Phenolic compounds in common buckwheat sprouts: composition, isolation, analysis and bioactivities

Abstract

Phenolic compounds in common buckwheat sprouts (CBSs) have gained research interest because of their multiple health benefits. Phenolic acids, flavanones, flavonols, flavan-3-ols, and anthocyanins are important bioactive components of CBS that exhibit biological activities, including anti-inflammatory, antioxidant, anti-proliferative, and immunomodulatory effects. The isolation and quantitative and qualitative analyses of these phenolic compounds require effective and appropriate extraction and analytical methods. The most recent analytical method developed for determining the phenolic profile is HPLC coupled with a UV–visible detector and/or MS. This review highlights the extraction, purification, analysis, and bioactive properties of phenolic compounds from CBS described in the literature.

Introduction

Sprouts, formed from germinated seeds, are among the most important vegetable foods rich in bioactive compounds, including flavonoids, phenolic acids, vitamins, and minerals (Mun et al., 2020). Additionally, sprouts are a source of functional ingredients. Because consumer perceptions of healthy foods have led to increased consumption of sprouts, various sprout-related products have emerged in the global food market. Sprouting is of interest as an effective strategy that allows rapid cultivation as well as manipulation of phenolic compounds in seeds (Jang et al., 2021). Buckwheat (Fagopyrum spp.) is a member of the Polygonaceae family mainly consumed as a pseudo cereal; the main buckwheat species are common buckwheat (F. esculentum Möench) and tartary buckwheat (F. tataricum Gaertner). Buckwheat sprouts are globally consumed as salads and fresh vegetables (Kim et al., 2004).

An increasing number of chemical, epidemiological, and pharmaceutical studies have shown that consumption of sprouts as edible plants is associated with improved health benefits (Guo et al., 2018; Huda et al., 2021). These beneficial effects, including antioxidant, antitumor, and anti-inflammatory effects, have been partially attributed to the action of bioactive compounds in buckwheat sprouts, which contain flavonoids and phenolic acids (Ishii et al., 2008; Thangaraj et al., 2019; Xiao et al., 2021). The representative phenolic compound in tartary buckwheat sprouts is quercetin-3-O-rutinoside (rutin), whereas various flavonoids including rutin and phenolic acids are detected in common buckwheat sprouts (CBSs) (Nam et al., 2015). Four C-glycosyl flavones (orientin, isoorientin, vitexin, and isovitexin) and two flavonols (rutin and quercetin-3-O-robinobioside) are the predominant flavonoids in CBSs (Horbowicz et al., 2013). CBS extracts exhibited inhibitory effects on the production of cytokines, including interleukin- (IL-) 6, IL-12, and tumor necrosis factor-α (TNF-α) in lipopolysaccharide (LPS)-induced peritoneal macrophages isolated from BALB/c mice (Nam et al., 2017). Ajaghaku et al. (2018) reported that a mixture of rutin and quercetin-3-O-robinobioside enhanced immune function through the upregulation of CD₄⁺ T-lymphocytes. In another study, yeast-fermented CBS showed strong anti-hyperlipidemic and antioxidant activities in a mouse model (Chen et al., 2020).

Considering the numerous health benefits of phenolic compounds in CBSs, the development of effective techniques for their efficient recovery/extraction and accurate determination is essential. Buckwheat extracts, including those of CBSs, usually contain a complex mixture of phenolic compounds, which are mainly flavonoids, phenolic acids, and their derivatives (Huda et al., 2021). Phenolic compounds in plants usually occur in both free and bound forms. Free phenolics are usually extracted from plant samples with organic solvents (e.g., methanol, ethanol, and acetone), whereas bound phenolics are usually isolated using acid and alkaline hydrolysis followed by solvent extraction. The isolated phenolics are generally analyzed using LC-coupled with various detection methods to determine their specific identities and/or concentrations (Dzah et al., 2020a; Kumar, 2017).

CBSs are considered rich sources of bioactive compounds, and have received continuous research attention. Although CBSs contain many phytochemicals that can confer health benefits, including vitamins, proteins, fibers, minerals, and polysaccharides, we focused specifically on phenolic compounds. This review provides a general overview of the types of phenolic compounds present in CBSs and their potential health effects, as well as the approaches applied for analytical sample preparation (material pre-treatments, extraction, and purification methods) and phenolics determination. It thus organizes and provides relevant information to assist researchers in comparing and selecting suitable methods for the future development of an effective and efficient procedure for the analysis of phenolic compounds from CBSs.

Methodology

“Buckwheat sprouts” was used as the keyword for an extensive literature search. The published literature selected for data analysis in this review included experimental studies that analyzed phenolic compounds from CBSs or germinated buckwheat seeds. Review articles, meeting abstracts, proceedings papers, corrections, and experimental studies that characterized only total phenolics, total flavonoids, or other non-phenolic bioactive compounds were excluded. The following information was verified as a criterion for data analysis: CBS part, specific name class of the phenolic compound, analytical sample preparation technique and determination methods used for the analysis of phenolic compounds.

Phenolics in common buckwheat sprouts

CBSs are a rich source of phenolic compounds. Generally, sprouts are consumed as ready-to-eat salad vegetables and as fresh vegetables with flour-based noodles (Nam et al., 2015). CBSs play a critical role in human diets because of their bioactive compounds. Phenolic compounds, categorized as flavonoids, phenolic acids, stilbenes, and tannins, consist of at least one aromatic ring with one or more hydroxyl groups (Jung et al., 2021). Phenolic compounds are concentrated in the leaf, stem, and root portions during sprouting (Abdel-Aty et al., 2021; Lim et al., 2021). Flavonoids and phenolic acids were chosen as the representative phenolic compounds reported in this study. Details of the individual phenolic compounds identified and the contents of these compounds are summarized in Table 1.

Table 1.

Phenolic compounds reported in common buckwheat sprouts

No	Compounds	Cultivar	Concentrations (mg/g dry weight)	References
Flavone
1	Orientin	Kitawase	1.5	Nam et al. (2018a)
		Kitawase	6.99	Lee et al. (2014)
		–	6.63	Jang et al. (2019)
		Hruszowska (cotyledons)	8.18	Wiczkowski et al. (2014)
		–	5.15	Chen et al. (2020)
		Hruszowska	5.28	Horbowicz et al. (2013)
2	Isoorientin	Kitawase	3.2	Nam et al. (2018a)
		Kitawase	8.13	Lee et al. (2014)
		–	12.63	Jang et al. (2019)
		Hruszowska (cotyledons)	11.82	Wiczkowski et al. (2014)
		–	6.86	Chen et al. (2020)
		Hruszowska	16.38	Horbowicz et al. (2013)
3	Vitexin	Kitawase	2.0	Nam et al. (2018a)
		Kitawase	5.05	Lee et al. (2014)
		–	5.90	Jang et al. (2019)
		Hruszowska (cotyledons)	6.25	Wiczkowski et al. (2014)
		–	1.63	Chen et al. (2020)
		Hruszowska	4.89	Horbowicz et al. (2013)
4	Isovitexin	Kitawase	2.4	Nam et al. (2018a)
		Kitawase	5.70	Lee et al. (2014)
		–	10.48	Jang et al. (2019)
		Hruszowska (cotyledons)	14.08	Wiczkowski et al. (2014)
		–	5.28	Chen et al. (2020)
		Hruszowska	10.66	Horbowicz et al. (2013)
5	Apigenin	–	0.1 (FW)	Almuhayawi et al. (2021)
Flavonol
6	Quercetin	Hruszowska (cotyledons)	0.15	Wiczkowski et al. (2014)
		–	0.33	Chen et al. (2020)
		–	0.05	Park et al. (2017)
		–	0.8 (FW)	Almuhayawi et al. (2021)
7	Quercetin-3-O-robinobioside	Kitawase	0.9a	Nam et al. (2018a)
		–	3.5a	Jang et al. (2019)
		Hruszowska (cotyledons)	2.94	Wiczkowski et al. (2014)
		Hruszowska	1.57a	Horbowicz et al. (2013)
8	Rutin	Kitawase	2.0	Nam et al. (2018a)
		–	9.98	Jang et al. (2019)
			2.94	Wiczkowski et al. (2014)
			7.19	Chen et al. (2020)
		Hruszowska (cotyledons)	7.60	Ren and Sun (2014)
		Hruszowska	7.46	Horbowicz et al. (2013)
9	Isoquercetin	–	0.04 (FW)	Almuhayawi et al. (2021)
Flavan-3-ol
10	Catechin	–	0.97 (FW)	Almuhayawi et al. (2021)
		–	0.06	Park et al. (2019)
		–	0.09	Park et al. (2017)
11	Epicatechin	–	0.04	Park et al. (2019)
		–	0.05	Park et al. (2017)
		Hruszowska (cotyledons)	0.4	Wiczkowski et al. (2014)
Anthocyanins
12	Cyanidin-3-O-galactoside	Kitawase	0.02	Kim et al. (2007)
		Hruszowska (hypocotyls)	0.11	Wiczkowski et al. (2014)
13	Cyanidin 3-O-galactopyranosyl-rhamnoside	Kitawase	0.1	Kim et al. (2007)
		Hruszowska (cotyledons)	0.55	Horbowicz et al. (2015)
		Hruszowska (hypocotyls)	0.81	Horbowicz et al. (2015)
		Hruszowska (cotyledons)	0.49	Wiczkowski et al. (2014)
		Hruszowska (hypocotyls)	1.57	Wiczkowski et al. (2014)
14	Cyanidin-3-O-glucoside	Nepal native	0.01	Kim et al. (2007)
		Hruszowska (hypocotyls)	0.05	Wiczkowski et al. (2014)
15	Cyanidin-3-O-rutinoiside	Nepal native	0.02	Kim et al. (2007)
		Hruszowska (cotyledons)	0.07	Wiczkowski et al. (2014)
		Hruszowska (hypocotyls)	0.11	Wiczkowski et al. (2014)
		Hruszowska (cotyledons)	0.08	Horbowicz et al. (2015)
		Hruszowska (hypocotyls)	0.07	Horbowicz et al. (2015)
Phenolic acids
16	Chlorogenic acid	Kitawase	2.76	Lee et al. (2014)
		Kitawase	1.1	Kim et al. (2008)
		–	0.12	Park et al. (2017)
		–	0.13	Zhang et al. (2015)
		–	0.06	Park et al. (2019)
17	Caffeic acid	–	0.02	Park et al. (2017)
		–	0.006	Zhang et al. (2015)
		–	0.08	Park et al. (2019)
		–	0.8	Almuhayawi et al. (2021)
18	Syringic acid	–	0.007	Zhang et al. (2015)
19	p-Coumaric acid	– –	0.006 1.2 (FW)	Zhang et al. (2015)
20	Ferulic acid	– –	0.006 0.34 (FW)	Zhang et al. (2015)
21	Sinapic acid	–	0.003	Zhang et al. (2015)
22	Gallic acid	–	0.006	Zhang et al. (2015)
		–	0.006	Park et al. (2019)
		–	0.02 (FW)	Almuhayawi et al. (2021)
23	Ellagic acid	–	0.08 (FW)	Almuhayawi et al. (2021)

