Comparison of the components of fresh Panax notoginseng processed by different methods and their anti-anemia effects on cyclophosphamide-treated mice.

ETHNOPHARMACOLOGICAL RELEVANCE: The traditional Chinese herb Panax notoginseng (PN) tonifies blood, and its main active ingredient is saponin. PN is processed by different methods, resulting in different compositions and effects. AIM OF THE STUDY: To investigate changes in the microstructure and composition of fresh PN processed by different techniques and the anti-anemia effects on tumor-bearing BALB/c mice after chemotherapy with cyclophosphamide (CTX). MATERIALS AND METHODS: Fresh PN was processed by hot-air drying (raw PN, RPN), steamed at 120 °C for 5 h (steamed PN, SPN), or fried at 130 °C, 160 °C, or 200 °C for 8 min (fried PN, FPN1, FPN2, or FPN3, respectively); then, the microstructures were compared with 3D optical microscopy, quasi-targeted metabolites were detected by liquid chromatography tandem mass spectrometry (LC‒MS/MS), and saponins were detected by high-performance liquid chromatography (HPLC). An anemic mouse model was established by subcutaneous H22 cell injection and treatment with CTX. The antianemia effects of PN after processing via three methods were investigated by measuring peripheral blood parameters, performing HE staining and measuring cell proliferation via immunofluorescence. RESULTS: 3D optical profiling revealed that the surface roughness of the SPN and FPN was greater than that of the other materials. Quasi-targeted metabolomics revealed that SPN and FPN had more differentially abundant metabolites whose abundance increased, while SPN had greater amounts of terpenoids and flavones. Analysis of the composition and content of the targeted saponins revealed that the contents of rare saponins (ginsenoside Rh1, 20(S)-Rg3, 20(R)-Rg3, Rh4, Rk3, Rg5) were greater in the SPN. In animal experiments, the RBC, WBC, HGB and HCT levels in peripheral blood were increased by SPN and FPN. HE staining and immunofluorescence showed that H-SPN and M-FPN promoted bone marrow and spleen cell proliferation. CONCLUSION: The microstructure and components of fresh PN differed after processing via different methods. SPN and FPN ameliorated CTX-induced anemia in mice, but the effects of PN processed by these two methods did not differ.

Selection and application of aptamers for p-hydroxybenzyl hydrogen sulfite after Gastrodia elata Bl. fumigated with sulfur.

Gastrodia elata Bl. is a widely used traditional Chinese medicine known for its medicinal properties. However, during the drying process, G. elata is often fumigated with sulfur to prevent corrosion and improve its appearance. Sulfur-fumigation can result in a reduction in the effective components of the herb and can also be hazardous to human health due to the remaining sulfur dioxide. Sulfur-fumigation of G. elata poses a significant challenge to both end-users and researchers. The detection of p-hydroxybenzyl hydrogen sulfite (p-HS) is a useful tool in determining whether G. elata has been fumigated with sulfur. Unfortunately, the current method for detecting p-HS is costly and requires sophisticated instruments. Therefore, there is a need to develop a more cost-effective and user-friendly method for the detection of p-HS. This study utilized the Capture-SELEX technique to screen high-affinity aptamers for p-HS, which were subsequently characterized by isothermal titration calorimetry (ITC). An aptamer sequence (seq 6) with a high affinity of Kd = 26.5 µM was obtained following 8 rounds of selection against p-HS. With the aptamer serving as the recognition element and gold nanoparticles as the colorimetric indicator, a simple and efficient colorimetric sensor was developed for the specific detection of p-HS. This detection method exhibited a limit of detection of 1 µg/ml, while the p-HS recoveries demonstrated a range of between 88.5 % and 105 % for samples of G. elata obtained in the market. In summary, the aptamer exhibited a high affinity for p-HS, and the sensor developed through the use of a colloidal gold detector based on nucleic acid aptamer can be utilized for rapid detection of sulfur-fumigated G. elata. With these findings, this research paper provides valuable scientific insights and highlights significant potential for future studies in this area.

A strategy for quality evaluation of complex herbal preparations based on multi-color scale and efficacy-oriented high-performance thin-layer chromatography characteristic fingerprint combined with chemometric method: Sanwujiao Pills as an example.

