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In this account, pectin components were obtained from akebia trifoliate koiaz peels with the acid extraction technology. The chemical constituent of the akebia trifoliate koiaz peels extract (ATKPE) was analyzed and its anti-corrosion performance for mild steel in HCl medium was investigated by electrochemical methods, surface analysis and theoretical calculations. Experimental results show that ATKPE exhibits excellent corrosion inhibition characteristic, and its inhibition efficiency can be as high as 90% when the concentration of ATKPE is 800 mg/L. Electrochemical measurements show that ATKPE is a mixed-type corrosion inhibitor consisting with Langmuir adsorption model. In addition, SEM, AFM and contact angle observations suggests that the ATKPE may attach firmly to the metal surface by forming a barrier film. XPS and ATR-FTIR results further verify the bonding interaction between the functional groups and steel substrate. Meanwhile, quantum chemical calculations and molecular dynamics (MD) simulations were performed to reveal the inhibition mechanism at molecular/atomic level. In brief, our findings demonstrate a novel environmentally benign, efficient corrosion inhibitor for steel protection in acidic media.

Akebia trifoliata (Thunb.) Koidz. is a perennial woody liana that belongs to Akebia Decne. Being high-value medicinal and economic crops, different parts of the plants are utilized for producing medicines, fruit, tea, wine, edible oil, etc. A. trifoliata is mainly wild, slow growth and low reproduction. However, the fragmented habitats caused by increasingly human activities led to a sharp decline yield of wild resources. Therefore, the domestication and cultivation of this plant could be the solution. Study on the genetic diversity among individuals or populations are the basis of making its conservation strategies and germplasm selection. In this study, 242 A. trifoliata accessions of 12 populations, collected from Qinba mountain area of China, were assessed using ISSR and SRAP markers, which generated 99.4% and 98.7% polymorphism, respectively. Both ISSR and SRAP revealed the high level of genetic diversity (H = 0.31, I = 0.47, ISSR; H = 0.33, I = 0.50, SRAP), and also revealed high genetic differentiation (Gst = 0.36 for ISSR, Gst = 0.34 for SRAP) and gene flow (Nm = 1.76 for ISSR, Nm = 1.44 for SRAP). Genetic variation observed within population was higher than of among population. The Mantel Test showed that the genetic variation among populations could be explained by the geographical distances (r2 = 0.1797, p = 0.020, ISSR marker; r2 = 0.2793, p = 0.010, SRAP marker;) The results of Structure analysis and PCoA also supported clustering model. Finally, based on ISSR and SRAP data, a core collection of 56 accessions by the LDSS method was selected. Collectively, our results lay the foundation for making A. trifoliata’s conservation strategies and germplasm selection.

Descriptions of the cambial variants of the lianoids in two families, Lardizabalaceae and Sabiaceae, were lacking in Taiwan. This study aimed to diagnose the stem characteristics of seven lianoid species from these two families to update existing knowledge. Specifically, the transverse sections of fresh stems were diagnosed to generate a key. The results showed that all seven species develop one of ten cambial variant types, viz. axial vascular elements in segments. Of these species, Sabia swinhoei Hemsley, Stauntonia obovata Hemsley, and S. obovatifoliola Hayata formed secondary rays. The thick and successive periderm was apparent in Stauntonia obovata and S. obovatifoliola at older stem. The sclerenchyma ring was continuous or discontinuous in two genera (Akebia and Stauntonia), but was absent in Sabia. Four diagnostic features could be used to distinguish Lardizabalaceae and Sabiaceae; namely, the periderm, cortical sclerenchyma, vessel arrangement, and cortex ducts. The cambial variations documented for these seven lianoid species in Taiwan update existing information, facilitating comparisons between Lardizabalaceae and Sabiaceae.

Akebia trifoliata is a fruit with rich nutritional properties, and its peel is produced as a by-product. In this research, we investigated the influences of microwave-assisted extraction parameters on antioxidant activity of the extract from Akebia trifoliata peels, and the ferric-reducing antioxidant power (FRAP) and Trolox equivalent antioxidant capacity (TEAC) as well as total phenolic contents (TPC) were used to optimize extraction parameters. The influences of ethanol concentration, microwave power and solvent-to-material ratio, as well as extraction temperature and time on TPC, FRAP and TEAC values, were assessed using single-factor tests. Three parameters with obvious effects on antioxidant capacity were selected to further investigate their interactions by response surface methodology. The optimal extraction parameters of natural antioxidants from Akebia trifoliata peels were ethanol concentration, 49.61% (v/v), solvent-to-material ratio, 32.59:1 mL/g, extraction time, 39.31 min, microwave power, 500 W, and extraction temperature, 50 °C. Under optimal conditions, the FRAP, TEAC and TPC values of Akebia trifoliata peel extracts were 351.86 ± 9.47 µM Fe(II)/g dry weight (DW), 191.12 ± 3.53 µM Trolox/g DW and 32.67 ± 0.90 mg gallic acid equivalent (GAE)/g DW, respectively. Furthermore, the main bioactive compounds (chlorogenic acid, rutin and ellagic acid) in the extract were determined by high-performance liquid chromatography. The results are useful for the full utilization of the by-product from Akebia trifoliate fruit.

