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The purpose of this study was to determine the variation of the stem anatomical structure (i.e. cortex: sclerenchyma, phloem; xylem: trachea, rays, interxylary phloem and wood parenchyma; and pith) of Gyrinops (Thymelaeaceae) in Sulawesi. The method of this research was descriptive exploration. Stem agarwood sample came from several regions areas of Sulawesi which were collected by Agarwood Research Center Laboratory, Faculty of Mathematics and Natural Sciences, University of Mataram. The slide methods were used fresh slides. The results of this research showed there were 1 species of Gyrinops decipiens and 3 species of Gyrinops sp., with various anatomical characters in G. decipiens and Gyrinops sp. in terms of the position, diameter and percentage of pith thickness; cortical sclerenchyma pattern and arrangement of wood parenchyma. There were also variations in cortical thickness; diameter, frequency and arrangement of trachea; frequency of rays; diameter, and interxylary phloem frequency; pith diameter; and the position of Ca-Oxalate in Gyrinops sp. 3 with Gyrinops sp. 1 and 2, while Gyrinops sp. 2 with Gyrinops sp. 1 there were found 5 variations such as the diameter, frequency and arrangement of trachea; arrangement of rays and interxylary phloem length.

The purpose of this study was to determine the variation of the stem anatomical structure (i.e. cortex: sclerenchyma, phloem; xylem: trachea, rays, interxylary phloem and wood parenchyma; and pith) of Gyrinops (Thymelaeaceae) in Sulawesi. The method of this research was descriptive exploration. Stem agarwood sample came from several regions areas of Sulawesi which were collected by Agarwood Research Center Laboratory, Faculty of Mathematics and Natural Sciences, University of Mataram. The slide methods were used fresh slides. The results of this research showed there were 1 species of Gyrinops decipiens and 3 species of Gyrinops sp., with various anatomical characters in G. decipiens and Gyrinops sp. in terms of the position, diameter and percentage of pith thickness; cortical sclerenchyma pattern and arrangement of wood parenchyma. There were also variations in cortical thickness; diameter, frequency and arrangement of trachea; frequency of rays; diameter, and interxylary phloem frequency; pith diameter; and the position of Ca-Oxalate in Gyrinops sp. 3 with Gyrinops sp. 1 and 2, while Gyrinops sp. 2 with Gyrinops sp. 1 there were found 5 variations such as the diameter, frequency and arrangement of trachea; arrangement of rays and interxylary phloem length.

Agarwood is a type of resinous wood found in the trunks of Aquilaria and some other genera. It is widely used as an herbal medicine for sedation, detoxification, and treatment of stomachaches, as well as for incense sticks. However, the number of source plants is decreasing, and in 2005, they were added to Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). To identify source species of agarwood, we previously developed a DNA-barcoding method using resin deposition sites. In this study, to identify additional agarwood source species, the barcoding method was applied to source plants and commercial agarwood samples collected from Sumbawa, Lombok, Sulawesi, and Kalimantan in Indonesia, a major agarwood-producing country. In addition, the method was also applied to incense stick samples labeled as agarwood. As a result, several samples were identified as Gyrinops, which is not currently listed as an agarwood source plant in the Japanese standards for non-Pharmacopoeial crude drugs 2018 (Non-JPS 2018). From the viewpoint of securing future resources, these findings suggest that Gyrinops species should, therefore, be added to the list of agarwood source species.

Agarwood is a type of resinous wood found in the trunks of Aquilaria, Gonystylus, and Gyrinops species [1]. High-quality agarwood is extraordinarily expensive and therefore its source plant species have become depleted due to exploitation. In 2005, these species were added to Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora [1]. Because these wild agarwood resources have become depleted, commercial production of agarwood has long been a desirable goal. In addition, inauthentic agarwood is sometimes produced from non-agarwood species. Few reports have attempted to identify source species in order to distinguish genuine from false agarwood. In this study, DNA was extracted from putative agarwood samples collected from Japanese, Indonesian, Thai, and Vietnamese markets. The trnL-trnF region and matK gene were amplified from each sample by PCR to serve as DNA barcodes for identifying the plant species to which each sample belonged. One of the wood samples did not originate from a genuine agarwood species. Although some species were identified, sequence data for agarwood source species currently available in GenBank is insufficient to identify the species to which all of these putative agarwood samples belonged. Thus, positive identification of remaining samples will require further exploration.

