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rvH1N1 Neuraminidase Inhibitory Activities of Phenolics from Perilla frutescens (L.) and Their Contents in Cultivars and Germplasm
Plant Breeding and Biotechnology 2018;6:404-412
Published online December 31, 2018
© 2018 Korean Society of Breeding Science.

Tae Joung Ha*, Myoung-Hee Lee, Chang-Hwan Park, Jung-In Kim, Eunyoung Oh, Suk-Bok Pae, Jae Eun Park, Sung-Up Kim, and Do-Yeon Kwak

Department of Southern Area Crop Science, National Institute of Crop Science, Rural Development Administration, 20th Jeompiljero, Miryang 50424, Korea
Corresponding author: *Tae Joung Ha, taejoung@korea.kr, Tel: +82-55-350-1222, Fax: +82-55-353-3050
Received July 23, 2018; Revised October 22, 2018; Accepted November 1, 2018.
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract

The influenza neuraminidase (NA, E.C. 3.2.1.18), an antiviral, has been the target of high pharmaceutical companies due to its essential role in viral replication cycle. Perilla frutescens (P. frutescens) is used in traditional Chinese medicine for various diseases, such as cold due to wind-cold, headache and cough. In this context, four major polyphenolic compounds including rosmarinic acid-3-O-glucoside (1), rosmarinic acid (2), luteolin (3), and apigenin (4) isolated from P. frutescens were evaluated for their inhibitory effect on recombinant virus H1N1 neuraminidase (rvH1N1 NA). Among the test compounds, rosmarinic acid and luteolin inhibited the rvH1N1 NA with an IC50 of 46.7 and 8.4 μM, respectively. The inhibition kinetics analyzed by the Dixon plots indicated that rosmarinic acid and luteolin were noncompetitive inhibitors and that the inhibition constant, KI, was established as 43.9 and 14.3 μM, respectively. In addition, 578 genetically diverse accessions and 39 cultivars of P. frutescens were analyzed using HPLC to characterize the diversity of polyphenolic composition and concentration. The individual and total compositions exhibited significant difference (P < 0.05), especially rosmarinic acid which was detected as the predominant metabolite in all accessions (58.8%) and cultivars (62.8%). Yeupsil and Sangback cultivars exhibited the highest rosmarinic acid (3,393.5 μg/g) and luteolin (383.3 μg/g) content respectively. YCPL177-2 with the high concentration (889.8 μg/g) of luteolin may be used as a genetic resource for breeding elite cultivars.

Keywords : Perilla frutescens, Neuraminidase, Luteolin, Rosmarinic acid
INTRODUCTION

The influenza virus, one of the most significant sources of viral respiratory infection in human, causes considerable morbidity and mortality and particularly fatal among young children, aged people, and patients with cardiopulmonary diseases (Baker and Mullooly 1980). In addition, influenza spreads around the world in seasonal epidemics killing numerous people in pandemic years. According to the World Health Organization (WHO) statistics, influenza causes an estimated 250,000–500,000 deaths and approximately three to five million cases of severe illness worldwide annually (Yang et al. 2016). Influenza virus, an RNA virus, belongs to the family of Orthomyxoviridae classified by the antigenic properties of two surface glycoproteins such as hemagglutinin (HA) and neuraminidase (NA). HA binds to the sialic acid-galactose of cell surface glycoproteins or glycolipids, while the NA releases virus by cleaving the sialic acid from the galactose on the cell surface (Moscona 2005). Thus, NA is believed to be necessary for the release of newly formed viruses from infected cells and for its efficient spread in the respiratory tract (von Itzstein 2007). Therefore, pharmacological strategies dealing with influenza pandemic is now based on antiviral drugs, in which NA inhibitors are the most important.

