Volatiles in Berries: Biosynthesis, Composition, Bioavailability, and Health Beneﬁts

: Volatile compounds in fruits are responsible for their aroma. Among fruits, berries contain many volatile compounds, mainly esters, alcohols, terpenoids, aldehydes, ketones, and lactones. Studies for volatile compounds in berries have increased extensively as the consumption of berry products rapidly increased. In this paper, we reviewed biosynthesis and proﬁles of volatiles in some berries (strawberry, blueberry, raspberry, blackberry, and cranberry) and their bioavailability and health beneﬁts, including anti-inﬂammatory, anti-cancer, anti-obesity, and anti-diabetic effects in vitro and in vivo. Each berry had different major volatiles, but monoterpene had an important role in all berries as aroma-active components. Volatile compounds were nonpolar and hydrophobic and rapidly absorbed and eliminated from our body after administration. Among them, monoterpenes, including linalool, limonene, and geraniol, showed many health beneﬁts against inﬂammation, cancer, obesity, and diabetes in vitro and in vivo. More research on the health beneﬁts of volatile compounds from berries and their bioavailability would be needed to conﬁrm the bioactivities of berry volatiles.


Introduction
Berries, one of the most common fruits in the human diet (strawberry, blueberry, red raspberry, black raspberry, blackberry, and cranberry in the United States), are rich in minerals, vitamins, dietary fibers, and especially polyphenols and volatiles [1][2][3].Volatile compounds in berries are responsible for the unique aroma of berries [4].Fruit volatile compounds are mainly comprised of diverse chemicals, including esters, alcohols, terpenoids, aldehydes, ketones, and lactones [5].The volatile composition of berries is complex and different by many factors, including the cultivar, ripeness, pre-and post-harvest storage conditions, fruit samples, temperature, and experimental conditions [6][7][8][9][10][11]. Blueberries (Vaccinium ashei) showed linalool increasing and α-terpineol and β-caryophyllene decreasing during the maturation of the blueberry [6].Full-red harvested strawberries contained more volatile compounds than 3 ⁄4-red harvested strawberries, regardless of the storage duration [12].Raspberries (raw, frozen, or frozen for a year) were examined to compare the long-term frozen storage.The changes in volatile composition during long-term frozen storage were negligible except for an increase in α-ionone and caryophyllene [8].
Volatile compounds are small and light molecules (below 250-300 Da) with low polarity and high vapor pressure [13].Plants synthesize and release volatile compounds to communicate and interact with environments, compensating for the immobility of plants [14].Volatile compounds play an important role in pollination by attracting pollinators, protecting from pathogens and herbivores, and even communicating with inter-and intra-plants [15].Volatiles can be divided into primary compounds and secondary compounds.Primary compounds are synthesized during maturation by anabolic or catabolic pathways of the plant [16].Secondary volatile compounds are produced from tissue disruption by autoxidation or enzyme catalyzing reactions [17][18][19].
A mixture of many different volatile compounds makes a unique aroma.Although a lot of compounds were found as volatile compounds in fruits, only a few compounds have been identified as aroma compounds of fruit flavor based on their quantitative abundance and olfactory thresholds [20].With the increasing consumption of berries and berry products such as fruits, juice, puree, jams, and other berry ingredients, there have been many studies conducted about identifying aroma volatile compounds of berries for developing consumer acceptability [21] and the studies about the health beneficial effect of berries, especially berry polyphenols.Berry polyphenols are composed of flavonoids, phenolic acids, tannins, stilbenes, lignans, and others and have been shown to possess many health effects, such as antioxidant, anti-inflammatory, and anti-cancer activities [22][23][24].Unlike berry polyphenols, although there have been extensive analyses of volatile berry composition, there is still a very limited number of studies on the bioavailability and health benefits of berry volatiles.Recently, volatile compounds in plants have been reported to have health-promoting activities such as anti-inflammatory effects [25,26].Many review articles mainly focused on the composition of berry volatiles and affecting factors such as different locations, ripeness, cultivar/genotypes, harvest and storage conditions, etc., but there is a lack of information about the bioavailability and biological activities of berry volatiles in our body.In this article, the biosynthesis of volatiles in plants, the chemical composition of some berries commonly consumed in the U.S. (blackberry, blueberry, cranberry, raspberry, and strawberry), bioavailability, and the health benefits of volatile compounds that are rich in berries were reviewed.

