ARTICLE   Open Access    

Metabolome provides new insights into the volatile substances in 'Ruidu Kemei' grapes under the two-crop-a-year cultivation system

  • # Authors contributed equally: Huan Yu, Rongrong Guo

More Information
  • Received: 29 May 2024
    Revised: 11 July 2024
    Accepted: 31 July 2024
    Published online: 04 November 2024
    Fruit Research  4 Article number: e035 (2024)  |  Cite this article
  • In subtropical regions, the implementation of a two-crop-a-year cultivation system depends on local climatic conditions. Grape volatile compounds vary greatly with the season, due to climate differences, which lead to extreme differences between summer grape fruits (SF) and winter grape fruits (WF). In the present study, a gas chromatography-mass spectrometer (GC-MS) was used to analyze volatile compounds from 'Ruidu Kemei' grapes grown under the two-crop-a-year cultivation system. Results showed that fruits in summer and winter contained 620 volatile compounds in 15 categories. Among them, terpenoids constituted the largest group, with 122 metabolites, followed by 115 esters. This indicated that the main volatile characteristic substances of 'Ruidu Kemei' were terpenoids and esters. Higher volatile compounds in SF might be associated with higher active accumulated temperatures in the summer growing season. In addition, terpenoids, heterocyclic compounds, esters, and aromatics showed greater differences than other compounds between SF and WF. Regarding terpenoids, WF exhibited superior performance, while SF performed better in esters and aromatics. For WF, higher solar radiation intensity promoted the biosynthesis of terpenoids, which lead to more floral characteristics than SF. According to the flavor omics analysis, 'Ruidu Kemei' was primarily characterized by green, fruity, herbal, woody, sweet, floral, fresh, fatty, citrus, and earthy. In the SF, green and fruity flavors were more prominent, while floral was the dominant fruity aroma in WF. This work provides new insights into the metabolism of volatiles in summer and winter grapes and reference for the selection and promotion of varieties with suitable aromas for a two-crop-a-year cultivation system.
  • Huangqin tea (HQT, Fig. 1), also called Huangjin tea in Chinese, has a long history of consumption in China[1]. It is a healthy herbal tea crafted from the aerial parts of Scutellaria baicalensis Georgi, S. scordifolia Fisch, S. amoena C. H. Wrigh, and S. viscidula Bung[24]. Unlike traditional tea made from Camellia sinensis (L.) O. Kuntze, HQT lacks stimulating components and is renowned for clearing heat and dry dampness, eliminating toxins, promoting digestion, and soothing fire[1,5]. It can be enjoyed both as a hot beverage and as a cold drink, and modern research suggests that HQT exhibits several pharmacological properties, such as anti-inflammatory, chemopreventive effects of colorectal cancer[6], anti-aging[7], cardiovascular protection, hypoglycemic effect, hypolipidemic effect, anti-tumor, anti-bacterial, anti-influenza virus, and enhance human resistance[812]. The predominant compounds found in HQT are flavonoids and essential oils and its potential health benefits are likely attributed to the presence of distinctive flavonoids, including isocarthamidin-7-O-β-D-glucuronide, carthamidin-7-O-β-D-glucuronide, apigenin-7-O-β-D-glucopyranside, chrysin-7-O-β-D-glucuronide, scutellarin, baicalin, wogonoside, and chrysin[1318].

    Figure 1.  The aerial parts of S. baicalensis Georgi, dried and brewed Huangqin tea.

    HQT is made using simple ingredients and has a straightforward preparation method. Traditionally, the above-ground portions of Scutellaria species have been utilized for crafting HQT, with the differentiation between stems and leaves remaining unessential. During the hot summer season (July to August), the aerial parts (stem, leaves, flowers) of these species of Scutellaria from a single source are collected and cut into small sections, which are then directly dried for later use. Alternatively, freshly harvested branch and leaf sections may undergo a transformative process through multiple rounds of steaming and subsequent drying within a steamer. Once suitably conditioned, they find their place within sealed containers, poised for extended storage periods. The brewing ritual commences by incorporating 7−8 g of this tea into 2 L of water, with subsequent replenishments of water at intervals 2−3 times. As the brewing ritual concludes, HQT graces the senses with a resplendent golden infusion—earning it another name, 'Huangjin tea'.

    HQT boasts a range of health advantages and is frequently gathered from the upper sections of Scutellaria species plants cultivated in the vicinity, intended for tea consumption. In recent years, large planting bases have been established in several regions, such as Beijing, Inner Mongolia Wuchuan, Yakeshi, Hebei Chengde, Shanxi Province, and Shandong Province, to comprehensively develop S. baicalensis resources. HQT has gained more attention and is now produced by specialized operating companies in various grades, such as bulk tea, bagged tea, and unique tea culture[12].

    To provide up-to-date information on the chemical composition, bioactivity, and safety aspects of the Scutellaria genus from HQT, this review paper has collected related literature from various databases, such as PubMed, Web of Science, Sci Finder, Scopus, Baidu Scholar, China National Knowledge Internet (CNKI), Wanfang and Weipu Data. This paper aims to draw attention to the need for further research and application of HQT in preventing and managing certain chronic diseases.

    HQT can be derived from several Scutellaria species. S. baicalensis is the most extensively cultivated, with a significant biomass in its aerial parts. Consequently, the primary source of HQT is the aerial part of S. baicalensis, and research focused on these aerial parts is also the most extensive.

    The aerial part and the root of S. baicalensis share similarities in their primary constituents, notably the significant presence of flavonoids, which are regarded as the main active components. However, there are specific chemical differences between the above-ground and underground parts of S. baicalensis. Our preliminary experiments have revealed the disparities in chemical composition between these two parts[18]. Notably, the root of S. baicalensis finds its primary use in medicinal applications, characterized by notably higher expression levels of specific 4′-deoxyflavones compared to the aerial organs. In contrast, the above-ground parts of the plant are employed to prepare tea. This differentiation in utilizing these plant components reflects the accumulated wisdom of generations cultivated through extensive periods of tasting and experience.

    This section provided an in-depth review of the chemical composition of the plant from which HQT is derived. Additionally, a specific emphasis on reviewing the aerial parts of S. baicalensis, which serve as the primary source of HQT has been placed.

    In the 1970s, researchers began to study the chemical composition of the aerial parts of S. baicalensis. The flavonoids are the most abundant chemical components in the aerial parts (flowers, stems, and leaves), and about 54 flavonoid compounds were identified (Table 1, Fig. 2). Most of them are flavonoids, dihydro flavonoids, and glycosides. The main glycoside-forming sugars are arabinose, glucose, and glucuronide, with the most abundant glucuronide glycosides. The optimization of extraction processes can yield a remarkable total flavonoid content of up to 5% in the stems and leaves of S. baicalensis[19].

    Table 1.  Flavonoids of HQT.
    NumberNameFormulaSpeciesRef.
    12',5-Dihydroxy-3',6,7,8-tetramethoxyflavoneC19H18O8S. baicalensis[17]
    22',6-DihydroxyflavoneC15H10O4S. baicalensis[16]
    33',4',5,5',7-PentamethoxyflavoneC20H20O7S. baicalensis[16]
    44',5-Dihydroxy-3',5',6,7-tetramethoxyflavoneC19H18O8S. baicalensis[22,27]
    54',5-Dihydroxy-7-methoxyflavanoneC16H14O5S. baicalensis[16]
    65,4′-Dihydroxy-6,7,8,3′-tetramethoxyflavoneC19H18O8S. baicalensis[17]
    75,2′-Dihydroxy-6,7,8-trimethoxyflavoneC18H16O7S. baicalensis[17]
    85,2′-Dihydroxy-6,7,8,3′-tetramethoxyflavoneC19H18O8S. baicalensis[17]
    95,2′-Dihydroxy-7,8-dimethoxyflavoneC17H14O6S. baicalensis[17]
    105,2′-Dihydroxy-7,8,6′-trimethoxyflavoneC18H16O7S. baicalensis[17]
    115,6,7,3',4'-Pentahydroxyflavone-7-O-glucuronideC21H20O13S. baicalensis[13]
    125,6,7,4'-TetrahydroxydihydroflavoneC15H12O6S. baicalensis[17]
    135,6,7-Trihydroxy-4'-methoxyflavoneC16H12O6S. baicalensis[15]
    145,7-Dihydroxy-6-methoxyflavanonC16H14O5S. baicalensis[17]
    155,7,4′-Trihydroxy-6-methoxyflavanoneC16H14O6S. baicalensis[22]
    165,7,4'-TrihydroxyflavanoneC15H12O6S. baicalensis[13]
    17(2S)-5,7,8,4'-Tetrahydroxyflavanone 7-O-β-D-glucuronopyranosideC21H20O12S. baicalensis[21]
    18(2S)-5,6,7,4'-Tetrahydroxyflavanone 7-O-β-D-glucuronopyranosideC21H20O12S. baicalensis[21]
    196-Hydroxyluteolin-7-O-glucuronideC21H18O13S. baicalensis[13]
    207-MethoxychrysinC16H14O4S. baicalensis[16]
    21ApigeninC15H10O5S. baicalensis, S. amoena,
    S. scordifolia, S. viscidula
    [17,2125]
    22Apigenin-4'-glucopyransideC21H20O10S. baicalensis[17]
    23Apigenin-6-C-glucoside-8-C-arabinosideC26H28O14S. baicalensis[13]
    24Apigenin-7-O-β-D-glucopyransideC21H20O10S. baicalensis[17]
    25Apigenin-7-O-β-D-glucuronideC21H18O11S. baicalensis, S. amoena,
    S. scordifolia
    [13,23,24]
    26Apigenin-7-O-methylglucuronideC22H20O11S. baicalensis[28]
    27BaicaleinC15H10O5S. baicalensis, S. amoena,
    S. scordifolia, S. viscidula
    [2225]
    28Baicalein-7-O-D-glucopyransideC21H20O10S. baicalensis[22]
    29BaicalinC21H18O11S. baicalensis, S. amoena,
    S. scordifolia, S. viscidula
    [16,2325]
    30CarthamidinC15H12O6S. baicalensis[13,20]
    31Carthamidin-7-O-β-D-glucuronideC21H20O12S. baicalensis[22]
    32ChrysinC15H10O4S. baicalensis, S. amoena,
    S. scordifolia
    [13,2124]
    33Chrysin-7-O-β-D-glucuronideC21H20O9S. baicalensis, S. amoena[23,29]
    34DihydrobaicalinC21H20O11S. baicalensis[22,28]
    35Dihydrooroxylin AC21H20O11S. baicalensis[13]
    36GenkwaninC16H12O5S. baicalensis[16]
    37IsocarthamidinC15H10O6S. baicalensis[20,28]
    38Isocarthamidin-7-O-β-D-glucuronideC21H20O12S. baicalensis[28]
    39IsoschaftsideC26H28O14S. baicalensis[13,16]
    40IsoscutellareinC15H10O6S. baicalensis[21,30]
    41Isoscutellarein 8-O-β-D-glucuronideC21H18O12S. baicalensis[21]
    42Kaempferol 3-O-β-D-glucopyranosideC21H20O11S. baicalensis[13,28]
    43LuteolinC15H10O6S. baicalensis[22,30]
    44Norwogonin-7-O-glucuronideC21H18O12S. baicalensis, S. amoena[13,17,23]
    45Oroxylin AC16H12O5S. baicalensis, S. amoena[17,23]
    46Oroxylin A-7-O-D-glucopyransideC22H22O10S. baicalensis[22,28]
    47Oroxylin A-7-O-β-D-glucuronideC22H20O11S. baicalensis, S. amoena[13,23]
    48PinocembrinC16H12O5S. baicalensis[13]
    49Pinocembrin-7-O-glucuronideC21H20O11S. baicalensis[13,16,22]
    50SalvigeninC18H16O6S. baicalensis[21]
    51ScutellareinC15H10O6S. baicalensis[28]
    52ScutellarinC21H18O12S. baicalensis, S. amoena,
    S. scordifolia, S. viscidula
    [2325,29]
    53WogoninC16H12O5S. baicalensis, S. amoena,
    S. scordifolia, S. viscidula
    [2125]
    54WogonosideC22H20O11S. baicalensis, S. viscidula[13,25]
     | Show Table
    DownLoad: CSV
    Figure 2.  Chemical structure of flavonoids in HQT.

