Network pharmacology and experimental evidence: MAPK signaling pathway is involved in the anti-asthma roles of Perilla frutescens leaf.

Perilla frutescens (PF) leaf is a traditional Chinese medicine and food with beneficial effects on allergic asthma. We sought to elucidate the active compounds, the targets, and underlying mechanisms of PF leaf in the treatment of allergic asthma by using experimental pharmacology and network pharmacology. An OVA-allergic asthma murine model was constructed to evaluate the effect of PF leaf on allergic asthma. And the network pharmacology and western blotting were performed to evaluate its underlying mechanisms in allergic asthma. PF leaf treatment significantly improved the lung function of OVA model mice and mitigated lung injury by significantly reducing of OVA-specific immunoglobulin E in serum, and interleukin 4, interleukin 5 and tumor necrosis factor alpha in the bronchoalveolar lavage fluid. 50 core targets were screened based on 8 compounds (determined by high performance liquid chromatography) through compound-target- disease network. Furthermore, MAPK signaling pathway was identified as the pathway mediated by PF leaf with the most potential against allergic asthma. And the WB results showed that PF leaf could down-regulate the expression of p-ERK, p-JNK and p-p38, which was highly consistent with the predicted targets and pathway network. In conclusion, this study provides the evidence to support the molecular mechanisms of PF leaf on the treatment of allergic asthma using network pharmacology and in vivo experiments.

Study on Purification, Identification and Antioxidant of Flavonoids Extracted from Perilla leaves.

The flavonoids from Perilla leaves were extracted using flash extraction assisted by ultrasonic extraction with ethanol. Subsequently, macroporous resin was employed for the isolation and purification of these flavonoids, followed by an investigation into their antioxidant activity. The process conditions for the extraction of flavonoids from Perilla leaves were designed and optimized using a one-way experiment combined with a response surface methodology. The optimal extraction conditions were determined as follows: the liquid-solid ratio was 20:1, ethanol volume fraction of 60%, ultrasound temperature of 60 °C, ultrasound time of 10 min and flash evaporation time of 60 s. The optimal extraction rate of flavonoids is 9.8 mg/g. In terms of separation and purification, a high-performance macroporous resin (HPD450 resin) with high purification efficiency was selected through static analysis and adsorption experiments. The optimal enrichment conditions were as follows: loading concentration of 0.06 mg/mL, optimal loading concentration of 20 mL, elution concentration of 70% and 76 mL, providing a reference for the further development and utilization of Perilla leaf flavonoids.

Effect of Extract of Perilla Leaves on the Quality Characteristics and Polycyclic Aromatic Hydrocarbons of Charcoal Barbecued Pork Patty.

This study aimed to investigate the effect of ethanolic extracts from perilla leaves (PLE) on the quality attributes and polycyclic aromatic hydrocarbons (PAHs) in charcoal-barbecued pork patties. The PLE addition and doneness had no significant effect on the pH of pork patties (p>0.05). Regardless of the concentration, the PLE significantly lower malondialdehyde concentrations and reduced the CIE L*, CIE a*, and CIE b* when compared to control. The addition of 0.2% of PLE did not adversely affect the organoleptic properties of doneness of medium and well-done pork patties. Addition of PLE at 0.4% to medium-cooked pork patties had stronger suppressing effect on the formation of light PAHs compare to control (p<0.05), also adding it to well-done pork patties had the lowest concentration of 4 PAHs and 8 PAHs, and a total of 16 PAHs (p<0.05). Therefore, PLE at 0.4% can be used for suppressing the formation of PAHs and lipid oxidation in well-cooked pork patty.

Microwave-ultrasonic technique development coupled with natural deep eutectic solvents in anthocyanin extraction from perilla leaves (Perilla frutescens var. Acuta).

