Sedative-Hypnotic Effect and Mechanism of Carbon Nanofiber Loaded with Essential Oils of Ligusticum chuanxiong (Ligusticum chuanxiong Hort.) and Finger Citron (Citrus medica L. var. sarcodactylis) on Mice Models of Insomnia.

(1) Background: Insomnia is a neurological illness that poses a significant threat to both physical and mental health. It results in the activation of neuroglial cells, heightened neuroinflammation, oxidative stress, and disruptions in the Hypothalamic-Pituitary-Adrenal (HPA) axis. Ligusticum Chuanxiong (CX) and Finger citron (FC) are frequently utilized botanicals for addressing sleeplessness. Both herbs possess notable anti-inflammatory properties in their volatile oils. However, their effectiveness is hindered by the nasal mucosal irritation and instability they exhibit. (2) Methods: This study involved the preparation of a nanofiber composite system using carbon nanofiber (CNF) suspensions containing essential oils of Ligusticum chuanxiong-Finger citron (CXEO-FCEO-CNF). The effects and mechanisms of these essential oils in improving insomnia were investigated using an insomnia mouse model after encapsulation. (3) Results: The CXEO-FCEO-CNF had an average particle size of 103.19 ± 1.64 nm. The encapsulation rates of essential oils of Ligusticum chuanxiong (CXEO) and essential oils of Finger citron (FCEO) were 44.50% and 46.15%, respectively. This resulted in a considerable improvement in the stability of the essential oils over a period of 30 days. The essential oils effectively decreased the irritation of the nasal mucosa following encapsulation. Furthermore, CXEO-FCEO-CNF enhanced voluntary activity and sleep in mice with insomnia, notably boosted the activity of superoxide dismutase (SOD), reduced the concentration of lipoxidized malondialdehyde (MDA), decreased the levels of hormones associated with the HPA axis, and regulated the levels of neurotransmitters, resulting in a beneficial therapeutic outcome. CXEO-FCEO-CNF contains a total of 23 active ingredients, such as alpha-Asarone, (E)-methyl isoeugenol, and Senkyunolide. These ingredients primarily work by modulating the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling system to decrease oxidative stress and inflammatory reactions. (4) Conclusions: This study presented initial evidence that the combination of CXEO and FCEO in nanofiber formulations effectively reduces the nasal mucosal irritation and instability of essential oils. Furthermore, it demonstrated the potential anti-neuroinflammatory and therapeutic effects of these formulations in treating insomnia. Overall, this study provides a theoretical foundation for developing new essential oil formulations derived from herbs.

Ligustilide alleviates oxidative stress during renal ischemia-reperfusion injury through maintaining Sirt3-dependent mitochondrial homeostasis.

BACKGROUND: Renal ischemia-reperfusion (I/R) injury is an inevitable complication during renal transplantation and is closely related to patient prognosis. Mitochondrial damage induced oxidative stress is the core link of renal I/R injury. Ligustilide (LIG), a natural compound extracted from ligusticum chuanxiong hort and angelica sinensis, has exhibited the potential to protect mitochondrial function. However, whether LIG can ameliorate renal I/R injury requires further investigation. Delving deeper into the precise targets and mechanisms of LIG's effect on renal I/R injury is crucial. PURPOSE: This study aimed to elucidate the specific mechanism of LIG's protective effect on renal I/R injury. METHODS: In this study, an in vivo model of renal ischemia-reperfusion (I/R) injury was developed in mice, along with an in vitro model of hypoxia-reoxygenation (H/R) using human proximal renal tubular epithelial cells (HK-2). To assess the impact of LIG on renal injury, various methods were employed, including serum creatinine (Cr) and blood urea nitrogen (BUN) testing, hematoxylin and eosin (HE) staining, and immunohistochemistry (IHC) for kidney injury molecule-1 (KIM-1). The effects of LIG on oxidative stress were examined using fluorescent probes dihydroethidium (DHE) and dichlorodihydrofluorescein diacetate (DCFH-DA), TdT-mediated dUTP Nick-End Labeling (TUNEL) staining, and flow cytometry. Additionally, the influence of LIG on mitochondrial morphology and function was evaluated through transmission electron microscopy (TEM), Mito Tracker Red CMXRos staining, adenosine triphosphate (ATP) concentration assays, and JC-1 staining. The potential mechanism involving LIG and Sirt3 was explored by manipulating Sirt3 expression through cell transfection. RESULTS: The results showed that LIG could provide protective function for mitochondria to alleviate oxidative stress induced by renal I/R. Further mechanistic studies indicated that LIG maintained mitochondrial homeostasis by targeting Sirt3. CONCLUSION: Our findings demonstrated that LIG alleviated oxidative stress during renal I/R injury through maintaining Sirt3-dependent mitochondrial homeostasis. Overall, our data raised the possibility of LIG as a novel therapy for renal I/R injury.

