Vasorelaxant activity of Sappan Lignum constituents and extracts on rat aorta and mesenteric artery.

We investigated the vasorelaxant activity of the methanolic extracts of Sappan Lignum (CSE) and its constituents, brazilin, sappanchalcone, and protosappanins A-E, on rat aorta and mesenteric artery. By comparing the vasorelaxant activity of CSE and brazilin on both blood vessels, we found that CSE contained active constituents other than brazilin. When added to brazilin, sappanchalcone and protosappanin D showed vasorelaxant activity on both blood vessels precontracted with phenylephrine. We clarified that the vasorelaxant activity of brazilin was endothelium-independent, while that of sappanchalcone was endothelium-dependent, on both blood vessels. On the other hand, the vasorelaxant activity of protosappanin D was independent of the endothelium of the aorta and dependent on the endothelium of the mesenteric artery. Experiments on sappanchalcone and protosappanin D using NG-nitro-L-arginine and indomethacin revealed the involvement of nitric oxide and prostaglandin as endothelium-derived relaxation factors (EDRFs). The anti-oketsu effect of Sappan Lignum might be attributable to the interaction of those compounds. We could partly evaluate the anti-oketsu activity of Sappan Lignum using both the aorta and the mesenteric artery. Through this study, we showed the importance of comparing the effects on the aorta and the mesenteric artery as we found that natural compounds showed different mechanisms of action on the two blood vessels.

Acylated anthocyanins from the violet-blue flowers of Orychophragonus violaceus.

Three acylated cyanidin 3-sambubioside-5-glucosides (1-3) were isolated from the violet-blue flowers of Orychophragonus violaceus, and their structures were determined by chemical and spectroscopic methods. Two of those acylated anthocyanins (1 and 3) were cyanidin 3-O-[2-O-(2-O-(4-O-(6-O-(4-O-(beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-xylopyranosyl)-6-O-(4-O-(beta-D-glucopyranosyl)-trans-acyl)-beta-D-glucopyranoside]-5-O-(6-O-malonyl-beta-D-glucopyranoside)s, in which the acyl groups were p-coumaric acid for 1, and sinapic acid for 3, respectively. The last anthocyanin 2 was cyanidin 3-O-[2-O-(2-O-(4-O-(6-O-(4-O-(beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-xylopyranosyl)-6-O-(4-O-(beta-D-glucopyranosyl)-trans-feruloyl)-beta-D-glucopyranoside]-5-O-beta-D-glucopyranoside. In these flowers, the anthocyanins 2 and 3 were present as dominant pigments, and 1 was obtained in rather small amounts.

[Study of plant DNA polymorphism--experiments for students].

Advances in molecular biology are being made in the fields of science, medicine, and pharmacy. The majority of students graduating from pharmaceutical university departments will be required to have knowledge of molecular biology techniques. Although it is not difficult to perform DNA experiments in a laboratory, they involve a series of operations. Therefore it is difficult to include such experiments in the curriculum for students at our university because of safety concerns and the experimental equipment and time required. This paper introduces a convenient experiment on plant DNA polymorphism using a polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) method that we developed. This experiment enables each student to handle DNA safely. Total DNA is extracted from a piece of leaf derived from Aconitum carmichaeli or Aquilegia flabellate, the nuclear 5.8S rRNA region is amplified by PCR, and the PCR product is digested by restriction enzymes. Different polymorphisms in the plants can be visualized on electrophoregrams. The total time required for the experiments is 2 days (about 4 h per day). In the field of pharmacognosy, authentication of herbal medicine using genetic analysis is increasingly important. Plant DNA polymorphisms required the same knowledge and techniques as genetic analysis and are part of the course content in medicinal plant science. The PCR-RFLP experiment proposed here is a suitable method to acquire molecular biological knowledge and techniques.

[Historical research on cinchona cultivation in Japan].

Cinchona is one of the most important medicinal plants as it contains quinine, a potent medicine for malaria. In this review, I reveal the history of cinchona introduction and cultivation in Japan. Cinchona was first introduced to Japan in 1876 from Java based on the proposal submitted by Takeaki Enomoto to the Meiji government. However, the cultivation attempt ended in failure. Later in 1922, Hoshi Pharmaceutical Co. succeeded for the first time in cultivating cinchona in Taiwan, which was then under Japanese colonial rule, and in manufacturing quinine from the cinchona tree in 1934. This was a historic feat in Japan, completing an entire process from cinchona cultivation to quinine manufacture all within the confines of the country. To commemorate this undertaking, the company dedicated a cinchona log harvested for the first time to the Imperial court. It was revealed that a log of unknown origin, which had been left untouched for years at Hoshi University, was the cinchona log from the time of commemoration. Yasusada Tashiro (1856-1928), who has made a great contribution to cinchona cultivation in Japan for over 50 years, led Hoshi Pharmaceutical Co. to success in cultivation.

