Perilla frutescens: A Rich Source of Pharmacological Active Compounds

Perilla frutescens (L.) Britton, an important pharmaceutical and nutraceutical crop, is widely cultivated in East Asian countries. In this review, we present the latest research findings on the phytochemistry and pharmacological activities of P. frutescens. Different databases, including PubMed, Scopus, CNKI, Agricola, Scifinder, Embase, ScienceDirect, DOAJ, and Web of Science, were searched to present the best review. In this review, we clearly represent the active constituents responsible for each and every pharmacological activity, plausible mechanism of action, and maximum inhibitory concentrations, as well as IC50 values. Approximately 400 different bioactive compounds, including alkaloids, terpenoids, quinines, phenylpropanoids, polyphenolic compounds, flavonoids, coumarins, anthocyanins, carotenoids, neolignans, fatty acids, polycosanols, tocopherols, and sitosterols, have been reported in the leaves, seeds, roots, and aerial parts of P. frutescens. The bioactive constituents of P. frutescens exhibited different enzyme-inhibition properties, including antihyaluronidase effects and aldose reductase inhibitory, α-glucosidase inhibitory, xanthine oxidase inhibitory, and tyrosinase inhibitory properties. P. frutescens showed strong anti-inflammatory, antidepressant, anti-spasmodic, anticancer, antioxidant, antimicrobial, insecticidal, neuroprotective, and hepatoprotective effects. Hence, the active constituents of P. frutescens used in the treatment of diabetes and diabetic complications (retinopathy, neuropathy, and nephropathy), prevention of hyperuricemia in gout patients, hyper pigmentation, allergic conditions, skin inflammation, skin allergy, atopic dermatitis, periodontosis, androgenic alopecia, gastric inflammation, oesophagitis, carcinogenesis, cardiovascular, Alzheimer’s, Parkinson’s, and cerebral ischemic disorders. Furthermore, we revealed the most active constituents and possible mechanisms of the pharmacological properties of P. frutescens.


Introduction
Perilla frutescens (L.) Britton, belonging to the family Lamiaceae, is widely distributed in East Asian countries, such as Japan, China, Korea, and Vietnam. Since ancient times, it has been used in traditional Chinese medicine and is cultivated as an edible crop in mainland China, Japan, and Korea. The leaves of Perilla were used in preparing vegetable curries, pickles, and chutneys. Several studies have reported the presence of rich polyphenolic compounds, which exhibit high antioxidant capacity, in Perilla leaves. Green tea prepared with Perilla leaves is highly popular in East Asian countries, due to its high antioxidant capacity, and has been used to treat fish and crab poisoning in these countries. Perilla essential oil (PEO), extracted from P. frutescens leaves, is a complex mixture of volatile components, constituting approximately 150 to 200 different compounds, exhibiting high antioxidant, anticancer, anti-inflammatory, insecticidal, and antimicrobial activities; 14.

Aldose Reductase Inhibitory Activity
Aldose reductase, or aldehyde reductase (AR), an important NADPH-dependent enzyme, is, ubiquitously, present at higher concentrations in the heart, eyes, neurons, and kidneys. This enzyme catalyses the reduction in monosaccharides, into their respective sugar alcohols. AR converts glucose into sorbitol, via the polyol pathway of glucose metabolism [61]. Excessive sorbitol is severely damaging, and its accumulation in the eyes, neurons, and kidneys of diabetic patients causes retinopathy, neuropathy, and nephropathy, respectively [62]. AR inhibitors are a class of drugs that averts sorbitol accumulation, by preventing or delaying the action of AR in tissues such as eyes, kidneys, and neurons, and are, consecutively, used to prevent the corresponding diabetic complications. Different bioactive compounds from plants possess AR inhibitory activity [63]. The rosmarinic acid, chlorogenic acid, caffeic acid, protocatechuic acid, and methyl rosmarinic acid contained in P. frutescens leaves possess significant AR-inhibitory activity. Figure 1 represents the different phytoconstituents of P. frutescens involved in aldosereductase-inhibitory activity. Rosmarinic acid exhibited strong inhibition against AR, with an IC 50 of 2.77 µM, followed by chlorogenic acid (IC 50 = 3.16 µM) and methyl rosmarinic acid (IC 50 = 4.03 µM). However, caffeic acid and protocatechuic acid exhibited weak AR-inhibitory activity (Paek et al., 2013). Luteolin, apigenin, and diosmetin, isolated from P. frutescens seeds, also, showed AR inhibitory activity. Luteolin was found to be a strong AR inhibitor, with an IC 50 of 1.89 µM, followed by apigenin (IC 50 = 4.18 µM) and diosmetin . Different glycosides isolated from P. frutescens leaves, including four monoterpene glycosides (perilloside A-D), five β-D-glucosides (eugenyl-β-D-glucoside, benzyl-β-D-glucoside, βsitosteryl β-D-glucoside, prunasin, and sambunigrin), and methyl-α-D-galactoside, significantly inhibited AR. In particular, perilloside A and perilloside C have been proven to be potent AR inhibitors that show competitive inhibition, as demonstrated by L-B plots [19]. Caffeic acid-3-O-glucoside and rosmarinic acid-3-O-glucoside isolated from P. frutescens seeds also exhibited AR inhibitory activity. Thus, these bioactive constituents in P. frutescens may be useful as a remedy to treat diabetic complications.

α-Glucosidase Inhibitory Activity
α-glucosidase is a hydrolytic enzyme that breaks down starch, oligosaccharides, and disaccharides into simple sugars, such as glucose molecules, by acting on α (1-4) bonds to facilitate intestinal absorption of carbohydrates. Their action drastically increases postmeal blood sugar levels in diabetes patients. α-Glucosidase inhibitors prevent or delay the breakdown of carbohydrates into simple sugars (glucose molecules), which delays the intestinal absorption of glucose, and consequently the increase in blood sugar levels after meals [64]. Hence, α-glucosidase inhibitors have been successfully used in treating type II diabetes mellitus. Figure 1 show the bioactive compounds involved in α-glucosidase inhibitory activity of P. frutescens. Ha et al. [23] reported that the five phenolic compounds isolated from P. frutescens seeds exhibited α-glucosidase inhibitory activity in a dose-dependent manner. Luteolin was found to be a potent α-glucosidase inhibitor with an IC50

