Integrated physiological, transcriptomics and metabolomics analysis revealed the molecular mechanism of Bupleurum chinense seedlings to drought stress.

Drought stress is a prominent abiotic factor that adversely influences the growth and development of Bupleurum chinense during its seedling stage, negatively impacting biomass and secondary metabolite production, thus affecting yield and quality. To investigate the molecular mechanism underlying the response of B. chinense seedlings under drought stress, this study employed comprehensive physiological, transcriptomic, and metabolomic analyses. The results revealed that under drought stress, the root soluble sugar and free proline content in B. chinense seedlings significantly increased, while the activities of SOD, POD, and CAT increased in the leaves. These findings indicate the presence of distinct response mechanisms in B. chinense to cope with drought stress. Integrated analysis further identified significant correlations between genes and metabolites related to amino acid biosynthesis in the leaves, as well as genes and metabolites associated with acetaldehyde and dicarboxylic acid metabolism. In the roots, genes and metabolites related to plant hormone signaling and the tricarboxylic acid (TCA) cycle showed significant correlations. These findings provide vital views into the molecular-level response mechanisms of B. chinense under drought stress. Moreover, this study establishes the groundwork for identifying drought-tolerant genes and breeding drought-resistant varieties, which could improve the drought tolerance of medicinal plants and have broader implications for agriculture and crop production in water-scarce areas.

Intercropping with maize and sorghum-induced saikosaponin accumulation in Bupleurum chinense DC. by liquid chromatography-mass spectrometry-based metabolomics.

Bupleuri Radix is an important medicinal plant, which has been used in China and other Asian countries for thousands of years. Cultivated Bupleurum chinense DC. (B. chinense) is the main commodity of Bupleuri Radix. The benefits of intercropping with various crops for B. chinense have been recognized; however, the influence of intercropping on the chemical composition of B. chinense is still unclear yet. In this study, intercropping with sorghum and maize exhibited little effect on the root length, root diameter, and single root mass of B. chinense. Only the intercropping with sorghum increased the root length of B. chinense slightly compared to the monocropping. In addition, 200 compounds were identified by UHPLC-Q-TOF-MS, and metabolomic combined with the Venn diagram and heatmap analysis showed apparent separation between the intercropped and monocropped B. chinense samples. Intercropping with sorghum and maize could both increase the saikosaponins, fatty acyls, and organic acids in B. chinense while decreasing the phospholipids. The influence of intercropping on the saikosaponin biosynthesis was probably related with the light intensity and hormone levels in B. chinense. Moreover, we found intercropping increased the anti-inflammatory activity of B. chinense. This study provides a scientific reference for the beneficial effect of intercropping mode of B. chinense.

Bupleurum exerts antiarrhythmic effects by inhibiting L-type calcium channels in mouse ventricular myocytes.

BACKGROUND: Bupleurum (Bup), is a traditional effective medicine to treat colds and fevers in clinics. Multiple studies have demonstrated that Bup exhibites various biological activities, including cardioprotective effects, anti-inflammatory, anticancer, antipyretic, antimicrobial, and antiviral effects, etc. Currently, the effects of Bup on cardiac electrophysiology have not been reported yet. METHODS: Electrocardiogram recordings were used to investigate the effects of Bup on aconitine-induced arrhythmias. Patch-clamp techniques were used to explore the effects of Bup on APs and ion currents. RESULTS: Bup reduced the incidence of ventricular fibrillation (VF) and delayed the onset time of ventricular tachycardia (VT) in mice. Additionally, Bup (40 mg/mL) suppressed DADs induced by high-Ca2+ and shortened action potential duration at 50 % completion of repolarization (APD50) and action potential duration at 90 % completion of repolarization (APD90) to 60.89 % ± 8.40 % and 68.94 % ± 3.24 % of the control, respectively. Moreover, Bup inhibited L-type calcium currents (ICa.L) in a dose-dependent manner, with an IC50 value of 25.36 mg/mL. Furthermore, Bup affected the gated kinetics of L-type calcium channels by slowing down steady-state activation, accelerating the steady-state inactivation, and delaying the inactivation-recovery process. However, Bup had no effects on the Transient sodium current (INa.T), ATX II-increased late sodium current (INa.L), transient outward current (Ito), delayed rectifier potassium current (IK), or inward rectifier potassium current (IK1). CONCLUSION: Bup is an antiarrhythmic agent that may exert its antiarrhythmic effects by inhibiting L-type calcium channels.

