[Effect of oxymatrine on Cryptosporidium parvum infection in mice based on the HMGB1-TLR2/TLR4-NF-κB pathway].

OBJECTIVE: To investigate the involvement of the high mobility group box protein B1 (HMGB1)-Toll-like receptor 2 (TLR2)/TLR4-nuclear factor κB (NF-κB) pathway in the intestinal mucosal injury induced by Cryptosporidium parvum infection, and to examine the effect of oxymatrine (OMT) on C. parvum infection in mice. METHODS: Forty SPF 4-week-old BALB/c mice were randomly divided into four groups, including the control group, infection group, glycyrrhizin (GA) group and OMT group. Each mouse was orally administered with 1 × 105 C. parvum oocysts one week in the infection, GA and OMT groups following dexamethasone-induced immunosuppression to model C. parvum intestinal infections in mice. Upon successful modeling, mice in the GA group were intraperitoneally injected with GA at a daily dose of 25.9 mL/kg for successive two weeks, and animals in the OMT group were orally administered OMT at a daily dose of 50 mg/kg for successive two weeks, while mice in the control group were given normal food and water. All mice were sacrificed two weeks post-treatment, and proximal jejunal tissues were sampled. The pathological changes of mouse intestinal mucosal specimens were observed using hematoxylin-eosin (HE) staining, and the mouse intestinal villous height, intestinal crypt depth and the ratio of intestinal villous height to intestinal crypt depth were measured. The occludin and zonula occludens protein 1 (ZO1) expression was determined in mouse intestinal epithelial cells using immunohistochemistry, and the relative expression of HMGB1, TLR2, TLR4, myeloid differentiation primary response gene 88 (MyD88) and NF-κB p65 mRNA was quantified in mouse jejunal tissues using quantitative real-time PCR (qPCR) assay. RESULTS: HE staining showed that the mouse intestinal villi were obviously atrophic, shortened, and detached, and the submucosal layer of the mouse intestine was edematous in the infection group as compared with the control group, while the mouse intestinal villi tended to be structurally intact and neatly arranged in the GA and OMT groups. There were significant differences among the four groups in terms of the mouse intestinal villous height (F = 6.207, P = 0.000 5), intestinal crypt depth (F = 6.903, P = 0.000 3) and the ratio of intestinal villous height to intestinal crypt depth (F = 37.190, P < 0.000 1). The mouse intestinal villous height was lower in the infection group than in the control group [(321.9 ± 41.1) µm vs. (399.5 ± 30.9) µm; t = 4.178, P < 0.01] and the GA group [(321.9 ± 41.1) µm vs. (383.7 ± 42.7) µm; t = 3.130, P < 0.01], and the mouse intestinal crypt depth was greater in the infection group [(185.0 ± 35.9) µm] than in the control group [(128.4 ± 23.6) µm] (t = 3.877, P < 0.01) and GA group [(143.3 ± 24.7) µm] (t = 2.710, P < 0.05). The mouse intestinal villous height was greater in the OMT group [(375.3 ± 22.9) µm] than in the infection group (t = 3.888, P < 0.01), and there was no significant difference in mouse intestinal villous height between the OMT group and the control group (t = 1.989, P > 0.05). The mouse intestinal crypt depth was significantly lower in the OMT group [(121.5 ± 27.3) µm] than in the infection group (t = 4.133, P < 0.01), and there was no significant difference in mouse intestinal crypt depth between the OMT group and the control group (t = 0.575, P > 0.05). The ratio of the mouse intestinal villous height to intestinal crypt depth was significantly lower in the infection group (1.8 ± 0.2) than in the control group (3.1 ± 0.3) (t = 10.540, P < 0.01) and the GA group (2.7 ± 0.3) (t = 7.370, P < 0.01), and the ratio of the mouse intestinal villous height to intestinal crypt depth was significantly higher in the OMT group (3.1 ± 0.2) than in the infection group (t = 15.020, P < 0.01); however, there was no significant