    In 1976, Takido et al.[20] isolated two flavanone derivatives, carthamidin and isocarthamidin, for the first time as natural products from the leaves of S. baicalensis. Later, Yukinori et al.[21] identified two new flavanones, (2S)-5,7,8,4'-tetrahydroxyflavanone 7-O-β-D-glucuronopyranoside and (2S)-5,6,7,4'-tetrahydroxyflavanone 7-O-β-D-glucuronopyranoside), in the leaves of S. baicalensis. Eight compounds of chrysin, wogonin, apigenin, salvigenin, scutellarein, isoscutellarein, apigenin 7-O-glucuronide, and isoscutellarein 8-O-glucuronide were also isolated. Wang et al.[15] used column chromatography to isolate seven flavonoids (wogonin, chrysin, 5,6,7-trihydroxy-4'-methoxyflavone, carthamindin, isocarthamidin, scutellarein, and chrysin 7-O-β-D-glucuronide) from a water extract of the leaves of S. baicalensis. Liu et al.[13] identified 21 flavonoids in the stems and leaves of S. baicalensis by HPLC-UV/MS and NMR, and found one flavonone (5,6,7,3',4'-Pentahydroxyflavone-7-O-glucuronide) was a new compound. Zhao[17] firstly isolated 5,6,7,4′-tetrahydroxyflavanone 7,5,7-dihydroxy-6-methoxyflavanone, oroxylin A, 5,4′-dihydroxy-6,7,8,3′-tetramethoxyflavone, 5,2′-dihydroxy-6,7,8,3′-tetramethoxyflavone, 5,2′-dihydroxy-7,8,6′-trimethoxyflavone, 5,2′-dihydroxy-7,8-dimethoxyflavone, 5,2′-dihydroxy-6,7,8-trimethoxyflavone, apigenin 4'-β-D-glucopyranoside, and apigenin-7-β-D-glucopyranoside from the aerial parts of S. baicalensis. Ma[22] firstly isolated 5,7,4'-trihydroxy-6-methoxyflavone, 5,4'-dihydroxy-6,7,3',5'-tetramethoxyflavone, from stems and leaves of S. baicalensis. Wang et al.[16] isolated 5,4'-dihydroxy-7-methoxyflavanone, genkwanin, 7-methoxychrysin, 3',4',5,5',7-pentamethoxyflavone from 60% ethanol extracts for stems and leaves of S. baicalensis for the first time. Also, the compounds of carthamidin-7-O-β-D-glucuronide, oroxylin A-7-O-β-D--glucuronide, and chrysin were isolated from this plant for the first time.

    The concentration of these chemical components in HQT varies depending on the plant part utilized. Employing the HPLC-DAD method, Shen et al.[18] established that the aerial parts (stems, leaves, and flowers) of S. baicalensis are rich in flavonoids, resembling the roots in composition but exhibiting significant disparities in content. The contents of isocarthamidin-7-O-β-D-glucuronide (106.66 ± 22.68 mg/g), carthamidin-7-O-β-D-glucuronide (19.82 ± 11.17 mg/g), and isoscutellarein-8-O-β-D-glucuronide (3.10 ±1.73 mg/g) were the highest in leaves. The content of apigenin-7-O-β-D-glucopyranoside (18.1 ± 4.85 mg/g) and chrysin-7-O-β-D-glucuronide (9.82 ± 5.51 mg/g) were the highest in flowers. HQT has a high content proportion of flavone glycosides, which is closely related to the activity of HQT. The concentrations of the nine main flavonoids in HQT infusions were measured using HPLC. The content of isocarthamidin-7-O-β-D-glucuronide (52.19 ± 29.81 mg/g) was the highest; carthamidin-7-O-β-D-glucuronide (31.48 ± 6.82 mg/g), chrysin-7-O-β-D-glucuronide (10.65 ± 0.40 mg/g) and apigenin-7-O-β-D-glucopyranside (5.39 ± 0.92 mg/g) were found at moderate levels in HQT samples. As for flavone aglycones, scutellarin (12.77 ± 1.14 mg/g), baicalin (1.88 ± 0.48 mg/g), isoscutellarein-8-O-β-D-glucuronide (2.84 ± 0.60 mg/g), wogonoside (0.23 ± 0.02 mg/g) and chrysin (0.03 ± 0.01 mg/g) has lower content in HQT[6].

    Although there are few studies on the chemical constituents of the aerial parts of S. amoena, S. scordifolia, and S. viscidula it has been shown that the compounds of the aerial parts are similar to S. baicalensis. The aerial parts of S. amoena contain the compounds of baicalein, baicalin, oroxylin A, oroxylin A-7-O-β-D-glucuronide, wogonin, chrysin, chrysin-7-O-β-D-glucuronide, norwogonin, 5,7-dihydroxy-6,8-dimethoxyflavone, scutellarin[23]. Zhang et al.[24] identified compounds of chrysin, wogonin, baicalein, apigenin, apigenin-7-O-β-D-glucoside, baicalin, and scutellarin in whole plants of S. scordifolia. The stems and leaves of S. viscidula all contain compounds of wogonoside, apigenin, baicalein, wogonin, baicalin, and scutellarin. The contents of baicalein, wogonoside, wogonin, and apigenin in the stem of S. viscidula were higher than those in the stem of S. baicalensis. In the leaves of the two species, the content of scutellarin was higher, while the content of other compounds was lower[25]. The content of scutellarin in S. viscidula was stem (2.30%) > leaf (1.78%) > flowers (0.38%)[26].

    The aerial parts of S. baicalensis are rich in essential oils, and the taste of HQT is closely related to this. The flowers of S. baicalensis are thought to have a Concord grape aroma, while HQT has a bitter flavor with distinctive herbal notes. Extensive analysis has identified 145 components in the essential oil obtained from the aerial parts of S. baicalensis. These components span various chemical classes, such as alkanes, carboxylic acids, fatty acids, monoterpenes/oxygenated monoterpenes, sesquiterpenes triterpenoids and Vitamins (Supplemental Table S1), which have demonstrated their efficacy in combatting bacteria, reducing inflammation and inhibiting tumor growth[2731]. Among these, major constituents include germacrene D (5.4%−39.3%), β-caryophyllene (29.0%), caryophyllene (18.9%), eugenol (18.4%), caryophyllene (15.2%), caryophyllene oxide (13.9%), (E)-β-caryophyllene (11.6%), 5-en-3-stigmasterol (11.3%), carvacrol (9.3%), thymol (7.5%), vitamin E (7.4%), neophytadiene (7.3%), γ-elemene (6.2%), 1-octen-3-ol (6.1%), allyl alcohol (5.5%), bicyclogermacrene (4.8%), myristicin (4.7%), acetophenone (4.6%), α-amyrin (4.6%), β-amyrin (4.4%), germacrene d-4-ol (4.3%), spathulenol (4.2%), β-pinene (4.1%), α-humulen (4.0%), 1-vinyl-1-methyl-2-(1-methylvinyl)-4-(1-methylethylidene)-cyclohexane (4.0%) are found in the aerial parts of S. baicalensis from different places[2731].

    Takeoka et al.[27] identified 64 components in volatile components of S. baicalensis flowers by solid-phase microextraction and analyzed them by GC and GC-MS. These flowers were collected at San Francisco State University (USA). Among the flower volatiles, the content of β-caryophyllene, germacrene D, δ-cadinene, γ-muurolene, and γ-cadinene were more than 3%. The essential oil obtained from the stem of S. baicalensis is mainly composed of diphenylamine, 2,2-methylenebis (6-tert-butyl-4-methylphenol), bornyl acetate, β-caryophyllene, germacrene D and 1-octen-3-ol.[32]. Gong et al.[28] analyzed and identified the specific chemical constituents of the aerial parts of S. baicalensis by using GC-MS technology and identified 37 compounds in total, such as allyl alcohol, acetophenone, caryophyllene, α-humulene, germacrene D, and γ-elemene. The plant material was collected in the Qinling Mountains in China. Lu et al.[29] found a big difference in essential oil components between the aerial and root of S. baicalensis from Kunming Botanical Garden, Yunnan Province (China). The aerial part of S. baicalensis mainly contained enols and sterols such as neophytadiene and vitamin E. However, it has the same compounds as the roots, such as nerolidol, hexadecanoic acid, 1,2-benzenedicarboxylic acid, squalene, stigmast-4-en-3-one, and partial alkanes. Recently, Wang et al.[31] found the essential oil level of the aerial parts of S. baicalensis was 0.09% (v/w, based on fresh weight) while its density was 0.93 g/mL, and obtained 31 components accounting for 97.64% of the crude essential oil, including sesquiterpenoid, monoterpenoids, phenylpropanoids, and others. It is also reported that the major components of the essential oil from the aerial parts of S. baicalensis were myristicin, eugenol, caryophyllene, caryophyllene oxide, germacrene D, spathulenol, and β-pinene, with eugenol as the most abundant. The sample of the aerial parts were harvested from Tangshan City (China). The composition of S. baicalensis essential oils varies according to the plant part used, geographical location, and growing conditions.

    Zgórka & Hajnos[33] identified the phenolic acid compounds of aerial parts of S. baicalensis by solid-phase extraction and high-speed countercurrent chromatography: p-coumaric acid, ferulic acid, p-hydroxybenzoic acid, and caffeic acid. Chirikova & Olennikov[34] found that the aerial part of S. baicalensis contains 11 kinds of saturated fatty acids and nine kinds of unsaturated fatty acids, among which the palmitic acid content is the highest. Chlorogenic acid, fernlic acid, protocatechuic acid, vanillic acid, rosmarinic acid, caffeic acid, p-hydroxybenzoic acid, and p-coumaric acid were also detected.

    Zhao [17] isolated four sterol compounds: β-sitosterol-3-O-β-D-glucoside, α-apinasterol, β-sitosterol, and four ester compounds: methoxyphaeophorbide, p-hydroxybenethyl ethanol hexadecanoic methyl ester, ethoxyphaeophorbide, and n-octadecanol, lutein from the aerial parts of S. baicalensis.

    It is reported that flavonoids and diterpenes are the two main groups of active constituents in the genus Scutellaria. However, only one diterpene (scutebaicalin) was identified in the stems and leaves of S. baicalensis[35].

    By atomic absorption spectrophotometry, Yuan et al.[36] determined the contents of 11 metal elements in different parts of S. baicalensis. It was found that the leaves and stems of S. baicalensis were rich in Mg, K, Cr, Ni, Co, Fe, Mn, and Pb. Meanwhile, Yan et al.[37] developed an inductively coupled plasma mass spectrometry method and determined 23 kinds of inorganic elements in the stems and leaves of S. baicalensis from eight regions. Although there were no differences in the types of inorganic elements in the stems and leaves of S. baicalensis from the different areas, the content of these elements varied significantly. Among these elements, Fe, Zn, Cu, Mn, Cr, Co, Ni, Sr, B, and Ni were essential human body elements. The content of Al (516.83 μg/g) and Fe (700.62 μg/g) was the highest, while the content of B (31.54 μg/g), Ti (23.10 μg/g), Mn (65.64 μg/g), Sr (62.27 μg/g), and Ba (89.68 μg/g) was relatively high.

