Determination of trans-resveratrol in Solanum americanum Mill. by HPLC

Abstract Solanum americanum Mill. is a plant that belongs to the Solanaceae family, its respective ripe fruit is dark purple. Ripe S. americanum Mill. fruits were submitted to physicochemical characterisation, and their trans-resveratrol contents were quantified by high-performance liquid chromatography. Such determination was executed with fruits at different stages of ripeness and freeze-stored fruits as well. In natura ripe fruit pulp and peel presented average trans-resveratrol amounts of 1.07 and 0.7960 μg per gram of sample, respectively. These amounts are significantly higher when compared to freeze-stored fruit (0.1353 μg of trans-resveratrol per gram of sample) and to other berries. All ripe fruits showed significant amounts of total anthocyanins and total antioxidants. Thus, for the first time, trans-resveratrol has been identified and quantified in S. americanum Mill. fruit samples.


Introduction
The Solanaceae family has approximately 2.700 species and 99 genera. Solanum, the largest and most complex genus in this family, is of great economic importance as foods, medicines and ornamental plants (Morais et al. 2015). Numerous studies have been carried out to identify and quantify biochemical compounds in various species of this genus (Nie et al. 2014; ABSTRACT Solanum americanum Mill. is a plant that belongs to the Solanaceae family, its respective ripe fruit is dark purple. Ripe S. americanum Mill. fruits were submitted to physicochemical characterisation, and their trans-resveratrol contents were quantified by high-performance liquid chromatography. Such determination was executed with fruits at different stages of ripeness and freeze-stored fruits as well. In natura ripe fruit pulp and peel presented average trans-resveratrol amounts of 1.07 and 0.7960 μg per gram of sample, respectively. These amounts are significantly higher when compared to freeze-stored fruit (0.1353 μg of trans-resveratrol per gram of sample) and to other berries. All ripe fruits showed significant amounts of total anthocyanins and total antioxidants. Thus, for the first time, trans-resveratrol has been identified and quantified in S. americanum Mill. fruit samples. Zhang et al. 2015;Mohan et al. 2013). Among the several biochemical compounds identified in plants, cis-and trans-resveratrol (3,4,5-trihydroxystilbene) are polyphenols that belong to the stilbene class; however, only the trans form is biologically active in the human body (Walle 2011). Resveratrol is mainly employed in researches which are aimed to minimise effects of free radicals in the human body and to prevent degenerative diseases, such as cancer (Stewart et al. 2003). Resveratrol exerts antioxidant (induces antioxidant enzymes), antidiabetic (effects on glucose metabolism) and oestrogenic effects (function as an oestrogen agonist). For cancer prevention, trans-resveratrol preserves the regular cell cycle, inhibits tumour invasion and angiogenesis, and for cardiovascular disease, trans-resveratrol reduces the expression of endothelial adhesion cells and inhibits cell apoptosis and platelet aggregation (Stewart et al. 2003;Gupta et al. 2011;lin et al. 2011;Szkudelski & Szkudelska 2011;li et al. 2013;Fuentes & Palomo 2014). These functions are important to maintain health and prevent inflammation, cardiovascular diseases and other health problems, like cancer (Tyihàk et al. 2011). A recommended resveratrol daily intake by humans still has to be established (lin et al. 2011). This study aimed to execute physicochemical characterisations and to quantify, by HPlC, trans-resveratrol in natura peel and pulp of Solanum americanum Mill. fruits at different ripeness stages, as well as to determine the effects of freezing on its content.

Ripe fruit global composition
S. americanum Mill. fruits are small (7.84 mm ± 0.81), shiny berries with colour varying from purple to black due to the presence of anthocyanins in fruits, the colour as confirmed by Cielab chromaticity coordinates close to zero, freezing did not affect fruit colour statistically (Table S1). The fruit sugar content is directly related to the total soluble solid content and the fruit ripeness stage. The soluble solid content decreased as it was used for respiration during freeze storage. In natura ripe fruit had 15% total soluble solids ( Table S2). The protein content 3.37 g/100 g (Table S2) is relatively high when compared to that of domesticated fruit, such as banana 1.0 g/100 g, avocado 2 g/100 g and tamarind 2.8 g/100 g, while the total lipid content was similar to that of most fruits, lower than 1 g/100 g (Rodrigues et al. 2011). The ascorbic acid content 6.59 mg 100 −1 (Table S2) of in natura fruit was similar to banana (nanica variety), 8.7 mg 100 −1 g, and watermelon (8.1 mg 100 −1 g) and higher than Argentinean apple 4.6 mg 100 −1 , but lower than blueberries (9.7 mg 100 −1 ); the vitamin content of frozen fruit decreased significantly due to its easy degradation. The antioxidant activity and total anthocyanin values did not differ significantly as a result of the freezing process (Table S3); however, the phenolic compound content of in natura fruit was greater than the same content type from frozen fruit. The total anthocyanin content of in natura fruit was 2.5 mg 100 −1 and in frozen fruit, 2.16 mg 100 −1 , similar to that found in other fruit. da Mota et al. (2010) investigated different grape cultivars and found similar results in cabernet sauvignon and pinot noir grapes, and the antioxidant activity of S. americanum Mill. fruits (Table S3) ranged from 94.07 to 95.66%, and is not significantly affected by freezing. Table S4 shows that the mineral content in natura and frozen fruit varied significantly. In natura fruit had significant amounts of iron, zinc and calcium.