This study provides a list of popular medicinal plants found in southern Benin (West Africa) with their mode of use, diseases treated, and thin-layer and high-performance liquid chromatography profiles. The list includes 10 of the most widely used plant species from Dantokpa Market (biggest market located in Cotonou) and Abomey-Calavi in the Republic of Benin. Species were identified by the Laboratory of Botany and Applied Ecology, University of Abomey-Calavi. Voucher specimens were deposited in the herbarium of the Experimental Station for Medicinal Plants, Graduate School of Pharmaceutical Sciences, Kyoto University, Japan, and in the National Herbarium of Benin, University of Abomey-Calavi. The list was as follows—Azadirachta indica (Meliaceae), Caesalpinia bonduc (Caesalpiniaceae), Catharanthus roseus (Apocynaceae), Garcinia kola (Clusiaceae), Khaya senegalensis (Meliaceae), Monodora myristica (Annonaceae), Moringa oleifera (Moringaceae), Talinum fruticosum (Talinaceae), Tridax procumbens (Asteraceae), and Xylopia aethiopica (Annonaceae).

Agarwood, which is used as medicine and incense, contains sesquiterpenes and chromones. Agarotetrol is a chromone derivative found in high concentrations in the water-extract fraction of agarwood and thus may be present in pharmaceutical products made from decoctions of agarwood. Agarotetrol has been reported to be present at the early stages of cell death in calli. We therefore examined the presence of agarotetrol in medical- and incense-grade agarwood, in agarwood-source plants lacking resin deposits, and in artificially made agarwood. Agarotetrol appeared as a large peak in the HPLC chromatograms of all samples of medical-grade and artificially made agarwood, and in most incense-grade agarwood samples. In contrast, agarwood samples lacking resin deposits did not contain agarotetrol. These results show that agarotetrol is characteristic of resin formation. Agarotetrol was also detected in decoctions of agarwood. A newly developed TLC method for the detection of agarotetrol in agarwood is described.

The essential oil of perilla (Perilla frutescens) contains volatile low molecular weight compounds such as monoterpenes and phenylpropenes. The composition of the essential oil is classified into about ten chemotypes. The biosynthesis of these constituents is strictly controlled genetically. Among the compounds contained in perilla essential oil, the bioconversion of pure compounds such as perillaldehyde, limonene, and citral has been reported, but that of many other components has not. In addition, changes in the volatile components of raw plant material during brewing have also been investigated for wine and beer. In this study, we examined the bioconversion of perilla essential oil components by Saccharomyces cerevisiae during the brewing of liquor with perilla leaves. S. cerevisiae was added to the ethanol–water extract of dried leaves of P. frutescens and P. citriodora for seven essential oil types: perillaldehyde type, piperitenone type, perillene type, perillaketone type, elsholtziaketone type, citral type, and phenylpropanoid type. Volatile compounds in the reaction mixtures were analyzed by solid-phase microextraction (SPME)-GC–MS, revealing bioconversion of perillaldehyde, isoegomaketone, neral, and geranial by S. cerevisiae. Analysis of the conversion products suggests that they were formed by the reduction of C=C bonds and aldehydes, as well as by esterification and dehydration reactions.

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.