Transcriptome Level Reveals the Triterpenoid Saponin Biosynthesis Pathway of Bupleurum falcatum L.

Bupleurum falcatum L. is frequently used in traditional herbal medicine in Asia. Saikosaponins (SSs) are the main bioactive ingredients of B. falcatum, but the biosynthetic pathway of SSs is unclear, and the biosynthesis of species-specific phytometabolites is little known. Here we resolved the transcriptome profiles of B. falcatum to identify candidate genes that might be involved in the biosynthesis of SSs. By isoform sequencing (Iso-Seq) analyses of the whole plant, a total of 26.98 Gb of nucleotides were obtained and 124,188 unigenes were identified, and 81,594 unigenes were successfully annotated. A total of 1033 unigenes of 20 families related to the mevalonate (MVA) pathway and methylerythritol phosphate (MEP) pathway of the SS biosynthetic pathway were identified. The WGCNA (weighted gene co-expression network analysis) of these unigenes revealed that only the co-expression module of MEmagenta, which contained 343 unigenes, was highly correlated with the biosynthesis of SSs. Comparing differentially expressed gene analysis and the WGCNA indicated that 130 out of 343 genes of the MEmagenta module exhibited differential expression levels, and genes with the most "hubness" within this module were predicted. Manipulation of these genes might improve the biosynthesis of SSs.

The Polysaccharides from the Aerial Parts of Bupleurum chinense DC Attenuate Epilepsy-Like Behavior through Oxidative Stress Signaling Pathways.

Bupleurum chinense DC. is a traditional Chinese medicine with a long medicinal history and is often used as the main ingredient in prescription drugs for epilepsy. The aerial parts of B. chinense DC. have similar efficacy and composition to B. chinense DC. Therefore, we speculated that the aerial parts of B. chinense DC. could be used in the treatment of epilepsy. Polysaccharides from the aerial parts of B. chinense DC. were selected to explore their therapeutic effects on epilepsy and their potential mechanism of action. The study is aimed at clarifying the antiepileptic effects of the polysaccharides from the aerial parts of B. chinense DC. and their potential underlying mechanisms. The chemical profile of the aerial parts of B. chinense DC. polysaccharides (ABP) was characterized by FT-IR spectrum and HPLC chromatogram. To determine the therapeutic effects of ABPs on epilepsy, we established a kainic acid- (KA-) induced rat model of epilepsy, and through H&E staining, Nissl staining, immunohistochemistry, biochemical analysis, ELISA, and Western blot analysis, we explored the mechanisms underlying the therapeutic effects of ABPs on epilepsy. The monosaccharide content of ABP included galacturonic acid (45.19%), galactose (36.63%), arabinose rhamnose (12.13%), and mannose (6.05%). Moreover, the average molecular weight of ABP was 1.38 × 103 kDa. ABP could improve hippocampal injuries and neuronal function in the KA-induced epilepsy rat model. ABP significantly inhibited oxidative stress in the hippocampus of KA-induced rats. More importantly, ABP could regulate TREM2 activation in the PI3K/Akt/GSK-3ß pathway to inhibit neuronal apoptosis, including increasing the expression of superoxide dismutase and lactate dehydrogenase and decreasing the expression of malondialdehyde. The current study defined the potential role of ABP in inhibiting the development of epilepsy, indicating that ABP could upregulate TREM2 to alleviate neuronal apoptosis, by activating the PI3K/Akt/GSK-3ß pathway and oxidative stress in epilepsy.

Overexpression of BcERF3 increases the biosynthesis of saikosaponins in Bupleurum chinense.