Biosynthesis of Volatiles in Plants
Plant volatiles can be grouped into terpenoids, phenylpropanoids/benzenoids, and fatty acid derivatives based on chemical structure and biochemical synthesis [14,27,28].Terpenes/terpenoids are the most abundant and diverse family of secondary plant metabolites and essential oils, including more than 30,000 compounds [29,30].Terpenes are classified by the amount of C5 isoprene units in the structure: isoprene (C5), monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), and so on, with polyterpenes (C5n where n can be ~30,000) [30].Monoterpenes and sesquiterpenes are the most abundant terpenes found in essential oils [31].Although they have diverse chemical structures, they all share common biosynthesis pathways, and they are synthesized in all parts of the plants, such as the leaves, fruits, flowers, stems, and roots [15].Since terpenes/terpenoids are the major class of berry volatiles, we focused more on the biosynthesis of terpenes, such as monoterpenes and sesquiterpenes, in this review.

Phenylpropanoids/Benzenoids
Phenylpropanoids/benzenoids are synthesized from the shikimate pathway [31].The shikimate pathway starts with the condensation of phosphoenolpyruvate and erythrose 4-phosphate [51].Chorismic acid is formed by the elimination of ring alcohol from shikimic acid, and this forms the phenylpropionic acid skeleton [29].Cinnamic acid and phydroxycinnamic acid undergo many enzymatic reactions to produce volatile compounds, including eugenol and benzyl benzoate [52].

Application of Plant Volatile Biosynthesis
Volatile compounds that are emitted from plants have an important role in many different functions, such as pollinator attraction, direct and indirect defenses against herbivores, insects, and microorganisms, and communication between and within plants [27,57,58].In addition, natural volatile compounds such as methyl jasmonate, allyl isothiocyanate, and tea tree oil have been used for modulating volatile biosynthesis and controlling the pre-and post-harvest quality of berries [9,59].Those volatile compounds also suppressed the decay in strawberries and blackberries stored at 10 • C [60].Sangiorgio et al. found a positive correlation between Lactobacillus, Paenibacillus spp., and norisoprenoids and a negative correlation between Enterobacteriaceae and monoterpenes [61].From these results, the raspberry microbiome can be selectively chosen for the overall better quality of fruit, including its aroma, shelf-life, and safety [61].Although there is still more research on the mechanisms required, accumulated results in metabolomic and genomic approaches can be used for making advances in fruit ripeness, quality, and consumer acceptability [62][63][64].

The Chemical Composition of Volatile Compounds in Berries
The major volatile compounds identified from five common berries in the U.S. (strawberry, blueberry, raspberry, blackberry, and cranberry) were summarized (Table 1).

Strawberry
Strawberries (Fragaria spp.) are the most consumed berry fruit for their sweet taste and unique aroma [86].The consumption of strawberry products, such as jams, juices, and puree, has significantly increased [87,88].Strawberries are rich in volatile compounds responsible for the strawberry flavor and aroma [89,90].Volatile compounds in strawberries have been extensively studied, and more than 360 volatile compounds have been identified [91].These compounds include esters, which were qualitatively and quantitatively dominant, terpenes, furanones, sulfur, lactones, alcohols, and carbonyls.In the study of Lu et al., a total of 42 volatiles were detected, with 19 esters, 10 alcohols, and 6 terpenes being the most abundant in the strawberry samples analyzed [65].

