Correlation between Antioxidant Activity and the Garcinol Content Released from Fruit Rinds of EndemicGarcinia quaesita Pierre on Different Cooking Conditions

+e antioxidant activity of garcinol has been identified as the basis for various bioactivities in it, and the genusGarcinia is the main source for the garcinol. G. quaesita, which is endemic to Sri Lanka, is a representative member of genus Garcinia, and the dried fruit rind of G. quaesita is used practically in all curry preparations to impart a sour flavor. In our study, garcinol was isolated (yield: 3.67%) from the dried fruit rind of G. quaesita, for the first time. Further, how cooking conditions enable the ingestion of garcinol during the consumption of curries was also examined. +e garcinol content released in different cooking conditions was positively correlated with the antioxidant activity in vitro.+e results revealed that boiling virgin coconut oil extract ofG. quaesita, simulating the common practice of oil frying during cooking, is the best method for obtaining the highest amount of garcinol into the curry medium.


Introduction
Oxidative stress-mediated events are considered to be the most important unifying pathogenic factors responsible for most of the noncommunicable diseases such as diabetes mellitus, coronary heart disease, and cancer [1]. e high antioxidant capacity of phytochemicals is considered to be the hidden miracle responsible for the health benefits associated with most of the food varieties including fruits, vegetables, and spices [2]. It has been shown that there is a strong relationship between consumption of plant-derived food and the protection against degenerative diseases as the antioxidants facilitate free-radical scavenging activity, transition-metal chelating activity, or singlet-oxygen quenching capacity [3][4][5][6]. ere is substantial evidence for the therapeutic role of spices that offer antioxidants, which prevent the oxidation of other compounds or neutralize free radicals [7]. Among the many natural antioxidants, garcinol has been identified as a good source of natural antioxidant as well as a potent bioactive compound that can be incorporated in the treatment of several degenerative diseases.
Garcinol is isolated from several species of the family Garcinia and was first isolated from the hexane extract of the dried fruit rind of G. indica [8]. Later, it was isolated from the bark of G. cambogia (G. gummi-gutta), fruits of G. cambogia (G. gummi-gutta), twig and leaves of G. bancana, bark of G. huillensis, pericarp of G. pedunculata, and leaves of G. travancorica [9][10][11][12][13]. e in vitro antioxidant activity studies done for garcinol by using electron spin resonance (ESR) spectrometry has shown that it has a potent radical scavenging activity [14]. Several studies on garcinol have revealed that it could exhibit the ability to fight many chronic diseases such as cardiovascular disorders, diabetes, neurodegenerative diseases, and asthma [15]. Further, it is found that garcinol inhibits growth of cancer cells and has the ability to modulate the expression of proinflammatory cytokines, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2, NF-κB, STAT3, Akt, FAK, death receptors, nicotinic receptors, cyclin D3, and histone acetyltransferases (p300 and PCAF) [16][17][18]. In addition, oral administration of garcinol has reduced acute ulceration of the stomach in laboratory animals [19]. Furthermore, garcinol showed a clear neuroprotective effect associated with the antioxidant activity by inhibiting iNOS induction in astrocytes [20]. Despite the fact that garcinol has several prominent bioactivities, very few successful paths for the synthesis of garcinol are reported to date and hence, it is important to discover plants that would be a source of garcinol [21].
In the present study, we have revealed that Garcinia quaesita Pierre (Clusiaceae), an endemic species native to Sri Lanka, as a new source of garcinol. G. quaesita is locally known as "Rathgoraka," and dried fruit rinds are used to impart a sour flavor in the preparation of traditional curries as spices. G. quaesita is similar in vegetative morphology to G.gummi-gutta, which belongs to the same family. e dried fruit rinds of G.gummi-gutta (L.) N. Robson (formerly known as Garcinia cambogia) is used in the cuisines of ailand, India, Burma, and Malaysia. Interestingly, in Sri Lanka, the dried rind of the fruit of G. quaesita has also been used in traditional medicine applications both as an astringent and as an antiseptic; ethnomedically, it is reported to act as a stomachic and is used in anorexia, chronic dyspepsia, hypertension, and dyslipidaemia [22]. Previously, Gunathilaka, Sriyani, and Sotheeswaran have reported the isolation of the bioactive compounds including quaesitol and harmonionic acid from the bark of G. quaesita [23]. However, to date, there is no published report on bioactivity studies and isolation of bioactive compound (s) from the fruit extract of G. quaesita grown in Sri Lanka.
Many changes occur in vegetables as well as in spices during the cooking process, and these cooking treatments soften the cell walls and facilitate the extraction of antioxidant molecules. Although published studies are available on the influence of cooking methods on the antioxidant activity of vegetables, there are limited studies on the effect of domestic cooking on spices [24]. e different cooking methods could encourage significant changes to the extraction of antioxidant molecules and affect the concentration of them in the cooking medium. e amount of the antioxidant active compounds released to curry medium depends on the conditions of the curry medium such as temperature, pH, and the polarity of the medium [25]. us, it is important to identify the effective cooking method of spices for the extraction of high concentration of antioxidant molecules into the curry medium. is is specially a concern since the fruit rinds of G. queasita are not consumed unless it is used as a paste. e investigation of correlation between concentration of antioxidant molecules and the antioxidant activity in particular of the curry medium is also advantageous to understand a better cooking treatment.
Herein, we report the total antioxidant activity of fruit extracts of G. quaesita, in different solvents, the isolation and characterization of antioxidant compound (garcinol) from the active fraction (s) using bioassay-guided fractionation, and the correlation studies between total antioxidant activities of its dried fruit under simulated cooking conditions and isolated garcinol.
UV spectra were recorded on a UV-Visible spectrophotometer (Shimadzu, Japan). 1 H and 13 C NMR spectra were recorded on a 400 Hz NMR spectrometer (Bruker, USA) using CDCl 3 . Melting points of the isolated compound were determined using a Stuart scientific electrothermal melting point apparatus and were uncorrected. e isolated compound was subjected to FTIR analysis for the determination of the functional groups present. A sample with KBr pelleted garcinol was used to obtain IR spectra in the scanning wave number ranging from 500 to 4000 cm − 1 with a resolution of 4 cm − 1 on FT-IR Spectrophotometer (Shimadzu, Japan). LC-MS spectra was recorded with LC-MS/ MS system (Waters, Milford, MA, USA) (4.6 × 150 mm, 5 μm) at flow rate 0.3 mL·min − 1 using acetonitrile: water (30%). Data were acquired for precursor masses from m/z 100− 1500 amu at 35 K resolution with data-dependent tandem mass spectrometry with both positive and negative modes. Digital polarimeter (M 241 Perkin-Elmer) was used to measure specific rotation of garcinol in ethanol. Further, a rotary evaporator (Buchi, Switzerland) was used in the preparation of plant extracts. e HPLC-UV/DAD analysis was carried for quantification of garcinol in different cooking methods on an Agilent 1100 (USA) (C 18 column (4.6 × 150 mm, 5 μm)).

