Antioxidant compounds and activities of Perilla frutescens var. crispa and its processed products

Abstract

Perilla frutescens var. crispa ‘Antisperill’ has stronger anti-inflammatory effects than the original cultivar (red perilla), but studies on its bioactive compounds are limited. This study aimed to evaluate the antioxidant compounds and activity of red perilla and ‘Antisperill’, as well as their processed products using freeze drying (FD) and microwave-assisted low temperature vacuum drying (MVD). Red perilla had higher antioxidant compounds and activities than ‘Antisperill’. MVD showed significantly greater antioxidant properties than FD among the processed products. Isoegomaketone, studied for its anti-obesity, antioxidant, anti-inflammatory, and anticancer properties, was only found in ‘Antisperill’ and its processed products, with the highest content in ‘Antisperill’ MVD samples. This study highlights that ‘Antisperill’ contains various antioxidants, and MVD is the optimal drying method to preserve and enhance its antioxidant content. As a result, ‘Antisperill’ could potentially serve as a natural antioxidant with both anti-inflammatory and antioxidant functions.

Introduction

The incidence of geriatric diseases, such as dementia, hypertension, diabetes, cerebrovascular disease, and degenerative arthritis, has recently been increasing due to the rise in the elderly population (Asif, 2012). Among them, steroidal and non-steroidal anti-inflammatory drugs are used to treat degenerative arthritis. However, studies have reported that they can cause side effects such as gastritis, nephritis, and heart disease. As a result, research on the development of anti-inflammatory substances based on natural products with fewer side effects has been actively conducted (Jin et al., 2016). Free radicals can act as a biological defense mechanism, but when they accumulate in the body beyond what is necessary, they are reported to cause oxidative stress, aging, metabolic syndrome, cancer, and chronic inflammation (Hwang et al., 2019).

Perilla frutescens var. crispa is a type of perilla in the Lamiaceae family that has a morphology similar to sesame leaf and is known as “Soyup” and “Chazugi” (Nam et al., 2019). The red color of perilla is due to anthocyanin pigment, and perilla has been reported to exhibit several physiological activities such as antioxidant, anti-inflammatory, anticancer, antibacterial, anti-allergic, antifungal, and anti-aging effects in various studies. The major components that contribute to its functional properties include rosmarinic acid, perillaldehyde, luteolin, apigenin, egomaketone, and isoegomaketone (Jin et al., 2016; Zhou et al., 2014). Perilla is cultivated and widely used in Asia, including Korea, China, and Japan. Its fruits, stems, and leaves are extensively utilized in traditional Chinese medicine for the treatment of bronchial diseases, indigestion, and insomnia (Wang et al., 2016). Perilla is cultivated in various cultivars within the same species, with notable cultivars being P. frutescens var. frutescens, P. frutescens var. acuta, and P. frutescens var. crispa (Nam et al., 2019). These cultivars can be differentiated by the color of their stems and leaves, which are primarily categorized as red and green (Liu et al., 2013). The cultivars used in this study are Perilla frutescens var. crispa, representing the traditional red color, and Perilla frutescens var. crispa, cv. Antisperill, representing the green color. ‘Antisperill’ is a transgenic variety that increased the isoegomaketone content through irradiation of P. frutescens var. crispa with 200 Gy of gamma radiation. Previous studies have reported higher anti-inflammatory effects in this transgenic variety compared to the original species, but research on antioxidant compounds and activities is lacking (Nam et al., 2019).

Microwave-assisted low temperature vacuum drying (MVD) is a drying technology that combines microwave heating (MH) and vacuum drying (VD). During drying, the vacuum condition causes water to vaporize at a temperature lower than its original boiling point, thereby minimizing chemical changes in the products (Kim et al., 2018; Wojdyło et al., 2014). Additionally, MVD offers a shorter total drying time compared to other drying methods, resulting in reduced energy and drying costs. Furthermore, drying under vacuum helps to reduce the oxidation reaction of the product (Kim et al., 2018). These advantageous features of MVD make it a widely used drying method as it contributes to improving the quality of the product by preserving various bioactive compounds and ensuring uniform heat distribution in the dried product (Hihat et al., 2017; Kagawa et al., 2019).

Therefore, in this study, we compared the antioxidant compounds and activities of perilla and its processed products. We also examined the differences in antioxidant compounds and activities based on the characteristics of each drying method, with the goal of establishing the most effective drying method for preserving the various bioactive compounds of ‘Antisperill’. Additionally, our aim is to expand the utilization of ‘Antisperill’ as a functional material in the future and contribute to the research and development of functional natural product materials.

Materials and methods

Plant samples

Red perilla and ‘Antisperill’ were grown in a smart farm located in Sangju-si, Gyeongsangbuk-do. The plants were cultivated under controlled conditions, with an average temperature of 29 °C and humidity of 56%. They were grown using 10 W LED lights and harvested on June 21, 2022 (Fig. 1). After washing and removing any foreign substances, we conducted a physicochemical analysis of color, soluble solid content (SSC), pH, and chlorophyll content on the day of harvest. The samples were then rapidly frozen in liquid nitrogen at − 196 °C and stored in a freezer at − 55 °C for antioxidant analysis.

Fig. 1.