Vadadustat (VAFSEO®) is a prolyl hydroxylase inhibitor being developed by Akebia Therapeutics, Inc. (Akebia) for the treatment of anaemia associated with chronic kidney disease (CKD). Akebia is collaborating with Mitsubishi Tanabe Pharma Corporation on the development and commercialization of vadadustat in Japan and with Otsuka Pharmaceutical Co. Ltd on the development and commercialization of vadadustat in the USA, the EU and certain other territories. The drug is approved in Japan for use in adult patients with anaemia associated with CKD and regulatory submissions are planned in the USA and the EU. This article summarizes the milestones in the development of vadadustat leading to this first approval.

Ethnopharmacological relevance 'Akebia stem' (Akebiae caulis) is one of the newest raw materials officially introduced into therapeutic practice from traditional Chinese medicine. A monograph on this material appeared for the first time in 2018 in Supplement 9.6 to the 9th edition of the European Pharmacopoeia. In the latest 10th edition of the European Pharmacopoeia, the monograph remained unchanged. The 'Akebia stem' monograph allows the use, as a raw material, of Akebia quinata (Houtt.) Decne., A. trifoliata (Thunb.) Koidz, or a mixture of the two species. Aim of the study The aim of this work is a detailed review of the scientific literature on the genus Akebia (family Lardizabalaceae), with particular emphasis on A. quinata and A. trifoliata, providing information on the botanical, ecological, and chemical characteristics of these species. Professional research on their biological activity has been reviewed. The attention is given to phytochemistry and cosmetology. The traditional use of Akebia species and their potential use in medicine and cosmetology are assessed. In addition, individual papers describing biotechnology research on in vitro cultures of the two Akebia species are presented. Materials and methods The presented botanical, ecological, phytochemical and biotechnological characterization is based on a thorough review of published scientific research. It is a compilation and evaluation of data on the chemical composition and biological activities of these Akebia species. Results This critical review of phytochemical studies demonstrates that triterpenoid saponins are dominant secondary metabolites from these species. A comparative analysis of phytochemical studies on A. quinata and A. trifoliata stems, roots, fruits, and seeds showed differences in metabolites based on the plant parts and species. The triterpenoid saponins mutongsaponin C and saponin Pj1 have been found only in A. trifoliata, whereas the phenolic glycoside 2-(3,4-dihydroxyphenyl)-ethyl-O-β-D-glucopyranoside has been found only in A. quinata. Biological activity studies of A. quinata stem, leaf and/or fruit extracts have confirmed diuretic, hepatoregenerative, neuroprotective, analgesic, anti-inflammatory, and anti-obesity effects and an influence on ethanol metabolism. Different action profiles have been demonstrated for A. trifoliata stem, leaf and/or fruit extracts, different profiles of action. Studies have proven the antibacterial and anticancer (liver and stomach) effects of these species. This review presents potential phytopharmacological applications of both species and detailed data on their broad applications in cosmetology. Attention is also drawn to information on the safety of using Akebia. Finally, an overview of biotechnology research on both species is presented. Conclusions This review provides comprehensive knowledge about the ethnopharmacological use of Akebia species. Moreover, new findings on the differences in the chemical composition and biological activity profiles are underlined.

The complete chloroplast genome of Akebia longeracemosa, a monoecious woody vine endemic to China, was determined. The total genome size is 158,020 bp, containing a large single copy region of 86,6...

Akebia species have been used for centuries in medicinal practices in a few Asian countries such as China and Japan. The dried stems of Akebia trifoliata are known as mutong in the Chinese pharmacopoeia (4) and mokutsu in Kampo, the traditional Chinese medicine developed in Japan (2). In China, the plant is grown in the provinces of Shandong, Hebei, Shanxi, Henan, Gansu, and some provinces in the south of the Yangtze River basin. During the summer of 2012, a leaf spot disease was detected on A. trifoliata grown in Nanning, Guangxi, China. The disease occurred and spread rapidly in July under conditions of high temperature and high humidity. The symptoms appeared on three sites that we inspected; disease incidences were higher than 80%. Initial symptoms consisted of small (less than 5 mm in diameter), circular, purple-brown leaf spots. Spots later enlarged and became elliptical, circular, or irregular with gray-white centers and dark brown rims. The centers were slightly concave. The spots could coalesce with each other, resulting in leaf desiccation and wilting. A fungal isolate was obtained from symptomatic leaf tissue that taken from a field (22°50′N, 108°22′E) in Nanning, Guangxi, China. Single-spore culture of the isolate was incubated on potato dextrose agar (PDA) for 7 days in the dark at 28°C. Conidiophores were straight to slightly curved, unbranched, and pale brown. Conidia (19.0 to 140.5 μm long and 7.0 to 11.0 μm wide) were formed singly or in chains, obclavate to cylindrical, straight or curved, pale brown, with a rounded apex and truncate base, and 1 to 13 pseudosepta. Morphological characteristics of the isolate were similar to the descriptions of Corynespora cassiicola (Berk. & M.A. Curtis) C.T. Wei (1). Genomic DNA of the isolate was extracted and used for PCR amplification of rDNA-ITS (internal transcribed spacer) sequence with primers ITS1 and ITS4. The PCR products were purified and sequenced. The sequence (GenBank Accession No. KC977496) was used in BLAST searches to interrogate GenBank for sequence similarity. High sequence similarity of 100% was obtained with several C. cassiicola strains. Pathogenicity of the isolate was investigated to demonstrate Koch's postulate. Young, healthy, fully expanded green leaves of A. trifoliata were surface sterilized. Fifteen leaves were inoculated with 10-μl drops of conidia suspension (105 conidia per ml) and 10 leaves were inoculated with the same volume of sterile water to serve as controls. All the leaves were placed in a humid chamber for 5 days. Spots with similar symptoms to those observed in the field developed on all inoculated leaves. The pathogen was reisolated and identified as C. cassiicola. The controls remained symptomless. According to previous reports, A. trifoliata was infected by Alternaria tenuissima in China and by Colletotrichum acutatum in Japan (3). To our knowledge, this is the first report of C. cassiicola found on Akebia species worldwide. Furthermore, this new disease primarily affects plantations and reduces the quality and yield of the medicine. Some effective measures should be taken to control this disease. References: (1) M. B. Ellis and P. Holliday. CMI Description of Pathogenic Fungi and Bacteria, 303, 1971. (2) F. Kitaoka et al. J. Nat. Med. 63:374, 2009. (3) Y. Kobayshi et al. J. Gen. Plant Pathol. 70:295, 2004. (4) L. Li et al. HortScience 45:4, 2010.