To rapidly evaluate the quality of complex herbal preparations, a new strategy was proposed based on multi-color scale and efficacy-oriented high-performance thin-layer chromatography (HPTLC) characteristic fingerprint combined with chemometric method. Firstly, effective components were screened through high-performance liquid chromatography with ultraviolet detection and evaporative light-scattering (HPLC-UV-ELSD), using multi-wavelength fusion combined with network pharmacology and molecular docking techniques. Subsequently, guided by the effective components, the targeted HPTLC characteristic fingerprint was established by multi-color scale scanning. Finally, combined with the chemometric method, the consistency of the preparation quality was evaluated, the marker components leading to quality differences were screened, and the quality control limit was established. Sanwujiao Pills (SWJPs) is a herbal preparation composed of six herbs for treating rheumatoid arthritis (RA). Through this strategy, four HPTLC characteristic fingerprints were established, they were derived from five herbs and guided by eight effective components in SWJPs. Through similarity, clustering heatmap, principal component analysis (PCA), and orthogonal partial least squares discriminant analysis (OPLS-DA), the quality distinctions among the 12 batches of SWJPs were determined. These batches were categorized into two groups based on their production time, and eight components affecting the quality of the preparation were identified. Meanwhile, the quality control threshold for SWJPs was determined based on Hotelling's T2 and DModX methods. This strategy aims to rapidly evaluate the quality of complex herbal preparations by HPTLC and extends the application of HPTLC fingerprint chromatography for identifying herbal medicine species and activity-related quality detection. The proposed strategy is also helpful for the quality control of other complex herbal preparations.

Isolation, characterization, and genomic analysis of a novel bacteriophage MA9V-1 infecting Chryseobacterium indologenes: a pathogen of Panax notoginseng root rot.

Chryseobacterium indologenes is one of the primary causative agents of root rot of Panax notoginseng, which significantly affected plant growth and caused economic losses. With the increasing incidence of antibiotic-resistant bacterial phytopathogens, phage therapy has been garnered renewed attention in treating pathogenic bacteria. However, the therapeutic potential of phage therapy on root rot of P. notoginseng has not been evaluated. In this study, we isolated a novel lytic phage MA9V-1 infecting C. indologenes MA9 from sewage and monitored the formation of clear and round plaques with a diameter of approximately 0.5-1.5 mm. Phage MA9V-1 exhibited rapid absorption (>75% in 8 min), a latency period of 20 min, and a burst size of 10 particles per cell. Transmission electron microscopy indicated that the phage MA9V-1 is a new myovirus hosting C. indologenes MA9. Sequencing of phage genomes revealed that phage MA9V-1 contained a linear double-stranded DNA genome of 213,507 bp with 263 predicted open reading frames, including phage structure, host lysing, and DNA polymerase/helicase but no genes of tRNA, virulence, and antibiotic resistance. Our proteomic tree and genomic analysis revealed that phage MA9V-1 shares identity with Sphingomonas phage PAU and Tenacibaculum phage PTm1; however, they also showed apparent differences. Further systemic evaluation using phage therapy experiments on P. notoginseng suggested that phage MA9V-1 can be a potential candidate for effectively controlling C. indologenes MA9 infection. Thus, we have presented a novel approach to solving root rot in P. notoginseng.

Viral infections inhibit saponin biosynthesis and photosynthesis in Panax notoginseng.

Virus-infected Panax notoginseng plants with chlorotic, mosaic, and pitted leaves are ubiquitous in the primary P. notoginseng-producing region in Wenshan autonomous prefecture, Yunnan province, China. However, the viruses that infect P. notoginseng and the effects of viral infections on the biosynthesis of secondary metabolites and photosynthesis remain unknown. This study identified a variety of viruses infecting P. notoginseng plants via deep-sequencing of small RNA (sRNA). Of the 10 identified viruses, seven had not previously been detected in P. notoginseng, including Cauliflower mosaic virus and Soybean chlorotic mottle virus. In addition, the simultaneous infection of P. notoginseng by Panax notoginseng virus A (PnVA), Panax cryptic virus 4 (PCV4), and Tomato yellow leaf curl China virus (TYLCCNV) was confirmed by PCR. Moreover, a quantitative PCR analysis showed that the expression levels of key genes related to saponin biosynthesis were generally down-regulated in the virus-infected P. notoginseng. Additionally, high-performance liquid chromatography results indicated the saponin content decreased in the roots of virus-infected P. notoginseng plants. The activities of photosynthesis-related enzymes, including ribulose-1,5-bisphosphate carboxylase/oxygenase, fructose 1,6-bisphosphatase, and fructose 1,6-biphosphate aldolase, decreased significantly in the virus-infected P. notoginseng plants. The viral infections also induced the expression of antioxidant genes and increased antioxidant enzyme activities. Furthermore, the expression levels of many resistance-related genes were up-regulated in P. notoginseng plants inoculated with a viral suspension. The study results provide the foundation for future research on P. notoginseng viral diseases, which may lead to the development of enhanced disease control measures.