    Olennikov et al.[38] studied the water-soluble polysaccharides from the aerial parts of S. baicalensis from Russia and found that the polysaccharides from S. baicalensis gradually accumulated before flowering and progressively decreased after flowering.

    Yan et al.[39] found that the stems and leaves of S. baicalensis were rich in amino acids, and there was no difference in the kinds of amino acids among different producing areas, but there was a significant difference in the contents of amino acids. The content of proline, threonine, glutamic acid, lysine, glutamine, and arginine was higher, and the content of methionine, hydroxyproline, and citrulline was low.

    Several studies have focused on the functional properties of HQT, with increasing attention given to the aerial parts of S. baicalensis as the main raw material for HQT production. S. baicalensis stems and leaves flavonoids (SSF) are considered the functional components of HQT. Modern pharmacology has shown that the flavonoids extracted from the stem and leaf of S. baicalensis have been found to possess anti-inflammatory, anti-bacterial, antiviral, antipyretic and analgesic, anti-tumor, hepatoprotective, antioxidant, hypoglycemic, hypolipidemic, detoxification, myocardial ischemia protection, brain injury protection, and immunomodulatory effects. However, few studies have been conducted on the individual flavonoid compounds in the total flavonoid extract from S. baicalensis. Therefore, this paper aims to summarize and supplement the current research on the functional properties of HQT and its primary raw material (S. baicalensis) extract.

    Injury and infection could lead to inflammation, which plays a key role in the accelerated pathogenesis of immune-mediated disease[40]. Tong et al.[41], Zhou et al.[42], and Zhao et al.[43] found that S. baicalensis stem-leaf total flavonoid (SSTF) could inhibit acute exudative inflammation caused by xylene, glacial acetic acid, and egg white and also have a significant inhibitory effect on chronic inflammatory of granulation tissue hyperplasia. Wang et al.[44] observed the effect of SSTF on the aerocyst synovitis of the rat model and found that it could reduce capillary permeability, reduce the aggregation of neutrophils and basophils in tissues, reduce histamine, bradykinin, and other substances that increase vascular permeability, which is conducive to the recovery of vascular permeability in inflammation. Studies have shown that the SSTF significantly inhibits specific and non-specific inflammatory responses and can regulate the body's cellular and humoral immune functions. The mechanism of action is closely related to the effective reduction of capillary permeability, inhibition of PGE2 and NO synthesis in vivo, reduction of TNF-α expression, and reduction of inflammatory exudation[45,46]. SSTF (200 mg/kg) could balance the CD+4 Tlymphocyte subsets Th1/Th2 cells and the related cytokines IL-10 and IFN-γ in the rheumatoid arthritis model[47]. SSTF (17.5, 35 and 70 mg/kg for 38 d) could significantly improve the impairment of relearning ability and retention ability on memory impairment and nerve inflammation in chronic cerebral ischemia rats, which might be due to the inhibition of the proliferation of astrocyte and balanced the expression of the inflammatory factors in the brain[48]. Besides, the extract of S. baicalensis stem-leaf shows anti-inflammation effects both in vitro and in vivo. In cultured macrophage cells (RAW 264.7), the extract of S. baicalensis stem-leaf showed a strong anti-inflammation effect, which inhibited the expression of IL-1β. Similarly, it suppressed the LPS-induced transcriptional activity mediated by NF-κB in fish aquaculture[49]. S. baicalensis stems and leaves (3, 6, and 12g /kg, gavage for 7 d, once per day) have anti-inflammatory effects on 2% carrageenan-induced acute pleurisy in rats, and the mechanism of action may be related to the reduction of the production of inflammatory factors and the down-regulation of TRPV1 signaling protein[50]. The combination of S. baicalensis stems- Polygonum cuspidatum (3.5, 7, and 14 g/kg, gavage for 7 d) has a protective effect on lipopolysaccharide-induced acute lung injury rats, and its mechanism may be related to down-regulating the expression of TRPV1 and inhibiting the levels of TNF-α and IL-1β in inflammatory cells[50]. It is also reported that S. baicalensis stem-leaf combined with Morus alba (4, 8, and 16 g/kg/d, gavage, 10 d) has a protective effect on rats with acute pneumonia induced by lipopolysaccharide, and the mechanism might be related to the reduction of inflammatory factors and the down-regulation of TRPV1 signaling pathway[51]. Besides, the network pharmacology showed that S. baicalensis stem-leaf could prevent and control COVID-19 by intervening in 30 targets and 127 pathways, potentially preventing and treating inflammation caused by COVID-19[52].

    Based on the studies, S. baicalensis stem-leaf extract shows promising anti-inflammatory properties. These effects are likely mediated through a combination of factors, including the modulation of immune responses, reduction of inflammatory mediators, and potential interactions with signaling pathways like TRPV1. However, it's important to note that while these studies provide valuable insights, further research, including clinical trials, is needed to establish the full extent of its benefits and its potential for therapeutic applications in humans.

    Scutellaria baicalensis stem and leaf aqueous extract exhibit different degrees of inhibition of 36 strains from 13 kinds of bacteria, such as Staphylococcus aureus, Staphylococcus, Streptococcus pneumoniae, alpha-hemolytic streptococcus, beta-hemolytic streptococcus and Escherichia coli. This shows that anti-bacterial activity against Staphylococcus aureus is strong (MIC50 0.94 g/L, MBC 0.94 g/L). In vivo (217 mg/kg), it protects against the death of mice infected by Staphylococcus aureus and shows a certain dose-dependence[53]. Zhang et al.[54] found that the stem and leaves of S. baicalensis against Staphylococcus aureus and Shigella dysenteriae with MIC values of 1 and 4 mg/mL, respectively. Besides, it is reported that the water extract of the aerial part of S. baicalensis could inhibit the growth of several common pathogenic bacteria in aquacultures, such as Aeromonas hydrophila, Edwardsiella tarda, Vibrio alginolyticus and V. harveyi[49].

    Zhao et al.[55] found that the active part of the stem and leaf of S. baicalensis could inhibit the cytopathic effect caused by 10 kinds of viruses such as Coxsackie B virus, influenza virus, parainfluenza virus, adenovirus, respiratory syncytial virus, and herpes simplex virus. It is suggested that the active parts of the stem and leaf of S. baicalensis can be used for the prevention and treatment of influenza virus, parainfluenza virus, coxsackievirus, and other related infectious diseases.

    These findings indicate that S. baicalensis stem and leaf extract possess anti-bacterial and antiviral properties, making it a potentially valuable natural resource for combating infections caused by various pathogens. However, while these results are promising, further research, including clinical trials, would be necessary to fully establish the effectiveness and safety of using S. baicalensis extract for preventing or treating bacterial and viral infections, including COVID-19.

    In a series of studies, Tong et al.[41] demonstrated that SSTF at a dosage of 20 mg/kg significantly reduced body temperature in rats with fever induced by subcutaneous injection of a 10% dry yeast suspension. Zhang et al.[56] conducted research on the antipyretic effect of scutellarin, an extract from S. baicalensis stems and leaves, in febrile rabbits and observed an antipyretic substantial impact induced by pyrogen. Yang et al.[57] conducted several animal experiments, where they discovered that intraperitoneal injection of effective doses of SSTF (42.2 and 84.4 mg/kg) exhibited a specific inhibitory effect on infectious fever in experimental animals. Moreover, intraperitoneal injection of appropriate SSTF doses (30.1, 60.3, and 120.6 mg/kg), as found in Yang et al.'s experiments[58], effectively inhibited the pain response in experimental animals. Furthermore, Zhao et al.[59] noted that SSTF exhibited a certain inhibitory effect on the pain response in experimental animals subjected to chemical and thermal stimulation.

    These findings suggest that S. baicalensis stem and leaf extract may have antipyretic and analgesic effects, particularly its total flavonoid component. These effects could be beneficial for managing fever and providing pain relief. However, as with any natural remedy, further research, including controlled clinical trials in humans, is necessary to fully understand the effectiveness, safety, and optimal dosing of S. baicalensis extract for these purposes.

    Amyloid protein (Aβ) has been widely recognized as the initiator of Alzheimer's disease (AD)[60]. The SSTF can improve cognitive function and delay the process of dementia. SSTF has been found to exert neuroprotective effects in AD animal models through various mechanisms. Ye et al.[61] demonstrated that oral administration of SSTF (50 mg/kg) could effectively improve cognitive function and reduce neuronal injury in Aβ25-35-3s -induced memory deficit rats. The underlying mechanisms may involve inhibiting oxidative stress and decreasing gliosis[62,63]. Furthermore, SSTF was shown to reduce Aβ-induced neuronal apoptosis by regulating apoptosis-related proteins Bax and Bcl-2[64]. Subsequent studies further validated the neuroprotective effects of SSTF. Cheng et al.[65] found that SSTF treatment inhibited neuronal apoptosis and modulated mitochondrial apoptosis pathway in composited Aβ rats. Ding & Shang[66] found that the SSF improves neuroprotection and memory impairment in rats due to its inhibition of hyperphosphorylation of multilocus Tau protein in rat brains. SSTF has also been found to exert neurogenesis-promoting effects by regulating BDNF-ERK-CREB signaling[12] and activating the PI3K-AKT-CREB pathway[67].

    In further studies, Ding et al.[11] proposed that the effect of SSF on promoting neurogenesis and improving memory impairment may be related to the regulation of abnormal expression of Grb2, SOS1, Ras, ERK, and BDNF molecules in the BDNF-ERK-CREB signaling pathway. Zhang et al.[68] found that SSF (25, 50, and 100 mg/kg) could significantly modulate okadaic-induced neuronal damage in rats, which provides a basis for evaluating SSF as a means to reduce tau hyperphosphorylation and Aβ expression in Alzheimer's disease. Cao et al.[69] found that SSTF (100 mg/kg, 60 d) may alleviate tau hyperphosphorylation-induced neurotoxicity by coordinating the activity of kinases and phosphatase after a stroke in a vascular dementia rat model. Gao et al.[70] demonstrated that the stems and leaves of S. baicalensis (SSF, 25, 50, and 100 mg/kg/d, 43 d) could inhibit the hyperphosphorylation of tau in rats' cerebral cortex and hippocampus induced by microinjection of okadaic acid, which may be related to the activities of protein kinase CDK5, PKA and GSK3β. Furthermore, Liu et al.[67] demonstrated that SSF (35, 70, and 140 mg/kg/d, 43 d) improved composited Aβ-induced memory impairment and neurogenesis disorder in rats through activated the PI3K-AKT-CREB signaling pathway and up-regulated the mRNA and protein expression of TRKB, PI3K, AKT, CREB and IGF2. More recently, a new study demonstrated that SSF (35, 70, and 140 mg/kg) alleviated myelin sheath degeneration in composited Aβ rats, potentially modulating sphingomyelin metabolism[71]. Collectively, these findings suggest that SSTF holds therapeutic potential for AD by targeting multiple Alzheimer's pathogenesis-related processes.

    Li et al.[72] confirmed that SSTF (5 mg/kg) could improve the behaviors and the numbers of dopaminergic neurons in the substantia nigra in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson's disease in mice, and these beneficial activities appear to be associated with the reduction of the level of serum malondialdehyde.

    These studies suggest that S. baicalensis stem and leaf extract have the potential to exert neuroprotective effects in various neurodegenerative conditions, including Alzheimer's disease and Parkinson's disease. These effects could be attributed to its ability to modulate oxidative stress, apoptosis, signaling pathways, and protein hyperphosphorylation. However, as with any potential therapeutic agent, further research is needed to establish the full extent of its benefits, optimal dosages, and mechanisms of action, as well as its potential applications in human patients.

