Effects of Ultrafine Powderization and Hydrogen-Rich Water Extraction on Antioxidant Activity of Red Ginseng

1. Preparation of ginseng powder

Five-year-old fresh ginseng was purchased from a local market in Yeongju, Korea, and washed with tap water. WG was produced by drying fresh ginseng at 50℃ for 3 days, using a dry oven (WFO-700W: Tokyo Rikakikai Co., Ltd., Tokyo, Japan). RG was produced by steaming fresh ginseng with distilled water for 7 hours, using OCOO (OC-S1170S; OCOO Co., Ltd., Seoul, Korea) and then drying at 50℃ for 3 days.

2. Coarse and ultrafine powderization

WG and RG were ground to coarse powder, using a food mixer (SHMF-3500SS; Hanil Electronics Co., Ltd., Seoul, Korea). Red ginseng ultrafine (RGU) powder was produced by grinding red ginseng coarse (RGC) powder, using a low-temperature turbo mill (LS-001; Mechano Korea Co., Ltd., Chuncheon, Korea). The low-temperature turbo mill has a high physical power (impact, compression, and shear strength). The speed of rotor was 10,500 rpm. The temperature of mill chamber was kept at -18℃, and the temperature of ground products was kept at 25℃ to 30℃ by a cooling device using R-22. Also, the classification of ground powder was performed at the same time with milling by the principle of centrifugal and drag force.

3. Particle size analysis

The particle sizes of RGC and RGU powder were analyzed in triplicate using a Laser Diffraction Particle Size Analyzer (Bettersizer 260; Bettersize Inc., Costa Mesa, CA, USA). Powder was dispersed in distilled water before measurements. D50 is a median particle diameter, which is an equivalent volume diameter at 50% cumulative volume.

4. Powder morphology

The morphologies of RGC and RGU powder were observed, using a microscope (DM IL LED Fluo; Leica Microsystems CMS GMBH, Wetzlar, Germany). RGC and RGU powder were observed at 10× magnification.

5. Water absorption capacity, water solubility index, and swelling power

Water absorption capacity (WAC), water solubility index (WSI), and swelling power (SP) were determined in triplicate using the method of Anderson et al. (1969). One gram of sample was suspended in 20 mL of distilled water at room temperature for 30 min, gently stirring during this period, and then centrifuged at 2,000 × g (14,520 rpm) for 10 min. The supernatant was decanted into an evaporating dish of known weight. WAC, WSI, and SP were calculated by the following equations:

$$ \begin{array}{c} \mathrm{W}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{ }\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{p}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{ }\mathrm{C}\mathrm{a}\mathrm{p}\mathrm{a}\mathrm{c}\mathrm{i}\mathrm{t}\mathrm{y}\left(\mathrm{W}\mathrm{A}\mathrm{C},\mathrm{ }\mathrm{g}/\mathrm{g}\right)=\\ \left(\frac{ wet sediment weight - dry sample weight }{ dry sample weight }\right)\end{array}$$

$$ \begin{array}{c} \mathrm{W}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{ }\mathrm{S}\mathrm{o}\mathrm{l}\mathrm{u}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}\mathrm{ }\mathrm{I}\mathrm{n}\mathrm{d}\mathrm{e}\mathrm{x} \left(\mathrm{W}\mathrm{S}\mathrm{I},\mathrm{\%}\right)= \\ \left(\frac{ dry supernatant weight }{ dry sample weight }\right)\times 100\end{array}$$

$$ \begin{array}{c} \mathrm{S}\mathrm{w}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{ }\mathrm{P}\mathrm{o}\mathrm{w}\mathrm{e}\mathrm{r} \left(\mathrm{S}\mathrm{P}\right)=\\ \left(\frac{ wet sediment weight }{ dry sample weight }\right)\times \left(\frac{1\times \left[\mathrm{W}\mathrm{S}\mathrm{I}\right(\%\left)\right]}{100}\right)\end{array}$$

6. Extraction

We purchased 99.99% pure H₂ gas from Dong-A Industrial Gas in Suwon, Korea, HPLC-grade solvents from Thermo Fisher Scientific, located in Waltham, MA, USA, and other reagent-grade chemicals from Daejung Chemical Co., in Seoul, Korea.