Rho2 involved in development, stress response and pathogenicity of Fusarium oxysporum.

Rho GTPases regulate the activity of cell wall biosynthesis, actin assembly and polar cell secretion. However, the function of Rho GTPase in filamentous fungi is poorly understood. To understand the role of Rho2 GTPase in Fusarium oxysporum, which is one of root rot pathogens of Panax notoginseng, △rho2 mutant was constructed. Phenotypes of △rho2, including conidiation, germination of spores, stresses (osmotic-, cell membrane-, cell wall disturbing-, metal-, and high temperature-) tolerance and pathogenicity were analyzed. The results showed that the growth of △rho2 was destroyed under cell wall disturbing stress and high temperature stress, suggesting that Rho2 regulated the response of F. oxysporum to cell wall synthesis inhibitors and high temperature stress. Germination of spores and pathogenicity to P. notoginseng were reduced in △rho2 mutant. Western blot results showed that rho2 deletion increased the phosphorylation level of Mpk1. To identify genes regulated by Rho2, transcriptome sequencing was carried out. 2477 genes were identified as upregulated genes and 2177 genes were identified as downregulated genes after rho2 was deleted. These genes provide clues for further study of rho2 function.

[Effects of propiconazole on physiological and biochemical properties of Panax notoginseng and dietary risk assessment].

To study the residue and dietary risk of propiconazole in Panax notoginseng and the effects on physiological and bioche-mical properties of P. notoginseng, we conducted foliar spraying of propiconazole on P. notoginseng in pot experiments. The physiolo-gical and biochemical properties studied included leaf damage, osmoregulatory substance content, antioxidant enzyme system, non-enzymatic system, and saponin content in the main root. The results showed that at the same application concentration, the residual amount of propiconazole in each part of P. notoginseng increased with the increase in the times of application and decreased with the extension of harvest interval. After one-time application of propiconazole according to the recommended dose(132 g·hm~(-2)) for P. ginseng, the half-life was 11.37-13.67 days. After 1-2 times of application in P. notoginseng, propiconazole had a low risk of dietary intake and safety threat to the population. The propiconazole treatment at the recommended concentration and above significantly increased the malondialdehyde(MDA) content, relative conductivity, and osmoregulatory substances and caused the accumulation of reactive oxygen species in P. notoginseng leaves. The propiconazole treatment at half(66 g·hm~(-2)) of the recommended dose for P. ginseng significantly increased the activities of superoxide dismutase(SOD), peroxidase(POD), and catalase(CAT) in P. notoginseng leaves. The propiconazole treatment at 132 g·hm~(-2) above inhibited the activities of glutathione reductase(GR) and glutathione S-transferase(GST), thereby reducing glutathione(GSH) content. Proconazole treatment changed the proportion of 5 main saponins in the main root of P. notoginseng. The treatment with 66 g·hm~(-2) propiconazole promoted the accumulation of saponins, while that with 132 g·hm~(-2) and above propiconazole significantly inhibited the accumulation of saponins. In summary, using propiconazole at 132 g·hm~(-2) to prevent and treat P. notoginseng diseases will cause stress on P. notoginseng, while propiconazole treatment at 66 g·hm~(-2) will not cause stress on P. notoginseng but promote the accumulation of saponins. The effect of propiconazole on P. notoginseng diseases remains to be studied.

[Physiological and biochemical mechanisms of brassinosteroid in improving anti-cadmium stress ability of Panax notoginseng].