Agarwood is known to generate a distinct fragrance upon heating and is used as both a medicine and a fragrant wood. Low molecular weight aromatic compounds (LACs) such as benzylacetone are emitted from agarwood on heating and have a sedative effect on mice. These are detected exclusively in the headspace vapor of heated agarwood and are absent in the wood itself; hence, some compounds in agarwood are thought to be converted to LACs by the process of heating. In this study, different fractions obtained from agarwood were analyzed to reveal the source compounds of LACs. Some LACs detected in the resinous agarwood were absent from the non-resinous parts and confirmed as characteristic of the resinous parts. The essential oil and hydrosol of agarwood obtained by distillation were analyzed by gas chromatography–mass spectrometry (GC–MS). Sesquiterpenes were detected in the essential oil, and sesquiterpenes and a variety of LACs were detected in the hydrosol. A hot water extract of agarwood remaining in the distillation flask after distillation was analyzed by high-performance liquid chromatography (HPLC), and agarotetrol was found to be the main compound. Purified agarotetrol was heated in a glass vial and its headspace vapor was analyzed by solid-phase microextraction GC–MS. Benzylacetone and other LACs were detected. These results indicate that agarotetrol, a chromone derivative, contributes to the fragrance of agarwood through the generation of LACs upon heating.

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.

The sedative effects of the essential oil released by living Ocimum basilicum (basil) plants were investigated using a mouse activity monitoring system. Ocimum basilicum plants were grown in a hydroponic chamber, and either the headspace air from the hydroponic chamber or the essential oil extracted from mature plants was administered by the inhalation route to mice in an open field test. The most effective dose of O. basilicum essential oil for reducing the locomotor activity of the mice was found to be 4.0 × 10⁻³ mg per cage. The headspace air was administered to mice held in a glass cage via a Teflon tube connected to a hydroponic chamber containing O. basilicum plants. A significant decrease in locomotor activity was observed when the hydroponic chamber contained nine plants. The results of this study demonstrate that the headspace air of living basil plants could effectively reduce the locomotor activity of mice.

Perilla produces the cyclohexanoid monoterpene perillaldehyde as a major constituent of an essential oil that is accumulated in its glandular trichomes. Perillaldehyde is a marker compound for quality control of soyo and has biological activities such as antibacterial, sedative, or vasodilatory effects. The predicted perillaldehyde formation involves the cyclization of geranyl diphosphate, hydroxylation, and oxidation, and cytochrome P450 plays a crucial role in perillaldehyde biosynthesis. In this study, a cytochrome P450-type enzyme with perillyl alcohol and perillaldehyde synthase activities was isolated by analyzing an expressed sequence tag library from several oil types of pure lines of perilla. A recombinant protein with a sequence that was highly specific for the type of perillaldehyde was expressed in Saccharomyces cerevisiae and evaluated by an in vitro enzymatic reaction. The recombinant protein catalyzed the hydroxylation and oxidation of limonene to perillyl alcohol and perillaldehyde. Cytochrome P450 limonene-7-hydroxylase cDNA from Perilla frutescens has been previously isolated. The cytochrome P450 isolated in this study shares 37% amino-acid identity with the previously isolated enzyme; however, it may have different characteristics.

Inhalation of scent compounds with phenylpropanoidal structures, such as trans-cinnamaldehyde, is expected to increase the appetite. The scent of curry powder is well known for its appetite-enhancing effect on humans. In this work, we show that the appetite of mice after inhalation of curry powder essential oil or benzylacetone showed a similar increase. The components of curry oil, trans-cinnamaldehyde, trans-anethole, and eugenol, each showed appetite-enhancing effects; therefore, these three scent compounds may be the active compounds in curry powder oil.

Fragrance in the air and odours of foods and drinks are reported to affect feeding behaviours of humans and other animals. Many previous studies focusing on the relationship between fragrance and appetite have described a reduction of food intake by fragrance administration to help prevent lifestyle diseases. Aromatic herbal medicines, such as cinnamon bark and fennel fruit, are considered to have appetite-enhancing effects and they are often blended in stomachics for relief of asitia and gastric distress in Japan. These fragrant herbal medicines contain many essential oils and their fragrances are hypothesised to be active substances. In this study, food intake and the expression of neuropeptide Y and proopiomelanocortin in the hypothalamus after inhalation of fragrant compounds or essential oils were investigated in mice. Food intake was increased 1.2-fold and the neuropeptide Y mRNA expression in the hypothalamus was increased significantly in mice that inhaled trans-cinnamaldehyde, benzylacetone or 1-phenyl-2-butanone, compared with the control group. These compounds might be effective for treating loss of appetite (anorexia) or eating disorders in elderly and infirm people via a non-invasive route of administration, namely, inhalation.

