Phylogenic Analysis and Evaluation of Ephedra Plants and Aconites for Medicinal Use.

Although Kampo medicine is now fully integrated into the modern Japanese healthcare system, most Kampo formulations depend on imported crude drugs from limited foreign areas. To prepare for possible shortages of crude drugs in the future, a wider scope for the supply of medicinal plants is necessary. We conducted field research and collaborated with international laboratories for phylogenic analysis and evaluation of medicinal plant resources. Our research on ephedra plants from a wide region of Eurasia has, for example, confirmed their phylogenic structure: based on DNA sequencing analysis of nuclear ribosomal internal transcribed spacer region 1 (ITS1) as well as the chloroplast intergenic spacer between trnL and trnF (trnL-F), the 8 major Chinese species and related plants grown on the continent could be divided into 3 groups. Additionally, Ephredra sinica was found to be synonymous with Ephredra dahurica and was reduced to a subspecies of Ephredra distachya. Furthermore, Ephredra likiangensis and Ephredra gerardiana, which are grouped in separate phylogenic trees, would be good candidates for medicinal material. Aconites from Hokkaido, as an example of domestic plants reviewed, were collected for phylogenic and aconitine alkaloid content analysis. The phylogenic analysis of nr ITSs revealed that the majority of specimens were genetically similar. However, the aconitine alkaloid content of the tuberous roots demonstrated that specimens from different habitats had varying alkaloid profiles. Environmental pressure of each habitat is presumed to have caused the morphology and aconitine alkaloid profiles of these genetically similar specimens to diversify.

Synthesis of a new glycosphingolipid, neurosporaside, from Neurospora crassa.

The glycosphingolipid neurosporaside (α-D-Glcp-(1 → 2)-ß-D-Galp-(1 → 6)-ß-D-Galp-(1 → 6)-ß-D-Galp-(1 →)-Cer) occurs in Neurospora crassa. We attempted to synthesize neurosporaside by block synthesis (route A) and linear synthesis (route B). Oligosaccharide derivatives were synthesized using trimethylsilyltrifluoromethanesulfonate and N-iodosuccinimide/trifluoromethane sulfonic acid as promoters. The target tetrasaccharide could not be attained via route A, but route B showed potential: glycosidic bonds (ß-D-Galp-(1 → 6)-ß-D-Galp-(1 → 6)-ß-D-Galp) were formed stereoselectively, leading to the synthesis of glycosphingolipid 2.

Diversity in aconitine alkaloid profile of Aconitum plants in Hokkaido contrasts with their genetic similarity.

Aconite tuber is a representative crude drug for warming the body internally in Japanese Kampo medicine and Chinese traditional medicine. The crude drug is used in major prescriptions for the aged. Varieties of Aconitum plants are distributed throughout the Japanese Islands, especially Hokkaido. With the aim of identifying the medicinal potential of Aconitum plants from Hokkaido, 107 specimens were collected from 36 sites in the summer of 2011 and 2012. Their nuclear DNA region, internal transcribed spacer (ITS), and aconitine alkaloid contents were analyzed. Phylogenic analysis of ITS by maximum parsimony analysis showed that the majority of the specimens were grouped into one cluster (cluster I), separated from the other cluster (cluster II) consisting of alpine specimens. The aconitine alkaloid content of the tuberous roots of 76 specimens showed 2 aspects-specimens from the same collection site showed similar aconitine alkaloid profiles, and cluster I specimens from different habitats showed various alkaloid profiles. Environmental pressure of each habitat is presumed to have caused the morphology and aconitine alkaloid profile of these genetically similar specimens to diversify.

The development of new molecular tools containing a chemically synthesized carbohydrate ligand for the elucidation of carbohydrate roles via photoaffinity labeling: carbohydrate-protein interactions are affected by the structures of the glycosidic bonds and the reducing-end sugar.

