Mass spectrometry-based ginsenoside profiling: Recent applications, limitations, and perspectives.

Ginseng, the roots of Panax species, is an important medicinal herb used as a tonic. As ginsenosides are key bioactive components of ginseng, holistic chemical profiling of them has provided many insights into understanding ginseng. Mass spectrometry has been a major methodology for profiling, which has been applied to realize numerous goals in ginseng research, such as the discrimination of different species, geographical origins, and ages, and the monitoring of processing and biotransformation. This review summarizes the various applications of ginsenoside profiling in ginseng research over the last three decades that have contributed to expanding our understanding of ginseng. However, we also note that most of the studies overlooked a crucial factor that influences the levels of ginsenosides: genetic variation. To highlight the effects of genetic variation on the chemical contents, we present our results of untargeted and targeted ginsenoside profiling of different genotypes cultivated under identical conditions, in addition to data regarding genome-level genetic diversity. Additionally, we analyze the other limitations of previous studies, such as imperfect variable control, deficient metadata, and lack of additional effort to validate causation. We conclude that the values of ginsenoside profiling studies can be enhanced by overcoming such limitations, as well as by integrating with other -omics techniques.

Efficient Somatic Embryogenesis, Regeneration and Acclimatization of Panax ginseng Meyer: True-to-Type Conformity of Plantlets as Confirmed by ISSR Analysis.

Panax ginseng Meyer grows in east Russia and Asia. There is a high demand for this crop due to its medicinal properties. However, its low reproductive efficiency has been a hindrance to the crop's widespread use. This study aims to establish an efficient regeneration and acclimatization system for the crop. The type of basal media and strength were evaluated for their effects on somatic embryogenesis, germination, and regeneration. The highest rate of somatic embryogenesis was achieved for the basal media MS, N6, and GD, with the optimal nitrogen content (≥35 mM) and NH4+/NO3- ratio (1:2 or 1:4). The full-strength MS medium was the best one for somatic embryo induction. However, the diluted MS medium had a more positive effect on embryo maturation. Additionally, the basal media affected shooting, rooting, and plantlet formation. The germination medium containing 1/2 MS facilitated good shoot development; however, the medium with 1/2 SH yielded outstanding root development. In vitro-grown roots were successfully transferred to soil, and they exhibited a high survival rate (86.3%). Finally, the ISSR marker analysis demonstrated that the regenerated plants were not different from the control. The obtained results provide valuable information for a more efficient micropropagation of various P. ginseng cultivars.

Development of a colorimetric enzymatic assay method for aromatic biogenic monoamine-producing decarboxylases.

Biogenic amines (BAs) produced by the action of bacterial amino acid decarboxylases in fermented foods cause various health problems in human. Despite the importance, detailed characterizations of the BA-producing decarboxylases are relatively less progressed than the studies on BA-producing bacteria, due to the time-consuming chromatography-based assay method. In this study, a simple and general colorimetric assay for aromatic amino acid decarboxylases coupled with an amine oxidase from Arthrobacter aurescens (AMAO) and horseradish peroxidase was developed using a tyrosine decarboxylase from Enterococcus faecium DSM20477 (EfmTDC) as a model enzyme. The activity profiles over pH and temperature and the kinetic analysis for EfmTDC revealed that the results by the colorimetric assay are compatible with those by the chromatographic assay. In addition, due to the broad substrate specificity of AMAO for histamine and 2-phenylethylamine, the colorimetric assay would be applicable to the characterization of other aromatic amino acid decarboxylases including histidine decarboxylases. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10068-021-00938-4.

Design of a native-like secreted form of the hepatitis C virus E1E2 heterodimer.

