*3.2. HPLC Analysis at 278 nm*

From the analysis of the data reported in Table 1 regarding the phenolic profiling of goji berries, we can first note that, among the 21 phenolic standards, vanillic acid, 4-hydroxy benzoic acid, 3-hydroxy-4-methoxy benzaldehyde, *p*-coumaric acid, *t*-ferulic acid, naringin, 2,3-dimethoxy benzoic acid, benzoic acid, *o*-coumaric acid, quercetin, harpagoside, and *t*-cinnamic acid were not detected or were present in trace amounts (below Limit Of Quantification or Limit Of Detection, data not shown). The inter- and intra-cultivar differences were huge and did not follow a specific trend. Generally speaking, Italian cultivars were richer in terms of phenolic compounds with respect to Romanian ones. Among Italian cultivars (especially for Wild), the last two harvesting dates gave berries richer in phenolic compounds regardless of the mixing treatment. Chlorogenic acid, carvacrol, and at lower extent, sinapinic acid were the most-detected metabolites. Conversely, gallic acid, catechin, and syringic acid were only present in some cultivars and their amounts differed a lot depending on the applied treatment. Lastly, a few samples were also characterized by low amounts of naringenin (P2D) and epicatechin (P4U).



The chromatograms of the flavonoid fraction, recorded at 360 nm, showed the presence of about 10–15 peaks, according to the different analyzed cultivars, among which the most representative was rutin. In Table 2, rutin and the total flavonoid contents are reported, calculated as rutin equivalents, after considering all the peaks whose areas were at least 10% of rutin area and taking into account the standard calibration curve of this molecule. Values are expressed in relation to dry weight (dw) for a comparison with the literature data and in μg/mg extract for a better evaluation of other data on bioactivity presented in this work and obtained by the extracts evaluation. Rutin accounted for about 185–400 μg/g dw of goji berries. In the literature, reported values of 281 [30], 296 [36], and 326 μg/g dw [37] are found, perfectly overlapping with our results. Wider ranges between 159–629 μg/g dw were reported in Zhang et al. [38] and higher values (730–1380 μg/g dw) were detected in Dong et al. [39]. Many different results were available in the literature regarding the other representative flavonoids recognized mainly as quercetin hexosides, and secondly as kaempferol and isorhamnetin derivatives. Zhang et al. [38] reported the quercetin-rhamno-di-hexoside content as 435–1065 μg/g, Donno et al. [14] reported a hyperoside content of 116 mg/100 g fresh weight, whereas Inbaraj et al. [30] studied different quercetin-rhamno-hexoside derivatives (in the range of 70–440 μg/g). In our chromatograms, many different peaks were detected in the flavonoid component, although their identification was not possible. These were reported as a sum and expressed as rutin equivalents on the basis of its calibration curve. According to this evaluation, rutin accounted for about 20–65% of the total flavonoid components. The selected samples did not show relevant differences among cultivars (rutin content mean values: P = 276 ± 8.3, W = 291 ± 8.7, E = 288 ± 8.6, and B = 255 ± 7.6 μg/g), nor among applied homogenization processes (D = 287 ± 8.6 vs. U = 279 ± 8.4 μg/g), whereas differences could be revealed in the rutin content along the maturation stage (i.e., rutin increased by 70%, from P1 to P3, collected a month apart in July–August 2015 and decreased by 45% from P4 to P5, collected fifteen days apart, in July–August 2016). The total flavonoid content ranged from 380–1600 μg/g with a slight difference between cultivars P and W (770 ± 38 vs. 910 ± 45 μg/g) and no differences after homogenization treatments D and U (815 ± 41 vs. 862 ± 43 μg/g).


**Table 2.** Rutin and flavonoid content of analyzed goji berries hydroalcoholic (HA) extracts. Flavonoids are expressed as rutin equivalent using the HPLC areas collected at 360 nm.

dw: dry weight.

As shown by the data, the rutin content increase was usually correlated with a decrease in the amount of other flavonoid components and vice versa, but this trend was not confirmed by the P4–P5 series. The comparison between the D and U series demonstrated differences between P4D and P4U samples only in terms of an increase of other flavonoids, whereas W5D and W5U samples were characterized by discrepancies in terms of rutin and flavonoid amount. Overall, the different ratios between rutin and other flavonoids in the goji phytocomplex depended on the cultivar type, but cultivars also seemed highly influenced by the maturation step, as is clearly shown by the sample P.