Photoaffinity labeling technology is a highly efficient method for cloning carbohydrate-binding proteins. When the carbohydrate probes are synthesized according to conventional methods, however, the reducing terminus of the sugar is opened to provide an acyclic structure. Our continued efforts to solve this problem led to the development of new molecular tools with an oligosaccharide structure that contains a phenyldiazirine group for the elucidation of carbohydrate-protein interactions. We investigated whether carbohydrate-lectin interactions are affected by differences in the glycosidic formation and synthesized three types of molecular tools containing Galp-GlcpNAc disaccharide ligands and a photoreactive group (1, 2, 3). Photoaffinity labeling validated the recognition of the new ligand by different glycosidic bonds. Photoaffinity labeling also demonstrated that both the reducing end sugar and non-reducing end sugar recognized the Erythrina cristagalli agglutinin.

Phylogenetic examination of crude drugs derived from Yunnanese Swertia plants.

Aiming to examine whether the genetic background of the crude drugs derived from four Yunnanese Swertia plants and their chemical constituent profiles correlate, we analyzed the nucleotide sequences of their nuclear ribosomal DNA regions including ITS1, 5.8S ribosomal RNA gene, and ITS2, together with those of Japanese S. japonica and S. pseudochinensis from Hebei Province. The result that two of the Yunnanese Swertia plants, S. binchuanensis and S. punicea, were genetically similar may explain their similarity in chemical constituent profiles. On the other hand, in spite of differences in chemical profile, S. decora and S. pseudochinensis were genetically close. The other Yunnanese Swertia plants, S. delavayi, and S. japonica, stood at intermediate positions between these two genetically similar pairs. The result suggests that although genetic background would have an influence, environmental factors, e.g., soil and weather conditions, might be critical for their production of secondary metabolites.

Difference in cultivation characteristics and genetic polymorphism between Chinese and Japanese strains of Wolfiporia cocos Ryvarden et Gilbertson (Poria cocos Wolf).

Poria, a dried sclerotium of Wolfiporia cocos Ryvarden et Gilbertson (Polyporaceae) has been used as a crude drug in both Chinese and Japanese (Kampo) traditional medicines. Recently, cultivated products of Chinese Poria strains have accounted for most of the market, while the cultivation of Japanese Poria strains has not been successful. Aiming to determine the relationship between the differences in cultivation characteristics and genetic polymorphism, we conducted a field cultivation experiment, a rot test, and rapid amplification of polymorphic DNA (RAPD) analysis of Poria strains collected from China and Japan: 3 Chinese and 7 Japanese strains. In field cultivation, although there was no marked inferiority of Japanese strains to Chinese ones in either mycelium propagation or the rate of sclerotium formation, Chinese strains formed whiter sclerotia with a mean size more than twice that of Japanese ones. Representatives of Chinese and Japanese strains, Yunnan and Kaimondake, respectively, were tested for wood-rotting ability. More wood was utilized and the wood color was darker in trials of the Yunnan strain. Amplifications of total DNA of these 10 fungal strains with 2 primers, PC-6 and PC-11, in RAPD analysis showed a difference in the amplicon profile between Japanese and Chinese strains, suggesting differences in their genetic background.

The effect of structural differences in the reducing terminus of sugars on the binding affinity of carbohydrates and proteins analyzed using photoaffinity labeling.

Because carbohydrates and proteins bind with such low affinity, the nature of their interactions is not clear. Photoaffinity labeling with diazirin groups is useful for elucidating the roles of carbohydrates in these binding processes. However, when carbohydrate probes are synthesized according to this conventional method, the reducing terminus of the sugar is opened to provide an acyclic structure. Because greater elucidation of carbohydrate-protein interactions requires a closed-ring carbohydrate in addition to the photoreactive group, we synthesized new molecular tools. The carbohydrate ligands were synthesized in three steps (glycosylation with allyl alcohol, deprotection, and ozonolysis). Specific binding proteins for carbohydrate ligands were obtained by photoaffinity labeling. Closed ring-type carbohydrate ligands, in which the reducing sugar is closed, bound to lectins more strongly than open ring-type sugars. Carbohydrate to protein binding was observed using AFM.

