Introduction

Herbs are rich sources of flavonoids and are used as natural spices and traditional medicine ingredients [1]. Salvia is a medicinally important genus under the family Lamiaceae that contains interesting health-promoting phytochemicals, notably polyphenolic compounds. At present, ~ 75 types of simple phenolic acids have been isolated from 23 Salvia species, and 50 types of flavonoid have been isolated from 51 Salvia species [2]. Common sage (Salvia plebeia R. Br.) is a medicinally important species under the genus Salvia that is widely distributed throughout Korea, China, and India. This plant is used in Asian traditional medicines for the treatment of several illnesses because of its antimicrobial, diuretic, antiblastic, antipyretic, anti-inflammatory, and antidynous activities [3, 4]. S. plebeia has also been selected as a natural drug candidate among various Chinese traditional herbs owing to its strong antioxidant properties [5, 6].

S. plebeia is known in Korean folk medicine as ‘Baem-Cha-Zu-Ki’ and is used for the prevention and treatment of inflammatory diseases. This traditional claim is backed by scientific evidence from several animal model-based experiments. For example, the polyphenolic extract showed sedative and gastro-protective activities against two gastric lesion assay-based methods in mice [7]. Some researchers had reported the inhibitory effects of the aqueous extracts on immediate-type allergic reactions in rat. Moreover, a potent anti-inflammatory activity was observed from a methanol extract [8,9,10]. Phenolic acids, flavones, isoflavones, and their glycosides play vital roles in the anti-inflammatory and other wide-ranging pharmacological activities of S. plebeia [3, 9, 11].

Flavonoids, known as a major group of phenolic compounds, affect the flavors and colors of plant-derived foods. They are categorized into flavanones, flavones, flavonols, isoflavones, and chalcones, and they possess protective and therapeutic effects against chronic diseases, such as cancer and coronary heart disease [12]. Many food resources, such as fruits, vegetables, and herbs, contain various flavonoids, such as quercetin, kaempferol, myricetin, luteolin, apigenin, naringenin, eriodictyol, and tangeretin [13]. Apigenin, luteolin, nepetin, hispidulin, and their glycosides are the major flavonoids found in the hydro-alcoholic extract of S. plebeia. The flavone classes are major phytochemicals responsible for the potent biological activity of the plant [3, 5, 6, 9, 14,15,16]. However, flavanone and other sub-classes of flavonoids are rarely reported, although they have significant pharmacological properties [17]. The previous studies revealed the potential biological activity of S. plebeia, and this effect is strongly related with the presence of polyphenolic compounds abundantly. Therefore, their component has been considered in developing nutraceutical ingredients as crude extracts and pure active compounds. The accurate quantification of the active compounds is a significant step for nutraceuticals and determining their physiological activities.

The aim of the present work was to characterize the phenolic compounds of S. plebeia using ultra-performance liquid chromatograph coupled with photodiode array detection and quadrupole time of flight mass spectrometry (UPLC-DAD-QTOF/MS). The flavonoids were quantified with an emphasis on the minor constituent flavanones. 5,6,7,4′-Tetrahydroxyflavanone 7-O-glucoside and 5,6,7,3′,4′-pentahydroxyflavanone 7-O-glucoside are proposed as new compounds, among the identified flavanones.

Materials and methods

Chemicals

HPLC-grade solvents (acetonitrile, methanol, and water) were obtained from Fisher Scientific (Fair Lawn, NJ, USA). Formic acid was purchased from Junsei Chemical (Tokyo, Japan). Galangin (Sigma Co., St. Louis, MO, USA) and 2,4,5-trimethoxycinnamic acid (Extrasynthese, Genay, France) were used as the internal standard for flavonoids and phenolic acids, respectively.

Plant material and extraction of phenolic compounds

The aerial part of common sage (S. plebeia R. Br.) was collected from Korea in 2015 and identified at the Paju Agricultural Technology Center, Korea. A voucher specimen (RDASPR14) was deposited in the Department of Agrofood Resources, National Institute of Agricultural Sciences, Rural Development Administration (Korea). The sample was freeze-dried in a deep freezer (PVTFD 10R, Ilsin Lab, Yangju, Korea), pulverized into powder and stored at − 60 °C prior to analysis.