FW fresh weight; –: not available

^aConcentration of quercetin-3-O-robinobioside is described as rutin equivalent

Flavonoids are plant secondary metabolites and an important group of phenolic compounds that are known to be strong antioxidants due to their high redox potential (Jung et al., 2021). Some flavonoids, including flavones, flavonols, flavan-3-ols, and anthocyanins, have been identified in CBSs (Table 1). Their concentrations vary depending on the cultivar, light conditions, and period of growth. Four C-glycosyl flavones, orientin, isoorientin, vitexin, and isovitexin are the predominant flavones detected in CBSs. In contrast, rutin is the most abundant flavonoid identified in tartary buckwheat sprouts (Nam et al., 2015). The concentrations of isoorientin and isovitexin in CBSs were in the range 3.2–16.38 mg/g dry weight (DW) and 2.4–14.08 mg/g DW, respectively (Chen et al., 2020; Horbowicz et al., 2013; Jang et al., 2019; Lee et al., 2014; Nam et al., 2015; Wiczkowski et al., 2014). Wiczkowski et al. (2014) reported that orientin, isoorientin, vitexin, and isovitexin are major flavones in CBSs, with their highest content observed in cotyledons. These flavones were present in traces or not detected in tartary buckwheat sprouts (Nam et al., 2015). However, although the level of rutin in CBSs was determined to be lower than that in tartary buckwheat sprouts (Kim et al., 2008; Nam et al., 2017), rutin was also an abundant flavonol in CBSs, with a concentration in the range 2.0–9.98 mg/g DW. Quercetin-3-O-robinobioside is a rutin epimer present in rooibos tea, saskatoon fruit, and CBSs (Beelders et al., 2012; Nam et al., 2015; Ozga et al., 2007). As quercetin-3-O-robinobioside elutes slightly faster than rutin in reversed-phase HPLC analysis, it is essential to achieve their optimum separation to distinguish the epimers. Quercetin-3-O-robinobioside occurs at a concentration of 0.9–2.94 mg/g DW in CBSs, whereas it was not detected in tartary buckwheat sprouts (Nam et al., 2015). Almuhayawi et al. (2021) have isolated 0.8 mg/g fresh weight (FW) of quercetin and isoquercetin (0.04 mg/g FW) from CBS extracts. Two flavan-3-ols, catechin and epicatechin, were also isolated from CBSs, although their concentrations were lower than those of other flavonoids (Almuhayawi et al., 2021; Park et al., 2017; Wiczkowski et al., 2014). Several researchers have also identified and quantified anthocyanins, which are water-soluble pigments usually present in fruits and vegetables, from CBSs. Kim et al. (2007) reported that cyanidin-3-O-galactoside, cyanidin-3-O-galactopyranosyl-rhamnoside, cyanidin-3-O-glucoside, and cyanidin-3-O-rutinoiside are the major anthocyanins in CBS. The highest contents of cyanidin-3-O-rutinoiside and cyanidin-3-O-galactopyranosyl-rhamnoside were detected in cv. Hruszowska (Wiczkowski et al., 2014). The concentrations of anthocyanins in the hypocotyls were higher than those in cotyledons (Horbowicz et al., 2013). The accumulation of anthocyanins during sprouting has been shown to strongly depend on light emission as an essential stimulus (Seo et al., 2015).

The phenolic acids isolated from CBSs were chlorogenic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, sinapic acid, gallic acid, and ellagic acid. Chlorogenic acid was the most abundant phenolic acid identified in CBSs, with a concentration in the range 0.06–2.76 mg/g DW (Kim et al., 2008; Lee et al., 2014; Park et al., 2017, 2019; Zhang et al., 2015), whereas only trace amounts of other phenolic acids were detected in CBS (Almuhayawi et al., 2021; Park et al., 2017, 2019; Zhang et al., 2015). In general, phenolic acids occur at a lower amount in CBSs than flavonoids.

Analytical sample preparation and determination of phenolic compounds in CBS

CBSs contain abundant amounts of bioactive phenolic compounds, particularly flavonoids and phenolic acids, which confer potential health benefits (Huda et al., 2021). Flavonoids, including orientin, isoorientin, vitexin, isovitexin, rutin, and quercetin-3-O-robinobioside, have been reported as the major phenolic compounds in CBSs (Table 1). For optimal utilization of the biological properties of flavonoids and other phenolic compounds from CBSs, it is essential to develop efficient methods for analytical sample preparation (extraction and purification), identification, and quantification of phenolic compounds. Studies in the past decade (Tables 2 and 3) have employed various sample preparation procedures and analytical techniques for qualitative and quantitative analysis of specific phenolic compounds in CBSs. This section discusses some of the advances made in methods to extract/recover and determine the free and bound specific phenolic compounds from CBSs.

Table 2.

Various sample preparation methods for the analysis of phenolics from common buckwheat sprouts