Plant Material and Preparation of Plant Extracts.
e dried fruit rinds of G. quaesita were collected from Kandy, Central Province, Sri Lanka, in March, 2016. e plant itself was taxonomically authenticated by comparing with specimens at the National Herbarium, National Botanical Gardens, Peradeniya, Sri Lanka.
A voucher specimen (No: 6/01/H/03) was deposited at National Herbarium, National Botanical Garden, Peradeniya, Sri Lanka. e dried fruit rinds were washed, oven dried (60°C, 30 minutes), and ground into a fine powder. e resulting powder (450.00 g) was sequentially extracted into hexane, ethyl acetate, and methanol (500 ml, 3 × 24 hours) using an electrical shaker (150 rpm, 28°C). Solvents were evaporated under reduced pressure below 40°C using a rotary evaporator. Concentrated extracts were freeze-dried and the resultant powder was stored at 4°C until further use.

Evaluation of Antioxidant Activity.
e DPPH radical scavenging activity was determined on the crude extracts of hexane, ethyl acetate, and methanol as mentioned below [26]. e 0.004% DPPH solution in methanol (200 μL) was added to a graded concentration of the plant extract mixture (2.8 mL), and the reactants were incubated at 25°C for 30 minutes in the dark. L-ascorbic acid in methanol was used as the positive control. e absorbance was measured at 517 nm using a UV-visible spectrophotometer.
e percentage of inhibition of DPPH radical scavenging activity of the tested samples was calculated as follows.
Percentage of inhibition (%) � (A 0 -A 1 /A 0 ) × 100%, where A 0 is the absorbance of the control and A 1 is the absorbance of the test (plant extract/reference compound). e concentration of plant extract mixture that inhibits the 50% of the DPPH radical formation (IC 50 ) was calculated using regression analysis.
e extract that showed the maximum antioxidant activity was used to isolate the antioxidant active compounds. Subsequently, DPPH radical scavenging activities of the isolated compound and cooked samples with dried rinds of G. quaesita were determined with same procedure as describe above.
Ferric reducing antioxidant power of isolated compound was estimated with the FRAP assay described by Benzie and Strain, with slight modifications [27]. e FRAP reagent was freshly prepared by adding 10 mM of 2,4,6-Tris (2-pyridyl)-1,3,5-triazine (TPTZ) dissolved in 40 mM of HCl, 20 mM of FeCl 3 in water, and 300 mM of acetate buffer (pH 3.6) in the ratio of 1 : 1 : 10. Garcinol (100 μL) was added to the FRAP reagent (3.0 mL) and mixed well. e absorbance of the resultant solution was measured at 593 nm using UV-visible spectrophotometer. FeSO 4 served as the standard compound.
e FRAP value was estimated using regression equation of FeSO 4 standard curve and expressed in terms of ferrous equivalent (FE; concentration of the extract or standard compound that expresses the same absorbance as 1 mmol of ferrous ion).

TLC Bioautography Analysis and Isolation of Antioxidant
Compounds.
e hexane extract of G. quaesita fruit rinds, which showed the highest antioxidant activity, was selected for the isolation of antioxidant compounds. An aliquot of crude hexane solution (1 mg/mL) was directly deposited as a spot on a TLC plate and developed with n-hexane:ethyl acetate (7.5 : 2.5). A solution of 0.004% (w/v) DPPH in methanol was sprayed on the developed TLC plate for derivatization [28].
Medium pressure liquid chromatography (MPLC) was performed on the crude hexane extract of G. quaesita (4.50 ± 0.05 g). Nine fractions were obtained based on the TLC patterns, and the combined fractions were subjected to TLC bioautography with DPPH. e 5 th combined fraction showed comparatively high antioxidant activity and it was further separated using flash column chromatography. e resultant two fractions were subjected to the TLC bioautography with DPPH, and the 2 nd fraction showed significant antioxidant activity. e most active target compound was isolated from the 2 nd fraction after subjecting to flash column chromatography (ethyl acetate: hexane � 7.5 : 2.5; yield:3.67% from the crude hexane extract of fruits of G. quaesita, 4.50 g). Based on comprehensive 1 H and 13 C NMR analysis, the structure of the isolated compound was elucidated as garcinol by comparing with reported data from literature [8]. In addition, it was subjected to the DPPH radical scavenging assay and ferric reducing antioxidant power (FRAP) assay.

Preparation of Differently Processed Samples Simulating
Cooking Conditions. Homogeneous pieces of fruit rinds of G. quaesita (50.0 g) were immersed in 80.0 mL of each distilled water, distilled water and acetic acid solution (pH = 4), distilled water and acetic acid solution (pH = 2), and boiled for one hour to simulate the boiling of vegetables, fish, or meat in different acidic (strong and weak) environments that are generated with other added cooking ingredients such as vinegar. Same procedure was followed for natural coconut milk and saturated salt solution (NaCl) to simulate the boiling of vegetables, fish, and meat with salt and coconut milk. e resultant aqueous extracts of G. quaesita were filtered and extracted into dichloromethane (50 ml × 3) by liquid-liquid partition. Another two samples of G. quaesita (50.0 g) were immersed in virgin coconut oil and treated with heat for one hour and stirred at room temperature for three hours to simulate pan frying (frying fish/meat) and mixing with oil without heating of vegetables. e resultant oil extractions were filtered and extracted into ethyl acetate (50 ml × 3). en, all organic solvents were evaporated using a rotary evaporator below 40°C. e DPPH assay was performed on the resultant dried samples, and IC 50 values were calculated. In addition, garcinol content was quantified using HPLC, and correlation analysis was done between the content of garcinol vs IC 50 of DPPH assay.

