Sustainable Processing of Floral Bio-Residues of Saffron (Crocus sativus L.) for Valuable Biorefinery Products

Tepals constitute the most abundant bio-residues of saffron (Crocus sativus L.). As they are a natural source of polyphenols with antioxidant properties, they could be processed to generate valuable biorefinery products for applications in the pharmaceutical, cosmetic, and food industries, becoming a new source of income while reducing bio-waste. Proper storage of by-products is important in biorefining and dehydration is widely used in the herb sector, especially for highly perishable harvested flowers. This study aimed to deepen the phytochemical composition of dried saffron tepals and to investigate whether this was influenced by the extraction technique. In particular, the conventional maceration was compared with the Ultrasound Assisted Extraction (UAE), using different solvents (water and three methanol concentrations, i.e., 20%, 50%, and 80%). Compared to the spice, the dried saffron tepals showed a lower content of total phenolics (average value 1127.94 ± 32.34 mg GAE 100 g−1 DW) and anthocyanins (up to 413.30 ± 137.16 mg G3G 100 g−1 DW), but a higher antioxidant activity, which was measured through the FRAP, ABTS, and DPPH assays. The HPLC-DAD analysis detected some phenolic compounds (i.e., ferulic acid, isoquercitrin, and quercitrin) not previously found in fresh saffron tepals. Vitamin C, already discovered in the spice, was interestingly detected also in dried tepals. Regarding the extraction technique, in most cases, UAE with safer solvents (i.e., water or low percentage of methanol) showed results of phenolic compounds and vitamin C similar to maceration, allowing an improvement in extractions by halving the time. Thus, this study demonstrated that saffron tepals can be dried maintaining their quality and that green extractions can be adopted to obtain high yields of valuable antioxidant phytochemicals, meeting the requirement for a sustainable biorefining.

Saffron, being a triploid (2n = 3x = 24) sterile species, is propagated through underground clonal corms [6]. In Mediterranean climate regions flowering occurs for few weeks from early to late autumn and is mainly controlled by seasonal thermoperiodicity, soil water content, and corm size [7][8][9]. There are one or several flowers per saffron plant, even up to 12 [6]. Flowers have a perianth of six violet tepals, three stamens, and a style culminating in three stigmas [10]. About 110 to 300 flowers are needed to obtain 1g of spice [11,12] and tepals constitute the most abundant bio-residue (~80% of total flower mass [13]).

Correlation Analysis
The correlation between TPC, TAC, and the antioxidant activity of the phytoextracts assessed with the FRAP, ABTS, and DPPH assays were statistically evaluated ( Table 2). Significant correlations were only found in the case of UAE, between TPC and both ABTS (−0.69) and DPPH (−0.45), and between TAC and FRAP (0.86).

Extractions
As regards ferulic acid, 9.65 ± 2.62 mg 100 g −1 DW was extracted through maceration with methanol at 50%, showing no significant differences to 20%. It was not obtained by the other extractions.
The yield of ellagic acid ranged from 1.32 ± 0.33 mg 100 g −1 DW for maceration with 80% methanol to 28.39 ± 4.32 mg 100 g −1 DW for UAE with 50% methanol, which was not significantly different to maceration with water and UAE with both 20% and 80% methanol. No extraction was performed by maceration with 50% methanol.
Hyperoside was obtained by all extractions. Its values varied from 4.35 ± 1.04 mg 100 g −1 DW regarding maceration with water to 27.26 ± 4.29 mg 100 g −1 DW for UAE with 50% methanol, which was not significantly different to the other methanol concentrations and maceration with both 20% and 80% methanol.
Isoquercitrin ranged from 0.22 ± 0.12 mg 100 g −1 DW for maceration with 20% methanol to 7.82 ± 3.09 mg 100 g −1 DW as regards maceration with 80% methanol, that not significantly differed to maceration with 80% methanol and UAE with both 20% and 50% methanol. It was not extracted by UAE with both water and 80% methanol.
Quercitrin was only achieved by means of maceration with all methanol levels and UAE with 50% methanol. Maceration with 50% methanol gave 9.27 ± 3.47 mg 100 g −1 DW, without significant differences with the other extractions.
The yield of rutin varied from 0.32 ± 0.31 mg 100 g −1 DW as regards maceration with 20% methanol to 37.61 ± 2.22 mg 100 g −1 DW for maceration with 80% methanol, which was not significantly different to UAE with water.
The value of epicatechin resulted 16.62 ± 15.89 for UAE with 20% methanol, without significant differences to UAE with 80% methanol. It was not attained by all other extractions.
Regarding vitamin C, 33.72 ± 0.89 mg 100 g −1 DW was gain through maceration with 20% methanol without significant differences to maceration with water and UAE with both water and 20% methanol. No extraction was achieved by all other extractions.
The phytochemical profile of the extracts depended on the extractions ( Table 2). For example, all the molecules except epicatechin were obtained using maceration with 20% methanol, while UAE with 20% methanol extracted all the chemicals except ferulic acid and quercitrin. However, no specific method-solvent combination performed a significant higher yield of these compounds altogether ( Figure 1).
Regarding vitamin C, 33.72 ± 0.89 mg 100 g −1 DW was gain through macerat 20% methanol without significant differences to maceration with water and UA both water and 20% methanol. No extraction was achieved by all other extraction The phytochemical profile of the extracts depended on the extractions (Tabl example, all the molecules except epicatechin were obtained using maceration w methanol, while UAE with 20% methanol extracted all the chemicals except fer and quercitrin. However, no specific method-solvent combination performed a cant higher yield of these compounds altogether ( Figure 1).