Hepatitis C virus (HCV) is a major worldwide health burden, and a preventive vaccine is needed for global control or eradication of this virus. A substantial hurdle to an effective HCV vaccine is the high variability of the virus, leading to immune escape. The E1E2 glycoprotein complex contains conserved epitopes and elicits neutralizing antibody responses, making it a primary target for HCV vaccine development. However, the E1E2 transmembrane domains that are critical for native assembly make it challenging to produce this complex in a homogenous soluble form that is reflective of its state on the viral envelope. To enable rational design of an E1E2 vaccine, as well as structural characterization efforts, we have designed a soluble, secreted form of E1E2 (sE1E2). As with soluble glycoprotein designs for other viruses, it incorporates a scaffold to enforce assembly in the absence of the transmembrane domains, along with a furin cleavage site to permit native-like heterodimerization. This sE1E2 was found to assemble into a form closer to its expected size than full-length E1E2. Preservation of native structural elements was confirmed by high-affinity binding to a panel of conformationally specific monoclonal antibodies, including two neutralizing antibodies specific to native E1E2 and to its primary receptor, CD81. Finally, sE1E2 was found to elicit robust neutralizing antibodies in vivo. This designed sE1E2 can both provide insights into the determinants of native E1E2 assembly and serve as a platform for production of E1E2 for future structural and vaccine studies, enabling rational optimization of an E1E2-based antigen.

Biochemical characterization and molecular docking analysis of novel esterases from Sphingobium chungbukense DJ77.

We identified three novel microbial esterase (Est1, Est2, and Est3) from Sphingobium chungbukense DJ77. Multiple sequence alignment showed the Est1 and Est3 have distinct motifs, such as tetrapeptide motif HGGG, a pentapeptide sequence motif GXSXG, and catalytic triad residues Ser-Asp-His, indicating that the identified enzymes belong to family IV esterases. Interestingly, Est1 exhibited strong activity toward classical esterase substrates, p-nitrophenyl ester of short-chain fatty acids and long-chain. However, Est3 did not exhibit any activity despite having high sequence similarity and sharing the identical catalytic active residues with Est1. Est3 only showed hydrolytic degradation activity to polycaprolactone (PCL). MOE-docking prediction also provided the parameters consisting of binding energy, molecular docking score, and molecular distance between substrate and catalytic nucleophilic residue, serine. The engineered mutEst3 has hydrolytic activity for a variety of esters ranging from p-nitrophenyl esters to PCL. In the present study, we demonstrated that MOE-docking simulation provides a valuable insight for facilitating biocatalytic performance.

Genetic diversity among cultivated and wild Panax ginseng populations revealed by high-resolution microsatellite markers.

BACKGROUND: Ginseng (Panax ginseng Meyer) is one of the world's most valuable medicinal plants with numerous pharmacological effects. Ginseng has been cultivated from wild mountain ginseng collections for a few hundred years. However, the genetic diversity of cultivated and wild ginseng populations is not fully understood. METHODS: We developed 92 polymorphic microsatellite markers based on whole-genome sequence data. We selected five markers that represent clear allele diversity for each of their corresponding loci to elucidate genetic diversity. These markers were applied to 147 individual plants, including cultivars, breeding lines, and wild populations in Korea and neighboring countries. RESULTS: Most of the 92 markers displayed multiple-band patterns, resulting from genome duplication, which causes confusion in interpretation of their target locus. The five high-resolution markers revealed 3 to 8 alleles from each single locus. The proportion of heterozygosity (He) ranged from 0.027 to 0.190, with an average of 0.132, which is notably lower than that of previous studies. Polymorphism information content of the markers ranged from 0.199 to 0.701, with an average of 0.454. There was no statistically significant difference in genetic diversity between cultivated and wild ginseng groups, and they showed intermingled positioning in the phylogenetic relationship. CONCLUSION: Ginseng has a relatively high level of genetic diversity, and cultivated and wild groups have similar levels of genetic diversity. Collectively, our data demonstrate that current breeding populations have abundant genetic diversity for breeding of elite ginseng cultivars.

CYPminer: an automated cytochrome P450 identification, classification, and data analysis tool for genome data sets across kingdoms.

BACKGROUND: Cytochrome P450 monooxygenases (termed CYPs or P450s) are hemoproteins ubiquitously found across all kingdoms, playing a central role in intracellular metabolism, especially in metabolism of drugs and xenobiotics. The explosive growth of genome sequencing brings a new set of challenges and issues for researchers, such as a systematic investigation of CYPs across all kingdoms in terms of identification, classification, and pan-CYPome analyses. Such investigation requires an automated tool that can handle an enormous amount of sequencing data in a timely manner. RESULTS: CYPminer was developed in the Python language to facilitate rapid, comprehensive analysis of CYPs from genomes of all kingdoms. CYPminer consists of two procedures i) to generate the Genome-CYP Matrix (GCM) that lists all occurrences of CYPs across the genomes, and ii) to perform analyses and visualization of the GCM, including pan-CYPomes (pan- and core-CYPome), CYP co-occurrence networks, CYP clouds, and genome clustering data. The performance of CYPminer was evaluated with three datasets from fungal and bacterial genome sequences. CONCLUSIONS: CYPminer completes CYP analyses for large-scale genomes from all kingdoms, which allows systematic genome annotation and comparative insights for CYPs. CYPminer also can be extended and adapted easily for broader usage.