Perilla [Perilla frutescens (L.)] is an annual short-day plant which belongs to the family Labiatae (Lamiaceae). The P. frutescens have been used as an antioxidant and traditional herbal medicine for treating various diseases including depression, anxiety, tumor, cough, allergy, intoxication, and some intestinal disorders (Makino et al. 2003; Yang et al. 2012) in East Asian countries such as Korea, China, and Japan. P. frutescens are taxonomically divided into two groups such as var. frutescens (“Deulkkae” in Korea) and var. crispa (“Chazogi” in Korea). The P. frutescens var. frutescens which is commonly known as “Deulkkae” in Korea has leaves that are widely used in sushi and herb salads as a spice, garnish or as food colorant. Perilla seeds are traditional source of oil produced in Korea. During the past three years, annual production and import of perilla seeds were approximately 48,730 and 24,760 metric tons, respectively. Moreover, perilla seeds contain considerably high levels (approx. 60%) of α-linolenic acid (ω-3), that possesses various potential biological activities such as beneficial effects on the vascular system (Shahidi and Miraliakbari 2005). In addition to α-linolenic acid, various flavonoids: apigenin, luteolin, scutellarein, shisonin; sterols: β-sitosterol, stigmasterol, campesterol; terpenoids: ursolic acid, oleanolic acid, tormentic acid; and phenolic acids: rosmarinic acid, caffeic acid, ferulic acid were isolated from the leaves and seeds of P. frutescens (Chen et al. 2003; Peng et al. 2005; Ȍztȕrk et al. 2010; Kang and Lee 2011). In previous reports, flavonoids were emphasized while phenolic compounds were generally overlooked with respect to their biological activities. It should be noted, however, that phenolic compounds isolated from P. frutescens can be expected to possess various biological activities. For example, rosmarinic acid is known as a highly efficient superoxide radical scavenger and inhibits epidermal inflammatory responses (Osakabe et al. 2004). Recently, our previous investigation on perilla seeds have led to the isolation and identification of nine phenolic compounds by ultra performance liquid chromatography with photodiode array detector and electrospray ionisation/mass analysis (Lee et al. 2013). In addition, several bioactivities such as antioxidant, α-glucosidase and aldose reductase inhibition effects were evaluated (Ha et al. 2012; Lee et al. 2013).

Though perilla seeds are widely used as traditional Chinese medicine for various diseases, its phytopharmacology and biological activity are not yet fully investigated. Reportedly, the investigation regarding NA inhibition of this plant has not been studied. Thus, this study examined the four major phenolic compounds (Fig. 1) isolated from perilla to gain new insights into their NA inhibitory action on a molecular level. The content of these compounds in selected Korean perilla germplasm and cultivars were also quantified.

MATERIALS AND METHODS

Plant materials

Thirty nine perilla cultivars (Table 1) including Anyu, Areum, Baegsang, Baekjin, Baekkwang, Bora, Chungsan, Daesil, Daesin, Daeyeop, Daeyu, Dami, Danjo, Dasil, Dayu, Deulhyang, Deulsaem, Donggeul1ho, Donggeul2ho, Hyangim, Ilyeop, Ipdeulkkae1, Joim, Kwangim, Manback, Namcheon, Neulbora, Okdong, Saebora, Saeyeupsil, Sangback, Sangyeup, Sodam, Soim, Whahong, Yangsan, Yeupsil, Youngho, and Yujin were studied and developed by the National Institute of Crop Science (NICS), Rural Development Administration (RDA), Korea. The perilla collection in NICS Genebank holds 1,896 germplasm, the seeds of which are preserved in cool dry storage. From this total, 578 P. frutescens accessions (Supplementary Table S1) were also used. These cultivars and germplasm were grown in the experimental field of the NICS, Milyang, Gyeongnam, in 2016. After harvesting, perilla seeds were immediately freeze-dried and stored at −20°C until further use and analysis.

Chemicals

Recombinant influenza A virus H1N1 (rvH1N1) neuraminidase (EC 3.2.1.18) was purchased from R&D Systems (Minneapolis, MN, USA). 2′-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid (4-MUNANA), acetic acid, CaCl2, DMSO, Tris, and NaCl were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Analytical grade acetonitrile and water were purchased from J.T. Baker (Phillipsburg, NJ, USA). Sep-Pak cartridges were purchased from Waters Co., (Milford, MA, USA). Ha et al. (2012) reported the quantitative analysis of the four major phenolic compounds. Herein, 1H and 13C NMR spectroscopic data were discussed below.

Rosmarinic acid-3-O-glucoside (1)

