraction of antioxidant compounds from rambutan (Nephelium lappaceum L.) peel as agricultural waste in Taiwan,” Journal of Tropical Crop Science, vol. 2, no. 2, pp. 10–16, 2015.

S. Silva, E. M. Costa, C. Calhau, R. M. Morais, and M. E. Pintado, “Anthocyanin extraction from plant tissues: a review,” Critical Reviews in Food Science and Nutrition, vol. 57, no. 14, pp. 3072–3083, 2017.

J. Sun, H. Peng, W. Su, J. Yao, X. Long, and J. Wang, “Anthocyanins extracted from rambutan (Nephelium lappaceum L.) pericarp tissues as potential natural antioxidants,” Journal of Food Biochemistry, vol. 35, no. 5, pp. 1461–1467, 2011.

J.-M. Kong, L.-S. Chia, N.-K. Goh, T.-F. Chia, and R. Brouillard, “Analysis and biological activities of anthocyanins,” Phytochemistry, vol. 64, no. 5, pp. 923–933, 2003.

M. J. Kaneria, M. B. Bapodara, and S. V. Chanda, “Effect of extraction techniques and solvents on antioxidant activity of pomegranate (Punica granatum L.) leaf and stem,” Food Analytical Methods, vol. 5, no. 3, pp. 396–404, 2012.

Q. D. Do, A. E. Angkawijaya, P. L. Tran-Nguyen et al., “Effect of extraction solvent on total phenol content, total flavonoid content, and antioxidant activity of Limnophila aromatica,” Journal of Food and Drug Analysis, vol. 22, no. 3, pp. 296–302, 2014.

E. Koffi, T. Sea, Y. Dodehe, and S. Soro, “Effect of solvent type on extraction of polyphenols from twenty three Ivorian plants,” Journal of Animal and Plant Sciences (JAPS), vol. 5, no. 3, pp. 550–558, 2010.

A. Trojanowska, I. Tsibranska, D. Dzhonova et al., “Ultrasound-assisted extraction of biologically active compounds and their successive concentration by using membrane processes,” Chemical Engineering Research and Design, vol. 147, pp. 378–389, 2019.

C. McDonnell and B. K. Tiwari, “Chapter five—ultrasound: A clean, green extraction technology for bioactives and contaminants,” in Comprehensive Analytical Chemistry, pp. 111–129, Elsevier, Amsterdam, Netherlands, 2017.

J. Quintero Quiroz, A. M. Naranjo Duran, M. Silva Garcia, G. L. Ciro Gomez, and J. J. Rojas Camargo, “Ultrasound-Assisted extraction of bioactive compounds from annatto seeds, evaluation of their antimicrobial and antioxidant activity, and identification of main compounds by LC/ESI-MS analysis,” International Journal of Food Science, vol. 2019, Article ID 3721828, 9 pages, 2019.

S. Singh and R. P. Singh, “In vitro methods of assay of antioxidants: an overview,” Food Reviews International, vol. 24, no. 4, pp. 392–415, 2008.

W.-y. Huang, H.-c. Zhang, W.-x. Liu, and C.-y. Li, “Survey of antioxidant capacity and phenolic composition of blueberry, blackberry, and strawberry in Nanjing,” Journal of Zhejiang University Science B, vol. 13, no. 2, pp. 94–102, 2012.

F. S. Hosseinian, W. Li, A. W. Hydamaka et al., “Proanthocyanidin profile and ORAC values of manitoba berries, chokecherries, and seabuckthorn,” Journal of Agricultural and Food Chemistry, vol. 55, no. 17, pp. 6970–6976, 2007.

C. Kevers, M. Falkowski, J. Tabart, J.-O. Defraigne, J. Dommes, and J. Pincemail, “Evolution of antioxidant capacity during storage of selected fruits and vegetables,” Journal of Agricultural and Food Chemistry, vol. 55, no. 21, pp. 8596–8603, 2007.

H. E. Miller, F. Rigelhof, L. Marquart, A. Prakash, and M. Kanter, “Antioxidant content of whole grain breakfast cereals, fruits and vegetables,” Journal of the American College of Nutrition, vol. 19, no. 3, pp. 312S–319S, 2000.

O. Martín-Belloso, I. Odriozola-Serrano, M. A. Rojas-Graü, and R. Soliva-Fortuny, “12—oxidative stability of antioxidants in fruits and vegetables,” in Oxidation in Foods and Beverages and Antioxidant Applications, pp. 391–423, Woodhead Publishing, 2010.

E. D. Cömert, B. A. Mogol, and V. Gökmen, “Relationship between color and antioxidant capacity of fruits and vegetables,” Current Research in Food Science, 2019.

PubChem, U.S. National Library of Medicine, National Center for Biotechnology Information Department of Health and Human Services, Bethesda, MD, USA, 2019.

C.-J. Yang, C.-S. Wang, J.-Y. Hung et al., “Pyrogallol induces G2-M arrest in human lung cancer cells and inhibits tumor growth in an animal model,” Lung Cancer, vol. 66, no. 2, pp. 162–168, 2009.

S. A. Avase, S. Srivastava, K. Vishal, H. V. Ashok, and G. Varghese, “Effect of pyrogallol as an antioxidant on the performance and emission characteristics of biodiesel derived from waste cooking oil,” Procedia Earth and Planetary Science, vol. 11, pp. 437–444, 2015.

