Akebiae Caulis Research Articles

Pharmacognostic standardization and machine learning-based investigations on Akebia quinata and Akebia trifoliata.

Currently, Akebiae Caulis is being used in clinical practice, but there are few reseaches on its different varieties. To ensure the accuracy and effectiveness of clinical practice,this study distinguished the Akebia quinata (Thunb.) Decne. and Akebia trifoliata (Thunb.) Koidz, using organoleptic evaluation, microscopic observation, fluorescence reaction, physicochemical properties, thin-layer chromatography, IR spectroscopy, HPLC, four machine learning models, and in vitro antioxidant methods. Analysis of the powders of these two varieties using optical microscopy revealed the presence of starch granules, cork cells, crystal fibers, scalariform vessels, and wood fibers. Scanning electron microscopy revealed the presence of scalariform vessels, pitted vessels, wood fibers, and calcium oxalate crystals. Several tissues, including the cork layer, fiber population, cortex, phloem, pith, xylem, and ray, were found in the transverse section. In addition, thin-layer chromatography was used to identify two components: oleanolic acid and calceolarioside B; 11 common peaks were identified in 15 batches of SAQ and 5 batches of SAT by using HPLC. Support vector machine, BP neural networks, and GA-bp neural networks were able to predict 100% accurately of the different origins of stem of Akebia quinate (Thunb.) Decne (SAQ) and Akebia trifoliata (Thunb.) Koidz (SAT). Extreme learning machine achieved a correct rate of 87.5%. Meanwhile, Fourier-transform infrared spectroscopy fingerprint identified nine characteristic absorption peaks of the secondary metabolites of SAQ and SAT. 2,2-Diphenyl-1-1-picrylhydrazyl experiment revealed that the IC50 values of SAQ and SAT extracts were 155.49 and 128.75 μg/ml, respectively. For the 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) assay, the IC50 value of SAT extract was found to be 269.24 μg/ml, which was lower than that of SAQ extract (IC50 = 358.99 μg/ml). This study successfully used different methods to differentiate between A.quinata (Thunb.) Decne. and A.trifoliata (Thunb.) Koidz., to help decide on which type to use for clinical application.

Droplet digital PCR for the identification of plant-derived adulterants in highly processed products

BackgroundThe high sensitivity of droplet digital PCR (ddPCR) contributes to its excellent performance in animal and microorganism identification, but the utilization of ddPCR is limited in plant adulterant identification of highly processed products for which effective methods are lacking. PurposeThis study investigated the feasibility of ddPCR in the identification of plant adulterants in Chinese patent medicine (CPM) as groundwork to develop ddPCR assays for other highly processed goods. MethodsThe original plant, processed and highly processed products of Mutong (Akebiae Caulis) and its two adulterants were used to analyze the specificity, sensitivity, and practical performance of the developed singleplex and triplex ddPCR assays. ResultsThe results revealed that the limit of detection (LOD) and limit of quantification (LOQ) for the selective ddPCR assays developed to identify Mutong and its adulterants were 0.00002 ng/μl and 0.00016 ng/μl, respectively, and that the regression equations representing the relationships between DNA concentration and target copy number all exhibited good linearity. Furthermore, the common adulterant of Mutong in three samples of Longdan Xiegan pills was successfully identified through ddPCR assays and confirmed by Sanger sequencing. ConclusionThis work comprehensively revealed the great ability of ddPCR technology in detecting plant adulterants in traditional Chinese medicine (TCM), providing a method for the quality control of highly processed plant products with complex components for commonly used goods.

Next-generation sequencing and targeted quantitative real-time polymerase chain reaction for detection of Akebiae Caulis in the traditional Chinese medical formula Longdan Xiegan Wan.