In this study, the effect of brassinosteroid(BR) on the physiological and biochemical conditions of 2-year-old Panax notoginseng under the cadmium stress was investigated by the pot experiments. The results showed that cadmium treatment at 10 mg·kg~(-1) inhibited the root viability of P. notoginseng, significantly increased the content of H_2O_2 and MDA in the leaves and roots of P. noto-ginseng, caused oxidative damage of P. notoginseng, and reduced the activities of SOD and CAT. Cadmium stress reduced the chlorophyll content of P. notoginseng, increased leaf F_o, reduced F_m, F_v/F_m, and PIABS, and damaged the photosynthesis system of P. notoginseng. Cadmium treatment increased the soluble sugar content of P. notoginseng leaves and roots, inhibited the synthesis of soluble proteins, reduced the fresh weight and dry weight, and inhibited the growth of P. notoginseng. External spray application of 0.1 mg·L~(-1) BR reduced the H_2O_2 and MDA content in P. notoginseng leaves and roots under the cadmium stress, alleviated cadmium-induced oxidative damage to P. notoginseng, improved the antioxidant enzyme activity and root activity of P. notoginseng, increased the content of chlorophyll, reduced the F_o of P. notoginseng leaves, increased F_m, F_v/F_m, and PIABS, alleviated the cadmium-induced damage to the photosynthesis system, and improved the synthesis ability of soluble proteins. In summary, BR can enhance the anti-cadmium stress ability of P. notoginseng by regulating the antioxidant enzyme system and photosynthesis system of P. notoginseng under the cadmium stress. In the context of 0.1 mg·L~(-1) BR, P. notoginseng can better absorb and utilize light energy and synthesize more nutrients, which is more suitable for the growth and development of P. notoginseng.

Effect of different cooking methods on saponin content and hematopoietic effects of Panax notoginseng-steamed chicken on mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax notoginseng-steamed chicken (PNSC) is a medicinal food with ethnic characteristics developed by the Miao ethnic group in the southeast of Yunnan Province, China. PNSC has been eaten for hundreds of years, and its tonic effect has been widely recognized by the people. However, its cooking conditions and scientific connotation of its effect of toning blood and supplementing deficiency are also lack of in-depth analysis. AIM OF THE STUDY: To optimize the cooking conditions of Panax notoginseng-steamed chicken (PNSC) and to explore its anemia-improving effects. MATERIALS AND METHODS: The ratio of P. notoginseng (PN) to chicken and the steaming time were systematically altered, and the PNSC cooking conditions was optimized with the response surface method. By establishing animal models of postpartum blood-deficiency anemia, acute hemorrhagic anemia and myelosuppressive anemia, the blood replenishing effect of PNSC was explored, and the blood replenishing mechanism of PNSC on myelosuppressive anemia was revealed by immunoblotting analyses and histopathological sectioning. RESULTS: The optimal processing conditions included a ratio of chicken to P. notoginseng of 100:5 and a steaming time of 5.5 h. The amounts of P. notoginseng polysaccharides (PNPS), total protein and blood-enriching P. notoginseng saponins were 44.3 mg/g, 2.48% and 2.04%, respectively. Freeze-dried powder of P. notoginseng steamed chicken soup (FPSC) was found to promote the recovery of routine blood factors and organ indexes in the three models of anemia and to activate the JAK2-STAT5 signaling pathway, induce phosphorylation of JAK2 and STAT5 and normalize the secretion of hematopoietic regulators EPO, IL-3, and TNF-α. CONCLUSION: FPSC improves the symptoms of anemia in mice, and it plays a role in tonifying blood by activating the JAK2-STAT5 signaling pathway and altering the expression of hematopoiesis-related factors.

From single- to multi-omics: future research trends in medicinal plants.

Medicinal plants are the main source of natural metabolites with specialised pharmacological activities and have been widely examined by plant researchers. Numerous omics studies of medicinal plants have been performed to identify molecular markers of species and functional genes controlling key biological traits, as well as to understand biosynthetic pathways of bioactive metabolites and the regulatory mechanisms of environmental responses. Omics technologies have been widely applied to medicinal plants, including as taxonomics, transcriptomics, metabolomics, proteomics, genomics, pangenomics, epigenomics and mutagenomics. However, because of the complex biological regulation network, single omics usually fail to explain the specific biological phenomena. In recent years, reports of integrated multi-omics studies of medicinal plants have increased. Until now, there have few assessments of recent developments and upcoming trends in omics studies of medicinal plants. We highlight recent developments in omics research of medicinal plants, summarise the typical bioinformatics resources available for analysing omics datasets, and discuss related future directions and challenges. This information facilitates further studies of medicinal plants, refinement of current approaches and leads to new ideas.