Akebia trifoliata (Thunberg) Koidzumi (three-leaf akebia), a climbing deciduous woody plant, grows wild in mountains of China and Japan. It has long been prized for its delicious sweet taste and medicinal value (Lu et al., 2019). Few pests and diseases reportedly affect this plant (Ye et al., 2013), but with more commercial planting of A. trifoliata in China, symptoms of anthracnose on leaves and fruits have increased. Between December 2018 and May 2019, typical anthracnose symptoms were first observed on A. trifoliata grown in Wuhan, China, with an incidence up to 15%. Diseased leaves exhibited irregular gray-brown spots with dark brown edges, and dark brown undersides, substantially affecting photosynthesis and growth. As disease progressed, white mycelium appeared on stems causing stem rot and fruit drop. Several round or needle-shaped dark brown spots formed on fruit peel, coalescing into irregular, slightly sunken blotches. Under high humidity, the whole fruit turned brown and the spots were covered by white mycelia, greatly affecting the fruits' ornamental quality. To isolate the pathogen, 5-mm2 pieces of symptomatic tissue from 10 infected leaves and fruits were surface-disinfected for 90 s in 1% sodium hypochlorite then 30 s in 75% ethanol, rinsed twice with sterile water, then incubated on potato dextrose agar (PDA, Oxoid) at 25°C under 12 h light/dark photoperiod. Pure cultures were obtained from hyphal tips of each colony. Initially, colonies produced white mycelia, turning gray after 5 days. The isolates produced abundant hyaline, single celled, straight and cylindrical conidia, with mean size 10.35 to 15.58 × 3.46 to 5.69 μm. Morphological characteristics were generally consistent with those of Colletotrichum gloeosporioides (Cannon et al. 2012). Genomic DNA of three isolates was extracted for PCR amplification of the internal transcribed spacer (ITS) region, and β-tubulin (TUB2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes (Weir et al. 2012). BLAST search identified all sequences (GenBank accession nos. MT451846 to MT451848 for ITS, MT573957 to MT573959 for TUB2, and MT573960 to MT573962 for GAPDH) as 100% matches to C. gloeosporioides (Penz.) Penz. & Sacc. CBS 112999 strain (JQ005152 for ITS, JQ005587 for TUB2, JQ005239 for GAPDH) (Damm et al. 2009). Identification was confirmed by maximum likelihood phylogenetic analysis using MEGA7 . To evaluate pathogenicity, isolates were inoculated onto one side of 10 wounded healthy leaves of 1-year-old pot-grown A. trifoliata plants and 10 nearly mature fruits, with 10 μl of conidial suspension (106/ml) and colonized PDA pieces (5 mm diam.) from 7-day-old cultures of the fungus in Petri dishes; control sides received 10 μl sterile distilled water and sterile agar pieces. The test was performed twice. After incubation at 25°C, 70% humidity under 12 h fluorescent illumination/12 h dark for 5 days, similar spots were observed on all inoculated leaves and fruits. Controls remained asymptomatic. The re-isolated pathogen was identified as C. gloeosporioides by biological characteristics and sequencing analysis, indicating that C. gloeosporioides was a causal agent of anthracnose of A. trifoliata. Anthracnose caused by C. acutatum has been reported on A. trifoliata in Japan (Kobayshi et al. 2004). To our knowledge, this is the first report of C. gloeosporioides found on Akebia species. The new disease primarily reduces the quality and yield of A. trifoliata. Effective measures should be taken to manage this disease. Funding: This study was supported by the National Natural Science Foundation of China (31701974; 31901980), Science and technology program funded by Wuhan Science and Technology Bureau (2018020401011307). References: Lu, W.L., et al. 2019. J. Ethnopharmacol. 234:204. Ye, Y.F., et al. 2013. Plant Dis. 97:1659. Kobayshi, Y., et al. 2004. J. Gen. Plant Pathol. 70:295. Cannon, P.F., et al. 2012. Stud. Mycol. 73:181. Weir, B.S., et al. 2012. Stud. Mycol. 73:115. Damm, U., et al. 2009. Fungal Divers. 39:45.