difference in the ratio of the mouse intestinal villous height to intestinal crypt depth between the OMT group and the control group (t = 0.404, P > 0.05). Immunohistochemical staining showed significant differences among the four groups in terms of occludin (F = 28.031, P < 0.000 1) and ZO1 expression (F = 14.122, P < 0.000 1) in mouse intestinal epithelial cells. The proportion of positive occluding expression was significantly lower in mouse intestinal epithelial cells in the infection group than in the control group [(14.3 ± 4.5)% vs. (28.3 ± 0.5)%; t = 3.810, P < 0.01], and the proportions of positive occluding expression were significantly higher in mouse intestinal epithelial cells in the GA group [(30.3 ± 1.3)%] and OMT group [(25.8 ± 1.5)%] than in the infection group (t = 7.620 and 5.391, both P values < 0.01); however, there was no significant differences in the proportion of positive occluding expression in mouse intestinal epithelial cells between the GA or OMT groups and the control group (t = 1.791 and 2.033, both P values > 0.05). The proportion of positive ZO1 expression was significantly lower in mouse intestinal epithelial cells in the infection group than in the control group [(14.4 ± 1.8)% vs. (24.2 ± 2.8)%; t = 4.485, P < 0.01], and the proportions of positive ZO1 expression were significantly higher in mouse intestinal epithelial cells in the GA group [(24.1 ± 2.3)%] (t = 5.159, P < 0.01) and OMT group than in the infection group [(22.5 ± 1.9)%] (t = 4.441, P < 0.05); however, there were no significant differences in the proportion of positive ZO1 expression in mouse intestinal epithelial cells between the GA or OMT groups and the control group (t = 0.037 and 0.742, both P values > 0.05). qPCR assay showed significant differences among the four groups in terms of HMGB1 (F = 21.980, P < 0.000 1), TLR2 (F = 20.630, P < 0.000 1), TLR4 (F = 17.000, P = 0.000 6), MyD88 (F = 8.907, P = 0.000 5) and NF-κB p65 mRNA expression in mouse jejunal tissues (F = 8.889, P = 0.000 7). The relative expression of HMGB1 [(5.97 ± 1.07) vs. (1.05 ± 0.07); t = 6.482, P < 0.05] 、TLR2 [(5.92 ± 1.29) vs. (1.10 ± 0.14); t = 5.272, P < 0.05] 、TLR4 [(5.96 ± 1.50) vs. (1.02 ± 0.03); t = 4.644, P < 0.05] 、MyD88 [(3.00 ± 1.26) vs. (1.02 ± 0.05); t = 2.734, P < 0.05] and NF-κB p65 mRNA [(2.33 ± 0.72) vs. (1.04 ± 0.06); t = 2.665, P < 0.05] was all significantly higher in mouse jejunal tissues in the infection group than in the control group. A significant reduction was detected in the relative expression of HMGB1 (0.63 ± 0.01), TLR2 (0.42 ± 0.10), TLR4 (0.35 ± 0.07), MyD88 (0.70 ± 0.11) and NF-κB p65 mRNA (0.75 ± 0.01) in mouse jejunal tissues in the GA group relative to the control group (t = 8.629, 5.830, 11.500, 4.729 and 6.898, all P values < 0.05), and the relative expression of HMGB1, TLR2, TLR4, MyD88 and NF-κB p65 mRNA significantly reduced in mouse jejunal tissues in the GA group as compared to the infection group (t = 7.052, 6.035, 4.084, 3.165 and 3.274, all P values < 0.05). In addition, the relative expression of HMGB1 (1.14 ± 0.60), TLR2 (1.00 ± 0.24), TLR4 (1.14 ± 0.07), MyD88 (0.96 ± 0.25) and NF-κ B p65 mRNA (1.12 ± 0.17) was significantly lower in mouse jejunal tissues in the OMT group than in the infection group (t = 7.059, 5.320, 3.510, 3.466 and 3.273, all P values < 0.05); however, there were no significant differences between the OMT and control groups in terms of relative expression of HMGB1, TLR2, TLR4, MyD88 or NF-κB p65 mRNA in mouse jejunal tissues (t = 0.239, 0.518, 1.887, 0.427 and 0.641, all P values > 0.05). CONCLUSIONS: C. parvum infection causes intestinal inflammatory responses and destruction of intestinal mucosal barrier through up-regulating of the HMGB1-TLR2/TLR4-NF-κB pathway. OMT may suppress the intestinal inflammation and repair the intestinal mucosal barrier through inhibiting the activity of the HMGB1-TLR2/TLR4-NF-κB pathway.

