Intranasal administration of the essential oil from Perillae Folium ameliorates social defeat stress-induced behavioral impairments in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Perillae Folium, the leaves and twigs of Perilla frutescens (L.) Britton, has been included in many traditional Chinese medicine herbal formulas to treat depression. However, the precise antidepressant mechanism of the essential oil from Perillae Folium (PFEO) has not been fully investigated. AIM OF THE STUDY: To assess the effects and potential mechanisms of PFEO on depression using animal models and network pharmacology analysis. MATERIALS AND METHODS: PFEO was intranasally administered to a mouse model of social defeat stress (SDS). The antidepressant effects of PFEO on SDS-induced mice were evaluated using behavioral tests. Enzyme-linked immunosorbent assay (ELISA) and western blot were performed to measure the levels of depression-related biomarkers in the hippocampus and serum of the mice. The chemical compounds of PFEO were determined using gas chromatography-mass spectrometry (GC-MS). Network pharmacology and molecular docking analyses were conducted to investigate the potential bioactive components of PFEO and the mechanisms underlying the antidepressant effects. To validate the mechanisms of the bioactive compounds, in vitro models using PC12 and BV2 cells were established and the blood-brain barrier (BBB) permeability was evaluated. RESULTS: The intranasal administration of PFEO suppressed SDS-induced depression in mice by increasing the time spent in the social zone and the social interactions in the social interaction test and by decreasing the immobility time in the tail suspension and forced swimming tests. Moreover, the PFEO treatment reduced the SDS-induced anxiety-like behavior, as inferred from the increased activity in the central zone observed in the open field test and in the open arms observed in the elevated plus maze test. PFEO administration recovered the SDS-induced decrease in the levels of 5-HT, NE, gamma-aminobutyric acid (GABA), and p-ERK in the hippocampus of mice. Furthermore, the increased serum corticosterone level was also attenuated by the PFEO treatment. A total of 21 volatile compounds were detected in PFEO using GC-MS, among which elemicin (15.52%), apiol (15.16%), and perillaldehyde (12.79%) were the most abundant ones. The PFEO compounds targeted 32 depression-associated genes, which were mainly related to neural cells and neurotransmission pathways. Molecular docking indicated good binding affinities between the bioactive components of PFEO (apiol, ß-caryophyllene, elemicin, and myristicin) and the key targets, including ACHE, IL1B, IL6, MAOB, SLC6A2, SLC6A3, SLC6A4, and tumor necrosis factor. Among the four compounds, ß-caryophyllene, elemicin, and myristicin were more effective in reducing neurotoxicity and neuroinflammation. Elemicin showed the highest BBB permeability rate. CONCLUSIONS: This study shows the antidepressant activities of PFEO in an SDS-induced mouse model and suggests its potential mechanisms of action: regulation of the corticosterone levels, hippocampal neurotransmitters, and ERK signaling. Apiol, ß-caryophyllene, elemicin, and myristicin may be the main contributors to the observed effects induced by PFEO. Further studies are needed to fully elucidate the underlying mechanisms and the main PFEO bioactive components.