Bioavailability of Berry Volatiles
The definition of bioavailability by the U.S. Food and Drug Administration (FDA) is "the rate and extent to which the active ingredient or active moiety is absorbed from a drug product and becomes available at the site of action" [108].More proper meaning is the part of ingested compound reaching the systemic circulation and specific site where it is available in the body [109].Investigating the bioavailability of a compound is important to find the clinical relevance of the health-promoting activities of the bioactive compound in our body [110].Thus, it is necessary to study the absorption, distribution, metabolism, and excretion of bioactive compounds.
Bioavailability can be variable due to many different factors, including chemical structure, physical state, solubility, route of administration, and distribution via biotransformation and excretion [111,112].Based on solubility, volatile compounds and essential oils are relatively more nonpolar hydrophobic compounds than polyphenols that are more polar hydrophilic nutraceuticals [113,114].Regarding the route of administration, volatile compounds are more suitable for pulmonary administration through inhalation, while polyphenols are normally administrated orally [115].Oral administration generally takes, on average, 30-90 min of action, while inhalation of gaseous compounds takes, on average, only 2-3 min [116].The bioavailability of volatiles is largely affected by volatility, instability, and hydrophobicity [117].
Although there are many types of research conducted about the identification and quantification of berry volatiles, information is still lacking on the bioavailability of berry volatiles in animals and humans [118].In this review, bioavailability studies of essential oils and herbal medicinal products that contain volatiles commonly present in berries were selected to estimate the bioavailability of volatile berry compounds.Unfortunately, the studies found are limited and mostly include animal models.
Most of the bioavailability studies of essential oils showed that the volatile compounds in essential oils are rapidly absorbed and eliminated after pulmonary, dermal, and oral administration [115].The compounds were mostly metabolized and eliminated within an hour of elimination half-life through the kidney after phase-II conjugation or CO 2 exhalation.
In Igimi et al., the absorption, distribution, and excretion of d-limonene, a monoterpene in many essential oils but also found in black raspberries and blackberries, were investigated in rats [119]. 14C-labeled d-limonene was orally administered to 21 male Wistar rats.The maximum radioactivity was obtained 2 h after administration in blood and 1-2 h after administration in tissues.High radioactivity in the liver, kidney, and blood became not significant after 48 h.The excretion of d-limonene was 60% in urine, 5% in feces, and 2% in expired CO 2 in 48 h.About 25% of administered d-limonene was eliminated in bile in 24 h in bile duct cannulated rats.Biotransformation studies of (+)-limonene in humans showed that the main metabolites of biotransformation were perillic acid, dihydroperillic acid, and limonene-10-ol, and their glucuronides, perillyl alcohol, p-mentha-1,8-dien-carboxylic acid, cis-and trans-dihydroperillic acid, limonene, 1,2-diol and limonene-8,9-diol [120].

Health Benefits of Berry Volatiles
Despite the sensory properties of fruits and vegetables, there are studies demonstrating that the role of aroma compounds is more than their odor impact [18,125].Recently, volatile compounds in plants have been reported to have health-promoting activities, including anti-inflammatory [25,126], anti-cancer [26], anti-obesity, and anti-diabetic effects [127].However, since there are not many studies on the health-promoting effects of berry volatiles, studies of volatile compounds from essential oils and other fruits and plants that berries commonly contain were used to review the potential health benefits of volatile compounds in berries (Figure 2).

Health Benefits of Berry Volatiles
Despite the sensory properties of fruits and vegetables, there are studies demonstrating that the role of aroma compounds is more than their odor impact [18,125].Recently, volatile compounds in plants have been reported to have health-promoting activities, including anti-inflammatory [25,126], anti-cancer [26], anti-obesity, and anti-diabetic effects [127].However, since there are not many studies on the health-promoting effects of berry volatiles, studies of volatile compounds from essential oils and other fruits and plants that berries commonly contain were used to review the potential health benefits of volatile compounds in berries (Figure 2).