    In recent years, ischemic cerebrovascular disease has seriously threatened human health. Cerebral ischemia is one of the leading causes of death. It can occur in focal or global ischemia, with most cases associated with ischemic stroke[73]. Neuronal protection against oxidative damage has been proposed as a potential therapeutic strategy to avoid damage during ischemic stroke[74]. It is reported that SSTF can reduce neuronal apoptosis and free radical damage caused by heart and brain ischemia. Zhao et al.[75] have found that the pretreatment of SSTF (100 mg/kg/d) can protect the ischemia-reperfusion myocardium by enhancing the activity of the anti-oxidative enzyme, inhibiting lipid peroxidation and attenuating the oxygen-free radicals-mediated damage to the myocardium in rats. In further studies, Zhao et al.[76] proposed that SSTF (50, 100, or 200 mg/kg/d, 7 d) pretreatment could alleviate the neuronal damage incurred by ischemia-reperfusion, demonstrating a neuroprotective effect in focal ischemia-reperfusion rat model, which may involve the prohibition of the apoptosis of the neurons. Yu et al.[77] confirmed that SSTF (17.5, 35, and 70 mg/kg/d, 7 d) could attenuate cardiomyocyte apoptosis during ischemia reperfusion injury by down-regulating the protein expression of the JAK2 gene. Qin et al.[78] found that SSF (17.5, 35, and 70 mg/kg/d, 38 d) can decrease the expression of the NMDAR in hippocampus, and increase the expression of VEGF in the cerebral cortex of chronic cerebral ischemia rats.

    Focal cerebral ischemia-reperfusion can result in neuronal loss but strongly promotes activation and proliferation of hippocampal glial cells. Losing hippocampal neurons is considered one of the basic pathological mechanisms of cognitive impairment[79]. Zhao et al.[79] found that the pretreatment with SSTF (100 and 200 mg/kg) could improve neurological function after focal cerebral ischemia-reperfusion injury, with preventive and protective effects. Shang et al. found flavonoids from S. baicalensis (35−140 mg/kg) could attenuate neuron injury and improve learning and memory behavior in rats with cerebral ischemia/reperfusion[80]. In further studies, Kong et al.[81] found that the mechanisms of the protective effects on the brain against cerebral ischemia/reperfusion injury of SSTF may involve decreasing the content of brain water, increasing microvascular recanalization, reducing the apoptosis of hippocampal neurons, and attenuating free radical damage. Bai et al.[82] proposed that SSTF (100 mg/kg/d, 7 d) could protect the neurological function in rats following I/R injury by alleviating the damage to the ultrastructure of cerebral cortex neurons and synapse. Yan et al.[83] found that SSTF (100 mg/kg/d, 7 d) pretreatment can exert preventive, protective effects on cerebral tissue by relieving brain edema, decreasing neural damage, promoting microvascular repatency, and increasing enzyme activity. It has been reported that the SSTF may protect neurons and their synaptic structures in multiple ways, but whether this mechanism enhances the resistance of neurons to damage or increases the repair function remains to be further explored.

    Essential hypertension is a common chronic cardiovascular disease, which can lead to multiple target organ damage, such as heart, brain, and blood vessels. It is a risk factor for coronary heart disease, heart failure, and other cardiovascular diseases. It is reported that SSTF (17.5, 35.0, and 70 0 mg/kg, 8 weeks) can inhibit myocardial remodeling in primary hypertensive rats, and the medium dose exerts the best inhibitory effect, and the mechanism may be related to inflammatory response induced by inhibiting the NF-κB signaling pathway[84].

    S. baicalensis stem and leaf extract have the potential to provide neuroprotective effects in conditions related to ischemic cerebrovascular disease and hypertension. Its ability to modulate oxidative stress, inflammation, and apoptotic pathways appears to contribute to its beneficial effects. However, further research is needed to fully understand the mechanisms and optimal usage of SSTF for these therapeutic purposes.

    Aging is associated with the deterioration of physiological function and the decline of cognitive ability[85]. It is reported that the alcohol extracts from roots, stems, leaves, and flowers of S. baicalensis (400 mg/kg, 7 weeks) could regulate the content of differential metabolites in urine samples of D-gal-induced aging-model rats to different degrees and play a certain role in improving the metabolic disorders of aging rats[7]. A further study investigated the anti-aging effects and potential mechanisms of S. baicalensis leaves and flower extract. S. baicalensis leaves (400 and 800 mg/kg, 7 weeks) have an anti-aging effect, which can improve the acquired alopecia, slow response, and other characteristics of aging rats, increase the spontaneous activity of aging rats, and reduce the damage of lipid peroxidation and glycosylation induced by D-galactose[86]. The S. baicalensis flowers extract (400 and 800 mg/kg, 7 weeks) could effectively reverse the cognitive decline and oxidative stress injury and alleviate liver pathological abnormalities in the D -galactose-induced aging rats, which are involved in the glutamine-glutamate metabolic pathway[85].

    S. baicalensis extracts, particularly those from leaves and flowers, may have anti-aging properties by regulating metabolic disorders, improving cognitive function, reducing oxidative stress, and alleviating aging-related physiological abnormalities. However, further research is necessary to fully understand the mechanisms underlying these effects and to determine the potential of these extracts for human applications in addressing age-related issues.

    The SSTF (200 mg/kg d, 35 d) can reduce the joint damage of collagen-induced arthritis mice and balance the CD+4 T lymphocyte subsets Th17 and Treg cells[87]. Besides, on the multiple sclerosis model, the SSTF (100, 200 mg/kg d, 16 d) displayed a protective effect on experimental autoimmune encephalomyelitis rats through a balance of the CD+4 Tlymphocyte subsets Th17 and Treg cells[88]. Zhang et al.[89] have found that SSTF attenuated EAE disease severity, accompanied by enhanced Treg frequency and level of Treg-associated cytokines (IL-10 and TGF-β), as well as downregulated Th17 frequency and expression of Th17-related cytokines (IL-17 and IL-23).

    It is reported that the essential oils from the aerial parts of S. baicalensis showed toxicity against booklice (Liposcelis bostrychophila) with an LC50 of 141.37 μg/cm2. The components of myristate, caryophyllene, eugenol, and caryophyllene oxide displayed dramatic toxicity against the L. bostrychophila, with LC50 values of 290.34, 104.32, 85.75, and 21.13 μg/cm2, respectively[31].

    Guo & Xu[90] have found that SSTF could inhibit the proliferation of Hela cells obviously (p< 0.01). Tang et al.[10] have found that SSTF could play an anti-colon cancer role by up-regulated the expressions of Cleaved Caspase-3 and the ratio of Bax/Bcl-2 (p < 0.05 and 0.01) and significantly down-regulated the expressions of MMP-2 and MMP-9 in HCT116 cells (p < 0.05 and 0.01). Recently, Shen et al.[6] studied the chemopreventive effects of HQT against AOM-induced preneoplastic colonic aberrant crypt foci in rats and found HQT inhibits AOM-induced aberrant crypt foci formation by modulating the gut microbiota composition, inhibiting inflammation and improving metabolomic disorders.

    Cardiovascular disease is one of the most important threats to human health. Hyperlipidemia is a major risk factor for atherosclerosis, which can cause various cardiovascular and cerebrovascular diseases. Different doses of SSTF (50, 100, and 200 mg/kg) can effectively reduce the body weight increase of rats with hypertriglyceridemia, reduce the serum levels of triglyceride(TG), total cholesterol(TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C), which indicate that SSTF has the effect of regulating blood lipid[91].

    Oxidative stress is important in developing tissue damage in several human diseases[92]. The antioxidant capacities of separated organs (flower, leaf, stem, and root) of S. baicalensis were conducted by DPPH, ABTS+, and RP methods, respectively. The results showed that the antioxidant activity of the root (66.9 ± 0.3, 121.6 ± 0.5, and 80.2 ± 0.4 μg/mL) was the highest, followed by the leaf (68.4 ± 1.3, 128.2 ± 2.1, and 135.8 ± 2.0 μg/mL), stem (127.8 ± 3.1, 199.2 ± 1.7, and 208.2 ± 8.3 μg/mL) and flower (129.8 ± 6.3, 285.7 ± 4.7, and 380.3 ± 14.2 μg/mL)[93]. The antioxidant activities of the extracts of the aerial part of S. baicalensis and separated organs were conducted by DPPH assay and reducing power method with the antioxidant ability 7.73–8.83 mg TE/g DW and 51.48–306.09 mg TE/g DW, respectively. The content of total polyphenols and total flavonoids were significantly positively correlated with the reducing power[94]. Liu et al.[95] found that the flavonoids extracted from the stems and leaves of S. baicalensis (SSF, 18.98, 37.36, and 75.92 μg/mL) could protect rat cortical neurons against H2O2-induced oxidative injury in a dose-dependent manner. Cao et al. found that SSTF could alleviate the damage of human umbilical vascular endothelial cells injured by H2O2 and reduce their apoptosis, which may be related to the increasing level of Bcl-2[96].

    Preventive treatment with SSTF (50, 100, and 200 mg/kg) could significantly inhibit the blood glucose increase induced by alloxan in mice, and SSTF treatment could reduce the blood glucose level in diabetic mice. Both the prevention group and the treatment group could increase the activity of serum superoxide dismutase and decrease the content of malondialdehyde[97]. Liu et al.[98] found that SSTF (75 and 150 mg/kg) could significantly reduce blood glucose and blood lipid and improve insulin resistance in type 2 diabetic rats with hyperlipidemia.

    Yang et al.[99] found that SSTF (35 mg/kg/d, 8 weeks) could resist hepatic fibrosis by inhibiting the expression of α-smooth muscle actin in Hepatic Stellate Cells. In vivo, it is reported that SSTF (50, 100, and 200 mg/kg) could significantly reduce alanine transaminase activities in serum, increase the expression of superoxide dismutase and reduce the content of malondialdehyde in acute hepatic injury mice induced by carbon tetrachloride and ethanol[100].

    These findings suggest that S. baicalensis stem and leaf extract hold promise in promoting various aspects of health, including immune modulation, antioxidant activity, anti-tumor effects, cardiovascular health, oxidative stress protection, and more. However, further research, including clinical studies, is necessary to better understand the full therapeutic potential and safety of these effects in human applications.

    Flavonoids are considered the main active components in HQT. As the active substance basis of HQT, the safety of SSTF has also been investigated. After 90 d of oral administration of SSTF (0.5, 1, and 2 g/kg) to rats, no abnormal changes were observed in all indexes, and no delayed toxic reactions or obvious toxic reactions were observed, indicating that the toxicity of SSTF is low[101]. The LD50 value of SSTF was 14.87 g/kg is equivalent to 68.5 times the maximum dose in the pharmacodynamic test of mice, and the experiment confirms the safety of oral administration of the SSTF. Intraperitoneal injection of SST showed certain toxicity in mice, with an LD50 value of 732.11 mg/kg[102]. Liu et al.[103] conducted a systematic safety assessment experiment on the aqueous extract of S. baicalensis stem and leaves based on the China National Standard 'Guidelines for the Safety Evaluation of Food Toxicology (GB15193-2014)'. The results indicated that S. baicalensis stem and leaves are non-toxic, non-teratogenic, and non-mutagenic. Acute toxicity tests in mice revealed a Maximum Tolerated Dose of 15.0 g/kg. A 90-d feeding trial showed no changes in toxicological damage in animals, even at a high dosage of 8.333 g/kg (equivalent to 100 times the recommended human daily intake), suggesting the safety and non-toxicity of consuming S. baicalensis stem and leaves. In addition, HQT has long been used in folklore, and no toxicity has been reported.

    These findings collectively indicate that HQT is generally safe for consumption. However, as with any herbal product, it's important to follow recommended dosages and consult healthcare professionals, especially for individuals with pre-existing health conditions or medications.