α-Glucosidase Inhibitory Activity
α-glucosidase is a hydrolytic enzyme that breaks down starch, oligosaccharides, and disaccharides into simple sugars, such as glucose molecules, by acting on α (1-4) bonds to facilitate intestinal absorption of carbohydrates. Their action drastically increases post-meal blood sugar levels in diabetes patients. α-Glucosidase inhibitors prevent or delay the breakdown of carbohydrates into simple sugars (glucose molecules), which delays the intestinal absorption of glucose, and consequently the increase in blood sugar levels after meals [64]. Hence, α-glucosidase inhibitors have been successfully used in treating type II diabetes mellitus. Figure 1 show the bioactive compounds involved in α-glucosidase inhibitory activity of P. frutescens. Ha et al. [23] reported that the five phenolic compounds isolated from P. frutescens seeds exhibited α-glucosidase inhibitory activity in a dosedependent manner. Luteolin was found to be a potent α-glucosidase inhibitor with an IC 50 value of 45.4 µM, and inhibited α-glucosidase in a non-competitive manner with an inhibition constant (K I ) of 45.0 µM and Michaelis-Menton's constant (Km) of 259.3 µM. Caffeic acid-3-O-glucoside, rosmarinic acid, rosmarinic acid-3-O-glucoside, and apigenin exhibited α-glucosidase inhibitory activity at concentrations higher than 100 µM. Owing to their α-glucosidase inhibitory activity, bioactive compounds of P. frutescens can be formulated into oral antidiabetic medications.

Tyrosinase Inhibitory Activity
Tyrosinases are oxidases involved in the initial stages of melanin biosynthesis. Overproduction of melanin causes hyper pigmentation and other skin disorders, such as melasma, solar melanosis, senile lentigos, and ephelides [71]. The modern lifestyle, junk foods, insomnia, industrial smoke, pollution, and synthetic drugs cause oxidative stress, which leads to aging and skin disorders. Hence, development of natural tyrosinase inhibitors with antioxidant capacity, to reduce hyper pigmentation, aging, and skin disorders, is essential. Several flavonoids and polyphenolic compounds possess both antioxidant and anti-tyrosinase activities (Figure 1). Kim et al. [22] reported that the methanolic extracts of P. frutescens seeds exhibit dose-dependent inhibition of tyrosinase and radical scavenging against DPPH and ABTS free radicals. The potential anti-tyrosinase activity of P. frutescens seeds is attributed to phenolic compounds, such as rosmarinic acid, rosmarinic acid-3-O-glucoside, luteolin, apigenin, chrysoeriol, and caffeic acid. Rosmarinic acid exhibited potential anti-tyrosinase activity, with an IC 50 value of 20.8 µM, followed by luteolin (24.6 µM), chrysoeriol (35.8 µM), apigenin (49.3 µM), rosmarinic acid-3-O-glucoside (57.9 µM), and caffeic acid (>300 µM). Furthermore, all of these compounds showed significant antioxidant activity, as evidenced by their DPPH and ABTS radical-scavenging capacities. Based on these activities, P. frutescens seeds can be used as health additives in food preparations.

Antispasmodic Effect
Vicenin-II, a bis C-glycosylflavonoid, displayed no direct spasmolytic effect, but significantly reduced Ba2+-or acetylcholine-induced smooth muscle contractions in the rat ileum (a part of the small intestine of the gastrointestinal tract), demonstrating an antispasmodic effect. The antispasmodic effect on smooth muscles relieves gastrointestinal discomfort and bowel disease symptoms, thus maintaining gut health. Hence, vicenin-II can be, prophylactically, used in maintaining and improving gut health [40].

