Effects of Different Solvents on the Extraction of Phenolic and Flavonoid Compounds, and Antioxidant Activities, in Scutellaria baicalensis Hairy Roots

Abstract

The hairy roots (HRs) of Scutellaria baicalensis are widely used and consumed worldwide as a medicine, especially in Asian countries, due to their biological and pharmacological activities. The HRs of this plant are rich in secondary metabolites. However, the optimal method and solvents for the extraction of secondary metabolites from S. baicalensis HRs have not been well studied. Therefore, in this study, S. baicalensis HRs were extracted with different solvents, including water (WE), 99.9% pure methanol (PM), 70% aqueous methanol (AM), 99.9% pure ethanol (PE), and 70% aqueous ethanol (AE). The phenolic and flavonoid compounds and various antioxidant activities of each extract were measured. The AE extract (16.85 ± 0.15%) had a higher yield, which led to the highest accumulation of total phenolic content (TPC) and total flavonoid content (TFC), and antioxidant activity. The TPC and TFC were highest in AE (66.03 ± 0.44 mg GAE/g and 40.11 ± 1.31 mg QE/g, respectively), whereas WE, PM, and PE showed lower values in all assays. In addition, the highest antioxidant activities, such as DPPH, ABTS, and SOD-like scavenging activities and reducing power, were achieved in the AE extract compared to the other solvent extracts. Based on these results, the AE extract showed the highest phenolic and flavonoid accumulation and antioxidant activities, highlighting its potential use in the manufacture of useful materials from S. baicalensis HRs and its novel applications.

1. Introduction

In recent years, an increasing number of scientists have reached the consensus that oxidants play an important role in the ageing process, supported by compelling evidence [1]. Consequently, several synthetic antioxidants have been formulated and developed. However, these synthetic antioxidants cause harmful damage to vital organs, including the circulatory system, kidneys, liver, and stomach, leading to conditions such as cancer [2]. Therefore, this growing concern has led to an increase in interest in natural dietary alternatives to synthetic antioxidants, highlighting the current research into vegetable sources and the investigation of raw materials to improve novel antioxidants.

Scutellaria baicalensis Georgi, otherwise called Chinese skullcap, has an extensive history of therapeutic use across East Asia, such as China and Korea, as well as in Europe and other regions. In China, its roots are often referred to as Huang-Qin, where ‘Huang’ represents its yellow colour and ‘Qin’ is an important type of reed-like herb [3]. This multipurpose plant can be used either whole or without the pericarp [4]. Scutellaria baicalensis has been widely used for detoxification and its antipyretic properties [5]. It also has several medicinal benefits in the treatment of various diseases, such as diarrhoea, jaundice, and inflammation [6]. The main active components in this plant are flavonoids, which are known for their free radical-scavenging activity [7]. In a previous study, it was stated that these compounds exhibit strong antioxidant properties, efficiently inhibiting the production of reactive oxygen species (ROS) during oxidative processes, as well as demonstrating anti-bacterial, anti-allergen, anti-inflammatory, anti-hypertensive, and anti-viral activities [8]. Remarkably, S. baicalensis has shown anti-cancer activity by hindering prostaglandin E2 (PGE2) production [9]. Moreover, Park et al. [10] showed that S. baicalensis extract could inhibit α-glucosidase, which is linked with elevated blood sugar levels, and defend against oxidative DNA damage produced by leukocytes. At the same time, S. baicalensis extract using methanol is emerging as a promising therapeutic agent for diabetes [11,12].

Hairy roots (HRs) can be obtained through Agrobacterium rhizogenes infection, which results in fast growth and the highest production of specialised natural compounds due to their biochemical and genetic stability [13,14]. Previous studies reported that HRs are one the best sources for the production of secondary metabolite biosynthesis in S. baicalensis. In addition, it has been reported that flavone production in S. baicalensis HRs is regulated by the expression of multiple genes, which shows that the enhancement of flavone compounds might be possible through the genetic engineering approach [15]. However, genetic engineering techniques are cost-effective and time-consuming. Using a suitable extraction method is one of the alternative strategies to enhance the secondary metabolites in S. baicalensis HR culture.