Phenolicsa	Analytical sample preparationb	Purificationb	Analytical instrument c	References
Orientin, isoorientin, vitexin and rutin	0.1 g sample + 1 mL 80% EtOH (3 h shaking) → centrifuged (10,000 g, 10 min) → supernatant was collected		UFLC	Lim et al. (2012)
Orientin, isoorientin, vitexin, isovitexin, rutin and Q-Gal-Rha	0.05 g sample + 1 mL 60% MeOH containing 0.4% TFA → 0.5 min UAE and 0.5 min vortexed (2x) → centrifuged (5,000 g at 4 °C) → supernatant was collected → re-extraction of residue (5 × with same procedure)		HPLC–UV	Horbowicz et al. (2013)
Orientin, isoorientin, vitexin, isovitexin and rutin	(1) Analysis of rutin: sample + 50% acetone (2 h agitation) → suction filtration → evaporated → dried residue used (2) Analysis of other flavonoids: sample + 50% MeOH (2 h agitation) → centrifuged (10,000 rpm, 15 min) → supernatant was collected → re-extraction of residue (1 × with same procedure) → pooled supernatants were dried		CEUV HPLC–UV HPLC–MS	Koyama et al. (2013)
Rutin	200 mg sample + 40 mL 80% EtOH (16 h stirring, 40 °C) → centrifuged (8,000 g, 10 min) → filtered		HPLC–UV	Tsurunaga et al. (2013)
Orientin, isoorientin, vitexin, isovitexin, rutin, quercetin and chlorogenic acid	10 mg sample + 1 mL MeOH containing 10% phosphoric acid → vortexed 5 min → incubated (3 h at 37 °C) → centrifuged (1,000 g, 5 min) and filtered		HPLC–UV	Arasu et al. (2014)
Orientin, isoorientin, vitexin, isovitexin, rutin, quercetin and chlorogenic acid	10 mg sample + 1 mL MeOH containing 10% phosphoric acid → vortexed 5 min → incubated (3 h at 37 °C) → centrifuged (10,000 g, 5 min) and filtered		HPLC–UV	Kim et al. (2014b)
Orientin, isoorientin, vitexin, isovitexin, rutin, quercetin and chlorogenic acid	10 mg sample + 1 mL MeOH containing 10% phosphoric acid → vortexed 5 min → incubated (3 h at 37 °C) with 0.5 min vortexing every 1 h → centrifuged (1,000 g, 5 min) and filtered		HPLC–UV	Lee et al. (2014)
Rutin and quercetin	1 g sample + 95% EtOH (1:20 w/v) → 6 h heating (70 °C) → vacuum filtered, dissolved in 95% EtOH, and stored (4 °C)		HPLC–UV	Ren and Sun (2014)
Cy-Gal, Cy-Glu, Cy-Rut, Cy-Gal-Rha, orientin, isoorientin, vitexin, isovitexin, rutin, Q-Gal-Rha, Q-Glu, quercetin, epicatechin and procyanidin B2	0.05 g sample + 1 mL 60% MeOH containing 0.4% → TFA 0.5 min UAE and 0.5 min vortexed (2x) → centrifuged (13,200 g at 4 °C, 10 min) → supernatant was collected → re-extraction of residue (5 × with same procedure) → pooled supernatants were centrifuged (13,200 g at 4 °C, 20 min)		HPLC–DAD HPLC–MS HPLC–CoulArray–ECD	Wiczkowski et al. (2014)
Cy-Gal, Cy-Glu, Cy-Gal-Rha, Cy-Glu-Rha, orientin, isoorientin, vitexin, isovitexin, rutin and Q-Gal-Rha	0.05 g sample + 1 cm3 60% MeOH containing 0.4% TFA → 0.5 min UAE and 0.5 min vortexed (2x) →  centrifuged (5,000 g at 4 °C, 5 min) → supernatant was collected → re-extraction of residue (5 × with same procedure) → pooled supernatants were centrifuged (5,000 g at 4 °C, 15 min)		HPLC–DAD HPLC–MS	Horbowicz et al. (2015)
Orientin, isoorientin, vitexin, isovitexin, Q3R and rutin	15 g sample + 150 mL 70% EtOH (48 h soaking) → filtered → re-extraction of residue (2 × with same procedure) → filtered solvent was pooled → evaporated → 840 mg obtained extract was dissolved with 40 mL water and 80 mL EtOAc/n-BuOH (3:1) → 250 mg EtOAc/n-BuOH fractionated extract was collected after separation and evaporation (2x) → suspended in 20 mL water and 40 mL EtOAc → aqueous fraction was collected and dried → 70 mg dried extract was obtained and stored (4 °C)		HPLC–PDA HPLC–Q–TOF/MS	Nam et al. (2015)
Orientin, isoorientin, vitexin, isovitexin, rutin, K3R, quercitrin, myricetin, luteolin, quercetin, kaempferol, gallic acid, DHBA, THBA, p-HBA, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, sinapic acid and THCA	3 g sample + 50 mL 70% MeOH (1 h UAE) → filtered and concentrated to 10 mL → filtered		HPLC–UV HPLC–MS	Zhang et al. (2015)
Orientin, isoorientin, vitexin, isovitexin, rutin, Q-Gal-Rha, Cy-Gal-Rha and Cy-Glu-Rha	Sample + 60% MeOH containing 0.4% TFA (1:4)		HPLC–UV	Dębski et al. (2016)
Rutin	Sample + 50% MeOH → lyophilized → dissolved in 1 mL 10% aqueous acetonitrile with 0.1% formic acid		HPLC–UV	Nakamura et al. (2016)
Orientin, isoorientin, vitexin, isovitexin, rutin, apigenin, quercetin, luteolin, kaempferol, naringenin, gallic acid and caffeic acid	10 g sample + 90 mL absolute MeOH → HAE (22,000 rpm; 2 × 30 s extraction, 10 s pause each round) → 3 min incubation → solvent/supernatant was filtered	● Sorbent (100 mg Strata-X RP cartridges) ● 1st conditioning (3 mL absolute MeOH); 2nd conditioning (3 mL 10% MeOH) ● 30 mL diluted methanol extract (1:9 in water v/v) was loaded → washed with 3 mL 10% MeOH → dried with vacuum pump → elution (3 mL absolute MeOH) ● Eluted extract was evaporated, dissolved in 1 mL 50% MeOH (ultrasound and vortexing), and filtered	HPLC–MS	Terpinc et al. (2016)
Derivatives of benzoic acid and cinnamic acid	800 mg sample + 20 mL water (adjusted to pH 2; 6 M HCl) → 5 × extraction with 20 mL DEE → evaporated under vacuum → neutralized and lyophilized → residue was hydrolyzed in 20 mL 2 M NaOH (4 h, nitrogen atmosphere) → acidified to pH 2 (6 M HCl) → 5 × extraction with 30 mL DEE → 15 mL 6 M HCl was added, placed under nitrogen atmosphere → hydrolyzed in a boiling water bath (1 h) → 45 mL DEE was added → ether extracts were evaporated → dissolved in 10 mL methanol and filtered		HPLC–DAD	Wiczkowski et al. (2016)
Orientin, isoorientin, vitexin and rutin	Sample + absolute MeOH (1:20 w/v) → extract was dissolved in EtOH		HPLC–UV	Yang et al. (2016)
4-HBA, catechin, chlorogenic acid, caffeic acid, epicatechin, rutin and quercetin	0.1 g sample + 5 mL 80% MeOH containing 10% acetic acid → UAE (10 min at 28 °C) → water bath (1 h at 37 °C) → centrifugation (3,000 rpm, 10 min) → re-extraction of residue (1 × with same procedure) → pooled supernatant was dried using nitrogen gas → dissolved with 5 mL absolute MeOH → diluted, filtered and stored		HPLC–UV HPLC–MS	Park et al. (2017)
Orientin, isoorientin, vitexin, isovitexin, rutin and quercetin	Sample + 1 mL MeOH containing 10% phosphoric acid → vortexed 5 min → incubation (3 h at 37 °C) with vortexing every 1 h → centrifuged (1,000 g, 5 min) and filtered		HPLC	Jeong et al. (2018)
Orientin, isoorientin, vitexin, isovitexin and rutin	100 mg sample + 1 mL MeOH (overnight at −20 °C) → centrifuged (17,000 g, 10 min) and filtered		UPLC–MS	Koja et al. (2018)
Orientin, isoorientin, vitexin, isovitexin, Q3R and rutin	15 g sample + 150 mL absolute MeOH (48 h soaking) → 10 min HAE → solvent containing extract was filtered → re-extraction of residue (2 × with same procedure) → filtered solvent was pooled → evaporated and stored at −20 °C		HPLC–PDA	Nam et al. (2018a)
Orientin, isoorientin, vitexin, isovitexin, Q3R and rutin	15 g sample + 150 mL absolute MeOH (24 h soaking) → solvent containing extract was filtered → re-extraction of residue (2 × with same procedure) → filtered solvent was pooled → evaporated		HPLC–PDA	Nam et al. (2018b)
Orientin, isoorientin, vitexin, isovitexin, Q3R and rutin	Sample + 90% MeOH (30 min UAE) → centrifuged (2,232 g, 10 min) and filtered → re-extraction of residue (1 × with same procedure) → filtered supernatant was pooled → filtrate/supernatant was evaporated and stored at −50 °C		HPLC–PDA	Jang et al. (2019)
Orientin, isoorientin, vitexin, isovitexin, Q3R and rutin	● Ultrasound-assisted DES-based extraction technique ● Optimum: 80% CCTG, extraction temperature of 56 °C, extraction time of 40 min ● Sample:solvent (100 mg:1 mL) ● Extract was centrifuged (10,000 rpm, 30 min) → supernatant was filtered	● Column containing sorbent (Sep-Pak C18 cartridges) ● 1st conditioning (2 × 5 mL absolute MeOH); 2nd conditioning (2 × 5 mL 0.01 N aqueous HCl) → evaporation ● Extract (0.5 mL) was loaded to column ● 1st elution with 6 mL 0.01 N aqueous HCl ● 2nd elution with 2 × 4 mL absolute MeOH ● Evaporated extract was dissolved (10 mL) with absolute MeOH	HPLC–PDA HPLC–Q–TOF/MS	Mansur et al. (2019)
Benzoic acid, caffeic acid, catechin, chlorogenic acid, epicatechin, gallic acid and rutin	0.2 g sample + 2 mL 80% MeOH → vortexed 0.5 min → UAE (1 h at 37 °C) → centrifuged (16,000 g, 15 min) → re-extraction of residue (1 × with same procedure) → pooled supernatant was evaporated → dissolved with 2 mL absolute MeOH and filtered		HPLC–UV	Park et al. (2019)
Orientin, isoorientin, vitexin, isovitexin, rutin and quercetin	1 g sample + 15 mL 70% EtOH (1 h UAE) → filtered		UPLC–MS	Chen et al. (2020)
Orientin, isoorientin, vitexin, isovitexin, Q3R and rutin	● MSPD-based extraction ● Optimum: C18 sorbent/sample (2:1; 100 mg) and 10 min static extraction with 5 mL 80% EtOH ● Extract was filtered		HPLC–PDA	Mansur et al. (2020)
Orientin, isoorientin, vitexin, isovitexin, rutin and quercetin	Sample + 1 mL MeOH containing 10% phosphoric acid → vortexed 5 min → incubated (3 h at 37 °C) with vortexing every 1 h → centrifuged (1,000 g, 5 min) and filtered		HPLC–UV	Sim et al. (2020)
Rutin	1 g sample + 95% EtOH (2 h) → filtered and stored (−20 °C)		HPLC–PDA	Witkowicz et al. (2020)
Gallic acid, caffeic acid, ferulic acid, protocatechuic acid, p-coumaric acid, catechin, OHD, luteolin, apigenin, quercetin, isoquercetin, ellagic acid, velutin orientin, isoorientin, vitexin, isovitexin and rutin	50 mg sample + acetone:water (4:1 v/v) (24 h shaking) → filtered and centrifuged (4,000 g, 10 min) → filtrate evaporation → dissolved with absolute MeOH		HPLC–DAD	Almuhayawi et al. (2021)
Epicatechin, luteolin, orientin, vitexin, apigenin, naringenin, kaempferol, iso-rhamnetin, quercetin, 4-HBA, caffeic acid, sinapic acid, syringic acid, p-coumaric acid, ferulic acid and chlorogenic acid	Sample + MeOH, water, and formic acid (5 × extraction) → extracts were acidified (pH 2) with 6 M HCl → extraction with DEE → residues were hydrolyzed with 4 M NaOH and 6 M HCl → extraction with DEE → DEE extracts were pooled, evaporated, and diluted in 80% MeOH		HPLC–MS/MS	Dębski et al. (2021)
Rutin and quercetin	1) Free phenolics (ethanol extract): 1 g sample + 10 mL 80% EtOH (20 min) → centrifuged (6,500 g, 10 min) → re-extraction of residue (2 × with same procedure) → 3 supernatants were pooled and evaporated → diluted (10 mL) with MeOH and stored 2) Bound phenolics (hydrolysis): solid residue (after 3 × EtOH extraction) + 20 mL 2 M NaOH (24 h shaking) → acidified (pH 2) with 6 M HCl → centrifuged → supernatant was extracted with DEE/EtOAc (1:1) → evaporated, diluted (10 mL) with MeOH and stored		HPLC–UV	Hung et al. (2021)
Orientin, isoorientin, vitexin, isovitexin, Q3R and rutin	Sample + 90% MeOH (30 min UAE) → centrifuged (2,232 g, 10 min) and filtered → re-extraction of residue (1 × with same procedure) → filtered supernatant was pooled → filtrate/supernatant was evaporated	● Column containing sorbent (100 g Diaion HP-20 resin) ● Extract was loaded to column ● 1st elution (1 L water); 2nd elution (1 L absolute MeOH) → evaporation ● Evaporated extract was freeze-dried and stored (−50 °C)	HPLC–PDA HPLC–MS	Jang et al. (2021)
Vitexin and isovitexin	0.25 g sample + 3 mL 70% MeOH (10 min UAE) → centrifuged (1,800 g, 15 min) → supernatant was stored (−18 °C)		HPLC–DAD	Kalinová et al. (2021)
Orientin, isoorientin, vitexin, rutin, catechin, epicatechin, hyperin and p-coumaric acid	1) Free phenolics (methanol extract): 0.5 g sample + 3 mL 70% MeOH (40 min shaking at 200 rpm in the dark) → centrifuged (8,709 g, 8 min, 10 °C) → re-extraction of residue (2 × with same procedure) → 3 supernatants were pooled → diluted (10 mL) with 70% MeOH, filtered, stored (2 °C) 2) Bound phenolics (hydrolysis): solid residue (after 3 × MeOH extraction) + 20 mL 2 M NaOH (4 h shaking at 200 rpm in the dark) → acidified (pH 3.2–3.4) with 3.5 mL formic acid centrifuged (8,709 g, 8 min, 10 °C) → filtered and stored (2 °C)	● Sorbent (100 mg Strata-X RP cartridges) ● 1st conditioning (3 mL absolute MeOH); 2nd conditioning (3 mL water) ● 30 mL diluted methanol extract (1:9 in water v/v) or 3 mL hydrolyzed extract was loaded → washed with 4 mL water → dried with vacuum pump → elution (2 mL 70% MeOH) ● Eluted extract was filtered and stored (−80 °C)	HPLC–MS	Živković et al. (2021)