Peyote identification on the basis of differences in morphology, mescaline content, and trnL/trnF sequence between Lophophora williamsii and L. diffusa.

Genus Lophophora (Cactaceae) has two species: Lophophora williamsii Coulter, which is called peyote, and L. diffusa Bravo. Although it was reported that L. williamsii contained mescaline and L. diffusa did not, we found L. williamsii specimens that did not contain mescaline. This finding indicated that the two species could not be differentiated in terms of mescaline content. Moreover, the relationship between mescaline content and morphology of the two species is also unknown. In this study, we attempted to clarify the difference in morphology, mescaline content, and DNA alignment of the chloroplast trnL/trnF region between L. williamsii and L. diffusa. As a result, L. williamsii specimens were classified into two groups. Group 1 had small protuberances on the epidermis, contained mescaline, and the analyzed region on the trnL/trnF sequence was 881 base pairs (bp) long in all except one (877 bp). Group 2 had large protuberances on the epidermis, did not contain mescaline, and the analyzed region was 893 bp long. On the other hand, L. diffusa had medium-sized protuberances on the epidermis, did not contain mescaline, and the analyzed region was 903 bp long. Also investigated was the potential application of the PCR-restriction fragment length polymorphism (RFLP) method as a means of identification based on the trnL/trnF sequence. By applying the PCR-RFLP method, the two species could be distinguished and L. williamsii specimens could be differentiated into group 1 and group 2.

[Historical research of cinchona cultivation in Japan (Part 3)--A proposal to introduce cinchona by Takeaki Enomoto].

We attempted to determine how one of the most valuable medicinal plants, cinchona, was brought to and cultivated in Japan. In the course of the study, we report that cinchona seedlings were brought to Japan for the first time in 1876, as per Takeaki Enomoto's proposal to the Meiji government. We also examine the details of his proposal in effort to clarify that the written proposal was submitted between February and March, 1874, and considered his motives leading to the submission. Cinchona is a very important medicinal plant that has saved human lives. However, there was no proof that Takeaki Enomoto's proposal made it introduction to Japan possible, nor was there any evidence that its cultivation was attempted. In addition, it was not clear that the details of the above has been found in document including the Nomutenmatsu (agricultural report published by the Meiji Government). It is significant that the details has been clarified by our series of studies not only from the standpoint of the history of pharmacy, but also from the accounts of the great predecessors involved, such as Takeaki Emonoto and Yasusade Tashiro.

[Historical research of cinchona cultivation in Japan (1): first trial cultivation in Japan in the early Meiji period recorded in Nomutenmatsu].

Cinchona is known as a magic bullet for malaria and its cultivation was dominated by Java on a global scale in the 19th century. In 1875, in accordance with a suggestion by Takeaki Enomoto, the Meiji government made a request to the Dutch government that cinchona seedlings be distributed to Japan. In response to that request, in 1876, 42 cinchona seedlings arrived in Yokohama from Java. It was the first time cinchona seedlings were shipped to Japan. After that, cinchona seeds and seedlings were shipped to Japan a total of three times between 1876 and 1883. The seeds shipped in 1878 were raised at the Nishigahara Agricultural Experiment Station and then planted at nine places in both Okinawa and Kagoshima Prefectures in 1882. The planter was Yasusada Tashiro. However, all of the planted seedlings had died by 1884. The first national farming plan of cinchona in Japan ended in failure. These matters were found in documents included in Nomutenmatsu compiled by the Ministry of Agriculture and Commerce of the Meiji government in 1888.

[Historical research of cinchona cultivation in Japan (Part 2). Useful tropical plants introduced from Java and India in the early Meiji era].