Metabolomics integrated with mass spectrometry imaging reveals novel action of tetramethylpyrazine in migraine.

Migraine as a common neurological disorder still lacks effective therapies. Tetramethylpyrazine (TMP) is the main bioactive component from Ligusticum chuanxiong hort., a traditional edible-medicinal herb. This study aimed to investigate the action of TMP on migraine by metabolomics with mass spectrometry imaging (MSI) analysis and molecular exploring, including random forest model analysis, KEGG enrichment analysis and metabolite-metabolite interaction network analysis. The results indicated that 26 key representative metabolic biomarkers were identified, especially γ-glu-cys, which were highly related to glutathione (GSH) metabolism. MSI found the abundance of eleven endogenous metabolites were modulated by TMP, particularly glucose, the most important energy metabolism molecule, and GSH were increased that maintains intracellular redox balance, which was consistent with activation of Nrf2 signals by TMP. These findings provide insights into the effectiveness of metabolomics integrated with MSI in explaining the metabolic mechanisms of TMP, and afford valuable information for healthy development of TMP in migraine.

Metabolomic profiling integrated with molecular exploring delineates the action of Ligusticum chuanxiong hort. on migraine.

INTRODUCTION: Ligusticum chuanxiong hort. (Chuanxiong, CX) is a well-known traditional edible-medicinal herb, especially in brain diseases. However, there is a lack of studies focusing on the action of CX in metabolites of migraine. HYPOTHESIS/PURPOSE: To investigate the action of the aqueous extract of CX (LCH) on nitroglycerin (NTG)-induced migraine. METHODS: The effects and mechanisms of LCH on migraine were evaluated in NTG-induced mice and rats. Laser speckle contrast imaging was performed to detect the cerebral blood flow. Metabolomic analysis in serum and mass spectrometry imaging in brain tissue from NTG-induced rats were conducted to explore the metabolites. The techniques including RT-qPCR, immunohistochemistry, and immunofluorescence were employed to estimate the molecular changes. RESULTS: Totally, 1480 metabolites were identified, among which, 50 and 49 differential metabolites were identified by t-test, fold change, and VIP score for NTG vs. CON and LCH+NTG vs. NTG, respectively. Next, 13 common real difference metabolites were revealed by comparative analysis, and KEGG annotation and enrichment analysis showed that the glutathione (GSH) metabolism pathway played important roles in migraine, whereas the glutamate could be metabolized to γ-glu-cys and converted to GSH. Molecular exploring further confirmed that LCH treatment increased the expression of essential components of GSH synthetase, such as GCLC and GCLM, and elevated the expression levels of Nrf-2 and its downstream targets including HO1 and NQO1. Moreover, the mass spectrometry imaging results found that LCH treatment promoted the synthesis of GSH and the spatial distribution of glucose as well as ATP metabolites to normal levels. CONCLUSION: To sum up, the present study firstly reveals that LCH plays a therapeutic role for migraine through glucose-glutamate-Nrf-2 axis, which might represent a promising approach in the development of advanced therapeutic strategies for migraine, and the LCH may be an effective drug or dietary supplement for relieving headache.

Antioxidant and anti-inflammatory effects of different ratios and preparations of Angelica sinensis and chuanxiong rhizoma extracts.

ETHNOPHARMACOLOGICAL RELEVANCE: Angelica sinensis (AS) and Chuanxiong rhizoma (CR) are frequently prescribed in clinical settings for their ability to enrich blood, regulate menstrual cycles, and alleviate pain. Despite their widespread use, there is a relative dearth of studies exploring their anti-inflammatory properties. AIM OF THE STUDY: To evaluate the antioxidant and anti-inflammatory effects of Angelica sinensis-Chuanxiong rhizoma (ASCR) extracts and investigate its anti-inflammatory mechanisms. MATERIALS AND METHODS: AS and CR were combined in six ratios and extracted using five solvents. The quality of the resulting ASCR extracts was assessed by determining the content of ferulic acid (FA) using HPLC. The antioxidant effects of the ASCR extracts were evaluated in vitro using the DPPH and ABTS assays, as well as in HUVECs exposed to H2O2-induced oxidative damage. Additionally, the anti-inflammatory effects of the extracts were investigated in vivo through the assays of ear edema in mice and paw edema in rats. Biochemical markers including NO, MDA, and SOD in paw tissues, as well as PGE2, TNF-α, and COX-2 in rat serum, were measured to further elucidate the anti-inflammatory mechanisms of ASCR extracts. RESULTS: The WA-2-1 was obtained by combining AS and CR in a 2:1 ratio through first water then ethanol extraction, and showed favorable antioxidant and anti-inflammatory activities. The extract demonstrated effective scavenging abilities against DPPH• and ABTS+• radicals while also protecting against H2O2-induced oxidative damage. Furthermore, in vivo studies revealed that WA-2-1 had significant inhibitory effects on ear and paw edema as well as the ability to decrease NO and MDA levels, enhance SOD activity, and downregulate the expression of COX-2, PGE2, and TNF-α. CONCLUSIONS: The combination of AS and CR exhibits favorable anti-inflammatory effects, attributed to its dual actions of mitigating oxidative stress and suppressing the production of inflammatory mediators in serum or tissues during the inflammatory process.