Chaihu, the dried roots of some species of Bupleurum L., is a famous Chinese herbal medicine for treatment of liver- and cold-related diseases, in which saikosaponins (SSs) are the major active compounds. Many of the genetic components upstream of SS biosynthetic pathways have been characterized; however, the regulatory mechanisms remain elusive. In this study we identified the APETALA2/Ethylene Responsive Factor family transcription factor gene BcERF3 from B. chinense. The expression of BcERF3 was induced in methyl-jasmonate-treated adventitious root of B. chinense; it was also expressed at higher levels in roots than in other tissues (stem, leaf, flower, and tender fruit of early fruiting plants). Transient expression of BcERF3 in the leaves of Nicotiana benthamiana resulted in intracellular localization of the protein in the nucleus. It was also demonstrated that the number of SSs was greater in BcERF3-overexpressing hairy roots of B. chinense than in plants treated with empty vector controls. This coincided with upregulation of ß-AS, which encodes a key enzyme involved with triterpenoid biosynthesis. In conclusion, BcERF3 plays a positive regulatory role in the biosynthesis of SSs.

Antitumor Effect of Saikosaponin A on Human Neuroblastoma Cells.

OBJECTIVE: Neuroblastoma (NB) is a highly metastatic tumor in children that develops in the sympathetic nervous system and has a low curative rate. Saikosaponin A (SSA), an active ingredient isolated from the root of Radix Bupleuri, is a natural compound with various pharmacological activities and shows good application prospects in antitumors. This study investigated the antihuman NB activity of SSA and underlying mechanisms associated with its actions. MATERIALS AND METHODS: The MTT method was used to detect the activity of SSA in inhibiting human NB cell SK-N-AS proliferation. Cell morphology was observed. The flow cytometry technology was used in analyzing the cell apoptosis rate. The Transwell assay evaluated cell migration and invasion following SSA treatment, apoptosis-related protein expression, and angiogenesis-related protein expression, and EMT-related proteins were detected by western blot analysis. RESULTS: SSA showed an inhibitory effect on SK-N-AS cells with the IC50 values of 14.14 µM at 24 h and 12.41 µM at 48 h. Results indicated that SSA has proapoptotic activity, and its proapoptotic activity is positively correlated with the Bax/Bcl-2/caspase-9/caspase-7/PARP pathway. Furthermore, SSA inhibited the invasion and migration of SK-N-AS cells via regulating the angiogenesis-related VEGFR2/Src/Akt pathway and the epithelial-mesenchymal transition- (EMT-) related protein expression. CONCLUSION: SSA exerts an antihuman NB effect and thus provides foundations for NB treatment.

Comparison of chemical constituents of Bupleurum marginatum var. stenophyllum and Bupleurum chinense DC. using UHPLC-Q-TOF-MS based on a metabonomics approach.

The overall chemical composition of Bupleurum marginatum var. stenophyllum and Bupleurum chinense DC. was compared in this study. Metabolites were identified using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. Multivariate statistical analysis techniques such as principal component analysis were used to conduct metabonomics analysis and study the correlation between different components. Principal component analysis results showed a clear distinction among medicinal materials of different origins and divided them into different categories, consistent with the results of hierarchical cluster analysis. Both partial least squares discriminant analysis (PLS-DA) and orthogonal partial least squares discriminant analysis (OPLS-DA) showed that the two materials could be distinguished clearly. Using PLS-DA and OPLS-DA combined with the S-plot and a variable importance in the projection (VIP) score >1, 24 differential metabolites were screened and identified; all of the metabolites were triterpenoid saponins. In addition, SPSS 25.0 and Metabo Analyst 4.0 were used to analyze significant differences in the relative contents of different compounds in the two materials. This study has successfully provided not only a new direction for research based on the chemical substances identified and the quality evaluation of Bupleuri Radix but also a better theoretical basis for the expansion of medicinal sources and their clinical application.

Recent Progress in Saikosaponin Biosynthesis in Bupleurum.