Photograph of PFC, PFCA (A) and PFCA processed products (B) evaluated in the present study. (PFC: Perilla frutescens var. crispa; PFCA: Perilla frutescens var. crispa (cv. Antisperill); FD: Freeze-Dried; MVD: Microwave assisted low-temperature Vacuum Dried; HV1: 1st harvest; HV2: 2nd harvest; HV3: 3rd harvest; P: Pill; T: Tea bag.)

The processed product ‘Antisperill’ used in this study was provided by SFC Bio (Industry-academic Cooperation Hall, Dankook University, Cheonan, Chungnam, Korea), as shown in Fig. 1. For the experiment, freeze-dried ‘Antisperill’ powder (FD-HV1 and FD-HV2) was prepared by freeze-drying the raw materials harvested on June 2, 2021, and June 17, 2021. The freeze-drying process was carried out using a freeze dryer (Freeze dryer, FD8508, Ilshin Lab Co. Ltd., Korea) at − 80 °C for 48–72 h, maintaining a pressure of 5–10 mTorr.

The microwave-assisted low temperature vacuum dried powder (MVD-HV3) was prepared by drying ‘Antisperill’ leaves harvested on August 9, 2021 in a microwave-assisted low temperature vacuum dryer (Microwave assisted low-temperature vacuum dryer, GDT Co., Ltd., Busan, Korea) at less than 40 °C, 20 ~ 40 torr, 1.5 kWh/kg, for less than 4 h, and then grinding to a size of 30 mesh using a grinder (JEIL TECH, 30 mesh, π 0.6, Korea). ‘Antisperill’ pills and tea bags (MVD-P and MVD-T) were processed from ‘Antisperill’ leaves harvested on September 16, 2020, by MVD followed by grinding and pill making. The samples were stored in a freezer at − 55 °C and used for extraction for analysis.

Physicochemical analysis

Color and chlorophyll content

The Hunter Lab values (L*, a*, b*) were measured using a color meter (Chroma Meter CR-400, Minolta, Tokyo, Japan) to assess outer color of Perilla frutescens var. crispa and its processed products.

The colors of the perilla and its processed products were expressed as L* (dark to light, on a scale of 0–100), a* (redness), and b* (yellowness) using a color meter (Chroma meter CR-400, Minolta, Japan). ‘Antisperill’ processed products were powdered by adding liquid nitrogen to a certain amount of the frozen sample in the mortar, and the average value was calculated by repeating three times each. Red perilla and ‘Antisperill’ raw materials were measured for front and back colors, and the average value was calculated by repeating three times each for nine samples.

Chlorophyll determination of red perilla and ‘Antisperill’ was measured with a chlorophyll meter (SPAD-502, Minolta, Japan) using the method of Markwell et al. (1995), and the front surfaces of nine samples were measured in three times and expressed as the mean value.

Organic acid contents

Organic acid composition profiling was analyzed by modified method of Tomsone et al. (2020). The extract was diluted using distilled water, and the dilution was filtered through a 0.45 μm syringe filter. The filtered sample was analyzed using high performance liquid chromatography (HPLC) (Agilent 1200 series, Agilent Technol., Wilmington, DE, USA) and separated at 25 °C using a prevail organic acid column (250 × 4.6 nm, 5 μm, Hichrom Ltd., Reading, UK). The mobile phase was 25 mM KH₂PO₄ adjusted to pH 2.5 with phosphoric acid, with a flow rate of 1.0 mL/min and a sample injection volume of 10 μL. A diode array detector (DAD) was used, and the wavelength was set at 210 nm.

For the calibration curves, the five different points (5, 12.5, 25, 50, 100 μg/mL for oxalate, 1, 2.5, 5, 10, 20 μg/mL for fumarate, 25, 62.5, 125, 250, 500 μg/mL for tartrate, malate, lactate, acetate, citrate, and succinate) were prepared with standard solutions, and the unit for the results was expressed in mg/100 g of fresh weight (FW) for red perilla and ‘Antisperill’ raw materials and mg/100 g of dry weight (DW) for ‘Antisperill’ processed products.

Antioxidant compounds and activities analysis

Sample extraction

Thirty grams of the sample was added to 300 mL of 80% ethanol, and then this mixture was homogenized in a commercial blender (JB 3060, Braun Co., Kronberg, Germany) three times for 3 min each time. The homogenized solution was filtered through a Whatman #2 paper filter, and then was concentrated in a rotary evaporator (N-1000, Eyela, Tokyo, Japan) at 45 °C. The extractions were stored at − 55 °C and used for antioxidant compound and activity measurements (Hwang et al., 2019).