Mercapto-functionalized palygorskite modified the growth of Ligusticum Chuanxiong and restrained the Cd migration in the soil-plant system.

Ligusticum Chuanxiong is an essential medicinal and edible plant, but it is highly susceptible to the enrichment of soil Cadmium (Cd), which seriously affects its medical safety. However, the control of Cd uptake by Ligusticum Chuanxiong is little reported. In this study, we reported that a green Mercapto-functionalized palygorskite (MPAL) effectively promoted Ligusticum Chuanxiong growth, and restrained the Cd uptake by Ligusticum Chuanxiong both in the mildly contaminated soil (M-Soil) and severely contaminated soil (S-Soil). The experimental results demonstrated that the application of MPAL significantly increased the biomass and antioxidant enzyme activity of Ligusticum Chuanxiong. In the M-Soil, the Cd content in the roots, stems, and leaves of Ligusticum Chuanxiong decreased markedly by 82.46-86.66%, 64.17-71.73%, and 64.94-76.66%, respectively, after the MPAL treatment. In the S-Soil, MPAL application decreased the Cd content in roots, stems, and leaves by 89.43-98.92%, 24.19-86.22%, and 67.14-77.90%, respectively. Based on Diethylenetriamine Pentaacetic Acid (DTPA) extraction, the immobilization efficiency of MPAL for Cd in soils ranged from 22.01% to 77.04%. Additionally, the HOAc extractable Cd was transformed into reducible and oxidizable fractions. Furthermore, MPAL enhanced the activities of soil alkaline phosphatase, and urease, but decreased sucrase activity. Environmental toxicological analysis indicated that MPAL reduced the potential ecological risk of Cd in the soil. These findings revealed that MPAL can effectively reduce Cd accumulation in Ligusticum Chuanxiong and promote plant growth, suggesting its potential as a viable amendment for remediating Cd-contaminated soils.

Identification of antidepressant constituents from Xiangfu-chuanxiong herbal medicine pair via spectrum-effect relationship analyses, molecular docking and corticosterone-induced PC12 cells.

Herbal medicine pair, composed of two single herbs, is a relatively fixed minimum prescription unit in the traditional Chinese medicine's formula and has special significance in clinic. The combination of Xiangfu (the rhizoma of Cyperus rotundus L, XF) and Chuanxiong (the rhizoma of Ligusticum chuanxiong Hort, CX) has been recoded as an herbal medicine pair XF-CX in the Yuan Dynasty (1347 CE) of China and widely used in traditional Chinese medicine formula, including Chaihu Shugan San, which has been clinically used for treatment of depression. However, the optimal ratio of the XF-CX herbal medicine pair and its antidepressant constituents are still unclear. Herein, the antidepressive-like effects of XF-CX herbal medicine pairs with different ratios of XF and CX (2:1, 1:1, 1:2) were evaluated using behavioral despair animal models in mice, and then its potential antidepressant constituents were recognized by spectrum-effect relationship analyses. Finally, the potential antidepressant constituents of the XF-CX herbal medicine pair were validated by molecular docking with glucocorticoid receptor and corticosterone (CORT)-induced PC12 cell injury model. The results indicated that different ratios of XF-CX pairs had antidepressive-like effects, and the XF-CX (2:1) exhibited a more significant effect. Thirty-two potential antidepressant constituents in the XF-CX herbal medicine pair were screened out from the spectrum-effect relationship combined molecular docking analyses. Among them, senkyunolide A, cyperotundone, Z-ligustilide, and levistilide A were validated to have protective effects against CORT-induced injury in PC12 cells. Our findings not only obtained the optimal ratio of XF-CX in the herbal medicine pair for the treatment of depression but also its potential antidepressant constituents, which will benefit in elucidating the mechanism of action and promoting the application of the herbal medicine pair in the clinic.

[Mechanism and compatibility efficacy of Astragali Radix-Chuanxiong Rhizoma drug pair in treating ischemic stroke based on network regulation].