A new chamigrane sesquiterpene from the rice fermentation of Antrodiella albocinnamomea.

A new chamigrane sesquiterpene, albocimea A (1), and one known compound, 6-hydroxy-8-methoxy-3S,5-dimethylisochroman (2), were isolated from the rice fermentation of the fungus Antrodiella albocinnamomea. The structure of new compound was elucidated by extensive spectroscopic analyses and electronic circular dichroism (ECD) calculations. Both compounds were evaluated for their cytotoxicity against five human cancer cell lines, but no significant cytotoxicity was found (IC50 values > 40 µM).

First report of a Panax notoginseng leaf disease caused by Alternaria alternata in China.

Panax notoginseng (Burk.) F. H. Chen is a perennial plant species in the family Araliaceae, and its roots and rhizome are precious materials for the production of traditional Chinese medicine. From April to June, 2018, new disease symptoms were detected on the mature leaves of 2- and 3-year-old Panax notoginseng (P. notoginseng) in Wenshan Autonomous Prefecture, Yunnan Province, China, and the disease incidence was about 10%-15% among the analyzed fields (3.6 ha, 23°49'46.99″ N, 104°06'12.99″ E, 1,631 m elevation). The diseased leaves had dark brown necrotic lesions (0.9-2.5 × 1.0-3.5 cm) and curled downward. As the disease progressed, the necrosis gradually spread along the vein to other leaf parts, eventually covering the whole leaf. In the late disease stage, the whole leaf was decayed and yellowed. For pathogen isolation, infected leaves (n=20) were surface sterilized in 1% sodium hypochlorite and washed with sterilized distilled water for 3 mins before being cut into smaller pieces (~1cm2), then placed onto potato dextrose agar (PDA) medium and incubated at 28°C under aseptic conditions for 3 days. The hypha around leaf discs were transferred onto the new PDA. A total of 20 colonies (SQ1~20) were obtained and purified by single spore culture for morphological characterization and molecular biological identification. The colonies of all isolates were nearly round, grayish white at the initial stage, and then turned to grayish brown. In addition, microscopic examination (100× magnification) of 20 isolates revealed dark, septate, and sparsely branched conidiophores as well as dark brown conidia with short conical beaks at their tip. Additionally, conidia (solitary or in short chains) were typically oval or club-shaped and had 0-2 longitudinal septa and 2-4 transverse septa (20-35 × 8-12 µm) (n = 50). Moreover, the conidia had a smooth or verrucose surface. Their morphological characteristics were similar to those descriptions given for members of section Alternaria by Lawrence et al. (2016). In order to further identify pathogenic species, genomic DNA was extracted from the colonies (SQ1~20) using a modified cetyltrimethylammonium bromide (CTAB) method (Loganathan et al. 2014). The sequences of internal transcribed spacer regions of ribosomal DNA (rDNA ITS) and partial RNA polymerase II second subunit gene (RPB2) were amplified by PCR using fungal universal primers ITS1/ITS4 (White et al. 1990) and fRPB2-5F/fRPB2-7cR (Liu et al. 1999), respectively. The DNA sequencing shows that ITS sequences from 20 isolates were totally same, and so did the RPB2 sequences (supplementary material). BLASTN analysis of NCBI database indicated that the RPB2 and ITS sequences have the highest nucleotide homology to A. Alternata ITS (MW008974) and RPB2 (LC132700), respectively. These two gene sequences were submitted to GenBank [Accession numbers ON075466 (ITS) and OP572232 (RPB2)]. Phylogenetic trees based on the combined ITS and RPB2 sequences were constructed by maximum parsimony method. The referenced ITS and RPB2 sequences of Alternaria were from three published articles (Rama et al. 2020; Sun et al. 2021; Wee et al. 2006). Phylogenetic analysis revealed that this isolate was clustered with A. alternata. Therefore, the morphology-based preliminary identification was verified by the phylogenetic analysis, and the isolate from diseased P. notoginseng leaves was A. alternata. To confirm its pathogenicity, the fungal isolate was assessed with 40 1-year-old healthy P. notoginseng plants in a greenhouse. Among them, the leaves of 20 of P. notoginseng plants were wounded using a sterile needle (seven or eight wounds per leaf) and then inoculated with 1mL conidial suspension (1 × 106 conidia/mL) prepared from 7-day-old fungal cultures grown on PDA medium. The inoculated plants were covered with plastic bags at 25°C for 24 h to maintain humidity, and then transferred to the greenhouse maintained at 28°C with a 16-h day/8-h night cycle and continuous misting. The other 20 control plants were only wounded and sprayed with sterile water. Typical necrotic lesions were detected on all of the inoculated P. notoginseng leaves cultivated in the greenhouse for 1 week post-inoculation. As the disease continued to develop, the necrotic lesions enlarged, and the infected leaves turned yellow and withered. These symptoms were similar to those observed on the naturally infected P. notoginseng. In contrast, the mock-inoculated control plants remained healthy. Furthermore, the fungus re-isolated from the infected P. notoginseng leaves in the pot experiment had similar morphological characteristics as the original strain. In addition, its genomic DNA was extracted for PCR analysis of ITS and RPB2 sequences, and the following DNA sequencing shows that the two DNA sequences were same as those of isolates SQ1~20, which confirmed that the re-isolated fungus was A. alternata. To the best of our knowledge, this is the first report of A. alternata causing a P. notoginseng leaf disease in China.