The effect of matrine and glycyrrhizic acid on porcine reproductive and respiratory syndrome virus in Vitro and in vivo.

Porcine reproductive and respiratory syndrome (PRRS) is endemic worldwide, seriously affecting the development of the pig industry, but vaccines have limited protective effects against PRRSV transmission. The aim of this study was to identify potential anti-PRRSV drugs. We examined the cytotoxicity of seven compounds formulated based on the mass ratio of glycyrrhizic acid to matrine and calculated their inhibition rates against PRRSV in vitro. The results showed that the seven compounds all had direct killing and therapeutic effects on PRRSV, and the compounds inhibited PRRSV replication in a time- and dose-dependent manner. The compound with the strongest anti-PRRSV effect was selected for subsequent in vivo experiments. Pigs were divided into a control group and a medication group for the in vivo evaluation. The results showed that pigs treated with the 4:1 compound had 100% morbidity after PRRSV challenge, and the mortality rate reached 75% on the 8th day of the virus challenge. These results suggest that this compound has no practical anti-PRRSV effect in vivo and can actually accelerate the death of infected pigs. Next, we further analyzed the pigs that exhibited semiprotective effects following vaccination with the compound to determine whether the compound can synergize with the vaccine in vivo. The results indicated that pigs treated with the compound had higher mortality rates and more severe clinical reactions after PRRSV infection (p < 0.05). The levels of proinflammatory cytokines (IL-6, IL-8, IL-1ß, IFN-γ, and TNF-α) were significantly greater in the compound-treated pigs than in the positive control-treated pigs (p < 0.05), and there was no synergistic enhancement with the live attenuated PRRSV vaccine (p < 0.05). The compound enhanced the inflammatory response, prompted the body to produce excessive levels of inflammatory cytokines and caused body damage, preventing a therapeutic effect. In conclusion, the present study revealed that the in vitro effectiveness of these agents does not indicate that they are effective in vivo or useful for developing anti-PRRSV drugs. Our findings also showed that, to identify effective anti-PRRSV drugs, comprehensive drug screening is needed, for compounds with solid anti-inflammatory effects both in vitro and in vivo. Our study may aid in the development of new anti-PRRSV drugs.

Isolation of Protoplasts from Tissues of Mexican Prickly Poppy (Argemone mexicana L.): An Alkaloid-Producing Medicinal Plant.

Protoplasts are plant cells from which the pectocellulosic cell wall has been removed, thus keeping the plasma membrane intact. For plant secondary metabolites research, this system is a powerful tool to study the metabolites' dynamics inside the cells, such as the subcellular localization of proteins, characterization of gene function, transcription factors involved in metabolite pathways, protein transport machinery, and to perform single-cell omics studies. Due to its lack of a cell wall, better images of the interior of the cell can be obtained compared to the whole tissue. This allows the identification of specific cell types involved in the accumulation of specialized metabolites, such as alkaloids, given their autofluorescence properties. Here is a simplified protocol to obtain protoplasts from leaves and in vitro cell cultures from Argemone mexicana, which produces the pharmacologically important alkaloids berberine and sanguinarine.

Combined analysis of the metabolome and transcriptome reveals the metabolic characteristics and candidate genes involved in alkaloid metabolism in Heuchera micrantha Douglas ex Lindl.