Based on the association network of "drug pair-disease", the effect characteristics of Astragali Radix-Chuanxiong Rhizoma drug pair in the treatment of ischemic stroke(IS) with Qi deficiency and blood stasis and the matching mechanism of the two were explored. Through Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) and SwissTargetPrediction Database, the effective chemical components of the drug pair were screened, and the candidate targets were predicted. Databa-ses such as GeneCards, DrugBank, Online Mendelian Inheritance in Man(OMIM), and Therapeutic Target Database(TTD) were searched to obtain gene targets related to IS. Through STRING and Cytoscape 3.9.1 software, the protein-protein interaction(PPI) network was constructed by using the interaction information of disease syndrome-related genes and candidate targets of drug pairs, and the core targets were screened according to the network topological feature values. Based on the Metascape platform and DAVID database, the biomolecular interaction information was integrated to analyze the Kyoto Encyclopedia of Genes and Genomes(KEGG) and mine biological functions, so as to further explore the mechanism of action and compatibility characteristics of Astragali Radix-Chuan-xiong Rhizoma. The results showed that the candidate biological process was mainly involved in the regulation of functional modules such as immune, blood circulation, neurotransmitter, and oxidative stress, and it was enriched in lipid and atherosclerosis, calcium signaling pathway, and platelet activation. Astragali Radix and Chuanxiong Rhizoma have their own characteristics. Astragali Radix has a regulatory response to growth factors while maintaining the body's immune balance, while Chuanxiong Rhizoma mainly improves the circulatory system and participates in hormone metabolism, so as to indicate the compatibility mechanism of Astragali Radix-Chuanxiong Rhizoma drug pair for multi-target and multi-pathway synergistic treatment of IS. Through further experimental verification, it was found that the Astragali Radix-Chuanxiong Rhizoma drug pair could significantly down-regulate the expression of key targets including TLR4, NF-κB, IL-1ß, F2R, PLCß1, and MYLK. This study preliminarily reveals that the Astragali Radix-Chuanxiong Rhizoma drug pair may play the three replenishing effects of promoting blood circulation, benefiting Qi, and clearing collaterals by correcting immune di-sorders, blood circulation disorders, and inflammation, which provide support for the clinical research on the subsequent improvement of Qi deficiency and blood stasis in the treatment of IS and provide a new idea for the analysis of modern biological connotation of the compatibility of seven emotions of traditional Chinese medicine.

Bibliometric analysis of research progress on tetramethylpyrazine and its effects on ischemia-reperfusion injury.

In recent decades, natural products have attracted worldwide attention and become one of the most important resources for pharmacological industries and medical sciences to identify novel drug candidates for disease treatment. Tetramethylpyrazine (TMP) is an alkaloid extracted from Ligusticum chuanxiong Hort., which has shown great therapeutic potential in cardiovascular and cerebrovascular diseases, liver and renal injury, as well as cancer. In this review, we analyzed 1270 papers published on the Web of Science Core Collection from 2002 to 2022 and found that TMP exerted significant protective effects on ischemia-reperfusion (I/R) injury that is the cause of pathological damages in a variety of conditions, such as ischemic stroke, myocardial infarction, acute kidney injury, and liver transplantation. TMP is limited in clinical applications to some extent due to its rapid metabolism, a short biological half-life and poor bioavailability. Obviously, the structural modification, administration methods and dosage forms of TMP need to be further investigated in order to improve its bioavailability. This review summarizes the clinical applications of TMP, elucidates its potential mechanisms in protecting I/R injury, provides strategies to improve bioavailability, which presents a comprehensive understanding of the important compound. Hopefully, the information and knowledge from this review can help researchers and physicians to better improve the applications of TMP in the clinic.

[Ligusticum cycloprolactam inhibits IL-1ß-induced apoptosis and inflammation of rat chondrocytes via HMGB1/TLR4/NF-κB signaling pathway].

Chondrocytes are unique resident cells in the articular cartilage, and the pathological changes of them can lead to the occurrence of osteoarthritis(OA). Ligusticum cycloprolactam(LIGc) are derivatives of Z-ligustilide(LIG), a pharmacodynamic marker of Angelica sinensis, which has various biological functions such as anti-inflammation and inhibition of cell apoptosis. However, its protective effect on chondrocytes in the case of OA and the underlying mechanism remain unclear. This study conducted in vitro experiments to explore the molecular mechanism of LIGc in protecting chondrocytes from OA. The inflammation model of rat OA chondrocyte model was established by using interleukin-1ß(IL-1ß) to induce. LIGc alone and combined with glycyrrhizic acid(GA), a blocker of the high mobility group box-1 protein(HMGB1)/Toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB) signaling pathway, were used to intervene in the model, and the therapeutic effects were systematically evaluated. The viability of chondrocytes treated with different concentrations of LIGc was measured by the cell counting kit-8(CCK-8), and the optimal LIGc concentration was screened out. Annexin V-FITC/PI apoptosis detection kit was employed to examine the apoptosis of chondrocytes in each group. The enzyme-linked immunosorbent assay(ELISA) was employed to measure the expression of cyclooxygenase-2(COX-2), prostaglandin-2(PGE2), and tumor necrosis factor-alpha(TNF-α) in the supernatant of chondrocytes in each group. Western blot was employed to determine the protein levels of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), caspase-3, HMGB1, TLR4, and NF-κB p65. The mRNA levels of HMGB1, TLR4, NF-κB p65, and myeloid differentiation factor 88(MyD88) in chondrocytes were determined by real-time fluorescent quantitative PCR(RT-qPCR). The safe concentration range of LIGc on chondrocytes was determined by CCK-8, and then the optimal concentration of LIGc for exerting the effect was clarified. Under the intervention of IL-1ß, the rat chondrocyte model of OA was successfully established. The modeled chondrocytes showed increased apoptosis rate, promoted expression of COX-2, PGE2, and TNF-α, up-regulated protein levels of Bax, caspase-3, HMGB1, TLR4, and NF-κB p65 and mRNA levels of HMGB1, TLR4, NF-κB p65, and MyD88, and down-regulated protein level of Bcl-2. However, LIGc reversed the IL-1ß-induced changes of the above factors. Moreover, LIGc combined with GA showed more significant reversal effect than LIGc alone. These fin-dings indicate that LIGc extracted and derived from the traditional Chinese medicine A. sinensis can inhibit the inflammatory response of chondrocytes and reduce the apoptosis of chondrocytes, and this effect may be related to the HMGB1/TLR4/NF-κB signaling pathway. The pharmacological effect of LIGc on protecting chondrocytes has potential value in delaying the progression of OA and improving the clinical symptoms of patients, and deserves further study.