The relationship between growth of the aerial part and alkaloid content variation in cultivated Aconitum carmichaeli Debeaux.

Processed root of aconite, Aconitum carmichaeli Debeaux--known as bushi in Japan--is indispensable for treating diseases among elderly persons in Japanese and Chinese traditional medicine. Its active component is bushi diester alkaloid (BDA), which consists of aconitine (ACO), mesaconitine (MES), hypaconitine (HYP), and jesaconitine (JES). Since an overdose of BDA results in severe side effects, the BDA content should be within safe limits. However, the BDA content of raw aconite root, even that produced by standard cultivation procedures, varies greatly. In this study, to clarify the cause of BDA variation, we examined the weight and BDA content of each part of cultivated A. carmichaeli: the aerial part, the mother tuberous root (MT), the daughter tuberous root (DT), and the rootlet (RL). We found the following positive relationships: between aerial part weight and DT weight, aerial part weight and BDA content in stem of apex, and BDA content in stem of apex and total BDA of DT attached to the plant. Furthermore, DT belonging to a higher weight group showed less BDA content variation. In addition, BDA of DT and those of MT and RL differ in both content and composition. In conclusion, it was suggested that the weight or the size of the aerial part was a good marker for monitoring BDA content and its variation in the tuberous root, and it was found to be desirable to prevent mixing MT and RL at harvest.

Studies of Ephedra plants in Asia. Part 6: Geographical changes of anatomical features and alkaloids content of Ephedra sinica.

Ephedra sinica Stapf is the main botanical origin of the Chinese herbal drug Mahuang, Ephedra Herb. Eighty-five samples of E. sinica, collected across eastern China, Mongolia, and Buryatia (Russia), were studied anatomically and chemically to elucidate the local variations and the relation between environmental factors and the variations. The results showed that samples grown in more arid conditions tended to have a more sinuous epidermis, more cuticular tubers, and more subepidermal fiber bundles anatomically, and contained more total ephedrine alkaloids. These samples also had a high pseudoephedrine content. These results imply that Ephedra Herb with good quality should be collected from arid fields, and the chemical quality can be estimated by observing the anatomical characteristics.

[Herbological studies on combination of rhubarb and mirabilite].

In traditional Chinese medicine, it has long been thought that the medicinal effect of a crude drug can be modified by combination with other crude drugs. One well-known example is the combination of mirabilite (a purgative) and rhubarb (an anti-inflammatory and essentially anti-blood stasis drug). One description in the medicinal literature states that mirabilite has to be added after rhubarb has been decocted. Another description states that rhubarb needs to be processed with liquor when both crude drugs are used together. However, the reason why rhubarb and mirabilite are used together, why mirabilite is added afterward, and why rhubarb needs to be processed with liquor have not been elucidated completely. Therefore, we performed a herbological study and found that rhubarb is expected to act as a purgative while mirabilite is expected to act as a stool softener when they are used together. We also found that they are used together to speed up the onset of a purgative effect in each other. Secondly, we decocted rhubarb (unprocessed or liquor-processed) and mirabilite together, and analyzed the content of principal compounds. We found that sennoside and anthraquinone contents of the rhubarb decoction were reduced by adding mirabilite. However, when mirabilite was added after rhubarb had been decocted, the decrease was smaller than when they were put in water at the same time. In addition, the decoction of liquor-processed rhubarb showed low sennoside content. Therefore we conclude that mirabilite is added after rhubarb has been decocted to prevent the decrease of active compounds, and we consider that unprocessed rhubarb is suitable for expecting a purgative effect.

Ephedra resource in Sichuan and Yunnan Provinces 2007.