Pharmacokinetic-pharmacodynamic (PK/PD) modeling to study the hepatoprotective effect of Perilla Folium on the acute hepatic injury rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Perilla Folium (PF), is a traditional medicinal material with the homology of medicine and food in China and has been widely used due to its rich nutritional content and medicinal value. The hepatoprotective effects of PF extract include their protection against acute hepatic injury, tert-butylhydroperoxide (t-BHP) induced oxidative damage, and Lipopolysaccharide (LPS) and D-galactosamine (D-GalN) induced hepatic injury have been well studied. However, there are few reports on the pharmacokinetics studies of PF extract in acute hepatic injury model rats, and the anti-hepatic injury activity of PF is still unclear. AIM OF THE STUDY: The differences in the plasma pharmacokinetic of 21 active compounds between the normal and model groups were compared， and established pharmacokinetics/pharmacodynamics (PK/PD) modeling was to analyze the hepatoprotective effects of PF. MATERIALS AND METHODS: The acute hepatic injury model was induced with an intraperitoneal injection of lipopolysaccharide (LPS) and D-galactosamine (D-GalN), and the plasma pharmacokinetics of 21 active compounds of PF were analyzed in the normal and model groups using ultra-high performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS). The correlation between plasma components and hepatoprotective effects indicators (the alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactic dehydrogenase (LDH)) in the model group was also investigated and established a Pharmacokinetic/pharmacodynamic (PK/PD) correlation analysis of the hepatoprotective effects of PF. RESULTS: The results revealed that organic acid compounds possessed the characteristics of faster absorption, shorter peak time and slower metabolism, while the flavonoid compounds had slower absorption and longer peak time, and the pharmacokinetics of various components were significantly affected after modeling. The results of PK/PD modeling analysis demonstrated that the plasma drug concentration of each component existed a good correlation with the three AST, ALT, and LDH, and the lag time of the efficacy of each component is relatively long. CONCLUSIONS: The plasma drug concentration of each component existed a good correlation with the three AST, ALT, and LDH, and the lag time of the efficacy of each component is relatively long in vivo.

Potential Mechanisms of Perillae folium Against COVID-19: A Network Pharmacology Approach.

In China, Perillae folium is widely used to treat colds, especially in the early stages of cold; the effect of taking P. folium is readily noticeable at that time. The active compounds and targets of P. folium were screened from Traditional Chinese Medicine Systems Pharmacology, Chinese Pharmacopoeia, and UniProt. Targets related to the initiation and progression of 2019 Coronavirus Disease (COVID-19) were retrieved from Online Mendelian Inheritance in Man and GeneCards. The potential therapeutic targets of P. folium on COVID-19 were the cross targets between them. Enrichment analysis of Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were conducted by using the Database for Annotation, Visualization and Integrated Discovery website. Molecular docking between key compounds and core targets was performed with AutoDock. The effects of P. folium extract and rosmarinic acid on inflammatory cytokines were tested by a cellular inflammatory model. The "Perillae folium-compound-target-COVID-19" network contained 11 kinds of compounds and 33 matching targets. There were 261 items in the GO functions (P < .05) and 67 items linked to the KEGG signaling pathways (P < .05). Luteolin and rosmarinic acid were key compounds of P. folium. Their docking with the core targets mitogen-activated protein kinase 1 (MAPK1) and chemokine (C-C motif) ligand 2 (CCL2), respectively, showed that they had good affinity with each other. Cell experiments demonstrated that P. folium extract had inhibitory effects on interleukin-6 and tumor necrosis factor (TNF)-α in cells, and was better than rosmarinic acid. Luteolin, rosmarinic acid, and other individual active compounds in P. folium, which may participate in PI3K-Akt, TNF, Jak-STAT, COVID-19, and other multisignaling pathways through multiple targets such as MAPK1 and CCL2, and play a therapeutic role in COVID-19.

Chemical compositions and bioactivities of essential oil from perilla leaf (Perillae Folium) obtained by ultrasonic-assisted hydro-distillation with natural deep eutectic solvents.

Natural deep eutectic solvents (NADESs) have received considerable attention for green extraction. In this study, ultrasonic-assisted natural deep eutectics were applied for hydrodistillation to extract essential oil from perilla leaves. Compared to hydrodistillation assisted with ultrasound, this novel method effectively raised the yield of perilla leaf EO from 0.21% to 0.69% (choline chloride: malic acid, molar ratio 2:1) and broadened the types of chemical compositions (71 in total), especially aliphatic and aromatic compounds. Stronger antimicrobial and antioxidant activity was confirmed, and phenolics were increased. Additionally, the pH of the final solution was detected. The leaf residues were dried, weighed and analyzed by scanning electron microscopy to show the influence of NADESs' strong extractability. This new extraction method may be applicable in edible natural product extraction and provide a reference for further exploration of NADESs.