The weighed powder (1.0 g) was suspended in 10 ml of extract solvents (two types of solvent systems; methanol: water: formic acid, 50:45:5, v/v/v for flavonoids and methanol: water: formic acid, 80:15:5, v/v/v for phenolic acids) containing the internal standard and extracted for 5 min at 200 rpm on an orbital shaker. The mixture was then centrifuged at 3000 rpm at 4 °C for 10 min. Each supernatant was immediately filtered through a syringe filter (0.2 µm, PVDF; Whatman, Kent, England), and 0.5 ml of phenolic extract was diluted by 4.5 ml of water, resulting in 5 ml of a phenolic-containing crude extract. The phenolic concentrate was then isolated from the crude extract using the solid phase extraction method with a Sep-pak C18 cartridge (Waters Co., Milford, MA, USA). The Sep-pak cartridges were activated by washing with 2 ml of methanol, followed by washing with 2 ml of water for conditioning. Then, the diluted flavonoid extract was loaded on a cartridge, and impurities were removed by washing with 2 ml of water. Finally, total phenolic mixtures were eluted from the cartridge using 3 ml of methanol. The phenolic eluent was concentrated using N2 gas, and then re-dissolved in 0.5 ml of the extract solvents prior to analysis with UPLC-DAD-QTOF/MS. Internal standards were used follow as; 20 µg/ml of galangin and 250 µg/ml of 2,4,5-trimethoxycinnamic acid. All experimental analyses were performed in triplicate.

UPLC-DAD-QTOF/MS conditions

The analysis of phenolic compounds was performed according to the Kim et al. [18] using UPLC-DAD-QTOF/MS system (Waters Co.) equipped with a Kinetex 1.7 µ XB-C18 100 Å column (150 × 2.1 mm i.d.; Phenomenex, Torrance, CA, USA) for flavonoids and a Cortecs UPLC T3 column (150 × 2.1 mm i.d.; Waters Co.) for phenolic acids, respectively. The separation of flavonoids was carried out under the following instrumental conditions: flow rate of 0.3 ml/min, the mobile phases 0.5% formic acid in water (solvent A) and 0.5% formic acid in acetonitrile (solvent B). The gradient condition used: 0 min 5% (B), 20 min 25% (B), 25 min 50% (B), 30–32 min 90% (B), 35–40 min 5% (B). The chromatograms for flavonoids were acquired at 280 and 350 nm wavelengths for flavanones and flavones, respectively. Mass spectra were recorded in the range of m/z 200–1,200 using electrospray ionization as the ionization source in positive ion mode. The MS settings used were: cone voltage, 30 V; source temperature, 120 °C; desolvation temperature 500 °C; and desolvation N2 gas flow of 1,050 l/h. Separation of phenolic acids was carried out under the following instrumental conditions: flow rate of 0.3 ml/min, mobile phases 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The gradient condition used: 2–4 min 3% (B), 15 min 4% (B), 20 min 6% (B), 30 min 11% (B), 40 min 15% (B), 42 min 25% (B), 47–48 min 50% (B), 50–52 min 90% (B), 55–60 min 3% (B). The chromatograms for phenolic acids were acquired at 280 nm and 320 nm wavelengths for hydroxybenzoic acid and hydroxycinnamic acid derivatives, respectively. Mass spectra were recorded in the range of m/z 50–800 using positive ion electrospray. The MS settings used were: cone voltage, 40 V; source temperature, 120 °C; desolvation temperature 500 °C; and desolvation N2 gas flow of 1,020 l/h. Instrument control, and data acquisition and evaluation for flavonoids and phenolic acids were performed using MassLynx V4.1 software. The identification of individual compounds from the sample was achieved by comparing UV-spectra and mass fragmentation patterns with the library database constructed from the literature (Table 1).

Table 1 LC/MS and NMR library of S. plebeia R. Br. from the literature
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Results and discussion

Identification and quantification of flavonoids from S. plebeia R. Br.

