*2.8. Statistical Analysis*

All experiments were conducted in triplicate unless otherwise indicated and the results were expressed as mean ± standard deviation (SD). The statistical analysis was conducted with SPSS (ver. 10.1) for Windows and a one-way analysis of variance (ANOVA). Duncan's multiple range tests were carried out to test any significant differences among various fruit maturity stages. Values with *p* < 0.05 were considered as significantly different

#### **3. Results and Discussion**

#### *3.1. Chemical Composition of the Steam-Distilled Essential Oil (SDEO) Fraction*

The yields of total SDEO and GBAF from *M. tricuspidata* fruit were 0.03 ± 0.01% and 0.37 ± 0.03%, respectively. Table 1 shows the volatile compounds identified in the SDEO and GBAF isolated from *M*. *tricuspidata* fruit along with their amounts and retention indices on DB-5MS (non-polar) and DB-WAX (polar) column. A total of 55 compounds including 17 tentatively identified compounds were identified in SDEO. The compounds that were found by only DB-5MS column but not by DB-WAX column were considered as tentatively identified. The compounds were 4 alcohols, 14 aldehyde and ketones, 7 terpenoids, 13 carotenoid-derived compounds, 6 aromatic and phenolic compounds, 11 acids and 3 miscellaneous. With the exception of aliphatic acids and their esters such as palmitic acid, linoleic acid, ethyl palmitate and linoleic acid, compounds with the highest concentration in the SDEO were *p*-cresol (393.50 ± 17.70), followed by δ-cadinene (147.67 ± 7.50), β-caryophyllene (145.67 ± 10.50), β-ionone (141.00 ± 4.40) and *n*-nonanal (140.33 ± 20.50 μg/g). In particular, 10 kinds of carotenoid-derived compounds were identified in the SDEO. These compounds have been found in various plants and are known to play an important role as characteristic aroma compounds of leaves, flowers or fruits of some plants [28,41,42]. Especially, theaspirane A and theaspirane B are present in green tea, black tea, grape and corn [43], and are believed to contribute to the unique aroma of *M. tricuspidata* fruit. Their chemical structures are presented in Figure 1.


**Table 1.** Concentration of compounds identified in steam-distilled essential oil (SDEO) and glycosidically bound aglycone fraction (GBAF) isolated from *M. tricuspidata* fruit.


.




1) Retention indices on DB-5MS column. 2) Retention indices on Suplecowax 10 column. 3) Values expressed as equivalents of *n*-decanol are given as mean ± standard deviation (*n* = 3). 4) Not detected or larger retention indices than 3000 in Supelcowax 10 column. 5) Not detected or less than 1.0 μg/100 g. 6) Tentatively identified based on mass spectral data only due to lack of authentic standard compound. 7) Compounds used for antioxidant activity assays.

**Figure 1.** Chemical structures of carotenoid-derived compounds identified in steam-distilled essential oil (SDEO) and glycosidically bound aglycone fraction (GBAF) isolated from *M. tricuspidata* fruit. Numbers in brackets indicate peak numbers as listed in Table 1.

In this study, the norisoprenoid compounds, 7,8-dihydro-α-ionone, 3-hydroxy-β-ionone, 4-oxo-7,8-dihydro-β-ionol and 9-hydroxymegastigma-4,6-dien-3-one (two isomers) were not detected in fractions separated by the steam-distillation and extraction (SDE) method but in the glycosidicaly bound volatiles fraction (GBAF). These results sugges<sup>t</sup> that most of the norisoprenoid compounds detected in the fruit are present in the form of glycosidic form rather than existing in free form in the maturing fruit or being formed in the process of preserving the fruits after harvesting [44]. These compounds can be derived from carotenoids by the action of related enzymes or chemical oxidation during processing or storage of *M. tricuspidata* fruit. It is considered that the carotenoid is decomposed in the process of separating volatile components by steam distillation. In particular, 3-hydroxy-β-ionone, 3,4-dihydro-α-ionone and two quantitatively detected 9-hydroxymegastigma-4,6-dien-3-one are present in glycosidic form in some plants [45,46]. In our previous study that analyzed phenolic compounds in the methanol extract of a fully matured fruit of the plant, we isolated a number of phenolic compounds including quercetin and parishin derivatives [25]. In this study, only 4-Hydroxybenzyl alcohol was able to be detected at a significant concentration suggesting most of the other phenolic compounds must have been degraded during the steam-distillation process.