1H NMR (500 MHz, DMSO-d6): δ 3.03 (1H, dd, J = 14.5, 8.5 Hz, H-7′α), 3.11 (1H, dd, J = 14.5, 4.5 Hz, H-7′β), 3.42 (1H, dd, J = 8.5, 8.5 Hz, glc-4), 3.54 (3H, m, glc-2, glc-3, and glc-5), 3.75 (dd, J = 12.0, 6.0 Hz, glc-6α), 3.99 (1H, J = 12.0, 2.0 Hz, glc-6β), 4.85 (1H, d, J = 6.6 Hz, glc-1), 5.20 (1H, dd, J = 8.0, 4.0 Hz, H-8′), 6.39 (1H, d, J = 16.0 Hz, H-8), 6.63 (1H, dd, J = 8.0, 2.0 Hz, H-6′), 6.72 (1H, d, J = 8.0 Hz, H-5′), 6.78 (1H, d, J = 2.0 Hz, H-2′), 6.88 (1H, d, J = 8.5 Hz, H-5), 7.20 (1H, dd, J = 8.0, 1.5 Hz, H-6), 7.55 (1H, s, H-2), 7.62 (1H, d, J = 16.0 Hz, H-7). 13C NMR (125 MHz, DMSO-d6): δ 38.3 (C-7′), 63.0 (glc-6), 71.9 (glc-4), 75.1 (C-8′), 75.3 (glc-2), 78.0 (glc-5), 78.9 (glc-3), 104.6 (glc-1), 115.6 (C-8), 116.8 (C-5′), 117.9 (C-5), 118.1 (C-2′), 118.5 (C-2), 122.3 (C-6′), 126.7 (C-6), 128.3 (C-1), 129.7 (C-1′), 145.7 (C-4′), 146.5 (C-3′), 147.5 (C-7), 147.6 (C-3), 151.6 (C-4), 168.7 (C-9), 173.8 (C-9′).

Rosmarinic acid (2)

1H NMR (500 MHz, CD3OD): δ 3.00 (1H, dd, J = 14.4, 8.5 Hz, H-7′α), 3.09 (1H, dd, J = 14.4, 4.28 Hz, H-7′β), 5.17 (1H, dd, J = 8.4, 4.2 Hz, H-8′), 6.26 (1H, d, J = 15.9 Hz, H-8), 6.61 (1H, dd, J = 8.1, 2.0 Hz, H-6′), 6.69 (1H, d, J = 8.0 Hz, H-5′), 6.74 (1H, d, J = 2.0 Hz, H-2′), 6.77 (1H, d, J = 8.1 Hz, H-5), 6.94 (1H, dd, J = 8.2, 2.0 Hz, H-6), 7.04 (1H, d, J = 2.0 Hz, H-2), 7.54 (1H, d, J = 15.9 Hz, H-7). 13C NMR (125 MHz, CD3OD): δ 38.4 (C-7′), 75.2 (C-8′), 114.9 (C-8), 115.6 (C-2), 116.7 (C-5′), 116.9 (C-5), 117.9 (C-2′), 122.2 (C-6′), 123.6 (C-6), 128.1 (C-1), 129.8 (C-1′), 145.7 (C-4′), 146.6 (C-3′), 147.2 (C-3), 148.1 (C-7), 150.1 (C-4), 168.9 (C-9), 174.1 (C-9′).

Luteolin (3)

1H NMR (500 MHz, CD3OD): δ 6.20 (1H, d, J = 2.1 Hz, H-8), 6.45 (1H, d, J = 2.1 Hz, H-6), 6.67 (1H, s, H-3), 6.90 (1H, d, J = 8.1 Hz, H-5′), 7.40 (1H, d, J = 2.3 Hz, H-2′), 7.42 (1H, dd, J = 8.2, 2.3 Hz, H-6′). 13C NMR (125 MHz, CD3OD): δ 94.3 (C-8), 99.2 (C-6), 103.2 (C-3), 104.1 (C-10), 113.8 (C-2′), 116.4 (C-5′), 119.3 (C-6′), 121.9 (C-1′), 146.1 (C-3′), 150.1 (C-4′), 157.7 (C-9), 161.9 (C-7), 164.3 (C-2), 164.5 (C-5), 182.0 (C-4).

Apigenin (4)

1H NMR (500 MHz, CD3OD): δ 6.19 (1H, d, J = 2.1 Hz, H-8), 6.47 (1H, d, J = 2.1 Hz, H-6), 6.76 (1H, s, H-3), 6.92 (2H, d, J = 9.7 Hz, H-2′ and H-6′), 7.92 (2H, d, J = 9.7 Hz, H-3′ and H-5′). 13C NMR (125 MHz, CD3OD): δ 94.3 (C-8), 99.2 (C-6), 103.2 (C-3), 104.1 (C-10), 116.3 (C-2′ and C-6′), 121.6 (C-2), 128.8 (C-3′ and C-5′), 157.7 (C-5), 161.5 (C-4′), 161.8 (C-9), 164.1 (C-1′), 164.5 (C-7), 182.1 (C-4).