Discussion
Conventional maceration and UAE are the most common extraction meth ployed to analyze the antioxidant molecules of saffron tepals (Table 4). Macera quires a high consumption of organic solvents and long extraction times [33]. U modern and green method based on ultrasonic waves that, by increasing the perm of plant cells through acoustic cavitation of the wall, improve the penetration of vent and the release of phytocompounds. UAE can allow the use of non-toxic sol work at room temperature, shorter extraction times compared to maceration [31 which needs the addition of heat to reduce the times [32], and a lower energy cos

Discussion
Conventional maceration and UAE are the most common extraction methods employed to analyze the antioxidant molecules of saffron tepals (Table 4). Maceration requires a high consumption of organic solvents and long extraction times [33]. UAE is a modern and green method based on ultrasonic waves that, by increasing the permeability of plant cells through acoustic cavitation of the wall, improve the penetration of the solvent and the release of phytocompounds. UAE can allow the use of non-toxic solvents, to work at room temperature, shorter extraction times compared to maceration [31,[34][35][36], which needs the addition of heat to reduce the times [32], and a lower energy cost [32].   Dried Microwave-assisted extraction Acidified (HCl) ethanol Anthocyanin content [46] All the extracts of dried saffron tepals presented phenolic compounds ( Table 1). As showed in Table 4, other authors reported their presence in dried and fresh tepals.
The total phenolic content (TPC) was not affected by the extractions (Table 1), meaning that UAE had similar performances to maceration in half the time. This result was consistent with the absence of a better extraction to obtain all the phytochemicals analyzed with the HPLC (Figure 1). Using extraction conditions similar to this study Caser et al. [15] found that for fresh saffron tepals 80% methanol gave higher TPC. Thus, dehydrated tepals may allow the use of more ecological solvents for the extraction of phenolic compounds. UAE with water could be selected to obtain non-toxic phytoextracts to be added to foods [13].
The total anthocyanins content (TAC) had an average value of 282.90 ± 71.82 mg G3G 100 g −1 DW (Table 1). Even in the case of anthocyanins, UAE achieved a yield similar to maceration in half the extraction time. A higher result was seen for UAE with water (413.30 ± 137.16 mg G3G 100 g −1 DW), showing a percentage variation from the average yield of +46%. This is in contrast with the results obtained from fresh saffron tepals [15], for which 80% methanol performed better meaning that even in the case of anthocyanins dehydration may allow the use of greener solvents. The values of TAC were lower than those obtained from saffron tepals by Lotfi et al. [43], which get 507.5 mg G3G 100 g −1 DW using maceration with acidified ethanol. TAC was also lower than that found in saffron spice (1867.3 mg G3G 100 g −1 DW) by Caser et al. [2], but greater compared to the contents of some highly pigmented vegetables [51], such as purple cauliflower (Brassica oleracea var. botrytis, 201 mg G3G 100g −1 DW) and red cabbage (Brassica oleracea var. capitata f. rubra, 199 mg G3G 100 g −1 DW), achieved through maceration with 0.1% HCl (v/v) in 80% methanol. Grape (Vitis vinifera) is one of the most common sources of these molecules [52] having a TAC ranging from 75.64 to 414.95 mg G3G 100 g −1 DW depending on the cultivars [53], which is comparable in its maximum values to that of our study. Since plant waste are usually used for the commercial preparation of these pigments [54], saffron tepals in both dried and fresh [15] forms could be considered a good candidate for their extraction.