Total anthocyanin contents

The total anthocyanin content was determined using the pH differential method (Hwang et al., 2019). The sample extracts were mixed with 0.025 M KCl buffer (pH 1.0) and 0.4 M sodium acetate buffer (pH 4.5), respectively, and the absorbance was measured at 510 nm and 700 nm wavelengths using a spectrophotometer (Optizen POP, Mecasys, Korea). The total anthocyanin content was expressed as mg cyanidin-3-glucoside equivalents (CGE)/100 g FW, according to the following equation.

$$\text{Total}\text{anthocyanin}\text{content}\left(\text{mg CGE}/1{00}^{\circ }\text{gFW}\right)=\frac{A\times MW\times DF\times 1000}{\epsilon }$$

$$\text{A}\left(\text{absorbance}\text{value}\right)={\left({\text{A}}_{510\text{nm}}-{\text{A}}_{700\text{nm}}\right)}_{\text{pH 1}.0}-{\left({\text{A}}_{510\text{nm}}-{\text{A}}_{700\text{nm}}\right)}_{\text{pH 4}.5}$$

$$\text{MW}\left(\text{cyanidin}-3-\text{O}-\text{glucoside}\text{molecular}\text{weight}\right)=449.2$$

$$\text{D}=\text{Dilution}\text{factor}$$

$$\epsilon \left(\text{cyanidin}-3-\text{O}-\text{glucoside}\text{molar}\text{absorptivity}\right)=26,900$$

Total flavonoids contents

Total flavonoid content was measured by colorimetric assay method (Hwang et al., 2019). In total, 0.3 mL of 5% NaNO₂ was added to a 1 mL aliquot of the diluted sample mixed with deionized water, thoroughly mixed, and allowed to react for 5 min. Then 0.3 mL of 10% AlCl₃ was added, vortexed to mix evenly, and reacted at room temperature for 6 min. Finally, 2 mL of 1 N NaOH and 2.4 mL of deionized water were added to make the total volume 10 mL, and the absorbance was measured at 510 nm using a spectrophotometer after vortexing. Catechin was used as a standard and the total flavonoid content was expressed as mg catechin equivalents (CE)/100 g.

Total phenolics contents

Total phenolic content was measured using the Folin–Ciocalteu colorimetric method (Hwang et al., 2019). 0.2 mL of sample extract and 2.6 mL of deionized water were mixed, 0.2 mL of Folin-Ciocalteu reagent was added, vortexed to mix thoroughly, and reacted at room temperature for 6 min. 2 mL of 7% Na₂CO₃ was added and reacted at room temperature dark for 90 min. The absorbance was then measured at a wavelength of 750 nm using a spectrophotometer. Gallic acid was used as a standard and the total phenolic compound content was expressed as mg gallic acid equivalents (GAE)/100 g.

Total ascorbic acid contents

Total ascorbic acid content was analyzed by dinitrophenylhydrazine (DNPH) method of Terada et al. (1978). Five grams of homogenized sample and 100 mL of 6% metaphosphoric acid buffer were mixed, and then centrifuged at 15,000 rpm for 20 min, and filtered with Whatman filter paper #2. The filtered extraction 1 mL and 2% DCIP 0.05 mL were mixed and reacted at room temperature for 1 h. After the reaction, 2% thiourea 1 mL and 2% DNPH 0.5 mL were added and reacted at 60 °C for 3 h. Then, the test tube was put into ice to make it cold and slowly added with 90% H₂SO₄ 2.5 mL. The absorbance of the sample was measured at 540 nm, and the standard calibration curve used ascorbic acid.

Analysis of polyphenol by HPLC

Polyphenol contents of sample extracts were analyzed using the method of Liu et al. (2013). The sample extracts were diluted with diluted solution (KH₂PO₄: methanol: D.W = 2: 3: 15) and filtered through a 0.45 μm syringe filter. The filtered sample was analyzed using HPLC (Agilent 1200 series, Agilent Technol., Wilmington, DE, USA), and analytical separation was performed using a Zorbax Eclipse XDB-C18 column (4.6 × 150 mm i.d., 5 µm, Agilent) at 40 °C. The mobile phase was 3% acetic acid (acetic acid: water = 30: 970) as mobile phase A and water as mobile phase B. The flow rate was 1.0 mL/min and the sample injection volume was set to 10 μL. The detector was a diode array detector (DAD) and the wavelength was set at 280 nm. The standard calibration curve was measured using the following as standards: chlorogenic acid, ( +)-catechin hydrate, (−)-epicatechin, (−)-epicatechin gallate, rutin hydrate, quercetin, and rosmarinic acid. The content was expressed as mg/100 g FW for red perilla and ‘Antisperill’ raw materials, and as mg/100 g DW for ‘Antisperill’ processed products.

Isoegomaketone contents

Isoegomaketone content was analyzed using HPLC according to the method of Nam et al. (2019). The sample extract was diluted with methanol and filtered through a 0.45 μm syringe filter, and the column was separated at 35 °C using an Eclipse XDB-C18 column (150 × 4.6 mm, 5 μm). Two solvents, water (A) and acetonitrile (B), were used as mobile phases, and the flow rate was 1.0 mL/min, the sample injection volume was 10 μL, and the detection wavelength was set to 254 nm. The standard calibration curve was prepared by using the standard substance isoegomaketone at concentrations of 10, 25, 50, 100, and 200 μg/mL.

DPPH radical scavenging activity

Antioxidant activity was used DPPH radical scavenging activity, and measured by modified method of Hwang et al. (2019). 50 μL of sample dilution and 2950 μL of 0.2 mM DPPH solution were mixed and reacted for 30 min at room temperature in a dark place. The absorbance was then measured at 517 nm using a spectrophotometer. The antioxidant activity of the extract was expressed as mg vitamin C equivalents (VCE)/100 g.

ABTS radical scavenging activity

Antioxidant activity was used ABTS radical scavenging activity and measured by modifying method of Hwang et al. (2019). 20 μL of sample dilution and 980 μL of ABTS solution were mixed and reacted at 37 °C for 10 min. The absorbance was then measured at 734 nm using a spectrophotometer. The antioxidant activity of the extract was expressed as mg VCE/100 g.

