**4. Factors that modulate mono and sesquiterpenic profile**

#### **4.1. Preharvest impact**

As evidenced in **Table 1**, of the 64 volatile terpenic compounds reported from elderflowers, 40 are oxygen-containing structures. As shown in **Figure 2**, the peak intensities of the monoterpenic metabolites predominate, representing up to 99 and 77% of the overall elderflowers and elderberries terpenic content, respectively [31, 37]. Linalool oxide (in the pyranoid form) is a major component from fresh elderflowers, accounting for up to 87% (relative to the overall GC peak area) [31]. Other authors reported that hotrienol (14%, w/w), rose oxide (5%, w/w), linalool (4%, w/w), and linalool oxide (furanic forms, 3%, w/w) were the major monoterpenic

**Figure 2.** GC × GC–ToFMS chromatogram contour plots from fresh elderflowers (A) and fresh ripe elderberries (B). The

chromatographic spaces corresponding to monoterpenic and sesquiterpenic compounds are highlighted.

66 Secondary Metabolites - Sources and Applications

metabolites from dried elderflowers [42] (chemical structures illustrated in **Figure 3**).

Regarding ripe elderberries, limonene and *p*-cymene are reported as the major monoterpenic components (**Figure 3**). Along with limonene (2.2–9.9 μg/kg of fresh berries), other authors reported as major components in fresh elderberries the monoterpenic compounds Crop quality could be defined as a set of agronomic/commercial, organoleptic, and nutritional qualities that are variable among (1) distinct species but also among different cultivars within the same species (genetic factors); (2) different climatic conditions, such as water availability and light exposition; and (3) different agronomic conditions, such as cultivation systems, fertilization, and harvesting date [46]. Altogether, these preharvest factors may have an impact on the final quality of the elderberry fruits and flowers; however, the information about these effects is scarce. The impact of preharvest factors is often focused on parameters with direct agronomic and commercial relevance, as plant yield, fruit size, sugar content and acidity (e.g., reviews on *S. nigra* plant [18, 23]), from which some nutritional quality parameters can be inferred. However, the comprehensive impact of these parameters on the chemical composition, specially in what concerns the target molecules with determining biological properties, still remains unknown. As relevant examples in the present appraisal, the impact of preharvest factors on *S. nigra* mono and sesquiterpenic compounds is still in the beginning and the available literature is mainly focused on ripening and cultivar effects. However, considering that these components, as plant secondary metabolites, play an important role in plant growth and development, in the interaction with surrounding environment, such as temperature, water, radiation, chemicals, mechanical (as wind or soil movement), pathogen attacks, and nutrient deficiencies [10, 47, 48], as well as in their potential health benefits, the interest in the detailed understanding of the impact preharvest parameters on their profile is of obvious interest.

During the ripening process, several other phenomena occur, namely biosynthesis and degradation of a wide range of secondary metabolites that may have direct relevance in elderberry sensorial characteristics. A recent metabolomics-based study that exploited the effects of the developmental stages of different cultivars on the volatile terpenic components [37] demonstrated that the variability of monoterpenic compounds (*β*-pinene, 1,3,8-*p*-menthatriene, terpinolene, dihydromyrcenol, fenchol, *α*-terpineol, and citral) and of the sesquiterpene *β*-elemene was linked to elderberries ripening. Overall, monoterpenic and sesquiterpenic content exhibited a similar trend of variation through ripening, that is, gradually decreased over the ripening stages, which was mainly ruled by the major components, namely limonene, *p*-cymene, *β*-caryophyllene, and aromadendrene. These components were proposed as qual-

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Plant cultivars generally differ in yield, organoleptic, and nutritional characteristics [23, 46], and their genetic background is a factor that influences quality traits [46]. In the particular case of elderberries, cultivars are classified based on their morphological characteristics and yield [52], as no definitive taxonomic DNA-based studies have been conducted in this species. Although, efforts have been made for their classification with molecular data. For instance, Portuguese *S. nigra* clones explored from local growers using different molecular characterization tools [52], and a genebank has been created for different *Sambucus* species and dozens of cultivars [53].