HPLC Quantification of Garcinol Content in Cooked
Samples.
e HPLC-UV/DAD analysis was carried out on an Agilent 1100 (USA) connected to a diode array detector and controlled by Agilent software. An aliquot of each cooked sample was filtered and injected into the HPLC C 18 column (4.6 × 150 mm, 5 μm) and eluted with a mobile phase containing acetonitrile: water in the ratio 7 : 3. e separation was carried out at room temperature (27°C) and monitored at 360 nm. e quantitative analysis of garcinol in each dried sample was achieved by peak area normalization.

Linearity.
e linearity was determined by correlation coefficients of the analytical curves generated by injections of working solutions at eight concentration levels. In the method of quantification of garcinol in different cooked samples, an external standard method was employed using a stock solution of garcinol (500.00 ± 0.01 mg/L) prepared in methanol. Eight working solutions with concentrations of 1.00-500.00 ± 0.01 mg/L of garcinol were prepared by diluting the standard stock solution. e analytical curves were built by triplicate injections.

Statistical Analysis.
All analyses/assays were done in triplicate. e results of the DPPH and FRAP assays were expressed as mean ± SD. Minitab statistical software was used for the regression analysis and Pearson's correlation coefficient analysis between the concentration of garcinol and DPPH radical scavenging activity.