Wild Ephedra plants growing near the Tibetan border of Yunnan and Sichuan Provinces and north-central Sichuan were surveyed and their DNA and ephedrine alkaloids content were analyzed. By analysis of internal transcribed spacer 1 (ITS) 1 DNA, E. likiangensis was found to be the dominant species in these regions, which clustered into 2 major groups in the phylogenic tree. Most Ephedra plants in these regions of ordinal size contained ephedrine and pseudoephedrine of more than 0.7%, the requirement for Japanese Pharmacopoeia 15th edition, suggesting that they have potential for crude drug production of Ephedra herbs.

Molecular characterization of akebia plants and the derived traditional herbal medicine.

The stems of Akebia plants, Akebiae Caulis, have long been used in traditional Chinese and Japanese medicines, and are mainly produced in western Japan. Three Akebia plants, Akebia quinata (AQ), A. trifoliata (AT), and A. pentaphylla (AP) grow wild in Japan. With the aim of carrying out molecular biological identification of Akebia plant species and discriminating Akebiae Caulis from other related crude drugs originating from non-Akebia plants, sequencing analysis of Akebia plants collected from various parts of Japan and the southern Korean Peninsula was performed. Specimens identified morphologically as AQ and AT had their respective common internal transcribed spacer one (ITS1) sequences, which could be distinguished. Cloning experiments of AP specimens showed that their ITS1 contained both common sequences of AQ and AT as well as their chimera. These chimeric sequences were not identical between AP specimens, suggesting that AP is not a species with uniform DNA sequences but a group of individuals with hybrid genomes of AQ and AT. Based on the sequences of Akebia species found here, we propose polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) methods to discriminate Akebiae Caulis from the related crude drugs and to distinguish three Akebia plants. Comparison of triterpene-rich fractions of extracts from Akebia plants by TLC showed that AP had an intermediate profile of AQ and AT.

Difference of ITS sequences of Akebia plants growing in various parts of Japan.

The stem of the Akebia plant, "Mokutsu", is a crude diuretic and antiphlogistic drug. Japanese products prepared from wild Akebia plants cover most of the Mokutsu market. Two Akebia plants, Akebia quinata Decaisne (Aq) and A. trifoliata Koidzumi (At) of Lardizabalaceae, are standardized as Mokutsu in Japanese pharmacopoeia. These two Akebia plants along with A. x pentaphylla Makino (Ap), which is considered a hybrid with the morphology of Aq and At, can be distinguished by DNA sequence analysis of internal transcribed spacers 1 and 2 (ITS) of nuclear ribosome DNA. Here, we report the results of molecular genetic analysis of Akebia plants grown in various wild habitats in Japan. We found that each of three Akebia plants could be distinguished in terms of their locality according to their nucleotide sequence in ITS, specifically at positions 91, 128, 133, 134, and 221. Plants with a comparable habitat had similar nucleotide sequences at these five points. We also found Aq with ITS and nucleotide deletion at position 86 that was distributed only around Awajishima in Shikoku (A), Harimanada (B), and Kinki (C), including the chief production center of Akebia Caulis. The results of these ITS sequences enabled discrimination of plants originating from Akebia Caulis.

Biotransformation of C-glucosylisoflavone puerarin to estrogenic (3S)-equol in co-culture of two human intestinal bacteria.

Puerarin and daidzein are the major naturally occurring isoflavones in leguminous plants. These two compounds are metabolized to equol by human intestinal flora. Here we isolated two intestinal bacteria capable of metabolizing puerarin and daidzein, respectively, from human feces. One of them, strain PUE, converted puerarin to daidzein by cleaving a C-glucosyl bond, whereas the other, strain DZE, converted daidzein to equol by reducing a double bond in ring C followed by elimination of an oxo group. Based on the 16S ribosomal RNA gene sequence, strain DZE showed 85% similarity with Eggerthella lenta. Equol produced by strain DZE was identified as (3S)-equol through several analytical methods. Moreover, we obtained (3S)-equol from puerarin by co-incubation with strain PUE and DZE. In addition, 5-hydroxyequol was obtained from genistein by incubation with strain DZE.