BackgroundAkebiae Caulis (Mu Tong) is commonly misused by Aristolochiae Manshuriensis Caulis (Guan Mutong) and Clematidis Armandii Caulis (Chuan Mutong), which are nephrotoxic and carcinogenic. However, in the Pharmacopoeia of the People’s Republic of China (2015 Edition), the method for determining Akebiae Caulis remains undefined.MethodsWe used DNA barcode-based next-generation sequencing (NGS) combined with quantitative real-time polymerase chain reaction (qPCR) to detect Akebiae Caulis in Longdan Xiegan Wan (LDXGW) for the first time. Compared with chromatographic studies, NGS enables better evaluation of the ingredient components of traditional Chinese medicine (TCM) preparations. The feasibility of qPCR using species-specific primers to determine the authenticity of species has been validated. In this study, the constituents of Akebiae Caulis in LDXGW from three different manufacturers were scanned by NGS. The independently developed qPCR detection primers of Akebiae Caulis, Aristolochiae Manshuriensis Caulis, and Clematidis Armandii Caulis were specifically used to analyze the LDXGW mentioned above.ResultsThe results showed that qPCR detected Clematidis Armandii Caulis in all commercial samples. Meanwhile, NGS detected the counterfeit species Clematis peterae (Tie-Xian Lian) in all samples. We found that qPCR shows a difference in detecting Akebiae Caulis, but it was not able to identify the unknown additives and adulterants for the primer pairs of Clematidis Armandii Caulis.ConclusionsHence, it is sensitive and rapid, qPCR is not suitable for detection alone. The NGS approaches offer important novel insights that complement the qPCR method. The combination of NGS and qPCR will be a powerful complement to traditional identification methods of TCM substances.

Modeling the Potential Distribution of Three Taxa of Akebia Decne. under Climate Change Scenarios in China

Akebia trifoliata (Thunb.) Koidz., Akebia trifoliata subsp. australis (Diels) T. Shimizu and Akebia quinata (Houtt.) Decne. are the source plants of the traditional Chinese medicines AKEBIAE CAULIS and AKEBIAE FRUCTUS, and have high pharmaceutical value. However, the resource reserve of these plants has dramatically declined due to habitat destruction, which has seriously affected their adequate supply and sustainable utilization. A poor knowledge of the potential distribution of these medicinal materials would seriously constrain the protective exploitation of wild resources and the establishment of new cultivations. In this study, based on the scenarios of SSP1-2.6, SSP2-4.5, SSP3-7.0 and SSP5-8.5, the maximum entropy model was used to predict the potential distribution of these three Akebia taxa under current and future (2030s, 2050s, 2070s and 2090s) climate conditions. Our findings showed that the potentially suitable areas of these three Akebia taxa were mainly distributed in China at 101.8–121.9° E and 23.5–34.6° N. Temperature played a more significant role than precipitation in affecting the distribution. The dominant bioclimatic variable that affected the distribution of A. trifoliata and A. quinata in China was the minimum temperature of the coldest month (BIO06). For A. trifoliata subsp. australis, the mean diurnal range (BIO02) was the dominant variable influencing its distribution. Compared with current conditions, the moderate- and high-suitability areas of these three Akebia taxa were predicted to shrink towards the core areas, while the low-suitability areas were all observed to increase from the 2030s to the 2090s. With the increase in radiative forcing of SSP, the low-impact areas of these three Akebia taxa showed a decreasing trend as a whole. Our results illustrate the impact of climate change on the distribution of Akebia, and would provide references for the sustainable utilization of Akebia’s resources.

Visualization of research on Chinese medicine-induced kidney injury by CiteSpace

This study aimed to provide researchers with a better understanding of the research status, progress and problems of Chinese medicine-induced kidney injury. The mapping knowledge domain(MKD) was chosen to visualize the research process, hotspots,and frontiers, thus providing a reference for follow-up research. The relevant articles were retrieved from China National Knowledge Infrastructure(CNKI) from its inception to November 20, 2020, and 683 were finally included for visualization, with a knowledge map covering the keywords, research institutions and authors plotted by Cite Space. Since 2006, the annual number of published articles regarding Chinese medicine-induced kidney injury has steadily declined. The top five keywords sorted by frequency of co-occurrence were tubulointerstitial nephritis(TIN), aristolochic acid(AA), Akebiae Caulis, experimental research, and descriptive research. Akebiae Caulis appeared at the highest frequency of co-occurrence among Chinese medicines and exhibited the highest burst intensity. The burst ended in 2020, when the Chinese medicine with the highest burst intensity changed to Tripterygii wilfordii and its preparations. It could be seen that Akebiae Caulis was the hotspot in the research of Chinese medicine-induced kidney injury, and T. wilfordii became the research frontier. The current research has mainly focused on " organic anion transporters" and " metabolomics". The decline in articles about Chinese medicine-induced kidney injury was attributed to the reduced clinical case reports after the in-depth study of Chinese medicine-induced kidney injury. However, more clinical studies need to be conducted based on the concept and method of pharmacovigilance to construct a pharmacovigilance system of Chinese medicine for actively and passively monitoring and collecting the clinical cases with Chinese medicine-induced kidney injury, figuring out the incidence and characteristics of adverse reactions caused by Chinese medicine, and carrying out comprehensive and multi-dimensional safety research.