Anti-Allergic Activity
Different bioactive components of P. frutescens, including rosmarinic acid, caffeic acid, luteolin, apigenin, methoxyflavanone and α-linolenic acid, were found to possess antiallegic activity (Figure 2) [73]. Kamei et al. [42] reported the presence of 8-hydroxy-5,7dimethoxyflavanone in P. frutescens leaves, and named it Perilla-derived methoxyflavanone (PDMF). PDMF may be used to prevent IgE-driven type I hypersensitivity reactions. Akt phosphorylation and intracellular Ca 2+ influx, the two critical molecular events involved in mast cell degranulation in allergic reactions, are suppressed by PDMF, which acts as a potent anti-allergic compound. The other polyphenols of P. frutescens, such as rosmarinic acid, luteolin, apigenin, and caffeic acid, also, suppress IgE-mediated type I hypersensitivity reactions. However, PDMF exhibits a more potent histamine-release inhibitory activity than known derived anti-inflammatory polyphenols. Further PDMF stimulation suppresses histamine release from RBL-2H3 cells in a dose-dependent manner, and the IC 50 value (68.5 mM) was found to be considerably lower than that of apigenin (96.8 mM), luteolin (174.1 mM), caffeic acid (620.4 mM), and rosmarinic acid (>1000 mM). Specific matrix proteins, such as matrix metalloproteinase (MMP), periostin, interleukin (IL-31), thymus, and activation-regulated chemokine (TARC), are sensitive markers for the clinical diagnosis of skin health [74]. These protein levels are drastically elevated during conditions of skin damage, such as skin injuries, skin inflammation, skin allergy, and atopic dermatitis; atopic dermatitis or atopic eczema is a type of severe skin inflammation, with symptoms such as swelling, redness, cracking, and itchy skin. Heo et al. [75] reported that an aqueous extract of P. frutescens mitigates DNFB (2,4-dinitrofluorobenzene)-induced atopic dermatitis, in a mouse model. The anti-dermatitis activity of the aqueous extract of P. frutescens is, possibly, due to the downregulation of matrix metalloproteinase-9 (MMP-9) and IL-31 expression levels, and upregulation of T-bet activity. Komatsu et al. [76] demonstrated that P. frutescens leaf extract prevents atopic dermatitis induced by house dust mite allergens (Dermatophagoides farina), in an NC/Nga AD mouse model. Moreover, P. frutescens leaf extract significantly decreased the serum levels of inflammatory markers, such as IgE, periostin, and TARC. Furthermore, P. frutescens leaf extract significantly decreased the allergen-stimulated CD4 + /CD8 + T cell ratio, in spleen lymphocytes.
Shin et al. [77] demonstrated that an aqueous extract of P. frutescens effectively inhibited immediate mast-cell-triggered allergy and inhibited the fatal systemic allergy, using Specific matrix proteins, such as matrix metalloproteinase (MMP), periostin, interleukin (IL-31), thymus, and activation-regulated chemokine (TARC), are sensitive markers for the clinical diagnosis of skin health [74]. These protein levels are drastically elevated during conditions of skin damage, such as skin injuries, skin inflammation, skin allergy, and atopic dermatitis; atopic dermatitis or atopic eczema is a type of severe skin inflammation, with symptoms such as swelling, redness, cracking, and itchy skin. Heo et al. [75] reported that an aqueous extract of P. frutescens mitigates DNFB (2,4-dinitrofluorobenzene)-induced atopic dermatitis, in a mouse model. The anti-dermatitis activity of the aqueous extract of P. frutescens is, possibly, due to the downregulation of matrix metalloproteinase-9 (MMP-9) and IL-31 expression levels, and upregulation of T-bet activity. Komatsu et al. [76] demonstrated that P. frutescens leaf extract prevents atopic dermatitis induced by house dust mite allergens (Dermatophagoides farina), in an NC/Nga AD mouse model. Moreover, P. frutescens leaf extract significantly decreased the serum levels of inflammatory markers, such as IgE, periostin, and TARC. Furthermore, P. frutescens leaf extract significantly decreased the allergen-stimulated CD4 + /CD8 + T cell ratio, in spleen lymphocytes.
Shin et al. [77] demonstrated that an aqueous extract of P. frutescens effectively inhibited immediate mast-cell-triggered allergy and inhibited the fatal systemic allergy, using compound 48/80 (a potent inducer of systemic allergy, by stimulating mast cell degranula-tion and promoting histamine release), in a dose-dependent manner. Furthermore, they reported that an aqueous extract of P. frutescens significantly inhibited the local allergic reaction, induced by anti-DNP IgE. The anti-allergic effects of the aqueous extract of P. frutescens may be due to the inhibition of histamine released from mast cells, TNF-α production, and passive cutaneous anaphylaxis (PCA). Jeon et al. [78] reported that luteolin obtained from the methanolic extracts of P. frutescens exhibited potential antiallergic and antipruritic effects. The antiallergic effect of luteolin is due to the inhibition of histamine release from mast cells, induced by compound 48/80. Luteolin significantly inhibited the scratching behaviour and vascular permeability induced by serotonin and compound 48/80, respectively, in an ICR mouse model. Hyaluronidase is one of the major enzymes regulating mast cell degranulation, and histamine release is, primarily, controlled by protein kinase C and Ca 2+ . Asada et al. [79] reported that a glycoprotein from P. frutescens hot-water extracts prevented the degranulation of mast cells, possibly due to inhibition of protein kinase C and hyaluronidase activity. Chen et al. [80] reported that the ethanolic extract of P. frutescens successfully exhibited an antiallergic effect in an ovalbumin (allergen)-sensitised asthma mouse model. The ethanolic extract suppressed serum IgE levels, downregulated allergenstimulated Th2 cytokines (IL-5 and IL-13), and decreased the secretion of allergic mediators (histamine and eotaxin). Furthermore, the ethanolic extract of P. frutescens inhibits airway hyper-responsiveness, by suppressing cell infiltration and reducing lung and bronchiole inflammation. Sanbongi et al. [81] reported that oral administration of rosmarinic acid extract of P. frutescens inhibits allergic inflammation, caused by the mite allergen D. farina. D. farina-sensitised C3H/He mice exhibited severe eosinophilic inflammation and elevated expression levels of IL-4, IL-5, and eotaxin in the lungs; rosmarinic acid extract significantly decreased the influx of eosinophils in the lungs and inhibited the lung tissue expression of IL-4, IL-5, and eotaxin proteins. In another study, by Takano et al. [82] rosmarinic acid-rich P. frutescens extract inhibited allergic rhinoconjunctivitis in humans, at least partly by inhibiting the infiltration of polymorphonuclear leukocytes (PMNL) into the nostrils. In this study, rosmarinic acid-rich extract significantly decreased the number of PMNL and inhibited the levels of inflammatory mediators, such as histamine, eotaxin, IL1-β, IL-8, and IgE levels, in nasal lavage fluid. Makino et al. [83] demonstrated that hot-water extracts of P. frutescens significantly suppressed the mice ear-PCA reaction, thereby concluding that rosmarinic acid is the active constituent, mainly, responsible for anti-allergic reactions. Dietary perilla oil, rich in α-linolenic acid, significantly suppressed serum lipid levels as well as IgG1 and IgA levels, in ovalbumin-sensitised mice. Based on these results, Chang et al. [84] reported that dietary perilla oil might, moderately, suppress asthmatic allergy. P. frutescens, PDMF, and rosmarinic acid could be, medially, useful in treating allergic diseases. Figure 2 represents the different bioactive components of P. frutescens exhibiting antidepressant activity. Rosmarinic acid and caffeic acid showed significant antidepressant activity; this was demonstrated by Takeda et al. [85] in a forced swimming test in mice, which is a well-accepted stress model of depression. The antidepressant activity was revealed via mechanisms other than the inhibition of monoamine transporters and monoamine oxidase. In neuropharmacological studies, neither rosmarinic acid nor caffeic acid affected either the uptake of monoamines to synaptosomes or mitochondrial monoamine oxidase activity in the mouse brain. The detailed mechanisms involved in the antidepressive-like properties of rosmarinic acid and caffeic acids are unclear. However, previous pharmacological studies have revealed that rosmarinic acid inhibits histamine release from mast cells [86], as well as that caffeic acid can activate the a1-adrenoreceptor system [87] and inhibit the production and release of nitric oxide (NO) [88,89]. Furthermore, previous studies using the forced swimming test have suggested that either the activation of α 1 -adrenoreceptors or the inhibition of NO production may be involved in the expression of antidepressive-like effects. Their effects may, reportedly, involve direct modulation of a second messenger system. Caffeic acid, reportedly, inhibits both protein kinase A and protein kinase C activity in vitro. Increased evidence suggests that the therapeutic effects of existing antidepressants are associated with adaptive changes in post-receptor signalling, rather than with their primary action. Furthermore, EOPF exhibited significant antidepressant activity in a chronic, unpredictable, mild stress (CUMS)-induced mouse model, a widely used rodent model of depression. Brain-derived neurotrophic factor (BDNF) is a nerve growth factor essential for neuronal survival, and promotes the growth, differentiation, and maintenance of neurons, as well as synaptic plasticity, cognitive function, and long-term memory. BDNF is present throughout the brain and spinal cord, and its levels are decreased in conditions of depression, aging, and neurological disorders, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and Lou Gehrig's disease. However, the hippocampal region of the brain shows high densities of BDNF. CUMS significantly decreased hippocampal BDNF protein levels, by downregulating the mRNA expression of BDNF. Administration of EOPF significantly elevates BDNF protein levels, by upregulating BDNF mRNA expression levels [90]. Thus, EOPF showed antidepressant activity, by enhancing BDNF protein levels. Chronic stress significantly decreases serotonin concentrations (5-hydroxytryptamine) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA), in the hippocampus of the brain. Furthermore, chronic mild stress could induce a proinflammatory response, by increasing the plasma levels of pro-inflammatory cytokines, including IL-1, IL-6, and TNF-α. CUMS also reduced open-field activity and sucrose consumption as well as increased the duration of immobility in the forced swimming and tail suspension tests. Administering EOPF effectively increased the concentrations of serotonin and 5-HIAA and reduced the levels of IL-6, IL-1β, and TNF-α. Furthermore, EOPF significantly enhanced open-field activity and sucrose consumption as well as reduced the duration of immobility. Ji et al. [91] demonstrated that perillaldehyde, a major constituent of EOPF, exhibited an antidepressant effect in an LPS-induced depression mouse model. In this experiment, perillaldehyde elevated the reduced levels of monoamines, such as norepinephrine and 5-hydroxytryptamine, in the prefrontal cortex of LPS-depressed mice. Furthermore, perillaldehyde decreased proinflammatory cytokine levels (IL-6 and TNF-α) in the prefrontal cortex of mice, and significantly reduced the LPS-enhanced immobility duration in the forced swimming and tail suspension tests.