All assays evaluated, namely, FRAP, ABTS, and DPPH, revealed the presence of antioxidant activity in the extracts with average values of 520.39 ± 34.48 mmol Fe 2+ Kg −1 DW for FRAP, 14.07 ± 0.73 µmol TE g −1 DW for ABTS, and 21.32 ± 3.45 µmol TE g −1 DW for DPPH (Table 1). Maceration with water obtained higher antioxidant activity measured with the FRAP method, which was not significantly different to UAE with water. This was in tune with that found by Caser et al. [2] for fresh saffron tepals. Interestingly, the antioxidant activity was higher compared to that found in saffron spice by Caser et al. [2] (338.2 mmol Fe 2+ Kg −1 DW for FRAP and 4.6 µmol TE g −1 DW for ABTS) and Caser et al. [15] (4.64 ± 0.50 µmol TE g −1 DW for ABTS). The ABTS values were greater than those achieved from some traditional Chinese medicinal plants [50], such as Chrysanthemum indicum (3.03 µmol TE g −1 DW for methanolic extract of inflorescence), Artemisia annua (6.29 µmol TE g −1 DW for aqueous extract of aerial parts), Campsis radicans (4.462 µmol TE g −1 DW for methanolic extract of flower), and also compared to extracts obtained from some common vegetables and fruits [50], such as wild cabbage (Brassica oleracea, 1.01 µmol TE g −1 DW) and tomato (Solanum lycopersicum, 1.49 µmol TE g −1 DW). The DPPH values were similar to those found in mengkudu (Morinda citrifolia, up to~23 µmol TE g −1 DW) by Thoo et al. [55].
The correlations among TPC, TAC, and the antioxidant activity were assessed ( Table 2). Significant correlations were only found in the case of UAE. Even though the FRAP, ABTS, and DPPH assays have been extensively applied, they do have some limitations which can cause an imprecise estimation [56]. Since the antioxidant activity of plant extracts mainly depends on the content of phenolic compounds, it was expected a strong positive correlation among TPC and the assays [50,[56][57][58]. However, TPC was either not correlated (FRAP) or negatively correlated (−0.69 ABTS and −0.45 DPPH) with the antioxidant capacity. No significant correlations among TPC and antioxidant activity was already found in extracts [59]. The negative correlation, already reported by other authors [59,60], suggests they might have opposite behavior when subjected to different solvents. The antioxidant capacity of samples may be influenced by extraction technique, solvent, pH, and metal ions [60]. Furthermore, synergistic and antagonistic interactions among the antioxidants in the extracts may interfere with the correlation, or there can be some nonphenolic molecules which can react with the Folin-Ciocalteu reagent without being free radical scavengers [59].
TAC was positively correlated with FRAP (0.86) ( Table 2), indicating that anthocyanins contributed more to antioxidant capacity than TPC.
Positive correlations among the antioxidant assays were assumed [56,58], since they are all based on the same principle of electron transfer [56]. However, other authors reported the absence of significant correlation [55,59]. This might be explained by the fact that plants extracts can contain antioxidants which may react in different ways [59].
The heterogeneity of the yields achieved for each molecule (Table 3) would explain the absence of a single better extraction for these compounds altogether (Figure 1). Overall, except for ferulic acid and quercitrin, UAE get results higher (for ellagic acid and epicatechin) or comparable (for vitamin C) to maceration using greener solvents, i.e., water (for hyperoside and rutin) or lower methanol concentrations (20% methanol for isoquercitrin) in half the extraction time (Table 3). A unique ideal extraction does not exist, but the choice should fall on that most environmentally and economically convenient.
The bioactive compounds here identified and quantified (Table 3) deepen the knowledge on the molecular composition of dried saffron tepals. The extracts showed the same molecules (ellagic acid, hyperoside, rutin, and epicatechin) as those found in fresh tepals by Caser et al. [15] using an analysis protocol similar to this study. In addition, in the dried tepals were also detected ferulic acid, isoquercitrin, quercitrin, and vitamin C. In Caser et al. [15] the absence in fresh tepals of the chemicals here obtained might be attributed to the drying procedure [61,62], which can facilitate extractions by disrupting cell walls and causing the formation of cavities and intercellular spaces [63]. Vitamin C and rutin (yield up to 37.61 ± 2.22 mg 100 g −1 DW) showed the highest yields among all the compounds analyzed with HPLC. Vitamin C (up to 33.72 ± 0.89 mg 100 g −1 DW) was lower than in the spice (73.2 mg 100 g −1 ; Caser et al. [15]), but higher or comparable than in some freeze-dried vegetables and fruits [64], such as cabbage (Brassica oleracea var. capitata, up to 28.3 mg 100 g −1 ), tomato (Solanum lycopersicum, up to 10.1 mg 100 g −1 ), apple Fuji (Malus domestica 'Fuji', up to 3.5 mg 100 g −1 ), mango (Mangifera indica, up to 18.6 mg 100 g −1 ), pineapple (Ananas comosus, 5.0 mg 100 g −1 ), and pomelo (Citrus maxima, up to 31.7 mg 100 g −1 ).