Cancer
Kim et al. demonstrated that geraniol inhibits human prostate cancer cell PC-3 proliferation in in vitro and in vivo xenograft mice models [164].Geraniol at 0.5 and 1 mM significantly suppressed the cell growth of PC-3 by increasing cell cycle arrest and apoptosis.Balb/C nude mice inoculated with PC-3 cells took intratumoral geraniol injection daily for 38 days at 0, 12, 60, or 300 mg/kg.The mice treated with 60 or 300 mg/kg geraniol showed significantly decreased tumor volume and weight.Injection of geraniol at 20 mg/kg also sensitized the chemotherapeutic agent Docetaxel (2 mg/kg) in the xenograft mice model.In Lee et al., geraniol inhibited prostate cancer growth by a down-regulating E2F8 transcription factor and inducing G2/M phase cell cycle arrest [165].In gastric adenocarcinoma AGS cells, geraniol showed cytotoxicity by inhibiting the JNK/ERK signaling pathway [166].Lavender essential oil, its active compounds linalool and linalyl acetate [167], and ethyl acetate fraction of Ajwa dates [168] also inhibited PC-3 cell proliferation by increasing apoptosis and cell cycle arrest.Linalool also reduced tumor growth in PC-3 xenograft mice [167] and the 22Rv1 xenograft mice model [169].Ethyl acetate exerted an anti-proliferative activity on human breast cancer MCF7 and SKBR3 cells [170] and human cervical cancer HeLa cells [171].α-terpineol [172] and linalool [173] also showed strong cytotoxicity on HeLa cells with apoptosis and cell cycle arrest.In human acute myeloid leukemia U937 cells [173], human oral cancer cells [174,175], and lung adenocarcinoma A549 cells [176], linalool significantly suppressed the cell growth.D-limonene [177] and d-limonene-rich blood orange volatile oils [178] inhibited the proliferation of lung cancer A549 and H1299 cells and human colon adenocarcinoma cells SW480 and HT-29 cells, respectively.Limonene (9 µM) significantly reduced the proliferation of human bladder cancer cell T24 after 24 h, showing induced apoptosis with increased G2/M cell cycle arrest and apoptotic markers (Bax, and cleaved caspase-3, 8, and 9) [179].In Yu et al., d-limonene induced apoptosis and autophagy-related genes in a lung cancer model [177].In an acetic acid-induced gastric ulcer rat model, 7-day oral administration of (-)-myrtenol at 50-100 mg/kg increased the healing of the ulcer [180].Myrcene640 µM) significantly decreased the proliferation of SCC9 oral cancer cells after 24 h [181].Myrcene also showed increased apoptosis with the concentration of 5-20 µM and significantly suppressed the migration of SCC9 cells at 10 µM myrcene treatment.The effects of volatile compounds rich in berries on cancer models were summarized in Table 3.

Obesity
A high-fat diet for obese humans and animals can increase endothelial dysfunction.It can lead to many other severe cardiovascular diseases and metabolic disorders.In Wang et al., geraniol was examined for the effect on endothelial function in high-fat diet (HFD)-fed mice [182].Forty mice were fed HFD for 8 weeks, while 20 mice had a normal diet.Then, HFD-fed mice were randomly assigned to intraperitoneal geraniol treatment (20 mice) or vehicle treatment (20 mice) group for 6 weeks.As a result, geraniol protected and improved HFD-induced endothelial dysfunction in HFD-fed mice by reducing aortic NADPH oxidases and ROS production.In Sousa et al., α-terpineol enantiomers were exam-ined for their effect on the biological markers in HFD-induced obese rats [183].Six weeks of daily α-terpineol supplementation (50-100 mg/kg of diet) suppressed pro-inflammatory cytokines (TNF-α and IL-1β), serum thiobarbituric acid reactive substances (TBARS), and recovered insulin sensibility.In Li et al., Microcapsules of d-limonene-rich sweet orange essential oil (SOEO) were orally administered to HFD-induced obese rats for 15 days [184].SOEO microcapsules decreased the body weight in obese rats by protecting the gut barrier, increasing Bifidobacterium, and reducing low-grade inflammation.While white adipocytes store the excessive energy in triglyceride forms, brown adipocytes burn the calories in heat form by non-shivering thermogenesis [185].Lone and Yun showed that limonene increased 3T3-L1 adipocytes browning through the activation of the β3-adenergenic receptor and ERK signaling pathway [185].In Ayala-Ruiz et al., male Wister rats (n = 6 per group) were administered with control, high-fat-sucrose diet (HFSD) and 0.6 mL of corn oil, HFSD with 1,8-cineole (0.88 mg/kg), limonene (0.43 mg/kg), α-terpineol (0.32 mg/kg), or the mixture of three terpenes per gavage for 15 weeks [186].Rats fed with HFSD with terpenes significantly reduced weight gain compared to the ones with only HFSD.In addition, all terpenes suppressed the fat deposition, serum glucose levels, and triacylglycerol levels.The effects of volatile compounds rich in berries against obesity were summarized (Table 4).[186] IL-1β = interleukin-1β; NADPH = nicotinamide adenine dinucleotide phosphate; ROS = reactive oxidative stress; TBARS = thiobarbituric acid reactive substances; TNF-α = tumor necrosis factor-α; ↑ = increase; ↓ = decrease.