BACKGROUND: Chaihu is a popular traditional Chinese medicine that has been used for centuries. It is traditionally used to treat cold fever and liver-related diseases. Saikosaponins (SSs) are one of the main active components of chaihu, in addition to essential oils, flavonoids, and polysaccharides. Considerable effort is needed to reveal the biosynthesis and regulation of SSs on the basis of current progress. OBJECTIVE: The aim of this study is to provide a reference for further studies and arouse attention by summarizing the recent achievements of SS biosynthesis. METHODS: All the data compiled and presented here were obtained from various online resources, such as PubMed Scopus and Baidu Scholar in Chinese, up to October 2019. RESULTS: A few genes of the enzymes of SSs participating in the biosynthesis of SSs were isolated. Among these genes, only the P450 gene was verified to catalyze the SS skeleton ß-amyrin synthase. Several UDP-glycosyltransferase genes were predicted to be involved in the biosynthesis of SSs. SSs could be largely biosynthesized in the phloem and then transported from the protoplasm, which is the biosynthetic site, to the vacuoles to avoid self-poisoning. As for the other secondary metabolites, the biosynthesis of SSs was strongly affected by environmental factors and the different species belonging to the genus of Bupleurum. Transcriptional regulation was studied at the molecular level. CONCLUSION: Profound discoveries in SSs may elucidate the mechanism of diverse the monomer formation of SSs and provide a reference for maintaining the stability of SS content in Radix Bupleuri.

Multi-scale Gaussian/Haar wavelet strategies coupled with sub-window factor analysis for an accurate alignment in nontargeted metabolic profiling to enhance herbal origin discrimination capability.

Metabolic dataset can provide an overview of different herbal origin, which is conducted by some statistical procedures. Such results often deviate to a certain degree, due to peaks shifts in chromatographic signals. In order to solve this problem, an improved algorithm of combining sub-window factor analysis with the mass spectrum information is proposed. The algorithm uses a peak detection approach derived either from multi-scale Gaussian function or Haar wavelet to locate the peaks with different application scope; the candidate drift points at each peak are estimated by Fast Fourier transform cross correlation; Specifically, the best drift points at each candidate peaks are confirmed by sub-window factor analysis and mass spectrum information in nontargeted metabolic profiling. Finally, the peak regions were aligned against a reference chromatogram, and the non-peak regions were used linear interpolation. The chromatographic signals of 30 Bupleurum samples were aligned as an illustration of this algorithm, and they could be well distinguished using some statistical procedures. The result demonstrates that the presented method is stronger than other mass-spectra based algorithms, when facing the alignment of some co-eluted peaks.

Bio-functionalization of phytogenic Ag and ZnO nanobactericides onto cellulose films for bactericidal activity against multiple drug resistant pathogens.

The present study describes the synthesis of silver and zinc oxide nanobactericides from the phytogenic source Bupleurum aureum. The synthesized nanobactericides were characterized and evaluated for bio-functionalization onto bacterial cellulose membrane which was synthesized by Komagataeibacterxylinus B-12068 culture strain. The synthesis of nanobacterides were initially confirmed using UV-Visible spectroscopy which indicated localized surface resonance (LSPR) peaks at 415 nm for silver nanobactericides and 280 nm for zinc nanobactericides. The nature of the capping agent for synthesized nanobactericides was predicted using FTIR which confirmed the presence of functional moieties. XRD analysis revealed their crystalline nature while morphological characteristics were studied using TEM which confirmed the polydispersity of nanobactericides with the average size in the range of 20-25 nm. The nanobactericides were tested for their antimicrobial activity against seven multi-drug resistant pathogens which were clinically isolated from patients suffering from a myriad of microbial infections. The tested pathogens had antimicrobial resistance to ten different antibiotics and have been reported to be the major cause of nosocomial infections. The nanobactericides displayed significant activity against the test pathogens. Silver nanobactericides showed the highest activity against Escherichia coli strain 55 with a 24 mm zone of inhibition while zinc oxide nanobactericides displayed the highest activity against methicillin-resistant Staphylococcus aureus (MRSA) with a 20 mm inhibition zone. The bio- functionalized cellulose films (BCF) were characterized using SEM along with physicochemical analysis. The BCF's were evaluated for antibacterial activity against test pathogens which resulted in marked antimicrobial potential against multi-drug resistant bacteria and therefore has the potential to be utilized as an efficient alternative to counter drug resistant pathogens.