^aQ-Gal-Rha quercetin-3-O-galactorhamnoside, Cy-Gal cyanidin-3-O-galactoside, Cy-Glu cyanidin-3-O-glucoside, Cy-Rut cyanidin-3-O-rutinoside, Cy-Gal-Rha cyanidin-3-O-galactorhamnoside, Q-Glu quercetin-3-O-glucoside, Cy-Glu-Rha cyanidin-3-O-glucorhamnoside, Q3R quercetin 3-O-robinobioside, K3R kaempferol-3-O-rutinoside, DHBA 3,4-dihydroybenzoic acid, THBA 2,3,4-trihydroxybenzoic acid, p-HBA p-hydroxybenzoic, THCA trans-3-hydroxycinnamic acid, 4-HBA 4-hydroxybenzoic acid, OHD ortho-hydroxydaidzein

^bThe extractions were performed under lab/room temperature unless otherwise mentioned. EtOH ethanol, MeOH methanol, TFA trifluoroacetic acid, UAE ultrasound-assisted extraction, EtOAc ethyl acetate, n-BuOH n-butanol, HAE homogenizer-assisted extraction, DEE diethyl ether, DES deep eutectic solvent, CCTG a DES composed of choline chloride and triethylene glycol, MSPD matrix solid-phase dispersion extraction

^cThe more detailed information is available in Table 3

Table 3.

Various analytical methods for the determination of phenolics from common buckwheat sprouts