Benzylacetone has appetite-enhancing and locomotor-reducing effects. The effective doses for these two outcomes overlap, and the weight gain of mice exposed to benzylacetone is caused by both appetite-enhancement and a reduction in locomotor activity. The appetite-enhancing effects of trans-cinnamaldehyde and benzylacetone have been reported previously. In this study, these appetite-enhancing effects were seen in mice after short-term, high-dose exposure.

Ophiopogon japonicus is a herbaceous perennial plant in Liliaceae, and its tubers are used in traditional Japanese medicine as Bakumondo, prescribed for treating cough, sputum, and thirst. Liriope is a genus of ornamental plants related to Ophiopogon, and its tubers are used in folk medicine as well. Although tubers from both genera are traded in Korean and Chinese markets, only O. japonicus is defined as the plant of origin for Bakumondo in the Japanese Pharmacopoeia [1], and Liriope tubers cannot legally be used as Bakumondo in Japan. Ophiopogon plants can be distinguished clearly from Liriope by their fruit color and by the morphological characteristics of their flowers. However, the tubers of both species are greatly similar, making it very difficult to differentiate the two genera by the appearance of their tubers. We, therefore, investigated the most appropriate DNA regions to use for practical and accurate identification of Ophiopogon and Liriope tubers. The sequence of the gene for the large subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase (rbcL) was found to be suitable for discriminating Ophiopogon and Liriope tubers. The identification procedure was simplified using restriction enzyme digestion of the amplified rbcL fragment. The detection limit for Liriope contamination was estimated by performing the procedure using mixed samples of powdered Ophiopogon and Liriope tubers.

Spikenard, the dried roots of Nardostachys chinensis, contains sesquiterpenoids and is widely used as an herbal tranquilizer. We previously demonstrated that spikenard vapor showed a sedative effect when administered by inhalation, and we identified hydrocarbon sesquiterpenoids as active components. Here we investigated the other components that contribute to the effects of spikenard. Six oxygenated sesquiterpenoids, including aristolane- and guaiane-types, were isolated from an acetone extract of spikenard. We evaluated the sedative activities of these oxygenated compounds using an inhalation administration method in a caffeine-treated excitatory mouse model. We identified aristolen-1(10)-en-9-ol and patchouli alcohol as highly effective sedative components. These compounds inhibited locomotion in mice by approximately 60 % at a dose of 300 µg/cage. In addition, aristolen-1(10)-en-9-ol prolonged pentobarbital-induced sleep to the same extent as 1 mg/kg diazepam. This effect completely disappeared with the administration of the GABAA-benzodiazepine receptor antagonist flumazenil (3 mg/kg), suggesting that the sedative effect of aristolen-1(10)-en-9-ol is expressed via the GABAergic system. Furthermore, differently from diazepam, inhalation of aristolen-1(10)-en-9-ol for 1 h did not affect the motor coordination in the rota-rod test. In the present study, we identified active components and provided evidence supporting the traditional sedative use of spikenard. Our research suggests that aristolen-1(10)-en-9-ol may be an effective aromatherapy, providing mild sedation.

The sedative activity of eight aromatic natural medicines that are traditionally used in Japanese scent sachets was examined using an open field test with mice. Galangal (Kaempferia galanga), patchouli (Pogostemon cablin), sandalwood (Santalum album), spikenard (Nardostachys chinensis), cinnamon (Cinnamomum cassia), clove (Syzygium aromaticum), star anise (Illicium verum), and borneol (Dryobalanops aromatica) distilled oils were used. These natural medicines have various pharmacological effects. For example, galangal has insecticidal activity and clove extracts possess strong total antioxidant activity. Aromatherapy, a well-known complementary medicine system that uses inhalation, has recently attracted much attention. The sedative activity of inhaled aromatic compounds or essential oils has been examined by measuring the spontaneous motor activity of mice in an open field test. The galangal, patchouli, sandalwood, spikenard, and borneol oils showed significant sedative effects. The effect was stronger for a mixture of the five oils than for any of the single oils. This suggests that the oil mixture may have synergistic activity. Sedative activity was not observed when inactive oils (cinnamon, clove, and star anise) were added to the mixture of the five active oils.