BACKGROUND: As potent antioxidants, anthocyanins can protect the body from free radicals. However, the traditional solvent extraction method has the disadvantages of requiring a high extraction temperature and long extraction time, so it is necessary to develop an efficient extraction method for anthocyanins. RESULTS: In this study, the technique of natural deep eutectic solvents (DESs) was applied to extract anthocyanins from purple perilla leaves with the aid of microwave-ultrasonic assisted extraction (MUAE). The response surface methodology (RSM), based on the Box-Behnken design (BBD), predicted the maximum extraction yield of anthocyanins to be 619.62 mg (100 g)-1 under the following conditions: x1 (ultrasonic extraction power) = 357.25 W, x2 (time) = 25.62 min, and x3 (temperature) = 57.80 °C. The biological activity of the extract obtained was evaluated by examining its radical-scavenging effect on 1,1-diphenyl-2-picrylhydrazyl, hydroxyl radical, and superoxide anion radicals. Its bacteriostatic impact was investigated on four typical bacteria: Shewanella putrefaciens (S. putrefaciens), Pseudomonas fluorescens (P. fluorescens), Escherichia coli (E. coli), and Staphylococcus aureus (S. aureus). CONCLUSION: The integrated extraction method of DESs with MUAE was efficient, energy-saving, green, and sustainable. © 2022 Society of Chemical Industry.

Leaf-associated microbiota on perilla (Perilla frutescens var. frutescens) cultivated in South Korea to detect the potential risk of food poisoning.

Perilla (Perilla frutescens) is a commonly consumed herb with various health benefits in Asia. However, the risks of food-borne illness owing to the presence of pathogens on perilla leaves have not been evaluated. In this study, we evaluated the microbiota of perilla leaves harvested in South Korea using Illumina MiSeq sequencing of the 16S rRNA gene. In total, 2,743,003 sequencing reads were obtained, and 92-437 operational taxonomic units were observed in all samples. Bacterial loads were quantified, and the diversity indices were compared. Differences in the microbiota among sampling times and regions were also investigated. Proteobacteria and Firmicutes were predominant phyla at both times. At the class level, the bacterial communities were composed primarily of Alphaproteobacteria, Bacilli, and Gammaproteobacteria. Diverse bacterial taxa, such as Bacillus, uncultured family Enterobacteriaceae, and Sphingomonas were detected, and the representative pathogenic species (i.e., Acinetobacter lwoffii, Klebsiella pneumoniae, and Staphylococcus aureus) were quantified by qRT-PCR. The results of the co-occurrence network analysis showed characteristics of bacterial taxa in the microbiome on perilla leaves and provided insights into the roles of correlations among diverse microbes, including potential pathogens. Based on these results, the potential risk of food-borne illness from consumption of perilla leaves may be higher in July than in April. In summary, the microbial compositions determined in this study can be used as a base data for food-safety management for prediction and prevention of future outbreaks.

[Study on morphological classification and chemical-type of Perilla frutescens cultivated germplasm].

Fifty cultivated Perilla seeds were collected all over the country and planted in Beijing experiment field for morphology and chemical-type researches. Twenty morphological characteristics were selected and observed, and the essential oil from leaves was extracted by steam distillation and analyzed by GC-MS to confirm chemical-types. There were significant diversities in plant height, leaf color and morphology, and fruit color and weight. Clustering analysis was carried out based on these morphological characteristics. Six types were divided with their chemical-type designated. Type Ⅰ: Six germplasms, attributed to P. frutescens var. crispa, with dwarf plants, thin creased purple leaf, named Crispa, their chemical types were diversified, including EK, PAPK, PA and PK. Type Ⅱ: Six germplasms, attributed to P. frutescens var. crispa, plants were taller than type I and with thin and creased green leaf, named Big Crispa, all PK type. Type Ⅲ: Seventeen germplasms, attributed to P. frutescens var. frutescens with leaf color upside green and underside purple, tall plant and wide distribution all over the China, named Ordinary Frutescens, all PK. Type Ⅳ: Four germplasms, attributed to P. frutescens var. acuta with tall plant and small seed, named Acuta, all PK. Type Ⅴ: Seven germplasms, attributed to P. frutescens var. frutescens with green leaves, tall plants and long clusters, named Long-spike Frutescens, all PK. Type Ⅵ: Ten germplasms, attributed to P. frutescens var. frutescens with big, thick and creased leaf, named Thick-leaf Frutescens, including PK, PP, PL and PA. The morphological classification of this paper would lay the foundation for the taxonomic naming and following evaluation of the Perilla germplasm resources.This study also showed that there was no correspondence but a certain correlation between volatile oil chemical-types and subspecies classification and morphological characteristics of Perilla.