Chemical Compositions, Extraction Optimizations, and In Vitro Bioactivities of Flavonoids from Perilla Leaves (Perillae folium) by Microwave-Assisted Natural Deep Eutectic Solvents.

Natural deep eutectic solvents (NADESs) have been gradually applied to green extraction of active ingredients. In this study, microwave-assisted NADESs were applied to the extraction of flavonoid compounds from perilla leaves. Through comparative experiments, NADES-3 (choline chloride and malic acid at a molar ratio of 1:1) was found to have the highest extraction efficiency of total flavonoids, including apigenin 7-O-caffeoylglucoside, scutellarein 7-O-diglucuronide, luteolin 7-O-diglucuronide, and scutellarein 7-O-glucuronide by HPLC-MS. The following optimal extraction parameters were obtained based on response surface design: water content in NADES of 23%, extraction power of 410 W, extraction time of 31 min, and solid-liquid ratio of 75 mg/mL, leading to the extraction yield of total flavonoids of 72.54 mg/g. Additionally, the strong antimicrobial and antiallergic activity, inhibition of nitrosation, and antioxidant activity of total flavonoids by using NADESs were confirmed. This new extraction method provides a reference for the further exploration of NADES systems and may be widely used for the green extraction of natural active ingredients.

[Quality standard of Perillae Folium based on multicomponent determination with HPLC method].

This research established the HPLC methods for the determination of perillaketone, perillaldehyde, caffeic acid, scutellarin, and rosmarinic acid in 33 batches of Perillae Folium. Kromasil C_(18)(4.6 × 250 mm, 5 µm) chromatographic column was used, and the mobile phase for determination of the perillaketone and perillaldehyde was methanol-water(55∶45) solution, at a flow rate of 1.0 mL·min~(-1), with the column temperature at 30 ℃. The mobile phase for the determination of caffeic acid, scutellarin and rosmarinic acid was methanol(A)-0.2% phosphoric acid aqueous solution(B) with gradient elution(0-20 min, 25%-30% A; 20-60 min, 30%-43% A). The flow rate was 1.0 mL·min~(-1) and the column temperature was set at 30 ℃. The results showed that the established method can achieve good separation of the five components in samples, with a good linear relationship and high accuracy, indicating that the methods can be used for the determination of Perillae Folium. The results showed that all samples contained five components. And the content of rosmarinic acid(0.04%-1.57%) > scutellarin(0.03%-0.77%) > perillaldehyde(0.02%-0.66%) > perillaketone(0.03%-0.30%) > caffeic acid(0.006%-0.07%). Thirty-three Batches of Perillae Folium can be grouped into 5 categories. There are certain content rules and region specificities under different clusters. Perillaketone, perillaldehyde, and rosmarinic acid can be used as the main markers to evaluate the quality of Perillae Folium.

Advances on Chemical Constituents of Essential Oils from Perillae Folium and Their Pharmacological Effect：A Review / 中国实验方剂学杂志

Perilla frutescens is a traditional medicinal and edible plant widely distributed in China and enjoys an extensive usage. P. frutescens contains multiple essential oils， which are composed of monoterpenes， sesquiterpenes， and their oxygen-containing derivatives. Compared with other parts of P. frutescens， Perillae Folium produce more oils， with volatile oils as the main constituents. There are many active substances in the volatile oils from Perillae Folium， mainly including perillaldehyde， perillaketone， perillaalcohol， D-limonene， β-caryophylene， etc. Such factors as germplasm， growth environment， extraction method， cultivation time， and harvest period all can trigger changes in volatile oil constituents and content from Perillae Folium. The volatile oils from Perillae Folium have diverse pharmacological effects like anti-oxidation， anti-bacteria， anti-inflammation， vasodilation， anti-tumor， and anti-depression， implying its high clinical application value. However， the chemical constituents in volatile oils from Perillae Folium are complex and unstable and their pharmacological activities are affected by many factors， so the safety and effectiveness of clinical medication fail to be guaranteed， which may has impeded the rational and effective use of these volatile oils. Many scholars in China and abroad have conducted a lot of research on the volatile oils from Perillae Folium， but there is currently no systematic and comprehensive research report on the chemical constituents of volatile oils from Perillae Folium and their pharmacological effects. This paper reviewed the relevant domestic and foreign literature， analyzed the development status of volatile oils from Perillae Folium， and summarized their extraction process， chemical constituents， and pharmacological actions， aiming to provide a reference for their further development， clinical application， and risk assessment.