Trans-resveratrol
The extracts obtained after clean-up were submitted to chromatographic separation by different methodologies, with and without addition of 2 mg l −1 of trans-resveratrol as standard sample, and the chromatograms were compared. The sample presented interferences, which are characteristic of this berry, that affect the separation of analyte. This problem probably occurred because the previously described chromatographic methodologies used for quantification of trans-resveratrol were developed for wine and grape samples. The methodology developed by Sun et al. (2006) shows a better resolution. The chromatograms show a large number of compounds which absorb at the same wavelength as trans-resveratrol however, with retention time different from that of the analyte. The compound was studied by analyses of spiked and unspiked samples with trans-resveratrol as a standard ( Figure S1). The calibration curve plotted by integrating the area of chromatographic peaks from standard trans-resveratrol solutions had a linear regression (y = 4,23,951x−18,772) with r 2 = 0.9916. In order to identify trans-resveratrol in fruit extracts, the retention times of the resveratrol extract, and of the extract with standard addition of each sample, were compared. In natura ripe peel of S. americanum Mill. fruit has about 0.7960 μg of resveratrol per gram of sample and ripe pulp has 0.2745 μg of resveratrol per gram of sample, a value higher than that found in other fruits which are traditionally considered sources of resveratrol. In addition to properties reported for trans-resveratrol, S. americanum Mill. fruit has other properties related to anthocyanins, as reported by Nawwar et al. (1989), similar to grape cultivars, which contain resveratrol and anthocyanin in their composition. The antioxidant activity associated with these compounds in foods contributes to their connotation in the market as functional foods. The trans-resveratrol content, determined in this study, was higher than that reported for the peel of some grape cultivars, which have an average of 24.06 μg g −1 , according to Romero-Pérez et al. (2001). It was equivalent to some Merlot (average of 4.1 mg/l) and Cabernet sauvignon (average of 1.7 mg/l of trans-resveratrol) grape cultivar samples according to Souto et al. (2001Souto et al. ( ). lyons et al. (2003 found approximately 6.82 and 16 mg g −1 of trans-resveratrol in blueberries and bilberries, respectively. Wang et al. (2002) reported that cranberry juice had about 0.24 μg g −1 of this compound, while grape juice had 0.35 μg g −1 .
Ripe in natura fruit peel and pulp had a significantly higher amount of this compound when compared to freeze-stored fruit (Table 5S). This compound is water soluble; therefore, its content may have decreased during sample thawing before analysis. In addition, it easily oxidises, is little resistant to light or biotransforms into the cis form, which is biologically inactive in the control of free radicals in the human body (Silva et al. 2013). The absence of trans-resveratrol in the initial fruit development stages is due to the fact that the synthesis of this compound, as well as other bioactive plant secondary-metabolism compounds, starts either upon exposure to ultraviolet light or to protect the plants against pathogens. This also explains its presence in ripe fruits (Ji et al. 2014). Semi-ripe fruit pulp samples did not show any significant difference in trans-resveratrol content after thawing, when compared to in natura samples, despite the higher values in frozen samples. This increase was most likely due to semi-ripe fruit sample inhomogeneity. Similar results were found for semi-ripe fruit peel in both treatments. S. americanum Mill. fruit had an amount of trans-resveratrol greater than the quantification limit of the method. It is worth pointing out that it was not possible to determine trans-resveratrol in green pulp samples due to precipitation and obstruction of the extraction cartridge, even after filtration. Its levels in green peel samples were not higher than the detection limit, as shown in Table S5. The values found in S. americanum Mill. fruit show that it is a promising source of resveratrol.

Conclusion
This is the first report regarding identification and quantification of trans-resveratrol in S. americanum Mill., with ripe and semi ripe fruit values ranging from 0.7960 to 0.1126 μg g −1 sample. The amount of this compound increases as the fruit ripens. The trans-resveratrol concentration in fruit peel and pulp decreased significantly after freezing. These results demonstrate that other means of conservation must be used to preserve this compound in S. americanum Mill. fruits. Additionally, besides the presence of trans-resveratrol, physicochemical and biochemical analyses of both in natura and frozen ripe S. americanum Mill. fruits revealed significant amounts of total anthocyanins, minerals and total antioxidant activity, as well as high vitamin C contents. This study gives evidence that S. americanum Mill. fruit is rich in nutrients.