Anti-Depressant Activity
Luteolin and apigenin, isolated from the fruit of Perilla frutescens (L.) Britton enhance monoamine uptake, either on monoamine-transporter transgenic Chinese hamster ovary (CHO) cells or on wild dopaminergic cell lines, with higher specificity for dopamine (DA) uptake than for norepinephrine (NE) and serotonin (5HT) uptake, with a greater potency and efficacy for luteolin than for apigenin. Furthermore, in the transgenic cells, the principal NE/DA uptake activated by luteolin was significantly prevented by the respective transporter inhibitor, and the transmitter-uptake-enhancing action was independent of its ligands, which supports the compounds as monoamine transporter activators. Furthermore, luteolin, markedly, inhibited the cocaine-targeted effect in CHO cells overexpressing the dopamine transporter. Thus, luteolin and apigenin function as monoamine transporter activators, which would improve several hypermonoaminergicneuropsychological disorders, especially cocaine dependence, by upregulating monoamine transporter activity [92].
Luteolin and apigenin, both isolated from FP, are novel monoamine transporter activators, and luteolin is the most potent DAT activator, which, strikingly, prevents in vitro cocaine-targeted action. Luteolin, therefore, could be effective for hyperfunctional monoamine-associated neuropsychological disorders (e.g., mania, schizophrenia, and drug problems, especially cocaine dependence). The mechanism of the enhancement of DAT/NET function is unknown and needs to be determined in future studies [93].

Hepatoprotective Activity
Rosmarinic acid, a major polyphenolic component of P. frutescens, reduces lipopolysaccharide-induced liver injury in d-galactosamine-sensitised mice. The mechanism of action of perilla-derived rosmarinic acid, in reducing liver injury induced by DGalN and LPS, is, mainly, attributed to the scavenging of superoxide or peroxynitirite. Furthermore, rosmarinic acid decreases the mRNA expression levels of the proinflammatory cytokine TNF-α, a prime cause of liver injury. Additionally, it decreases the mRNA expression levels of inducible nitric oxide synthase (iNOS), an important enzyme that produces nitric oxide (NO), which plays an important role in the apoptosis of injured hepatocytes [93].
Caffeic acid and rosmarinic acid from P. frutescens leaves exhibit significant hepatoprotection against tert-butylhydroperoxide (t-BHP)-induced oxidative stress damage in the liver and suppress oxidative stress damage in different ways. They increase the levels of intracellular GSH, by enhancing the activity of γ-GCS, and enhance the activity of various antioxidant enzymes, such as SOD, GPx, and CAT. They, further, reduce the levels of oxidative stress biomarker enzymes, such as aspartate transaminase (AST), alanine transaminase (ALT), lactate dehydrogenase (LDH), and lipid peroxidation. Their combination showed synergistic hepatoprotective activity, compared to their individual action [94].

Hair Growth Promotion Activity
The butanolic fraction of P. frutescens extract (BFPE) promoted hair regrowth in C57BL/6 mice; BFPE successfully induced the anagen phase of hair growth in mice seven days after shaving. Furthermore, BFPE stimulated hair elongation through the proliferation and differentiation of hair follicles and prevented androgenic alopecia. The hair growthpromoting activity of BFPE is, mainly, due to its active constituent, rosmarinic acid, which increased the viability of PHFC (primary hair follicle fibroblast) cells. Furthermore, rosmarinic acid reduced testosterone-and dihydrotestosterone-induced androgenic alopecia. The antimicrobial, anti-inflammatory, anti-leukotriene B4, and anti-androgenic alopecia activities of rosmarinic acid in P. frutescens leaves, synergistically, promote hair growth [95].

Hypolipidemic Activity
The total flavonoid extract of P. frutescens (TFP), mainly, contains apigenin and luteolin. TFP inhibited hyperlipidemia in rats fed with a high-fat diet. TFP decreases lipid accumulation in adipose tissues and serum levels of triacylglycerols, total cholesterol, and low-density lipoprotein cholesterol (bad cholesterol), accompanied by increased levels of high-density lipoprotein cholesterol (good cholesterol). Furthermore, TFP suppressed oxidative stress in hyperlipidemic rats, by increasing the levels of antioxidant enzymes, such as SOD and GPx, following the inhibition of lipid peroxidation, by decreasing serum malondialdehyde (MDA) levels in the serum of high-fat-diet-fed rats. Based on this evidence, P. frutescens can be used as a food additive to prevent atherosclerosis [96].