To the best of our knowledge, 13 carotenoid-derived compounds (isophorone, 4-oxoisophorone, theaspiranes A, theaspiranes B, 7,8-dihydro-α-ionone, β-ionone, β-ionone epoxide, dihydroactinidiolide, 3-hydroxy-β-ionone, β-cyclocitral, β-homocyclocitral and two 9-hydroxymegastigma-4, 6-dien-3-one isomers) are being identified for the first time from *M. tricuspidata* fruit oil. These compounds are related to carotenoids [44]. *M. tricuspidata* fruit contains several carotenoids including α-carotene, β-carotene, zeaxanthin, ruboxanthin, and lutein [47]. As described in the introduction section above, Bajpai and colleagues have previously identified 29 compounds with 1,1-difluoro-4-vinylspiropentane, scyllitol, 1-phenyl-1-cyclohexylethane, diethyl phthalate and 4,4-diphenyl-5-methyl-2-cyclohexenone as major constituents in the essential oil obtained from *M. tricuspidata* fruit by microwave-assisted extraction [26]. However, most of these compounds were not detected in this study. We believe that the difference in detected components is caused by the difference in extraction method and plant samples. In the present study, we used a fresh fruit instead of a dried one.

#### *3.2. Chemical Composition of Glycosidically Bound Aglycone Fraction (GBAF)*

It is well established that volatile components in plants and foods are present in free form while some components exist in glycosidically bound forms [41,48,49]. The volatile components in the form of glycoside in association with saccharides have a hydroxyl group in the molecule and are bonded in the form of a β-glycoside. These glycosides can be hydrolyzed by β-glycosidases produced by microorganisms to produce free form of volatiles [28,33,41]. The enzyme preparation used for such a purpose are enzymes with glycosidase activities such as β-<sup>d</sup>-glucosidase, <sup>α</sup>-<sup>l</sup>-arabinopyranosidase, <sup>α</sup>-<sup>l</sup>-arabinofuranosidase and <sup>α</sup>-<sup>l</sup>-rhanosidase.

In this experiment, GBV fractions were isolated from an Amberlite XAD-2 column and then *Asp. niger* cellulase was used to release aglycones from their conjugates. Compared with the gas chromatograms of the volatile components separated by the SDE method, the number of components detected in the GBV fraction (Supplementary Figure S1) was smaller. However, it can be clearly seen that the intensities of the peaks are significantly higher in the GBV fraction. These results indicate that the overall compositions of the volatile components constituting the GBV fraction are clearly di fferent from the volatile components present in the free form. Identities of individual compounds identified in the SDEO and GBAF are presented in Table 1.

Regarding aldehydes and ketones which belong to the oxygenated compounds, 14 components were detected in the volatile components fraction separated by the SDE method while only a small amount of phenylacetaldehyde was detected in the GBAF (Table 1). These results sugges<sup>t</sup> that aldehydes and ketones present in fruits are not combined with saccharides in the form of glycosides.