Large-scale analysis of small RNAs derived from traditional Chinese herbs in human tissues.

Plant-derived microRNAs have recently been reported to function in human blood and tissues. Controversy was immediately raised due to possible contamination and the lack of large sample sizes. Here, we report thousands of unique small RNAs derived from traditional Chinese medicine (TCM) herbs found in human blood cells and mouse lung tissues using a large-scale analysis. We extracted small RNAs from decoctions of 10 TCM plants (Ban Zhi Lian, Chai Hu, Chuan Xin Lian, Di Ding Zi Jin, Huang Qin, Jin Yin Hua, Lian Qiao, Pu Gong Ying, Xia Ku Cao, and Yu Xing Cao) and obtained millions of RNA sequences from each herb. We also obtained RNA-Seq data from the blood cells of humans who consumed herbal decoctions and from the lung tissues of mice administered RNAs from herbal decoctions via oral gavage. We identified thousands of unique small RNA sequences in human blood cells and mouse lung tissues. Some of these identified small RNAs from Chuan Xin Lian and Hong Jing Tian could be mapped to the genomes of the herbs, confirming their TCM plant origin. Small RNAs derived from herbs regulate mammalian gene expression in a sequence-specific manner, and thus are a superior novel class of herbal drug components that hold great potential as oral gene-targeted therapeutics, highlighting the important role of herbgenomics in their development.

Triple herbal extract DA-9805 exerts a neuroprotective effect via amelioration of mitochondrial damage in experimental models of Parkinson's disease.

Moutan cortex, Angelica Dahurica root, and Bupleurum root are traditional herbal medicines used in Asian countries to treat various diseases caused by oxidative stress or inflammation. Parkinson's disease (PD) has been associated with mitochondrial dysfunction, but no effective treatment for mitochondrial dysfunction has yet been identified. In this study we investigated the neuroprotective effects of the triple herbal extract DA-9805 in experimental models of PD. DA-9805 was prepared by extracting three dried plant materials (Moutan cortex, Angelica Dahurica root, and Bupleurum root in a 1:1:1 mixture) with 90% ethanol on a stirring plate for 24 h at room temperature and fingerprinted using high-performance liquid chromatography. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite 1-methyl-4-phenylpyridinium (MPP+), which both exert neurotoxic effects on dopaminergic neurons by inhibiting mitochondrial oxidative phosphorylation (OXPHOS) complex I, were used to make experimental models of PD. In MPP+-treated SH-SY5Y cells, DA-9805 ameliorated the suppression of tyrosine hydroxylase expression and mitochondrial damage on OXPHOS complex 1 activity, mitochondrial membrane potential, reactive oxygen species (ROS) generation, and oxygen consumption rate. In the MPTP-induced subacute PD model mice, oral administration of DA-9805 recovered dopamine content as well as bradykinesia, as determined by the rotarod test. DA-9805 protected against neuronal damage in the substantia nigra pars compacta (SNpc) and striatum. In both in vitro and in vivo models of PD, DA-9805 normalized the phosphorylation of AKT at S473 and T308 on the insulin signaling pathway and the expression of mitochondria-related genes. These results demonstrate that the triple herbal extract DA-9805 showed neuroprotective effects via alleviating mitochondria damage in experimental models of PD. We propose that DA-9805 may be a suitable candidate for disease-modifying therapeutics for PD.

Radix Bupleuri: A Review of Traditional Uses, Botany, Phytochemistry, Pharmacology, and Toxicology.