Development of ITS sequence based SCAR marker and multiplex-SCAR assay for the rapid authentication of Tetrapanacis Medulla and Akebiae Caulis

Development of ITS sequence based SCAR marker and multiplex-SCAR assay for the rapid authentication of Tetrapanacis Medulla and Akebiae Caulis

Rapid Analysis of Aristolochic Acid Analogues in Traditional Chinese Patent Medicine by LC-MS/MS

Aristolochic acids have been demonstrated to have renal toxicity, cause carcinogenesis, and may cause gene mutations. A series of risk control measurements have been adopted worldwide since 1990s. Some varieties of traditional Chinese medicine with high content of aristolochic acids have been banned in China. However, some species containing aristolochic acids in microscale are still in use. In recent years, with the continuous awareness of drug safety, the aristolochic acid analogues were generally considered to be of potential safety risks. Among these constituents, aristolochic acid I is still the one with most studies. Therefore, in addition to aristolochic acid I, it is necessary to establish an accurate and rapid method to determine other aristolochic acid analogues. LC-MS/MS methods based on multireaction monitoring mode was established to simultaneously determine 9 aristolochic acid analogues including 5 aristolochic acids and 4 aristolactams for the first time. Furthermore, the method was applied for Long dan Xie gan Pill, a traditional complex compound preparation with a long history for treatment of diseases including hepatochlic hygropyrexia, dizziness, tinnitus, and deafness. It has attracted widespread attention because of the aristolochic acid nephropathy. The crude drug Caulis Aristolochiae manshuriensis (Guanmutong) collected in the prescription was replaced by Akebiae Caulis (Mutong), and the established method helps to understand the product safety on market. As a result, aristolochic acid I, aristolochic acid Iva, and aristolactam I were detected and determined in one batch of Long dan Xie gan Pill among 25 batches of samples. It provided practical approach to demonstrate trace aristolochic acids and aristolactams. It was beneficial to control the safety of related traditional Chinese medicine products.

Research Trends in Pharmacopuncture Treatment for Lateral Epicondylitis

The aim of this study was to examine pharmacopuncture treatment for lateral epicondylitis, and to contribute to developing a standardized treatment regimen by reviewing trends in clinical trials. Five randomized controlled trials, 1 case-control study, and 8 cohort studies published after 1999, that involved pharmacopuncture for lateral epicondylitis, were selected from Korean and international online databases (<i>n</i> = 8). The type of pharmacopuncture, dose, frequency, efficacy, and adverse events were analyzed. Seven types of pharmacopuncture were used, namely Bee Venom, Illicium henryi Diels, Akebiae Caulis, Angelicae sinensis Diels, Ligusticum chuanxiong Hort, Hominis Placenta, and Salviae Miltiorrhizae Radix. Dose, treatment duration, and treatment frequency varied widely. One study assessed the treatment efficacy according to frequency. Nine studies lacked data on adverse events. The quality of 5 randomized controlled trials was low. Although pharmacopuncture treatment appeared to be effective for lateral epicondylitis, it was difficult to standardize the regimen for lateral epicondylitis.