    In recent years, HQT, a non-Camellia tea with a long history in China, has attracted attention due to its diverse pharmacological activities. Among various Scutellaria species, S. baicalensis is the most extensively studied and cultivated source for HQT. The aerial parts, including flowers, stems, and leaves, serve as the principal source of HQT preparation. HQT are rich in flavonoids and volatile components with various beneficial effects. To date, about 295 compounds have been identified from HQT, including approximately 54 flavonoid compounds and 145 volatile components identified online. The current research on the activity of HQT primarily focuses on flavonoid compounds, with limited studies on the larger quantity of volatile oil compounds.

    Additionally, the processing and brewing techniques used to prepare HQT may influence the bioactivity of its flavonoid content, although few studies have investigated this. More research is necessary to optimize the processing and brewing techniques to maximize the health benefits of HQT. Comparative studies reveal that the aerial parts of S. baicalensis, while sharing similarities with the roots, contain varying flavonoid compositions. Limited research on S. scordifolia, S. amoena, and S. viscidula, indicates the presence of comparable flavonoid compounds in their aerial parts. Although individual flavonoids like baicalin, wogonin, and scutellarin have demonstrated various therapeutic properties, it is essential to consider the synergistic effects of these compounds when consumed together in the form of tea. These findings contribute to laying the groundwork for quality assessment of HQT and offer insights into potential health benefits.

    HQT is mainly derived from the aerial parts of S. baicalensis. Recent studies have increasingly recognized the pharmacological value of the aerial parts of S. baicalensis. Preliminary pharmacological studies have shown that the aerial parts of S. baicalensis may possess beneficial activities in antioxidant, anti-tumor, antiviral, anti-bacterial, protection of ischemia-reperfusion injured neural function, neuroprotective effects against brain injury, and blood lipid regulation. These findings suggest that the value of using HQT may be attributed to these pharmacological activities. Although HQT is generally safe for consumption, further investigation is required to understand its safety profile, particularly in special populations such as pregnant or lactating women, children, and individuals with pre-existing medical conditions. Additionally, potential interactions between the flavonoids in HQT and conventional medications must be examined to ensure their safe and effective use with pharmaceutical treatments. In addition, although the safe dose of HQT on rodents has been studied, the safe dose for humans has yet to be determined.

    In conclusion, these initial research results support the potential health benefits of HQT and encourage more in-depth studies on its raw materials. Further studies are necessary to elucidate the synergistic effects of the flavonoids in HQT, optimize the processing and brewing techniques for maximum bioactivity, and investigate the safety profile and potential interactions with conventional medications. A comprehensive understanding of HQT will contribute to developing evidence-based recommendations for promoting health and well-being.

    The authors confirm contribution to the paper as follows: Conceptualization and writing: Quan Y, Li Z, Meng X, Li P, Shen J; Figure and table modification: Quan Y, Li Z, Meng X, Li P; review and editing: Wang Y, He C, Shen J. All authors reviewed the results and approved the final version of the manuscript.

    The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

    This research was funded by the Shandong Provincial Natural Science Foundation, China (ZR2022QH147, ZR2022QH165) and the Innovation Team and Talents Cultivation Program of National Administration of Traditional Chinese Medicine (No. ZYYCXTD-D-202005).

  • The authors declare that they have no conflict of interest.

  • Supplemental Table S1 The information of all detected metabolites.
    Supplemental Table S2 49 of the 620 metabolites arnotated to 20 KEGG pathways.
    Supplemental Table S3 143 differential volatile compounds amnotated to 159 sensory flavors.
  • [1]

    Romero I, Vazquez-Hernandez M, Maestro-Gaitan I, Escribano MI, Merodio C, et al. 2020. Table grapes during postharvest storage: a review of the mechanisms implicated in the beneficial effects of treatments applied for quality retention. International Journal of Molecular Sciences 21(23):9320

    doi: 10.3390/ijms21239320

    CrossRef   Google Scholar

    [2]

    Wu Y, Duan S, Zhao L, Gao Z, Luo M, et al. 2016. Aroma characterization based on aromatic series analysis in table grapes. Scientific Reports 6:31116

    doi: 10.1038/srep31116

    CrossRef   Google Scholar

    [3]

    Lu HC, Chen WK, Wang Y, Bai XJ, Cheng G, et al. 2021. Effect of the seasonal climatic variations on the accumulation of fruit volatiles in four grape varieties under the double cropping system. Frontiers in Plant Science 12:809558

    doi: 10.3389/fpls.2021.809558

    CrossRef   Google Scholar

    [4]

    Ubeda C, Cortiella MGI, Villalobos-González L, Gómez C, Pastenes C, et al. 2020. Ripening and storage time effects on the aromatic profile of new table grape cultivars in Chile. Molecules 25:5790

    doi: 10.3390/molecules25245790

    CrossRef   Google Scholar

    [5]

    Xu XQ, Liu B, Zhu BQ, Lan YB, Gao Y, et al. 2015. Differences in volatile profiles of Cabernet Sauvignon grapes grown in two distinct regions of China and their responses to weather conditions. Plant Physiology and Biochemistry 89:123−33

    doi: 10.1016/j.plaphy.2015.02.020

    CrossRef   Google Scholar

    [6]

    El Hadi MAM, Zhang FJ, Wu FF, Zhou CH, Tao J. 2013. Advances in fruit aroma volatile research. Molecules 18(7):8200−29

    doi: 10.3390/molecules18078200

    CrossRef   Google Scholar

    [7]

    Yang C, Wang Y, Liang Z, Fan P, Wu B, et al. 2009. Volatiles of grape berries evaluated at the germplasm level by headspace-SPME with GC-MS. Food Chemistry 114:1106−14

    doi: 10.1016/j.foodchem.2008.10.061

    CrossRef   Google Scholar

    [8]

    Chen H, Zhang Z, Zhang L, Bai S, Ning P, et al. 2024. Comparative analysis of the evolution of green leaf volatiles and aroma in six Vitis vinifera L. cultivars during berry maturation in the Chinese Loess Plateau region. Foods 13(8):1207

    doi: 10.3390/foods13081207

    CrossRef   Google Scholar

    [9]

    Scafidi P, Pisciotta A, Patti D, Pasquale T, Di Rosario L, et al. 2013. Effect of artificial shading on the tannin accumulation and aromatic composition of the Grillo cultivar (Vitis vinifera L.). BMC Plant Biology 13:175

    doi: 10.1186/1471-2229-13-175

    CrossRef   Google Scholar

    [10]

    Sun Q, Zhao Y, Zhu S, Du F, Mao R, et al. 2022. Rain-shelter cultivation affects the accumulation of volatiles in 'Shuijing' grape berries during development. HortScience 57(8):877−88

    doi: 10.21273/HORTSCI16567-22

    CrossRef   Google Scholar

    [11]

    Xie S, Lei Y, Wang Y, Wang X, Ren R, et al. 2019. Influence of continental climates on the volatile profile of Cabernet Sauvignon grapes from five Chinese viticulture regions. Plant Growth Regulation 87:83−92

    doi: 10.1007/s10725-018-0455-8

    CrossRef   Google Scholar

    [12]

    Rodríguez-Lorenzo M, Mauri N, Royo C, Rambla JL, Diretto G, et al. 2023. The flavour of grape colour: anthocyanin content tunes aroma precursor composition by altering the berry microenvironment. Journal of Experimental Botany 74(20):6369−90

    doi: 10.1093/jxb/erad223

    CrossRef   Google Scholar

    [13]

    Mencarelli F, Bellincontro A. 2020. Recent advances in postharvest technology of the wine grape to improve the wine aroma. Journal of the Science of Food and Agriculture 100(14):5046−55

    doi: 10.1002/jsfa.8910

    CrossRef   Google Scholar

    [14]

    Ding S, Su P, Wang D, Chen X, Tang C, et al. 2023. Blue and red light proportion affects growth, nutritional composition, antioxidant properties and volatile compounds of Toona sinensis sprouts. LWT 173:114400

    doi: 10.1016/j.lwt.2022.114400

    CrossRef   Google Scholar

    [15]

    He L, Xu XQ, Wang Y, Chen WK, Sun RZ, et al. 2020. Modulation of volatile compound metabolome and transcriptome in grape berries exposed to sunlight under dry-hot climate. BMC Plant Biology 20:59

    doi: 10.1186/s12870-020-2268-y

    CrossRef   Google Scholar

    [16]

    Pons A, Allamy L, Schüttler A, Rauhut D, Thibon C, et al. 2017. What is the expected impact of climate change on wine aroma compounds and their precursors in grape? OENO One 51:141−46

    doi: 10.20870/oeno-one.2017.51.2.1868

    CrossRef   Google Scholar

    [17]

    González-Barreiro C, Rial-Otero R, Cancho-Grande B, Simal-Gándara J. 2015. Wine aroma compounds in grapes: a critical review. Critical Reviews in Food Science and Nutrition 55(2):202−18

    doi: 10.1080/10408398.2011.650336

    CrossRef   Google Scholar

    [18]

    Reynolds AG, Wardle DA, Dever M. 1996. Vine performance, fruit composition, and wine sensory attributes of gewürztraminer in response to vineyard location and canopy manipulation. American Journal of Enology and Viticulture 47(1):77−92

    doi: 10.5344/ajev.1996.47.1.77

    CrossRef   Google Scholar

    [19]

    Kovalenko Y, Tindjau R, Madilao LL, Castellarin SD. 2021. Regulated deficit irrigation strategies affect the terpene accumulation in Gewürztraminer (Vitis vinifera L.) grapes grown in the Okanagan Valley. Food Chemistry 341:128172

    doi: 10.1016/j.foodchem.2020.128172

    CrossRef   Google Scholar

    [20]

    Leng F, Zhou J, Wang C, Sun L, Zhang Y, et al. 2022. Post-veraison different frequencies of water deficit strategies enhance Reliance grapes quality under root restriction. Food Chemistry 390:133181

    doi: 10.1016/j.foodchem.2022.133181

    CrossRef   Google Scholar

    [21]

    Khalil-Ur-Rehman M, Wang W, Dong Y, Faheem M, Xu Y, et al. 2019. Comparative transcriptomic and proteomic analysis to deeply investigate the role of hydrogen cyanamide in grape bud dormancy. International Journal of Molecular Sciences 20:3528

    doi: 10.3390/ijms20143528

    CrossRef   Google Scholar

    [22]

    Lu G, Zhang K, Que Y, Li Y. 2023. Grapevine double cropping: a magic technology. Frontiers in Plant Science 14:1173985

    doi: 10.3389/fpls.2023.1173985

    CrossRef   Google Scholar

    [23]

    Guo R, Wang B, Cheng G, Lin L, Cao X, et al. 2016. Research advances in regionalization for two-crop-a-year grape cultivation in China. Journal of Southern Agriculture 47:2091−97

    doi: 10.3969/j:issn.2095-1191.2016.12.2091

    CrossRef   Google Scholar

    [24]

    Cheng G, Zhou S, Liu J, Feng Q, Wei R, et al. 2023. Widely targeted metabolomics provides new insights into the flavonoid metabolism in 'Kyoho' grapes under a two-crop-a-year cultivation system. Horticulturae 9(2):154

    doi: 10.3390/horticulturae9020154

    CrossRef   Google Scholar

    [25]

    Li F, Jia J, Song X, Liu X, Zhu X, et al. 2021. Selection of grape varieties suitable for double cropping a year in northern greenhouse. Agricultural Biotechnology 10(5):5−11

    Google Scholar

    [26]

    Ma Y, Gao Z, Du W, Xie F, Ren G, et al. 2023. Integrated metabolomic and transcriptomic analyses reveal that bagging delays ripening of 'Ruidu Kemei' grape berries. Scientia Horticulturae 317:112058

    doi: 10.1016/j.scienta.2023.112058

    CrossRef   Google Scholar

    [27]