Radix Bupleuri (Chaihu) has been used as a traditional medicine for more than 2000 years in China, Japan, Korea, and other Asian countries. Phytochemical studies demonstrated that this plant contains essential oils, triterpenoid saponins, polyacetylenes, flavonoids, lignans, fatty acids, and sterols. Crude extracts and pure compounds isolated from Radix Bupleuri exhibited various biological activities, such as anti-inflammatory, anticancer, antipyretic, antimicrobial, antiviral, hepatoprotective, neuroprotective, and immunomodulatory effects. However, Radix Bupleuri could also lead to hepatotoxicity, particularly in high doses and with long-term use. Pharmacokinetic studies have demonstrated that the major bioactive compounds (saikosaponins a, b2, c, and d) were absorbed rapidly in rats after oral administration of the extract of Radix Bupleuri. This review aims to comprehensively summarize the traditional uses, botany, phytochemistry, pharmacology, toxicology, and pharmacokinetics of Radix Bupleuri reported to date with an emphasis on its biological properties and mechanisms of action.

[Expression of key enzyme genes and content of saikosaponin in saikosaponin biosynthesis under drought stress in Bupleurum chinense].

To research the expression of key enzymes in saikosaponin biosynthesis and the content of saikosaponin under the drought stress, the study focused on the gene-level and the end product responses to environmental change. Taking the five months of Bupleurum chinense as research materials, the contents of saikosaponin A and saikosaponin D under different stress levels were measured by HPLC. The drought was simulated by poly ethylene glycol. The real-time fluorescence quantitative PCR was used to analyze the expression of four key enzymes genes HMGR, IPPI, FPS, ß-AS and the expression of ß-tubulin was set as a reference gene. The results showed that drought stress significantly improved the content of saikosaponin. The contents of SSa and SSd were highest researching 0.648% and 0.781%, respectively when the concentration of PEG was 10%. Meanwhile, the results reflected that the expression of four key enzymes had risen differently and FPS, ß-AS raised significantly(P<0.01). In addition, the results of correlation analysis showed that there was a significant positive correlation between the expression of the four key enzymes genes and the content of saikosaponin. In a word, the contents of secondary metabolites were regulated by the expression of key enzymes genes under the drought stress in B. chinense.

[Effect of combined application of zinc, boron and molybdenum on yield and saikosaponin a, saikosaponin d contents of Bupleurum chinense].

This research use "3414" fertilizer effect experiments to handle zinc, boron and molybdenum trace element fertilizer, determined the dry matter accumulation and content of saikosaponion a and d, to investigate the different ratio of zinc, boron and molybdenum on yield and saikosaponin a, saikosaponin d contents of Bupleurum chinense. Found The suitable ratio of zinc, boron and molybdenum play an active role on dry matter accumulation and distribution, the treatment Zn2B2Mo3 is the best one to promote the dry matter accumulation and transfer to the underground part; in a certain range, only use zinc or molybdenum can promote the yield of B. chinense, the yield of treatment Zn2B2Mo1 is the highest one. According to the results of regression analysis: in accordance with Zn 48.45 g x hm(-2), B 355.05 g x hm(-2), Mo 86.40 g x hm(-2), can obtain the yield with 3313.05 kg x hm(-2); the treatment Zn2BMo2 is most effective to promote the total saikosaponin a and d accumulated, according to the results of regression analysis: in accordance with Zn 36.15 g x hm(-2), B 343.05 g x hm(-2), Mo 106.35 g x hm(-2), the content of total saikosaponin a and d can reach 1.23%. This research first discovered the suitable ratio of zinc, boron and molybdenum can promote the yield and saikosaponin a, saikosaponin d contents on B. chinense.

[Studies on high efficient plant regeneration system from anther callus of Bupleurum chinense].

The callus of Bupleurum chinense with anthers at the stage of uninucleate was induced. After several subcultures, anther calli of B. chinense were cultured at 20 MS culture mediums with different plant hormones to differentiate into plantlets. Differentiation of callus was detected after 21 and 49 days to select the most effective medium. There were 19 culture mediums in which anther callus could differentiate into plantlets with differentiation rate range from 3% to 60% , and most less than 20%. MS + KT 0.5 mg x L(-1) + sucrose 30 g c L(-1) + phytagel 5 g x L(-1) was the best differentiation medium with the differentiation rate of 60%, followed by MS + ZT 1.0 mg x L(-1) + sucrose 30 g x L(-1) + phytagel 5 g x L(-1) with the differentiation rate of 58%. Then plantlets were transferred to rooting medium to obtain whole plant. All plantlets could root in the rooting medium of MS + sucrose 30 g x L(-1) + phytagel 5 g x L(-1) and 1/2 MS + NAA 0.5 mg x L(-1) + sucrose 30 g x L(-1) + phytagel of 5 g L(-1) with the rooting rate of 100%. As a result, the high efficient and stable plant regeneration system was established from anther callus of B. chinense.