application/pdf, We investigated the leaf demography of a temperate woody liana, Akebia trifoliata, in a temperate forest in Japan, Akebia is semi-evergreen: some leaves are shed before winter, while others remain through the winter. Previous studies of semi-evergreen species found that variation in leaf life span was caused by variation in the timing of leaf emergence, Leaves that appeared just before winter over-wintered, while leaves appearing earlier were shed, However, it is unclear whether leaves of the same cohort (i.e., leaves that appear at the same time within a single site) show variation in life span under the effect of strong seasonality. To separate variation in life span among the leaves in each cohort from variation among cohorts, we propose a new method - the single leaf diagram, which shows the emergence and death of each leaf. Using single leaf diagrams, our study revealed that Akebia leaves within a cohort showed substantial variation in life span, with some over-wintering and some not. In addition, leaves on small ramets in the understory showed great variation in life span, while leaves on large ramets, which typically reach higher positions in the forest canopy, have shorter lives, As a result, small ramets were semi-evergreen, whereas large ramets were deciduous, The longer lives of leaves on small ramets can be interpreted as a shade-adaptive strategy in understory plants.

Based on the first-hand data of field investigation, existing literature, books and plant specimens database resources, the complete and available spatial database of wild Akebia trifoliate in China was established firstly. After analyzing the geographical distribution of Akebia trifoliate and its ecological factors, such as climate, soil and topographic characteristics, based on the existing literature, we selected 16 climatic indicators and used statistical analysis to study key climatic variables affecting its growth from a macro perspective. The results show that: (1) According to the characteristic roots and contribution rate obtained by principal component method, three principal factors are determined. Their corresponding value of three principal factors were 6.0538 and 37.84%, 4.7487 and 29.68%, 3.3822 and 21.14% respectively. All the characteristic roots indicate that the three principal factors are playing a dominant role in climate variables of influencing the distribution of Akebia trifoliate. (2) The first principal factor is called “heat factor”, the most important role determining geographical distribution of Akebia trifoliate, including the variables with larger factor load such as active accumulated temperature (≥10℃), warm index, biological temperature and annual average temperature. It shows that Akebia trifoliate tends to grow in areas with higher heat conditions. The second principal factor is called “humidity factor”, the second role for Akebia trifoliate, including the variables with larger factor load such as evapotranspiration rate, humidity index, annual precipitation, growing season precipitation and relative humidity. So it shows that Akebia trifoliate tends to be in areas with sufficient precipitation and high humidity. The third principal factor is called “illumination factor”, including the variables with larger load such as annual sunshine hours, growing season sunshine hours and extreme low temperature. So Akebia trifoliate likes the areas with relatively short sunshine hours. (3) Heat factor and humidity factor are the main limiting factors for its growth. The significant climatic variables offer a basis for the selection of ecological factors and the determination of weight in the suitability analysis of Akebia trifoliate. These results could provide a theoretical basis for the artificial cultivation of Akebia trifoliate. The ideas and methods for wild Akebia trifoliate in this paper could give a reference for the study of other valuable wild plants.

Akebia trifoliata belongs to the Lardizabalaceae family of flowering plants, has tremendous potential as a new fruit crop for further domestication and commercialization in China. However, the selection of A. trifoliata is extremely limited due to lack of information about basic inheritance of fruit traits. In this study, 11 key fruit characters, including single fruit weight (SFW), fruit length (FL), fruit width (FW), peel thickness (PT), peel weight (PW), soluble solids content (SSC), seed weight (SW), pulp weight (PuW), edible ratio (ER), hundred-grain weight (HGW) and seed number (SN) were evaluated, and genetic parameters were analyzed. The results showed that the 11 tested traits have high repeatability range from 0.87 to 0.95. Correlation analysis showed that SFW was positively correlated with FL, FW, PT, PW and PuW, but negatively correlated with ER. The Smith–Hazel-index based multitrait selection was used to select superior lines. The SH selection index identified superior lines with high SFW and ER, and low SN. Taken together, our results provide important information for domestication and breeding program of Akebia trifoliate.

Here, the potential use of herbal residues of Akebia as feedstock for ethanol production is evaluated. Additionally, five deep eutectic solvents from hemicellulose-derived acids were prepared to overcome biomass recalcitrance. Reaction temperatures had more significant influences on solid loss and chemical composition than the molar ratios of choline chloride (ChCl) to derived acids. Glycolic acid resulted in the maximum levels of lignin, xylan and glucan removal, which were 60.0%, 100% and 71.5%, respectively, at 120°C with a 1:6M ratio of ChCl-glycolic acid. In contrast, ChCl-formic acid resulted in the greatest level of glucan retention, at 97.8%, with a lignin removal rate of 40.7% under the same pretreatment conditions. Moreover, ChCl loading could significantly enhance the selectivity of carboxylic acid for lignin dissolution. A 98.0% level of subsequent enzymatic saccharification and a 100% ethanol yield were achieved after ChCl-formic acid pretreatments of Akebia' herbal residues.