Previous studies by Huang et al. [16] and Kim [17] showed that the physiologically active constituents of S. baicalensis wild-type root have low solubility in hot water and benzene extraction, but can be readily extracted using solvents, such as methanol, ethanol, and acetone; however, the selection of a suitable solvent based on the polarity of the compounds is an important task [18]. The yield and function of the dissolved bioactive compounds’ composition might be affected depending on the extraction solvent employed. Therefore, the main objective of this study was to extract S. baicalensis HRs using various solvents, quantify the phenolic and flavonoid content, and compare the respective antioxidant properties of each solvent. Simultaneously, high-performance liquid chromatography (HPLC) was employed to identify individual phenolic and flavonoid compounds. To date, no studies have conducted analyses of the secondary metabolites and antioxidant activities of S. baicalensis HRs by using different solvents. This investigation aimed to identify the most effective solvents for extracting functional materials from S. baicalensis HRs and to promote the efficient use of S. baicalensis HRs.

2. Materials and Methods

2.1. Plant Materials

The seed of S. baicalensis was obtained from the Rural Development Administration, South Korea. HR cultures of S. baicalensis were established as described previously by Park et al. [19]. In brief, HRs from S. baicalensis were subcultured on a solidified MS medium [20], and then, transferred to an MS liquid culture medium for further experimental analysis. The basal MS medium contained vitamins, salts, and 30 g/L sucrose. The pH of the medium was adjusted to 5.8, and the medium was autoclaved at 121 °C for 20 min. HR cultures were grown at 25 °C on a gyratory shaker (100 rpm) in an incubator with a 16 h photoperiod under cool white fluorescent lights (flux rate of 35 µmol/s/m²). HR cultures were maintained in an MS liquid medium and subcultured every 10 days (Figure 1). The HR cultures were harvested, freeze-dried, and ground to fine a powder for further experiments.

2.2. Preparation of the Sample Extract

2.2.1. Hot Water Extraction

For sample extraction in hot water, 1 g of dried powder of S. baicalensis HRs was placed in a 50 mL tube along with 20 mL of distilled water (WE). The sample was then incubated in an 80 °C water bath for 12 h. Following incubation, the mixture was centrifuged at 12,000 rpm for 20 min at 4 °C, and the supernatant was filtered using Whatman No. 2 filter paper. The collected liquid extract was freeze-dried to transform it into powder. Dimethyl sulfoxide (DMSO) with a purity of 50% was added to prepare the stock solution of the extracts [21], which was then employed for further experimental investigation at a concentration of 25 mg/mL.

2.2.2. Alcoholic Solvent Extraction

To 1 g of S. baicalensis HR dried powder, 20 mL of 99.9% pure methanol (PM), 70% aqueous methanol (AM), 99.9% pure ethanol (PE), or 70% aqueous ethanol (AE) was added, and the mixture was sonicated for 1 h at room temperature. The mixture was then centrifuged at 12,000 rpm for 20 min at 4 °C. The supernatant of the samples was filtered using a 0.45 µm PTFE hydrophilic syringe filter. The filtered alcoholic liquid extract was concentrated at 50 °C using a rotary evaporator (Heidolph Laborota 4000 efficient, Germany). After evaporation, the powdered sample, in a round-bottom flask, was washed with distilled water and transferred into a falcon tube. Then, the extract was freeze-dried, and 50% DMSO was added to the freeze-dried sample to make up a concentration of 25 mg/mL. The stock solution was kept at 4 °C until further use. The yield of all samples obtained through the above process was calculated using the formula reported by El Mannoubi [22].

$$\mathrm{Yield} \left(\%\right)=\frac{\mathrm{The} \mathrm{mass} \mathrm{of} \mathrm{the} \mathrm{lyophilised} \mathrm{sample}}{\mathrm{The} \mathrm{mass} \mathrm{of} \mathrm{the} \mathrm{initial} \mathrm{dried} \mathrm{sample} \mathrm{powder}}\times 100$$

(1)

2.3. Analysis of Total Phenolic Content (TPC) and Total Flavonoid Content (TFC)

The TPC was determined according to the protocol described by Park et al. [23], with slight modifications. First, to create a working solution, all sample extracts were diluted with 50% DMSO to a sample extract concentration of 5000 ppm. We mixed 0.1 mL of the above solution with 0.5 mL of 2N Folin and Ciocalteu’s phenol reagent (Junsei Honsha Co., Tokyo, Japan). The mixture was incubated for 3 min at room temperature, and 4 mL of Na₂CO₃ (10%, w/v) was added; the solution was mixed gently, and incubated in the dark for 90 min. The absorbance of each sample was determined using a UV–Vis spectrophotometer (SPECTROstar Nano plate reader, BMG LABTECH) at 760 nm. The equivalent calibration curve of gallic acid (ranging from 0 to 493.75 μg/mL; y = 0.0012x + 0.0558, R² = 0.998) was used to quantify the TPC in the samples. The results of the TPC are presented as the average of three replications and expressed in terms of milligrams of gallic acid equivalent per gram of dry weight (mg gallic acid equivalent (GAE)/g DW).