The UPLC-DAD chromatogram revealed the presence of 10 peaks at 350 nm and 6 peaks at 280 nm (Fig. 1a, b). The chromatogram analysis, when compared with the constructed flavonoid library of S. plebeia showed possible novel compounds, glycosylation sites, and mass fragmentation patterns. The glycosylation of flavones and flavanones in S. plebeia occurred at the A-ring’s 7-position, except for in luteolin 5-O-glucoside. Furthermore, the hydroxylated and methylated derivatives of apigenin and luteolin were found to be conjugated at the 6-positions of the flavonoid backbones. The four flavone aglycones were detected and assigned as luteolin (peak 12), nepetin (peak 13), apigenin (peak 15), and hispidulin (peak 16) based on previously reported MS data [19, 20]. At retention times of 13.31 and 13.47 min, peaks 4 and 5 were produced and showed similar ion fragment patterns (m/z 471, 449, 287) by losing m/z 162, corresponding to a glucose or galactose moiety [21]. Conformation of peak 4 and 5 identification was performed by comparing the elution order of them with the literature. The 5-position glycosylation in the flavonoid ring system favor moderate polarity than that of the 7-position and result for faster retention time, hence, peak 4 and 5 were identified as luteolin 5-O-glucoside and luteolin 7-O-glucoside, respectively [22,23,24]. Peak 8 was assigned as apigenin 7-O-glucoside (cosmosiin) based on the parent ion m/z 433, sodium adducts ion m/z 455, and the characteristic loss of the hexose fragment to obtain m/z 271 as the fragment ion [25]. Thus, apigenin 7-O-glucoside is reported for the first time from S. plebeia.

Fig. 1
figure 1

UPLC-DAD chromatograms of phenolic compounds in S. plebeia R. Br. a at 350 nm for flavones; and b at 280 nm for flavanones. (1) 5,6,7,3′,4′-pentahydroxyflavanone 7-O-glucoside (naasalvinin B), (2) 6-hydroxyluteolin 7-O-glucoside, (3) 5,6,7,4′-tetrahydroxyflavanone 7-O-glucoside (naasalvinin C), (4) luteolin 5-O-glucoside (galuteolin), (5) luteolin 7-O-glucoside (cynaroside), (6) 5,7,3′,4′-tetrahydroxy-6-methoxyflavanone 7-O-glucoside (naasanone), (7) nepetin 7-O-glucoside (nepitrin), (8) apigenin 7-O-glucoside (cosmosiin), (9) 5,7,4′-trihydroxy-6-methoxyflavanone 7-O-glucoside (naasalvinin A), (10) hispidulin 7-O-glucoside (homoplantaginin), (11) 5,7,3′,4′-tetrahydroxy-6-methoxyflavanone, (12) luteolin, (13) nepetin (6-methoxyluteolin), (14) 5,7,4′-trihydroxy-6-methoxyflavanone, (15) apigenin, (16) hispidulin (6-methoxyapigenin)

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Peak 2 was a predominant peak observed on the UV spectrum, producing parent ion m/z 465 and fragment ion m/z 303 after losing a glucose moiety (Fig. 2a). Study of hydroxyl flavones confirmed 6-hydroxyluteolin 7-O-glucoside existed in S. plebeia using nuclear magnetic resonance spectroscopy (NMR) [19]. The fragmentation pattern was similar to that of peak 2, resulting in its identification as 6-hydroxyluteolin 7-O-glucoside.

Fig. 2
figure 2

MS/UV spectra and structures of four representative flavanones in S. plebeia R. Br. a 5,6,7,3′,4′-pentahydroxyflavanone 7-O-glucoside (naasalvinin B), b 5,6,7,4′-tetrahydroxyflavanone 7-O-glucoside (naasalvinin C), c 5,7,3′,4′-tetrahydroxy-6-methoxyflavanone 7-O-glucoside (naasanone), d 5,7,4′-trihydroxy-6-methoxyflavanone 7-O-glucoside (naasalvinin A)

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Fig. 3
figure 3

UPLC-DAD chromatogram and MS/UV spectra of rosmarinic acid in S. plebeia R. Br. a at 320 nm for RA; and b MS/UV spectra

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A total of six flavanones were identified from the chromatogram of S. plebeia. In previous studies, the flavone class of flavonoids was the most abundant flavonoid compounds to be presented in S. plebeia; however, in the present study, unlike to the previous reports significant concentration of flavanones class were identified. A comparison of the molecular weights of peaks 6 and 9 showed 16 Da increments that corresponded to peaks 1 and 3 (Table 2), respectively, owing to hydroxyl moiety substitutions at the position 3′ of the B-ring (Fig. 2c, d). Peaks 6 and 9 were assigned by comparing their spectroscopic data [14].