Identification of Toxic Herbs Using Deep Learning with Focus on the Sinomenium Acutum, Aristolochiae Manshuriensis Caulis, Akebiae Caulis

Toxic herbs are similar in appearance to those known to be safe, which can lead to medical accidents caused by identification errors. We aimed to study the deep learning models that can be used to distinguish the herb Aristolochiae Manshuriensis Caulis (AMC), which contains carcinogenic and nephrotoxic ingredients from Akebiae Caulis (AC) and Sinomenium acutum (SA). Five hundred images of each herb without backgrounds, captured with smartphones, and 100 images from the Internet were used as learning materials. The study employed the deep-learning models VGGNet16, ResNet50, and MobileNet for the identification. Two additional techniques were tried to enhance the accuracy of the models. One was extracting the edges from the images of the herbs using canny edge detection (CED) and the other was applying transfer learning (TL) to each model. In addition, the sensitivity and specificity of AMC, AC, and SA identification were assessed by experts with a Ph.D. degree in herbology, undergraduates and clinicians of oriental medicine, and the ability was compared with those of MobileNet-TL′s. The identification accuracies of VGGNet16, ResNet50, and MobileNet were 93.9%, 92.2%, and 95.6%, respectively. After adopting the CED technique, the accuracy was 95.0% for VGGNet16, 63.9% for ResNet50, and 80.0% for MobileNet. After using TL without the CED technique, the accuracy was 97.8% for VGGNet16-TL, 98.9% for ResNet50-TL, and 99.4% for MobileNet-TL. Finally, MobileNet-TL showed the highest accuracy among three models. MobileNet-TL had higher identification accuracy than experts with a Ph.D. degree in herbology in Korea. The result identifying AMC, AC, and SA in MobileNet-TL has demonstrated a great capability to distinguish those three herbs beyond human identification accuracy. This study indicates that the deep-learning model can be used for herb identification.

Study on assay strategy of total tritepenoid saponins in traditional Chinese medicines using total saponins from Akebiae Caulis as an example

Due to lack of reference substances,the content of triterpenoid saponins in traditional Chinese medicines is usually characterized by colorimetric determination of total saponins. However,the specificity of colorimetric method is poor,and the determination result is not accurate enough. So,in this paper,the content determination method of total triterpenoid saponins was studied by taking Akebiae Caulis saponins as an example. The contents of three main saponin aglycones,including arjunolic acid,hederagenin and oleanolic acid,were determined by HPLC method. Referring to the content determination method of total flavonol glycosides in Ginkgo biloba leaves in the 2015 edition of Chinese Pharmacopoeia,the content of Akebiae Caulis saponins was obtained by multiplying the total content of the three above-mentioned aglycones with conversion coefficient. LC-MS/MS analysis results showed that mutongsaponin C and aponin PJIwere the two main triterpene saponins in Akebiae Caulis,and they shared the same molecular formula. So,the average value of the ratios of the molecular weight between mutongsaponin C and the three aglycones was defined as the conversion coefficient.The three aglycones were separated on an ACE Excel 3 C18-AR column( 4. 6 mm×150 mm,3 μm),and methanol-water( containing0. 04% glacial acetic acid and 0. 02% triethylamine) was used as mobile phase with gradient elution. The detection wavelength was set at 210 nm,and the flow rate was 0. 5 m L·min-1. The results showed that there was a good linearity among the ranges of 1. 053-16. 84,0. 200-3. 200 and 1. 515-24. 24 μg for arjunolic acid,hederagenin and oleanolic acid,respectively. Their average recoveries were97. 90%,97. 50% and 100. 5%,with RSD of 2. 0%,2. 9% and 2. 9%,respectively. The results of methodological investigation met the requirements of content determination. The conversion coefficient was 2. 31. This method is simple and reliable,and can be used for the determination of total triterpenoid saponins in Akebiae Caulis. The assay strategy can be used for the determination of total triterpenoid saponins in other traditional Chinese medicines.

Precise species detection of traditional Chinese patent medicine by shotgun metagenomic sequencing