[Morphological study of sexual reproductive disorders in Ligusticum chuanxiong].

Chuanxiong Rhizoma is a well-known Sichuan-specific herbal medicine. Its original plant, Ligusticum chuanxiong, has been cultivated asexually for a long time. L. chuanxiong has sexual reproductive disorders, which restricts its germplasm innovation. However, there is little research on the reproductive system of L. chuanxiong. This study is based on a comparative anatomical research approach, using morphological dissection, paraffin sectioning, staining and compression, and combined with scanning electron microscopy technology, to observe and compare the flowers, fruits, and seeds at various stages of reproductive growth of L. chuanxiong and its wild relative L. sinense. The results showed that the meiosis of pollen mother cells is abnormal in L. chuanxiong anthers, and the size and number of microspores are uneven and inconsistent in the tetrad stage. tapetum cells are not completely degenerated during anther development. During the pollen ripening stage, there are fine cracks in the anther wall, while most anthers could not release pollen normally. The surface of mature pollen grains is concave and partially deformed, and the pollens are all inactive and cannot germinate in vitro. The starch, polysaccharides, and lipids in the pollen were insufficient. The filaments of L. chuanxiong are short at the flowering stage and recurved downward. Double-hanging fruits were observed in the fruiting stage, being wrinkled; with shriveled seeds. Compared with L. sinense at the same stage, the anthers of L. sinense developed normally, and the pollen grains are vigorous and can germinate in vitro. The double-hanging fruits of L. sinense are full and normal; at the flowering period, the filaments are long and erect, significantly higher than the stigma. Mature blastocysts are visible in the ovary of both L. chuanxiong and L. sinense, and there is no significant difference in stigmas. The conclusion is that during the development of L. chuanxiong stamens, the meiosis of pollen mother cells is abnormal, and tetrad, tapetum, filament and other pollen structures develop abnormally. L. chuanxiong has the characteristic of male infertility, which is an important reason for its sexual reproductive disorders.

Structurally diverse phthalides from fibrous roots of Ligusticum chuanxiong Hort. and their biological activities.

Falonolide A (1) and B (2), two novel polyyne hybrid phthalides resulting from unprecedented carbon skeleton polymerized by Z-ligustilide and falcarindiol, along with six new related phthalides (3-8), were isolated from Ligusticum chuanxiong Hort. Their structures were elucidated by spectroscopic analysis, computer-assisted structure elucidation (CASE) analysis, DP4+ probability analysis and electronic circular dichroism (ECD) calculations. A plausible biosynthetic pathway for 1-8 was proposed, and the production mechanism of 2 was revealed by density functional theory (DFT) method. Compounds 4 and 6 exhibited significant vasodilatory activity with EC50 of 8.00 ± 0.86 and 6.92 ± 1.02 µM, respectively. Compound 4 also displayed significant inhibitory effect of NO production with EC50 value of 8.82 ± 0.30 µM. Based on the established compounds library, structure-activity relationship analysis of phthalides was explored to provide insights into the drug development of vasodilators and anti-flammatory.

Synergistic amelioration between Ligusticum striatum DC and borneol against cerebral ischemia by promoting astrocytes-mediated neurogenesis.