Statistical analysis

For the statistical analysis of each experimental result, the SPSS 20 program (SPSS Inc. Chicago, IL, USA) was used to perform the analysis of variance (ANOVA). Independent samples t-test was used to test the significance of differences between red perilla and ‘Antisperill’ raw materials, Duncan’s multiple range test was used to test the significance of differences among ‘Antisperill’ processed products (p < 0.05). The correlation of the mean values of each factor was analyzed using Pearson’s correlation coefficient, and the analytical measurements were repeated three times and expressed as the mean ± standard deviation.

Results and discussion

Color value

The hunter L*, a* and b* value and chlorophyll content were shown in Table 1. The L* values, which represent lightness, were 31.79 ± 1.30 and 33.68 ± 0.72 for the front and back sides of red perilla, respectively. For ‘Antisperill’, the L* values were 34.98 ± 1.15 and 45.32 ± 1.00 for the front and back sides of ‘Antisperill’, respectively. It showed that the front side of both red perilla and ‘Antisperill’ had significantly lower L* values than the back side, and ‘Antisperill’ had significantly brighter lightness than red perilla. The a* values of the front (0.96 ± 0.68) and back (8.32 ± 1.11) of red perilla were significantly higher than the front (− 13.55 ± 0.73) and back (− 12.15 ± 0.86) of ‘Antisperill’. Red perilla contains anthocyanin pigment, which gives it a reddish-purple color, so the a* value, which represents redness, was measured higher than that of ‘Antisperill’, which has a greenish color.

Table 1.

Hunter L*, a*, b* color and SPAD value of Perilla frutescens var. crispa (red perilla), Perilla frutescens var. crispa (cv. Antisperill) and its processed products

Cultivar	L* (Lightness)	a* (Redness)	b* (Yellowness)	SPAD
PFC-front	31.79 ± 1.30d	0.96 ± 0.68b	1.67 ± 1.15c	35.37 ± 0.71b
PFC-back	33.68 ± 0.72c	8.32 ± 1.11a	− 2.32 ± 0.95d
PFCA-front	34.98 ± 1.15b	− 13.55 ± 0.73d	13.32 ± 1.37b	45.74 ± 2.99a
PFCA-back	45.32 ± 1.00a	− 12.15 ± 0.86c	15.63 ± 1.37a
FD-HV1	66.14 ± 0.33a	− 18.92 ± 0.10d	32.86 ± 0.18a	N.A
FD-HV2	66.46 ± 0.82a	− 19.28 ± 0.33d	31.45 ± 0.43b	N.A
MVD-HV3	55.82 ± 0.41b	− 8.78 ± 0.06b	25.22 ± 0.03c	N.A
MVD-P	43.14 ± 0.33d	− 4.64 ± 0.02a	18.96 ± 0.18e	N.A
MVD-T	54.41 ± 0.97c	− 10.81 ± 0.64c	23.38 ± 0.88d	N.A

Results are the mean values ± standard deviation from three measurements (n = 3); Means in the same column with different letters (a, b, c, d, and e) are significantly different at p < 0.05; PFC: Perilla frutescens var. crispa; PFCA: Perilla frutescens var. crispa (cv. Antisperill); FD: Freeze-Dried; MVD: Microwave assisted low-temperature vacuum dried; HV1: 1st harvest; HV2: 2nd harvest; HV3: 3rd harvest; P: Pill; T: Tea bag; N.A: Not available

The color measurement results of the ‘Antisperill’ processed products showed that the L* values of FD-HV1 and FD-HV2 were 66.14 ± 0.33 and 66.46 ± 0.82, respectively, which were significantly higher than those of MVD-HV3 (55.82 ± 0.41), MVD-P (43.14 ± 0.33), and MVD-T (54.41 ± 0.97). The brightness of the microwave low temperature vacuum dried samples decreased in the order of powder, tea bags, and pills. The a* value of processed products was highest in MVD-P (− 4.64 ± 0.02) followed by MVD-HV3 (− 8.78 ± 0.06), MVD-T (− 10.81 ± 0.64), FD-HV1 (− 18.92 ± 0.10), and FD-HV2 (− 19.28 ± 0.33). Finally, the b* value, which indicates yellowness, was highest in FD-HV1 (32.86 ± 0.18) and lowest in MVD-P (18.96 ± 0.18).

These results can be interpreted as differences in color depending on the drying method, and FD powder showed significantly higher lightness than MVD powder. This is similar to the results of Samakradhamrongthai et al. (2022), which measured the color of okra powder for three drying methods (hot air dying, freeze drying, and microwave vacuum drying), and found that freeze drying powder had a significantly higher L* value than microwave vacuum-dried powder. In this study, the redness of MVD samples is higher than that of FD samples, which is due to the decomposition of chlorophyll by heat during the drying process to form brown pheophytin pigment (An et al., 2022). Therefore, it is considered that the relatively higher temperature of MVD than that of FD led to the decomposition of chlorophyll, which represents green color, resulting in the higher a* value.