rvH1N1 NA inhibitory activities

Throughout the experiment, 4-MUNANA was used as a substrate. In a fluorescence experiment, the cleaving activity of the NA was monitored at 37°C by Spectra MAX plus spectrophotometer (Molecular Devices, Sunnyvale, CA). The inhibitory activities of NA were performed by following the methodology used by We et al. (2009) with slight modification. In general, 10 μL of an ethanolic inhibitor solution was mixed with 50 μL of 1.2 mM stock solution of 4-MUNANA and 2.93 mL of 50 mM Tris buffer (5 mM CaCl2, 200 mM NaCl, pH 7.5) in a quartz cuvette. Then, 10 μL of a 50 mM Tris buffer solution of rvH1N1 NA was added. The resultant solution was mixed well, and monitored with a fluorescence spectrometer at an excitation of 365 nm and emission of 445 nm in a kinetic mode. The linear increase of emission at 445 nm, which expresses the formation of 4-methylumbelliferone(4-MU) was measured continuously. Two concentrations (100 and 20 μM) of 4-MUNANA were selected for Dixon plots.

The assay was conducted in triplicate of separate experiments. The data analysis was performed by using Sigma Plot 13.0 (SPSS Inc, Chicago, IL). The inhibitory concentration leading to 50% activity loss (IC50) was obtained by fitting experimental data to the logistic curve by the equation as follows (Copeland 2000):

Activity (%)=100[1/(1+([I]/IC50))]

Inhibition mode was analyzed by Enzyme Kinetics Module (SPSS Inc.) equipped with Sigma Plot 13.0.

HPLC conditions for quantification

The quantification of four major phenolic compounds in the seeds of perilla germplasm and cultivars were carried out using Ultimate 3000 HPLC (Dionex Softron GmbH, Germering, Germany) analysis. A 20 μL sample of the 80% methanol extract was injected into an analytical Eclipse XDB-C18 column (150 × 4.6 mm I.D., 5 μm; Agilent Technologies, Palo Alto, CA, USA). Gradient elution was carried out with 0.1% acetic acid in water (eluent A) and 0.1% acetic acid in acetonitrile (eluent B). The gradient elution program was as follows: 0–3 minutes, 5% B; 5 minutes, 10% B; 20 minutes, 30% B; 30 minutes, 50% B, and then held for 10 minutes before returning to the initial conditions. The total running time was 30 minutes with a flow rate of 1.0 mL/minute. Moreover, the column temperature was maintained at 25°C and the detection was made at 330 nm.

Preparation of sample and calibration curve

The dried seeds of perilla were pulverised (60 mesh) for 3 minutes using a HR 2860 coffee grinder (Philips, Drachten, Netherlands). Each sample (1.0 g) was extracted in 30 mL of 80% methanol for 6 hours at room temperature in a shaking incubator. The supernatant was centrifuged at 3000×g for 3 minutes and then filtered through a 0.45 μm syringe filter (Whatman Inc., Maidstone, UK) prior to HPLC analysis. For quantification, the peak areas of the isolated compounds were integrated from the HPLC chromatogram at 330 nm using Dionex software. The stock solutions were prepared by dissolving in methanol to obtain a 1 mg/mL concentration. Calibration curves were obtained with methanol at eight different concentrations (0.5, 1, 2, 5, 10, 25, 50, and 100 μg/mL). All calibration curves had coefficients of linear correlation r2 > 0.998.

Statistical analysis

Statistical data analysis was performed using the analysis of variance (ANOVA) and the least significant difference (LSD) test to determine statistically different values at a significance level of α ≤ 0.05. All statistical analyses were performed using the SAS EG (SAS Institute Inc., Cary, NC, USA).