Hydrogen-rich water was prepared as follows. A 100 mL bottle was filled with 10 mL of distilled water, and the container was sealed with an aluminum lid and rubber stopper. Two needles were used to puncture the rubber stopper; one was positioned below water surface, while the other did not come into contact with water. For three minutes, H₂ gas was bubbled via the needle that touched the water at a rate of 1 L/min. Both needles were removed from the rubber stopper at the same time after 3 min.

One gram of RGU powder was added to 10 mL of HPLC-grade distilled water or hydrogen-rich water, and then vortexed. Each sample was sonicated at 35℃ for 30 min using a POWERSONIC Ultrasonic Cleaner (POWERSONIC 420; Hwashin Tech Co., Ltd., Seoul, Korea). The mixture was filtered, using a syringe filter PTFE-H (SH25P045NL; Hyundai Micro Co., Ltd., Seoul, Korea). Then, the sample was centrifuged at 10,000 × g for 20 min, and the supernatant was used for antioxidant activity analysis.

7. Determination of total phenolic content

Total phenolic content (TPC) was determined in triplicate with a slight modification of the Folin-Denis method (Folin and Denis, 1912). The prepared extract of 0.5 mL was added to 0.5 mL of 1 N phenol reagent (Sigma-Aldrich Co., Saint Louis, MO, USA) and reacted at room temperature for 5 min, and then 1 mL of 10% sodium carbonate was added. The vortexed mixture was incubated at room temperature under dark conditions for 30 min. Absorbance values were measured at 725 nm, using a UV spectrophotometer (U-2001; Hitachi Ltd., Tokyo, Japan). TPC was expressed as gallic acid equivalent (mgGAE/g) on dry weight (DW).

8. Determination of total flavonoid content

Total flavonoid content (TFC) was determined in triplicate with a slight modification of the Moreno method (Moreno et al., 2000). The prepared extract of 0.1 mL was placed in a test tube, and 5 mL of 10% aluminum nitrate solution and 0.1 mL of 1 M potassium acetate solution were added. A total volume of 4 mL was made, adding 3.3 mL of tertiary distilled water. The vortexed mixture was incubated at room temperature under dark condition for 30 min. Absorbance values were measured at 415 nm, using a UV spectrophotometer (U-2001; Hitachi Ltd., Tokyo, Japan). TFC was expressed as quercetin equivalent (mgQE/g).

9. DPPH radical scavenging activity assay

DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity was determined in triplicate by Blois' method with a slight modification (Oh et al., 2015; Ka et al., 2016). The prepared extract of 1 mL was added to 3 mL of DPPH (Sigma-Aldrich Co., Ltd., Saint Louis, MO, USA) reagent. The vortexed mixture was incubated at room temperature under dark conditions for 30 min. Absorbance values were measured at 517 nm using a UV spectrophotometer (U-2001; Hitachi Ltd., Tokyo, Japan). DPPH radical scavenging activity was calculated according to the following equation:

$$ \begin{array}{c} \mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{n}\mathrm{ }\mathrm{o}\mathrm{f}\mathrm{ }\mathrm{D}\mathrm{P}\mathrm{P}\mathrm{H} \left(\mathrm{\%}\right)= \\ \left(\frac{ absorbance of control-absorbance of sample }{ absorbance of control }\right)\times 100\end{array}$$

10. ABTS radical scavenging activity assay

ABTS (2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) radical scavenging activity was determined in triplicate by the method described by Re et al. (1999) with a slight modification. ABTS radicals were produced by reacting 7 mM ABTS stock solution with 2.45 mM potassium persulfate and allowing the mixture to stand at room temperature under dark condition for 12 hr before use. The mixture was diluted with ethanol to get an absorbance of 0.700 ± 0.02 at 734 nm. The prepared extract of 0.2 mL was mixed with 3.8 mL of diluted solution, and absorbance values were determined at 734 nm using a spectrophotometer (U-2001; Hitachi Ltd., Tokyo, Japan) after 6 min of incubation. ABTS radical scavenging activity was calculated according to the following equation:

$$ \begin{array}{c} \mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{n}\mathrm{ }\mathrm{o}\mathrm{f}\mathrm{ }\mathrm{A}\mathrm{B}\mathrm{T}\mathrm{S}\mathrm{ }\left(\mathrm{\%}\right)=\\ \left(1-\frac{ absorbance of sample }{ absorbance of control }\right)\times 100\end{array}$$

11. Statistical analysis

Data from all experiments were statistically analyzed via ANOVA and Duncan’s multiple range test, using SPSS software program (SPSS Inc., Chicago, IL, USA). Other data were analyzed statistically by independent-paired t-test, using the SPSS software (SPSS Inc., Chicago, IL, USA). A p value < 0.05 was considered significant.

