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Perilla frutescens var. crispa (Lamiaceae) is an annual plant that is the botanical origin of the natural medicine "Soyo" listed in the Japanese Pharmacopoeia and is also used as a fragrant vegetable. Its characteristic components are essential oils and anthocyanins. Cyanogenic glycosides have also been isolated from perilla, but no reports have clarified which cyanogenic glycosides are abundant or differences in cyanogenic glycoside content according to the extent of perilla leaf growth or growth stage. Here, for the first time we determined the content and distributions of cyanogenic glycosides in perilla. The picric acid test, a common qualitative test for cyanogenic compounds, was used to quickly and semi-quantitatively detect cyanogenic compounds in perilla. Prunasin was the most abundant cyanogenic glycoside. The prunasin content per unit mass of perilla leaves varied by strain, regardless of leaf color or the main compound in the essential oils of each strain. Prunasin was higher in fresh leaves than in dried leaves and higher in young leaves than in mature leaves. When perilla was cultivated in an outdoor field, the prunasin content was initially high during the vegetative stage in summer before decreasing and then increasing until flower buds were beginning to form, and then gradually decreased again after flowering., (© 2022. The Author(s) under exclusive licence to The Japanese Society of Pharmacognosy.)

Caffeic acid and rosmarinic acid are common components of Labiatae plants, such as shiso (Perilla frutescens Britton var. crispa W. Deane) and Boraginaceae plants. These compounds have various pharmacological activities, such as anti-inflammatory, anti-anxiety, and anti-depressive activities, but the content of these compounds in perilla has not been studied in detail. This study investigated the caffeic acid and rosmarinic acid contents of several pure strains in genus Perilla. Perilla plants cultivated under a certain set of conditions had different caffeic acid and rosmarinic acid contents. For example, their contents were higher in P. setoyensis ("Setoegoma"), suggesting that the genetic background of the species greatly affects caffeic acid and rosmarinic acid contents. Several strains of P. frutescens var. crispa were cultivated at the Experimental Station for Medicinal Plants, Graduate School of Pharmaceutical Sciences, Kyoto University and differences in their caffeic acid and rosmarinic acid contents were also observed. The total content of anthocyanins, which are closely related to the leaf color of perilla, was measured as cyanidin-3-glucoside equivalents, and a weak positive correlation was observed between the content of rosmarinic acid, and the total content of total anthocyanins. Furthermore, the results suggest that luminosity and photon flux density of light during cultivation can affect rosmarinic acid content.

High-quality perilla leaves are defined as those having purple upper and lower surfaces and a pleasant smell. The Japanese Pharmacopoeia specifies the content of essential oils in perilla leaves but not the content of rosmarinic acid. Rosmarinic acid is a common component of Labiatae plants such as shiso (Perilla frutescens Britton var. crispa W. Deane). Rosmarinic acid has been reported to exhibit anti-inflammatory and anti-oxidant activity but the factors affecting the content of rosmarinic acid in plants remain unknown. This study describes a simple and reproducible method for quantifying rosmarinic acid. We elucidated the main causes for the different rosmarinic acid contents of plants by examining various samples of perilla using the proposed method. Significant differences in rosmarinic acid content between varieties and cultivators were observed. The rosmarinic acid content was higher in green perilla compared with red perilla, in wild species compared with cultivated species, and in plants cultivated in outdoor nurseries compared with in indoor nurseries. The proposed quantitative method was used to examine the rosmarinic acid content in a Kampo formula, Hangekobokuto, and was found to be higher in decoctions prepared using the Kouge method compared with the typical preparation method. We examined the chlorophyll and caffeic acid contents of several samples and their relationship with the rosmarinic acid content.