Table 2 Phenolic compounds and their mass spectrometric data in S. plebeia R. Br.
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Peak 6 was identified as 5,7,3′,4′-tetrahydroxy-6-methoxyflavanone 7-O-glucoside, and peak 9 as 5,7,4′-trihydroxy-6-methoxyflavanone 7-O-glucoside. Compared with peaks 6 (m/z 481) and 9 (m/z 465), the parent ion of peaks 1 (m/z 467) and 3 (m/z 451) showed increases of 14 Da, respectively, suggesting an additional CH3 group instead of an H atom. Thus, these two compounds were presumed to be 5,6,7,3′,4′-pentahydroxyflavanone 7-O-glucoside and 5,6,7,4′-tetrahydroxyflavanone 7-O-glucoside, respectively. Their characteristic product ions, as shown in Table 1, also demonstrated increases of 14 Da, suggesting that the methyl group attached to the phenolic hydroxyl group to form a methoxy group. Peaks 11 and 14 were the aglycone forms of peaks 6 and 9, respectively. These peaks were presumed to be 5,7,3′,4′-tetrahydroxy-6-methoxyflavanone and 5,7,4′-trihydroxy-6-methoxyflavanone. The detected flavanones were found as aglycones and glycosides, and showed various hydroxyl and methoxyl patterns (Supplementary Table 1). These two flavanone glycosides (peaks 1 and 3) were tentatively identified for the first time as new flavonoids from S. plebeia. We further proposed a common name for these flavanones as 5,7,4′-trihydroxy-6-methoxyflavanone 7-O-glucoside (naasalvinin A), 5,6,7,3′,4′-pentahydroxyflavanone 7-O-glucoside (naasalvinin B), 5,6,7,4′-tetrahydroxyflavanone 7-O-glucoside (naasalvinin C) and 5,7,3′,4′-tetrahydroxy-6-methoxyflavanone 7-O-glucoside (naasanone). Finally, flavonoids containing 6-hydroxyluteolin 7-O-glucoside and a series of naasalvinin were further required to separate single compounds and identify chemical structures using the NMR technique. However, further NMR data are required to clearly confirm the structures of the compounds.

In Salvia species, several flavanones have been reported, including 6-methoxynaringenin, 6-methoxynaringenin 7-O-glucoside, and filifolin, which have methoxy patterns at the 6-position of the flavanone backbone, except for eriodictyol [3, 26]. However, in our study, we did not detect these compounds, perhaps because of agro-ecological variations in plant growth, maturation stage, and/or the analytical methods used to extract the flavonoids [26,27,28].

As shown in Table 1, 6-hydroxyluteolin 7-O-glucoside, hispidulin 7-O-glucoside (homoplantaginin), and nepetin 7-O glucoside (nepetrin) were the major flavone compounds quantified, having concentrations of 2,452.7, 2,281.0, and 2,002.6 mg/100 g dry weight (DW), respectively, which accounted for 58.4% of the total flavonoids in the plant. The higher concentration of homoplantaginin, which indicates that this flavonoid is the major flavone in S. plebeia is inconsistence with previous reports [9]. 6-Hydroxyluteolin 7-O-glucoside was previously reported in plants of the Lamiaceae family as a major flavone glycoside [29, 30]. Luteolin 5-O-glucoside (galuteolin), naasanone, and naasalvinin B were also identified in significant amounts and quantified as 1,101.1, 938.3, and 775.1 mg/100 g DW, respectively.