[Comprehensive yield and quality comparison of Bupleurum chinense' s seeds of different harvesting time from Qingchuan County with Topsis approach].

In order to investigate the comprehensive quality differences of the seeds harvested in different growing time, and offer theoretical guide for the optimization of seeds' production technology, we analyzed the apparent size, 1 000-grain weight, water absorbency, germination indexes, postharvest embryo rate change, relatively contents of coumarins and the yield of single plant of its seeds of different harvesting time, and compared their comprehensive quality by Topsis analysis. The results showed that there existed obvious differences in yield and quality between seeds of 3 harvesting times. While the harvesting time postponed, the yield of single plant raised, and the shrunken seeds rate decreased, the quality of seedlings increased, while the contents of coumarins showed a steady increase, and the germination rate decreased. The comprehensive quality of the seeds harvested in the black ripe time rank the first place, followed by the brown ripe time and the yellow ripe time. As the harvesting time delays, the seeds' comprehensive quality increases, therefore, we could put off the seeds' harvesting time properly for the high efficient seed production.

Metabolic fingerprinting by 1HNMR for discrimination of the two species used as Radix Bupleuri.

Radix Bupleuri is a traditional Chinese medicine harvested from two Bupleurum species (B. chinense and B. scorzonerifolium). It is widely used and is sourced from different regions of China. 1H NMR spectroscopy and multivariate data analysis were applied to 67 Radix Bupleuri samples to discriminate the two species, and explore the influences of habitat and culture method on the quality of Radix Bupleuri based on their metabolomics profiles. Metabolites responsible for the differences between the two species were higher levels of arginine, citric acid, sucrose, saikosaponin b1/b2 analogs, volatile oil with an (E)-2-olefin aldehyde fragment, and fatty acids in B. scoreonerifolium, and more saikosaponin a/c/d analogs in B. chinense. The variances of two cultivation areas were observed due to the higher amount of saikosaponins a/c/d in samples from Shaanxi and lipidsin samples from Shanxi. No obvious difference was detected between cultivars and wild type. 1HNMR metabolomics can simultaneously detect saikosaponins and hydrocarbon aldehydes, and also differentiate the two main saikosaponin skeletons, making it a suitable tool for the species discrimination and quality evaluation of Radix Bupleuri.

Large-scale production of saikosaponins through root culturing of Bupleurum falcatum L. using modified airlift reactors.

Modification of internal configuration of a bubble column, airlift and stirred tank reactor (10-200 L) was made for root cultures of Bupleurum falcatum L. Agitation with an impeller covered with partition mesh was ineffective for a 10-L modified reactor, because it caused intensive foaming and subsequent overflow of the culture medium even at a low rotation speed of 50 rpm and a low aeration rate of 0.1 vvm (volume per volume of medium). In contrast, efficient aeration through a ceramic sparger placed at the bottom of a 20-L bubble column reactor yielded approximately 25 g/L of dry roots and 500 mg/L of saikosaponin-a and saikosaponin-d over 42 days. On a 200-L scale, however, the roots became flocculated under the upper perforated plate initially positioned near the middle of the reactor, forming a firm disk of roots and a large empty space between the disk and the medium. Thus, the roots had poor contact with the medium, which severely suppressed their growth. To avoid this flocculation, a bottom perforated plate and draft tube were installed as a partitioning device separating the culturing area (outside the draft tube) from the aeration area (inside the draft tube). The draft tube was made of a stainless steel mesh rather than a solid material, and the tube greatly increased the root yield in the 20-L reactor. This configuration was successfully applied at the 200-L scale, yielding 500-600 mg/L of saikosaponin-a and saikosaponin-d over 56 days.