RESULTS

rvH1N1 NA inhibitory activities

Numerous studies demonstrated that natural products in traditional Chinese medicine have been given unprecedented attention due to their biological effects such as antiviral activities (Kitazato et al. 2007; Yang et al. 2016). It is well-known that influenza virus is one of the major pandemic diseases worldwide because it can lead to serious threat to human health. Neuraminidase (NA) inhibitors have the potential to treat influenza epidemics because they block the secretion of virions from the surface of infected cells. Thus, four phenolic compounds including rosmarinic acid-3-O-glucoside (1), rosmarinic acid (2), luteolin (3), and apigenin (4) as shown in Fig. 1 were isolated from the seeds of P. frutescens and were evaluated for their inhibitory activities against rvH1N1 NA. In the NA inhibitory assay, the enzyme assay was performed using fluorescence spectrophotometer to detect the increase at 445 nm associated with 4-MU released from the substrate. As a result, luteolin showed a dose-dependent inhibitory effect on rvH1N1 NA, as shown in Fig. 2. As luteolin increased, the enzyme activity rapidly decreased until it was completely suppressed. The inhibitor concen tration leading to 50% activity lost (IC50) was estimated to be 8.4 μM. Rosmarinic acid also showed a dose-dependent inhibitory effect on this hydrolysis. The IC50 of rosmarinic acid was obtained as 46.7 μM. Interestingly, rosmarinic acid-3-O-glucoside and apigenin did not show any inhibitory activity up to 100 μM, indicating that protocatechuic moiety was an essential element in eliciting the inhibitory activity.

Subsequently, the inhibition kinetics of rvH1N1 NA using luteolin was investigated. The kinetic behavior of rvH1N1 NA during the enzyme liberation of 4-MU was studied first. Under the conditions employed in the present investigation, the liberation of 4-MU catalyzed by rvH1N1 NA follows Michaelis-Menten kinetics. The kinetic parameters obtained from a Dixon plot show that Km is equal to 143.5 μM and that Vmax is equal to 333.6 RFU/min. The estimated value of Km obtained with a fluorometric method was similar to the previously reported value (Potier et al. 1979). In addition, comparable result was also obtained using rosmarinic acid with different kinetic parameters.

The kinetic and inhibition constants obtained are listed in Table 1. As illustrated in Fig. 2, the inhibition kinetics analyzed by Dixon plots show that rosmarinic acid and luteolin are noncompetitive inhibitors because increasing substrates resulted in a family of lines with a common intercept on the −[I] axis but with different slopes. The equilibrium constant for inhibitor binding, KI, was obtained from −[I] value at the intersection of two lines. The inhibition kinetics analyzed by Lineweaver-Burk plots also confirmed that rosmarinic acid and luteolin are noncompetitive inhibitors (data not shown).

Quantification of phenolic compounds in perilla cultivars and germplasm

In general, plant foods serve as an important source of phenolic compounds such as phenolic acids, flavonoids, and condensed tannins. For example, millet has many bioactive compounds, including hydroxybenzoic acid, the derivatives of which showed the highest content with 87–98%, hydroxycinnamic acid, and flavonoid. Among them, p-coumaric acid and trans-ferulic acid were observed to be the main compounds in various millet cultivars (Chandrasekara and Shahidi 2011). Apigenin derivatives of flavonoid type were also present in high amounts in comparison with other phenolic compounds (Chandrasekara and Shahidi 2012). Isoflavones were derived from the phenylpropanoid pathway and malonylglucoside isoflavones were predominant in soybean (Lee et al. 2005). Many researchers have reported that perilla contains several phenolic compounds, including flavones, anthocyanins, and phenolic acids (Kosuna and Haga 1997). More recently, the isolation, identification and quantitification of nine phenolic compounds from the seed of perilla were reported (Lee et al. 2013). To date, there is no fully comparative study on phenolics composition conducted in all Korean-bred perilla cultivars and germplasm. Moreover, their contents have not been extensively investigated in various perilla germplasm. Thus, for this study, the chemical compositions and contents of four major phenolics obtained from 39 perilla cultivars and 578 perilla germplasm accessions were analyzed using HPLC. As shown in Table 2, phenolic compounds such as rosmarinic acid-3-O-glucoside, rosmarinic acid, luteolin, and apigenin varied in all Korean-bred perilla cultivars and are statistically significant at (P < 0.05) differences. Among the phenolic compounds, rosmarinic acid was detected as the predominant (62.8%) metabolite in all cultivars, followed by rosmarinic acid-3-O-glucoside (32.9%), and luteolin (3.2%). Total phenolic contents were also observed with significant differences (P < 0.05) ranging from 2,188.5 to 5,669.8 μg/g. Among the 39 cultivars, Yeupsil showed the highest total phenolic content with 5,669.8 ± 33.7 μg/g, while the lowest was observed in cultivar Donggeul1ho (2188.5 ± 74.4 μg/g) (Table 2). In previous results, both rosmarinic acid and luteolin were found to inhibit the rvH1N1 NA indicating that these two compound contents are important factors in determining the quality and functionality of perilla. Results showed that the highest rosmarinic acid content was found in Yeupsil with 3,393.5 ± 19.8 μg/g, while the lowest was observed in cultivar Areum (1,333.8 ± 5.8 μg/g) (Table 2). On the other hand, the highest luteolin content was found in Snagback with 383.3 ± 8.1 μg/g followed by Bora (343.7 ± 11.7 μg/g) and Sebora (320.0 ± 12.1 μg/g). This indicates that cultivars Yeupsil and Snagback can be used to further improve the perilla hybridization program to obtain more desired varieties.