BACKGROUND: Alkaloids, important secondary metabolites produced by plants, play a crucial role in responding to environmental stress. Heuchera micrantha, a well-known plant used in landscaping, has the ability to purify air, and absorb toxic and radioactive substances, showing strong environmental adaptability. However, there is still limited understanding of the accumulation characteristics and metabolic mechanism of alkaloids in H. micrantha. RESULTS: In this study, four distinct varieties of H. micrantha were used to investigate the accumulation and metabolic traits of alkaloids in its leaves. We conducted a combined analysis of the plant's metabolome and transcriptome. Our analysis identified 44 alkaloids metabolites in the leaves of the four H. micrantha varieties, with 26 showing different levels of accumulation among the groups. The HT and JQ varieties exhibited higher accumulation of differential alkaloid metabolites compared to YH and HY. We annotated the differential alkaloid metabolites to 22 metabolic pathways, including several alkaloid metabolism. Transcriptome data revealed 5064 differentially expressed genes involved in these metabolic pathways. Multivariate analysis showed that four key metabolites (N-hydroxytryptamine, L-tyramine, tryptamine, and 2-phenylethylamine) and three candidate genes (Cluster-15488.116815, Cluster-15488.146268, and Cluster-15488.173297) that merit further investigation. CONCLUSIONS: This study provided preliminarily insight into the molecular mechanism of the biosynthesis of alkaloids in H. micrantha. However, further analysis is required to elucidate the specific regulatory mechanisms of the candidate gene involved in the synthesis of key alkaloid metabolites. In summary, our findings provide important information about how alkaloid metabolites build up and the metabolic pathways involved in H. micrantha varieties. This gives us a good starting point for future research on the regulation mechanism, and development, and utilization of alkaloids in H. micrantha.

Identification of Urine Metabolic Markers of Stroke Risk Using Untargeted Nuclear Magnetic Resonance Analysis.

Stroke remains the second leading cause of mortality worldwide, and the third leading cause of death and morbidity combined, affecting more than 12 million people every year. Stroke pathophysiology results from complex interactions of several risk factors related to age, family history, gender, lifestyle, and the presence of cardiovascular and metabolic diseases. Despite all the evidence, it is not possible to fully prevent stroke onset. In recent years, there has been an exploration of innovative methodologies for metabolite analysis aimed at identifying novel stroke biomarkers. Utilizing Nuclear Magnetic Resonance (NMR) spectroscopy, we investigated small molecule variations in urine across different stages of stroke risk. The Framingham Stroke Risk Score was used in people over 63 years of age living in long-term care facilities (LTCFs) to calculate the probability of suffering a stroke: low stroke risk (LSR, control), moderate stroke risk (MSR), and high stroke risk (HSR). Univariate statistical analysis showed that urinary 4-hydroxyphenylacetate levels increased while glycolate levels decreased across the different stroke risk groups, from the LSR to the HSR groups. Trimethylamine N-oxide (TMAO) had average concentration values that were significantly higher in elderly people in the HSR group, while trigonelline levels were significantly lower in the MSR group. These metabolic markers can be used for early detection and to differentiate stages of stroke risk more efficiently.

[Effect of different initial processing treatments on commercial characters and chemical components of Fritillaria taipaiensis].

To investigate the effects of different initial processing methods on the quality of Fritillaria taipaiensis, this study explored the effects of anti-browning treatment, drying methods, and drying temperatures on the commercial characters, chromaticity values, and alkaloid and nucleoside components of Fritillariae Taipaiensis Bulbus. The results were comprehensively evaluated through correlation analysis(CA), principal component analysis(PCA), and hierarchical clustering analysis(HCA). Compared with those of the direct drying group(WD60), the chromaticity values(ΔE*) of the groups with scraped outer skin( FHB1) and mixed lime powder treatments(FHB2) were significantly reduced, indicating the inhibition of the browning process. The total alkaloid content of the group with mixed raw soil treatment(FHB3) and the FHB2 group showed no significant change, whereas that of the group with 5%Na Cl O solution rinse treatment(FHB4) was the lowest. Compared with air-blast dried(WD50) samples, the ΔE* values of freezedried(FS6) and vacuum-dried(FS5) samples were significantly decreased, with an increase in total alkaloid contents. Conversely,the ΔE* values of shade-dried(FS1) and sun-dried(FS2) samples were significantly increased, with severe browning and low total alkaloid contents. The total alkaloid contents of heat-pump-dried(FS4) samples showed no significant change, and their ΔE* value was significantly decreased, with a light degree of browning and favorable commercial characters. The total alkaloid content of air-blast dried samples initially increased and then decreased within the range of 40-80 ℃, and the highest content was recorded at 70 ℃. The ΔE* values of high-temperature air-blast dried samples(70-80 ℃) were smaller with a light degree of browning, whereas their texture was compact and lacked powder. CA revealed a significant relationship between the uracil content and chromaticity value of the samples(P< 0. 05). The clustering relationships among samples subjected to different treatments were visualized via PCA and HCA. The results showed that FHB2 and air-blast drying(50-60 ℃) were more suitable for large-scale production, and heat pump drying could be a promising direction for future development. This study provides a scientific basis for optimizing the initial processing methods of Fritillaria taipaiensis.