In the early Meiji era, Takeaki Enomoto made a proposal to the government that cinchona and coffee seedlings be introduced to Japan. In response, the Meiji government dispatched Masatsugu Takeda of the Ministry of Internal Affairs to Java and India from March to August 1878 for the purpose of investigating useful plants of tropical origin and introducing them to Japan. This paper clarifies the route to those destinations and the plants obtained locally. Using the seeds obtained from India during his travels, the cultivation of cinchona was attempted in 1882 for the first time in Japan. In Ogasawara, coffee cultivation was conducted, again for the first time in Japan, using coffee seeds brought back from Java. The cultivation of coffee was successful and served as the foundation of the Ogasawara coffee that exists to this day. Takeda also introduced a number of books and materials related to useful tropical plants available as a result of his travels, which contributed to the promotion of new industries and businesses in the Meiji era.

Anti-inflammatory constituents of Sappan Lignum.

We performed an in vitro assay for seven compounds from methanolic extract of Sappan Lignum (CSE) that inhibit the chemical mediators of inflammation using the J774.1 cell line: brazilin (1), sappanchalcone (2), protosappanin A (3), protosappanin B (4), protosappanin C (5), protosappanin D (6), and protosappanin E (7). Those compounds were evaluated for their inhibitory effects on nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production and their suppressive effects on tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) mRNA expression. As a result, we clarified that 1 inhibited NO production, and almost no inhibition in PGE(2). On the contrary, 2, 6, and 7 inhibited both NO and PGE(2) production and suppressed TNF-alpha, IL-6, COX-2, and iNOS mRNA expression. An examination of carrageenin-induced mouse paw edema suggested that the CSE contained active compounds other than 1, the main constituent in CSE. It was thus revealed that several compounds and mechanisms contributed to the anti-inflammatory effect of CSE.

Rapid and sensitive detection of Lophophora williamsii by loop-mediated isothermal amplification.

We have developed a convenient method for the detection of Lophophora williamsii using loop-mediated isothermal amplification (LAMP). We designed six species-specific primers for L. williamsii, including two loop primers. This L. williamsii-specific primer set was used for LAMP of total DNA extracted from L. williamsii and from L. diffusa. Real-time monitoring of LAMP was achieved by measuring turbidity due to the formation of magnesium pyrophosphate. Amplification occurred in samples mixed with total DNA from L. williamsii, but not in those mixed with total DNA from L. diffusa. We could also visually observe L. williamsii by adding fluorescent detection reagent to the reaction tube and exposing it to UV light. This new method amplified L. williamsii selectively and is expected to be applicable to the detection of peyote.

Rapid detection of Panax ginseng by loop-mediated isothermal amplification and its application to authentication of Ginseng.

We have developed a novel method called loop-mediated isothermal amplification (LAMP) to detect Panax ginseng, the botanical source of Ginseng (Ginseng Radix), and to distinguish P. ginseng from Panax japonicus. Six allele-specific primers (two outer primers, two inner primers, and two loop primers) were designed based on the 18S ribosomal RNA gene sequence of P. ginseng, and LAMP was performed using those primers and total DNA extracted from P. ginseng as template. Amplifications were observed from approximately 30 min onwards at DNA concentrations of 0.5 to 10.0 ng. The presence of loop primers shortened the reaction time considerably. In contrast, in the reactions using total DNA from P. japonicus as template, no amplifications were observed. LAMP also enabled us to distinguish Ginseng from Japanese Ginseng (Panacis Japonici Rhizoma). LAMP was proven to be a rapid, highly sensitive, and specific method for the detection of P. ginseng and Ginseng.

Rapid identification of Curcuma longa and C. aromatica by LAMP.

We have developed a novel method for the identification of Curcuma longa and C. aromatica called "loop-mediated isothermal amplification (LAMP)," based on trnK gene sequences. LAMP employs four primers that recognize six regions on the target DNA. Cycling elongation was initiated when the four primers were annealed to the target DNA. Amplifications were detected by measuring turbidity due to the formation of magnesium pyrophosphate. We designed allele-specific primer sets for C. longa and C. aromatica, respectively. LAMP using a primer set for C. longa and total DNA extracted from C. longa rhizome (0.5-10.0 ng) as template was detected up to 70 min. On the other hand, in the reaction using a primer set for C. longa and total DNA from C. aromatica as template, no amplifications were detected. The same tendency could be seen in the reactions using a set of primers for C. aromatica. LAMP enabled not only identification but also detection with high specificity. This rapid, specific, sensitive, and convenient method is expected to be applicable to the identification of the botanical origin of commercially available herbal products.

Vasorelaxant effects of Acer nikoense extract and isolated coumarinolignans on rat aortic rings.