The composition and contents of four major phenolics in 578 perilla germplasm were also investigated by HPLC. The frequency distribution and median of individual phenolics in the perilla germplasm are shown in Fig. 3. The mean contents for rosmarinic acid-3-O-glucoside, rosmarinic acid, luteolin and apigenin were 539.9 (25.5%), 1,257.1 (58.8%), 163.2 (7.6%) and 178.9 (8.4%) μg/g, respectively. The concentration of rosmarinic acid ranged from 280.8 to 3,230.3 μg/g, which is a 10-fold difference between the lowest and the highest concentrations. Among the germplasm, YCPL601 showed the highest values for rosmarinic acid. In addition, luteolin and apigenin contents ranged from 0 to 889.8 and μg/g, from from 0 to 2,657.4 μg/g, respectively. The contents of luteolin and apigenin in germplasm showed 2.3 and 12.7 times higher than those of higher cultivars (cv. Sangback and cv. Bora). The highest luteolin and apigenin contents were found in YCPL177-2 and YCPL205-1 with 889.8 and 2,657.4 μg/g, respectively. Moreover, the frequency distribution and median of individual phenolics between P. frutescens var. frutescens and P. frutescens var. crispa are shown in Fig. 4. The average contents of rosmarinic acid-3-O-glucoside and rosmarinic acid in P. frutescens var. frutescens (893.6 and 1,451.7 μg/g, respectively) were significantly (P < 0.05) higher than that in P. frutescens var. crispa (158.3 and 1,047.1 μg/g, respectively). The average contents of apigenin, on the other hand, in P. frutescens var. crispa (196.0 μg/g) was significantly (P < 0.05) higher than that in P. frutescens var. frutescens (87.9 μg/g). Therefore, the selection of germplasm accessions for perilla breeding may also facilitate the development of lines with desirable levels of phenolic compounds. This research was the first to demonstrate the comparison and determination of phenolic compounds in the seeds of various perilla germplasm.

DISCUSSION

In this study, the inhibitory activity of the four major phenolic compounds isolated from the seeds of P. frutescense against recombinant virus H1N1 neuraminidase (rvH1N1 NA) was described. Among them, rosmarinic acid and luteolin had noncompetitive inhibitory effects on rvH1N1 NA with an IC50 of 46.7 and 8.4 μM, respectively. Thus, these compounds could be useful in further investigation for potential therapeutic agents in the treatment of influenza. However, the biological significance of rosmarinic acid and luteolin as neuraminidase inhibitors in living systems is still largely unknown. Hence, it is not clear if ingested rosmarinic acid and luteolin are absorbed into the system through the intestinal tract and delivered to the places where neuraminidase inhibitors are needed. Moreover, findings showed that there is great variability in the rosmarinic acid and luteolin composition in all Korean-bred perilla cultivars and germplasm. In particular, rosmarinic acid was the predominant component in all cultivars and germplasm. Cultivars Yeupsil and Snagback were found to have higher content of rosmarinic acid and luteolin than the other cultivars. The significant differences in luteolin content among germplasm accessions were also observed and it ranged between 0.0 and 889.8 μg/g. Maximum contents of luteolin was observed in YCPL177-2 germplasm that is 2.3 times higher than those of higher cultivar (cv. Sangback). Therefore, germplasm accessions with high content of rosmarinic acid and luteolin could be further used in crop improvement program for quality and functionality of perilla.

Supplementary Information
ACKNOWLEDGEMEMTS

This work was supported by a grant of AGENDA Program (No. PJ010159042018), Rural Development Administration, Republic of Korea.