Inotropic and Lusitropic Effects
Korotkich et al. [97] reported that P. frutescens extract (PFE) exhibited positive inotropic and lusitropic effects on the myocardium of rabbits. PFE increased myocardial contraction (inotropic) and relaxation (lusitropic) effects in a dose-dependent manner and demonstrated that the inotropic and lusitropic effects were attributed to the metabolism of calcium in cardiac muscle cells. An increase in the influx of calcium ions, through L-type membrane channels, increases the velocity of contraction (inotropic), and the acceleration of the sarcoplasmic reticulum uptake of calcium ions increases the relaxation velocity (lusitropic).

Neuroprotective Activity
Luteolin from the ripe seed of P. frutescens exhibits neuroprotective effects against ROSinduced cytotoxicity in primary cortical neurons. Luteolin enhanced the viability of H 2 O 2 intoxicated primary neurons [98] inhibited ROS production in primary neurons treated with H 2 O 2 , and enhanced mitochondrial membrane potential in a concentration-dependent manner. Furthermore, luteolin decreased oxidative stress in primary cortical neurons, by enhancing the levels of antioxidant enzymes, such as CAT and GSH. Hence, luteolin can be used as a dietary supplement to prevent neurodegenerative disorders such as Alzheimer's, Parkinson's, and cerebral ischemia disorders. Kim et al. [99] reported that luteolin, from the alcoholic extract of P. frutescens, inhibited NO production in microglial cells, by suppressing the mRNA expression levels of iNOS. Rosmarinic acid from the methanolic extract of P. frutescens exhibited neuroprotective effects, against oxidative stress induced by H 2 O 2 in C6 glial cells [100]. H 2 O 2 -intoxicated glial cells showed elevated levels of ROS, NO production, and lipid peroxidation, which are associated with apoptosis of glial cells. H 2 O 2 intoxication upregulated the mRNA expression levels of iNOS and COX-2. Rosmarinic acid treatment protects glial cells, by reducing ROS, NO, and lipid peroxidation. The neuroprotective effect of rosmarinic acid was due to the downregulation of protein and mRNA expression levels of iNOS and COX-2. Lee et al. [101] demonstrated that the methanolic extract of P. frutescens and its active constituent rosmarinic acid effectively improved cognitive function and exhibited objective discrimination in an amyloid β (Aβ [25][26][27][28][29][30][31][32][33][34][35] )-induced mouse model of Alzheimer's disease (AD). The methanolic extract of P. frutescens and rosmarinic acid inhibited the production of NO (neurotoxicant) and MDA (causing cell membrane damage by lipid peroxidation) in Aβ 25-35 -injected mouse brain. Perilla seed oil and its active constituent α-linolenic acid (ALA) protect SH-SY5Y (human neuroblastoma) cells from H 2 O 2 -induced oxidative stress and apoptosis; the cell death of SH-SY5Y cells is attenuated, by downregulating the Bax/Bcl-2 ratio, cleaved PARP, cleaved caspase-3, and caspase-9 (Lee et al., 2018). β-site amyloid precursor protein cleaving enzyme 1 (BACE 1) is one of the three enzymes required for the production of β-amyloid, which is a neurotoxic peptide that plays a major role in Alzheimer's disease. Inhibition of BACE 1 limits or stops the production of β-amyloid, which, consequently, slows the pathology of Alzheimer's disease. Choi et al. [102] reported that luteolin and rosmarinic acid from methanolic extracts of P. frutescens inhibited the BACE 1 enzyme activity, in a dose-dependent manner, with IC 50 values of 0.5 and 21 µM, respectively, and inhibited the BACE 1 enzyme in a noncompetitive manner. Perilla seed oil and leaf extract showed neuroprotective effects against β-amyloid-peptide-induced toxicity in pheochromocytoma (PC12) cells, reducing oxidative stress and inhibiting hyperphosphorylation of tau protein. Tau protein is a microtubuleassociated protein that maintains the normal shape and structure of neurons, which is hyperphosphorylated in neurodegenerative diseases and is incapable of maintaining the normal neuron morphology. Perilla seed oil and leaf extract successfully inhibited tau protein phosphorylation and enhanced neurite-outgrowth-bearing cells [103].
Isoegomaketone, isolated from P. frutescens essential oil, exhibited anti-inflammatory effects in LPS-induced mouse macrophages (RAW 264.7 cells) and suppressed the production of NO (inflammatory mediators), IL-6 (inflammatory cytokine), and monocyte chemoattractant type-1 (MCP-1, a protein that attracts monocytes and macrophages to the areas of inflammation). Park et al. [54] prepared different synthetic derivatives of isoegomaketone. One of the isoegomaketone derivatives inhibited NO, MCP-1, and IL-6 more effectively than its precursor. Park et al. [54] also, demonstrated that isoegomaketone and its derivatives suppressed the NF-kB pathway and activator protein-1 (AP-1), which are two inflammation-associated genes involved in the production of inflammatory mediators and inflammatory cytokines. Moreover, 9-hydroxy-isoegomaketone from P. frutescens leaves inhibited NO production in the LPS-stimulated mouse macrophage cell line RAW 264.7, as 9-hydroxy-isoegomaketone inhibits NO production, in a dose-dependent manner, with an IC 50 value of 14.4 µM [55].
Yang et al. [20] reported that rosmarinic acid from P. frutescens leaves showed antiinflammatory effects in cecal ligation and puncture (CLP)-induced septic mice and in human endothelial cells. Rosmarinic acid suppresses phorbol myristateacetate (PMA), TNF-α, IL-1β, and CLP-mediated endothelial protein C receptor (EPCR) shedding, by inhibiting the expression of TNF-α and converting enzyme (TACE). Furthermore, rosmarinic acid suppressed the phosphorylation of p38, ERK1/2, and JNK stimulated by PMA [106]. High-mobility group box-1 (HMGB-1) is a DNA-binding nuclear protein that acts as a proinflammatory cytokine, which plays an important role in infections, injury, and sepsis-related inflammation. Rosmarinic acid suppressed CLP-or LPS-stimulated HMGB1 release, and inhibited HMGB1 mediated inflammation in endothelial cells and sepsisrelated mortality. Based on the above studies, rosmarinic acid can be used as a potential therapeutic agent for treating vascular inflammatory diseases, by inhibiting the HMGB-1 signalling pathway.
Luteolin reported from the P. frutescens alcoholic extracts inhibits neuro-inflammatory response in LPS-activated microglial (BV-2) cells, through the suppression of NO production [99]. Luteolin suppressed NO production in a dose-dependent manner, with an IC 50 value of 6.9 µM, and inhibited NO production by suppressing the mRNA expression levels of iNOS. Deep-mechanism studies demonstrated that luteolin suppressed the degradation of Ik-Bα, an inhibitor of NF-kB, which is a protein family that mediates inflammatory responses, by producing inflammatory mediators and proinflammatory cytokines. Thus, luteolin inhibits the neurotoxic effector (NO) in the central nervous system, by suppressing the mRNA expression levels of iNOS [99].
The essential oil of P. frutescens (EOPF) suppressed chronic stress-induced neuroinflammatory responses, by inhibiting the plasma levels of proinflammatory cytokines, including IL-1, IL-6, and TNF-α. Ji et al. [91] reported that perillaldehyde, a major constituent of EOPF, could reduce neuroinflammation by inhibiting IL-6 and TNF-α levels in the prefrontal cortex of an LPS-induced mouse model. Reflux of gastric contents from the stomach into the oesophagus causes inflammation of mucosal tissue and oxidative stress, a condition called reflux oesophagitis. Arya et al. [107] demonstrated that P. frutescens fixed oil successfully inhibited experimental reflux oesophagitis in Wistar albino rats. In this experiment, P. frutescens fixed oil exhibited different activities, such as antioxidant, lipoxygenase-inhibitory, anticholinergic, and antihistamine activities. These activities of P. frutescens fixed oil are, mainly, due to the presence of α-linolenic acid. Together, these activities provide protection against reflux oesophagitis in experimental rats.