ETHNOPHARMACOLOGICAL RELEVANCE: Ligusticum chuanxiong Hort (LCH), with the accepted name of Ligusticum striatum DC in "The Plant List" database, is a widely used ethnomedicine in treating ischemic stroke, and borneol (BO) is usually prescribed with LCH for better therapy. Our previous study confirmed their synergistic effect on neurogenesis against cerebral ischemia. However, the underlying mechanism is still unclear. AIM OF THE STUDY: More and more evidence indicated that astrocytes (ACs) might be involved in the modulation of neurogenesis via polarization reaction. The study was designed to explore the synergic mechanism between LCH and BO in promoting astrocyte-mediated neurogenesis. MATERIALS AND METHODS: After primary cultures and identifications of ACs and neural stem cells (NSCs), the oxygen-glucose deprivation (OGD) model and the concentrations of LCH and BO were optimized. After the OGD-injured ACs were treated by LCH, BO, and their combination, the conditioned mediums were used to culture the OGD-injured NSCs. The proliferation, migration, and differentiation of NSCs were assessed, and the secretions of BDNF, CNTF, and VEGF from ACs were measured. Then the expressions of C3 and PTX3 were detected. Moreover, the mice were performed a global cerebral ischemia/reperfusion model and treated with LCH and (or) BO. After the assessments of Nissl staining, the expressions of Nestin, DCX, GFAP, C3, PTX3, p65 and p-p65 were probed. RESULTS: The most appropriate duration of OGD for the injury of both NSCs and ACs was 6 h, and the optimized concentrations of LCH and BO were 1.30 µg/mL and 0.03 µg/mL, respectively. The moderate OGD environment induced NSCs proliferation, migration, astrogenesis, and neurogenesis, increased the secretions of CNTF and VEGF from ACs, and upregulated the expressions of C3 and PTX3. For the ACs, LCH further increased the secretions of BDNF and CNTF, enhanced PTX3 expression, and reduced C3 expression. Additionally, the conditioned medium from LCH-treated ACs further enhanced NSC proliferation, migration, and neurogenesis. The in vivo study showed that LCH markedly enhanced the Nissl score and neurogenesis, and decreased astrogenesis which was accompanied by downregulations of C3, p-p65, and p-p65/p65 and upregulation of PTX3. BO not only decreased the expression of C3 in ACs both in vitro and in vivo but also downregulated p-p65 and p-p65/p65 in vivo. Additionally, BO promoted the therapeutic effect of LCH for most indices. CONCLUSION: A certain degree of OGD might induce ACs to stimulate the proliferation, astrogenesis, and neurogenesis of NSCs. LCH and BO exhibited a marked synergy in promoting ACs-mediated neurogenesis and reducing astrogenesis, in which LCH played a dominant role and BO boosted the effect of LCH. The mechanism of LCH might be involved in switching the polarization of ACs from A1 to A2, while BO preferred to inhibit the formation of A1 phenotype via downregulating NF-κB pathway.

Cadmium induced changes in rhizosphere microecology to enhance Cd intake by Ligusticum sinense cv. Chuanxiong.

As the most famous and widely used traditional Chinese medicine (TCM), Ligusticum sinense cv. Chuanxiong (L. Chuaniong) has been affected by cadmium (Cd) exceeding with high ability of Cd accumulation. There is relatively little research on Cd absorption and storage process in L. Chuanxiong, which is an important reason for the poor remediation efficiency. Hence, this study takes L. Chuanxiong as the point of penetration to explore how L. Chuanxiong affects rhizobacteria through root exudates to alter soil Cd intake, as well as to explore the migration and storage of Cd in its body with 0.10 (T0), 5.00 (T5), 10.00 (T10) mg/kg Cd contaminations. The results showed that the relative abundance of amino acids and phospholipids secreted from L. Chuanxiong root noticeably increased with increasing Cd levels, which directly activated soil Cd or extremely significantly (P < 0.01) recruited bacteria such as Bacillus, Arthrobacter to indirectly increase Cd availability. Under the interaction of root exudates and rhizobacteria, Cd bioavailability increased by 80.00% in rhizosphere soil and Cd accumulation in L. Chuanxiong increased 5.44-6.65 mg/kg. Cd subcellular distribution analysis demonstrated that Cd was mainly stored in the root (10-fold more than in the leaf), whose Cd content was cytoderm>cytoplasm>organelle in tissues. The sequential extraction results found that non-soluble phosphate and protein-chelated Cd dominated (85.00-90.00%) in the cell, while Cd cheated with alcohol soluble protein, amino acid salts, water-soluble organic acid in cell was minimal (5.50%). The phenomenon indicated that L. Chuanxiong fixed Cd in root (the medical part) with low translocation ability. This study can provide theoretical support for the high-quality production of L. Chuanxiong and other root medical plant in heavy metal influenced sites.

Haplotype-phased genome unveils the butylphthalide biosynthesis and homoploid hybrid origin of Ligusticum chuanxiong.