High-quality perilla leaves are purple on upper and lower surfaces and have a good aroma. The Japanese Pharmacopoeia specifies the content of essential oils in perilla leaves but not that of anthocyanins. Several reports have described the chemical species of anthocyanins in red perilla, but a complete analysis of anthocyanins in perilla has not been reported. In this study, the anthocyanins in the leaves of cultivated and wild species of perilla and those in commercially available perilla herbs were studied. Red perilla and most P. citriodora strains accumulate cyanidin derivatives that differ in the acyl group on the glucose moiety at the 3-O- and 5-O-positions of the anthocyanins. Several strains of P. citriodora contain cyanidin derivatives that are different from those in red perilla and most P. citriodora species. Green perilla and wild species other than P. citriodora do not contain foliar anthocyanins. The anthocyanins in commercially available perilla herbs and natural dyes made from red perilla were in agreement with those in fresh red perilla leaves and most P. citriodora samples. The amounts and types of anthocyanins were not associated with place of cultivation, although some changes occurred due to degradation during storage. These results provide clues regarding the biosynthesis of anthocyanins in perilla and the evolution of red perilla. The characteristics and stability of anthocyanins are discussed., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Studies on the biosynthesis of oil compounds in Perilla will help in understanding regulatory systems of secondary metabolites and in elucidating reaction mechanisms for natural product synthesis. In this study, two types of alcohol dehydrogenases, an aldo-keto reductase (AKR) and a geraniol dehydrogenase (GeDH), which are thought to participate in the biosynthesis of perilla essential oil components, such as citral and perillaldehyde, were isolated from three pure lines of perilla. These enzymes shared high amino acid sequence identity within the genus Perilla, and were expressed regardless of oil type. The overall reaction from geranyl diphosphate to citral was performed in vitro using geraniol synthase and GeDH to form a large proportion of citral and relatively little geraniol as reaction products. The biosynthetic pathway from geranyl diphosphate to citral, the main compound of citral-type perilla essential oil, was established in this study., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Geraniol and linalool are acyclic monoterpenes found in plant essential oils that have attracted much attention for their commercial use and in pharmaceutical studies. They are synthesized from geranyl diphosphate (GDP) by geraniol and linalool synthases, respectively. Both synthases are very similar at the amino acid level and share the same substrate; however, the position of the GDP to which they introduce hydroxyl groups is different. In this study, the mechanisms underlying the regiospecific hydroxylation of geraniol and linalool synthases were investigated using a domain swapping approach and site-directed mutagenesis in perilla. Sequences of the synthases were divided into ten domains (domains I to IV-4), and each corresponding domain was exchanged between both enzymes. It was shown that different regions were important for the formation of geraniol and linalool, namely, domains IV-1 and -4 for geraniol, and domains III-b, III-d, and IV-4 for linalool. These results suggested that the conformation of carbocation intermediates and their electron localization were seemingly to be different between geraniol and linalool synthases. Further, five amino acids in domain IV-4 were apparently indispensable for the formation of geraniol and linalool. According to three-dimensional structural models of the synthases, these five residues seemed to be responsible for the different spatial arrangement of the amino acid at H524 in the case of geraniol synthase, while N526 is the corresponding residue in linalool synthase. These results suggested that the side-chains of these five amino acids, in combination with several relevant domains, localized the positive charge in the carbocation intermediate to determine the position of the introduced hydroxyl group., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Studying the biosynthesis of oil compounds in Perilla will help to elucidate regulatory systems for secondary metabolites and reaction mechanisms for natural product synthesis. In this study, two types of alcohol dehydrogenases, isopiperitenol dehydrogenases 1 and 2 (ISPD1 and ISPD2), which are thought to participate the oxidation of isopiperitenol in the biosynthesis of perilla, were isolated from three pure lines of perilla. Both ISPD1 and ISPD2 oxidized isopiperitenol into isopiperitenone with an oxidized form of nicotinamide adenine dinucleotide (NAD(+)) cofactor. ISPD1 used both isopiperitenol diastereomers, whereas ISPD2 used cis-isomer as a substrate. However, only ISPD2 was isolated from piperitenone-type perilla. These results suggests that in perilla, ISPD2 is related to the biosynthesis of piperitenone, which was formed via (-)-cis-isopiperitenol.

Periodic update of stored seeds is indispensable for sustainable use and preservation of plant genetic resources, and performance of this task requires precise data on the relationship between storage period and the germination rate of seeds after long-term storage. In this study, germination rates of pure strains of perilla (Perilla frutescens (L.) Britton var. crispa) were investigated for three strains by using the mericarps stored for 1-20 years at 4 degrees C under dry conditions. The germination rates were fairly good (65-70%) for 5-8 years after harvest, but dropped to almost 0% for mericarps stored more than 9 years. Mericarps that did not germinate were analyzed for viability by using 2,3,5-triphenyltetrazolium chloride. For old mericarps (stored more than 9 years), the germination rates and seed viability did not correlate. Further study on days required for germination of mericarps harvested each year was derived from our observation record, suggesting that germination rates and longevity of mericarp life were increasingly influenced by individual difference as the storage period lengthened.

Geraniol and linalool synthases were isolated from three pure strains of Perilla hirtella and Perilla setoyensis, which are wild species of perilla. Their amino acid sequences were very similar to those of Perilla citriodora and Perilla frutescens that were reported previously. However, comparison of the sequences of the same functional synthases derived from different species of Perilla demonstrated that the similarities were high among P. citriodora, P. hirtella and P. frutescens, but low between P. setoyensis and any of the others. This result corresponds well with our previous results showing that P. setoyensis is remotely related to the other perilla species. Both geraniol and linalool synthases utilize geranyl diphosphate (GDP) as their catalytic substrate and they were expressed simultaneously in perilla. The linalool synthase is considered to be the enzyme whose metabolite seems not to be oxidized nor reduced in the plant body and the geraniol and limonene synthases are the initial-step-catalyzing enzymes for a variety of oil compounds. The regulation of the substrate flow between them would be interesting for further study., (2010 Elsevier Ltd. All rights reserved.)