Extraction Methods
Two extraction procedures, i.e., maceration and UAE, with deionized water or 20%, 50%, and 80% methanol in deionized water (v:v) as solvents were applied. The extractions were conducted at room temperature (ca. 21 • C) and using a powdered sample-solvent ratio of 1:50 g ml −1 .
As regards maceration, the samples were soaked in each solvent type and kept into a glass tube under stirring (1000 rpm) in the dark for 30 min, whereas for UAE the sample tubes were inserted into an ultrasonic extractor (Sarl Reus, Drap, France) using a frequency Plants 2021, 10, 523 9 of 15 of 23 kHz for 15 min. The extracted solutions were all filtered with one-layer of filter paper (Whatman No. 1, Maidstone, UK) and then with a 0.45 µm PVDF syringe filter (CPS Analitica, Milano, Italy). The extracts were then stored at −20 • C for analyses. All the extractions were carried out in triplicate for each solvent and method used.

Spectrophotometric Analysis
All the analysis were conducted in three replicates and performed as reported by Caser et al. [15].

Total Phenolic Content
Total phenolic content was estimated using the Folin-Ciocalteu method. In each plastic tube 200 µL of each phytoextract were mixed with 1000 µL of diluted (1:10) Folin-Ciocalteu reagent. After 10 min of incubation at dark and room temperature, 800 µL of Na 2 CO 3 7.5% (w/v) were added to each tube. The samples were incubated at dark and room temperature for 30 min. The absorbance at 765 nm was measured by means of a UV-Vis spectrophotometer (Cary 60 UV-Vis, Agilent Technologies, Santa Clara, CA, USA). The results were expressed as mg of gallic acid equivalents (GAE) per 100 g of dry weight (mg GAE 100 g −1 DW).

Total Anthocyanin Content
Total anthocyanins content was measured using the pH-differential method. Buffer solution at pH 1 (4.026 g KCl + 12.45 mL HCl 37% in a 1 L water volume) was added to 500 µL of phytoextract reaching 5 mL in each flask. The same was made in a second flask using a buffer solution at pH 4.5 (32.82 g C 2 H 3 NaO 2 + 18 mL C 2 H 4 O 2 in a 1 L water volume). The samples were left in the dark at room temperature for 20 min. The absorbance of both flasks was read at 515 and 700 nm at a UV-Vis spectrophotometer (Cary 60 UV-Vis, Agilent Technologies, Santa Clara, CA, USA). The total anthocyanin content was calculated using the following formula: [(A × sample dilution factor × 1000)/(molar absorptivity × 1)] where A is [(Absorbance 515 nm − Absorbance 700 nm) at pH 1] − [(Absorbance 515 nm − Absorbance 700 nm) at pH 4.5]. The results were expressed as milligrams of cyanidin 3-O-glucoside (C3G) per 100 g of dry weight (mg C3G 100 g −1 DW).

Antioxidant Activity
The antioxidant activity was determined using the following methods: Technologies, Santa Clara, CA, USA). The ABTS radical-scavenging activity was calculated as where Abs0 is the absorbance of the control (solution without phytoextract) and Abs1 is the absorbance of the sample. The results were expressed as µmol of Trolox equivalents per gram of dry weight (µmol TE g −1 DW).

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The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method. To obtain 100 µM of DPPH radical cation (DPPH·) 2 mg of DPPH were mixed up with 50 mL of MeOH, up to have an absorbance of 1.000 (±0.005) at 515 nm. Then, 1.5 mL of diluted DPPH· was added to 20 µL of phytoextract and the reaction was left in the dark at room temperature for 30 min. The absorbance was read at 515 nm at a spectrophotometer (Cary 60 UV-Vis, Agilent Technologies, Santa Clara, CA, USA). The DPPH radical-scavenging activity was calculated as where Abs0 is the absorbance of the control (solution without phytoextract) and Abs1 is the absorbance of the sample. Results were expressed as µmol of Trolox equivalents per gram of dry weight (µmol TE g −1 DW).