The TFC was quantified according to the protocol described by Kim et al. [24], with slight modifications. For TFC analysis, to make a working solution, all sample extracts were diluted with 50% DMSO to a sample extract concentration of 5000 ppm. Initially, 2.0 mL of distilled water was mixed with 0.5 mL of working solution, and then, 0.15 mL of NaNO₂ (5%, w/v), and the mixture was allowed to react at room temperature for 5 min. Finally, we added 0.15 mL of AlCl₃ (10%, w/v) and allowed the mixture to stay at room temperature for 15 min. The absorbance of each sample was measured at 415 nm using a UV–Vis spectrophotometer. The equivalent calibration curve of quercetin (ranging from 62.5 to 500 μg/mL; y = 0.002x + 0.0839, R² = 0.9996) was used to quantify the TFC in the samples. The results are presented as the average of three replications and expressed in terms of milligrams of quercetin equivalent per gram of dry weight (mg quercetin equivalent (QE)/g DW).

2.4. Analysis of Individual Phenolic and Flavonoid Contents by High-Performance Liquid Chromatography (HPLC)

To analyse the phenolic and flavonoid compounds using HPLC, we dissolved our extracts with 50% DMSO to a concentration of 25 mg/mL. Phenolic and Flavonoid compounds were analysed from S. baicalensis HRs according to the protocol described by Park et al. [19,25]. The analysis was carried out using HPLC (NS-4000, Futecs, Daejeon, Korea) and RP-HPLC columns (C18, 250 × 4.6 mm, 5 μm, RStech, Daejeon, Republic of Korea) with a UV–vis detector and autosampler at 30 °C, and water containing 0.2% (v/v) acetic acid or formic acid (solvent A) and pure methanol (solvent B) was used in a gradient as the mobile phase at a flow rate of 1 mL/min, as shown in Tables S1 and S2. Phenolic and flavonoid compounds were chromatographically detected at 280 and 275 nm, and the injection volumes were 100 and 60 µL, respectively. Retention time comparison and spike tests were used to identify the phenolic and flavonoid compounds, and calibration curve formulae were used to quantify the phenolic and flavonoid compounds (Tables S3 and S4). Three biological replicates were analysed to determine the phenolic and flavonoid content in each sample. The results for phenolic and flavonoid contents are represented as mg/g DW.

2.5. Antioxidant Activities

2.5.1. 2,2-Di Phenyl-1-Picryl Hydrazyl (DPPH) Free Radical-Scavenging Activity

The DPPH radical-scavenging activity of the S. baicalensis HR extract was determined as previously reported by Kim et al. [26]. A volume of 40 μL of different concentrations of S. baicalensis HR extract (31.25, 62.5, 125, 250, 500, and 1000 μg/mL) was mixed with 160 μL of 0.2 mM DPPH solution (prepared using 99.9% methanol). The mixtures were incubated in the dark for 30 min, followed by measurement using a UV-Vis spectrophotometer at 517 nm. As a blank, we used a sample mixed with 99.9% methanol, whereas 50% DMSO mixed with 0.2 mM DPPH was used as a control. All analyses were performed using three biological replicates, and the DPPH-scavenging activity was calculated using the following equation:

$$\mathrm{DPPH}-\mathrm{scavenging} \mathrm{activity}\left(\%\right)=\frac{{\{\mathrm{A}}_{\mathrm{c}}-{(\mathrm{A}}_{\mathrm{s}}-{\mathrm{A}}_{\mathrm{b}}\left)\right\}}{{\mathrm{A}}_{\mathrm{c}}}\times 100$$

(2)

where A_c is the absorbance of the control; A_s is the absorbance of the sample; and A_b is the absorbance of the sample blank.

The absolute value of the 50% inhibitory concentration of 0.2 mM DPPH (AIC₅₀) of the sample was calculated using the plotted curve and expressed in mg/mL, which was based on the previous report by de Menezes et al. [27].