Quality standard of Perillae Folium based on multicomponent determination with HPLC method / 中国中药杂志

This research established the HPLC methods for the determination of perillaketone, perillaldehyde, caffeic acid, scutellarin, and rosmarinic acid in 33 batches of Perillae Folium. Kromasil C_(18)(4.6 × 250 mm, 5 μm) chromatographic column was used, and the mobile phase for determination of the perillaketone and perillaldehyde was methanol-water(55∶45) solution, at a flow rate of 1.0 mL·min~(-1), with the column temperature at 30 ℃. The mobile phase for the determination of caffeic acid, scutellarin and rosmarinic acid was methanol(A)-0.2% phosphoric acid aqueous solution(B) with gradient elution(0-20 min, 25%-30% A; 20-60 min, 30%-43% A). The flow rate was 1.0 mL·min~(-1) and the column temperature was set at 30 ℃. The results showed that the established method can achieve good separation of the five components in samples, with a good linear relationship and high accuracy, indicating that the methods can be used for the determination of Perillae Folium. The results showed that all samples contained five components. And the content of rosmarinic acid(0.04%-1.57%) > scutellarin(0.03%-0.77%) > perillaldehyde(0.02%-0.66%) > perillaketone(0.03%-0.30%) > caffeic acid(0.006%-0.07%). Thirty-three Batches of Perillae Folium can be grouped into 5 categories. There are certain content rules and region specificities under different clusters. Perillaketone, perillaldehyde, and rosmarinic acid can be used as the main markers to evaluate the quality of Perillae Folium.

Chemotype Study of Volatile Components from Perillae Folium of Different Germplasms / 中国药房