Comparative Analysis of Panax ginseng Berries from Seven Cultivars Using UPLC-QTOF/MS and NMR-Based Metabolic Profiling.

The commercial use of Panax ginseng berries is increasing as P. ginseng berries are known to contain large amounts of ginsenosides, and many pharmacological activities have been reported for the various ginsenosides. For the proper use of P.ginseng berries, it is necessary to study efficient and accurate quality control and the profiling of the overall composition of each cultivar. Ginseng berry samples from seven cultivars (Eumseung, Chung-buk Province, Republic of Korea) were analyzed using ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-QTOF/MS) for profiling of the ginsenosides, and high-resolution magic-angle-spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy for profiling of the primary metabolites. Comparing twenty-six ginsenoside profiles between the variant representatives and between the violet-stem variant, Kumpoong and Sunwon were classified. In the case of primary metabolites, the cultivars Kumpoong and Gopoong were classified. As a result of correlation analyses of the primary and secondary metabolites, in the Gopoong cultivar, the metabolism was found to lean toward energy metabolism rather than ginsenoside synthesis, and accumulation of osmolytes was low. The Gopoong cultivar had higher levels of most of the amino acids, such as arginine, phenylalanine, isoleucine, threonine, and valine, and it contained the highest level of choline and the lowest level of myo-inositol. Except for these, there were no significant differences of primary metabolites. In the Kumpoong cultivar, the protopanaxatriol (PPT)-type ginsenosides, ginsenoside Re and ginsenoside Rg2, were much lower than in the other cultivars, while the other PPT-type ginsenosides were inversely found in much higher amounts than in other cultivars. The Sunwon cultivar showed that variations of PPT-type ginsenosides were significantly different between samples. However, the median values of PPT-type ginsenosides of Sunwon showed similar levels to those of Kumpoong. The difference in primary metabolites used for metabolism for survival was found to be small in our results. Our data demonstrated the characteristics of each cultivar using profiling data of the primary and secondary metabolites, especially for Gopoong, Kumpoong, and Sunwon. These profiling data provided important information for further research and commercial use.

A new immunodeficient retinal dystrophic rat model for transplantation studies using human-derived cells.

PURPOSE: To create new immunodeficient Royal College of Surgeons (RCS) rats by introducing the defective MerTK gene into athymic nude rats. METHODS: Female homozygous RCS (RCS-p+/RCS-p+) and male nude rats (Hsd:RH-Foxn1mu, mutation in the foxn1 gene; no T cells) were crossed to produce heterozygous F1 progeny. Double homozygous F2 progeny obtained by crossing the F1 heterozygotes was identified phenotypically (hair loss) and genotypically (RCS-p+ gene determined by PCR). Retinal degenerative status was confirmed by optical coherence tomography (OCT) imaging, electroretinography (ERG), optokinetic (OKN) testing, superior colliculus (SC) electrophysiology, and by histology. The effect of xenografts was assessed by transplantation of human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE) and human-induced pluripotent stem cell-derived RPE (iPS-RPE) into the eye. Morphological analysis was conducted based on hematoxylin and eosin (H&E) and immunostaining. Age-matched pigmented athymic nude rats were used as control. RESULTS: Approximately 6% of the F2 pups (11/172) were homozygous for RCS-p+ gene and Foxn1mu gene. Homozygous males crossed with heterozygous females resulted in 50% homozygous progeny for experimentation. OCT imaging demonstrated significant loss of retinal thickness in homozygous rats. H&E staining showed photoreceptor thickness reduced to 1-3 layers at 12 weeks of age. Progressive loss of visual function was evidenced by OKN testing, ERG, and SC electrophysiology. Transplantation experiments demonstrated survival of human-derived cells and absence of apparent immune rejection. CONCLUSIONS: This new rat animal model developed by crossing RCS rats and athymic nude rats is suitable for conducting retinal transplantation experiments involving xenografts.