1. Properties of red ginseng powders

Cryogenic grinding, which was used in the ultrafine powderization of RGC powder, is a dry milling process to grind at low temperatures. It increases hardness and decreases the extensibility of ginseng cell wall matrix, thus making it brittle, which causes particle size to decrease quickly (Lee et al., 2012). The conditions of low-temperature turbo mill to produce RGU powder are described in Table 1. The photographs of WGC, RGC, and RGU powder are shown in Fig. 1. The effect of ultrafine powderization on particle size distribution is shown in Table 2. D50 of RGC powder was 195 μm, and that of RGU powder was 79 μm. In another study, the particle size of RGU powder was similar to the particle size in this study (Kim et al., 2018). D50 of RGU powder was abruptly decreased. There was 2.5 times reduction in the particle size of RGU powder, compared to that of RGC powder.

Table 1.

Conditions of the low-temperature turbo mill to produce red ginseng ultrafine powder.

Fig. 1.

Photographs of (A) white ginseng coarse, (B) red ginseng coarse, and (C) red ginseng ultrafine powder.

Table 2.

Particle size distribution and surface area of red ginseng coarse and ultrafine powder.

The surface areas of RGC and RGU powder were 17 m²/cm³ and 62 m²/cm³, respectively. The RGU powder containing relatively small particles had a larger surface area than the RGC powder containing relatively large particles. This was in a good agreement with the results of the particle size distributions of RGC and RGU powder. Ultrafine powderization decreased particle size distribution toward small size and produced ultrafine powder with smaller particles. In general, the surface area of powder with smaller particles is larger than that of powder with larger particles (Kim et al., 2018).

RGC and RGU powder were observed by a microscope (DM IL LED Fluo; Leica Microsystems CMS GMBH, Wetzlar, Germany). The changes in the structures of RGC and RGU powder are illustrated in Fig. 2. The microscopic images demonstrated that the particle size of RGU powder was decreased, compared to that of RGC powder. The microscopic image of RGC powder showed a mixture of large and small particles, whereas that of RGU powder showed uniform particle sizes. The particle size of RGC powder was considerably larger than that of RGU powder. Smaller particles that seemed to have broken off from larger particles indicated the impact of ultrafine powderization on RGC powder. According to particle sizes, the appearance of RGC powder was rougher because particle size was large. The color of RGC powder, in which particle size is larger, was darker than that of RGU powder, as shown in Fig. 1.

Fig. 2.

Microcsope images of (A) red ginseng coarse and (B) ultrafine powder.

2. Effect of ultrafine powderization on hydration properties of red ginseng powders

The hydration properties of RGC and RGU powder are shown in Table 3, and Fig. 3, Fig. 4, and Fig. 5. In RGC powder, WAC and SP were decreased after steaming, compared to those of WGC powder. WAC and SP of RGC powder were 5.0 g/g and 2.0, respectively, and those of WGC powder were 7.3 g/g and 2.7, respectively. WSI, which is often used as an indicator of the degradation of molecular components (Kirby et al., 1988), measures the amount of soluble components released from plant materials. The result showed that WSI was increased by steaming. WSI was 39.3% in RGC powder and 36.9% in WGC powder. WSI of RGC powder was increased by 7%, compared to that of WGC powder.

Table 3.

Water absorption capacity, water solubility index, and swelling power of white and red ginseng powders.

Fig. 3.

Water absorption capacity of white and red ginseng powders.Different subscript letters indicate a significantly different (p < 0.05). WGC: White ginseng coarse powder; RGC: Red ginseng coarse powder; RGU: Red ginseng ultrafine powder.

Fig. 4.

Water solubility index of white and red ginseng powders.Different subscript letters indicate a significantly different (p < 0.05). WGC: White ginseng coarse powder; RGC: Red ginseng coarse powder; RGU: Red ginseng ultrafine powder.

Fig. 5.