Instrument typea	Stationary phaseb	Mobile phasec	Flow rate; injection	Identification and/or quantificationd	Phenolics analytee	References
UFLC	C18 RP (5 μm, 250 × 4.6 mm)	A: 2% acetic acid in water B: 2% acetic acid in 45% acetonitrile	1 mL/min; 10 μL	355 nm External standard (RT) External standard calibration	Flavonoid	Lim et al. (2012)
HPLC–UV	C18 RP (3 μm, 250 × 2 mm); 45 °C	A: acetonitrile/water/formic acid (6:89:5) B: acetonitrile/water/formic acid (80:15:5)	0.2 mL/min	350 nm External standard (RT) External standard calibration	Flavonoid	Horbowicz et al. (2013)
CE–UV	Silica NP (75 μm); 25 °C	50 mM boric acid buffer containing 0.1 M SDS (pH 9.0)	Pressure injection (20 psi × 10 s)	280 nm External standard (RT) External standard calibration	Flavonoid	Koyama et al. (2013)
HPLC–UV	C18 RP (150 × 4.6 mm); 40 °C	A: 0.1% formic acid in water B: 0.1% formic acid in acetonitrile	0.8 mL/min; 10 μL	280 nm External standard (RT) External standard calibration
HPLC–MS	C18 RP (150 × 4.6 mm); 40 °C	A: 0.1% formic acid in water B: 0.1% formic acid in acetonitrile	0.8 mL/min; 10 μL	ESI positive mode Scanning (100–1000 m/z) External standard (RT) + MS spectra
HPLC–UV	C18 RP (150 × 4.6 mm); 40 °C	A: 0.1% formic acid in water B: 0.1% formic acid in acetonitrile	0.8 mL/min; 10 μL	280 nm External standard (RT) Rutin standard calibration	Flavonoid	Nakamura et al. (2013)
HPLC–UV	C18 RP (250 × 4.6 mm)	Acetonitrile/0.1% formic acid (16:84)	1 mL/min	370 nm External standard (RT) External standard calibration	Flavonoid	Tsurunaga et al. (2013)
HPLC–UV	C18 RP (5 μm, 250 × 4.6 mm); 40 °C	A: methanol/water/acetic acid (5:92.5:2.5) B: methanol/water/acetic acid (95:2.5:2.5)	1 mL/min; 10 μL	350 nm External standard (RT) External standard calibration	Flavonoid and phenolic acid	Arasu et al. (2014)
HPLC–UV	C18 RP (5 μm, 250 × 4.6 mm); 40 °C	A: methanol/water/acetic acid (5:92.5:2.5) B: methanol/water/acetic acid (95:2.5:2.5)	1 mL/min; 10 μL	350 nm External standard (RT) External standard calibration	Flavonoid and phenolic acid	Kim et al. (2014b)
HPLC–UV	C18 RP (5 μm, 250 × 4.6 mm); 40 °C	A: methanol/water/acetic acid (5:92.5:2.5) B: methanol/water/acetic acid (95:2.5:2.5)	1 mL/min; 10 μL	350 nm External standard (RT) External standard calibration	Flavonoid and phenolic acid	Lee et al. (2014)
HPLC–UV	C18 RP (5 μm, 250 × 4.6 mm); 50 °C	A: 0.2% acetic acid in water B: 0.2% acetic acid in methanol	5 μL	350 nm External standard (RT) External standard calibration	Flavonoid	Ren and Sun (2014)
HPLC–DAD	C18 RP (3 μm, 250 × 2 mm); 45 °C	A: acetonitrile/water/formic acid (6:89:5) B: acetonitrile/water/formic acid (80:15:5)	0.2 mL/min	350 nm (flavonoid); 520 nm (anthocyanin) External standard (RT) External standard calibration	Flavonoid	Wiczkowski et al. (2014)
HPLC–MS	C18 RP (3 μm, 250 × 2 mm); 45 °C	A: acetonitrile/water/formic acid (6:89:5) B: acetonitrile/water/formic acid (80:15:5)	0.2 mL/min	ESI positive mode External standard (RT) + MS spectra
HPLC–CoulArray–ECD	C18 RP (3 μm, 250 × 2 mm); 45 °C	Acetonitrile/water/phosphoric acid (9.99:89.99:0.02)	0.2 mL/min	+ 800–950 mV External standard (RT) External standard calibration
HPLC–DAD	C18 RP (3 μm, 250 × 2 mm); 45 °C	A: acetonitrile/water/formic acid (6:89:5) B: acetonitrile/water/formic acid (80:15:5)	0.2 mL/min	350 nm (flavonoid); 520 nm (anthocyanin) External standard (RT) External standard calibration	Flavonoid	Horbowicz et al. (2015)
HPLC–MS	C18 RP (3 μm, 250 × 2 mm); 45 °C	A: acetonitrile/water/formic acid (6:89:5) B: acetonitrile/water/formic acid (80:15:5)	0.2 mL/min	ESI positive mode External standard (RT) + MS spectra
HPLC–PDA	C18 RP (5 μm, 250 × 4.6 mm); 25 °C	A: 0.1% formic acid in water B: 0.1% formic acid in acetonitrile	0.8 mL/min; 20 μL	350 nm External standard (RT)	Flavonoid	Nam et al. (2015)
HPLC–Q–TOF/MS	C18 RP (5 μm, 250 × 4.6 mm); 40 °C	A: 0.1% formic acid in water B: 0.1% formic acid in acetonitrile	0.8 mL/min; 20 μL	ESI positive and negative mode Scanning (50 – 1700 m/z) External standard (RT) + MS spectra
HPLC–UV	C18 RP (5 μm, 250 × 4.6 mm); 30 °C	1) C-glycosyl flavones analysis: A: 0.1% formic acid in water B: methanol 2) Other flavonoid analysis: A: 0.15% phosphoric acid in water B: acetonitrile 3) Phenolic acid analysis: A: 0.15% phosphoric acid in water B: methanol	1 mL/min (flavonoid), 0.7 mL/min (phenolic acid); 20 μL	355 nm (flavonoid); 270 nm (phenolic acid) External standard (RT)	Flavonoid and phenolic acid	Zhang et al. (2015)
HPLC–MS	C18 RP (5 μm, 250 × 4.6 mm); 30 °C	1) C-glycosyl flavones analysis: A: 0.1% formic acid in water B: methanol 2) Other flavonoid analysis: A: 0.15% phosphoric acid in water B: acetonitrile 3) Phenolic acid analysis: A: 0.15% phosphoric acid in water B: methanol	1 mL/min (flavonoid), 0.7 mL/min (phenolic acid); 20 μL	ESI negative mode Scanning (50 – 1000 m/z) External standard (RT) + MS spectra External standard calibration
HPLC–UV	C18 RP (3 μm, 250 × 2 mm); 45 °C	A: acetonitrile/water/formic acid (6:89:5) B: acetonitrile/water/formic acid (80:15:5)	0.2 mL/min	350 nm (flavonoid); 520 nm (anthocyanin) External standard (RT) External standard calibration	Flavonoid	Dębski et al. (2016)
HPLC–UV	C18 RP (150 × 4.6 mm); 40 °C	A: 0.1% formic acid in water B: 0.1% formic acid in acetonitrile	0.8 mL/min; 10 μL	280 nm External standard (RT) Rutin standard calibration	Flavonoid	Nakamura et al. (2016)
HPLC–MS	C18 RP (2.6 μm, 100 × 2 mm); 25 °C	A: 0.1% formic acid in water B: acetonitrile	0.3 mL/min; 10 μL	ESI negative mode External standard (RT) + MS spectra External standard calibration	Flavonoid and phenolic acid	Terpinc et al. (2016)
HPLC–DAD	C18 RP (3 μm, 250 × 2 mm); 45 °C	A: acetonitrile/water/formic acid (6:89:5) B: acetonitrile/water/formic acid (80:15:5)	0.2 mL/min	280 and 320 nm External standard (RT) External standard calibration	Phenolic acid	Wiczkowski et al. (2016)
HPLC–UV	C18 RP (5 μm, 250 × 4.6 mm); 35 °C	A: acetic acid/water (2:98) B: acetic acid/ acetonitrile/water (2:45:53)	1 mL/min; 10 μL	355 nm External standard (RT) External standard calibration	Flavonoid	Yang et al. (2016)
HPLC–UV	C18 RP (5 μm, 250 × 4.6 mm); 30 °C	A: acetic acid/water (0.15:99.85) B: methanol	1 mL/min; 20 μL	280 nm External standard (RT) External standard calibration	Flavonoid and phenolic acid	Park et al. (2017)
HPLC–MS	C18 RP (5 μm, 250 × 4.6 mm); 30 °C	A: acetic acid/water (0.15:99.85) B: methanol	1 mL/min; 20 μL	ESI negative mode Scanning (100–1300 m/z) External standard (RT) + MS spectra External standard calibration
HPLC	C18 RP (5 μm, 250 × 4.6 mm)	A: water B: acetic acid/methanol (5:95)	1 mL/min; 5 μL	254 nm External standard (RT) External standard calibration	Flavonoid	Jeong et al. (2018)
UPLC-MS	C18 RP (1.7 μm, 50 × 2.1 mm); 40 °C	A: 0.1% formic acid in water B: 0.1% formic acid in methanol	0.25 mL/min	ESI negative mode External standard (RT) + MS spectra External standard calibration Internal standard (chrysin)	Flavonoid	Koja et al. (2018)
HPLC–PDA	C18 RP (5 μm, 250 × 4.6 mm); 40 °C	A: 0.1% formic acid in water B: 0.1% formic acid in acetonitrile	0.8 mL/min; 20 μL	350 nm External standard (RT) External standard calibration	Flavonoid	Nam et al. (2018a)
HPLC–PDA	C18 RP (5 μm, 250 × 4.6 mm); 40 °C	A: 0.1% formic acid in water B: 0.1% formic acid in acetonitrile	0.8 mL/min; 20 μL	350 nm External standard (RT) External standard calibration	Flavonoid	Nam et al. (2018b)
HPLC–PDA	C18 RP (5 μm, 250 × 4.6 mm); 40 °C	A: 0.1% formic acid in water B: 0.1% formic acid in acetonitrile	1 mL/min; 5 μL	360 nm External standard (RT) External standard calibration	Flavonoid	Jang et al. (2019)
HPLC–PDA	C18 RP (5 μm, 250 × 4.6 mm); 40 °C	A: 0.1% formic acid in water B: 0.1% formic acid in acetonitrile	0.8 mL/min; 10 μL	350 nm External standard (RT) External standard calibration	Flavonoid	Mansur et al. (2019)
HPLC–Q–TOF/MS	C18 RP (1.8 μm, 150 × 2.1 mm); 40 °C	A: 0.1% formic acid in water B: 0.1% formic acid in acetonitrile	0.3 mL/min; 2 μL	ESI negative mode Scanning (100–1000 m/z) External standard (RT) + MS spectra
HPLC–UV	C18 RP (5 μm, 250 × 4.6 mm); 30 °C	A: acetic acid/water (0.15:99.85) B: methanol	1 mL/min; 20 μL	280 nm External standard (RT) External standard calibration	Flavonoid and phenolic acid	Park et al. (2019)
UPLC-MS	C18 RP (1.7 μm, 150 × 2.1 mm); 40 °C	A: 0.1% formic acid in water B: 0.1% formic acid in acetonitrile	0.3 mL/min; 1 μL	ESI positive mode External standard (RT) + MS spectra External standard calibration	Flavonoid	Chen et al. (2020)
HPLC–PDA	C18 RP (5 μm, 250 × 4.6 mm); 40 °C	A: 0.1% formic acid in water B: 0.1% formic acid in acetonitrile	0.8 mL/min; 10 μL	350 nm External standard (RT) External standard calibration	Flavonoid	Mansur et al. (2020)
HPLC–UV	C18 RP (5 μm, 250 × 4.6 mm)	A: water B: 5% acetic acid in methanol	1 mL/min; 20 μL	254 nm External standard (RT) External standard calibration	Flavonoid	Sim et al. (2020)
HPLC–PDA	C18 RP (5 μm, 250 × 4.6 mm); 25 °C	A: 1% formic acid in water B: 1% formic acid in acetonitrile	1 mL/min	370 nm External standard (RT) External standard calibration	Flavonoid	Witkowicz et al. (2020)
HPLC–DAD	Silica NP (7 μm, 150 × 3 mm)	A: water-formic acid (90:10) B: acetonitrile/water/formic acid (85:10:5)	0.8 mL/min; 20 μL	External standard (RT) External standard calibration Internal standard (3,5-dichloro-4-hydroxybenzoic)	Flavonoid and phenolic acid	Almuhayawi et al. (2021)
HPLC–MS/MS	C18 RP (2.7 μm, 50 × 0.5 mm); 45 °C	A: water/formic acid (99.05:0.95) B: acetonitrile/formic acid (99.05:0.95)	0.015 mL/min	ESI negative mode (MRM) External standard (RT) + MS spectra External standard calibration	Flavonoid and phenolic acid	Dębski et al. (2021)
HPLC–UV	C18 RP (5 μm, 150 × 4.6 mm); 35 °C	A: 1% phosphoric acid in water B: methanol	1 mL/min	280 nm External standard (RT) External standard calibration	Flavonoid	Hung et al. (2021)
HPLC–PDA	C18 RP (5 μm, 250 × 4.6 mm); 40 °C	A: 0.1% formic acid in water B: 0.1% formic acid in acetonitrile	1 mL/min; 5 µL	360 nm External standard (RT) External standard calibration	Flavonoid	Jang et al. (2021)
HPLC–MS	C18 RP (5 μm, 250 × 4.6 mm); 40 °C	A: 0.1% formic acid in water B: 0.1% formic acid in acetonitrile	1 mL/min; 5 µL	ESI negative and positive mode Scanning (200–1200 m/z) External standard (RT) + MS spectra
HPLC–DAD	C18 RP (3 μm, 150 × 2 mm); 25 °C	A: 5% acetonitrile + 0.1% o-phosphoric acid B: 80% acetonitrile + 0.1% o-phosphoric acid	0.25 mL/min	350 nm External standard (RT) External standard calibration	Flavonoid	Kalinová et al. (2021)
HPLC–MS	C18 RP (2.7 μm, 150 × 2.1 mm); 30 °C	A: 0.1% aqueous formic acid B: acetonitrile	0.25 mL/min; 20 µL	ESI negative mode External standard (RT) + MS spectra External standard calibration	Flavonoid and phenolic acid	Živković et al. (2021)