2.5.2. 2,2′-Azino-bis 3-Ethylbenzo Thiazoline-6-Sulfonic Acid (ABTS) Free Radical-Scavenging Activity

The ABTS-scavenging activity of S. baicalensis HR extracts was analysed according to the protocol described by Zheleva-Dimitrova et al. [28]. To prepare the ABTS buffer solution, 7 mM ABTS powder was completely dissolved in 2.5 mM potassium persulfate solution (prepared using distilled water), and then, incubated for 16 h. For each experiment, the ABTS buffer solution was prepared fresh. After incubation, the absorbance of the ABTS buffer solution was adjusted to 0.7 ± 0.002 at 734 nm using distilled water. To analyse the ABTS activity in the HR samples, 20 μL of different concentrations of S. baicalensis HR extract, ranging from 31.25 to 1000 μg/mL, was placed in a 96-well titre plate, and 180 μL of the ABTS buffer solution was added, followed by incubation in the dark for 2.5 min. After incubation, the absorbance of each sample was immediately read three times at 734 nm using a UV–Vis spectrophotometer. As a blank, we used a sample mixed with distilled water, whereas 50% DMSO mixed with ABTS buffer solution was used as a control. The ABTS-scavenging activity was calculated similarly to the formula described in the DPPH-scavenging assay.

2.5.3. Super Oxide Dismutase (SOD)-like-Scavenging Activity

SOD-like activity was measured based on the method described by Marklund et al. [29]. A volume of 200 μL of serially diluted S. baicalensis HR extract samples ranging from 31.25 to 1000 μg/mL was placed in a test tube, and 50 μL of 50 mM Tris-HCl buffer containing 10 mM EDTA at pH 8.5 was added. Then, 50 μL of 7.2 mM pyrogallol dissolved in distilled water was added to initiate the reaction, followed by incubation in the dark at room temperature for 10 min. Subsequently, the inhibition of the auto-oxidation of pyrogallol was measured at 420 nm. As a blank, we used a sample mixed with distilled water, whereas 50% DMSO mixed with 7.2 mM pyrogallol was used as a control. The SOD-like activity and AIC₅₀ of 7.2 mM pyrogallol were calculated using a similar formula applied to the DPPH radical-scavenging activity. Each experiment was carried out in triplicate.

2.5.4. Reducing Power Assay

The reducing power assay was performed based on the method described by Gülçin et al. [30], with slight modification. In detail, 300 μL of 0.2 M phosphate buffer (pH 6.6) was added to 300 μL of different concentrations of S. baicalensis HR extracts and mixed gently, and 300 μL of 1% C₆N₆FeK₃ was added following incubation at 50 °C for 20 min. Then, 300 μL of 10% trichloroacetic acid was added, and all mixtures were vortexed and centrifuged for 10 min at 10,000 rpm. After centrifugation, 500 μL of the supernatant was transferred to a new Eppendorf tube, and 500 μL of distilled water and 100 μL of 0.1% FeCl₃ were added and vortexed gently. The absorbance of the samples was measured at 700 nm, and an increase in absorbance at this wavelength indicated that each sample had more reducing power. Each experiment was carried out in triplicate.

2.6. Statistical Analysis

All results were expressed as the mean ± standard deviation (SD). Statistical analysis of the data was performed via analysis of variance (ANOVA) in SPSS 20 (SPSS Inc., Chicago, IL, USA), and significant differences were verified at the p < 0.05 level using Duncan’s multiple range test. Pearson’s correlation analysis was performed using MetaboAnalyst 6.0.

3. Results

3.1. Analysis of Yield (%), TPC, and TFC

As for the extraction yield, the samples extracted using AE and AM had the highest yields, which were approximately 10% higher than the samples extracted with PM and PE. At the same time, higher yields corresponded to higher TPC and TFC. The AE showed the highest yield, which resulted in the highest TPC and TFC content. However, the AM showed a relatively higher yield, which led to the second highest TPC and TFC content. The yields of PM-extracted samples and PE samples were relatively low, and their TPCs and TFCs were significantly lower than those of aqueous alcohol extracts. Although WE showed the second highest yield value, its TPC and TFC were lower. This indicates that WE is not suitable for the extraction of TPC and TFC from S. baicalensis HRs (

Table 1. Extraction yield (%), TPC, and TFC of the extract from S. baicalensis HRs.

	Yield (%)	Total Phenolic Content (mg GAE/g DW)	Total Flavonoid Content (mg QE/g DW)
WE	16.7 ± 0.2 a	17.37 ± 0.29 e	8.71 ± 0.36 e
PM	8.3 ± 0.1 c	24.48 ± 0.1 d	19.91 ± 0.26 d
PE	7.35 ± 0.05 d	32.81 ± 0.25 c	25.41 ± 0.5 c
AM	15.6 ± 0.1 b	57.64 ± 0.25 b	35.14 ± 1.34 b
AE	16.85 ± 0.15 a	66.03 ± 0.44 a	40.11 ± 1.31 a

Different letters in the values denoted represent statistically significant differences among the means according to Duncan’s multiple range test (ANOVA, p < 0.05). Values are means ± SD.