Antioxidant Activity
Oxidative stress is, mainly, referred to as an imbalance between reactive oxygen species (free radicals) and antioxidant capacity, occurring when ROS production exceeds antioxidant capacity. It causes severe cellular damage, by disrupting cellular defence systems by interfering with cellular survival mechanisms, and is associated with several human diseases, including aging, neurodegenerative, cardiovascular, carcinogenesis, hepatic, and kidney diseases [108]. Oxidative stress-induced cell death can be weakened, by suppressing intracellular ROS production and by inducing the expression of antioxidant enzymes and cytoprotective proteins, such as superoxide dismutase (SOD), glutathione peroxidase (GP X ), thioredoxin, catalase, γ-glutamylcysteine synthetase (g-GCS), glutathione S-transferase (GST), hemeoxygenase-1 (HO-1), and NAD(P)H: quinone oxidoreductase-1 (NQO1). Moreover, the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway controls the expression of these proteins and enzymes, and its activation protects cells against oxidative stress, thus functioning as a cellular defence system [109].
In addition, 2 1 ,3 1 -dihydroxy-4 1 ,6 1 -dimethoxychalcone (DDC), a flavonoid isolated from the leaves of green perilla, suppresses intracellular ROS production. Furthermore, it stimulates the expression of antioxidant enzymes, such as γ-GCS, NQO1, and HO-1. DDC increases glutathione content and antioxidant response element (ARE) activity in a concentration-dependent manner. Nrf2 induces the expression of antioxidant genes, by binding to AREs, which is crucial in the antioxidant genes expression, to overcome the cellular damage caused by oxidative stress. DDC significantly induced Nrf2-dependent ARE activation. Furthermore, phosphorylation of Nrf2 plays an important role in nuclear translocation and transcriptional activation, through AREs. The Nrf2-ARE pathway is regulated by different signalling kinases, including p38 MAPK (p38 mitogen-activated protein kinase), PI3K, and protein kinase C. DDC, effectively, increased the p38 MAPK and PI3K/Akt pathways. Thus, DDC from green Perilla acts as an effective antioxidant [43]. Figure 4 depicts a schematic representation of Nrf2-dependent ARE pathway activation, by the DDC of P. frutescens. γ-Glutamylcysteine synthetase (γ-GCS) catalyses the major regulatory (rate-limiting) step in the de novo synthesis of glutathione (GSH), a major antioxidant tripeptide that protects cells from cellular damage caused by ROS. Park et al. [26] reported that caffeic acid from the leaves of green perilla increased γ-GCS activity in a dose-dependent manner. Similarly, GSH content increased, according to γ-GCS activity. This result revealed that the elevation of γ-GCS activity by caffeic acid is responsible for the de novo increase in GSH synthesis.

Anticancer Activity
The ethanolic leaf extract of P. frutescens (PFLE) inhibited the viability of HL-60 human leukemia cells, in a concentration-dependent manner. PFLE caused DNA defragmentation and arrested the cell cycle in the G1-phase. Its treatment increased the activities of γ-Glutamylcysteine synthetase (γ-GCS) catalyses the major regulatory (rate-limiting) step in the de novo synthesis of glutathione (GSH), a major antioxidant tripeptide that protects cells from cellular damage caused by ROS. Park et al. [26] reported that caffeic acid from the leaves of green perilla increased γ-GCS activity in a dose-dependent manner. Similarly, GSH content increased, according to γ-GCS activity. This result revealed that the elevation of γ-GCS activity by caffeic acid is responsible for the de novo increase in GSH synthesis.