Characterization of metabolite profiles from the leaves of green perilla (Perilla frutescens) by ultra high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry and screening for their antioxidant properties.

The objective of this research was to access the determination of metabolite profiles and antioxidant properties in the leaves of green perilla (Perilla frutescens), where these are considered functional and nutraceutical substances in Korea. A total of 25 compositions were confirmed as six phenolic acids, two triterpenoids, eight flavonoids, seven fatty acids, and two glucosides using an ultra high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-Q-TOF-MS/MS) technique from the methanol extract of this species. The individual and total compositions exhibited significant differences, especially rosmarinic acid (10), and linolenic acids (22 and 23) were detected as the predominant metabolites. Interestingly, rosmarinic acid (10) was observed to have considerable differences with various concentrations in three samples (Doryong, 6.38 µg/g; Sinseong, 317.60 µg/g; Bongmyeong, 903.53 µg/g) by UPLC analysis at 330 nm. The scavenging properties against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radicals also showed potent effects with remarkable differences at a concentration of 100 µg/mL, and their abilities were as follows: Sinseong (DPPH, 86%; ABTS, 90%) > Bongmyeong (71% and 84%, respectively) > Doryong (63% and 73%, respectively). Our results suggest that the antioxidant activities of green perilla leaves are correlated with metabolite contents, especially the five major compositions 10 and 22-25. Moreover, this study may be useful in evaluating the relationship between metabolite composition and antioxidant activity.

Structural characterization of phenolic antioxidants from purple perilla (Perilla frutescens var. acuta) leaves.

The antioxidant activities of various extracts from purple perilla (Perilla frutescens var. acuta) leaves based on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability, 2,2-azino-bis-(3-ethylenebenzothiozoline-6-sulfonic acid) (ABTS) radical cation scavenging ability, and reducing power were investigated. Purple perilla leaves were initially extracted with 80% ethanol and then sequentially fractionated according to solvent polarity. Of all fractions, the ethyl acetate fraction had the highest antioxidant activity. This fraction was separated into four sub-fractions (sub-fractions 1-4) by Sephadex LH-20 chromatography. Of the four sub-fractions, sub-fraction 3 had the highest antioxidant activity. The EC50 values of sub-fraction 3 for DPPH radical scavenging activity and reducing power were 2.4 and 1.7 times lower than those of butylated hydroxytoluene (BHT), respectively. Based on HPLC analysis, the most abundant phenolic acid in sub-fraction 3 of purple perilla leaves was rosmarinic acid, at 314.3 mg/g. The structure of rosmarinic acid was confirmed by ESI-IT-TOF MS and NMR analysis.

Comparison of Growth Kinetics of Various Pathogenic E. coli on Fresh Perilla Leaf.

Growth kinetics for Escherichia coli O157:H7 in perilla leaves were compared to those of pathogenic E. coli strains, including enteropathogenic (EPEC), enterotoxigenic (ETEC), enteroinvasive (EIEC) and other enterohemorrhagic (EHEC) at 13, 17, 24, 30 and 36 °C. Models for lag time (LT), specific growth rate (SGR) and maximum population density (MPD) as a function of temperature were developed. The performance of the models was quantified using the ratio method and an acceptable prediction zone method. Significant differences in SGR and LT among the strains were observed at all temperatures. Overall, the shortest LT was observed with E. coli O157:H7, followed by EPEC, other EHEC, EIEC and ETEC, while the fastest growth rates were noted in EPEC, followed by E. coli O157:H7, ETEC, other EHEC and EIEC. The models for E. coli O157:H7 in perilla leaves was suitable for use in making predictions for EPEC and other EHEC strains.