Swelling power of white and red ginseng powders.Different subscript letters indicate a significantly different (p < 0.05). Abbreviations: WGC: White ginseng coarse powder; RGC: Red ginseng coarse powder; RGU: Red ginseng ultrafine powder

After ultrafine powderization, the WAC and SP of RGU powder were 3.2 g/g and 1.4, respectively. WAC and SP of RGU powder were decreased by 35% and 30%, respectively, compared to those of RGC powder. However, the WSI of RGU powder with the D50 of 79 μm was 42.1%. WSI of RGU powder was increased by 7%, compared with that of RGC powder. WSI of RGU powder was found to be significantly higher than that of RGC powder (p < 0.05). Other studies had shown similar results that when using wheat bran and wheat straw powder, WAC was decreased in ultrafine powder (Rosa et al., 2015; Yang et al., 2014), and when using white ginseng powder, SP was decreased and WSI was increased in ultrafine powder (Lee et al., 2013).

If ginseng is steamed, the physical structure of ginseng is affected, and so the hydration properties of ginseng, such as WAC, WSI, and SP, are changed. WAC is related to the surface area and porous structure of plant material, which promote the bonding of water molecules to hydrophilic groups. After steaming, the porous structure of plant material may be destroyed and surface area may be reduced, both leading to a decrease in WAC (Li et al., 2019).

SP of RGC powder was decreased with steaming, compared to that of WGC powder. The original structure of ginseng cell wall matrix was destroyed, and the amount of macromolecules was reduced, both leading to decrease in SP (Li et al., 2019).

Unlike WAC and SP decreased, the WSI of RGC powder was increased. It may be due to the fact that steaming loosens the tight structure of ginseng cell wall matrix, exposes more hydrophilic groups, and degrades some insoluble fiber and converts it into the soluble small oligosaccharides (Li et al., 2019). This result is also explained by dextrinization, wherein starch structure is destroyed by high heat (Guha et al., 1997). Amylose (a soluble starch) is split from amylopectin (an insoluble starch), thus increasing the amount of soluble material (Guha et al., 1997). In addition, glycosidic linkages are broken down at high temperature (approximately 80℃), resulting in the release of glucose (a soluble dietary fiber) (Zavareze and Dias, 2011).

WAC is related to the water absorption behavior and particle structures, such as mean particle size, surface area, total pore volume, porosity, and mean pore radius (Yang et al., 2014; Jacobs et al., 2015). Larger particle and rougher surface have larger intraparticulate or interparticulate spaces, which absorb more interstitial water (Zhao et al., 2009; Phat et al. 2015). Furthermore, water accumulated in the pores of plant powders is weakly bonded and can be easily released by centrifugal force, while the water accumulated in the nanopores of plant cell wall matrix or water strongly associated by cell wall polysaccharides through hydrogen bonding can be retained (Jacobs et al., 2015). After ultrafine powderization, porous structure may be destroyed, which leads to decrease in the WAC of RGU powder (Li et al., 2019). The shorter chain length of RGU powder lowers SP. Ultrafine powderization destroys the original plant cell wall matrix of RG destroyed, and decreased the amount of intact macromolecules, both leading to decrease in SP (Tester and Karkalas, 1996).

The increased WSI of RGU powder suggested that ultrafine powderization could increase the solubility of phytochemicals in RGU powder. Ultrafine powderization removes cellulose barrier in plant material, resulting in a smaller particle size, and increases WSI (Lee et al., 2012; Hermansson, 1982). WSI of RGU powder can also be improved by shortening the diffusion time of water soluble molecules in particle (Zhao et al., 2015). In addition, many cracks on the uneven surface of RGU powder may be another important reason for the higher WSI of RGU powder, compared to that of RGC powder (Zhao et al., 2017). Also, the increased WSI of RGU powder is due to the increased surface area, which could expose more polar groups, hydrophilic cellulose, and hemicellulose groups to water (Hong and Zhang, 2005).

3. Effect of ultrafine powderization and hydrogen-rich water extraction on antioxidant activities of red ginseng powders

The antioxidant activities of WGC, RGC, RGU, and RGUH powder were analyzed by TPC, TFC, DPPH, and ABTS. Their results are shown in Table 4.

Table 4.

Total phenolic and total flavonoid contents, and DPPH and ABTS radical scavenging activity of white and red ginseng powders.