    Bindi M, Miglietta F, Gozzini B, Orlandini S, Seghi L. 1997. A simple model for simulation of growth and development in grapevine (Vitis vinifera L.) II. model validation. Vitis 36:73−76

    Google Scholar

    [28]

    Cheng G, Zhou S, Zhang J, Huang X, Bai X, et al. 2019. Comparison of transcriptional expression patterns of phenols and carotenoids in 'Kyoho' grapes under a two-crop-a-year cultivation system. PLoS ONE 14(1):e0210322

    doi: 10.1371/journal.pone.0210322

    CrossRef   Google Scholar

    [29]

    Chen WK, Bai XJ, Cao MM, Cheng G, Cao XJ, et al. 2017. Dissecting the variations of ripening progression and flavonoid metabolism in grape berries grown under double cropping system. Frontiers in Plant Science 8:1912

    doi: 10.3389/fpls.2017.01912

    CrossRef   Google Scholar

    [30]

    Wang H, Wang X, Yan A, Liu Z, Ren J, et al. 2023. Metabolomic and transcriptomic integrated analysis revealed the decrease of monoterpenes accumulation in table grapes during long time low temperature storage. Food Research International 174:113601

    doi: 10.1016/j.foodres.2023.113601

    CrossRef   Google Scholar

    [31]

    Alessandrini M, Gaiotti F, Belfiore N, Matarese F, D'Onofrio C, et al. 2017. Influence of vineyard altitude on Glera grape ripening (Vitis vinifera L.): effects on aroma evolution and wine sensory profile. Journal of the Science of Food and Agriculture 97(9):2695−705

    doi: 10.1002/jsfa.8093

    CrossRef   Google Scholar

    [32]

    Beekwilder J, Alvarez-Huerta M, Neef E, Verstappen FWA, Bouwmeester HJ, et al. 2004. Functional characterization of enzymes forming volatile esters from strawberry and banana. Plant Physiology 135(4):1865−78

    doi: 10.1104/pp.104.042580

    CrossRef   Google Scholar

    [33]

    Friedel M, Frotscher J, Nitsch M, Hofmann M, Bogs J, et al. 2016. Light promotes expression of monoterpene and flavonol metabolic genes and enhances flavour of winegrape berries (Vitis vinifera L.cv. Riesling). Australian Journal of Grape and Wine Research 22:409−21

    doi: 10.1111/ajgw.12229

    CrossRef   Google Scholar

    [34]

    Kwasniewski MT, Vanden Heuvel JE, Pan BS, Sacks GL. 2010. Timing of cluster light environment manipulation during grape development affects C13 norisoprenoid and carotenoid concentrations in Riesling. Journal of Agricultural and Food Chemistry 58(11):6841−49

    doi: 10.1021/jf904555p

    CrossRef   Google Scholar

    [35]

    Zhang E, Chai F, Zhang H, Li S, Liang Z, et al. 2017. Effects of sunlight exclusion on the profiles of monoterpene biosynthesis and accumulation in grape exocarp and mesocarp. Food Chemistry 237:379−89

    doi: 10.1016/j.foodchem.2017.05.127

    CrossRef   Google Scholar

    [36]

    Yao H, Jin X, Feng M, Xu G, Zhang P, et al. 2021. Evolution of volatile profile and aroma potential of table grape Hutai-8 during berry ripening. Food Research International 143:110330

    doi: 10.1016/j.foodres.2021.110330

    CrossRef   Google Scholar

    [37]

    Dunlevy J, Kalua C, Keyzers R, Boss P. 2009. The production of flavour & aroma compounds in grape berries. In Grapevine Molecular Physiology & Biotechnology, ed. Roubelakis-Angelakis KA. Dordrecht: Springer. pp. 293−340. doi: 10.1007/978-90-481-2305-6_11

  • Cite this article

    Yu H, Guo R, Liu J, Shi X, Huang G, et al. 2024. Metabolome provides new insights into the volatile substances in 'Ruidu Kemei' grapes under the two-crop-a-year cultivation system. Fruit Research 4: e035 doi: 10.48130/frures-0024-0029
    Yu H, Guo R, Liu J, Shi X, Huang G, et al. 2024. Metabolome provides new insights into the volatile substances in 'Ruidu Kemei' grapes under the two-crop-a-year cultivation system. Fruit Research 4: e035 doi: 10.48130/frures-0024-0029

Figures(5)  /  Tables(2)

Article Metrics

Article views(1168) PDF downloads(244)

ARTICLE   Open Access    

Metabolome provides new insights into the volatile substances in 'Ruidu Kemei' grapes under the two-crop-a-year cultivation system

Fruit Research  4 Article number: e035  (2024)  |  Cite this article

Abstract: In subtropical regions, the implementation of a two-crop-a-year cultivation system depends on local climatic conditions. Grape volatile compounds vary greatly with the season, due to climate differences, which lead to extreme differences between summer grape fruits (SF) and winter grape fruits (WF). In the present study, a gas chromatography-mass spectrometer (GC-MS) was used to analyze volatile compounds from 'Ruidu Kemei' grapes grown under the two-crop-a-year cultivation system. Results showed that fruits in summer and winter contained 620 volatile compounds in 15 categories. Among them, terpenoids constituted the largest group, with 122 metabolites, followed by 115 esters. This indicated that the main volatile characteristic substances of 'Ruidu Kemei' were terpenoids and esters. Higher volatile compounds in SF might be associated with higher active accumulated temperatures in the summer growing season. In addition, terpenoids, heterocyclic compounds, esters, and aromatics showed greater differences than other compounds between SF and WF. Regarding terpenoids, WF exhibited superior performance, while SF performed better in esters and aromatics. For WF, higher solar radiation intensity promoted the biosynthesis of terpenoids, which lead to more floral characteristics than SF. According to the flavor omics analysis, 'Ruidu Kemei' was primarily characterized by green, fruity, herbal, woody, sweet, floral, fresh, fatty, citrus, and earthy. In the SF, green and fruity flavors were more prominent, while floral was the dominant fruity aroma in WF. This work provides new insights into the metabolism of volatiles in summer and winter grapes and reference for the selection and promotion of varieties with suitable aromas for a two-crop-a-year cultivation system.

    • Grape (Vitis vinifera L.) is one of the most popular fruits in the world. Table grapes account for approximately 36% of global grape production[1]. In China, table grapes account for 80% of total grape production[2]. In light of this, it is very important to study the aroma of table grapes. The volatile compounds in grapes affect sensory evaluation, which could be the reason that consumers choose certain grapes over others[3,4]. Volatile compounds in fruits are responsible for defining their aroma and flavor. We can obtain grapes with distinct aromas and characteristics for the varying volatile combinations and concentrations[5]. Fruit volatile compounds are mainly comprised of esters, alcohols, aldehydes, ketones, lactones, terpenoids, and apocarotenoids[6]. Many factors affect volatile composition, including the genetic diversity[7,8], viticultural techniques[9,10], degree of maturity[4], climatic conditions[3,11,12], and postharvest storage conditions[4,13]. Among these factors, climate conditions (sunlight, temperature, water status, etc.) were often considered an important factor for grape volatile compounds for the same cultivar[11].

      Light is the primary climatic factor affecting volatile composition. As we all know, intensity, quality, and photoperiod are the main factors of light regulation[14]. Sunlight promotes the accumulation of terpenoids and monoterpene, which are the typical aroma components in Muscat grapes[3,12]. Modified canopy management (basal leaf removal) and exposure to appropriate proportions of blue and red light were effective strategies to improve the characteristic aroma[14,15]. Temperature is another important climate factor affecting volatile composition. Generally, excessively high temperature is deemed to have negative effects on fruit metabolism[16]. For example, high temperature in the winter season inhibited most VviCCDs expression than in summer grape berries, which was associated with norisoprenoid accumulation[3]. Temperate zones are more conducive to the formation of aroma substances[17]. Compared with grapes grown under cool conditions, the same grape variety presented a higher concentration of monoterpenes when cultivated under warm conditions[18]. The other factor that influences grape development is water availability. Proper water deficit has been proven to be available for increasing the characteristic aroma contents, especially terpenes and esters[19,20]. Therefore, improving water use efficiency can increase fruit flavor.

      Different terrains forms different aroma characteristics. The southern subtropical region of China was not a traditional viticultural area due to the sticky rainy weather and inadequate low-temperature accumulation[3,21]. With the application of grape two-crop-a-year cultivation technology, the above-mentioned problems have been conquered[22], and Guangxi (a province in southern China) has become a unique advantage viticulture area[23]. Due to the plentiful sunlight and temperature accumulation, grape berries could be harvested twice a year[3,22]. Summer grape fruits are the name of grapes harvested in the first growing season, while winter grape fruits are the name of grapes harvested in the second growing season[24]. 'Ruidu Kemei', breeding from a cross between 'Italy' and 'Muscat Louis', is a new table grape variety appropriate to two-crop-a-year cultivation[25,26]. At present, there are few reports about grape volatiles under two-crop-a-year cultivation systems. Recently, Lu et al. compared the volatile profiles of 'Riesling', 'Cabernet Sauvignon', 'Victoria', and 'Muscat Hamburg' grape berries under two-crop-a-year cultivation[3]. However, knowledge about the volatile profiles of two-crop grapes is still very rare. More work needs to be carried out to establish the aroma substance characteristics, and to provide a theoretical basis for improving aroma under two-crop-a-year cultivation systems.

      To distinguish grape volatiles under the two-crop-a-year cultivation system, the volatiles in summer and winter berries of 'Ruidu Kemei' were qualitatively and quantitatively analyzed by headspace solid phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS). Meanwhile, two crops' volatiles were also conducted in relation to climate factors in the present study.

    • This experiment was conducted during two growing seasons in 2022 on 3-year-old 'Ruidu Kemei' grapevines in the vineyards of the Grape and Wine Research Institute, Guangxi Academy of Agricultural Sciences, located in Nanning, Guangxi Province, China (22°36'39" N, 108°13'51" E). In this vineyard, the vines were managed on a canopy frame with a single trunk and were planted in north-south-oriented rows spaced 1.5 m (between vines) × 2.5 m (between rows). Nutrition, pest, water, and fertilizer management was carried out by uniform standards for two-crop-a-year as previously described[3].

      The key techniques of two-crop-a-year cultivation systems was described by Cheng et al.[24]. Summer grape fruits (SF) were harvested on July 15th, and winter grape fruits (WF) were harvested on December 31st.

    • Six vines for sampling were chosen based on their relatively consistent growth status. Six biological replicates were conducted in this study, and each biological replicate comprised 90 berries from six clusters of different vines, then sampled berries of each biological replicate were mixed and put into a 50 mL centrifuge tube, and immediately frozen in liquid nitrogen, and stored at −80 °C until needed.

      Temperature (°C), relative humidity (%), and solar radiation intensity (W/m2) were acquired according to Cheng et al.[24]. Growing degree days (base 10 °C) were calculated from bloom to harvest according to Bindi et al.[27].

    • Samples of each biological replicate were ground to powder in liquid nitrogen, and 500 mg powder was transferred immediately to a 20 mL head-space vial (Agilent, Palo Alto, CA, USA), containing NaCl-saturated solution, to inhibit any enzyme reaction. The vials were sealed using crimp-top caps with TFE-silicone headspace septa (Agilent). At the time of SPME analysis, each vial was placed at 60 °C for 5 min, then a 120 μm DVB/CWR/PDMS fiber (Agilent) was exposed to the headspace of the sample for 15 min at 60 °C.