Results and Discussion
In the present study, the antioxidant activity for hexane, ethyl acetate, and methanol extracts of dried fruit rinds of G. quaesita was determined using DPPH radical scavenging assay, and the IC 50 values of crude extracts were 8.74 ± 0.75, 77.36 ± 0.82, and 340.8 ± 1.02 ppm, respectively. Interestingly, the highest antioxidant activity was observed in the crude hexane extract; hence, it was assumed that nonpolar compounds might be responsible for its activity. Even though the hexane extract of fruits of G. quaesita showed the highest antioxidant activity even better than the standard ascorbic acid, Shivapriya et al has reported that hexane extract of fruits of G. cambogia, which is similar in vegetative morphology to G. quaesita, showed very insignificant antioxidant activity [29]. e hexane extract that showed the highest antioxidant activity was selected for bioactivity-guided fractionation and isolation of antioxidant compounds in the present study. e active compounds present in the hexane extract were monitored using TLC bioautography in order to guide the separation as it allows rapid access for detection and localization of the active compounds in a plant extract. In this method, DPPH radical scavenging activity was observed visually as yellow spots on a purple background.
is is a straightforward method to identify active fractions and to isolate antioxidant compounds. e fraction that showed the highest antioxidant activity was then further separated using silica gel flash column chromatography. Finally, one pure compound was isolated and subjected to structure elucidation. e yellow crystalline compound isolated from hexane extract was identified as garcinol by UV, 1 H NMR, 13 C NMR, and LC-MS spectral data and by comparison with the literature data, mp 126°C, UV in EtOH (log ϵ) 256 nm, 327 nm; IR 3200-3500, 1730, 1640 cm − 1 ; LC-MS was m/z 602 and [α] 27 � -140°, Figure 1 [8].
Even though garcinol has been isolated from several species of the family Garcinia, this is the first report on the isolation of garcinol from the hexane fruit extract of G. quaesita, an endemic plant native to Sri Lanka.
Garcinol showed concentration-dependent radical scavenging activity in the DPPH assay (expressed as IC 50 values). e IC 50 value of garcinol was 0.24 ± 0.13 μg/mL, while the standard antioxidant compound L-ascorbic acid achieved 10.10 ± 0.07 μg/mL. Although garcinol is known as an antioxidant compound, its comprehensive antioxidant activity in relation to its bioavailability under processing conditions has not been reported. e FRAP value of the isolated compound was found to be 740.85 ± 0.01 μM (Fe(II)/g compared to ascorbic acid with an FRAP value of 1206.00 ± 0.06 μM(Fe(II)/g. Garcinol is a polyisoprenylated benzophenone and chemically it is a tri-isoprenylated chalcone, and the antioxidant nature of garcinol largely depends on its polyphenolic structure. e dried fruit of G. quaesita is a well-known spice used extensively in Sri Lankan and Asian daily curry preparations. e presence of large amounts of highly antioxidant active garcinol in the fruits of G. quaesita (yield; 3.67%) from the crude hexane extract of fruits of G. quaesita can be used to unleash its health benefits. erefore, our investigation is also focused on studying the scientific validation of the use of dried fruits of G. quaesita in curry preparations. e fruits of G. quaesita have been used in the several different simulated cooking methods in order to understand the effects of cooking conditions on the antioxidant efficiency of dried fruit rinds of G. quaesita. In addition, since garcinol is the main antioxidant compound in dried fruits of G. quaesita, quantification of garcinol content present was also carried out. Table 1 and Figure 2 show the IC 50 values of differently processed samples of G. quaesita simulating cooking conditions applicable in the DPPH assay.