Akebiae Caulis, ‘Mutōng’ in Chinese, refers to the stem of Akebia quinata in the Korean modern pharmacopoeia. The stems of Aristolochia manshuriensis and Clematis armandii have also been traditionally used for clinical purposes, and are often marketed as ‘Mutōng’ under the names ‘Guān-Mutōng and Chuān-Mutōng’, respectively. Furthermore, due to the morphological similarity of the herbal medicines, their correct identification by traditional subjective methods is difficult. Therefore, to develop a reliable method for discriminating these three herbal medicines, we applied the tools of molecular genetics, including random amplified polymorphic DNA (RAPD) and sequence characterized amplified region (SCAR) marker development using samples of Akebia quinata, Akebia trifoliata, Aristolochia manshuriensis, and Clematis armandii. By comparative analysis of RAPD polymorphisms, we identified several potential PCR products that can be used to distinguish the herbal materials of each species. Using these RAPD products, we developed unique sequence characterized amplified region (SCAR) markers for determination of the species of plant materials; the products were 590 bp for A. quinata, 300 bp for A. trifoliata, 373 bp for A. manshuriensis, and 432 bp for C. armandii. Furthermore, we optimized SCAR markers for simultaneous discrimination of the four species by multiplex-PCR. These markers allow efficient identification of closely related herbal medicines originating from Akebia, Aristolochia, and Clematis species, and have potential uses in preventing the distribution of adulterants, as they can be used to identify each species and distinguish them from inauthentic substitutes.

This study investigates the quality characteristics of muffins prepared with various concentrations of akebia leaves powder. Muffins are prepared by additions of 0%, 2%, 4%, 6% and 8% powder to the flour for basic formulation. Heights decrease significantly when the amount of Akebia leaves powder increases. When the weight is being measured, there are no meaningful differences between the samples. As a result from measuring the colors, the lightness, redness and yellowness of muffins decrease as the concentrations of the powder increase. When measuring the texture of akebia leaves muffins, the hardness, cohesiveness, gumminess and chewiness decrease, but the springiness and adhesiveness increase as the concentrations of Akebia leaves powder increase. When the sensual qualities of the muffins are measured, the color, sweetness, softness and overall quality of all of the samples have shown significant differences, and the muffins containing 4% of Akebia leaves powder reveal the highest sensual quality indexes.Key words: Akebia quinata leaves, muffins, quality characteristics

Morphology. Most Lardizabalaceae are woody vines forming dense mats covering other plants, frequently climbing into the canopy of trees. Only Decaisnea are erect shrubs. Stems are usually covered in lenticels. The wood anatomy of Lardizabalaceae is unique in having very wide pith surrounded by a ring of vascular bundles, separated by wide intervascular rays. Decaisnea has long vessel elements and scalariform pitting on lateral walls and vessels with scalariform perforation plates. The vines have comparatively short and wide vessel elements with elliptic alternate pitting and vessels with simple perforation plates. Reduced short shoots are common in the East Asian taxa, but absent in the South American ones. Leaves are alternate or fasciculate on short shoots, usually palmately compound (imparipinnate in Decaisnea, biternate in Lardizabala), without stipules. Inflorescences are always axillary racemes or derived from that. Usually they emerge together with the leaves from short shoots on old growth. Typical simple racemes are found in Akebia, Lardizabala, Sinofranchetia and Stauntonia, usually with the larger female flowers at the base of the inflorescence. When the basal pedicels are elongated, corymbose racemes are formed, which are common in Stauntonia. When the racemes are branched, panicles are formed, and these are common in Decaisnea and some Japanese forms of Akebia quinata. Umbels are formed in Boquila, and through a

Akebia trifoliata (Thunb) koidz contains a variety of active ingredients, exerting the promising pharmacological actions. Its stems and fruits are legally recorded as herbal medicines in the Chinese Pharmacopeia. However, different parts of Akebia trifoliate stems have different effects. In this study, the literature in the last decade was retrieved to summarize the pure compounds and pharmacological effects of Akebia trifoliata stems. It showed that Akebia trifoliata stems mainly contain a variety of oleanolic acids, ivy saponin-type triterpenoid saponins, which have antitumor, anti-inflammatory, diuretic and antibacterial actions.

The Akebia Decne belongs to the Lardizabalaceae family of flowering plants, which contains approximately nine genera and 50 species (Delectis Florae Reipublicae Popularis Sinicae Agendae Academiae Sinicae Edita, 2001). The family is widespread in East Asia, whereas there are two monotypic genera occurring in Chile. The plants of the genus Akebia are perennial, deciduous vines producing large edible fruits commonly known as ‘‘wild bananas’’ in China. The fruit resembles a small bunch of thick bananas that crack open longitudinally when ripe in Chinese lunar August. It is therefore called ‘‘Bayuezha’’ (August crack) by the local people and has been prized for its delicious sweet taste for centuries in China. Species of Akebia are commonly referred to as ‘‘chocolate vine’’ in United States, are grown by gardeners as ornamental climbing vines, are well-known medicinal plants, and have been used in Chinese herbalism for at least 2000 years. Three species (subspecies) (A. quinata, A. trifoliata ssp. trifoliata, and A. trifoliata ssp. australis) are listed in Chinese Pharmacopoeia (Pharmacopoeia Commission of PRC, 2005). The value of the Akebia fruit and its natural compounds with antineoplastic, diuretic, and antiphlogistic properties (Jiangsu New Medical College, 1985) is of great potential for development of an alternative high-value crop. However, few studies on Akebia breeding and cultivation have been conducted and limited information is available apart from a few reports on phytochemical analyses (Gao and Wang, 2006; Kawasaki and Higuchi, 1976a, 1976b). Synthesis of knowledge on these species is needed to provide insights for developing management options that enhance their conservation and contribution to fruit production. Recent investigations suggest that Akebia is worthy of being exploited as a new high-value fruit crop in China for its health benefits and other newly found fruit properties. This article provides a broad review on the most economically important Akebia species and discusses the value of exploring these species for domestication and commercial development.