Figures
Fig. 1. Chemical structures of compound 1–4 isolated from seed of P. frutescens.
Fig. 2. Effects of rosmarinic acid (2) and luteolin (3) on the activity of rvH1N1 neuraminidase. (A) Dose dependent inhibition of rvH1N1 NA activity. (B) Dixon plots of 4-MU releasing by rvH1N1 NA in the presence of rosmarinic acid (2) and luteolin (3) in Tris buffer (pH 7.5) at 37°C.
Fig. 3. Box plot of the phenolic constituents in the seeds of 578 perilla germplasm. The horizontal lines in the interior of the box are the median values. The height of a box is equal to the interquartile distance, indicating the distribution for 75% of the data. Approximately 90% of the data falls inside the whiskers (the lines extending from the top and bottom of the box). RA-3-glc: rosmarinic acid-3-O-glucoside, RA: rosmarinic acid, Lut: luteolin, Api: apigenin.
Fig. 4. Range and distribution of the phenolic contents between P. frutescens var. furtescens and P. frutescens var. crispa. RA-3-glc: rosmarinic acid-3-O-glucoside, RA: rosmarinic acid, Lut: luteolin, Api: apigenin.
Tables

Kinetics and inhibition constants of luteolin and rosmarinic acid on rvH1N1 neuraminidase.

Compounds

Luteolin Rosmarinic acid
IC50 8.4 μM 46.7 μM
Km 143.5 μM 133.1 mM
Vmax 333.6 RFU/min 325.7 RFU/min
Inhibition type noncompetitive noncompetitive
KI 14.3 μM 43.9 μM

Comparison of rosmarinic acid-3-O-glucoside (1), rosmarinic acid (2), luteolin (3), and apigenin (4) contents in the seed of perilla cultivars.

Cultivars Contents (μg/g)z)