).

3.2. Analysis of Individual Phenolics (Including Flavanols and Flavonols) Using HPLC

For the analysis of individual phenolics, we identified 10 compounds, and the results showed that the AM extraction method resulted in the highest accumulation of individual phenolics (Figure 2 and Figures S1–S12). Among the individual phenolics, kaempferol showed the highest content in the AM (6.19 ± 0.86 mg/g DW), followed by AE (3.72 ± 0.08 mg/g DW), PE (1.36 ± 0.05 mg/g DW), PM (0.68 ± 0.05 mg/g DW), and WE (0.25 ± 0.02 mg/g DW). Next to kaempferol, quercetin showed the second highest content, which was highest in AM (1.24 ± 0.12 mg/g DW), whereas the lowest content was obtained in WE. The third highest content was achieved in rutin, and it was highest in PM (0.8 ± 0.03 mg/g DW), in which it was 1.07-, 1.13-, 1.27-, and 1.51- times higher than that in PE, AM, AE, and WE, respectively. In the case of chlorogenic acid, AE showed the highest content, followed by AM, WE, PE, and PM. The lowest content was obtained from gallic acid, which ranged from 0.04 to 0.05 mg/g DW. The (-)-epicatechin (0.23 ± 0.04 mg/g DW) and benzoic acid (0.55 ± 0.03 mg/g DW) contents were high in the AM extract. Most of the individual phenolic compounds were highest in AM, followed by AE extract, whereas the other extracts (WE, PM, and PE) did not show significant differences. In conclusion, aqueous alcoholic extracts (AM and AE) were more effective for the extraction of phenolics from S. baicalensis HRs, whereas the hot water extraction method showed low efficiency.

3.3. Analysis of Individual Flavonoids Using HPLC

To quantify the flavonoids, a linear calibration curve graph obtained at a wavelength of 275 nm for each standard (baicalin, baicalein, and wogonin) was used. Our results indicate that different solvents exert different effects on flavonoid content. The highest level of baicalin (mg/g DW) was attained in the AM (10.21 ± 0.09), followed by the PM (4.78 ± 0.03), AE (3.49 ± 0.04), PE (3.38 ± 0.03), and WE (3.23 ± 0.03). The baicalein content varied significantly among the various solvent extracts. The AE content (15.99 ± 0.67 mg/g DW) was 1.55-, 2.22-, 4.11-, and 266.5 times higher than that in AM, PE, PM, and WE, respectively. The wogonin content was highest in AM (6.74 ± 0.14 mg/g DW), followed by AE (6.66 ± 0.23 mg/g DW), PM (3.93 ± 0.16 mg/g DW), and PE (3.79 ± 0.1 mg/g DW), whereas it was not detected in WE. Except baicalin, the aqueous–alcoholic solvents showed the highest content of flavonoid compounds (baicalein and wogonin), although the baicalin content was significantly higher in the AM extract (Figure 3).

3.4. In Vitro Antioxidant Activities of S. baicalensis HR Extracts

Several antioxidant activity assays, including DPPH, ABTS, SOD-like-scavenging activity, and reducing power assays, were performed to determine the effect of the extraction solvents on S. baicalensis HR extracts. Among these assays, the DPPH and ABTS radical-scavenging activities are among the most efficient methods for evaluating antioxidant capacity by measuring free radical-scavenging activity. As shown in Figure 3, concentrations ranging from 31.25 to 1000 μg/mL showed a slight increase with increasing concentrations. In the DPPH assay, at 1000 μg/mL, the AE sample of the S. baicalensis HR extract showed the highest scavenging activity (83.86 ± 0.21%), followed by AM (76.59 ± 1.05%), PM (46.05 ± 0.21%), PE (42.47 ± 4.69%), and WE (19.49 ± 2.49%) (Figure 4A). As shown in

Table 2. AIC₅₀ values of the DPPH, ABTS, and SOD assays from different solvent extracts of S. baicalensis HRs.

