Phenolic compounds
Table 1 shows the chromatographic characteristics, tentative identification, and individual quantification of phenolic compounds in R. idaeus leaf extracts obtained by HPLC-DAD/ESI-MS. Sixteen phenolic compounds were tentatively identified: five phenolic acids (ellagic and chlorogenic acid derivatives), three flavonoids (one flavonol and two flavan-3-ols), and eight hydrolysable tannins.
Peak 1 ([M−H]– at m/z 633) and peaks 8-10 ([M−H]– at m/z 935) were assigned as hexahydroxydiphenoyl group, being identified as galloyl-HHDP-glucose and galloyl-bis-HHDP-glucose, respectively. The fragment at m/z 301 represented evidence of the presence of an (HHDP) group [ellagic−H]− in the molecule [16]. Raspberry leaf extracts have revealed the presence of compounds that showed an UV spectrum coherent with galloyl and HHDP derivatives [17], therefore confirming these identifications. Peaks 5 and 6 ([M−H]− at m/z 783) were assigned to pedunculagin isomers according to their pseudomolecular ion and fragmentation pattern as described by [18, 19]. Similarly, peak 7 ([M−H]− at m/z 1251) was identified as punicalagin gallate [20].
Peak 2 ([M−H]– at m/z 355) was tentatively identified as dihydrocaffeoylquinic based on the parent ion, leading to the MS2 base peak of m/z 191 corresponding to dihydrocaffeic acid mainly by the neutral loss of 164 u.
Peaks 11-14 were tentatively identified as ellagic acids derivatives. Thus, peaks 11 and 14 ([M−H]− at m/z 433) were assigned as ellagic acid pentoside, producing a base fragment at m/z 301, corresponding to a deprotonated ellagic acid, revealing the loss of a pentose moiety (−132 u). Similarly, peak 12 ([M−H]− at m/z 477) was tentatively identified as a methyl ellagic acid hexoside. Peak 13 was positively identified as ellagic acid according to its retention time, mass spectra and UV–Vis characteristics by comparison with standards found in the market. According to the literature, Rubus leaves are rich in ellagic acid derivatives [21, 22], where ellagic acid may occur in its free form, but also be released through hydrolysis of ellagitannins [17, 23, 24].
Flavan-3-ols were also detected in raspberry leaves, revealing peaks 3 ([M−H]− at m/z 577) and 4 ([M−H]− at m/z 865) a λmax spectra around 276–281 nm, characteristic of this group of compounds, these compounds were tentatively assigned to a procyanidin dimer and procyanidin trimer, respectively. The only flavone (peak 15) detected was positively identified compared to a commercial standard.
Hydrolysable tannins, including peaks 1, 5-10, and 16, were the primary phenolic compounds in R. idaeus leaf extracts.Considering the different extraction methods, ultrasound-assisted extraction was more effective in extraction in terms of total phenolic compounds (109 ± 3 mg/g of extract), while for the flavonols and flavan-3-ols class, the most efficient extraction method was infusion and maceration.
Altogether, the extracted phenolic compounds are abundant, giving those extracts the characteristics sought for cosmetic use. Previous studies show that high polyphenols content extracts are useful for treating and preventing oxidative stress-induced premature skin aging. The advantageous characteristics of polyphenols, which are especially pertinent for topical administration, include antioxidant activity, protection against UV damage, inhibition of skin proteinases, anti-microbial activity, and anti-carcinogen action [25].
Ellagic acid may lead to improvement of hyperpigmentation and dark spots, melasma, increased UV damage protection, and seems to have antiwrinkle activity according to Sharifi-Rad et al. [26]. Chlorogenic acids skin-related benefits are described as antioxidant and anti-aging; photoprotective and antitumor; anti-inflammatory, and antibacterial [27]. Procyanidin possesses antioxidant, antitumor, anti-inflammatory, immunosuppressive, and antiallergy properties and protective properties against chronic diseases and metabolic disorders [28]. The identified flavone, luteolin-7-O-glucuronide, may be an ideal candidate to alleviate severe inflammatory responses and oxidative stress according to Cho et al. [29].
The major phenolic compound present, galloyl-bis-HHDP-glucose, is described in the literature as exerting anti-inflammatory effects [30, 31]. Pedunculagin isomers, other hydrolyzable tannins, are described as antioxidants and anticancer [32].
Insert Table 1
Bioactive potential
Antibacterial activity
The microdilution method was used to study the antibacterial properties against pathogenic bacteria. All extracts inhibited most of the tested bacterial strains, with UAE extracts being the most effective. UAE samples inhibited the Gram-negative bacteria E. coli, K. pneumoniae, M. morganii, and P. mirabilis with a 5 mg/mL MIC. In a report by Khalifa et al. [33] when studying raspberry aqueous extracts to inhibit E. coli and P. aeruginosa, concentrations of 25 mg/mL and 50 mg/mL were used. Also, whilst in that work, a MIC of 25 mg/mL against L. monocytogenes was found, in the present work, 5 mg/mL was determined.
Regarding the Gram-positive bacteria, it was achieved for raspberry UAE extract a MIC of 2.5 mg/mL against E. faecalis and the methicillin-resistant S. aureus. In contrast, previous works have achieved a MIC of 50 mg/mL for this last strain with aqueous raspberry extracts [33].