A garcinol calibration curve was developed acquiring chromatograms at 360 nm in the linearity range of 1-500 ppm. A reverse-phase HPLC analysis was performed in order to quantify the garcinol content. e garcinol content of the dried samples prepared from liquid-liquid partition was calculated using regression analysis (Y � 5.84X + 37.37) and is shown in Table 2 and Figure 3.
According to the observed data, the highest antioxidant activity was obtained for the nonpolar coconut oil medium, which is again correlated with the observed higher antioxidant activity of the crude hexane extract. e garcinol content showed a significant positive correlation with antioxidant activity of curry medium, with Pearson's correlation coefficients of 0.880 (R 2 � 87.0%, p < 0.05). It suggests that antioxidant activity of the curry medium that contains dried fruits of G. quaesita depends on the released garcinol content into the curry medium. erefore, it reflects a strong relationship between antioxidant activity and the nature of the conditions in the curry medium. is linear relationship quantitatively confirms the significance of the garcinol content in the curry medium to the antioxidant activity. Further, in comparison to the antioxidant activity obtained for crude extracts of G. quaesita, hexane extract showed the highest antioxidant activity and garcinol was present there as the major compound. us, it can be rationalized that garcinol is responsible for the antioxidant activity in G. quaesita. e extract obtained from oil medium showed the highest antioxidant activity in the DPPH assay (8.64 ± 1.15 and 6.73 ± 1.29 respectively) suggesting that the oil medium is more favorable to release nonpolar garcinol to the curry medium. Interestingly, the antioxidant activity in neutral aqueous medium also turned out to be relatively high (IC 50 � 15.86 ± 0.1), suggesting that garcinol has the ability to be released into    Journal of Food Quality polar aqueous medium through its hydrophilic functional groups. Importantly, among the cooking conditions mentioned above, boiling virgin coconut oil extract simulating the oil frying method is the best method to obtain the highest antioxidant activity. However, since oil frying of curry mixtures is invariably laced with water, traditional cooking methods using G. quaesita could bring out garcinol both by oil and water. Hence, traditional cooking with G. quaesita using oil can be appreciated as a best practice to obtain the highest amount of garcinol leading to potentially higher bioavailability and positive health benefits. Unless it is a paste made out of fruit rinds of G. quaesita, the traditional practice is to remove the fruit rind of G. quaesita after cooking without consumption. Significantly, if an oil medium is used, garcinol may be released to the diet whether used as a paste or as the whole fruit rind. is is the first report on correlation study between the antioxidant activity of the different curry mediums that contain dried fruit rinds from Garcinia family and the garcinol content.

Conclusions
e results revealed that antioxidant activity is the highest in the hexane extract of dried fruit rinds of G. quaesita. It was confirmed that the predominant antioxidant compound in the hexane extract is garcinol. Garcinol exhibits a statistically significant in vitro antioxidant activity in DPPH and FRAP assays. e antioxidant activity of curry medium is greatly influenced by the nature of cooking conditions, and the best method to obtain highest amount of garcinol into the curry medium is the oil frying method followed closely by neutral aqueous medium. ere is a positive correlation between garcinol content in different cooking conditions and the antioxidant activity in vitro.
us, these results validated the traditionally practiced aqueous/oil frying for releasing maximum amount of garcinol leading to potent antioxidant activity-related health benefits.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare no conflicts of interest.

Supplementary Materials
e yellow crystalline compound isolated from hexane extract was identified as garcinol by UV, 1 H NMR, 13    Journal of Food Quality