Organic acid contents

The organic acid content was analyzed by HPLC, and were shown in Table 2. The results showed that organic acid of perilla and its processed products had varying compositions of oxalic acid, citric acid, malic acid, lactic acid, tartaric acid, and fumaric acid. Compared to the organic acid content of red perilla and ‘Antisperill’ raw materials, total organic acid content of ‘Antisperill’ (3385.61 mg/100 g FW) was higher than that of red perilla (2647.09 mg/100 g FW). Oxalic acid was found to be the main organic acid in red perilla and ‘Antisperill’ with 84.12 and 88.55% of total organic acids, respectively. Compared to the organic acid content of ‘Antisperill’ processed products (MVD-HV3, MVD-P, MVD-T), the total organic acid content was higher in MVD-HV3 (1909.48 mg/100 g DW), MVD-P (1823.49 mg/100 g DW), and MVD-T (1361.77 mg/100 g DW). Similar to red perilla and ‘Antisperill’ raw materials, oxalic acid was significantly higher in MVD-HV3, MVD-P, and MVD-T than other organic acids, with a ratio of 74.87, 68.83, and 58.55%, respectively.

Table 2.

Organic acid contents of Perilla frutescens var. crispa (red perilla), Perilla frutescens var. crispa (cv. Antisperill) and its processed products

	Oxalic acid	Citric acid	Malic acid	Lactic acid	Tartaric acid	Fumaric acid	Total sum (mg/100 g)
PFC	2226.78 ± 218.07b	119.70 ± 12.48a	115.54 ± 9.11a	115.51 ± 26.24a	67.52 ± 6.44a	2.04 ± 0.27a	2647.09
PFCA	2997.97 ± 222.83a	121.31 ± 20.20a	135.17 ± 12.04a	74.46 ± 5.60a	54.10 ± 5.42a	2.60 ± 0.24a	3385.61
MVD-HV3	1429.62 ± 62.02a	N.D	156.56 ± 10.78b	252.80 ± 5.81b	66.68 ± 5.08c	3.82 ± 0.34b	1909.48
MVD-P	1255.07 ± 24.10b	N.D	172.41 ± 7.44a	216.32 ± 6.17c	174.89 ± 6.22a	4.80 ± 0.69a	1823.49
MVD-T	797.38 ± 60.30c	N.D	150.58 ± 3.96b	317.16 ± 11.17a	93.02 ± 6.11b	3.63 ± 0.11b	1361.77

Results are the mean values ± standard deviation from three measurements (n = 3); Means in the same column with different letters (a, b, and c) are significantly different at p < 0.05; The units of PFC and PFCA were expressed in mg/100 g Fresh Weight (FW); The units of MVD processed products were expressed in mg/100 g Dry Weight (DW); PFC: Perilla frutescens var. crispa; PFCA: Perilla frutescens var. crispa (cv. Antisperill); MVD: Microwave assisted low-temperature vacuum dried; HV3: 3rd harvest; P: Pill; T: Tea bag.; N.D: Not detected

In the present study, the organic acid content of red perilla and ‘Antisperill’ raw material, except for oxalic acid, did not show any significant difference. This is similar to a study by Yamazaki et al. (2003) on metabolite profiles and gene expression in red and green perilla, which reported that the content of citric acid, malic acid, and other organic acids did not differ based on perilla color. In addition, after MVD treatment of ‘Antisperill’, the organic acid content decreased by 43.60% in MVD-HV3, 46.14% in MVD-P, and 59.78% in MVD-T, respectively. Tomsone et al. (2020), which analyzed the organic acid content of horseradish leaves (Armoracia rusticana) after FD and MVD treatment, reported that the organic acid content decreased significantly after drying treatment, but overall, MVD treatment was an effective method for organic acid preservation.

Total flavonoids contents

Total flavonoids content of perilla and its processed products are shown in Table 3. Total flavonoids contents of red perilla and ‘Antisperill’ raw materials were 166.83 ± 11.46 and 69.72 ± 4.79 mg CE/100 g FW, respectively, indicating that red perilla had a statistically higher total flavonoids content than ‘Antisperill’ with green color.

Table 3.

Antioxidant compounds and antioxidant activities of Perilla frutescens var. crispa (red perilla), Perilla frutescens var. crispa (cv. Antisperill) and its processed products

	Total anthocyanins	Total flavonoids	Total phenolics	Total ascorbic acid	DPPH	ABTS
PFC	42.33 ± 5.80	166.83 ± 11.46a	280.38 ± 5.38a	54.32 ± 2.73a	157.36 ± 5.73a	299.98 ± 12.79a
PFCA	N.D	69.72 ± 4.79b	111.92 ± 3.96b	30.17 ± 4.70b	55.49 ± 3.61b	93.07 ± 4.84b
FD-HV1	N.A	1406.31 ± 6.24d	2038.16 ± 9.93d	N.A	1627.41 ± 7.62d	2062.88 ± 35.62 cd
FD-HV2	N.A	1822.52 ± 24.52c	2454.39 ± 3.04c	N.A	2046.62 ± 18.33c	2570.86 ± 74.96c
MVD-HV3	N.D	4259.05 ± 261.62b	4493.33 ± 267.45b	82.80 ± 5.49b	4863.50 ± 147.09b	5022.84 ± 685.62b
MVD-P	N.D	927.78 ± 41.99e	1415.00 ± 51.34e	32.75 ± 5.10c	1089.82 ± 119.36e	1781.14 ± 56.52d
MVD-T	N.D	4676.19 ± 64.92a	5623.33 ± 217.13a	134.55 ± 10.79a	5639.57 ± 132.10a	6540.14 ± 172.02a