It is reported that elderberry yield ranges anywhere from 1 to over 30 kg per bush, depending on cultivar [54, 55]. This aspect, together with the fact that several cultivars are nowadays explored for the formulation of various products, where formula standardization is required, implying the comparison of cultivars' composition, can play a significant role in their application (e.g., [56]) and then become important decision tool for producers. The fact that mono and sesquiterpenic synthesis is encoded by a variety or cultivar-related genes implies that their levels can be cultivar-dependent, which, on the one hand, might be used to trace its varietal origin [57] and, on the other hand, can be used to better manage their final product and to maximize the commercial value of the crop. An exploratory study, suggested a possible cultivar effect over the mono and sesquiterpenic compounds profile from fresh elderflowers [31]; however, more consolidated data is still required to sustain the stated remarks, namely

in what concerns the number of analyzed samples and different harvesting years.

The specific cultivar metabolite profile may imply differences at the sensorial level in *S. nigra*based products, as shown for elderflower- and elderberry-based products obtained from different cultivars [32, 34, 56]. In a study that merges the results from the sensory evaluation and information on the aroma of the individual volatile compounds, the results highlighted that different elderberry cultivars had specific sensory characteristics (as fresh-fruity-sweet aroma) and, hence, volatile composition [38]. Differences in linalool and α-terpineol (ranging from 2.8 to 21.7 and from 213.6 to 2699.6 μg/kg, respectively) were reported for thawed ripe elderberries from different cultivars [41]. Likewise, the terpenic alcohols and oxides in elderflowers from different cultivars ranged from 0.8 to 3870 ng/mL for hotrienol; from 1.2 to 2320 ng/mL for *cis*-rose oxide; from 2.3 to 1840 ng/mL for linalool; and from 1.3 to 1100 ng/mL

Despite the studies reported earlier, a more comprehensive understanding of the influence of preharvest parameters will require their analysis in an integrated approach, including,

ity markers to follow-up the ripening process [37].

for linalool oxide (furanic form) [32, 34].

The production of terpenic metabolites depends on the physiological and developmental stage of the plant [10, 37]. Fruit ripening, in particular, is a crucial phenomenon that affects different physiological and biochemical processes, which are determinant to the development of nutritional and organoleptic characteristics [30]. The fruit organoleptic characteristics such as taste, color, and aroma are important quality and consumer acceptance-determining features [30, 49].

During ripening, major events occur, including cell expansion and softening, dismantling of the photosynthetic apparatus, and degradation of chlorophyll [11]. Elderberry ripening takes place from the 1 to 2-month period, starting with a green appearance and they ripen over a period of 6–8 weeks from July to September (depending on the geographic location). When elderberries become ripe, they have a characteristic deep purple color [23]. The accumulation of sugars (expressed as total soluble solids [TSS]) and decrease in acidity (pH and titratable acidity [TA]) have been routinely used by growers as a decision-making parameter to establish the harvesting moment and even the commercial price of the berries [18, 23, 37]. The ripe elderberries' pH ranges from 3.8 to 4.8; TA ranges from 0.48 to 1.43 g citric acid/100 g FW berries, while TSS ranges from 10.1 to 17.5°Brix [37, 38, 50, 51]. **Figure 4** illustrates the impact of ripening in those tree parameters on elderberries harvested in a Portuguese location (Tarouca, Távora and Varosa Valley), in the harvest season of 2013.

**Figure 4.** Total soluble solids (TSS), titratable acidity (TA), and pH from elderberries at five ripening stages [37], harvested in a Portuguese plantation (Tarouca, Távora and Varosa Valley), in the harvest season of 2013. The harvesting date assigned with \* represents the ripe stage.