In the volatile fractions separated by the SDE method, few aromatic alcohol and phenolic compounds including constituents such as *p*-cresol, estragole, 2-methyl-5-(1-methylethyl) phenol, methoxy-2-methylphenol, 2,4,6-trimethylbenzaldehyde and 2-hydroxy-4-methylbenzaldehyde were detected in lower concentrations. By contrast, in the GBAF fraction, a large amount of aromatic alcohols and phenolic compounds were detected. Among them, benzyl alcohol, 2-phenylethyl alcohol, resorcinol, α-methoxy-*p*-cresol, *p*-hydroxybenzyl alcohol, *p*-hydroxybenzaldehyde, 4-methylsalicylaldehyde, methyl *p*-hydroxybenzoate, ferulic acid, methyl ca ffeate, pyrocatechol, *p*-hydroxyphenylethyl alcohol, vanillyl alcohol, *p*-hydroxybenzoic acid, methyl vanillate, vanillic acid, and *p*-(*p*-hydroxybenzyl) phenol were detected only in the GBAF (Table 1). The chemical structures of the phenolic compounds detected in the GBAF are shown in Figure 2. As shown in the figure, one or more hydroxyl groups are contained in the molecular structure, and thus the β-glycoside bond is hydrolyzed by treating β-glucosidase in the presence of sugar in the form of β-glycoside in the hydroxyl group. These compounds are smaller in molecular weight and simple in structure compared to other phenolic compounds, but are widely distributed in plants and are known to contribute to various physiological activities. Interesting biological activities have been reported for tyrosol, *p*-hydroxybenzyl alcohol and *p*-hydroxybenzaldehyde including anti-oxidant activities, improving functional blood flow, preventing memory deficits, and providing protective e ffects on the blood–brain barrier [50–53].

**Figure 2.** *Cont.*

**Figure 2.** Chemical structures of aromatic and phenolic compounds identified in glycosidically bound aglycone fraction (GBAF) isolated from *M. tricuspidata* fruit. Numbers in brackets indicate peak numbers as listed in Table 1.

#### *3.3. Total Phenol Contents of Fractions*

The total phenol contents of the SDEO, FV and GBAF were also determined and comparisons of the results are presented in Figure 3. Among all, the highest total phenol content was obtained from the GBAF while the SDEO showed the lowest total phenol content ( <10 mg/g dw). The total phenol content of the FV fraction was slightly lower than the GBAF while it was much higher than that of SDEO. The relatively higher total phenol contents in the GBAF and FV is due to the solvents used as the e fficiency of the phenolics extraction depends on the type of the solvent. During isolation of the GBAF, extraction of the aglycones liberated by enzymatic hydrolysis employed a more polar solvent (ethyl acetate) while only *n*-pentane-diethyl ether (1:1) was used in the case of SDEO. It is well established that phenolic compounds are extracted more e fficiently with polar solvents [54].

**Figure 3.** Total phenol contents of fractions isolated from *M. tricuspidata* fruit. SDEO, steam-distilled essential oil; FV, free volatile; GBAF, glycosidically bound aglycone fraction liberated from GBV by *Asp. nige*r cellulose; GBV, glycosidically bound volatile fraction.

#### *3.4. Antioxidant Activity of SDEO and GBAF*

Antioxidant activities of fruit extracts have been characterized extensively [55]. In this study, antioxidant capacities of each fraction expressed in percent of radical (DPPH and ABTS) scavenging activities and reducing power as measured by FRAP assay, and EC50 as compared to the positive controls BHA and BHT, are presented in Figure 4 and Table 2. In all the antioxidant property measurement methods, the GBAF showed the highest antioxidant activity while the SDEO showed the lowest. Considering the total yields of these fractions and their respective total phenol content results described above, it can be said that there is a strong positive correlation between their concentrations and their respective antioxidant activities. Maximum antioxidant activities of the GBAF were obtained in the DPPH and FRAP methods where its activity was even higher or equivalent to those of the synthetic antioxidants BHA and BHT. While the antioxidant properties of phenolic compounds are

extensively demonstrated in the literature, some of the volatile compounds exclusively detected in the GBAF might also have greatly contributed to its considerable antioxidant capacity observed in this study. It should also be noticed that the volatile aroma components detected in higher concentrations in the GBAF including *p*-Hydroxybenzyl alcohol, *p*-hydroxybenzaldehyde and tyrosol are well known to have strong biological activities [56–58]. However, while antioxidant activity estimations based on synthetic radicals are indispensable tools, many people raise concerns about their substantiation through in vivo and clinical trials which also have more safety issues [59].