Twenty-eight samples of mericarps of Perilla frutescens var. frutescens were collected through fieldwork performed in Phongsali and Xieng Khouang provinces in northern Laos. No perilla samples were collected from Savannakhet province in the south although more than 20 sites were investigated. Perilla plants are mostly grown mixed with dry-paddy rice by slash-and-burn cultivation in Laos. The most popular local name for perilla mericarps in the area was "Ma Nga Chan". Weight of 1,000 grains and hardness of the mericarps were measured, and all mericarps were found to be large (weight of 1,000 grains around 2 g) and soft (limit load weight under 300 g), which were preferred for culinary use in Laos. The composition of the essential oils obtained from the herbaceous plants raised from the mericarps was divided into five types, perillaketone, elemicine plus myristicine, shisofuran, piperitenon, and myristicine, and GC-MS analysis of these Laotian perilla samples showed that they were similar to those of corresponding types of known Japanese perilla strains. One of the shisofuran-type perilla contained large amounts of putative alpha-naginatene, which is likely to be an intermediate of the biosynthesis of naginataketone. The farmers' indifference to the oil type of the leaf seems to leave Laotian perilla as a good genetic resource for studies of the biosynthesis of oil compounds.

Geraniol synthases were isolated from five pure strains of Perilla citriodora and Perilla frutescens which vary in essential oil type, the main compounds of which were citral, elsholtziaketone, perillaketone, and perillene, respectively. This result supports the putative biosynthetic pathways of these three furylalkenes which are all produced by way of citral. Nucleotide sequences of geraniol synthases from three oil types of P. citriodora were identical, and almost the same as the sequence from P. frutescens, a species with twice the chromosome number of P. citriodora. This identity in sequence between P. citriodora and P. frutescens, together with other previous results, indicates that P. frutescens was formed as an amphidiploid of P. citriodora and an unknown wild species.

Phenylpropanoid volatile components in plants are useful and valuable not only as flavorings, but also as medicines and food supplements. The pharmacological actions and toxicities of these compounds have been well studied but their synthetic pathways are generally unclear. In this study, we mined expressed sequence tag libraries of pure strains of perilla maintained for over 30 years for their oil type and conducted gas chromatography-mass spectrometry analyses of the perilla oils to confirm the presence of monohydrates speculated to be intermediates of the phenylpropene synthetics pathways. These putative monohydrate intermediates and their regioisomers were synthesized to identify the reaction products of assays of heterologously expressed enzymes. An enzyme involved in the synthesis of a phenylpropanoid volatile component was identified in perilla. Expression of this enzyme in Saccharomyces cerevisiae showed that it is a member of the cytochrome P450 family and catalyzes the introduction of a hydroxy group onto myristicin to form an intermediate of dillapiole. The enzyme had high sequence similarity to a CYP71D family enzyme, high regiospecificity, and low substrate specificity. This study may aid the elucidation of generally unexploited biosynthetic pathways of phenylpropanoid volatile components. [ABSTRACT FROM AUTHOR]

Abstract: Cytochrome P450 mono-oxygenases from peppermint, spearmint and perilla (all members of the family Lamiaceae) mediate the regiospecific hydroxylation of the parent olefin (−)-limonene to produce essential oil components oxygenated at C3, C6 and C7, respectively. Cloning, expression and mutagenesis of cDNAs encoding the peppermint limonene-3-hydroxylase and the spearmint limonene-6-hydroxylase have allowed the identification of a single amino acid residue which determines the regiospecificity of oxygenation by these two enzymes. A hybridization strategy provided a cytochrome P450 limonene hydroxylase cDNA from perilla with which to further evaluate the structural determinants of regiospecificity for oxygenation of the common substrate (−)-limonene. The perilla cDNA was a partial clone of 1550bp (lacking the N-terminal membrane insertion domain), and shared 66% identity with the peppermint 3-hydroxylase and spearmint 6-hydroxylase at the amino acid level. The perilla cytochrome P450 was expressed in Escherichia coli as a chimeric protein fused with the N-terminal membrane insertion domain of the limonene-3-hydroxylase. The kinetically competent recombinant protein was characterized and shown to produce a mixture of C3-, C6- and C7-hydroxylated limonene derivatives with a distribution of 33%, 14% and 53%, respectively. [Copyright &y& Elsevier]