During the ripening process, several other phenomena occur, namely biosynthesis and degradation of a wide range of secondary metabolites that may have direct relevance in elderberry sensorial characteristics. A recent metabolomics-based study that exploited the effects of the developmental stages of different cultivars on the volatile terpenic components [37] demonstrated that the variability of monoterpenic compounds (*β*-pinene, 1,3,8-*p*-menthatriene, terpinolene, dihydromyrcenol, fenchol, *α*-terpineol, and citral) and of the sesquiterpene *β*-elemene was linked to elderberries ripening. Overall, monoterpenic and sesquiterpenic content exhibited a similar trend of variation through ripening, that is, gradually decreased over the ripening stages, which was mainly ruled by the major components, namely limonene, *p*-cymene, *β*-caryophyllene, and aromadendrene. These components were proposed as quality markers to follow-up the ripening process [37].

available literature is mainly focused on ripening and cultivar effects. However, considering that these components, as plant secondary metabolites, play an important role in plant growth and development, in the interaction with surrounding environment, such as temperature, water, radiation, chemicals, mechanical (as wind or soil movement), pathogen attacks, and nutrient deficiencies [10, 47, 48], as well as in their potential health benefits, the interest in the detailed understanding of the impact preharvest parameters on their profile is of obvious

The production of terpenic metabolites depends on the physiological and developmental stage of the plant [10, 37]. Fruit ripening, in particular, is a crucial phenomenon that affects different physiological and biochemical processes, which are determinant to the development of nutritional and organoleptic characteristics [30]. The fruit organoleptic characteristics such as taste, color, and aroma are important quality and consumer acceptance-determining features [30, 49]. During ripening, major events occur, including cell expansion and softening, dismantling of the photosynthetic apparatus, and degradation of chlorophyll [11]. Elderberry ripening takes place from the 1 to 2-month period, starting with a green appearance and they ripen over a period of 6–8 weeks from July to September (depending on the geographic location). When elderberries become ripe, they have a characteristic deep purple color [23]. The accumulation of sugars (expressed as total soluble solids [TSS]) and decrease in acidity (pH and titratable acidity [TA]) have been routinely used by growers as a decision-making parameter to establish the harvesting moment and even the commercial price of the berries [18, 23, 37]. The ripe elderberries' pH ranges from 3.8 to 4.8; TA ranges from 0.48 to 1.43 g citric acid/100 g FW berries, while TSS ranges from 10.1 to 17.5°Brix [37, 38, 50, 51]. **Figure 4** illustrates the impact of ripening in those tree parameters on elderberries harvested in a Portuguese location (Tarouca,

**Figure 4.** Total soluble solids (TSS), titratable acidity (TA), and pH from elderberries at five ripening stages [37], harvested in a Portuguese plantation (Tarouca, Távora and Varosa Valley), in the harvest season of 2013. The harvesting

Távora and Varosa Valley), in the harvest season of 2013.

date assigned with \* represents the ripe stage.

interest.

68 Secondary Metabolites - Sources and Applications

Plant cultivars generally differ in yield, organoleptic, and nutritional characteristics [23, 46], and their genetic background is a factor that influences quality traits [46]. In the particular case of elderberries, cultivars are classified based on their morphological characteristics and yield [52], as no definitive taxonomic DNA-based studies have been conducted in this species. Although, efforts have been made for their classification with molecular data. For instance, Portuguese *S. nigra* clones explored from local growers using different molecular characterization tools [52], and a genebank has been created for different *Sambucus* species and dozens of cultivars [53].

It is reported that elderberry yield ranges anywhere from 1 to over 30 kg per bush, depending on cultivar [54, 55]. This aspect, together with the fact that several cultivars are nowadays explored for the formulation of various products, where formula standardization is required, implying the comparison of cultivars' composition, can play a significant role in their application (e.g., [56]) and then become important decision tool for producers. The fact that mono and sesquiterpenic synthesis is encoded by a variety or cultivar-related genes implies that their levels can be cultivar-dependent, which, on the one hand, might be used to trace its varietal origin [57] and, on the other hand, can be used to better manage their final product and to maximize the commercial value of the crop. An exploratory study, suggested a possible cultivar effect over the mono and sesquiterpenic compounds profile from fresh elderflowers [31]; however, more consolidated data is still required to sustain the stated remarks, namely in what concerns the number of analyzed samples and different harvesting years.