Deep Eutectic Systems as Novel Vehicles for Assisting Drug Transdermal Delivery.

In recent years, deep eutectic systems (DES) emerged as novel vehicles for facilitating the transdermal delivery of various drugs, including polysaccharides, proteins, insulin, vaccine, nanoparticles, and herb extracts. The objective of this study is to conduct a comprehensive review of the application of DES to transdermal drug delivery, based on previous work and the reported references. Following a brief overview, the roles of DES in TDDS, the modes of action, as well as the structure-activity relationship of DES are discussed. Particularly, the skin permeation of active macromolecules and rigid nanoparticles, which are the defining characteristics of DES, are extensively discussed. The objective is to provide a comprehensive understanding of the current investigation and development of DES-based transdermal delivery systems, as well as a framework for the construction of novel DES-TDDS in the future.

Production of Minor Ginsenosides from Panax notoginseng Flowers by Cladosporium xylophilum.

Panax notoginseng flowers have the highest content of saponins compared to the other parts of Panax notoginseng, but minor ginsenosides have higher pharmacological activity than the main natural ginsenosides. Therefore, this study focused on the transformation of the main ginsenosides in Panax notoginseng flowers to minor ginsenosides using the fungus of Cladosporium xylophilum isolated from soil. The main ginsenosides Rb1, Rb2, Rb3, and Rc and the notoginsenoside Fa in Panax notoginseng flowers were transformed into the ginsenosides F2 and Rd2, the notoginsenosides Fd and Fe, and the ginsenoside R7; the conversion rates were 100, 100, 100, 88.5, and 100%, respectively. The transformation products were studied by TLC, HPLC, and MS analyses, and the biotransformation pathways of the major ginsenosides were proposed. In addition, the purified enzyme of the fungus was prepared with the molecular weight of 66.4 kDa. The transformation of the monomer ginsenosides by the crude enzyme is consistent with that by the fungus. Additionally, three saponins were isolated from the transformation products and identified as the ginsenoside Rd2 and the notoginsenosides Fe and Fd by NMR and MS analyses. This study provided a unique and powerful microbial strain for efficiently transformating major ginsenosides in P. notoginseng flowers to minor ginsenosides, which will help raise the functional and economic value of the P. notoginseng flower.

Panax notoginseng WRKY Transcription Factor 9 Is a Positive Regulator in Responding to Root Rot Pathogen Fusarium solani.