WGC, RGC, and RGU powder were extracted with water, but only RGUH powder was extracted with hydrogen-rich water to check the effect of MH dissolved in water. TPCs of WGC and RGC powder were 1.6 and 4.0 mg tannic acid equivalent/g, respectively, and TFCs were 1.2 and 1.6 mg rutin equivalent/g, respectively, as shown in Fig. 6 and Fig. 7. Therefore, the amount of phenolic compounds including flavonoids in the extract of RGC powder was significantly higher than those of WGC powder (p < 0.05). TPCs of RGU and RGUH powder were increased to 6.2 and 7.3 mg tannic acid equivalent/g, respectively, and TFCs to 2.1 and 3.0 mg rutin equivalent/g, respectively.

Fig. 6.

Total phenolic contents of white and red ginseng powders.Different subscript letters indicate a significantly different (p < 0.05). WGC: White ginseng coarse powder; RGC: Red ginseng coarse powder; RGU: Red ginseng ultrafine powder; RGUH: hydrogen-rich water extraction of red ginseng ultrafine powder.

Fig. 7.

Total flavonoid contents of white and red ginseng powders.Different subscript letters indicate a significantly different (p < 0.05). WGC: White ginseng coarse powder; RGC: Red ginseng coarse powder; RGU: Red ginseng ultrafine powder; RGUH: hydrogen-rich water extraction of red ginseng ultrafine powder.

The antioxidant activities of WGC and RGC powder determined by DPPH radical scavenging activity were 51.0% and 86.2%, respectively, and those determined by ABTS radical scavenging activity were 40.7% and 44.9%, respectively. The antioxidant activities of RGU and RGUH powder determined by DPPH radical scavenging activity were increased to 72.7% and 78.6%, respectively, and those determined by ABTS radical scavenging activity to 96.8% and 98.7%, respectively. The antioxidant activities showed significant differences among WGC, RGC, RGU, and RGUH powder (p < 0.05). The antioxidant activities in RGU and RGUH powder were increased markedly with ultrafine powderization and hydrogen-rich water extraction.

DPPH radical scavenging activity has been widely used in determining the antioxidant activities of pure antioxidant compounds and fruit extracts (Shih et al., 2006). DPPH radical scavenging activities in WGC and RGC powder are shown in Table 4 and Fig. 8. In DPPH radical scavenging activity, the WGC and RGC powder scavenged 27.2% and 68.3% of DPPH radicals, respectively. DPPH radical scavenging activity of RGC powder was increased by 1.5 times, compared to that of WGC powder. DPPH radical scavenging activity of RGC powder was significantly (p < 0.05) higher than that of WGC powder.

Fig. 8.

DPPH radical scavenging activity of white and red ginseng powders.Different subscript letters indicate a significantly different (p < 0.05). WGC: White ginseng coarse powder; RGC: Red ginseng coarse powder; RGU: Red ginseng ultrafine powder; RGUH: hydrogen-rich water extraction of red ginseng ultrafine powder; DPPH: 2,2-diphenyl-1-picryl-hydrazyl.

ABTS radical scavenging activity has been commonly used to measure the antioxidant activities of various biological specimens (Rufiáan and Morales, 2007). ABTS radical scavenging activities of WGC and RGC powder are shown in Table 4 and Fig. 9. In ABTS radical scavenging activity, WGC and RGC powder scavenged 40.7% and 44.9% of the ABTS radicals, respectively. ABTS radical scavenging activity of RGC powder was increased by 10%, compared to that of WGC powder. ABTS radical scavenging activity of RGC powder was significantly (p < 0.05) higher than that of WGC powder. The antioxidant activity measured by ABTS radical scavenging activity showed the same relationships as DPPH radical scavenging activity did, but antioxidant values were different. This difference in antioxidant activity could be due to the different reaction mechanisms involved. Moreover, it is assumed that the difference in radical scavenging activity is due to different reaction media; ABTS is soluble in both aqueous and organic solvents, allowing for testing in various media, including simulated biological conditions, but DPPH is primarily used in alcoholic solutions (Shalaby and Shanab, 2013).

Fig. 9.

ABTS radical scavenging activity of white and red ginseng powders.Different subscript letters indicate a significantly different (p < 0.05). WGC: White ginseng coarse powder; RGC: Red ginseng coarse powder; RGU: Red ginseng ultrafine powder; RGUH: hydrogen-rich water extraction of red ginseng ultrafine powder; ABTS: 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid).