The organic extract of the heartwood of Acer nikoense Maxim. (Aceraceae) showed vasorelaxant activity on rat aorta with or without endothelium. Coumarin [scopoletin (1)] and coumarinolignans [cleomiscosin A (2) and aquillochin (3)] were isolated as major constituents from the organic extract of the heartwood of A. nikoense. Compounds 1-3 exhibited moderate vasorelaxant effects on rat aorta, while 2 and 3 showed vasorelaxant effects in the norepinephrine-stimulated and also in high K+-depolarized preparations.

In vitro study for inhibition of NO production about constituents of Sappan Lignum.

In the course of our screening, we found that the methanolic extract of Sappan Lignum showed strong activity against lipopolysaccharide (LPS)-induced nitric oxide (NO) production by macrophages in vitro. As it was reported that Brazilin inhibited inducible NO gene, we conducted to similar tests for six known compounds isolated from Sappan Lignum, namely, brazilein, sappanchalcone, protosappanin A, protosappanin B, protosappanin C besides brazilin. And six compounds were also subjected to six tests to speculate their properties: (1) inhibition of NO production by cultured J774.1 (macrophage-like) cell line, (2) suppression of inducible NO synthase (iNOS) gene expression, (3) inhibition of NO production by murine peritoneal macrophages, (4) DPPH radical scavenging activity, (5) reduction of ferric ion and (6) antioxidant activity. Brazilein and sappanchalcone showed significant inhibition of lipopolysaccharide (LPS)-induced NO production by J774.1 cell line like Brazilin; 100% inhibition at 30 microM in test (1) and at 10 microM in test (3). The mechanisms underlying the inhibition of NO production by the compounds were investigated in test (2). As a result, brazilin was found to almost completely suppress iNOS gene expression at 100 microM as reported, and brazilein and sappanchalcone also suppressed iNOS gene expression. But strong activities were not observed for protosappanins A, B and C. So, we conducted tests (4), (5) and (6) to investigate other properties about six compounds. Protosappanin A and Brazilin demonstrated high antioxidant activity compared with Vitamin E in tests (4) and (5). Protosappanin A and B inhibited the oxidation of linoleic acid in test (6). Among the dibenzoxocin derivatives, only protosappanin C did not show significant activity in all the tests. We found that sappanchalcone showed same activity as brazilin, and six compounds isolated from Sappan Lignum showed various properties.

Genetic profiling of Sasa species by analysis of chloroplast intron between rbcL and ORF106 and partial ORF106 regions.

Kuma-zasa is Japanese folk medicine derived from plants of genus Sasa, family Bambusaceae. Although the plants of origin of Kuma-zasa were reported to be Sasa palmata, S. senanensis, S. yahikoensis, and S. kurilensis, authentication of those plants was difficult because of similarity in morphology. Several methods for the classification of genus Sasa are available, but none involve a genetic approach. Here, we performed the genetic profiling of genus Sasa, including the four species used medicinally. Thirteen sequences were observed in chloroplast DNA intron between rbcL and ORF106 and partial ORF106 regions of 34 specimens of 16 Sasa species and one specimen of Phyllostachys pubescens. We observed differences in alignment in this region among the specimens. The analyzed lengths varied from 759 to 821 bp depending on the specimen. There were nine base substitutions, eight successive thymines or adenines, and one to three repeat units of 31 bp. Moreover, we could not find species-specific alignment: different alignments were observed in specimens of the same species, while the same alignment was observed in specimens of different species. In the phylogenetic tree reconstructed by maximum parsimony analysis, medicinally used species did not form a cluster, although most of them were positioned close to each other. The genetic profiling of Sasa species would be of use in determining the botanical origin of the herbal medicine derived from the leaves of Sasa plants.

Chemopreventive potential of cyclic diarylheptanoids.

Eleven cyclic diarylheptanoids and seven related compounds were screened as potential antitumor promoters by using the in vitro short-term 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced Epstein-Barr virus early antigen (EBV-EA) activation assay. In addition, the cyclic diarylheptanoid myricanone (2) was examined for antitumor initiating activity in a two-stage carcinogenesis assay of mouse skin tumors induced by peroxynitrite as an initiator and TPA as a promoter. Myricanone (2) exhibited significant antitumor-initiating effect on mouse skin. These data suggest that cyclic, as well as linear, diarylheptanoids might be valuable chemopreventors.