1 2 3 4 total
Anyu 1,259.5 ± 22.1j 2,283.1 ± 25.7e,f,g 39.8 ± 0.7k,l 41.9 ± 13.4h,i,j 3,624.3 ± 33.6d
Areum 1,266.4 ± 13.3j 1,333.8 ± 5.8u 9.7 ± 0.2q 27.6 ± 0.1j,k 2,637.6 ± 19.1p
Baegsang 1,126.1 ± 24.8l,m 2,295.5 ± 40.8d,e,f - - 3,421.6 ± 65.6e,f,g
Baekjin 1,805.5 ± 9.4c 2,095.7 ± 2.3i,j,k,l,m,n - - 3,901.2 ± 7.1c
Baekkwang 1,488.7 ± 7.7f 2,413.9 ± 18.0d - - 3,902.6 ± 25.7c
Bora 484.0 ± 6.6u 2,366.1 ± 10.5d,e 343.7 ± 11.7b 208.9 ± 27.7a 3,402.8 ± 14.9e,f,g,h
Chungsan 1,358.8 ± 25.1h,i 2,134.2 ± 27.5h,I,j,k,l 11.0 ± 0.1o,p 27.1 ± 0.1j,k 3,531.1 ± 52.5d,e
Daesil 1,633.5 ± 3.6e 1,994.0 ± 7.4m,n,o,p,q 86.9 ± 3.6i 85.9 ± 13.9f 3,800.2 ± 28.4c
Daesin 539.5 ± 13.8t 1,940.7 ± 20.5o,p,q,r 271.0 ± 4.0d 119.4 ± 1.8c,d 2,870.5 ± 32.0n,o
Daeyeop 1,173.4 ± 6.6k 2,214.4 ± 25.6f,g,h,i - - 3,387.8 ± 32.1e,f,g,h
Daeyu 919.6 ± 10.9q 2,251.6 ± 17.8e,f,g,h - - 3,171.2 ± 6.9i,j,k
Dami 1,065.5 ± 22.9n 2,150.9 ± 42.4g,h,I,j,k,l 18.8 ± 0.6m,n 31.0 ± 0.1j 3,266.2 ± 64.8g,h,i,j
Danjo 1,089.6 ± 8.3m,n 2,054.8 ± 22.2k,l,m,n,o - - 3,144.3 ± 30.5j,k,l
Dasil 967.2 ± 13.9o,p 2,082.7 ± 39.8i,j,k,l,m,n 10.3 ± 0.1p,q 26.5 ± 0.1j,k 3,086.8 ± 53.8k,l,m
Dayu 1,334.3 ± 17.1i 2,156.1 ± 25.3g,h,I,j,k,l 27.8 ± 3.6l,m 37.8 ± 1.2i,j 3,556.0 ± 37.6d,e
Deulhyang 1,167.1 ± 28.0k,l 1,878.0 ± 7.4q,r - - 3,045.0 ± 10.2k,l,m
Deulsaem 1,232.4 ± 9.8j 1,587.8 ± 8.6s 94.1 ± 1.7i 62.9 ± 4.6g,h 2,977.1 ± 12.1l,m,n
Donggeul1ho 355.0 ± 1.4v 1,474.9 ± 83.2s,t 233.8 ± 19.2e 124.9 ± 26.4c,d 2,188.5 ± 74.4r
Donggeul2ho 470.3 ± 2.1u 2,026.4 ± 23.1l,m,n,o,p 116.9 ± 2.1h 90.7 ± 1.5e,f 2,704.4 ± 21.7o,p
Hyangim 1,427.0 ± 18.1g 2,916.3 ± 19.1b 11.5 ± 0.1o,p 27.1 ± 0.1j,k 4,381.9 ± 37.1b
Ilyeop 986.2 ± 22.5o 2,206.6 ± 8.4f,g,h,I,j 225.0 ± 1.5e 78.1 ± 6.5f,g 3,495.9 ± 10.2d,e,f
Ipdeulkkae1 832.0 ± 3.9r 1,968.5 ± 1.5n,o,p,q 124.2 ± 3.3g,h 115.7 ± 3.1c,d 3,040.4 ± 2.2k,l,m
Joim 1,434.3 ± 13.5g 1,897.3 ± 6.5p,q,r - - 3,331.6 ± 20.0f,g,h,i
Kwangim 1,722.5 ± 47.4d 2,072.0 ± 5.8j,k,l,m,n,o 14.3 ± 0.3n 29.5 ± 0.1j 3,838.3 ± 10.5c
Manback 937.0 ± 28.2p,q 2,181.1 ± 30.2f,g,h,I,j,k 12.8 ± 0.2o 29.4 ± 0.2j 3,160.3 ± 38.1i,j,k
Namcheon 790.5 ± 40.4r 1,912.2 ± 12.4p,q,r 133.5 ± 3.3f,g 112.6 ± 2.1d, e 2,948.8 ± 16.5m,n
Neulbora 373.3 ± 19.2v 2,118.0 ± 14.6h,I,j,k,l,m 89.8 ± 2.0i 91.7 ± 2.0e,f 2,672.8 ± 16.9p
Okdong 899.0 ± 36.4q 1,413.5 ± 47.0t,u 11.2 ± 0.4o,p 27.2 ± 0.1j,k 2,350.8 ± 83.9q
Saebora 548.4 ± 5.1t 2,350.4 ± 69.2d,e 320.0 ± 12.1c 153.6 ± 12.2b 3,372.4 ± 98.4f,g,h
Saeyeupsil 1,231.4 ± 40.9j 1,373.1 ± 54.2t,u 10.2 ± 0.1p,q 26.7 ± 0.1j,k 2,641.4 ± 95.2p
Sangback 644.6 ± 42.0s 2,058.9 ± 12.3k,l,m,n,o 383.3 ± 8.1a 151.5 ± 13.4b 3,238.3 ± 14.4h,i,j
Sangyeup 457.5 ± 29.2u 2,110.9 ± 12.3i,j,k,l,m 141.6 ± 3.2f 137.8 ± 17.4b,c 2,847.8 ± 17.3n,o
Sodam 1,895.3 ± 8.0b 1,391.5 ± 4.6t,u 87.0 ± 1.9i 90.2 ± 0.9e,f 3,464.0 ± 15.4d,e,f
Soim 629.8 ± 8.3s 1,835.0 ± 22.9r 142.9 ± 0.7f 124.0 ± 7.3c,d 2,731.7 ± 39.2o,p
Whahong 653.5 ± 1.4s 1,810.5 ± 0.5r 80.8 ± 0.5i,j 50.4 ± 0.9h,i 2,595.3 ± 2.3p
Yangsan 1,130.7 ± 27.2k,l,m 2,354.8 ± 65.4d,e 68.2 ± 1.2j 59.8 ± 0.7g,h,i 3,613.5 ± 90.7d
Yeupsil 2,216.3 ± 14.5a 3,393.5 ± 19.8a 29.6 ± 0.7l,m 30.4 ± 0.1j 5,669.8 ± 33.7a
Youngho 1,397.5 ± 9.7g,h 2,159.7 ± 22.7g,h,I,j,k,l - - 3,557.2 ± 32.4d,e
Yujin 1,526.5 ± 7.2f 2,776.5 ± 17.8c 49.2 ± 0.5k 49.3 ± 1.7h,i,j 4,401.6 ± 22.7b

Data are expressed as mean ± standard error of triplicate samples.

Different superscripts in the same column represent significant differences between samples (P < 0.05).


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