Diabetes
In Bacanlı et al., streptozotocin (STZ) (60 mg/kg) was injected into Wistar rats to induce type 1 diabetes [187].Diabetic rats were orally treated with d-limonene (50 mg/kg body weight) for 28 days.D-limonene treatment significantly reduced DNA damage and induced the level of antioxidant enzymes (catalase, superoxide dismutase, and total glutathione).D-limonene also altered hepatic enzyme and lipid profile, suggesting the potential of d-limonene being protective against diabetes in the liver and kidney in rats.D-limonene showed potential antihyperglycemic activities [188,189] and reduced lipid peroxidation with increased antioxidant activity [190].In El-Bassossy et al., geraniol (150 mg/kg) was orally treated in STZ-induced obese rats for 7 weeks [191].Geraniol significantly reduced systolic cardiac function related to diabetes by alleviating oxidative stress.Geraniol treatment also reduced GLUT 2 transporter [192], hyperglycemia [193], diabetic nephropathy [194], and improved impaired vascular reactivity [195] in STZ-induced diabetic rats.Linalool also exerted a reduction in fasting blood glucose level, insulin resistance, glycation oxidative stress [196], and nephropathic changes in kidneys [197] on STZ-induced diabetic rats.Xuemei et al. investigated the effect of myrtenol on STZ-induced gestational diabetes mellitus (GDM) in rats [198].GDM is diabetes that occurs only during pregnancy.Twenty-five mg/kg of STZ was injected into the pregnant rats to induce GDM.Myrtenol (50 mg/kg) was orally administered for 2 weeks.Myrtenol oral administration helped decrease blood glucose levels and pro-inflammatory markers.It also increased high-density lipoprotein (HDL) and antioxidant status in diabetic pregnant rats.The effects of volatile compounds rich in berries against diabetes were summarized in Table 5.

Conclusions
As berry consumption through the fruit and products, including berry-flavored water, juice, and others, have rapidly increased, many studies about monitoring and improving overall berry quality, including flavor, aroma, appearance, shelf-life, and safety, were conducted to target consumer acceptability.However, the beneficial health effects of berry volatiles have not been extensively studied.In this article, we looked into the biosynthesis of plant volatiles, volatile composition, and possible bioavailability and health benefits of some berry volatiles were reviewed.Major terpene volatiles were synthesized via MVA and MEP pathways.Major chemical classes in berries were esters, alcohols, terpenoids, aldehydes, ketones, and lactones.Berries had different profiles of volatiles, but monoterpene showed a crucial role in characterizing the unique berry aroma in all five berries.Volatile compounds were nonpolar and hydrophobic and rapidly absorbed and eliminated from our body after administration.Among them, monoterpenes, including linalool, limonene, and geraniol, showed many health benefits associated with inflammation, cancer, obesity, and diabetes in vitro and in vivo, suggesting potential health beneficial effects of berry volatiles.More research on animal and human models of the health benefits of berry volatiles and bioavailability would be needed to confirm their bioactivities.

Table 1 .
The major volatile compounds in berries.

Table 2 .
The effect of volatile compounds rich in berries on inflammation models.

Table 3 .
The effect of volatile compounds rich in berries on cancer models.

Table 4 .
The effect of volatile compounds rich in berries on obesity models.

Table 5 .
The effect of volatile compounds rich in berries on diabete models.