Influence of Secondary Metabolites According to Maturation of Perilla (Perilla frutescens) on Respiratory Protective Effect in Fine Particulate Matter (PM2.5)-Induced Human Nasal Cell

Abstract

Fine particulate matter (PM2.5) exposure worsens chronic respiratory diseases through oxidative stress and inflammation. Perilla frutescens (L.) has potential respiratory protective properties, but the impact of growth stages on its beneficial metabolites is unclear. We aimed to evaluate how different growth stages affect phenolic acids, flavonoids, and polycosanols in perilla seeds and flowers and their efficacy in countering PM2.5-induced damage. Perilla seeds and flowers from five varieties at 10, 20, 30, and 40 days post-flowering were analyzed for metabolite content. Their antioxidant, anti-inflammatory, and respiratory protective effects were tested in RPMI 2650 cells. Our findings indicated that perilla flowers contained higher levels of functional components than seeds and exhibited significant variation with maturation. Phenolic acids of perilla flowers were highest at the early stages of maturation after flowering. However, individual flavones of perilla flowers were the highest at the late maturation stages after flowering. Extracts from perilla flowers harvested 20 days after flowering exhibited significant respiratory protection, effectively inhibiting inflammatory cytokines, mucus secretion, and oxidative stress markers. In conclusion, the flower parts of perilla, particularly those harvested 20 days after flowering, are useful materials for obtaining phenolic compounds, including rosmarinic acid, with high antioxidant and respiratory enhancement effects.

Keywords: fine particulate matter; growth periods; perilla flower; phenolics; respiratory disease.

Figure 1

Flowering stage and growth periods of five varieties of perilla seeds and flowers. (A) Perilla frutescens plant. (B) Perilla flower of Anyu cultivar at 10, 20, 30, and 40 days after flowering stage. (C) Flowering stage and harvesting times according to 5 different varieties.

Figure 2

Distribution of extraction yield [EY], antioxidant compounds (TPC, TFC), antioxidant activities (ABTS and DPPH radical scavenging activity), total individual phenolics [TIP], caffeic acid [CA], rosmarinic acid [RA], Luteolin [LT], Apigenin [APG], total policosanol [TPCC], Hexacosanol [HC], Octacosanol [OC], and Triacosanol [TC] in perilla seeds and flowers, based on varieties and growth periods. Values are the mean ± SD of 3 replicates. Different small letters in the same items indicate a significant difference (p < 0.05) among different growth periods. *** p < 0.001, ** p < 0.01 and * p < 0.05 represent significant differences between perilla seed and perilla flower.

Figure 3

Phenolic acid (caffeic acid and rosmarinic acid) and flavonoid (apigenin and luteolin) metabolic pathway of perilla according to maturation stage. Values are the mean ± SD of 3 replicates. Different small letters in the same items indicate a significant difference (p < 0.05) among different growth periods. *** p < 0.001 represent significant differences between perilla seed and perilla flower.

Figure 4

Respiratory disease enhancement effects of perilla seed and flower extracts with different varieties and growth periods in PM2.5-induced human nasal cell. (A) Cytotoxicity of extracts in human nasal (RPMI 2650) cell, (B) cell viability in PM2.5-exposed human nasal (RPMI 2650) cells. Values are the mean ± SD of 3 replicates. Different small letters in the same items indicate a significant difference (p < 0.05) among different growth periods. *** p < 0.001 represents significant difference between perilla seed and perilla flower. ^### p < 0.001 represents significant difference compared to PM2.5 treated control.

Figure 5

Respiratory disease enhancement effects of perilla seed and flower extracts with different varieties and growth periods in PM2.5-induced human nasal cell. (A) nitric oxide (NO), and (B) mucin 5AC (MUC5AC) secretion in PM2.5-exposed human nasal (RPMI 2650) cells. Values are the mean ± SD of 3 replicates. Different small letters in the same items indicate a significant difference (p < 0.05) among different growth periods. *** p < 0.001 represents significant difference between perilla seed and perilla flower. ^### p < 0.001 represents significant difference compared to PM2.5 treated control.

Figure 6

Correlation heat map analysis of the assigned antioxidant compounds, antioxidant activities, individual phenolic compounds, policosanol, and respiratory disease enhancement effects obtained from perilla (Perilla frutescens L.) seed and flower in various varieties and growth periods.

Figure 6

Correlation heat map analysis of the assigned antioxidant compounds, antioxidant activities, individual phenolic compounds, policosanol, and respiratory disease enhancement effects obtained from perilla (Perilla frutescens L.) seed and flower in various varieties and growth periods.

Figure 7

PM2.5-induced respiratory protective effect of selected cultivars (Anyu) at 20 days after flowering of perilla flowers in RPMI 2650 cells compared to perilla seed. Assessment of cytoprotective effect, reactive oxygen species (ROS) levels, lipid peroxidation of malonaldehyde (MDA; nmol mg⁻¹ protein), nitric oxide (NO), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, mucin 5AC (MUC5AC), and matrix metalloproteinase-9 (MMP-9) concentrations. Values are the mean ± SD of 3 replicates. Different small letters in the same items indicate a significant difference (p < 0.05) among different concentrations of extracts. *** p < 0.001, ** p < 0.01 and * p < 0.05 represent significant differences between perilla seed and perilla flower. ^### p < 0.001 represents significant difference compared to PM2.5 treated control.