Results are the mean values ± standard deviation from three measurements (n = 3); Means in the same column with different letters (a, b, c, d, and e) are significantly different at p < 0.05; The units of PFC and PFCA were expressed in mg/100 g FW; The units of FD and MVD processed products were expressed in mg/100 g DW; PFC: Perilla frutescens var. crispa; PFCA: Perilla frutescens var. crispa (cv. Antisperill); FD: Freeze-Dried; MVD: Microwave assisted low-temperature vacuum dried; HV1: 1st harvest; HV2: 2nd harvest; HV3: 3rd harvest; P: Pill; T: Tea bag; N.D: Not detected; N.A: Not available

Jiang et al. (2020) reported that total flavonoids content of red perilla (45.9 mg/g DW) is significantly higher than that of green perilla (30.2 mg/g DW). In addition, in the study by Chu et al. (2000) measured the total flavonoids content of purple and green leaves of sweet potato, and found that purple leaves had significantly higher content compared to green leaves. The purple color of sweet potato leaves is due to anthocyanin pigment, and the significantly higher content of total flavonoids in red perilla compared to ‘Antisperill’ in this study also suggests that anthocyanin in red perilla contributed to the total flavonoids content (Vishnu et al., 2019).

The total flavonoid content of ‘Antisperill’ processed product by FD was 1406.31 ± 6.24 mg CE/100 g DW for FD-HV1 and 1822.52 ± 24.52 mg CE/100 g DW for FD-HV2. The total flavonoid content of FD-HV2 was significantly higher than that of FD-HV1.

While the total flavonoids content of ‘Antisperill’ processed product by MVD was 4259. 05 ± 261.62 mg CE/100 g DW for MVD-HV3, 927.78 ± 41.99 mg CE/100 g DW for MVD-P, and 4676.19 ± 64.92 mg CE/100 g DW for MVD-T, which were significantly higher in the order of MVD-T, MVD-HV3, and MVD-P. According to the drying method, the total flavonoid content of MVD samples was significantly higher than that of FD samples. Studies on cherries and carrots have also reported that vacuum-microwave drying results in a higher total flavonoid content than freeze drying (Nguyen and Le, 2018; Wojdyło et al., 2014). These studies suggest that microwave drying can preserve bioactive compounds in products as effectively as freeze-drying if the appropriate power, temperature, and pressure are applied.

The total flavonoid content of the ‘Antisperill’ processed products was significantly higher than that of the raw ‘Antisperill’. This difference is believed to be attributed to the pressure applied inside the plant by microwaves, which disrupts plant cells and enhances the extraction of flavonoid and phenolic compounds (Hihat et al., 2017). Among the processed products, MVD-P (927.78 ± 41.99 mg CE/100 g DW) showed significantly the lowest total flavonoids content. This difference can be attributed to the processing methods applied. Ioannou et al. (2012) reported that processing method and formulation of the product significantly affect flavonoid content, the results of this study suggest that the total flavonoid content decreased as processing progressed.

Total phenolics contents

Total phenolics content of perilla and its processed products are shown in Table 3. The total phenolics contents of red perilla and ‘Antisperill’ raw materials were 280.38 ± 5.38 and 111.92 ± 3.96 mg GAE/100 g FW, respectively, indicating that red perilla exhibited significantly higher values compared to green perilla. This finding is consistent with the results reported by Meng et al. (2008), who observed a significantly higher total phenolic content in red perilla compared to green perilla. Additionally, Nguyen and Le (2018) also reported a 1.4-fold higher total phenolic content in red perilla compared to green perilla.

The total phenolic contents of FD-HV1 and FD-HV2 were 2038.16 ± 9.93 and 2454.39 ± 3.04 mg GAE/100 g DW, respectively. For MVD-HV3, MVD-P, and MVD-T, the total phenolic contents were 4493.33 ± 267.45, 1415 ± 51.43, and 5623.33 ± 217.13 mg GAE/100 g DW, respectively. When comparing the drying methods, MVD-HV3 and MVD-T showed significantly higher values than FD powder. Previous studies have reported that microwave energy can influence the release of phenolic compounds by causing structural changes due to rapid pressure increases (Hihat et al., 2017). This is believed to be the same reason for the observed difference in total flavonoid content.

Total phenolics content of ‘Antisperill’ processed products was significantly higher in the order of MVD-T, MVD-HV3, and MVD-P. This can be attributed to the temperature increase resulting from friction during the processing procedure. Vidović et al. (2013) reported that industrial processing, such as sorting, cutting, and grinding, can disrupt plant cellular tissues and release oxidizing enzymes, leading to a reduction in total phenolic content. Among the ‘Antisperill’ processed products, MVD-P had significantly lower total phenolics content than MVD-HV3 and MVD-T, which may be due to the complexity of the pills manufacturing process compared to powders and tea bags, based on the study by Ioannou et al. (2012), which reported that the process applied to the raw material can affect the phenol content.