The specific cultivar metabolite profile may imply differences at the sensorial level in *S. nigra*based products, as shown for elderflower- and elderberry-based products obtained from different cultivars [32, 34, 56]. In a study that merges the results from the sensory evaluation and information on the aroma of the individual volatile compounds, the results highlighted that different elderberry cultivars had specific sensory characteristics (as fresh-fruity-sweet aroma) and, hence, volatile composition [38]. Differences in linalool and α-terpineol (ranging from 2.8 to 21.7 and from 213.6 to 2699.6 μg/kg, respectively) were reported for thawed ripe elderberries from different cultivars [41]. Likewise, the terpenic alcohols and oxides in elderflowers from different cultivars ranged from 0.8 to 3870 ng/mL for hotrienol; from 1.2 to 2320 ng/mL for *cis*-rose oxide; from 2.3 to 1840 ng/mL for linalool; and from 1.3 to 1100 ng/mL for linalool oxide (furanic form) [32, 34].

Despite the studies reported earlier, a more comprehensive understanding of the influence of preharvest parameters will require their analysis in an integrated approach, including, among others, climate, agricultural practices, soil, and harvesting year to fully understand how these affect the biochemical mechanisms involved in the formation of mono and sesquiterpenic metabolites from elderflowers and berries and also to improve its valorization potential, particularly when related with health benefits and relevant sensorial characteristics. Also, the influence of climate change on the *S. nigra* plant response should be a noteworthy issue.

temperatures [5]. Elderberries can also be pulse-light treated and further crushed and mashed to produce concentrate juices [64–67]. These later steps promote the degradation of berry cell walls, contributing to the alteration of metabolites' profile, namely increasing the anthocyanin content of juices [63]. All these handling and storage processes may have an impact on the

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The knowledge of the impact of handling and storage conditions on the terpenic metabolites of *S. nigra* is still scarce for both, berries and flowers. However, more information is available regarding postharvest effects over the elderflowers' matrix. For instance, the impact of freezing, freeze-drying, air-drying, and vacuum packing over the volatile terpenic compounds was

After 1 year of storage, a decrease of the total terpenic content up to 47% for frozen elderflowers; up to 67 and 71% when vacuum packed and kept under light exposure and without light exposure, respectively; up to 82% for air-dryed elderflowers; and up to 85% for freeze-dried elderflowers (**Figure 6**) [31]. Under vacuum packing, there was no significant impact from light exposure. Linalool oxides were suggested as markers of the impact of the studied post-

Drying methodologies, as air-drying or freeze-drying, often fail to completely preserve volatile aroma compounds [68], as reported in dried elderflowers, mainly due to diffusion and evaporation losses [31, 33, 69, 70]. Drying of elderberries or their products promotes a water activity reduction, contributing to the preservation of the samples against microbial contamination and also decreases the degradation of anthocyanins [60], by increasing their stability

**Figure 6.** Variation trends of the abundance of oxygen-containing monoterpenes (A), hydrocarbon monoterpenes (B) and sesquiterpenes (C) toward the different handling and storage conditions for up to 1 year, based on the corresponding GC

peak areas (au: Arbitrary units). Adapted with permission [31].

harvest conditions over the volatile terpenic metabolites of elderflowers [31].

chemical composition of these matrices, as discussed as follows.

monitored for up to 1 year (**Figure 6**) [31].

#### **4.2. Postharvest impact**

Postharvest management includes a set of postproduction practices comprising, among others, cleaning to eliminate undesirable elements and improve product appearance, sorting, cooling, control of variables such as temperature and relative humidity, and packing, ensuring that the product complies with the established quality standards for fresh and processed products [58, 59]. Postharvest practices may deeply affect the quality of a product in many aspects such as chemical and sensorial characteristics but also their potential health benefits, and ultimately, it may affect product's acceptability and marketability [30]. Therefore, reliable and objective quality-control tools to measure the impact of postharvest practices (ideally integrated with preharvesting conditions) over product quality and in the present appraisal on sensory quality are essential.

Elderflowers and elderberries go through different postharvest handling and storage conditions that precede processing, to prepare stable formulations for commercialization. **Figure 5** illustrates the main steps from harvesting for the storage of elderberries and elderflowers and the main chemical changes that may occur throughout these processes [31, 33, 42, 60–63].