    • After sampling, desorption of the VOCs from the fiber coating was carried out in the injection port of the GC apparatus (Model 8890; Agilent) at 250 °C for 5 min in the splitless mode. The identification and quantification of VOCs was carried out using an Agilent Model 8890 GC and a 7000D mass spectrometer (Agilent), equipped with a 30 m × 0.25 mm × 0.25 μm DB-5MS (5% phenyl-polymethylsiloxane) capillary column. Helium was used as the carrier gas at a linear velocity of 1.2 mL/min. The injector temperature was kept at 250 °C and the detector at 280 °C. The oven temperature was programmed from 40 °C (3.5 min), increasing at 10 °C/min to 100 °C, at 7 °C/min to 180 °C, at 25 °C/min to 280 °C, hold for 5 min. Mass spectra was recorded in electron impact (EI) ionization mode at 70 eV. The quadrupole mass detector, ion source, and transfer line temperatures were set, respectively, at 150, 230, and 280 °C. The MS with selected ion monitoring (SIM) mode was used for the identification and quantification of analytes.

    • Unsupervised PCA was performed by the statistics function prcomp within R (www.r-project.org). The data was unit variance scaled before unsupervised PCA.

    • The HCA results of samples and metabolites were presented as heatmaps with dendrograms, while Pearson correlation coefficients (PCC) between samples were calculated by the cor function in R and presented as only heatmaps. Both HCA and PCC were carried out by the R package ComplexHeatmap. For HCA, normalized signal intensities of metabolites (unit variance scaling) are visualized as a color spectrum.

    • For two-group analysis, differential metabolites were determined by VIP (VIP > 1) and absolute Log2FC (|Log2FC| ≥ 1.0). VIP values were extracted from OPLS-DA results, which also contain score plots and permutation plots, and was generated using R package MetaboAnalystR. The data was log transform (log) and mean centering before OPLS-DA. To avoid overfitting, a permutation test (200 permutations) was performed.

    • Identified metabolites were annotated using the KEGG Compound database (www.kegg.jp/kegg/compound, accessed on April 2nd, 2022), annotated metabolites were then mapped to the KEGG Pathway database (www.kegg.jpkegg/pathway.html, accessed on April 2nd, 2022). Pathways with significantly regulated metabolites mapped then fed into MSEA (metabolite sets enrichment analysis), their significance was determined by hypergeometric test's p-values.

    • Volatiles in grape berries were affected by meteorological parameters under the double cropping system[3]. Significant differences in meteorological parameters between the two crop growing seasons are shown in Table 1. The summer growing season was from 1 March to 15 July, and the winter growing season was from 1 September to 31 December. In the present study, the active accumulated temperatures for both growing seasons were greater than 3,100 °C (Table 1), meaning that the active accumulated temperatures were sufficient to guarantee normal grape maturity[28]. The active accumulated temperature, the effective accumulated temperature, and the daily average temperature for the summer growing season was higher than those of the winter growing season. However, there were 83.33 h of high temperatures over 35 °C during the summer growing season, which was less than the winter growing season (127.17 h). Moreover, the relative humidity during the summer growing season showed a higher value than the winter growing season. For the solar radiation intensity and cumulative solar radiation, the winter growing season was higher than the summer growing season.

      Table 1.  Phenology and climatic factors during the two crop-growing seasons in Nanning (China) in 2022.

      Meteorological data Summer Winter
      Phenology 1 Mar−15 Jul 15 Aug−31 Dec
      Active T (°C) 3,393.53 3,149.98
      Effective T (°C) 2,023.53 1,769.98
      Average daily temperature (°C) 24.78 21.92
      High temperature (> 35 °C) (°C) 83.33 127.17
      Relative humidity (%) 86.94 80.78
      Solar radiation Intensity (W/m2) 93.86 108.65
      Cumulative solar radiation (W/m2) 3,703,264.3 3,805,233.3
    • To figure out the difference between SF and WF, volatile metabolite analysis was applied in this study. A total of 620 metabolites in 15 categories were detected, including 122 terpenoids, 115 esters, 99 heterocyclic compounds, 60 hydrocarbons, 52 ketones, 48 alcohols, 47 aldehydes, 31 aromatics, 11 amines, 11 acids, eight phenols, seven nitrogen compounds, three halogenated hydrocarbons, two sulfur compounds, and four others (Fig. 1a, Supplementary Table S1). There was no difference between SF and WF for 12 categories (Fig. 1b). WF had more terpenoids and heterocyclic compounds than SF. Conversely, SF had more esters (Fig. 1b).

      Figure 1. 

      (a) Categorical all metabolite statistics. (b) Categorical metabolite statistics for SF & WF. (c) All metabolites for hierarchical cluster analysis (HCA). (d) The relative content of classified metabolites for SF & WF.

      For the relative metabolite contents, it was found that the metabolites were divided into two clusters, and significant differences could be observed in the substances between SF and WF. The metabolite relative contents in Cluster I were higher in SF, while WF exhibited higher relative contents in Cluster II metabolites (Fig. 1c). Phenols showed little difference between SF and WF, but SF was richer in the 14 other categories than that of WF (Fig. 1d).

      SF and WF were evidently distinguished by PCA (Fig. 2a), the explanation rate of the first five principal components reached 87.1% (Fig. 2b). The cluster dendrogram divided SF and WF into two groups, which was consistent with PCA (Fig. 2c). The results indicated that the volatile compounds differed greatly between SF and WF.

      Figure 2. 

      (a) Principal component analysis (PCA). (b) Grouped principal component analysis explanation rate plot. (c) Sample hierarchical clustering tree.

    • To better distinguish volatile compounds between summer fruits and winter fruits, metabolites with fold change ≥ 2 and fold change ≤ 0.5 were selected as significant differences. The comparison SF_vs_WF showed a total of 143 different metabolites accounted for 23.18 % of the total detected substances, including 85 up-regulated metabolites and 58 down-regulated metabolites (Fig. 3a). The metabolites with a higher number for up-regulated were terpenoids, ketone, hydrocarbons, ester, aldehyde, alcohol, halogenated hydrocarbons, acids, and others (Fig. 3b). The metabolites with a higher number for down-regulated were amine, aromatics, nitrogen compounds, phenol, and heterocyclic compounds. It's worth noting that no sulfur compounds showed a statistically significant difference between SF and WF (Fig. 3a & b). For relative content, terpenoids, heterocyclic compounds, esters, and aromatics showed greater difference than other compounds (Fig. 3c).

      Figure 3. 

      (a) Volcanic plot of differential volatile compounds. (b) Bar chart of the number of volatile compounds classified for up-regulation & down-regulation. (c) Scatter plot of differential volatile compounds. (d) Bar chart of the top 20 differential volatile compounds.

      To determine the metabolites with large differences for the SF_vs_WF comparison, a list of the top 20 substances using Log2FC was made, including 10 up-regulation substances and 10 down-regulation substances (Fig. 3d). There were obvious distinctions between the SF and WF. The top 20 substances using Log2FC contained five categories: terpenoids (8), aromatics (4), heterocyclic compounds (3), esters (3), and ketones (2). The top 10 up-regulation substances contained seven terpenoids, one aromatic, one heterocyclic compound, one ketone, while three esters, three aromatics, two heterocyclic compounds, and one ketone in the top 10 down-regulation substances. These results suggested that, for the top 20 substances using Log2FC, terpenoids were mainly up-regulated in WF, while esters and aromatics were up-regulated in SF. Specifically, the up-regulated and down-regulated substances with the largest Log2FC for SF_vs_WF comparison were [1α,4aα,8aα]-1,2,4a,5,6,8a-hexahydro-4-7-dimethyl-1-[1-methylethyl]naphthalene (terpenoid) and 3-Hexen-1-ol, acetate, (Z)-(ester).

    • Fourty-nine of the 620 metabolites were annotated to 20 KEGG pathways (Supplementary Table S2). Additionally, 13 differential volatile compounds out of 143 differential volatile compounds between SF and WF were primarily annotated and enriched in the following seven pathways: biosynthesis of secondary metabolites, metabolic pathways and sesquiterpenoid and triterpenoid biosynthesis, monoterpenoid biosynthesis, limonene and pinene degradation, terpenoid backbone biosynthesis, and α-Linolenic acid metabolism (Fig. 4a & b; Table 2). Among them, the top three KEGG pathway types were biosynthesis of secondary metabolites, metabolic pathways and sesquiterpenoid, and triterpenoid biosynthesis, accounting for 53.85%, 46.15%, and 38.46% of the total differential volatile compounds annotated in KEGG respectively (Fig. 4a). KEGG annotations and enrichment showed that sesquiterpenoid and triterpenoid biosynthesis, monoterpenoid biosynthesis, limonene and pinene degradation were the main KEGG pathways for the differential volatile compounds between SF and WF (Fig. 4b). Significantly except sesquiterpenoid and triterpenoid biosynthesis, the other six pathways were mainly down-regulated (Table 2). These 13 differential volatile compounds were nine terpenoids, three aldehydes, and one ester (Table 2). Only four terpenoids were more in WF when compared with SF, including (E)-β-Famesene, Naphthalene,1,2,3,5,6,8a-hexahydro-4,7-dimethyl-1-(1-methylethyl)-, (1S-cis)-, α-Farnesene, and (E)-1-Methyl-4-(6-methylhept-5-en-2-ylidene)cyclohex-1-ener (Table 2). All the remaining nine differential volatile compounds were less in WF than in SF (Table 2).

      Figure 4. 

      The classification of the KEGG enrichment pathway. (a) KEGG enrichment analysis of differential volatile compounds. (b) KEGG annotations and enrichment of differential volatile compounds for SF_vs_WF comparison.

      Table 2.  KEGG functional annotation and enrichment of differential volatile compounds between SF and WF.

      Formula Compounds KEGG_pathway Class Odor SF vs WF
      C10H18O L-α-Terpineol Metabolic pathways, Biosynthesis of secondary metabolites, Monoterpenoid biosynthesis Terpenoids Lilac, floral, terpenic Down
      C7H6O BenzAldehyde Metabolic pathways Aldehyde Sweet, bitter, almond, cherry Down
      C8H8O BenzAldehyde, 2-methyl- Metabolic pathways Aldehyde Mild floral, sweet Down
      C10H18O Bicyclo[3.1.0]hexan-2-ol,
      2-methyl-5-(1-methylethyl)-, (1α,2β,5α)-
      Metabolic pathways, Biosynthesis of secondary metabolites, Monoterpenoid biosynthesis Terpenoids Balsam Down
      C15H24 Naphthalene, 1,2,3,5,6,8a-hexahydro-4,7-dimethyl-1-(1-methylethyl)-, (1S-cis)- Metabolic pathways, Biosynthesis of secondary metabolites, Sesquiterpenoid and triterpenoid biosynthesis Terpenoids Thyme, herbal, woody, dry Up
      C7H6O2 2-hydroxy-BenzAldehyde Metabolic pathways Aldehyde Medical, spicy, cinmon, wintergreen, cooling Down
      C8H14O2 3-Hexen-1-ol, acetate, (Z)- Biosynthesis of secondary metabolites, α-Linolenic acid metabolism Ester Fresh, green, sweet, fruity, ba--, apple, grassy Down
      C15H24 α-Farnesene Biosynthesis of secondary metabolites, Sesquiterpenoid and triterpenoid biosynthesis Terpenoids Citrus, herbal, lavender, bergamot, myrrh, neroli, green Up
      C15H24 (E)-1-Methyl-4-(6-methylhept-5-
      en-2-ylidene)cyclohex-1-ene
      Biosynthesis of secondary metabolites, Sesquiterpenoid and triterpenoid biosynthesis Terpenoids Up
      C15H24O 2,6,10-Dodecatrienal,
      3,7,11-trimethyl-, (E,E)-
      Biosynthesis of secondary metabolites, Terpenoid backbone biosynthesis, Sesquiterpenoid and triterpenoid biosynthesis Terpenoids Down
      C15H24 (E)-β-Famesene Sesquiterpenoid and triterpenoid biosynthesis Terpenoids Woody, citrus, herbal, sweet Up
      C10H16O Bicyclo[3.1.1]hept-2-ene-2-methanol, 6,6-dimethyl- Limonene and pinene degradation Terpenoids Woody, minty Down
      C10H16O 3-Oxatricyclo[4.1.1.0(2,4)]octane, 2,7,7-trimethyl- Limonene and pinene degradation Terpenoids Green Down
      − indicates no annotation of substance.
    • One hundred and fourty-three differential volatile compounds were annotated to 159 sensory flavors (Supplementary Table S3). The top 10 sensory flavors with the highest number of annotations were green (23), fruity (21), herbal (14), woody (14), sweet (13), floral (9), fresh (8), fatty (8), citrus (8), and earthy (7) (Fig. 5a), which were the most important sensory flavors for SF and WF. The top 10 differential volatile compounds with high numbers of sensory flavor features annotation were Hexanoic acid, propyl ester (Ester), 3-Hexen-1-ol,acetate,(Z)-(Ester), Butanoic acid,hexyl ester (Ester), Butanoic acid, octyl ester (Ester), Fenchone (Terpenoids), Isocyclocitral (Aldehyde), Pyrazine, 2-methyl-5-(1-methylethyl)-(Heterocyclic compound), Heptanal (Aldehyde), and Geranyl isobutyrate (Ester), which were the most important differential volatile compounds of sensory flavors for SF and WF (Fig. 5b).