金沢大学医薬保健研究域薬学系, 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. × 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. © 2009 The Japanese Society of Pharmacognosy and Springer.

Eleven polymorphic microsatellite loci were isolated and characterized from an AC-enriched genomic library of Akebia trifoliate ssp. australis. The number of allele per locus ranged from 3 to 14. The observed heterozygosity and expected heterozygosity at population level were 0.196–1.000 and 0.522–0.902, respectively. In addition, this set of microsatellites produced robust cross-species amplification in other two related taxa, suggesting these microsatellite markers should provide a useful tool for genetic and conservation studies of the Akebia species.

Two new triterpenoid saponins, hederagenin 3-O-α-L-arabinopyranosyl-(1→2)-α-L-arabinopyranoside named akeboside La (compound 1), oleanolic acid 3-O-α-L-arabinopyranosyl-(1→2)-β-D-glucopyranoside named akeboside Lb (compound 2), along with five known saponins, oleanolic acid 3-O-α-L-rhamnopyranosyl-(1→2)-α-Larabinopyranoside (compound 3), hederagenin 3-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside (compound 4), oleanolic acid 3-O-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside (compound 5),3-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl oleanolic acid 28-O-α-L-rhamnopyranosyl-(1→4)-α-Dglucopyranosyl-(1→6)-β-D-glucopyranoside (compound 6), 3-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl hederagenin 28-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside (compound 7) were isolated from the n-butanol part of the 80% ethanol extracts of the dried stems of Akebia quinata (Thunb.) Decne.Compound 5 was isolated from plants of genus Akebia for the first time. The structures were elucidated on the basis of physicochemical properties and spectral data.

The stems of Akebia quinata have been analyzed for their triterpene glycoside constituents, resulting in the isolation of six new triterpene glycosides, along with 19 known ones. On the basis of extensive spectroscopic analysis, including 2D NMR data, and chemical evidence, the structures of the new compounds were deter-mined to be 3beta-[(O-beta-D-glucuronopyranosyl-(1-->3)-alpha-L-arabinopyranosyl)oxy]olean-12-en-28-oic acid O-alpha-L-rhamnopyranosyl-(1-->4)-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl ester, 3beta-[(O-beta-D-glucuronopyranosyl-(1-->3)-O-[alpha-L-rhamnopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl)oxy]olean-12-en-28-oic acid O-alpha-L-rhamnopyranosyl-(1-->4)-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl ester, 3beta-[(O-beta-D-glucuronopyranosyl-(1-->3)-alpha-L-arabinopyranosyl)oxy]-23-hydroxyolean-12-en-28-oic acid O-alpha-L-rhamnopyranosyl-(1-->4)-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl ester, 3beta-[(O-beta-D-glucuronopyranosyl-(1-->3)-O-[alpha-L-rhamnopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl)oxy]-23-hydroxyolean-12-en-28-oic acid O-alpha-L-rhamnopyranosyl-(1-->4)-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl ester, 3beta-[(O-beta-D-glucopyranosyl-(1-->3)-O-[alpha-L-rhamnopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl)oxy]-29-hydroxyolean-12-en-28-oic acid, and 3beta-[(O-beta-D-glucopyranosyl-(1-->3)-O-[alpha-L-rhamnopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl)oxy]-23,29-dihydroxyolean-12-en-28-oic acid, respectively. The main triterpene glycosides contained in the stems of A. quinata were found to have two sugar units at C-3 and C-28 of the aglycone in this study, whereas those of Akebia trifoliate were reported to possess one sugar unit at C-28 of the aglycone. It may be possible to distinguish between A. quinata and A. trifoliate chemically by comparing their triterpene glycoside constituents.

The pharmacological evaluation demonstrated that the extracts from the stem ofS. brachyantheracould significantly increase the outputs of urine of rats compared to those of furosemide treated group, and the effect could last for a longer period of time. The best effect appeared in the first two hours, which scientifically confirmed the diuretic effect of the plant. The comparative pharmacognosy study showed that the characters of the crude drugs of the stem ofS. brachyantherawere similar to those ofAkebia caulis. Further systemic work on its chemical constituents by chromatographic methods and NMR elucidations led to the isolation of 10 triterpenoids, 6 flavonoids, 4 lignanoids, and 3 phenylethanoid glycosides, whose structural types were much similar to those ofA. quinata. Among them, 7 compounds were firstly reported in the genus ofStauntoniaand calceolarioside B was the common characteristic constituent in both plants. From the similar pharmacognosy characters, pharmacological effects, and chemical constituents, it could be concluded thatS. brachyantherahave a great possibility to be a succedaneum ofAkebia caulis, whose supply is extremely short in recent years.