Total ascorbic acid contents

Total ascorbic acid content of perilla and its processed products is shown in Table 3. The total ascorbic acid content of red perilla was significantly higher than that of ‘Antisperill’. The total ascorbic acid content of ‘Antisperill’ was 82.8 ± 5.49 mg/100 g DW for MVD-HV3, 32.75 ± 5.10 mg/100 g DW for MVD-P, and 134.55 ± 10.79 mg/100 g DW for MVD-T, with significantly higher contents followed by MVD-T, MVD-HV3, and MVD-P. In general, vitamin C in vegetables is known to be highly destroyed during the drying process (Kim et al., 2018). The results of this study show that ascorbic acid was degraded in the powdered and pill products due to heat generation and exposure to air during the grinding and dehydration processes (Zia and Alibas, 2021).

DPPH radical scavenging activity

Total antioxidant activity using DPPH radical of perilla and its processed products is shown in Table 3. DPPH radical scavenging activity of red perilla and ‘Antisperill’ raw materials was determined to be 157.36 ± 5.73 mg VCE/100 g FW and 55.49 ± 3.61 mg VCE/100 g FW, respectively. In several studies, red perilla showed significantly higher antioxidant capacity compared to green perilla (Meng et al., 2008; Saita et al., 2012). Asif (2012) reported that phenolic contents and antioxidant activity were correlated with leaf color in perilla, which supports the findings of this study that total phenolics content and DPPH radical scavenging activity of red perilla are significantly higher than ‘Antisperill’.

DPPH radical scavenging activity of ‘Antisperill’ processed products was significantly higher in MVD samples than in FD samples. Possible reasons for this could be the release of significant quantities of phenolic compounds due to the disruption of cell walls induced by microwave treatment, as reported by Hihat et al. (2017). The significantly lowest value of DPPH radical scavenging activity of MVD-P among MVD products was due to the fact that several food additives and excipients were added in addition to ‘Antisperill’ MVD powder during the preparation of pills.

ABTS radical scavenging activity

ABTS radical scavenging activity of red perilla was significantly 2.4 times higher than that of ‘Antisperill’. This suggests that anthocyanin, which represents the red color, contributed to the antioxidant activity of red perilla. According to Zheng et al. (2020), the antioxidant activity of red perilla was higher than that of green perilla, suggesting that flavonoids and anthocyanins contributed to the antioxidant activity of red perilla. Skowyra et al. (2014) reported that the ABTS radical scavenging activity of red perilla was 65.03 ± 2.98 mg TE/100 g DW, which was lower than our result. This may be due to differences in cultivar, growing region, and environment factor. Several studies have suggested that total flavonoids and total phenolics contents are highly correlated with antioxidant activity, and the results of our study also showed that ABTS radical scavenging activity tended to be similar to total flavonoids and total phenolics contents. Several studies have shown that MVD treatment of fruits and vegetables results in higher antioxidant activity compared to FD treatment. These findings support the results of the present study, which demonstrated significantly higher ABTS radical scavenging activity in MVD powder and tea bags compared to FD powder (An et al., 2022; Nguyen and Le, 2018; Wojdyło et al., 2014).

Total anthocyanin contents

The total anthocyanin content of perilla and its processed products is presented in Table 3. Red perilla exhibited a total anthocyanin content of 42.33 ± 5.80 mg CGE/100 g FW, whereas no anthocyanin was detected in the green-colored ‘Antisperill’ and its processed products. Anthocyanin, a water-soluble flavonoid responsible for the red, purple, and blue colors in plants, is known to be a key factor in distinguishing between red and green perilla. Meng et al. (2008) reported that HPLC analysis of green perilla samples showed no detectable anthocyanin content and Yamazaki et al. (2003) stated that anthocyanin accumulation is specific only to red perilla.

Polyphenol contents

Polyphenol content of perilla and its processed products is shown in Table 4. Rosmarinic acid content of red perilla was higher than that of ‘Antisperill’. Rosmarinic acid is a type of polyphenol abundant in plants such as perilla, sesame leaf, oregano, basil, rosemary, and others. It exhibits various biological activities, including antioxidant, anti-inflammatory, anti-allergic, antibacterial, antidepressant, and anticancer properties (Hossan et al., 2014; Kagawa et al., 2019).

Table 4.

Polyphenol contents of Perilla frutescens var. crispa (red perilla), Perilla frutescens var. crispa (cv. Antisperill) and its processed products

	Rosmarinic acid	Chlorogenic acid	( +)-Catechin hydrate	(−)-Epicatechin	(−)-Epicatechin gallate	Rutin hydrate	Quercetin	Total sum
PFC	20.57 ± 2.47a	2.63 ± 0.56b	0.58 ± 0.02b	4.60 ± 0.88a	N.A	N.D	10.01 ± 0.16a	38.39
PFCA	7.65 ± 2.15b	5.81 ± 0.46a	1.42 ± 0.03a	1.38 ± 0.06b	N.A	2.88 ± 0.30	4.19 ± 0.37b	23.33
FD-HV1	N.A	17.29 ± 0.02d	N.A	9.14 ± 0.86e	15.52 ± 0.25e	38.31 ± 0.35e	N.A	80.26
FD-HV2	N.A	17.32 ± 0.08d	N.A	12.39 ± 0.37d	22.50 ± 0.45d	48.10 ± 1.16d	N.A	100.31
MVD-HV3	2616.81 ± 142.40b	45.93 ± 0.22b	28.60 ± 1.84a	83.01 ± 2.15b	83.13 ± 4.70b	139.41 ± 3.61a	278.55 ± 4.97b	3275.44
MVD-P	314.06 ± 22.25c	32.85 ± 0.18c	19.82 ± 0.59b	60.41 ± 1.11c	58.93 ± 2.70c	89.73 ± 0.96c	136.97 ± 1.43c	712.77
MVD-T	2912.66 ± 60.15a	47.87 ± 0.19a	28.53 ± 0.38a	86.01 ± 1.33a	92.08 ± 2.61a	115.36 ± 5.70b	649.54 ± 36.16a	3932.05