	AIC50 of DPPH	AIC50 of ABTS	AIC50 of SOD
WE	3.53 ± 0.53 c	1.1 ± 0.07 e	0.66 ± 0.07 bc
PM	1.04 ± 0.01 ab	0.8 ± 0.01 d	0.84 ± 0.11 c
PE	1.2 ± 0.21 b	0.62 ± 0.01 c	1.09 ± 0.16 d
AM	0.73 ± 0.09 ab	0.38 ± 0.02 b	0.54 ± 0.12 b
AE	0.49 ± 0.04 a	0.29 ± 0.01 a	0.29 ± 0.04 a

Different letters in the values denoted represent statistically significant differences among the means according to Duncan’s multiple range test (ANOVA, p < 0.05). Values are means ± SD.

, based on the AIC₅₀ value, showing the concentration required for 50% inhibition of a specific substance, the required concentrations of AM (0.73 ± 0.09 mg/mL) and AE (0.49 ± 0.04 mg/mL) showed the highest efficiency, whereas PM (1.04 ± 0.01 mg/mL), PE (1.2 ± 0.21 mg/mL), and WE (3.53 ± 0.53 mg/mL) showed the highest AIC₅₀ values, indicating the lowest scavenging activity.

Similarly, the ABTS radical-scavenging activity of S. baicalensis HR extracts showed a similar trend to that of DPPH. The AE (96.24 ± 1.56%) showed the highest ABTS-scavenging activity at 1000 μg/mL, followed by AM (94.13 ± 0.51%), PE (69.47 ± 0.08%), PM (59.75 ± 0.29%), and WE (47.36 ± 1.65%) (Figure 4B). As shown in Table 2, the lowest required concentration to scavenge ABTS was achieved in AE (0.29 ± 0.01 mg/mL), followed by AM (0.38 ± 0.02 mg/mL), PE (0.62 ± 0.01 mg/mL), PM (0.8 ± 0.01 mg/mL), and WE (1.1 ± 0.07 mg/mL), which showed that the WE extract had the lowest scavenging activity and was approximately four times lower than the AE. Based on the DPPH and ABTS-scavenging activity assay results, AM and AE had high scavenging activity.

SOD is one of the most important antioxidant enzymes in all living cells. It reacts with ROS and transforms it into hydrogen peroxide (H₂O₂), which is harmless to the body. The SOD assay was performed to determine whether these extracts inhibited the auto-oxidation reaction of pyrogallol. As shown in Figure 5A, at 1000 μg/mL, both AM (69.1 ± 2.81%) and AE (79.7 ± 3%) showed the highest levels of SOD-like activity; these findings were consistent with the DPPH and ABTS results. In contrast to the DPPH and ABTS results, WE (58.15 ± 1.15%) showed SOD-like-scavenging activity similar to PM and PE (54.99 ± 5.88% and 42.75 ± 5.64%, respectively). At the same time, the AIC₅₀ value of pyrogallol auto-oxidation showed that AE (0.29 ± 0.04 mg/mL) showed the highest efficiency, followed by AM (0.54 ± 0.12 mg/mL), WE (0.66 ± 0.07 mg/mL), PM (0.84 ± 0.11 mg/mL), and PE (1.09 ± 0.16 mg/mL) (Table 2).

Reducing power, which measures the conversion of Fe₃⁺/ferricyanide complexes to Fe₂⁺/potassium ferrocyanide, is also one of the most efficient methods for evaluating antioxidant activity. There was a gradual increase in reducing power with an increase in the concentration of the sample. The AE (0.37 ± 0.01) showed the most powerful reducing power at 1000 µg/mL, followed by AM (0.32 ± 0.01), PE (0.22 ± 0.00), PM (0.19 ± 0.01), and WE (0.16 ± 0.00) (Figure 5B).

3.5. Pearson Correlation Analysis between the Phenolic and Flavonoid Compounds and Antioxidant Activities

Pearson’s correlation analysis was carried out to know the interrelationship among phenolic compounds, total phenolics, total flavonoids, and antioxidant activities (

Table 3. Pearson’s correlations analysis among the phenolic and flavonoid compounds and antioxidant activities.