An HPLC-UV-MS method for the analysis of aristolochic acids A, B, C and D, 7-OH-aristolochic acid A, and aristolic acid in a number of plant materials and their commercial products has been developed. HPLC with photodiode array detection and electrospray ionisation-MS in the selected ion monitoring mode allowed the identification of the target compounds and increased the selectivity of complex analyses such as those associated with multi-botanical preparations. The presented method was used to analyse 10 plant samples and six commercial products that possibly contained aristolochic acids. The resulting chromatographic profiles of the samples were significantly different from each other, and the method was directly transferred to HPLC-MS, which was used to confirm the presence of the six aristolochic acids mentioned above.

Akebia Fructus has long been used for hepatocellular carcinoma (HCC) in China, while the molecular mechanism remains obscure. Our recent work found thatAkebia trifoliate (Thunb.) Koidzseed extract (ATSE) suppressed proliferation and induced endoplasmic reticulum (ER) stress in SMMC-7721. The present study aimed to throw more light on the mechanism. ER stress occurred after ATSE treatment in HepG2, HuH7, and SMMC-7721 cells, manifested as ER expansion, and SMMC-7721 was the most sensitive kind in terms of morphology. Cell viability assay showed that ATSE significantly inhibited cells proliferation. Flow cytometry analysis indicated that ATSE leads to an upward tendency of G0/G1 phase and a reduced trend of the continuous peak after G2/M phase in HepG2; ATSE promoted apoptosis in HuH7 and a notable reduction in G0/G1 phase; ATSE does not quite influence cell cycles of SMMC-7721. Western blot analysis showed an increased trend of the chosen ER stress-related proteins after different treatments but nonsignificantly; only HYOU1 and GRP78 were decreased notably by ATSE in HuH7. Affymetrix array indicated that lots of ER stress-related genes’ expressions were significantly altered, and downward is the main trend. These results suggest that ATSE have anticancer potency in HCC cells via partly inducing ER stress.

The Chinese crude drug “Mu-tong, ” “Mokutsu” in Japanese, is standardized as the stem of Akebia quinata Decne. and A. trifoliata Koidz. of Lardizabalaceae in the modern Japanese pharmacopoeia. On the other hand, the botanical origin of Mu-tong is rather complicated in China, and many different plants among several families are used as a kind of Mu-tong. The herbological study clarified that Chinese doctors in the Tang dynasty had changed the original name “Tong-tsao” to “Mu-tong, ” meaning “woody Tong-tsao, ” to distinguish it from another type of Tong-tsao derived from the soft pith of Tetrapanax papyrifera Koch of the family Araliaceae plant. The classical botanical origin of Mu-tong is Akebia plants. Though the pith of Tetrapanax papyrifera is used as “Tong-tsao” in modern China, Akebiae Caulis is the true botanical source of ancient prescriptions such as those found in Shang-han-lun, an ancient Chinese medical book.

An exploration for Akebia in Gunma and Yamagata prefectures was conducted from September 24th to 26th and from September 31th to October 2nd, 1998. A total of 63 samples were collected from forest and farms. Farmers chose the superior varieties from wild Akebia and cultivate them. We collected a sample which had tolerance to powdery mildew that is an important blight of the Akebia.

Eleven species belonging to seven genera in the Lardizabalaceae were analyzed in terms of restriction fragment length polymorphism (RFLP) of chloroplast DNA and the sequence of the chloroplast gene,rbcL, of Lardizabalaceae and its related families. Phylogenetic trees inferred from parsimony, neighbor joining and maximum likelihood methods based on RFLP data showed that two South American genera,Boquila andLardizabala, and three East Asian genera,Akebia, Holboellia andStauntonia are closely related to each other, respectively. On the other hand, the parsimony, neighbor joining and maximum likelihood trees constructed using sequence data of therbcL gene showed thatAkebia, Stauntonia, Boquila andLardizabala clustered as(((Akebia, Stauntonia), Boquila), Lardizabala). This difference may be attributable to fewer informative sites inrbcL genes than in RFLP in this family.Decaisnea diverges at the very base of the Lardizabalaceae.

Akebia saponin D is a typical bioactive triterpenoid saponin isolated the rhizome of Dipsacus asper Wall. Our previous studies demonstrated that the oral bioavailability of akebia saponin D was very low, but the underlying mechanisms remained unknown. The present study aims to investigate the intestinal absorptive characteristics of akebia saponin D as well as the absorptive transport behavior influenced by co-administration of three absorption-enhancing agents and three efflux protein inhibitors using an in vitro everted gut sac method and an in situ intestinal perfusion model. The results showed that akebia saponin D had a quite limited intestinal permeability, and there was a non-linear increase in transepithelial transportation with increasing concentrations of akebia saponin D. The absorption of akebia saponin D was intestinal segment selective and the small intestine was the best absorptive site. Among three absorption promoters, borneol could significantly improve the permeability of akebia saponin D across ileum, while Tween-80 and DMSO had almost no absorption-enhancing effect. In addition, verapamil, probenecid and pantoprazole in the perfusates were used in this study as modulators of transporters such as P-glycoprotein, MRPs and BCRP in the intestinal mucosa, respectively. The results exhibited that the ileal permeability of akebia saponin D was markedly elevated by the co-administration of probenecid, indicating that akebia saponin D may be likely a substrate of MRPs. The above-mentioned results suggest that akebia saponin D has a poor intestinal absorption not only due to its poor transepithelial permeability but also owing to the contribution of efflux transporters such as MRPs in the intestine.