The amount of flavonoids existing in the plant in aglycone forms was small, accounting for only 4.3% of the total flavonoids. In the genus Salvia, reasonable amounts of different types of flavonoids were identified. For example, apigenin (250 mg/100 g dry weight extracts; DWE) and its glycoside (360 mg/100 g DWE), isorhamnetin (290 mg/100 g DWE), and hispidulin (630 mg/100 g DWE) were reported from the ethyl acetate extract of S. officinalis [31]. Similarly, nepitrin, homoplantaginin, nepetin, and hispidulin were also reported 1,215, 1,050, 792, and 788 mg/100 g DWE, respectively, from the methanol extract of the plant [9]. Moreover, higher concentrations of cynaroside, nepitrin, homoplantaginin, nepetin, and hispidulin were identified from the ethanol extract of S. plebeia [16].

The abundance of luteolin 7-O-glucoside was found to be twofold higher than that of luteolin 5-O-glucoside. The natural glycosylation process in the plant was most likely to occur at 7′-, 3′-, and 4′-O-glucoside positions than the 5′-O-glucoside position. However, the present study indicated that the A-ring of the flavonoid is potentially conjugated at the 7-hydroxyl group of the sugar molecule [32]. The O-methoxylated flavonoids are also substituted at the 6-position of aglycones, such as nepetin. Based on the percentages of flavones (76.5%) and flavanones (23.5%) from total flavonoids, the former was predominant. Flavanones are natural primary biosynthetic intermediates for the synthesis of flavones [33]. Thus, the abundance of flavones may represent the utilization of flavanones as precursors for the biosynthesis of the flavones identified in S. plebeia.

Pharmacological activities of flavones have been reported in cell and animal model-based evaluations of individual compounds from S. plebeia. For example, luteolin and hispidulin had inhibitory effects on cytokine production in a postmenopausal osteoporosis model [28]. The 6-methoxyflavones, such as hispidulin and nepetin, were also reported as having antiviral activities against influenza A neuraminidase [27]. In particular, hispidulin, a marker compound in S. plebeia exhibited strong antioxidant, anti-fungal, anti-mutagenic, and anti-neoplastic properties [34, 35]. Even though, there was no report on the medicinal effect related to flavanone from S. plebeia; antioxidant [36], anti-tumoral [17], anti-diabetic [37], and anti-obesity [38] activities have been reported on this compound identified from Citrus spp. Flavanones could be a potential nutraceutical candidates [17, 39]. Thus, flavanones, such as naasalvinin B and C from Korean common sage, create unique flavanone derivatives that might be candidate compounds to treat chronic diseases.

Identification and quantification of phenolic acids from S. plebeia R. Br.

The hydrocinnamic derivative of phenolic acids besides the flavonoids was also quantitatively determined from S. plebeia. This peak was a predominant peak in the chromatogram compared to the other flavonoid peaks (Fig. 1a, b). The mass fragmentation revealed pseudomolecular ion [M + Na]+ at m/z 383, [M + H] + at m/z 361, and three product ions at m/z 181(− 180 mu, caffeic acid), m/z 163(− 198 mu, further loss of a H2O), and m/z 135(− 226 mu, further loss of a CO). Hence comparing its MS data with the literature this peak is identified as rosmarinic acid (RA) (Fig. 3) [3, 23, 26, 33]. RA and its derivatives were reported to be widely distributed in Lamiaceae family [40]. The previous studied were also stated that the presence of phenolic acids and its derivatives such as, protocatechuic acid, caffeic acid, syringic acid, vanillic acid, ethyl caffeate, and methyl rosmarinate [3, 11, 19, 26,27,28, 30]. In similar with previous studies higher concentration of RA was determined (6806.0 mg/100 g DW). The higher concentration of RA in this plant could make S. plebeia as a potential functional ingredient in cosmetic industry [40].

Conclusions

A total of 16 flavonoid compounds and 1 phenolic acid were quantitatively identified from S. plebeia. Flavonoids were classified into 10 flavones and 6 flavanones. Apigenin, hispidulin, and luteolin were identified from this plant. The hydroxy- and methoxy-derivatives of luteolin were also determined. 6-Hydroxyluteolin 7-O-glucoside was predominant flavonoids and assumed to be a chemo-marker for this plant. Four new compounds among the flavanones were identified from S. plebeia for the first time and named as naasanone and naasalvinin A, B, and C. RA was also identified as representative phenolic acid. Thus, from the data the presence of higher concentration of polyphenolic constitutes in S. plebeia supported the potential medicinal capabilities of the plant.