Panax notoginseng (Burk) F.H. Chen is a rare and valuable Chinese herb, but root rot mainly caused by Fusarium solani severely affects the yield and quality of P. notoginseng herbal materials. In this study, we isolated 30 P. notoginseng WRKY transcription factors (TFs), which were divided into three groups (I, II, and III) on the basis of a phylogenetic analysis. The expression levels of 10 WRKY genes, including PnWRKY9, in P. notoginseng roots increased in response to a methyl jasmonate (MeJA) treatment and the following F. solani infection. Additionally, PnWRKY9 was functionally characterized. The PnWRKY9 protein was localized to the nucleus. The overexpression of PnWRKY9 in tobacco (Nicotiana tabacum) considerably increased the resistance to F. solani, whereas an RNAi-mediated decrease in the PnWRKY9 expression level in P. notoginseng leaves increased the susceptibility to F. solani. The RNA sequencing and hormone content analyses of PnWRKY9-overexpression tobacco revealed that PnWRKY9 and the jasmonic acid (JA) signaling pathway synergistically enhance disease resistance. The PnWRKY9 recombinant protein was observed to bind specifically to the W-box sequence in the promoter of a JA-responsive and F. solani resistance-related defensin gene (PnDEFL1). A yeast one-hybrid assay indicated that PnWRKY9 can activate the transcription of PnDEFL1. Furthermore, a co-expression assay in tobacco using ß-glucuronidase (GUS) as a reporter further verified that PnWRKY9 positively regulates PnDEFL1 expression. Overall, in this study, we identified P. notoginseng WRKY TFs and demonstrated that PnWRKY9 positively affects plant defenses against the root rot pathogen. The data presented herein provide researchers with fundamental information regarding the regulatory mechanism mediating the coordinated activities of WRKY TFs and the JA signaling pathway in P. notoginseng responses to the root rot pathogen.

Herb Polysaccharide-Based Drug Delivery System: Fabrication, Properties, and Applications for Immunotherapy.

Herb polysaccharides (HPS) have been studied extensively for their healthcare applications. Though the toxicity was not fully clarified, HPS were widely accepted for their biodegradability and biocompatibility. In addition, as carbohydrate polymers with a unique chemical composition, molecular weight, and functional group profile, HPS can be conjugated, cross-linked, and functionally modified. Thus, they are great candidates for the fabrication of drug delivery systems (DDS). HPS-based DDS (HPS-DDS) can bypass phagocytosis by the reticuloendothelial system, prevent the degradation of biomolecules, and increase the bioavailability of small molecules, thus exerting therapeutic effects. In this review, we focus on the application of HPS as components of immunoregulatory DDS. We summarize the principles governing the fabrication of HPS-DDS, including nanoparticles, micelles, liposomes, microemulsions, hydrogels, and microneedles. In addition, we discuss the role of HPS in DDS for immunotherapy. This comprehensive review provides valuable insights that could guide the design of effective HPS-DDS.

Steamed Panax notoginseng and its Saponins Inhibit the Migration and Induce the Apoptosis of Neutrophils in a Zebrafish Tail-Fin Amputation Model.

Panax notoginseng (PN) is a Chinese medicinal herb that is traditionally used to treat inflammation and immune-related diseases. Its major active constituents are saponins, the types and levels of which can be changed in the process of steaming. These differences in saponins are causally relevant to the differences in the therapeutic efficacies of raw and steamed PN. In this study, we have prepared the extracts of steamed PN (SPNE) with 70% ethanol and investigated their immunomodulatory effect using a zebrafish tail-fin amputation model. A fingerprint-effect relationship analysis was performed to uncover active constituents of SPNE samples related to the inhibitory effect on neutrophil number. The results showed that SPNE significantly inhibited the neutrophil number at the amputation site of zebrafish larvae. And SPNE extracts steamed at higher temperatures and for longer time periods showed a stronger inhibitory effect. Ginsenosides Rh1, Rk3, Rh4, 20(S)-Rg3, and 20(R)-Rg3, of which the levels were increased along with the duration of steaming, were found to be the major active constituents contributing to the neutrophil-inhibiting effect of SPNE. By additionally investigating the number of neutrophils in the entire tail of zebrafish larvae and performing TUNEL assays, we found that the decreased number of neutrophils at the amputation site was due to both the inhibition of their migration and apoptosis-inducing effects of the ginsenosides in SPNE on neutrophils. Among them, Rh1 and 20(R)-Rg3 did not affect the number of neutrophils at the entire tail, suggesting that they only inhibit the migration of neutrophils. In contrast, ginsenosides Rk3, Rh4, 20(S)-Rg3, and SPNE did not only inhibit the migration of neutrophils but also promoted neutrophilic cell death. In conclusion, this study sheds light on how SPNE, in particular the ginsenosides it contains, plays a role in immune modulation.

Increasing Expression of PnGAP and PnEXPA4 Provides Insights Into the Enlargement of Panax notoginseng Root Size From Qing Dynasty to Cultivation Era.