*S. nigra* flowers or berries are typically collected during the morning and transported to processing facilities in specific plastic crates, avoiding damage caused by their own weight [5]. Flowers are often frozen or air-dried and then the stems are removed, while elderberries are sun dried or refrigerated, and the stems are removed and stored in silos at subzero

**Figure 5.** From harvesting to elderberry and elderflower storage. The main chemical changes that may occur through different steps are included [31, 33, 42, 60–63].

temperatures [5]. Elderberries can also be pulse-light treated and further crushed and mashed to produce concentrate juices [64–67]. These later steps promote the degradation of berry cell walls, contributing to the alteration of metabolites' profile, namely increasing the anthocyanin content of juices [63]. All these handling and storage processes may have an impact on the chemical composition of these matrices, as discussed as follows.

among others, climate, agricultural practices, soil, and harvesting year to fully understand how these affect the biochemical mechanisms involved in the formation of mono and sesquiterpenic metabolites from elderflowers and berries and also to improve its valorization potential, particularly when related with health benefits and relevant sensorial characteristics. Also, the influence of climate change on the *S. nigra* plant response should be a noteworthy issue.

Postharvest management includes a set of postproduction practices comprising, among others, cleaning to eliminate undesirable elements and improve product appearance, sorting, cooling, control of variables such as temperature and relative humidity, and packing, ensuring that the product complies with the established quality standards for fresh and processed products [58, 59]. Postharvest practices may deeply affect the quality of a product in many aspects such as chemical and sensorial characteristics but also their potential health benefits, and ultimately, it may affect product's acceptability and marketability [30]. Therefore, reliable and objective quality-control tools to measure the impact of postharvest practices (ideally integrated with preharvesting conditions) over product quality and in the present appraisal

Elderflowers and elderberries go through different postharvest handling and storage conditions that precede processing, to prepare stable formulations for commercialization. **Figure 5** illustrates the main steps from harvesting for the storage of elderberries and elderflowers and the main chemical changes that may occur throughout these processes [31, 33, 42, 60–63].

*S. nigra* flowers or berries are typically collected during the morning and transported to processing facilities in specific plastic crates, avoiding damage caused by their own weight [5]. Flowers are often frozen or air-dried and then the stems are removed, while elderberries are sun dried or refrigerated, and the stems are removed and stored in silos at subzero

**Figure 5.** From harvesting to elderberry and elderflower storage. The main chemical changes that may occur through

**4.2. Postharvest impact**

70 Secondary Metabolites - Sources and Applications

on sensory quality are essential.

different steps are included [31, 33, 42, 60–63].

The knowledge of the impact of handling and storage conditions on the terpenic metabolites of *S. nigra* is still scarce for both, berries and flowers. However, more information is available regarding postharvest effects over the elderflowers' matrix. For instance, the impact of freezing, freeze-drying, air-drying, and vacuum packing over the volatile terpenic compounds was monitored for up to 1 year (**Figure 6**) [31].

After 1 year of storage, a decrease of the total terpenic content up to 47% for frozen elderflowers; up to 67 and 71% when vacuum packed and kept under light exposure and without light exposure, respectively; up to 82% for air-dryed elderflowers; and up to 85% for freeze-dried elderflowers (**Figure 6**) [31]. Under vacuum packing, there was no significant impact from light exposure. Linalool oxides were suggested as markers of the impact of the studied postharvest conditions over the volatile terpenic metabolites of elderflowers [31].

Drying methodologies, as air-drying or freeze-drying, often fail to completely preserve volatile aroma compounds [68], as reported in dried elderflowers, mainly due to diffusion and evaporation losses [31, 33, 69, 70]. Drying of elderberries or their products promotes a water activity reduction, contributing to the preservation of the samples against microbial contamination and also decreases the degradation of anthocyanins [60], by increasing their stability

**Figure 6.** Variation trends of the abundance of oxygen-containing monoterpenes (A), hydrocarbon monoterpenes (B) and sesquiterpenes (C) toward the different handling and storage conditions for up to 1 year, based on the corresponding GC peak areas (au: Arbitrary units). Adapted with permission [31].