**Figure 4.** Antioxidant activities of steam-distilled essential oil (SDEO), free volatile (FV) and glycosidically bound aglycone fraction (GBAF) isolated from *M. tricuspidata* fruit. (**a**) 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) free radical scavenging activity, (**b**) 2,2-Azino-Bis(3-Ethylbenzothiazoline-6- Sulfonic Acid (ABTS) free radical scavenging activity; (**c**) Ferric reducing antioxidant power (FRAP). Samples, 1000 ug/mL; \* Butylated hydroxyltoluene BHA, Butylated hydroxyanisole (BHT), 200 ug/mL.


**Table 2.** Antioxidant activity of SDEO, FV and GBAF isolated from *M. tricuspidata* fruit.

1 EC50 (μg/mL) values were calculated from the regression lines using six different concentrations (10–100 μg/mL) in triplicate and data represent 50% scavenging activity. 2 Ferric-reducing antioxidant power (FRAP) were calculated from the regression lines using six different concentrations (10–100 μg/mL in triplicate and the values were presented by sample concentration at 0.5 of absorbance at 517 nm DPPH, 2,2-Diphenyl-1-Picrylhydrazyl; ABTS, 2,2-Azino-Bis(3-Ethylbenzothiazoline-6-Sulfonic Acid; SDEO, steam-distilled essential oil; FV, free volatile; GBAF, glycosidically bound aglycone fraction liberated from glycosidically bound volatile fraction by *Asp. niger* cellulase. Different superscripts in the same column indicate significant differences (*p* < 0.05). +, cation.

Even though antioxidant activity of the SDEO was found to be much lower than the other fractions, it is suggested that its observed antioxidant property is related to the compounds detected in it. Compounds such as palmitic acid, linoleic acid and *p*-cresol that were detected in relatively higher concentrations in the SDEO are not important antioxidants [60,61]. Generally, the antioxidant capacity of volatile compound fractions from *M. triscuspidata* fruit extracted with the SDE method and that of GBAF are attributed to the individual components identified. The antioxidant activities expressed in EC50 of some individual phenolic compounds evaluated in this study are also presented in Table 3. Based on these results, it can be suggested that as most potent bioactive compounds are glycosidically bound forms in *M. triscuspidata* fruit, enzymatic processing like fermentation can play an important role in enhancing its biological activities.


**Table 3.** Antioxidant activity of phenolic compounds identified in GBAF.

1 EC50 (μg/mL) values were calculated from the regression lines using six different concentrations (10–100 μg/mL) in triplicate and data represent 50% scavenging activity. 2 FRAP were calculated from the regression lines curve using six different concentrations (10–100 μg/mL) of authentic standards in triplicate and the values were presented by sample concentration at 0.5 of absorbance at 517 nm. Different superscripts in the same column indicate significant differences (*p* < 0.05).

#### *3.5. Antioxidant Activity of Individual Phenolic Compounds in GBAF*

In order to evaluate the antioxidant activities of individual compounds, EC50 of 12 compounds identified in the GBAF was determined and the results are presented in Table 3. In all the three assay methods, methyl ca ffeate displayed by far the strongest antioxidant activity expressed in EC50. Pyrocatechol also showed the highest DPPH scavenging activity and was even higher than the synthetic

antioxidants BHA and BHT. As can be seen from Table 3, several phenolic compounds including pyrocatechol, vanillyl alcohol, methyl ca ffeate and ferulic acid have shown antioxidant potencies higher than that of positive controls. Ferulic acid and methyl ca ffeate, the two compounds that showed the highest DPPH-scavenging activities in this study, have been previously reported to have antioxidant activities expressed in EC50 of DPPH scavenging activity of 22 and 10.64 μg/mL for ferulic acid methyl ca ffeate, respectively [50,62].

Therefore, it can be assumed that these compounds have greatly contributed to the overall higher antioxidant activity observed in the GBAF. As described above, only a few phenolic compounds were detected in lower concentrations in the SDEO fraction. Considering this, proper processing techniques are required before application of *M. triscuspidata* fruit for its biological activity. While processing techniques such as specific enzymatic treatments can help release some compounds, processing methods like fermentation with microorganisms may give more e fficient results. A previous study has demonstrated an increase in the levels of phenolic compounds such as kaempferol and quercetin after lactobacillus-mediated fermentation of *M. triscuspidata* leaf [63].