Results are the mean values ± standard deviation from three measurements (n = 3); Means in the same column with different letters (a, b, c, d, and e) are significantly different at p < 0.05; The units of PFC and PFCA were expressed in mg/100 g FW; The units of FD and MVD processed products were expressed in mg/100 g DW; PFC: Perilla frutescens var. crispa; PFCA: Perilla frutescens var. crispa (cv. Antisperill); FD: Freeze-Dried; MVD: Microwave assisted low-temperature vacuum dried; HV1: 1st harvest; HV2: 2nd harvest; HV3: 3rd harvest; P: Pill; T: Tea bag; N.D: Not detected; N.A: Not available

Meng et al. (2008) reported a high content of rosmarinic acid in red and red-green perilla, while green perilla had low or no content of this compound. Additionally, Liu et al. (2013) reported that purple with green perilla exhibited the highest rosmarinic acid content. These studies support the findings of the present study, which demonstrated that the rosmarinic acid content in red perilla was significantly higher than that in ‘Antisperill’. Rosmarinic acid content of ‘Antisperill’ processed products was significantly higher in MVD-T, MVD-HV3, and MVD-P. The rosmarinic acid content of perilla and its processed products showed a similar trend to the total phenolic content, which is due to rosmarinic acid being the main phenolic compound in perilla (Zhou et al., 2014).

Polyphenol content in the processed products of ‘Antisperill’ varied depending on the drying method used. The levels of chlorogenic acid, (−)-epicatechin, (−)-epicatechin gallate, and rutin were found to be significantly higher in MVD samples compared to FD samples. Wojdyło et al. (2014) found that MVD drying method preserved the highest amounts of polyphenols in sour cherries. These results support our study, which also showed that the polyphenol content in MVD samples was significantly higher than in FD samples.

The ‘Antisperill’ MVD samples exhibited significantly higher contents of chlorogenic acid, (−)-epicatechin, (−)-epicatechin gallate, and quercetin, followed by MVD-T, MVD-HV3, and MVD-P samples. These contents were observed to decrease with each processing step and were found to be similar to the total phenolics content observed in this study.

Isoegomaketone contents

Isoegomaketone content of perilla and its processed products is shown in Fig. 2. Isoegomaketone was not detected in red perilla, and isoegomaketone content of ‘Antisperill’ raw material was 2.09 ± 0.42 mg/100 g FW. Isoegomaketone content of ‘Antisperill’ processed products was 103.70 ± 8.85, 56.91 ± 1.64, and 87.10 ± 7.48 mg/100 g DW for MVD-HV3, MVD-P, and MVD-T respectively, and it was significantly higher followed by MVD-HV3, MVD-T, and MVD-P.

Fig. 2.

Isoegomaketone contents of PFC, PFCA (A), and PFCA processed products (B). Vertical bars indicate standard deviation. Different letters are significant differences by Duncan’s multiple range test (p<0.05). N.D, Not detected.

Perilla contains essential oil components such as isoegomaketone, perilla ketone, perillaldehyde, esholtzia ketone, and limonene. These aromatic substances give perilla its distinctive fragrance. Isoegomaketone, in particular, has been studied for its various functions, including anti-obesity, antioxidant, anti-inflammatory, and anti-cancer properties (Jin et al., 2016). According to Martinetti et al. (2017), isoegomaketone was not detected in the red-colored P. frutescens var. crispa, and perillaldehyde emerged as the primary aromatic compound in red perilla, which supports the findings of our study where isoegomaketone was also not detected in red perilla. Several studies have reported lower or undetectable levels of isoegomaketone in red perilla compared to green perilla. These studies have also noted that the difference in color may influence the accumulation of various phytochemicals in perilla (Chen et al., 2022; Martinetti et al., 2017). The isoegomaketone content of the processed products of ‘Antisperill’ revealed that MVD-P had significantly lower isoegomaketone content than MVD-HV3 and MVD-T. This difference is attributed to the volatility of isoegomaketone, an aromatic compound that is highly sensitive to high temperature, moisture, and oxygen. Consequently, the content in MVD-P was lower due to the inclusion of more processing steps compared to MVD-HV3 and MVD-T.

In conclusion, ‘Antisperill’ exhibits a range of antioxidant compounds, and the findings in this study indicate that the MVD method is the optimal drying technique for preserving and enhancing the antioxidant compounds and activities of ‘Antisperill’. Therefore, ‘Antisperill’ is expected to play various roles as a functional food and a natural antioxidant with anti-inflammatory as well as antioxidant functions.

Declarations

Conflict of interest

The authors declare no conflict of interest.