BackgroundCurrent quality control methods for traditional Chinese patent medicines (TCPMs), e.g., microscopy, thin-layer chromatography (TLC), and high-performance liquid chromatography (HPLC), cannot detect herbal species composition with adequate precision. To address this issue, more effective detection methods should be explored. Hypothesis/PurposeWe hypothesized that shotgun metagenomic sequencing can fulfill the requirements for the species detection of multi-ingredient TCPMs. MethodsLongdan Xiegan Wan (LDXGW), once thought to be the chief culprit in aristolochic acid nephropathy (AAN), was selected to establish the method. It was used for both reference and commercial LDXGW samples. The precision authentication of herbal species contained in multi-ingredient TCPM is based on the shotgun metagenomic sequencing of genomic DNA without PCR amplification. Chemical analyses were also conducted as a contrast test. ResultsOver 100 G of raw data was obtained, and this value represented more than 0.75 billion reads. After assembling and filtering all the reads, a total of 261 contigs were obtained, which belonged to the ITS2, psbA-trnH, and matK regions of the reference and commercial samples. Because the homology of the rbcL region was high, it was not analyzed in the HTS data. Bioinformatics analysis indicated that the ITS2 region, as a DNA barcode, showed the highest identification efficiency. It could successfully detect all prescribed species, including four processed herbal ingredients, in the lab-made reference samples. The commercial samples all met the requirements of the Chinese Pharmacopoeia according to the TLC and HPLC tests. However, the shotgun metagenomic sequencing detected the substitution of Akebiae Caulis (Mutong) in the commercial samples, while the chemical analyses could not distinguish. ConclusionThe results highlight that shotgun metagenomic sequencing is a complementary method for the precise species detection of TCPMs.

Oleanane triterpenoids from Akebiae Caulis exhibit inhibitory effects on Aβ42 induced fibrillogenesis.

Previous phytochemical investigations of Akebiae Caulis resulted in the isolation of triterpenes, triterpene glycosides, phenylethanoid glycosides and megastigmane glycoside. Amyloid beta (Aβ), the main component of the senile plaques detected in Alzheimer's disease, induces cell death. However, only a limited number of studies have addressed the biological and pharmacological effects of Akebiae Caulis. In particular, the inhibitory activity of Akebiae Caulis against Aβ42 fibrillogenesis remains unclear. Herein, a new triterpene glycoside, akequintoside F (1), along with nine known compounds pulsatilla saponin A (2), collinsonidin (3), akebonic acid (4), hederagenin (5), 1-(3',4'-dihydroxycinnamoyl) cyclopentane-2,3-diol (6), asperosaponin C (7), leontoside A (8), quinatic acid (9), and quinatoside A (10) were isolated from Akebiae Caulis using repeated column chromatography with silica gel, LiChroprep RP-18, and MCI gel. The chemical structures of compounds 1-10 were illustrated based on 1D and 2D NMR spectroscopy, including 1H-1H COSY, HSQC, HMBC and NOESY spectroscopic analyses. Compound 1 a novel compound and known compounds 6 and 7 were isolated for the first time from this plant. Among these compounds, 1, 3, 4, 5 and 7 displayed significant inhibitory effects on Aβ42 induced fibrillogenesis. We present the first report of new compound 1 and the inhibitory effects of components from Akebiae Caulis on Aβ42 fibrillogenesis.

Rapid Identification of Officinal Akebiae Caulis and Its Toxic Adulterant Aristolochiae Manshuriensis Caulis (Aristolochia manshuriensis) by Loop-Mediated Isothermal Amplification.

Mu-tong (Akebiae Caulis) is a traditional Chinese medicine commonly used as a diuretic and antiphlogistic. A common adulterant of Mu-tong is Guan-mu-tong (Aristolochiae Manshuriensis Caulis), which is derived from the stem of Aristolochia manshuriensis Komarov, and contains carcinogenic aristolochic acids. We used an alternative technique, loop-mediated isothermal amplification (LAMP), to differentiate Mu-tong from Guan-mu-tong because LAMP is quick, highly sensitive, and specific. We designed a set of four common primers (G-F3, G-B3, G-FIP, and G-BIP) and a loop primer (G-LB) for LAMP based on the internal transcribed spacer 2 sequence of Ar. manshuriensis. We successfully amplified the LAMP assays and visual detection occurred within 60 min at isothermal conditions of 65°C. The LAMP reaction exhibited a tenfold increase in detection (4.22 pg/μl DNA) over conventional polymerase chain reaction demonstrating that LAMP is a useful technique to detect Guan-mu-tong. We conclude that the LAMP technique is a potentially valuable safety control method for simple and efficient discrimination of Mu-tong from its adulterant Guan-mu-tong.