Anticancer Activity
The ethanolic leaf extract of P. frutescens (PFLE) inhibited the viability of HL-60 human leukemia cells, in a concentration-dependent manner. PFLE caused DNA defragmentation and arrested the cell cycle in the G1-phase. Its treatment increased the activities of apoptotic proteins, including caspase-3, caspase-8, and caspase-9, in a dose-dependent manner, and induced the cleavage of poly (ADP-ribose) protein (PARP), a DNA-repair enzyme. Furthermore, PFLE upregulated the levels of the proapoptotic protein Bax and downregulated the levels of the anti-apoptotic protein Bcl-2. PFLE treatment increased the levels of glucose-regulated protein-78 (GRP-78), phosphorylated eIF-2α, and JNK and p21 levels, in a concentration-dependent manner. Kwak et al. (2009) demonstrated that PFLE strongly induced the apoptosis of HL-60 through different mechanisms, including mitochondria-mediated (upregulation of caspase-9 by releasing cytochrome C from mitochondria), death-receptor-mediated (upregulation of key initiator caspase-8), and endoplasmic-reticulum-stress-mediated (upregulation of GRP78 and activation of eIF-2α and JNK by phosphorylation) pathways. The activity of PFLE towards apoptosis of HL-60 might be attributed to its active constituent luteolin.
P. frutescens leaf extract (PLE) inhibited hepatic carcinoma (Hep-G2) in a dose-dependent manner, with an IC 50 value of 105 µg/mL, and exhibited antiproliferative activity in Hep-G2 cells, by inducing apoptosis in both caspase-dependent and caspase-independent pathways. PLE upregulated the mRNA expression levels of pro-apoptotic proteins caspase-8 and Bax, whereas the mRNA expression levels of the anti-apoptotic protein Bcl-2 were downregulated by PLE treatment. PLE upregulated apoptotic inducers, including c-Jun, Jun-B, and Fos-B, in the FOS/JUN-pathway, and NFkBIA and TNFSF9, through the autocrine pathway. The apoptotic effect of PLE on Hep-G2 cells was attributed to its active constituents, such as rosmarinic acid, caffeic acid, luteolin, and triterpene acids [110].
Kwak et al. [111] demonstrated that the ethanol extract of perilla leaf (PLE) inhibited colorectal cancer (HCT116) and non-small cell lung cancer (H1299), in a dose-dependent manner, with IC 50 values of 50 µg/mL and 67 µg/mL, respectively. The PLE-treated cells exhibited nuclear fragmentation and chromatin condensation. PLE treatment significantly enhanced the sub-G1 cell population and inhibited the migration of non-small cell lung cancer cells and their adhesion.
Banno et al. [14] reported that lipophilic triterpene acids, such as tormentic acid and oleanolic acid from the ethanolic extracts of Perilla leaves, showed inhibitory activity against skin carcinoma, through the protein kinase C signalling pathway. Lipophilic pentacyclic triterpene acids, such as ursolic acid, oleanolic acid, maslinic acid, 3-epimaslinic acid, corosolic acid, and 3-epicorosolic acid, inhibit skin carcinoma promoted by 12-Otetradecanoylphorbol-13-acetate (TPA) in an ICR mouse model. All pentacyclic triterpene acids inhibited the TPA-induced epidermal proliferation. Furthermore, they inhibited skin inflammation by suppressing the expression of inflammatory genes. However, maslinic acid and 3-epicorsolic acid were more effective against TPA-mediated skin tumour promotion. They inhibited epidermal proliferation and skin inflammation compared to other pentacyclic triterpene acids. Maslinic acid and 3-epicorsolic acid, effectively, inhibited skin carcinoma, by suppressing the expression of Cox-2 and Twist-1 proteins, and inhibited the phosphorylation (activation) of epidermal signalling pathway proteins, such as IGF-1βR, Src, STAT3, JNK1/2, and c-Jun. Furthermore, they enhanced the levels of the tumour-suppressor protein, Pdcd4 [80].
Isoegomaketone, from the aerial parts of green perilla, inhibited the viability of mouse melanoma (B16) cells, in a concentration-dependent manner. It inhibits melanoma growth in tumour-transplanted mice. The possible effects of the antitumor/anticancer activity of isoegomaketone are, also, explained. Isoegomaketone induces apoptosis of melanoma cells, via both caspase-dependent and caspase-independent pathways. It induces ROS generation, nuclear fragmentation, cell shrinkage, and cell membrane blebbing, and increases caspase-3 and caspase-9 activities, in a concentration-dependent manner. Isoegomaketone upregulates Bax (pro-apoptotic protein) and downregulates Bcl-2 (anti-apoptotic protein). Furthermore, isoegomaketone significantly increases Bax/Bcl-2 [9].
Dietary perilla oil inhibits methyl nitrosourea (MNU)-induced colon cancer in F344/ NSlc rats. Narisawa et al. [112] demonstrated that perilla oil inhibits colonic tumour development by MNU, mainly because of the presence of α-linolenic acid, a ω-3 PUFA. α-Linolenic acid modified the sensitization of colonic cell membranes to carcinogens, such as MNU.
Perillaldehyde, a major volatile component of the essential oil of P. frutescens, inhibited the viability of mouse gastric cancer cell lines (MFCs). Perillaldehyde inhibits MFCs by inducing autophagy in MFCs, which is dependent on adenosine monophosphate-activated protein kinase (AMPK), and AMPK activation is, LKB1 dependent, a primary upstream kinase of AMPK. Zhang et al. [113] reported that PAH activates AMPK, via phosphorylation of the Thr172 residue. Phosphorylated AMPK induces autophagy in gastric cell lines. Furthermore, perillaldehyde upregulated the levels of apoptotic proteins, such as caspase-3 and p53, and promoted apoptosis of gastric cancer cells in vivo.
Perilla essential oil showed antifungal activity against phytopathogenic fungi, including A. niger, A. flavus, A. oryzae, Rhizopus oryzae, and Alternaria alternata [3]. Rosmarinic acid, and its extracts from P. frutescens leaves, exhibited strong antimicrobial activity against different bacterial and fungal pathogens, including E. coli, S. aureus, B. subtilis, A. niger and C. albicans. Rosmarinic acid exhibits antibacterial activity, by targeting deformylase, which is a bacterial enzyme peptide that is essential in synthesising bacterial functional polypeptides. Rosmarinic acid shows fungal inhibitory activity, by targeting the fungal co/post-translational enzyme N-myristoyltransferase [116].
Among all compounds reported from P. frutescens, rosmarinic acid and luteolin were found to show a wide variety of biological applications. Figure 5 shows the different biological functions of the rosmarinic acid and luteolin of P. frutescens.
Among all compounds reported from P. frutescens, rosmarinic acid and luteolin were found to show a wide variety of biological applications. Figure 5 shows the different biological functions of the rosmarinic acid and luteolin of P. frutescens. Perilla frutescens shows no toxicity and no side effects to human. However, it shows few side effects for cattle and livestock. However, the side effects are negligible, when the cattle eat limited P. frutescens. Perilla frutescens contains a pneumotoxin in the leaves and seeds that when metabolised in the rumen produces toxic intermediaries. Perilla ketone, from the essential oil of Perilla frutescens, is a pulmonary edemagenic agent for laboratory animals and livestock, when they take a high amount of P. frutescens.