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.

Ethnopharmacological relevance Longdan Xieganwan, which contains Aristolochia species, is a traditional Chinese prescription. It has been used for thousands of years to “enhance liver”. However, many cases of Longdan Xieganwan induced nephropathy were reported recently. Aim of the study This study was designed to compare the possible toxic effects of Longdan Xieganwan and three different Aristolochia species, i.e. Akebia trifoliate (Thunb.) koid (Akebia trifoliate), Akebia quinata (Thunb.) Decne. (Akebia quinata) and Caulis aristolochiae manshuriensis (Aristolochia manshuriensis). Materials and methods Mice were orally administered these drugs for 28 days. Clinical signs, body weights, serum biochemistry, organ weights and histopathology were examined. Results Significantly decreased body weights and obvious nephropathy were noticed in the Aristolochia manshuriensis groups at doses higher than 0.24 g/kg/d. A few endothelial cell degenerations in renal glomerulus were observed in the Akebia trifoliate group at a high-dose of 2.00 g/kg/d. No significant changes were observed in the other groups. Conclusions The no-observed-adverse-effect levels (NOAELs) for Aristolochia manshuriensis, Akebia trifoliate, Akebia quinata and Longdan Xieganwan in this study for mice were 0.06 g/kg/d, 0.40 g/kg/d, higher than 3.00 g/kg/d and higher than 10.00 g/kg/d, which were equivalent to 0.25 times, 5 times, 25 times and 10 times of normal human dose in clinical prescription, respectively.

There is evidence that gene duplication and diversification within the MADS-box gene family had significant impact on floral architecture. In this study, we report the isolation of four class B homologous genes from Akebia trifoliata, termed AktAP3_1, AktAP3_2, AktAP3_3, and AktPI. Phylogenetic analysis indicates that the three AktAP3 paralogs were produced by two gene duplication events and AktAP3_2 and AktAP3_3 are recent paralogs, which are yielded by the duplication before the origin of the genus Akebia. In situ hybridization demonstrates that these genes are mainly expressed in the stamens and carpels of A. trifoliata, but in differential patterns, similar to those in other basal eudicot and basal angiosperm species. AktAP3_3 and AktPI are expressed in the developing petaloid perianth, suggesting that the petaloidy of the perianth is caused by the expression of class B genes. Reverse transcriptase polymerase chain reaction analyses indicate that these genes are expressed in both male and female flowers, but at different levels. We explore the interaction behavior of the class B proteins in the basal eudicots using yeast two-hybrid system for the first time. The AktAP3_1/2/3 proteins and the AktPI protein can form obligate heterodimers, but at different strength. From the mRNA expression and protein interaction patterns of the duplicated copies of the AktAP3 genes, we conclude that subfunctionalization very likely contributes to the maintenance of multiple AP3-like gene copies in A. trifoliata.

The U.S. Food and Drug Administration (FDA) is concerned about botanicals and botanical containing products that may contain aristolochic acid, either naturally or through adulteration. Some of these botanicals are known to contain, or are suspected of containing, aristolochic acid. These botanicals include Aristolochia species (spp.), Asarum spp., and Bragantia spp. Botanicals that may be adulterated with aristolochic acid include Akebia spp., Clematis spp., Coculus spp., Diploclisia spp., Menispernum spp., Sinomenium spp., and Stephania spp. Additional names associated with these species include Mu tong, Fang ji, Guang fang ji, Fang chi, Kan-Mokotsu, and Mokutsu. The similarity of names, as well as the identification and handling of botanicals in preparation of traditional Chinese medicines, makes it difficult to avoid inadvertent adulteration of both botanicals and dietary supplements. Reported serious adverse effects in humans include nephropathy and urothelial carcinomas. Rodents administered aristolochic acid developed lymphoma and cancers in the kidney, bladder, stomach, and lung, implying a potential increased risk of developing malignancies in humans (1, 2). The test materials used in the validation were Aristolochia spp. root, Aristolochia manschuriensis stem, Akebia trifoliata stem, Clematis armandii stem at 20 ppm high fortification, Stephania tetrandra root at 10 ppm low fortification, tablets containing a 20 ppm high fortification, and tablets with a 10 ppm low fortification. The fortified materials were spiked with ground Aristolochia spp. root. All test materials were authenticated by voucher or analysis for aristolochic acid I (Aristolochia spp. root). Due to the amount of Aristolochia spp. root needed for some experiments in the single laboratory validation (SLV), the ground Aristolochia spp. root was further diluted, as necessary, with ground Stephania tetrandra root (negative control) to produce the necessary amounts of Aristolochia spp. root containing aristolochic acid I.