More than 10 phenolics in ginseng, including ferulic, gentisic, cinnamic, syringic, and p-hydroxybenzoic acids, have been reported (Dixon and Paiva, 1995). The increased TPC of RGC powder is attributed to the increase of free and conjugated phenolic contents due to the release of phenolic compounds which are bonded with glucosides or amine functionalities during steaming (Dewanto et al., 2002).

TPC and antioxidant activity have a highly positive correlation in many plant species (Oktay et al., 2003). It is known that the antioxidant potentials of phenolic compounds are affected by the quantity and nature of phenolic compounds, such as molecular weight, number of aromatic rings, and nature of hydroxyl group substitution (Li et al., 2008). For these reasons, RGC powder examined in this study demonstrated better antioxidant activity than WGC powder did.

Decrease in particle size results in an increase in macromolecule breakage, specific surface area, exposure of inner pores, and depolymerized cell wall components on particle surfaces (Zhang et al., 2017). Therefore, some phenolic compounds are released or exposed by them (Zhu et al., 2015). Also, this phenomenon is explained by particle size-extraction relationship. Phenolic compounds are easily released, if surface area is increased as particle size is decreased (Cho, 2014) and if ultrafine powderization changes the phenolic compounds to a free form, which is easier to be released from the ginseng cell wall matrix (Kim et al., 2013). When the water extracts reach extraction equilibrium, the microstructural changes affect the diffusion process of phytochemicals, leading to increased extractable macromolecules (Zhang et al., 2017). RGU powder presented higher antioxidant activity than that of RGC powder, which was very likely related to the higher content of phenolic compounds in RGU powder. In this study, the DPPH and ABTS radical scavenging activities of RGU powder showed a similar trend to the results of TPC and TFC, indicating that the DPPH and ABTS radical scavenging activity of RGU powder was related to the amount of phenolic compounds (Rockenbach et al., 2011). It confirms that TPC and TFC were the key factors in antioxidant activities.

When MH was incorporated into water, the TPC and TFC of RGUH powder were increased, compared to those of RGU powder. It can be considered that the increases of TPC and TFC in RGUH powder are due to the protection of phenolic compounds from the oxidative reactions and the liberation of the conjugated and cell wall-bound phenolic compounds (Alwazeer et al., 2023).

Phenolic compounds contain one or more hydroxyl groups (-OH) attached to aromatic ring. These hydroxyl groups are highly susceptible to oxidation reactions. The presence of multiple hydroxyl groups in the phenolic compounds can enhance their susceptibility to oxidation, because each hydroxyl group can act as a potential site of oxidation (Waterhouse and Laurie, 2006). MH has a thermodynamic potential-reducing property that makes it a potent antioxidant. It is involved in the biological electron transfer system at pH 7, with a standard reduction potential of (2H⁺/H₂) that equals -420 mV. Thus, it can shield phenolic compounds from oxidative reactions, making them less susceptible to breakdown (Hu et al., 2021). Therefore, the presence of MH in solvent could potentially protect phenolic compounds from oxidation reactions during the extraction process. This is particularly crucial in cases where the plant materials are subjected to grinding, which can destroy their structure and make them more susceptible to oxygen and oxidation reactions in extraction (Jha and Sit, 2022).

Saturated hydrogen in water easily forms gas bubbles. Hydrogen bubbles burst at solid–liquid interface, leading to the degradation of plant cell wall matrix. Phytochemicals, such as phenolic compounds bound to cell wall, can be liberated after this explosion of bubbles, leading to the dissolution of phenolic compounds in water (Blicharski and Oniszczuk, 2017). In addition, the infusion of MH would have removed any dissolved oxygen. Then it is possible that fewer oxygen radicals (e.g., •$$ {\mathrm{O}}_2^{-}$$, •OH) are produced because there is not enough O₂ to react (Duried, 2024). Therefore, MH in solvent can help extract more phytochemicals. For this reason, it can be expected that DPPH and ABTS radical scavenging activities in RGUH powder were improved by the increase of TPC and TFC, compared to those of RGU powder.

In this study, we demonstrate that ultrafine powderization and hydrogen-rich water extraction can be utilized to enhance the antioxidant activity of red ginseng powder by extracting more phenolic compounds. Therefore, both technologies can be applied to other medicinal plants.