Qualitative and quantitative analysis of Swertia herbs by high performance liquid chromatography-diode array detector-mass spectrometry (HPLC-DAD-MS).

We evaluated the composition of Swertia herbs using high performance liquid chromatography-diode array detector-mass spectrometry (HPLC-DAD-MS). Eleven peaks of 6 species were unequivocally identified by comparing their retention times, UV spectra, on-line electrospray ionization mass (ESI-MS) spectra, and collision-induced dissociation mass spectrometry/mass spectrometry (CID-MS/MS) data with those of authentic compounds. We adopted wavelengths of 254 nm, 340 nm and 230 nm to simultaneously determine these 11 compounds. By comparing the overall DAD and total ion current (TIC) profiles of various samples, the 6 species were differentiated in terms of the occurrence and/or relative concentrations of the eleven compounds. Our novel validated HPLC-DAD-MS method not only facilitates quality control and identification of Swertia herbs, but is also applicable to systematic investigations of the distribution of secoiridoids, flavonoids, and xanthones in the genus Swertia.

Enantioselective dehydroxylation of enterodiol and enterolactone precursors by human intestinal bacteria.

During the course of experiments on the transformation of lignans to phytoestrogenic substances, such as enterodiol (END) and enterolactone (ENL), a previously isolated bacterium, Eubacterium (E.) sp. strain SDG-2, capable of phenolic p-dehydroxylation in the biotransformation of secoisolariciresinol diglucoside to END and ENL, was concluded to be Eggerthella (Eg.) lenta (Eg. sp. SDG-2) on the basis of 16S rRNA gene sequence analysis. The bacterium could transform (+)-dihydroxyenterodiol (DHEND, 3a) to (+)-END (1a), but not for (-)-DHEND (3b) to (-)-END (1b) under anaerobic conditions. By incubation of a mixture of (+)- and (-)-dihydroxyenterolactone (DHENL, 4a and 4b) with Eg. sp. SDG-2, only (-)-DHENL (4b) was converted to (-)-ENL (2b), selectively. On the other hand, we isolated a different bacterium, strain ARC-1, capable of dehydroxylating (-)-DHEND (3b) to (-)-END (1b) from human feces. Strain ARC-1 could transform not only (-)-DHEND (3b) to (-)-END (1b), but also (+)-DHENL (4a) to (+)-ENL (2b). However, the bacterium could not transform (+)-DHEND (3a) and (-)-DHENL (4b). Both bacterial strains demonstrated different enantioselective dehydroxylation.

Enantioselective oxidation of enterodiol to enterolactone by human intestinal bacteria.

In the course of our experiments on the metabolic conversion of lignans to the estrogenic substances enterodiol (END) and enterolactone (ENL) by human intestinal flora, we isolated two anaerobes, Ruminococcus sp. END-1 and strain END-2, capable of oxidizing END. The former selectively converted (-)-END to (-)-ENL, while the latter selectively converted (+)-END to (+)-ENL, indicating enantioselective oxidation by intestinal bacteria.

DNA sequencing analysis of ITS and 28S rRNA of Poria cocos.