	GA	CAT	CA	CFA	EPC	EG	BA	RUT	QUE	KAE	Baicalin	Baicalein	WOG	TPC	TFC	DPPH	ABTS	SOD	RPA
GA	1	0.319	0.848	0.630	0.473	−0.629	0.541	−0.525	−0.014	0.523	0.311	0.243	0.165	0.432	0.190	0.231	−0.162	−0.808	0.442
CAT		1	0.005	0.793	0.945	−0.021	0.955	0.153	0.730	0.823	0.982	0.285	0.576	0.486	0.455	−0.371	−0.467	−0.200	0.425
CA			1	0.557	0.164	−0.529	0.262	−0.430	−0.272	0.463	−0.008	0.556	0.342	0.621	0.431	−0.035	−0.396	−0.889	0.658
CFA				1	0.844	−0.181	0.891	−0.014	0.483	0.984	0.780	0.691	0.810	0.850	0.758	−0.517	−0.756	−0.649	0.824
EPC					1	−0.051	0.973	0.095	0.752	0.845	0.967	0.249	0.531	0.465	0.396	−0.290	−0.415	−0.326	0.413
EG						1	−0.176	0.649	0.584	−0.163	0.091	−0.073	0.181	−0.159	0.027	−0.460	−0.027	0.291	−0.141
BA							1	0.029	0.640	0.896	0.949	0.359	0.590	0.573	0.486	−0.316	−0.499	−0.408	0.521
RUT								1	0.446	0.062	0.214	0.086	0.287	0.018	0.200	−0.498	−0.236	0.247	0.010
QUE									1	0.477	0.828	−0.001	0.407	0.135	0.178	−0.382	−0.195	−0.036	0.105
KAE										1	0.796	0.720	0.847	0.861	0.802	−0.585	−0.809	−0.544	0.826
Baicalin											1	0.227	0.555	0.429	0.399	−0.366	−0.417	−0.220	0.373
Baicalein												1	0.909	0.962	0.971	−0.784	−0.959	−0.540	0.967
WOG													1	0.925	0.965	−0.890	−0.961	−0.456	0.913
TPC														1	0.965	−0.719	−0.947	−0.643	0.996
TFC															1	−0.856	−0.989	−0.469	0.957
DPPH																1	0.863	0.170	−0.712
ABTS																	1	0.433	−0.935
SOD																		1	−0.680
RPA																			1

GA—gallic acid; CAT—catechin; CA—chlorogenic acid; CFA—caffeic acid; EPC—(-)-epicatechin; EG—epicatechin gallate; BA—benzoic acid; RUT—rutin; QUE—quercetin; KAE—kaempferol; WOG—wogonin; TPC—total phenolic content; TFC—total flavonoid content; DPPH—2,2-Di phenyl-1-picryl hydrazyl free radical-scavenging activity; ABTS—2,2′-Azino-bis 3-ethylbenzo thiazoline-6-sulfonic acid free radical-scavenging activity; SOD—super oxide dismutase-like-scavenging activity; RPA—reducing power assay.

). In this study, TPC and TFC had a strong positive relationship with reducing power assay. Among the individual identified phenolic compounds, except epicatechin gallate, all other compounds showed a positive correlation with TPC; among these, kampferol and caffeic acid showed a strong positive correlation. Interestingly TFC showed a positive correlation with all the identified individual phenolic and flavonoid compounds, whereas compounds such as kaempferol, caffeic acid, baicalein, and wogonin showed a strong positive correlation with TFC. On the other hand, all the identified phenolic and flavonoid compounds exhibited a negative correlation with ABTS values, whereas in the DPPH except gallic acid all other phenolic compounds showed a negative correlation. Phenolic compounds such as epicatechin gallate and rutin showed a positive correlation, whereas the other identified compounds showed a negative correlation with SOD. Interestingly, except epicatechin gallate, all other identified phenolic and flavonoid compounds showed a positive correlation with the reducing power assay. Among these, individual compounds such as baicalein, wogonin, TPC, and TFC showed a strong correlation with the the reducing power assay. From these results, it is shown that baicalein, caffeic acid, epicatechin gallate, gallic acid, kaempferol, rutin, and wogonin are the main contributors to antioxidant activity (DPPH, SOD, and reducing power assay).

4. Discussion

Extraction yield is affected by various factors, such as water proportion and polarity, which play an important role in improving the recovery of phenolic and flavonoid compounds [31]. In this study, the highest yield was achieved with AE, followed by WE and AM, whereas AE and AM showed the highest TPC and TFC. Even though WE showed the second highest yield, it showed the lowest TPC and TFC. This is consistent with the results of Lim et al. [32]. Similarly, other studies have reported that hot water extraction in S. baicalensis is ineffective for bioactive compounds [16,17,32]. According to a previous report by Yu et al. [33], aqueous alcoholic solvents (80% methanol and ethanol) were more efficient than water for TPC extraction from peanut shells. Turkmen et al. [34] reported that mate tea and black tea showed the highest polyphenol content in 50% ethanol, which further supports the findings of our study.