In another report, Krauze-Baranowska et al. [34] studied the antimicrobial activity of raspberry shoot aqueous extracts reporting a MIC of 60 mg/mL against K. pneumoniae and 120 mg/mL against E. faecalis, whilst in this work, a MIC of 5 mg/mL and 2.5 mg/mL respectively, were determined. That report presented better results using the MRSA, where a MIC of less than 1 mg/mL was achieved compared with the present work’s best result of 2.5 mg/mL using the UAE extract. In another study, Bauza-Kaszewska et al., [35] found that all three R. idaeus preparations tested (seedless, seeds, and pomace) had no or low antibacterial activity against the tested microorganisms, one of which being L. monocytogenes, which was also studied in the current study in which the infusion extract presented a MIC of 5 mg/mL.
Regarding Minimum Bactericidal Concentration (MBC) results, none of the studied extracts presented bactericide effects. Khalifa et al. [33] determined bactericide results against the Gram-negative strain P. aeruginosa and the Gram-positive L. monocytogenes, finding a 100 mg/mL result for both.
Krauze-Baranowska et al. [34], obtained a MBC of 120 and 60 mg/mL alongside E. faecalis and K. pneumoniae, respectively. Again, the present work obtained a better result against MRSA, with an MBC of 1 mg/mL. Thus, the tested extracts could be used as bioactive compounds capable of inhibiting the bacterial growth of some pathogenic strains.
Antimicrobial activity is an important bioactivity in the development of a cosmetic, as it is a parameter that will contribute to increasing the shelf life of the final product. The antimicrobial activity of extracts from raspberry residues can be explained by the presence of dihydrocaffeoylquinic acid [27].
Insert Table 2
Antioxidant activity
The study evaluated the antioxidant activity of raspberry extracts from its aerial parts, using two methods - OxHLIA and TBARS (Table 3). Among the extracts, the one obtained through decoction showed the highest antioxidant activity after 60 minutes, followed by macerations. However, there were no significant differences found between the antioxidant activities of the extracts obtained through infusion and UAE. At 120 minutes, the only statistical difference found was between infusion and UAE. At 60 minutes, none of the samples showed better activity than the positive control, Trolox, although decoction had a similar response, while at 120 minutes, both the decocted and macerated samples showed better activity than the pure compound (Trolox). Comparing the two analysis times, for all extraction types, the best antioxidant activity was found after 60 minutes, with a statistically significant decrease at 120 minutes. Considering the TBARS assay, the best extract was the one of infusion, showing a statistically lower EC50 than the maceration. In contrast, the decoction and UAE did not show significant differences. The differences in the antioxidant activity between the two assays are mostly related to the antioxidant mechanisms of each assay.
As reported in a previous work involving the study of leaf extracts from seven different raspberry cultivars concerning their antioxidant activity, they showed values of 88.1 mmol of Trolox equivalents for the most active sample [21]. Vara et al. [36] studied red raspberry hydroethanolic extracts, which presented an IC50 of 298 mg/mL for OxHLIA and an EC50 of 122 mg/mL for TBARS assay. Thus, R. idaeus aerial parts presented better antioxidant activity than its fruits.
UV light and pollutants may also produce oxidative stress, which can deplete the natural antioxidant reserve. This can lead to loss of cellular integrity, increased matrix metalloproteinases, wrinkle formation, metastases, and peroxidation of fatty acids. Natural polyphenols have shielding effects against radical oxygen species, making them promising for antiaging applications. Antioxidant activity manifested by raspberry waste extracts can be explained by the presence of dihydrocaffeoylquinic acid, luteolin-7-O-glucuronide, pedunculagin isomers, and mostly due to procyanidin.
Insert Table 3
Cytotoxicity
Insert Table 4
The UAE and infusion extracts showed the lowest IC50 values in the anti-inflammatory assay, indicating stronger activity. Still, the ones obtained by infusion did not show statistically significant differences towards the maceration sample. Finally, the decoction was the least effective extraction type in terms of anti-inflammatory activity. The UAE was also the extract with higher antitumor activity against all tested cell lines, showing statistically significant lower IC50 values than the extracts obtained by the other techniques. This shows that UAE preserves polyphenols with higher biological properties towards cell lines. Still, none of the samples showed an activity close to the positive controls. In terms of cytotoxicity to normal cell lines, none of the extraction techniques showed extracts with toxicity, representing more than 400 cells per plate.
Proof of the cytotoxic safety of extracts from raspberry bioresidues allows taking advantage of their excellent antitumor and anti-inflammatory properties. The antitumor behavior can be mainly attributed to dihydrocaffeoylquinic acid and procyanidin isomers and, in a more residual way, to pedunculagin. Inflammation is a tissue's defence mechanism against pathogen invasions, cell injury, and irritation, and acts as a means of removing injured and necrotic cells. It can be acute or chronic, and chronic inflammation is thought to be a major factor in the emergence of chronic illnesses. The studied extracts have been shown to have relevant anti-inflammatory properties. They may have been evidenced by the major presence of the compound galloyl-bis-HHDP-glucose, as well as dihydro caffeoylquinic acid, procyanidin isomers, and luteolin-7-O-glucuromide, also present in the extracts.