OBJECTIVE：To analyze the chemotypes of volatile components from Perillae Folium of different germplasms ，and to investigate the relationship of germplasm and leaf color with chemotype. METHODS ：The fingerprints of volatile components from 30 batches of Perillae Folium were prepared by GC-MS with P 4 peak as reference. Similarity Evaluation System for TCM Chromatographic Fingerprint （2004A edition ）was applied to evaluate the similarity and confirm common peaks. The volatile components of Perillae Folium were determined by the same GC-MS method. Qualitative Navigator （B.08.00）software was used to analyze and compare with NIST 17.0 standard mass spectrum database. The compounds corresponding to the peak were analyzed ； clustering analysis was carried out with Origin 2018 software. RESULTS ：There were 13 common peaks in the fingerprints of volatile components from 30 batches of Perillae Folium . The similarities were 0.13-1.00. Totally 54 components were identified from 30 batches of Perillae Folium of different germplasm. Cluster analysis showed that 30 batches of Perillae Folium samples could be clustered into three categories ；among them ，SCY-1，YNT-9，YNX-17，YN-28 were clustered into one category ，with phenylpropanoid-elemicin（PP-e as ）the main volatile component ，being PP-e type ；GS-4，GS-7，GS-11，GS-19，HBA-14， HBA-20，GZZ-8，LN-39，GSL-27，GSQ-32，GSQ-33，GST-31，YNW-12，LN-38 were clustered into one category ，and the content of perilla ketone （PK）in them was the highest except for LN- 38， being PK type [the content of phenylpropanoid-apiol（PP-a）in LN- 38 was higher than that of perilla ketone ，being PP-a type] ；HBS-2，HBS-3，HBS-6， C201859）HBS-15，HBS-16，HBS-24，HBS-25，GX-26，SXS-30，SCC- 36，RB-37，SC-29 were clustered into one category ，and thecontent of perillaldehyde （PA）was the highest ，being PA type.The color characteristics of Perillae Folium of different germplasm showed that Perilla frutescens （L.） Britt. var.frutescens with green leaves on both sides was PK type ，while P. frutescens （L.）Britt. var. arguta with purple leaves on one or both sides was PA type ，and P. frutescens （L.） Britt var. auriculato-dentata C. Y. Wu et Hsuan ex H. W. Li was PP-e type. CONCLUSIONS：The chemotype of volatile components in Perillae Folium have a certain corresponding relationship with their leaf colors. Most of P. frutescens （L.）Britt. var. arguta with purple leaves on one side or both sides are PA type. P. frutescens （L.） Britt. var. acuta （Thunb.）Kudo，P. frutescens （L.）Britt var. auriculato-dentata C. Y. Wu et Hsuan ex H. W. Li and P. frutescens （L.）Britt. var. frutescens with green leaves on both sides do not belong to PA type ，among which P. frutescens （L.）Britt var. frutescens is PK type ，while P. frutescens （L.）Britt var. auriculato-dentata C. Y. Wu et Hsuan ex H. W. Li is mostly PP-e type.

Comprehensive Comparison of Two Color Varieties of Perillae Folium Using Rapid Resolution Liquid Chromatography Coupled with Quadruple-Time-of-Flight Mass Spectrometry (RRLC-Q/TOF-MS)-Based Metabolic Profile and in Vivo/in Vitro Anti-Oxidative Activity.

Perillae Folium (PF), which is extensively used as a dietary vegetable and medicinal herb, contains two varietal forms corresponding to purple perilla leaf (Perilla frutescens var. crispa) and green perilla leaf (Perilla frutescens var. frutescens). However, the components and efficacy of different PF varieties remain underexplored so far. In the present work, a nontargeted rapid resolution liquid chromatography coupled with quadruple-time-of-flight mass spectrometry (RRLC-Q/TOF-MS)-based metabolomics approach was developed to investigate the difference in the chemical compositions between green PF and purple PF. A total of 71 compounds were identified or tentatively identified, among which 7 phenolic acids, 10 flavonoids, and 9 anthocyanins were characterized as differential metabolites. In addition, heatmap visualization and ultraperformance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UPLC-TQ-MS/MS)-based quantitative analysis revealed that flavonoids and anthocyanins especially had higher contents in purple PF. Furthermore, the anti-oxidative activities of two varietal PFs were evaluated in vivo zebrafish and in vitro human umbilical vein endothelial cells (HUVECs). The results showed that the purple PF had more pronounced anti-oxidative activities than did the green PF, which may be due to the presence of anthocyanins and a higher concentration of flavonoids in its phytochemical profile. The outcome of the present study is expected to provide useful insight on the comprehensive utilization of a PF resource.

Protective Effect and Mechanism of Aqueous Extract of Perillae Folium on Adriamycin-induced Oxidative Injury in HK-2 Cell / 中国实验方剂学杂志