^aUFLC ultra-fast liquid chromatography, CE capillary electrophoresis, UPLC ultra-high performance liquid chromatography, DAD diode array detector, ECD electrochemical array detector, PDA photodiode array detector, Q-TOF/MS quadrupole time-of-flight/MS

^bRP reversed-phase, NP normal phase

^cSDS: sodium dodecyl sulfate; C-glycosyl flavones: orientin, isoorientin, vitexin and isovitexin

^dRT: retention time comparison; ESI: electron spray ionization; MS spectra: data from MS analysis

^eThe specific name for polyphenol analyte is described in Table 2

The preparation of analytical samples usually involves several steps, including sample dehydration and size reduction, extraction, hydrolysis, purification, and storage (Dzah et al., 2020a). Table 2 summarizes the various techniques used to prepare analytical CBS samples for the determination of their phenolic compounds. In most studies, CBS samples were first subjected to dehydration using oven drying or freeze-drying (lyophilization), followed by particle size reduction by milling or grinding combined with sieving as required. Reduced moisture after dehydration minimizes the rate of lipid oxidation (Kim et al., 2014a) and enzyme activity in the sample (George et al., 2014). Moreover, reduced particle size leads to an increase in surface area, resulting in increased interaction with solvent and mass transfer of phenolic analytes, while sieving enhances the homogeneity and extraction efficiency (Dzah et al., 2020a).

Various factors, including the chemical nature of the isolated analytes, methods employed for extraction and purification, storage conditions, and interfering analytes, may influence the extraction and recovery yields of specific phenolic analytes from the extracts (Naczk and Shahidi, 2004). As shown in Table 2, methods used for preparation of analytical CBS samples vary widely among studies. For instance, most studies used low sample weights between 0.01 and 1 g, with only a few studies using high sample weights between 3 and 15 g (Nam et al., 2015, 2018a, b; Terpinc et al., 2016; Zhang et al., 2015). The lowest sample-to-solvent ratio was approximately 0.05:10 (w/v) (Tsurunaga et al., 2013) and the highest was approximately 1.67:10 (w/v) (Živković et al., 2021). Conventional organic solvents, such as methanol (50–100%), ethanol (70–100%), and aqueous acetone (50–80%) have been frequently used for the solid–liquid extractions of free phenolic compounds and their derivatives from CBS, in the order methanol > ethanol > aqueous acetone. In some cases, the extraction solvents were acidified with aqueous phosphoric acid (Arasu et al., 2014; Jeong et al., 2018; Kim et al., 2014b; Lee et al., 2014; Sim et al., 2020), acetic acid (Park et al., 2017), trifluoroacetic acid (Dębski et al., 2016; Horbowicz et al., 2015; Wiczkowski et al., 2014) or formic acid (Dębski et al., 2021). In addition, the use of deep eutectic solvents (DESs), a new group of organic solvents developed as promising environmentally friendly alternatives to conventional solvents, has been investigated for the extraction of major flavonoids from CBS. A DES composed of choline chloride (hydrogen bond acceptor) and triethylene glycol (hydrogen bond donor) (80% in water v/v) was found to be more effective than methanol for the extraction of vitexin and quercetin-3-O-robinobioside (Mansur et al., 2019).

Solid–liquid extractions of phenolics from CBSs (Table 2) were performed either under static conditions (conventional soaking with solvent, with or without heating) or under dynamic conditions using classical techniques such as vortexing, agitation, shaking, and stirring. In some studies, more advanced techniques such as ultrasound-assisted extraction (UAE) and homogenizer-assisted extraction (HAE) have also been used to increase the extraction yield and efficiency, with UAE being employed more often than HAE. A comparative assessment based on literature by Dzah et al. (2020b) revealed that the UAE method not only contributes to increased extraction yield of phenolic compounds, but also better preserves and increases their biological activity. The solid–liquid extractions of phenolics from CBSs (Table 2) were performed mostly at room temperature. Heating treatments ranging from 28 to 70 °C were applied in some studies; Ren and Sun (2014) performed heat treatment at 70 °C for 6 h using a water boiler. Alternatively, Koja et al. (2018) applied overnight extraction at a freezing temperature of −20 °C.