Medial comminution as a risk factor for the stability after lateral-only pin fixation for pediatric supracondylar humerus fracture: an audit.

BACKGROUND AND PURPOSE: Closed reduction and lateral-only pin fixation is one of the common treatment methods for displaced supracondylar fracture in children. However, several risk factors related to the stability have been reported. The aim of this study was to evaluate the medial comminution as a potential risk factor related to the stability after appropriate lateral-only pin fixation for Gartland type III supracondylar humerus fracture. METHODS: Sixty-seven patients with type III supracondylar fractures who were under the age of 12 years were included. Immediate postoperative and final Baumann and humerocapitellar angles were measured. Pin separation at fracture site was evaluated to estimate the proper pin placement. Presence of the medial comminution was recorded when two pediatric orthopedic surgeons agreed to the loss of cortical contact at the medial column by the small butterfly fragment or comminuted fracture fragments. Factors including age, sex, body mass index, pin number, pin separation at fracture site, and medial comminution were analyzed. RESULTS: Medial comminution was noted in 20 patients (29.8%). The average pin separation at fracture site was significantly decreased in patients with medial comminution compared to patients without medial comminution (P=0.017). A presence of medial comminution was associated with a 4.151-fold increase in the log odds for the Baumann angle changes of more than average difference between immediate postoperative and final follow-up angle (P=0.020). CONCLUSION: When lateral-only pin fixation is applied for Gartland type III supracondylar humerus fracture in children, the medial comminution may be a risk factor for the stability because of the narrow pin separation at fracture site. We recommend additional medial pin fixation for supracondylar humerus fracture with medial comminution.

Molecular Docking Analysis and Biochemical Evaluation of Levansucrase from Sphingobium chungbukense DJ77.

Bacterial exopolymer Levan (ß-(2,6) polyfructan) synthesized by levansucrase has attracted interest for various applications due to its low intrinsic viscosity compared with other polysaccharides. We report a novel levansucrase (Lsc) isolated from Sphingobium chunbukense DJ77 and verify its biochemical characteristics by comparative analysis of molecular docking analysis (MOE) and catalytic residue analysis. The complete sequence of the Lsc encoding gene ( lsc) was cloned under the direction of the T7 promoter and purified in an Escherichia coli BL21 (DE3) protein expression system. The enzyme activity analysis and ligand docking MOE study of S. chungbukense DJ77 Lsc revealed that Arg 77, Ser112, Arg 195, Asp196, Glu257, and Gln275 were involved in the sucrose binding and splitting as well as transfructosylation activity. A catalytic comparison of Lsc of S. chungbukense DJ77 with the results of site-directed mutational analysis indicated that Gln275 may coordinate a favorable substrate binding environment, offering broad pH resistance in the range of 5-10. The results suggest that the recombinant E. coli carrying S. chungbukense DJ77 Lsc might produce levan under the regular growth conditions with less need for pH manipulation.

Genome and evolution of the shade-requiring medicinal herb Panax ginseng.

Panax ginseng C. A. Meyer, reputed as the king of medicinal herbs, has slow growth, long generation time, low seed production and complicated genome structure that hamper its study. Here, we unveil the genomic architecture of tetraploid P. ginseng by de novo genome assembly, representing 2.98 Gbp with 59 352 annotated genes. Resequencing data indicated that diploid Panax species diverged in association with global warming in Southern Asia, and two North American species evolved via two intercontinental migrations. Two whole genome duplications (WGD) occurred in the family Araliaceae (including Panax) after divergence with the Apiaceae, the more recent one contributing to the ability of P. ginseng to overwinter, enabling it to spread broadly through the Northern Hemisphere. Functional and evolutionary analyses suggest that production of pharmacologically important dammarane-type ginsenosides originated in Panax and are produced largely in shoot tissues and transported to roots; that newly evolved P. ginseng fatty acid desaturases increase freezing tolerance; and that unprecedented retention of chlorophyll a/b binding protein genes enables efficient photosynthesis under low light. A genome-scale metabolic network provides a holistic view of Panax ginsenoside biosynthesis. This study provides valuable resources for improving medicinal values of ginseng either through genomics-assisted breeding or metabolic engineering.