[60]. Other strategies have been used to preserve the elderberries' bioactive components or to enhance their nutritional value, as for instance, their processing with pulsed ultraviolet light to enhance the phenolic content [61]. However, no studies were performed so far on mono and sesquiterpenic fractions of elderberries.

for several years, the agronomic and organoleptic qualities were the main market drivers, nowadays, the importance of the nutritional value is strongly increasing, thanks to increased consumer awareness on the dietary health effects of plant consumption [46]. It is for this reason that understanding how *S. nigra* mono and sesquiterpenic metabolites respond to exposures of different biotic and abiotic stresses is of major importance. Understanding and managing the effects of preharvest and postharvest factors are critical for further economical exploitation of these natural products, namely to (1) provide the growers of robust decision-making tools, (2) fulfill the current standardization requirements for production of plant-based extracts and products, and (3) contribute to assure the high-quality *S. nigra*-

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Thanks to FCT/MEC for the financial support to the QOPNA Research Unit (FCT UID/ QUI/00062/2013), through national funds and where applicable co-financed by the FEDER, within the PT2020 Partnership Agreement. This work was developed within the scope of the project CICECO - Aveiro Institute of Materials, POCI-01-0145-FEDER-007679 (FCT Ref. UID/ CTM/50011/2013), financed by national funds through the FCT/MEC and when appropriate co-financed by FEDER under the PT2020 Partnership Agreement. Â. Salvador thanks the grant AgroForWealth: Biorefining of agricultural and forest by-products and wastes: integrated strategic for valorization of resources toward society wealth and sustainability (CENTRO-01- 0145-FEDER-000001), funded by Centro2020, through FEDER and PT2020, and Operational Group Sambucus Valor: integrated valorization of elderberry plant according to the patterns of healthy consumption: from the plant to the formulation of new value-added food products, funded by PDR 2020, Measure 1, "Innovation," Formation of Operational Groups (PDR2020-

341).

2 CICECO-Aveiro Institute of Materials, Department of Chemistry, University of Aveiro,

[1] World Health Organization. WHO Traditional Medicine Strategy 2014-2023. Vol. 1.

and Sílvia M. Rocha<sup>1</sup>

\*

146/Initiative no

1 QOPNA, Department of Chemistry, University of Aveiro, Aveiro, Portugal

based products.

**Acknowledgements**

101-031117, Partnership no

Ângelo C. Salvador1,2, Armando J. D. Silvestre2

\*Address all correspondence to: smrocha@ua.pt

Geneva, Switzerland: WHO Press; 2013. pp. 1-78

**Author details**

Aveiro, Portugal

**References**

Storage time also plays an important role in the mono and sesquiterpenic composition illustrated by the fact that 15 compounds, including rose oxides, hotrienol, linalool, *α*-terpineol, hydroxylinalool, and limonene, partially or completely vanished during storage of dried elderflowers [33]. Cellular disruption might explain the release of volatile compounds in certain postharvest conditions, namely freezing storage [31, 71, 72]. Likewise, the packaging material might affect their profile, being reported that elderflower's tea bags made of aluminum had the highest average concentrations of rose oxide, linalool oxide, nerol oxide, and hotrienol, when compared to paper and plastic bags [33].

Some components, such as hotrienol, were observed to increase during storage of elderflowers, which could be associated with the action of enzymes, such as glucosidases, that unbound the volatile components from glycosides present in the matrix [33]. Non-oxygen-containing structures, that is, monoterpenes and sesquiterpenes, also increased under certain postharvest conditions (**Figure 6**), again assuming that *de novo* biosynthesis of terpenes may play a key role in this phenomenon [31, 33].

The modifications in *S. nigra* terpenic profile upon different postharvest conditions have a significant impact on sensorial characteristics of its products, being linked to a dynamic and complex network of enzymatic and physicochemical phenomena. Understanding the postharvest impact is a step forward to manage and control the production of elderflower and elderberry formulations [31].