Phenolic compounds extracted from CBSs in most of the studies summarized in Table 2 contained the only free phenolics and not the bound fraction, which could be partly attributed to the residues being discarded after extraction with organic solvents. Some studies used the residues to perform re-extraction up to one to five times using the same procedure as that for the first extraction, to enhance the extraction yields of free phenolic compounds. Moreover, after re-extraction, simultaneous fractionation of the residue using water, ethyl acetate/n-butanol (EtOAc/n-BuOH, 3:1 v/v), water, and EtOAc was performed by Nam et al. (2015). To extract bound phenolic compounds from the residues of CBS extracts, some studies combined alkaline (NaOH) and acid (HCl or formic acid) hydrolysis with diethyl ether (DEE) (Debski et al., 2021; Wiczkowski et al., 2016; Živković et al., 2021) or DEE/EtOAc (1:1 v/v) (Hung et al., 2021). The combination of acid and alkaline hydrolysis allows the extracts to be neutralized and prevents further hydrolytic degradation, thereby allowing successful separation and purification of the phenolic compounds (Dzah et al., 2020a).

The unwanted compounds or fractions that may affect the final extraction/recovery yield and interfere in the determination of the target compounds can be removed by purifying the extract samples (Kumar, 2017). In some studies, the solid-phase extraction (SPE)-based purification technique was employed (Table 2). The CBS extracts containing phenolic compounds were retained in a specific sorbent (C18 or resin) with or without column preconditioning using organic solvents (methanol, aqueous methanol, aqueous HCl, or water). The unwanted fractions in the extracts were washed with aqueous methanol, aqueous HCl, or water, and the retained phenolic compounds were eluted with methanol. An alternative to traditional SPE is the matrix solid-phase dispersion extraction technique, which has the advantage of simultaneously extracting and purifying major flavonoids (Mansur et al., 2020). The CBS extracts were obtained using centrifugation (1,000–17,000 × g; 3,000–22,000 rpm), filtration (using filter paper, vacuum filter or membrane filter), and drying (via vacuum evaporation, nitrogen gas, or freeze-drying) during and/or after the extraction and/or purification steps. The obtained extracts were stored either in liquid form dissolved in organic solvents (methanol, ethanol, and acetonitrile) or in dried form at 4 or −50 °C. The preparation times for the CBS analytical sample varied widely among studies, from a few minutes up to days to complete the procedure. Sample preparation procedures that required the longest time used conventional extraction techniques such as soaking or shaking (Nam et al., 2015, 2018a, b; Almuhayawi et al., 2021).

Various analytical methods have been used for the qualitative and quantitative determination of multiple phenolic compounds in plant extracts. Among them, LC-based techniques have been the most widely applied (Dzah et al., 2020a; Kumar, 2017), especially for the analysis of buckwheat extracts (Huda et al., 2021). Regarding the analysis of CBS extracts, LC [HPLC, ultra-fast liquid chromatography (UFLC), and ultra-high performance liquid chromatography (UPLC)] systems equipped with an UV detector, diode array detector (DAD), or photodiode array detector (PDA) have been frequently applied for the separation and determination of phenolic compounds (Table 3). To improve the sensitivity of the analytical method, some studies employed LC coupled with MS techniques, such as MS, tandem MS/MS, and quadrupole time-of-flight/MS (Q–TOF/MS).

The stationary phase (column) is a major component of LC, and is used mainly for the separation of analytes. The non-polar C18 reverse-phase (RP) column was most frequently used for the analysis of phenolic compounds in CBSs, although its specifications varied across studies, including particle size (1.7–5 µm), length (50–250 mm), and inner diameter (ID; 0.5–4.6 mm). The UPLC and MS-based techniques used columns with lower particle sizes, lengths, and IDs. According to Žuvela et al. (2019), although hundreds of RP-LC column types are available in the market, none of them are almost identical. The differences between columns are mainly in the medium type (monolithic, porous, or non-porous), geometry (particle size and shape, diameter and pore volume, and area of the bed), bed chemical properties (type of attached ligands and their density), and stationary-phase carrier composition (carbon, polymers, or silica). Therefore, there is no universal RP-LC column that can cover a variety of analytes.

The polarity and pH of mobile phase solvents, as well as column temperature, are also important LC parameters for the separation of analytes (Dzah et al., 2020a). Various mobile phase systems (Table 3) composed of polar solvents, such as water and methanol or acetonitrile (either in absolute or aqueous forms), with or without the addition of formic acid (0.1–10% v/v), acetic acid (0.15–5% v/v), or phosphoric acid (0.02–1% v/v), have been used for the elution and separation of phenolic compounds from CBS extracts by LC with binary gradient elution (elution flow rates of 0.015–1 mL/min) and column temperatures of 25–50 °C. According to Bligh et al. (2013), binary gradient elution systems help separate compounds from different classes of flavonoids using RP-LC columns. The addition of acids to mobile phase solvents serves to avoid a rise in pH and ionization, as well as a reduced retention time during RP-LC separation, thereby enhancing the resolution and reproducibility of each separation.

The detector in the LC system is responsible for turning a physical or chemical attribute into a measurable signal corresponding to the identity and/or concentration of the analyte. The UV, DAD, and PDA detectors are the most common LC detectors in use today because many analytes of interest absorb in the UV and visible regions between 190 and 600 nm (Swartz, 2010). Table 3 shows that phenolic acids, anthocyanins, and flavonoids other than anthocyanin were identified at 270–320 nm, 520 nm, and 254–370 nm, respectively. The phenolic analytes were identified based on the consistency in retention time of detected analytes in the sample with that of the corresponding reference standards, and quantified using an external calibration curve derived from the reference standards analyzed at different concentration levels. The LC method provides only limited structural information (UV spectrum) and tentative identification of the eluted analyte peaks in the samples by comparison with reference standards (Kumar, 2017). Thus, complete peak separation is necessary for both qualitative and quantitative determination using LC analysis to enhance its reliability.

More advanced detectors, including MS, MS/MS, and Q–TOF/MS, have also been applied for the identification and quantification of phenolic compounds in CBS extracts. Electron spray ionization (ESI) modes, either negative, positive, or both positive and negative, are usually applied to determine the fragmentation patterns of the ions. After ionization of the sample, the molecular masses of the phenolic compounds were mostly calculated based on the m/z in the range 50–1700. The analytes were identified based on their retention times and MS spectra, and their concentrations were calculated relative to the corresponding external standards from the calibration curves (Table 3). Several MS-based analytical techniques have become popular choices in the analysis of buckwheat extracts (Huda et al., 2021) because their sensitivity and selectivity facilitate the analysis of phenolic compounds present at low concentrations in plant extracts (Dzah et al., 2020a; Kumar, 2017). The ESI–MS analysis is also highly compatible with the polar solvents used as mobile phase in RP-LC because they readily undergo electrochemical reactions in the spraying nozzle, thus enabling the combination of the LC system with MS detectors (Banerjee and Mazumdar, 2012; Lim and Lord, 2002). The use of LC–MS techniques also allows simultaneous monitoring of the retention time of the isolated compound peak and determination of their MS data during the analysis (Santi and Dipjyoti, 2013). In addition, LC–MS techniques are widely considered more powerful than conventional LC because they can be used for qualitative analysis of phenolic compounds even when baseline separation is not achieved. However, flavonoid isomers (e.g., orientin, isoorientin, vitexin, isovitexin, rutin, and quercetin-3-O-robinobioside) need to be fully separated (Fig. 1) to enable their accurate quantitative analysis in food, as they exhibit identical molecular masses and similar fragment patterns, and cannot be distinguished based on only MS information (Nam et al., 2015; Jang et al., 2019; Mansur et al., 2019).

Fig. 1.

HPLC chromatogram of major flavonoids in common buckwheat sprouts at 350 nm. Peak 1: orientin; 2: isoorientin; 3: vitexin; 4: isovitexin; 5: quercetin-3-O-robinobioside; 6: rutin

The determination of phenolic compounds in CBS is a complex process that requires a combination of multiple analytical approaches, such as analytical sample preparation, separation, identification, and quantification of analytes. It is difficult to recommend a universally suitable combined method from the available studies because none of the methods are comprehensive enough to cover all the analytes (e.g., flavonoids, phenolic acids, anthocyanin, free or bound forms, isomers), and each method has its own advantages and disadvantages. Nevertheless, by considering the nature of the target analytes and the characteristics, advantages, and disadvantages of the analytical techniques, a combination of effective and efficient approaches can be utilized for better outcomes.