Conclusions
P. frutescens is an annual herb, belonging to the family Lamiaceae, and is mainly cultivated in China, Japan, Korea, and India. P. frutescens has gained attention from researchers worldwide, owing to its emerging economic importance. The enormous phytochemical constituents, their medicinal properties, and their nutraceutical properties make P. frutescens a unique crop. The leaves, aerial parts, and seeds of P. frutescens are used for culinary and non-culinary purposes. Approximately 400 compounds, including alkaloids, terpenoids, quinines, phenylpropanoids, glycosides, benzoxipen derivatives, polyphenolic compounds, flavonoids, chalcones, coumarins, carotenoids, anthocyanins, neolignans, fatty acids, policosanols, tocopherols, and phytosterols, were reported from P. frutescens. Rosmarinic acid, luteolin, apigenin, caffeic acid, and their derivatives, which are present in leaves, seeds, and aerial parts, were the most active constituents of P. frutescens. Rosmarinic acid, luteolin, apigenin, and caffeic acid are the key bioactive constituents responsible for many biological activities, including antimicrobial, antioxidant, anticancer, antidepressant, anti-allergic, anti-inflammatory, antidiabetic, and hypolipidemic activities. Rosmarinic acid, luteolin, apigenin, and caffeic acid exhibited neuroprotective, hepatoprotective, and cardioprotective effects. Further Rosmarinic acid and luteolin exhibited Perilla frutescens shows no toxicity and no side effects to human. However, it shows few side effects for cattle and livestock. However, the side effects are negligible, when the cattle eat limited P. frutescens. Perilla frutescens contains a pneumotoxin in the leaves and seeds that when metabolised in the rumen produces toxic intermediaries. Perilla ketone, from the essential oil of Perilla frutescens, is a pulmonary edemagenic agent for laboratory animals and livestock, when they take a high amount of P. frutescens.

Conclusions
P. frutescens is an annual herb, belonging to the family Lamiaceae, and is mainly cultivated in China, Japan, Korea, and India. P. frutescens has gained attention from researchers worldwide, owing to its emerging economic importance. The enormous phytochemical constituents, their medicinal properties, and their nutraceutical properties make P. frutescens a unique crop. The leaves, aerial parts, and seeds of P. frutescens are used for culinary and non-culinary purposes. Approximately 400 compounds, including alkaloids, terpenoids, quinines, phenylpropanoids, glycosides, benzoxipen derivatives, polyphenolic compounds, flavonoids, chalcones, coumarins, carotenoids, anthocyanins, neolignans, fatty acids, policosanols, tocopherols, and phytosterols, were reported from P. frutescens. Rosmarinic acid, luteolin, apigenin, caffeic acid, and their derivatives, which are present in leaves, seeds, and aerial parts, were the most active constituents of P. frutescens. Rosmarinic acid, luteolin, apigenin, and caffeic acid are the key bioactive constituents responsible for many biological activities, including antimicrobial, antioxidant, anticancer, antidepressant, anti-allergic, anti-inflammatory, antidiabetic, and hypolipidemic activities. Rosmarinic acid, luteolin, apigenin, and caffeic acid exhibited neuroprotective, hepatoprotective, and cardioprotective effects. Further Rosmarinic acid and luteolin exhibited xanthine oxidase, tyrosinase, alpha-glucosidase, hyaluronidase, and aldose reductase inhibitory activities. The antimicrobial, anti-inflammatory, anti-leukotriene B4, and anti-androgenic alopecia activities of rosmarinic acid and luteolin were, synergistically, involved in the hair growth promotion of P. frutescens leaf. Rosmarinic acid was found at very high concentrations in P. frutescens, compared to caffeic acid, luteolin, and apigenin. The essential oil of P. frutescens (EOPF) exhibited stronger insecticidal, antiplasmodial, antifungal, antibacterial, and antiviral activities, and demonstrated anticarcinogenic effects, in different cancer cell lines. EOPF suppresses the neuroinflammatory response, inhibits inflammation of the mucosal layer of the oesophagus, and exhibits anti-inflammatory effects. Furthermore, EOPF shows radical scavenging, lipoxygenase inhibitory, anticholinergic, and antihistamine activities. All of these EOPF activities were due to the presence of 200 different volatile substances, including monoterpenes, sesquiterpenes, and phenylpropanoids. Isoegomaketone and perillal were found to be the most active constituents of EOPF, with anti-inflammatory, insecticidal, antimicrobial, and anticarcinogenic effects. P. frutescens leaves constitute triterpene acids, anthocyanins, coumarins, carotenoids, and neolignans. Nine different types of triterpene acids present in P. frutescens exhibited anticarcinogenic effects against different types of human cancer cell lines. Seven different types of anthocyanins present in P. frutescens showed anti-inflammatory and anticancer activities. Coumarins and carotenoids present in P. Frutescens demonstrated xanthine-oxidase-inhibitory, anti-inflammatory, antioxidant, and anticancer activities. Two neolignans present in P. frutescens can, potentially, treat endotoxemia and inflammation. Perilla leaves are of industrial importance because of their medicinal and nutraceutical properties. Perilla leaves are edible as well as nutritious and are used as leafy vegetables and spicy herbs in Asian cuisine. The leaves are used in pickles, soups, salads, condiments, garnishes, food flavours, and wraps. Perilla seed oil, extracted from toasted seeds, is used for cooking in China, Japan, and Korea. Perilla seed oil is an important edible oil used for flavour enhancement, cooking, roasting, and dipping sauce. The seed oil is rich in polyunsaturated fatty acids, policosanols, phytosterols, and tocopherols. α-linolenic acid constitutes about 55-65% of the polyunsaturated fatty acids of P. frutescens. Perilla seed oil contains the highest proportion of omega-3-fatty acids, compared to any other plant oil worldwide. Perilla seed oil and α-linolenic acid exhibit strong neuroprotective effects, and tocopherols show strong antioxidant effects. Perilla seeds are, also, rich in flavonoids and polyphenolic compounds. Perilla seed oil, extracted from untoasted seeds, has been used for fueling lamps, printing ink, varnishes, paints, linoleum, lacquers, and waterproof coatings. The seed cake obtained after oil extraction has been used as an animal feed and a biofertiliser. Thus, in this review, we discuss the phytochemical constituents, biological properties, medicinal uses, and pharmaceutical and nutraceutical importance of P. frutescens. This review can serve as a ground work for subsequent studies on P. frutescens.

Conflicts of Interest:
The authors declare no conflict of interest.