Baicalin, baicalein, and wogonin are well-known representative compounds present in S. baicalensis and have various physiological properties [7]. The S. baicalensis HR produced by Agrobacterium rhizogenes A4 strain showed that the baicalin was dominated when grown on liquid culture [35]. In another study, it was reported that baicalin was highest in the hydrogen peroxide-treated transgenic S. baicalensis HR produced by A. rhizogenes A4 [36]. Previously, several studies reported that in the HR produced through transformation with A. rhizogenes A4, an equal or higher amount of wogonoside content is usually detected than that of baicalin [37,38,39,40]. However, in our study, we produced the transgenic HR line via the A. rhizogenes R1000 strain, thus resulting in the highest baicalein content. From this result, it is shown that the difference in the individual flavone content might be due to the growth conditions and the Agrobacterium strain used to produce transgenic HR lines.

Phenylpropanoids are among the most important groups of secondary metabolites in plants, and these compounds have a protective role against environmental stress due to their strong antioxidant properties [41]. In addition, they are considered a precursor for the production of flavonoid compounds [42]. From this study, we found that except for chlorogenic acid and baicalein, most of the individual phenolic and flavonoid contents were highest in the AM extract, whereas the antioxidant activities were higher in the AE extract. A previous study reported that baicalein is involved in the cell’s protection against oxidative damage [35,36]. The highest antioxidant activity in the AE extract might be due to the high baicalein content. These results indicate that AE extract might contribute to antioxidant, anti-inflammatory, and cardiac function [43].

The number of aromatic and hydroxyl group phenolic compounds and their exact positions also have a considerable impact on antioxidant activity [44]. Depending on the circumstances and substances under investigation, intermolecular communications that can be antagonistic or synergistic regulate the antioxidant activity of phenolic compounds. Albishi et al. [45] reported that phenolic compounds were correlated with the DPPH and the ferric-reducing antioxidant power (FRAP) assay. Similar results were obtained in another study whereby DPPH positively correlated with the phenolic compounds [46]. This result was consistent with our study results, which showed that some of the phenolic compounds showed a positive correlation with DPPH, SOD, and the reducing power assay.

According to a previous report [32], AE of S. baicalensis roots showed the highest TPC, TFC, DPPH, ABTS, FRAP, and reducing power. This supports our study’s results, showing that the aqueous ethanol (AE) had the highest TPC, TFC, and antioxidant activities. In addition, previous studies have shown that baicalein, chlorogenic acid, TPC, and TFC have strong antioxidant activity [43,47,48]. In this study, a significant amount of baicalein and chlorogenic acid was achieved in AE, which led to the highest antioxidant activities in the AE extract. These results indicate that baicalein, chlorogenic acid, TPC, and TFC play a significant role in the antioxidant activities of S. baicalensis HR. Fu et al. [49] revealed correlations between TPC and the antioxidant activity of 62 other plant species; the results showed that an increase in TPC led to the highest antioxidant activity. This is consistent with our study’s results, which showed that the highest TPC had the highest antioxidant activities. In addition, according to our results, the AE extraction method showed a higher TPC and TFC than the AM extract of S. baicalensis HRs. At the same time, the TPC and TFC showed a significant difference between the AE and AM extraction methods, which suggests that the AE method is among the most efficient methods for extracting TPC and TFC. Therefore, it was concluded that the AE extraction method was effective in achieving the highest yield and antioxidant activities.

5. Conclusions

In this study, we developed an appropriate extraction method to obtain the highest yields of TPC, TFC, and individual phenolic and flavonoid compounds from S. baicalensis HRs. Among the different solvents tested, AE showed the highest extraction yield of compounds from S. baicalensis HRs. Thus, we concluded that the aqueous–alcoholic solvent is one of the most efficient solvents for extracting valuable compounds from S. baicalensis HRs. Despite notable significant differences occurring between the AM and AE methods concerning the presence of compounds such as TPC, TFC, and individual phenolic and flavonoid compounds, both methods exhibited similar antioxidant activities. Thus, the best choice between AM and AE can be made based on precise requirements and conditions. This study on the extraction of a suitable method for obtaining the highest yield of secondary metabolites and antioxidant activities is a good starting point for future research on increasing the production of therapeutically valuable secondary metabolites. In addition, it must be noted that the conducted experiment concerning the small scale of S. baicalensis HR cultures is preliminary and it requires further testing in large-scale experiments.