Objective: To investigate the protective effect of Perillae Folium with aqueous extract (PFAE) on some key factors of Adriamycin (ADR)-induced oxidative injury in human renal tubular epithelial cells(HK-2), including the survival rate, oxidative injury indexes and cell apoptosis,in order to define the underlying mechanism. Method: A model of ADR-induced HK-2 cells oxidative injury was established in vitro, then cell viability was detected by cell counting kit-8 (CCK-8) after intervention with positive reference N-acetylcysteine (NAC) or PFAE (5,15,45 g·L-1) at different concentrations. According to the morphological changes under microscopy, the optimum concentration of PFAE was screened out for the follow-up experiments. Then, the experiments were divided into six groups:blank group, ADR (0.05 g·L-1) group, PFAE (15 g·L-1) group, ADR+PFAE (0.05+15) g·L-1 group, NAC (0.81 g·L-1) group, and ADR+NAC (0.05+81) g·L-1 group. After that, malondialdehyde (MDA), superoxide dismutase (SOD), total antioxidant capacity(TAC) were measured in the cell homogenate after 24 h administration. The level of reactive oxygen species (ROS) was detected by 2',7'-dichloroflurescin diacetate (DCFH-DA) fluorescence probe. Flow cytometry and TdT-mediated dUTP Nick-End Labeling (TUNEL) were used to monitor the cell apoptosis. Western blot was used to observed the expressions of mitochondrial apoptosis-associated proteins, like B lymphocyte tumor-2 gene (Bcl-2), Bcl-2 related X protein (Bax), cysteine aspartate protease-9 (Caspase-9), cysteine aspartate protease-3 (Caspase-3) and poly ADP-ribose polymerase (PARP), as well as their shear bodies. In addition, the phosphorylation protein expressions of p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK) in mitogen-activated protein kinase (MAPK) signaling transduction pathway were detected by Western blot. Result: Compared with blank group, ADR group showed a decreased cell viability (PPPPPPPP-1. The ATC and SOD levels were increased in ADR+PFAE group and ADR+NAC group (PPConclusion: PFAE could alleviate the oxidative injury of HK-2 cells induced by ADR, and have an antioxidant effect, which inhibited cell apoptosis through mitochondrial apoptotic pathway and ERK/p38 MAPK signaling pathway.

Optimization of extraction technology for Xiaochuan Decoction based on information entropy theory / 中草药

Objective: To optimize the extraction technology of Saposhnikoviae Radix, Perillae Folium, Magnoliae Flos, Armeniacae Amarum Semen, and honey-fried Ephedrae Herba in Xiaochuan Decoction by information entropy theory. Methods: With the contents of prem-O-glucosylcimifugin, 4'-O-beta-glucopyranosyl-5-O-methylvisamminol, amygdalin, and the yield of extract as comprehensive evaluation indexes in order to optimize the extraction process parameters of orthogonal test, the weight coefficient of each index was determined by the information entropy weight method. Results: Optimum extraction technology was as follows: reflux extraction for 3 times with 10 fold water, for 1.5 h each time. Conclusion: The optimized method is stable and reliable, and can provide the reference for further development and utilization of the formula.

Optimization of Clarification and Purification Technology of Perillae folium Extract / 中国药房

OBJECTIVE:To optimize the clarification and purification technology of Perillae folium extract. METHODS:The effects of 3 clarification and purification methods as chitosan flocculation clarification,ZTC 1+1-Ⅱflocculation clarification,water precipitation on retention rate of total flavonoids and removal rate of solid of Perillae folium extract were compared to screen suit-able clarification and purification technology. With the retention rate of total flavonoids and removal rate of solid as comprehensive evaluation index,single factor and orthogonal test were designed to investigate the optimal value of concentration proportion,the amount of the flocculant,flocculation temperature and whisking speed in optimal clarification and purification method. RESULTS:Among 3 methods,the chitosan flocculation clarification was the best with concentration proportion of 1∶4,chitosan of 1.0 g/L, flocculation temperature at 60 ℃,whisking speed of 100 r/min,whisking time of 4 min,standing time of 12 h. Under the condi-tion of optimal processing,the retention rate of total flavonoids was (85.1 ± 0.75)%,and the removal rate of solid was (24.6 ± 1.33)%(n=5). CONCLUSIONS:Chitosan flocculation can be used to effectively remove the impurity of Perillae folium extract, and optimized clarification and purification technology is stable and feasible.