Genomic and Proteomic Evidences for Exopolysaccharide Biosynthesis in Sphingobium chungbukense DJ77.

Sphingobium chungbukense DJ77 is a Gram-negative bacterium has metabolic capability of producing exopolysaccharide (EPS) as a potential reducing and stabilizing agent for metallic nanoparticle synthesis. In this study, we investigated the genomic and proteomic analysis to verify metabolic pathway and involved genes and enzymes related to EPS biosynthesis in S. chungbukense DJ77. End-sequencing results of randomly selected fosmid library, which were prepared from high molecular weight DNA of S. chungbukense DJ77, showed identity to sequences from genes related the EPS biosynthesis pathways in several bacteria. We also observed that proteomic responses in S. chungbukense DJ77 by heterogeneously expressing gelA and gelN involved in gellan biosynthesis in Sphingomonas elodea. Comparative two-dimensional gel electrophoresis revealed that both GelA and GelN altered internal expression levels of proteins involved in EPS biosynthesis in S. chungbukense DJ77. The results might provide the genomic and proteomic evidences for presence of EPS biosynthesis pathways in S. chungbukense DJ77.

The Genome-Based Virus Taxonomy with ICTV Database Extension.

In 1966, the International Classification of Viruses (ICNV) was established to standardize the naming of viruses. In 1975, the organization was renamed 'ICTV', by which it is still known today. The primary virus classification provided by ICTV in 1971 was for viruses infecting vertebrates, which includes 19 genera, 2 families, and a further 24 unclassified groups. Presently, the 10th virus taxonomy has been published. However, early classification of viruses was based on the clinical results of "in vivo" and "in vitro", as well as on the shape of PhynoType virus. However, due to the development of next-generation sequencing (NGS) and the accompanying bioinformatics analysis pipelines, a reconstruction of the classification system has been proposed. At a meeting held in Boston, USA between June 9-11, 2016, even there was an in-depth discussion regarding the classification of viruses using Metagenomic data. One suggested activity that arose from the meeting was that viral taxonomy should be reconstructed, based on genotype and bioinformatics analysis "in silico". This article describes our efforts to achieve this goal by construction of a web-based system and extension of an associated database, based on ICTV taxonomy. This virus taxonomy web system was specifically designed to extend the virus taxonomy up to strain and isolation, which was then connected with the NCBI database to facilitate searching for specific viral genes; there are also links to journals provided by the EMBL RESTful API that improve accessibility for academic groups.

Identification of Viral Taxon-Specific Genes (VTSG): Application to Caliciviridae.

Virus taxonomy was initially determined by clinical experiments based on phenotype. However, with the development of sequence analysis methods, genotype-based classification was also applied. With the development of genome sequence analysis technology, there is an increasing demand for virus taxonomy to be extended from in vivo and in vitro to in silico. In this study, we verified the consistency of the current ICTV taxonomy using an in silico approach, aiming to identify the specific sequence for each virus. We applied this approach to norovirus in Caliciviridae, which causes 90% of gastroenteritis cases worldwide [1]. First, based on the dogma "protein structure determines its function," we hypothesized that the specific sequence can be identified by the specific structure. Firstly, we extracted the coding region (CDS). Secondly, the CDS protein sequences of each genus were annotated by the conserved domain database(CDD) search. Finally, the conserved domains of each genus in Caliciviridae are classified by RPS-BLAST with CD database. The analysis result is that Caliciviridae has sequences including RNA helicase in common. In case of Norovius, Calicivirus coat protein C terminal and viral polyprotein N-terminal appears as a specific domain in Caliciviridae. It does not include in the other genera in Caliciviridae. If this method is utilized to detect specific conserved domains, it can be used as classification keywords based on protein functional structure. After determining the specific protein domains, the specific protein domain sequences would be converted to gene sequences. This sequences would be re-used one of viral bio-marks.

Anterior Dislocation of the Radial Head Combined with Plastic Deformity of the Ulnar Shaft in an Adult: A Case Report.