G. Upadhyay, S. P. Gupta, O. Prakash, and M. P. Singh, “Pyrogallol-mediated toxicity and natural antioxidants: triumphs and pitfalls of preclinical findings and their translational limitations,” Chemico-Biological Interactions, vol. 183, no. 3, pp. 333–340, 2010.

D. Y.-W. Lee, X. Lin, E. E. Paskaleva et al., “Palmitic acid is a novel CD4 fusion inhibitor that blocks HIV entry and infection,” AIDS Research and Human Retroviruses, vol. 25, no. 12, pp. 1231–1241, 2009.

C. Santos and D. F. Weaver, “Topically applied linoleic/linolenic acid for chronic migraine,” Journal of Clinical Neuroscience, vol. 58, pp. 200-201, 2018.

M. A. Belury, R. M. Cole, D. B. Snoke, T. Banh, and A. Angelotti, “Linoleic acid, glycemic control and Type 2 diabetes,” Prostaglandins, Leukotrienes and Essential Fatty Acids, vol. 132, pp. 30–33, 2018.

M. K. Sang and K. D. Kim, “The volatile-producing Flavobacterium johnsoniae strain GSE09 shows biocontrol activity against phytophthora capsici in pepper,” Journal of Applied Microbiology, vol. 113, no. 2, pp. 383–398, 2012.

K. K. Varsha, L. Devendra, G. Shilpa, S. Priya, A. Pandey, and K. M. Nampoothiri, “2,4-Di-tert-butyl phenol as the antifungal, antioxidant bioactive purified from a newly isolated Lactococcus sp,” International Journal of Food Microbiology, vol. 211, pp. 44–50, 2015.

Y. W. Song, Y. Lim, and S. K. Cho, “2,4-Di-tert-butylphenol, a potential HDAC6 inhibitor, induces senescence and mitotic catastrophe in human gastric adenocarcinoma AGS cells,” Biochimica et Biophysica Acta (BBA)—Molecular Cell Research, vol. 1865, no. 5, pp. 675–683, 2018.

N. M. Saeed, E. El-Demerdash, H. M. Abdel-Rahman, M. M. Algandaby, F. A. Al-Abbasi, and A. B. Abdel-Naim, “Anti-inflammatory activity of methyl palmitate and ethyl palmitate in different experimental rat models,” Toxicology and Applied Pharmacology, vol. 264, no. 1, pp. 84–93, 2012.

E. El-Demerdash, “Anti-inflammatory and antifibrotic effects of methyl palmitate,” Toxicology and Applied Pharmacology, vol. 254, no. 3, pp. 238–244, 2011.

M. A. H. Altaie, R. B. Janius, Y. H. Taufiq-Yap, and U. Rashid, “Basic properties of methyl palmitate-diesel blends,” Fuel, vol. 193, pp. 1–6, 2017.

H. W. Lin, I. Saul, V. L. Gresia, J. T. Neumann, K. R. Dave, and M. A. Perez-Pinzon, “Fatty acid methyl esters and Solutol HS 15 confer neuroprotection after focal and global cerebral ischemia,” Translational Stroke Research, vol. 5, no. 1, pp. 109–117, 2014.

M. D. Larrañaga, R. J. Lewis, and R. A. Lewis, “M,” in Hawley’s Condensed Chemical Dictionary, R. J. L. a. R. A. L. M. D. Larrañaga, Ed., pp. 849–948, John Wiley & Sons, Inc., Hoboken, NJ, USA, 16th edition, 2016.

M. Yousry, M. Manal, K. Magdy, and M. Khouloud, “Characterization of marine Burkholderia cepacia antibacterial agents,” Journal of Natural Products, vol. 3, pp. 86–94, 2010.

C. Bu, D. Duan, Y. Wang, L. Ma, Y. Liu, and G. Shi, “Acaricidal activity of ethyl palmitate against tetranychus cinnabarinus,” in Information Technology and Agricultural Engineering. Advances in Intelligent and Soft Computing, Springer, Berlin, Germany, 2012.

National Center for Biotechnology Information, “PubChem database: ethyl palmitate,” National Center for Biotechnology Information, Bethesda, MD, USA, September 2019, https://pubchem.ncbi.nlm.nih.gov/compound/Ethyl-palmitate.

A. R. Othman, N. Abdullah, S. Ahmad, I. S. Ismail, and M. P. Zakaria, “Elucidation of in-vitro anti-inflammatory bioactive compounds isolated from Jatropha curcas L. plant root,” BMC Complementary and Alternative Medicine, vol. 15, p. 11, 2015.

M. D. Larrañaga, R. J. Lewis, and R. A. Lewis, “E,” in Hawley’s Condensed Chemical Dictionary, R. J. L. a. R. A. L. M. D. Larrañaga, Ed., pp. 543–605, John Wiley & Sons, Inc., Hoboken, NJ, USA, 16th edition, 2016.

M. D. Larrañaga, R. J. Lewis, and R. A. Lewis, “D,” in Hawley’s Condensed Chemical Dictionary, R. J. L. a. R. A. L. M. D. Larrañaga, Ed., pp. 411–544, John Wiley & Sons, Inc., Hoboken, NJ, USA, 16th edition, 2016.