We determined the DNA sequences of the internal transcribed spacer 1 and 2 (ITS 1 and 2), the 5.8S rRNA gene and most of the 28S rRNA gene of Poria cocos for the first time, and conducted analysis of 20 samples including cultured mycelias and crude drug materials obtained from various localities and markets. Direct sequencing of the ITS 1 and 2 regions of the samples, except for four wild samples, showed that they had identical DNA sequences for ITS 1 and 2 with nucleotide lengths of 997 bps and 460 bps, respectively. By cloning, the four wild samples were found to have combined sequences of common ITS sequences with 1 or 2-base-pair insertions. Altogether both ITS 1 and 2 sequences were substantially longer than those of other fungal crude drugs such as Ganoderma lucidum and Polyporus umbellatus. Thus, Poria cocos could be distinguished from these crude drugs and fakes by comparing the nucleotide length of PCR products of ITS 1 and 2. Contrary to the basic homogeneity in ITS 1 and 2, three types (Group 1, 2, 3) of the 28S rRNA gene with distinctive differences in length and sequence were found. Furthermore, Group 1 could be divided into three subgroups depending on differences at nucleotide position 690. Products with different types of 28S rRNA gene were found in crude drugs from Yunnan and Anhui Provinces as well as the Korean Peninsula, suggesting that the locality of the crude drugs does not guarantee genetic uniformity. The result of DNA typing of Poria cocos may help discrimination of the quality of the crude drug by genotype.

Isolation and characterization of a human intestinal bacterium, Eubacterium sp. ARC-2, capable of demethylating arctigenin, in the essential metabolic process to enterolactone.

Plant lignans, such as pinoresinol diglucoside, secoisolariciresinol diglucoside and arctiin, are metabolized to mammalian lignans, enterolactone or enterodiol, by human intestinal bacteria. Their metabolic processes include deglucosylation, ring cleavage, demethylation, dehydroxylation and oxidation. Here we isolated an intestinal bacterium capable of demethylating arctigenin, an aglycone of arctiin, to 2,3-bis(3,4-dihydroxybenzyl)butyrolactone (1) from human feces, and identified as an Eubacterium species (E. sp. ARC-2), which is similar to Eubacterium limosum on the basis of morphological and biochemical properties and 16S rRNA gene sequencing. By incubating with E. sp. ARC-2, arctigenin was converted to 1 through stepwise demethylation. Demethylation of arctigenin by E. sp. ARC-2 was tetrahydrofolate- and ATP-dependent, indicating that the reaction was catalyzed by methyltransferase. Moreover, E. sp. ARC-2 transformed secoisolariciresinol to 2,3-bis(3,4-dihydroxybenzyl)-1,4-butanediol by demethylation.

Studies on cultivated ephedra plants in inner mongolia autonomous region and ningxia hui autonomous region.

Progression of the desertification in northern China has been causing damage to wild Ephedra plants on which we depend for most of supply of the traditional herbal medicine, "Ma huang." The Chinese government encourages the cultivation of Ephedra plants, and Ephedra fields have been reclaimed in the original Ephedra habitats in recent years. We surveyed 7 Ephedra fields that have been recently developed in the Inner Mongolia Autonomous Region and Ningxia Hui Autonomous Region to collect information on Ephedra plant cultivation, especially pertaining to crop species. Specimens taken from those Ephedra fields were genetically and morphologically analyzed, and their ephedrine alkaloid content was examined. DNA analyses of Ephedra specimens, including DNA sequencing of ITS (internal transcribing sequence of nuclear ribosomal DNA) and trn L/F (intron of trnL and intergenic spacer between the trnL and trnF of chloroplast DNA) region and species-specific amplification of trn L/F were conducted to identify Ephedra species. Based on the results of DNA sequencing and morphological determination, the crops grown in 6 fields ware identified as Ephedra sinica, while co-planting of E. sinica and E. intermedia was found in one field where a higher appearance rate of plants with varied morphology from wild Ephedra plants was observed. Furthermore, direct sequencing of the PCR product of the trn L/F region of some specimens from the field and their species-specific PCR showed ambivalent result. Cloning and sequencing of the PCR product of the trn L/F region of those specimens DNA suggested their heteroplasmy, containing both E. sinica- and E. intermedia-type chloroplasts. On the other hand, the profile of the ephedrine alkaloid content was clearly correlated with the result of direct sequencing of the trn L/F region; the specimens showing the E. sinica-type sequence contained more ephedrine than pseudoephedrine, and the specimens of the E. intermedia-type more pseudoephedrine.