Dried Nardostachys chinensis roots contain sesquiterpenoids that are widely used as herbal tranquilizers. We previously identified the highly sedative sesquiterpenoid valerena-4, 7(11)-diene (VLD) from this plant. In the present study, we investigated stress reducing effects of VLD and the associated mechanisms of action. Application of 15-min restraint stresses induced excitatory behaviors in mice. Immobility times in the forced swim test and sleeping times in the pentobarbital sleep test were shortened in the stressed group by 47% and 43%, respectively, compared with the control group. Furthermore, restraint stress increased serum corticosterone levels by 75%, and cerebral serotonin (5-HT) and dopamine (DA) levels. Inhaled VLD (300 µg/cage) suppressed stress-induced excitatory behaviors and significantly reduced stress-induced blood corticosterone, cerebral 5-HT, and DA levels. These results suggest that VLD interacts with the hypothalamic–pituitary–adrenal axis and the sympathetic-adrenomedullary system. These interactions appear to involve GABAergic and D2 antagonist activities. Moreover, tests in anosmic and intravenously treated mice showed that the sedative effect of inhaled VLD was expressed via olfactory stimulation and pulmonary absorption. Although more studies are required to further elucidate the properties of this compound, our studies suggest that VLD may be an effective anti-stress aromatherapy for humans.

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.

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.

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.

The aromatherapeutical potential of Piper guineense essential oil was investigated in mice via inhalation administration, and the active compounds were identified. An open field test and light/dark transition test were used to evaluate the sedative and anxiolytic activities of this essential oil, respectively. P. guineense essential oil showed significant sedative activity at an effective dose of 4.0×10[−5] mg per cage compared to the control group. It also showed potent anxiolytic effect at a dose of 4.0×10[−6] mg per cage. The main compounds of P. guineense essential oil were linalool (41.8%) and 3, 5-dimethoxytoluene (10.9%). These two main compounds were shown to play a major role in the sedative activity of P. guineense essential oil. These results suggest that inhalation of P. guineense essential oil might induce a mild tranquilizing effect.

Benzylacetone is released by heated agarwood, when inhaled it has a potent effect on reducing the locomotor activity of mice. This study investigated the relationships between the sedative activities of benzylacetone and its derivatives as well as the chemical structures of these compounds by comparing their activities in mice treated with a series of compounds. It was demonstrated that benzylacetone-like compounds had sedative activities and their intensities varied depending on the functional group in the carbon chain, the substituent in the benzene ring, and their combinations. A quantitative structure–activity relationship study was carried out using a series of 17 benzylacetone derivatives to determine the structural features with significant for the sedative activity.

The present study investigated the gastroprotective effects of a cinnamon diet using different gastric ulcer mouse models. Dose dependency and the effective dose period of administration of a cinnamon powder diet were established using the water immersion stress gastric ulcer model. A cinnamon powder diet significantly protected mice against ulceration by stress, ethanol, HCl and oral administration of aspirin, but not against ulceration by oral administration of indomethacin or subcutaneous administration of indomethacin or aspirin. Such a diet conferred protection against gastric ulcers at an effective concentration of 100 mg cinnamon powder per gram of food after administration for 4 weeks and the active compound of cinnamon powder for gastroprotective activity was identified as cinnamaldehyde. These findings indicate that regular ingestion of cinnamon powder offers gastroprotection presumably through a cytoprotective mechanism but the efficacy against NSAIDs-induced gastric ulcers may be limited.