Butylphthalide is one of the first-line drugs for ischemic stroke therapy, while no biosynthetic enzyme for butylphthalide has been reported. Here, we present a haplotype-resolved genome of Ligusticum chuanxiong, a long-cultivated and phthalide-rich medicinal plant in Apiaceae. On the basis of comprehensive screening, four Fe(II)- and 2-oxoglutarate-dependent dioxygenases and two CYPs were mined and further biochemically verified as phthalide C-4/C-5 desaturases (P4,5Ds) that effectively promoted the forming of (S)-3-n-butylphthalide and butylidenephthalide. The substrate promiscuity and functional redundancy featured for P4,5Ds may contribute to the high phthalide diversity in L. chuanxiong. Notably, comparative genomic evidence supported L. chuanxiong as a homoploid hybrid with Ligusticum sinense as a potential parent. The two haplotypes demonstrated exceptional structure variance and diverged around 3.42 million years ago. Our study is an icebreaker for the dissection of phthalide biosynthetic pathway and reveals the hybrid origin of L. chuanxiong, which will facilitate the metabolic engineering for (S)-3-n-butylphthalide production and breeding for L. chuanxiong.

Essential oil from Ligusticum chuanxiong Hort. Alleviates lipopolysaccharide-induced neuroinflammation: Integrating network pharmacology and molecular mechanism evaluation.

ETHNOPHARMACOLOGICAL RELEVANCE: Chuanxiong, the rhizome of Ligusticum chuanxiong Hort., is an ancient herbal medicine that has gained extensive popularity in alleviating migraines with satisfying therapeutic effects in China. As the major bioactive component of Chuanxiong, the essential oil also exerts a marked impact on the treatment of migraine. It is widely recognized that neuroinflammation contributes to migraine. However, it remains unknown whether Chuanxiong essential oil has anti-neuroinflammatory activity. AIM OF THE STUDY: To explore the anti-neuroinflammatory properties of Chuanxiong essential oil and its molecular mechanisms by network pharmacology analysis and in vitro experiments. MATERIALS AND METHODS: Gas chromatography-mass spectrometry (GC-MS) was used to identify the chemical components of Chuanxiong essential oil. Public databases were used to predict possible targets, build the protein-protein interaction network (PPI), and perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Moreover, cytological experiments, nitric oxide assay, enzyme-link immunosorbent assay, western blotting, and immunofluorescence assay were adopted to prove the critical signaling pathway in lipopolysaccharide (LPS)-induced BV2 cells. RESULTS: Thirty-six compounds were identified from Chuanxiong essential oil by GC-MS, and their corresponding putative targets were predicted. The network pharmacology study identified 232 candidate targets of Chuanxiong essential oil in anti-neuroinflammation. Furthermore, Chuanxiong essential oil was found to potentially affect the C-type lectin receptor, FoxO, and NF-κB signaling pathways according to the KEGG analysis. Experimentally, we verified that Chuanxiong essential oil could significantly reduce the overproduction of inflammatory mediators and pro-inflammatory factors via the NF-κB signaling pathway. CONCLUSION: Chuanxiong essential oil alleviates neuroinflammation through the NF-κB signaling pathway, which provides a theoretical foundation for a better understanding of the clinical application of Chuanxiong essential oil in migraine treatment.

LcSAO1, an Unconventional DOXB Clade 2OGD Enzyme from Ligusticum chuanxiong Catalyzes the Biosynthesis of Plant-Derived Natural Medicine Butylphthalide.

Butylphthalide, a prescription medicine recognized for its efficacy in treating ischemic strokes approved by the State Food and Drug Administration of China in 2005, is sourced from the traditional botanical remedy Ligusticum chuanxiong. While chemical synthesis offers a viable route, limitations in the production of isomeric variants with compromised bioactivity necessitate alternative strategies. Addressing this issue, biosynthesis offers a promising solution. However, the intricate in vivo pathway for butylphthalide biosynthesis remains elusive. In this study, we examined the distribution of butylphthalide across various tissues of L. chuanxiong and found a significant accumulation in the rhizome. By searching transcriptome data from different tissues of L. chuanxiong, we identified four rhizome-specific genes annotated as 2-oxoglutarate-dependent dioxygenase (2-OGDs) that emerged as promising candidates involved in butylphthalide biosynthesis. Among them, LcSAO1 demonstrates the ability to catalyze the desaturation of senkyunolide A at the C-4 and C-5 positions, yielding the production of butylphthalide. Experimental validation through transient expression assays in Nicotiana benthamiana corroborates this transformative enzymatic activity. Notably, phylogenetic analysis of LcSAO1 revealed that it belongs to the DOXB clade, which typically encompasses genes with hydroxylation activity, rather than desaturation. Further structure modelling and site-directed mutagenesis highlighted the critical roles of three amino acid residues, T98, S176, and T178, in substrate binding and enzyme activity. By unraveling the intricacies of the senkyunolide A desaturase, the penultimate step in the butylphthalide biosynthesis cascade, our findings illuminate novel avenues for advancing synthetic biology research in the realm of medicinal natural products.