Development of FTIR fingerprint for identification of armand clematis stem (Chuanmutong) and related herbs

Armand clematis stem (Clematidis Armandii Caulis, Chuanmutong) is a widely used Chinese herb to disinhibit urine and relieve stranguria. It is difficult to be identified owing to its various macroscopic feature and unknown characteristic compounds. Thus, total of 24 Chuanmutong samples and 7 related herbs including four manshurian aristolochia stem (Aristolochiae Manshuriensis Caulis, Guanmutong) and three akebia stem (Akebiae Caulis, Mutong) samples were collected and analyzed in the range of 4 000 - 400 cm⁻¹ by Fourier Transform Infrared (FTIR) and two-dimensional infrared correlation spectroscopy (2D-FTIR) techniques. The FTIR spectra of 24 Chuanmutong samples are consistent in the spectrum profiles, position and intensity of characteristic peaks. 20 of the 24 Chuanmutong samples were randomly selected as calibration samples to calculate and simulate mean spectrum. This mean spectrum is named as FTIR fingerprint of Chuanmutong with characteristic peaks at 3 412, 2 932, 1 739, 1 639, 1 509, 1 456, 1 426, 1 376, 1 332, 1 261, 1 159, 1 035, 897 ,609 cm⁻¹. Meanwhile, the limited level (Mean-3σ=0.992 6) to identify true or false Chuanmutong by correlation coefficient of FTIR spectra was calculated based on the 20 Chuanmutong calibration samples. Then, the rest 4 Chuanmutong, 4 Guanmutong and 3 Mutong samples were used as validation samples to evaluate the identification efficacy. The result shows that the FTIR spectra of 4 Chuanmutong validation samples were similar to the fingerprint. Their correlation coefficients of FTIR spectra were over the limited level and accepted as Chuanmutong. However, the spectra of Guanmutong and Mutong were significantly different from Chuanmutong fingerprint. The correlation coefficients of Guanmutong (0.902 1-0.940 4, n=4) and Mutong (0.954 9-0.978 9, n=3) FTIR spectra were less than the limited level and rejected from Chuanmutong. Furthermore, the number, position and intensity of auto-peaks on the 2D-FTIR were drastically different among the three herbs. It is concluded that the developed FTIR fingerprinting can be rapidly and accurately identify Chuanmutong and differentiate from related herbs.

Anti-oxidant Activity of Phenolic Compounds and Simultaneous Contents Determination using HPLC-UV from the Akebiae caulis

Anti-oxidant Activity of Phenolic Compounds and Simultaneous Contents Determination using HPLC-UV from the Akebiae caulis

Akebiae Caulis Inhibits Oxidative Stress through AMPK Activation

Akebiae Caulis Inhibits Oxidative Stress through AMPK Activation

Authentication of Akebia quinata DECNE. from its common adulterant medicinal plant species based on the RAPD-derived SCAR markers and multiplex-PCR

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.

Molecular identification and cytotoxicity study of herbal medicinal materials that are confused by Aristolochia herbs

Herbal materials derived from Aristolochia species contain carcinogenic aristolochic acids (AAs) and have been used as traditional Chinese medicines (TCMs) or adulterants of other TCMs. The purpose of this study is to identify the TCMs Stephaniae Tetrandrae Radix, Akebiae Caulis, Aucklandia Radix and Aristolochiae Fructus by sequencing the matK, rbcL, trnH-psbA and trnL-trnF DNA regions. The cytotoxicities of AAs and these TCMs were also studied in COS-7 and HEK-293 cells. Diagnostic polymorphic sites were identified in all the four DNA loci for the differentiation of genuine herbs from their adulterants/substitute. The 48h IC50 of AAI were 78μM (COS-7) and 70μM (HEK-293) while the IC50 of AAII were higher than 100μM in both cell lines. Except Aucklandia Radix, cytotoxicity study also showed that AA-containing herbs were more toxic than their corresponding genuine herbs and substitute.

Studies on Mutong, Akebiae Caulis (3) Herbological Study on Change in the Origin of Crude Drugs Categorized as Akebiae Caulis

Studies on Mutong, Akebiae Caulis (3) Herbological Study on Change in the Origin of Crude Drugs Categorized as Akebiae Caulis

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.