*2.5. Anti-Inflammatory Test*

Anti-inflammatory activity (AI) was evaluated determining the cyclooxygenase activity (COX). The COX-1 and COX-2 inhibitory assay was carried out using COX Inhibitor Screening Assay Kit (Catalogue N◦ 560131, Cayman Chemicals, Ann Abor, MI, USA) according to the instructions provided by the manufacturer and as previously reported [27]. The AI was determined using the phenolic extract and results are expressed as inhibitory activity (IC50, μg/mL).

## *2.6. Statistical Analysis*

Data are reported as the mean ± SD and three biological replicates are carried out for each sample. Statistical evaluation was conducted by ANOVA, followed by multicomponent Duncan's test (*p* < 0.05) to discriminate among the mean values.

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

#### *3.1. Simple Sugars, Organic Acids and Total Phenolics Content*

The moisture and quantity of simple sugars, malic and citric acids, TPC and ODC were reported in Table 1. The moisture content in the fruits is very close, about 78%, according to the literature [1,11]. Sucrose was completely absent in all the samples analyzed; *M. nigra* had 7.66 g/100 g FW of sugars whereas the glucose and fructose contents were similar and equal to 3.94 and 3.72 g/100 g FW, respectively.

**Table 1.** Amounts of moisture (%), glucose, fructose, malic and citric acid (expressed as g/100 g FW), total phenolic compounds (TPC) (mg Gallic Acid Equivalent (GAE)/100 g FW) and *o*-diphenolic compounds (ODC) (mg of chlorogenic acid /100 g FW) in different mulberry fruit varieties. Values represent the results of three determinations ± SD; means with different letters in the same column are significantly different from each other (*p* < 0.05) according to the multicomponent Duncan's test.


The fruits of other varieties showed a similar trend and although the glucose and fructose were equivalent, the total amount of sugars was lower corresponding to about 6 g/100 g FW. These values are in agreemen<sup>t</sup> as reported for the species of mulberry (*M. nigra*, *M. alba* and *M. laevigata*) cultivated in Pakistan [1], but they are notably inferior when compared to *M. nigra*, *M. alba* and *M. rubra* grown in Turkey for which the content of glucose and fructose was corresponding to about 6–7 g/100 g FW, respectively [30]. The organic acid content (malic and citric) was low and ranged between 0.13 and 1.02 g/100 g FW for *M. alba cv Nello* and *M. nigra*; *M. nigra* showed a greater quantity of citric acid of 0.92 g/100 g FW and this was in accordance with what was found in different varieties of Turkish mulberries where the amount of citric acid ranged between 0.39 and 1.08 g/100 g FW [30].

The differences between species in terms of citric and malic acid content might be caused by genetic factors as well as agronomic practices and ecological factors (temperature, soil conditions, humidity, ect.). In fact, Koyuncu [31] reported a variable amount of citric and malic acid that ranged between 0.5 to 2.3 g/100 g FW and 3.5 to 19.8 g/100 g FW for different genotypes of mulberry fruits cultivated in Turkey. These differences are also evident in fruits grown in different localities of the same country; Koyuncu [31] reported different amounts in citric and malic acid for black mulberry fruits from two locations ranged between 0.8 to 1.3 g/100g FW and 5.7 to 9.9 g/100g FW, respectively. In other Turkish mulberry genotypes, the content of malic and citric acid was very different, varying from 12.9 to 21.8 g/100 g FW and from 2.1 to 4.1 g/100 g FW, respectively [11].

Organic acids are water soluble and together with the sugars contribute to the taste of vegetables and fruits. The ratio of the total acid amount to the content of sugars in fruits is a criterion for the maturity; moreover, organic acids have a high impact on taste because of their conditioning (a reduction) on sweetness and their favoring effect on sourness. In addition, the type and the amount of acidity could be used for food decay; if the fruit is molded during the wait, there is an increment of some organic acids which seems also to have a significant impact on the purity control [30].

The amount of TPC and ODC, evaluated as mg of gallic acid equivalent (GAE) and mg of chlorogenic acid, respectively, is also reported in Table 1. The data showed that in *M. nigra* and

*M. alba Legittimo* the content of TPC was equal to about 485 and 423 mg GAE/100 g FW, respectively and corresponding to more than three times the TPC present in *M. alba* cv *Nello*. *M. nigra* showed an ODC content of 102.21 mg/100g FW, an amount about 1.8 and 4 times higher than that present in *M. alba* cv *Legittimo nero* and *M. alba* cv. *Nello*, respectively.

Mulberry fruits are a good source of phenolic compounds and the results clearly showed that fruit analyzed had high total phenolic content, nevertheless they showed wide differences in comparison to the literature data. In fact, TPC in mulberry fruits was reported which ranged from 104.8 to 213.5 GAE mg/100 g FW for eight Thai genotypes [32], from 158.4 to 249 mg/GAE 100g FW and from 100.5 to 348.8 mg GAE/100g FW, respectively for mulberries harvested in different Turkish sites [33,34], and from 76.7 to 180 mg GAE/100g FW in fruits grown in Spain [35]. However, data reported by Imran et al. [1] and by Ercisli et al. [12] showed that the TPC content in fruits collected in the northern region of Pakistan and in North-East Anatolia (Turkey) was very high, ranging from 880 to 1650 and from 1943 to 2237 mg GAE/100g FW, respectively. This grea<sup>t</sup> variability in the content of total phenols is related to the genotype, the conditions of growth and cultivation [22] and could be influenced by fruit moisture, too. It is known, in fact, that the plant can accumulate phenolic compounds under various stress conditions (heat, UV light, pathogen attack, etc.); in particular, climatic changes like low or high-temperature stress can promote the production of the phenolic compounds [36–38].