A 25-year-old woman presented to the emergency room with a painful and swollen right forearm. She had just sustained an injury from an accident during which her arm was tightly wound by a rope as she was lowering a net from a fishing boat. Before being released, her arm was rigidly trapped in the rope for approximately ten minutes. Radiographs revealed anterior dislocation of the radial head that was accompanied by plastic deformation of the proximal ulna, manifested as a reversal of the proximal dorsal angulation of the ulna (PUDA); suggested a Monteggia equivalent fracture. With the patient under general anesthesia, we reduced the radial head by posterior compression at 90° of elbow flexion and at neutral rotation of the forearm. However, the reduction was easily lost and the elbow re-dislocated with even slight supination or extension of the arm. After the osteotomy of the ulnar deformity to restore the PUDA to normal, the reduction remained stable even with manipulation of the arm. We found that the patient could exercise a full range of motion without pain at the 3-month follow-up, and neither residual instability nor degenerative changes were observed at the final 3-year follow-up.

Comprehensive Profiling and Quantification of Ginsenosides in the Root, Stem, Leaf, and Berry of Panax ginseng by UPLC-QTOF/MS.

The effective production and usage of ginsenosides, given their distinct pharmacological effects, are receiving increasing amounts of attention. As the ginsenosides content differs in different parts of Panax ginseng, we wanted to assess and compare the ginsenosides content in the ginseng roots, leave, stems, and berries. To extract the ginsenosides, 70% (v/v) methanol was used. The optimal ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry (UPLC-QTOF/MS) method was used to profile various ginsenosides from the different parts of P. ginseng. The datasets were then subjected to multivariate analysis including principal component analysis (PCA) and hierarchical clustering analysis (HCA). A UPLC-QTOF/MS method with an in-house library was constructed to profile 58 ginsenosides. With this method, a total of 39 ginsenosides were successfully identified and quantified in the ginseng roots, leave, stem, and berries. PCA and HCA characterized the different ginsenosides compositions from the different parts. The quantitative ginsenoside contents were also characterized from each plant part. The results of this study indicate that the UPLC-QTOF/MS method can be an effective tool to characterize various ginsenosides from the different parts of P. ginseng.

Applications of molecular markers in the discrimination of Panax species and Korean ginseng cultivars (Panax ginseng).

The development of molecular markers is one of the most useful methods for molecular breeding and marker-based molecular associated selections. Even though there is less information on the reference genome, molecular markers are indispensable tools for determination of genetic variation and identification of species with high levels of accuracy and reproducibility. The demand for molecular approaches for marker-based breeding and genetic discriminations in Panax species has greatly increased in recent times and has been successfully applied for various purposes. However, owing to the existence of diverse molecular techniques and differences in their principles and applications, there should be careful consideration while selecting appropriate marker types. In this review, we outline the recent status of different molecular marker applications in ginseng research and industrial fields. In addition, we discuss the basic principles, requirements, and advantages and disadvantages of the most widely used molecular markers, including restriction fragment length polymorphism, random amplified polymorphic DNA, sequence tag sites, simple sequence repeats, and single nucleotide polymorphisms.

Cytohistological study of the leaf structures of Panax ginseng Meyer and Panax quinquefolius L.

BACKGROUND: Both Panax ginseng Meyer and Panax quinquefolius are obligate shade-loving plants whose natural habitats are broadleaved forests of Eastern Asia and North America. Panax species are easily damaged by photoinhibition when they are exposed to high temperatures or insufficient shade. In this study, a cytohistological study of the leaf structures of two of the most well-known Panax species was performed to better understand the physiological processes that limit photosynthesis. METHODS: Leaves of ginseng plants grown in soil and hydroponic culture were sectioned for analysis. Leaf structures of both Panax species were observed using a light microscope, scanning electron microscope, and transmission electron microscope. RESULTS: The mesostructure of both P. ginseng and P. quinquefolius frequently had one layer of noncylindrical palisade cells and three or four layers of spongy parenchymal cells. P. quinquefolius contained a similar number of stomata in the abaxial leaf surface but more tightly appressed enlarged grana stacks than P. ginseng contained. The adaxial surface of the epidermis in P. quinquefolius showed cuticle ridges with a pattern similar to that of P. ginseng. CONCLUSION: The anatomical leaf structure of both P. ginseng and P. quinquefolius shows that they are typical shade-loving sciophytes. Slight differences in chloroplast structure suggests that the two different species can be authenticated using transmission electron microscopy images, and light-resistant cultivar breeding can be performed via controlling photosynthesis efficiency.