HPLC Analysis
Qualitative and quantitative analyses of the phytoextracts were carried out using an Agilent 1200 High-Performance Liquid Chromatography coupled with an Agilent UV-Vis diode array detector (Agilent Technologies, Santa Clara, CA, USA). The chromatographic separation was made with a Kinetex C18 column (4.6 × 150 mm 2 , 5 µm, Phenomenex, Torrance, CA, USA) using several mobile phases and recording UV spectra at different wavelengths [65], as described in Table 5. Each compound was determined comparing the retention times and UV spectra with the standards under the same chromatographic conditions as reported by Donno et al. [57]. The standards, purchased from Sigma-Aldrich (Saint Louis, MO, USA), were the following: flavonols (hyperoside, isoquercitrin, quercetin, quercitrin, and rutin), catechins (catechin and epicatechin), benzoic acids (ellagic and gallic acids), cinnamic acids (caffeic, chlorogenic, coumaric, and ferulic acids), and vitamin C (ascorbic and dehydroascorbic acids). All the analyses were performed in three replicates.

Statistical Analysis
All data were log transformed before the statistical analysis. As regards the HPLC-DAD analysis, the limit of quantitation (LOQ) [57] was added when the treatments had data for only one or two replicates out of three, while the value 0 was added for the treatments without data.
The data were analyzed with the R-studio software to identify the statistically supported differences between the different extractions. Significant mean differences were verified with one-way ANOVA (p < 0.05) and Tukey's post-hoc test after checking the data for normality and homoscedasticity through Shapiro-Wilk's test (p > 0.05) and Levene's test (p < 0.05), respectively. The compounds non respecting the ANOVA assumptions were analyzed with Kruskal-Wallis non-parametric test (p < 0.05) and the post-hoc Dunn's comparison test.
As regards the data resulting from the spectrophotometric analysis, statistical comparisons were performed using ANOVA for TPC, TAC, and FRAP and Kruskal-Wallis test for DPPH and ABTS (p > 0.05 in Levene's test). Regarding the data resulting from the HPLC analysis, ANOVA was made for ellagic acid, hyperoside, and rutin, while Kruskal-Wallis test for the other compounds (p < 0.05 in Shapiro-Wilk's test).
A correlation analysis between the content of total phenolics (TPC) and anthocyanins (TAC), and the assays FRAP, ABTS, and DPPH used to measure the antioxidant activity of the extracts was made with the R-studio software. The Pearson correlation was adopted when the variables were normally distributed according to the Shapiro-Wilk's test (p > 0.05), otherwise the non-parametric Kendall correlation.

Conclusions
Processing food wastes and by-products to generate high-value products for industrial application has been attractive great economic and scientific interest [66,67]. The sustainable processing of biomass, including that resulting from agriculture, into valuable and safe biobased products falls within the definition of biorefining [68]. The use of renewable resources can help farmers to expand their sources of income and create new job opportunities while reducing bio-waste and the environmental footprint in a circular bioeconomy perspective [69].
The economic use of a by-product is related to its intrinsic, nutritional, or utility value. The dried saffron tepals are largely produced after stigma are separated from the flowers and are a natural source of antioxidant compounds. This study demonstrated that the phytochemical composition and the antioxidant capacity of extracts of dried saffron tepals is influenced by the extraction technique. In most cases, UAE with water or low percentage of methanol showed results of phenolic compounds and vitamin C similar to maceration, allowing an improvement in extractions by halving the time. Compared to the spice, the dried saffron tepals showed a lower content of total phenolics and anthocyanins, but a higher antioxidant activity.
To produce waste-derived products, bio-waste shall either be separated and recycled at source or collected separately before undergoing treatments, which gives storage an important role (Directive 2008/122/EC). The cost of biomass and its storage are some essential factors for the economic viability of biorefineries [67]. The drying technique has always been widely used in the herbs sector to stabilize and preserve plant material, and in particular flowers, which have high perishability due to the elevated moisture content [31]. The drying treatment can preserve tepals until their possible use in food or pharmacological-cosmetic industries, for example in the preparation of soaps and cosmetic products where they can act as antioxidants or colorants in place of synthetic excipients, meeting the demand of the cosmetic sector to use products of natural origin [70].
This study allowed to compare the results with those previously attained for fresh tepals by Caser et al. [15], which performed a similar extraction protocol. This work has highlighted that, after drying, saffron tepals retain bioactive phenolics. The dehydration procedure may improve extractions as safer solvents gave higher yield of phenolic compounds. Furthermore, the HPLC-DAD analysis detected some phenolic compounds (i.e., ferulic acid, isoquercitrin, and quercitrin) not previously found in fresh tepals. Vitamin C, already discovered in the spice, was interestingly detected also in dried tepals.
Taken together, these results assess that floral bio-residues of saffron can be sustainably processed to obtain high yields of valuable phytochemicals with potential applications in the pharmaceutical, cosmetic, and food industries, meeting the requirement for a sustainable biorefining and opening new possibilities for this by-product to become an important income source.