Health benefits of CBS and its phenolic compounds

Antioxidant activity

Sprouts contain a wide range of antioxidants (Guo et al., 2018), including flavonoids, which possess many biological properties, such as anti-cancer, anti-aging, and anti-inflammatory effects (Lee et al., 2018; Nam et al., 2017; Rha et al., 2020). Regular consumption of natural antioxidants has been shown to significantly reduce and delay reactive oxygen species (ROS)-related diseases (Ravula et al., 2021). The in vitro antioxidant activity of CBSs was evaluated using the commonly used DPPH and ABTS radical scavenging assays. The results are expressed as the percentage of radical scavenging activity, Trolox equivalents, or vitamin C equivalents. CBS ethanol extracts showed approximately 88% DPPH scavenging activity at a concentration of 5 mg/mL (Liu et al., 2008). Jeong et al. (2018) and Witkowicz et al. (2020) reported DPPH scavenging activity in CBS extracts to be 8000 mg Trolox equivalents/100 g DW and 46.88 μM Trolox equivalents/g DW, respectively. The antioxidant activity of common buckwheat seeds against DPPH was 1138 mg Trolox equivalents/100 g (Kim et al., 2019), indicating that the sprouts had significantly higher antioxidant activity than the seeds. The DPPH scavenging activity of CBSs grown in the dark was 25 mg vitamin C equivalents/g DW (Nam et al., 2018a, b). With the ABTS radical scavenging assay, CBS ethanol extracts exhibited approximately 2 μM Trolox equivalents/g FW (Almuhayawi et al., 2021). Sim et al. (2020) reported that the CBS methanol extract produced approximately 5 mg Trolox equivalents/g FW. Against the ABTS radical, the flavonoid isomers exhibited scavenging activity in the order isoorientin > rutin > orientin > isovitexin > vitexin (Nam et al., 2018a, b). Accumulation of intracellular ROS is a significant cause of numerous metabolic diseases. Jeong et al. (2018) found that CBS extracts decreased ROS generation in a dose-dependent manner in a tert-butyl hydroperoxide-induced HepG2 cell model. CBSs also significantly reduced intracellular oxidative stress, including peroxides and superoxide anions, in HepG2 cells (Liu et al., 2008). Although the intracellular antioxidant activities of CBSs are not well described, CBSs and their flavonoids are potential sources of antioxidants that can play a critical role in the prevention of ROS-related diseases.

Anti-inflammatory activity

Significant amounts of inflammatory mediators are produced during inflammation, and excessive production of mediators is a major cause of chronic inflammatory diseases (Lee et al., 2018). Biomedicine research has focused on finding natural therapeutic agents for inflammation-associated diseases with minimal toxicity and high potency. The anti-inflammatory activities of CBSs and their flavonoids are listed in Table 4. CBS extracts have been reported to show strong inhibitory effects on the inflammatory mediators [nitric oxide (NO), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2)] and cytokines including tumor TNF-α, IL-6, and IL-12 in LPS-induced RAW 264.7 and peritoneal macrophages (Nam et al., 2017). Almuhayawi et al. (2021) reported that CBS extracts directly inhibited COX-2 and lipoxygenase activities. Ishii et al. (2008) demonstrated the suppressive effects of CBS extracts on human colon cancer cells and mice via the inhibition of cytokine upregulation. The activation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) is closely linked to cytokine production (Kim et al., 2020). Isoorientin attenuated LPS-induced pro-inflammatory responses by downregulating the MAPKs and NF-κB signaling pathways (Yuan et al., 2014). Yoo et al. (2014) found that rutin downregulated high-mobility group box 1 protein-dependent inflammatory responses in human umbilical vein endothelial cells. Orientin significantly reduced the inflammatory response in LPS-stimulated acute lung injury in mice by suppressing the activation of NF-κB and the nucleotide-binding domain-like receptor protein 3 inflammasome (Xiao et al., 2021). Thus, CBSs and their flavonoids are potential natural therapeutic agents against inflammation.

Table 4.

Health benefits of common buckwheat sprout extracts and their phenolics

Extract/phenolicsa	Beneficial effects	Mechanismb	References
80% (v/v) ME	Anti-inflammatory effect	Inhibition of cytokines, COX-2, NO production, NF-κB activation, and MAPK in LPS-induced RAW 264.7 and peritoneal macrophages	Nam et al. (2017)
80% (v/v) EE	Anti-inflammatory effect	Suppression of NO and cytokines production in LPS-induced RAW 264.7 cells	Kim et al. (2019)
80% (v/v) EE	Anti-inflammatory effect	Inhibition of COX-2 and lipoxygenase activities	Almuhayawi et al. (2021)
70% (v/v) EE	Anti-inflammatory effect	Inhibition of upregulation of inflammation cytokines in LPS-stimulated CoLoTC cells and mice model	Ishii et al. (2008)
Isoorientin	Anti-inflammatory effect	Down-regulation of MAPK and NF-κB signaling pathway in LPS-induced mouse microglial cells	Yuan et al. (2014)
Rutin	Anti-inflammatory effect	Down-regulation of high mobility group box 1 protein-dependent inflammatory responses	Yoo et al. (2014)
Orientin	Anti-inflammatory effect	Suppression of NF-κB and nucleotide-binding domain-like receptor protein 3 inflammasome; Upregulation of nuclear factor erythroid 2-related factor 2	Xiao et al. (2021)
Rutin and quercetin-3-O-robinobioside	Immune-enhancing properties	Upregulation of CD4+ T-lymphocytes	Ajaghaku et al. (2018)
Orientin	Anti-proliferation of cancer cells	Anti-proliferative effects against colorectal carcinoma cells	Thangaraj et al. (2019)
Orientin	Anti-proliferation of cancer cells	Suppression of colonic cell proliferation via down-regulation of proliferative antigens	Thangaraj and Vaiyapuri (2017)
Vitexin and isovitexin	Neuroprotective effect	Neuroprotective effects on amyloid β-induced PC-12 cells	Guimarães et al. (2015)
Isoorientin	Neuroprotective effect	Protective effect against 6-hydroxydopamine-induced neurotoxicity in SH-SY5Y cells by increasing Nrf2-antioxidant signaling pathway	Ma et al. (2020)
Vitexin	Antidiabetic effect	Protective effect on pancreatic β-cells against high-glucose induced damages	Ganesan et al. (2020)

^aME methanol extract, EE ethanol extract

^bCOX-2 cyclooxygenase-2, NF-κB nuclear factor-kappa B, MAPK mitogen-activated protein kinases, LPS lipopolysaccharide, NO nitric oxide, Nrf2: nuclear factor erythroid 2 related factor 2

Other biological activities

The biological activities of CBS extracts have not been extensively described. Orientin exhibited anti-proliferative effects against colorectal carcinoma cells by arresting the cell cycle in a dose-dependent manner and regulating cyclin and cyclin-dependent protein kinases (Thangaraj et al., 2019). It downregulated proliferative markers, including proliferative cell nuclear antigen and Ki67, which are associated with tumor initiation (Thangaraj and Vaiyapuri, 2017). Neuroprotective effects of isoorientin were observed against 6-hydroxydopamine-induced neurotoxicity in SH-SY5Y cells by activation of the nuclear factor erythroid 2-related factor 2 antioxidant signalling pathway (Ma et al., 2020). Vitexin exhibited a protective effect on pancreatic β-cells against high-glucose-induced damage and enhanced insulin release (Ganesan et al., 2020). Isovitexin attenuated amyloid β-induced neuronal death in PC-12 cells (Guimarães et al., 2015). Vitexin and isovitexin were also detected in the brain using LC–MS/MS, indicating their ability to penetrate the blood–brain barrier (Bai et al., 2017).

Summary and future research approaches

CBS extracts and their phenolic compounds have been shown to exhibit potent antioxidant and anti-inflammatory activities both in vitro and in vivo, as detailed in this review. However, other biological activities of CBS extracts are not as well described as those of tartary buckwheat sprouts. The biological activity of CBSs is closely linked to the amount and class of phenolic compounds. Studies have focused on the bioactivities of major flavonoids (C-glycosyl flavones and rutin) in CBS, while the health benefits of quercetin-3-O-robinobioside have been rarely reported. Greater attention must be paid to the further discovery of bioactivities of CBS extracts and phenolics that have been less studied. Effective sample preparation and analytical techniques are needed to determine the health benefits of CBS phenolics. In the past decade, various sample preparation procedures and analytical techniques have been employed for qualitative and/or quantitative analysis of specific phenolic compounds, both free and bound, in CBS. Solid–liquid extractions using methanol (50–100%), performed at room temperature and under static or dynamic conditions, were the most frequently applied for the isolation of phenolics from CBS. The phenolics isolated from the CBS extracts were further purified using SPE-based approaches. The phenolics in the CBS extracts were mostly determined by LC. Thus, there does not exist any consolidated method that can cover all phenolic analytes in CBS. However, from available studies, an effective and efficient combined analytical approach can be developed by considering the nature of the target phenolics and the characteristics of the analytical methods, as well as their advantages and disadvantages.

Declarations

Conflict of interest

The author declares that there is no conflict of interest.