Antifungal activity of Ligusticum chuanxiong essential oil and its active composition butylidenephthalide against Sclerotium rolfsii.

BACKGROUND: Peanut stem rot caused by Sclerotium rolfsii is an epidemic disastrous soil-borne disease. Recently, natural products tend to be safe alternative antifungal agents to combat pathogens. RESULTS: This work determined the preliminary antifungal activity of 29 essential oils against S. rolfsii and found that Ligusticum chuanxiong essential oil (LCEO) showed the best antifungal activity, with an EC50 value of 81.79 mg L-1 . Sixteen components (98.78%) were identified in LCEO by gas chromatography-mass spectrometry analysis, the majority by volume comprising five phthalides (93.14%). Among these five phthalides, butylidenephthalide was the most effective compound against S. rolfsii. Butylidenephthalide not only exhibited favorable in vitro antifungal activity against the mycelial growth, sclerotia production and germination of S. rolfsi, but also presented efficient in vivo efficacy in the control of peanut stem rot. Seven days after application in the glasshouse, the protective and curative efficacy of butylidenephthalide at 300 mg L-1 (52.02%, 44.88%) and LCEO at 1000 mg L-1 (49.60%, 44.29%) against S. rolfsii were similar to that of the reference fungicide polyoxin at 300 mg L-1 (54.61%, 48.28%). Butylidenephthalide also significantly decreased the oxalic acid and polygalacturonase content of S. rolfsii, suggesting a decreased infection ability on plants. Results of biochemical actions indicated that butylidenephthalide did not have any effect on the cell membrane integrity and permeability but significantly decreased nutrient contents, disrupted the mitochondrial membrane, inhibited energy metabolism and induced reactive oxygen species (ROS) accumulation of S. rolfsii. CONCLUSION: Our results could provide an important reference for understanding the application potential and mechanisms of butylidenephthalide in the control of S. rolfsii. © 2023 Society of Chemical Industry.

Unveiling differential mechanisms of chuanxiong cortex and pith in the treatment of coronary heart disease using SPME-GC×GC-MS and network pharmacology.

Ligusticum chuanxiong Hort (LCH) is a well-known traditional Chinese medicinal herb for treating coronary heart disease (CHD). This study investigated the differential preventive mechanisms of Rhizome Cortex (RC) and Rhizome Pith (RP) of LCH. Solid-phase microextraction combined with comprehensive two-dimensional gas chromatography-tandem mass spectrometry analysis identified 32 differential components, and network pharmacology revealed 11 active ingredients and 191 gene targets in RC, along with 12 active ingredients and 318 gene targets in RP. Primary active ingredients in RC were carotol, epicubenol, fenipentol, and methylisoeugenol acetate, while 3-undecanone, (E)- 5-decen-1-ol acetate, linalyl acetate, and (E)- 2-Methoxy-4-(prop-1-enyl) phenol were dominant in RP. KEGG mapping analysis associated 27 pathways with RC targets and 116 pathways with RP targets. Molecular docking confirmed the efficient activation of corresponding targets by these active ingredients. This study provides valuable insights into the preventive and therapeutic effects of RC and RP in CHD.

Mechanism of ligusticum cycloprolactam against neuroinflammation based on network pharmacology and experimental verification.

Ligustilide, a natural phthalide mainly derived from chuanxiong rhizomes and Angelica Sinensis roots, possesses anti-inflammatory activity, particularly in the context of the nervous system. However, its application is limited because of its unstable chemical properties. To overcome this limitation, ligusticum cycloprolactam (LIGc) was synthesized through structural modification of ligustilide. In this study, we combined network pharmacological methods with experimental verification to investigate the anti-neuroinflammatory effects and mechanisms of ligustilide and LIGc. Based on our network pharmacology analysis, we identified four key targets of ligustilide involved in exerting an anti-inflammatory effect, with the nuclear factor (NF)-κB signal pathway suggested as the main signalling pathway. To verify these results, we examined the expression of inflammatory cytokines and inflammation-related proteins, analysed the phosphorylation level of NF-κB, inhibitor of κBα (IκBα) and inhibitor of κB kinase α and ß (IKKα+ß), and evaluated the effect of BV2 cell-conditioned medium on HT22 cells in vitro. Our results, demonstrate for the first time that LIGc can downregulate the activation of the NF-κB signal pathway in BV2 cells induced by lipopolysaccharide, suppress the production of inflammatory cytokines and reduce nerve injury in HT22 cells mediated by BV2 cells. These findings suggest that LIGc inhibits the neuroinflammatory response mediated by BV2 cells, providing strong scientific support for the development of anti-inflammatory drugs based on natural ligustilide or its derivatives. However, there are some limitations to our current study. In the future, further experiments using in vivo models may provide additional evidence to support our findings.