Root size is a key trait in plant cultivation and can be influenced by the cultivation environment. However, physical evidence of root size change in a secular context is scarce due to the difficulty in preserving ancient root samples, and how they were modified during the domestication and cultivation stays unclear. About 100 ancient root samples of Panax notoginseng, preserved as tribute in the Palace Museum (A.D. 1636 to 1912, Qing dynasty), provided an opportunity to investigate the root size changes during the last 100 years of cultivation. The dry weight of ancient root samples (~120 tou samples, tou represents number of roots per 500 g dry weight) is 0.22-fold of the modern samples with the biggest size (20 tou samples). Transcriptome analysis revealed that PnGAP and PnEXPA4 were highly expressed in 20 tou samples, compared with the 120 tou samples, which might contribute to the thicker cell wall and a higher content of lignin, cellulose, and callose in 20 tou samples. A relatively lower content of dencichine and higher content of ginsenoside Rb1 in 20 tou samples are also consistent with higher expression of ginsenoside biosynthesis-related genes. PnPHL8 was filtrated through transcriptome analysis, which could specifically bind the promoters of PnGAP, PnCYP716A47, and PnGGPPS3, respectively. The results in this study represent the first physical evidence of root size changes in P. notoginseng in the last 100 years of cultivation and contribute to a comprehensive understanding of how the cultivation environment affected root size, chemical composition, and clinical application.

Market access for Chinese herbal medicinal products in Europe-A ten-year review of relevant products, policies, and challenges.

BACKGROUND: With increased consumer demand in Europe for natural and efficacious health products, the use of herbal products in the market is rising. Products of Chinese herbal medicine (CHM) could greatly expand European consumer options; however, only seven herbal medicinal products (HMPs) based on CHM formulae have been registered in the European Union (EU) since 2012. PURPOSE: This study reviews the ten-year registration status of HMPs based on CHM formulae in Europe and identifies major challenges and possible solutions for pharmaceutical companies seeking market access for new HMPs. METHODS: An overview of relevant EU regulations identifies pathways to market access in EU countries for CHM products. A discussion of successful attempts to register HMPs based on CHM formulae since 2012 highlights specific challenges that applicants can expect to face. RESULTS: CHM products can enter the EU market as HMPs through the full or well-established use marketing authorization, or through the simplified registration procedure. Alternatively, some CHM products have entered the market as dietary supplements, nutritional foods, and agricultural products; however, under these categories, claims for medicinal use cannot be advertised. Since the registration of the first CHM product, Diao Xin Xue Kang (with the single component of Dioscorea nipponica rhizome), in 2012, only six other HMPs based on CHM formulae have been successfully registered. Among these, four are mono-component products. The remaining two products contain combinations of several herbal ingredients. It is more difficult to register combination products than mono-component products, due to their more complex composition and differences in registration requirements (esp. concerning establishing indications) in China and Europe. CONCLUSIONS: To promote the successful registration of CHM products in Europe, pharmaceutical companies are advised to: demonstrate full control of, and the ability to test, their supply chain and manufacturing procedures following the guidance of European competent authorities; carefully adhere to all steps of the registration process and advices from European competent authorities; take the medication habits and pharmaceutical needs of European market into consideration; and establish collaboration with European local organizations, as appropriate.

Highly Regioselective Biotransformation of Protopanaxadiol-type and Protopanaxatriol-type Ginsenosides in the Underground Parts of Panax notoginseng to 18 Minor Ginsenosides by Talaromyces flavus.

The transformation of major ginsenosides to minor ginsenosides by microorganisms was considered to be an environmentally friendly method. Compared with GRAS (generally recognized as safe) strains, non-food-grade microorganisms could transform polar ginsenosides to various minor ginsenosides. In this study, Talaromyces flavus screened from the P. notoginseng rhizosphere was capable of transforming PPD-type and PPT-type ginsenosides in the underground parts of P. notoginseng to 18 minor ginsenosides. The transformation reactions invovled deglycosylation, epimerization, and dehydration. To the best of our knowledge, this transformation characteristic of T. flavus was first reported in fungi. Its crude enzyme can efficiently hydrolyze the outer glucose linked to C-20 and C-3 in major ginsenosides Rb1, Rb2, Rb3, Rc, Rd, and 20(S)-Rg3 within 48 h. The transformation of major ginsenosides to minor ginsenosides by T. flavus will help raise the functional and economic value of P. notoginseng.