      Figure 5. 

      (a) Radar map for analysis of differential metabolite sensory flavor characteristics. (b) Sankey diagram of flavor omics.

      Compared with WF, SF mainly showed green, fruity, herbal, woody, sweet, and earthy, the relevant substances were Hexanoic acid, propyl ester (Ester), 3-Hexen-1-ol,acetate,(Z)- (Ester), Butanoic acid,hexyl ester (Ester), Butanoic acid,octyl ester (Ester), Fenchone (Terpenoids), Isocyclocitral (Terpenoids), Pyrazine, 2-methyl-5-(1-methylethyl)-(Terpenoids), etc (Fig. 5b). WF mainly showed more floral, fresh, fatty, and citrus than SF, according to a higher number of up-regulated metabolites for SF_vs_WF comparison, including 2-Undecenal,E-(Aldehyde), 2-Octen-1-ol,(E)-(Alcohol), 2-Dodecenal,(E)-(Aldehyde), (E)-β-Famesene (Terpenoids), etc (Fig. 5b).

    • Meteorological data differ greatly between the two crop seasons in Guangxi (China). The active accumulated temperature, the effective accumulated temperature, the daily average temperature, and the relative humidity for the summer growing season were higher than those of the winter growing season[3,24,29], which were in line with the present research. However, there were more hours of high temperatures over 35 °C in the winter growing season than that in the summer growing season. This result is the opposite of other study findings[3,24]. Consistently with previous studies[24,29], the solar radiation intensity and cumulative solar radiation was higher in the winter growing season. According to the results of this research and literary references, meteorological data for two crop seasons varies by year.

      In the present research, 620 volatile compounds in 15 categories were detected in summer and winter fruits, including 122 terpenoids, 115 esters, 99 heterocyclic compounds, 60 hydrocarbons, 52 ketones, 48 alcohols, 47 aldehydes, 31 aromatics, 11 amines, 11 acids, eight phenols, seven nitrogen compounds, three halogenated hydrocarbons, two sulfur compounds, and four others. These results indicated that terpenoids were the main volatile characteristic substances of 'Ruidu Kemei', followed by esters. It has been confirmed that terpenoids were the characteristic aroma components of muscat flavored varieties, which was consistent with the present study[30].

      Grape cultivation in the field was greatly impacted by climate conditions. Berries were influenced greatly by their growing environment in terms of chemical composition. Due to variations in climate between the summer and winter growing seasons, the most important metabolites of grapes perform differently under a two-crop-a-year cultivation system, such as flavonoids[24,29], phenols, carotenoids[28], and volatiles[3]. The present study showed clear differences in the concentration of volatile compounds in response to meteorological data for two crop seasons, which verified the findings of previous research[3], while the compounds of volatiles mainly remained similar for the volatile compounds depending largely on the genotype of the grape cultivar rather than the growing environment[11]. However, when compared with WF, higher volatile compound concentration was observed in berries of summer, which would be caused by more hours of high temperatures over 35 °C during the winter growing season. Generally, lower temperatures were conducive to the accumulation of aromatic substances. Furthermore, this data appears to be related to the higher active accumulated temperatures in the summer growing season, which favored the grape ripening and volatile accumulation in the grape berries[10,31].

      To determine the distinction of volatile compounds between summer fruits and winter fruits, 143 significant different metabolites were selected. For number and relative content, terpenoids, heterocyclic compound, ester and aromatics showed greater differences than other compounds. In particular, for the top 20 substances using Log2FC, terpenoids (such as [1α,4aα,8aα]-1,2,4a,5,6,8a-hexahydro-4-7-dimethyl-1-[1-methylethyl]naphthalene) were mainly up-regulated in WF, while esters (such as 3-Hexen-1-ol, acetate, (Z)-) and aromatics (such as Benzene, (1-methoxypropyl)-) were up-regulated in SF, since heat and sunlight stress can reduce the aromatic content of grapes, while less solar radiation intensity favored the higher level of aromatics[9]. The most likely precursors for the esters were lipids and amino acids. Their metabolism during ripening will therefore play an important role in determining both the levels and types of esters formed[32]. It has been reported that cluster sunlight exposure in viticulture in dry-hot climates caused a notable decrease in esters, including ethyl hexanoate and hexyl acetate[15]. Sunlight was advantageous for accumulating terpenoids[3], the activation of terpene synthase genes (VvTPS54 and VvTPS56) and the synthesis of carotenoids in grapes, subsequently leading to the accumulation of terpenoids and norisoprenoids[33,34]. As reported, higher solar radiation intensity and cumulative solar radiation enhanced accumulation of terpenes[12]. Zhang et al. indicated that VvDXS2 and VvDXR were partially linked to differential terpene accumulation for different illumination conditions[35]. Sun et al. found that grape berries grown in rain shelters contain lower levels of terpenoids and norisoprenoids during development, possibly as a result of less light, inhibiting isoprenoids during development[10]. In this study, more hours of high temperatures over 35 °C in the winter growing season than that in the summer growing season, higher solar radiation intensity, and cumulative solar radiation still promoted sesquiterpenoid and triterpenoid biosynthesis, monoterpenoid biosynthesis, limonene, and pinene degradation (Fig. 4b). These results reconfirmed that the increased light exposure was beneficial for terpene accumulation, which could infer that the negative effect of the elevated berry temperature might be surpassed by the beneficial effect of increased synthesis of terpenes induced by light. However, Friedel et al. made an opposite judgment[33]. Thus, grape cultivars might respond differently to climate. In summary, for 'Ruidu Kemei', WF performed better in terpenoids, whereas SF displayed better in esters and aromatics. Based on previous studies of other grape varieties[3], it could be concluded that WF probably always forms higher concentrations of terpenes than SF under a two-crop-a-year cultivation system in the Guangxi region of South China, which has a typical subtropical humid monsoon climate.

      Among other qualities, aroma flavor contributes to consumers' acceptance of table grapes[30]. Table grapes' flavor was generally determined by their free volatiles since they were directly detectable and tasteable[36]. Flavors varied from different ingredients and different concentrations of volatile substances. In the present study, the different relative content of volatile substances was the reason for the different flavors of grapes in two growing seasons. Fruit aroma profile visually displayed that the most important sensory flavors for 'Ruidu Kemei' were green, fruity, herbal, woody, sweet, floral, fresh, fatty, citrus, and earthy. Green and fruity were the most critical aroma for 'Ruidu Kemei', due to most volatiles annotated. Green, fruity, herbal, woody, sweet, and earthy were more prominent in the SF, the relevant substances were Hexanoic acid, propyl ester, 3-Hexen-1-ol,acetate,(Z)-, Butanoic acid,hexyl ester, Butanoic acid, octyl ester, Fenchone, Isocyclocitral, Pyrazine, 2-methyl-5-(1-methylethyl)-, etc (Fig. 5b). Compared with SF, for WF, floral was the most prominent fruit smell, followed by fresh and fatty smell, and then citrus smell, the metabolites with the greatest contribution were 2-Undecenal,E-, 2-Octen-1-ol,(E)-, 2-Dodecenal,(E)-, (E)-β-Famesene, etc (Fig. 5b). In general, seasonal differences can be observed in the sensory properties of grape berries from the same variety[37]. Floral dominated in WF for performing better in terpenoids than SF. Due to their association with floral scents, terpenoids may attract appropriate pollinators and facilitate reproduction[37].

    • In the present research, 620 volatile compounds in 15 categories were detected in summer and winter fruits by a GC-MS/MS-based metabolomics approach. Terpenoids were the main volatile characteristic substances of 'Ruidu Kemei', followed by esters. Meteorological data for two crop seasons varied by years. The variational climatic factors in the summer and winter growing seasons were responsible for the difference in volatile metabolites between the two crops of grapes. Compared with the WF, higher active accumulated temperatures in the summer growing season contributed to higher volatile compound concentration in SF. Moreover, terpenoids, heterocyclic compounds, esters, and aromatics showed greater differences than other compounds between SF and WF. In addition, it was demonstrated that the winter cropping cycle promoted the biosynthesis of terpenoids by higher solar radiation intensity and cumulative solar radiation, which lead to more floral fruit smell than SF. On the contrary, more esters and aromatics were observed in SF in response to less solar radiation intensity, cumulative solar radiation and higher active accumulated temperatures in the summer growing season. Flavor omics analysis presented that the most important sensory flavors for 'Ruidu Kemei' were green, fruity, herbal, woody, sweet, floral, fresh, fatty, citrus, and earthy. Green and fruity were the most critical aroma for 'Ruidu Kemei', due to the most volatiles annotated. Green, fruity, herbal, woody, sweet, and earthy were more prominent in the SF. Floral was the most prominent fruit smell in WF. Clarification the characteristics of aroma substances of grape berries in two growing seasons can provide a basis for the scientific control of grape aroma, the improvement of grape quality, and the optimization of grape cultivation technology.

    • The authors confirm contribution to the paper as follows: study conception and design: Guo R, Lin L, Zhang Y; data collection: Lin L, Yu H, Liu J, Shi X; analysis and interpretation of results: Yu H, Huang G; draft manuscript preparation: Yu H; review & editing, project administration and funding acquisition: Guo R, Lin L. All authors reviewed the results and approved the final version of the manuscript.

    • All data generated or analyzed during this study are included in this published article and its supplementary information files.

      • This work was supported by grants from the Guangxi Key Research and Development Program (GuikeAB19245031), the special project for basic scientific research of Guangxi Academy of Agricultural Sciences (Guinongke2021YT127 & Guinongke2023YM111).

      • The authors declare that they have no conflict of interest.

      • # Authors contributed equally: Huan Yu, Rongrong Guo

      • Copyright: © 2024 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (5)  Table (2) References (37)
  • About this article
    Cite this article
    Yu H, Guo R, Liu J, Shi X, Huang G, et al. 2024. Metabolome provides new insights into the volatile substances in 'Ruidu Kemei' grapes under the two-crop-a-year cultivation system. Fruit Research 4: e035 doi: 10.48130/frures-0024-0029
    Yu H, Guo R, Liu J, Shi X, Huang G, et al. 2024. Metabolome provides new insights into the volatile substances in 'Ruidu Kemei' grapes under the two-crop-a-year cultivation system. Fruit Research 4: e035 doi: 10.48130/frures-0024-0029

Catalog

  • About this article

/

DownLoad:  Full-Size Img  PowerPoint
Return
Return