Effectiveness of smoking cessation interventions among adults: an overview of systematic reviews.

BACKGROUND: This overview of reviews aims to identify evidence on the benefits (i.e. tobacco use abstinence and reduction in smoking frequency) and harms (i.e. possible adverse events/outcomes) of smoking cessation interventions among adults aged 18 years and older. METHODS: We searched Medline, Embase, PsycINFO, Cochrane Database of Systematic Reviews, Database of Abstracts of Reviews of Effects, the CADTH Health Technology Assessment Database and several other websites for grey literature. Searches were conducted on November 12, 2018, updated on September 24, 2020, with publication years 2008 to 2020. Two reviewers independently performed title-abstract and full-text screening considering pre-determined inclusion criteria. Data extraction and quality assessments were initially completed by two reviewers independently (i.e. 73% of included studies (n = 22)) using A Measurement Tool to Assess Systematic Reviews-2 (AMSTAR 2), and the remainder done by one reviewer and verified by another due to resources and feasibility. The application of Grading of Recommendations Assessment, Development and Evaluation (GRADE) was performed by one independent reviewer and verified by another. RESULTS: A total of 22 Cochrane systematic reviews evaluating the impact of smoking cessation interventions on outcomes such as tobacco use abstinence, reduction in smoking frequency, quality of life and possible adverse events were included. Pharmaceutical (i.e. varenicline, cytisine, nicotine replacement therapy (NRT), bupropion) and behavioural interventions (i.e. physician advice, non-tailored print-based self-help materials, stage-based individual counselling, etc.) showed to have increased smoking cessation; whereas, data for mobile phone-based interventions including text messaging, hypnotherapy, acupuncture, continuous auricular stimulation, laser therapy, electrostimulation, acupressure, St John's wort, S-adenosyl-L-methionine (SAMe), interactive voice response systems and other combination treatments were unclear. Considering harms related to smoking cessation interventions, small/mild harms (i.e. increased palpitations, chest pain, nausea, insomnia, headache) were observed following NRT, varenicline and cytisine use. There were no data on harms related to behavioural therapies (i.e. individual or group counselling self-help materials, internet interventions), combination therapies or other therapies (i.e. laser therapy, electrostimulation, acupressure, St John's wort, SAMe). CONCLUSION: Results suggest that pharmacological and behavioural interventions may help the general smoking population quit smoking with observed small/mild harms following NRT or varenicline. Consequently, evidence regarding ideal intervention strategies and the long-term impact of these interventions for preventing smoking was unclear. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42018099691.

Comprehensive Quality Assessment of Brassica napus L. Seeds via HPTLC, LC-QToF, and Anatomical Investigation.

The Brassicaceae family, commonly referred to as cruciferous plants, is globally cultivated and consumed, with the Brassica genus being particularly renowned for its functional components. These vegetables are rich sources of nutrients and health-promoting phytochemicals, garnering increased attention in recent years. This study presents a comprehensive microscopic, chromatographic, and spectroscopic characterization of Brassica napus L. seeds from Kazakhstan aimed at elucidating their morphological features and chemical composition. Microscopic analysis revealed distinct localization of flavonoids, total lipids, and alkaloids. High-performance thin-layer chromatography (HPTLC) analysis of seed extracts demonstrated a complex chemical profile with significant quantities of non-polar compounds in the hexane extracts. Additionally, methanolic extracts revealed the presence of diverse chemical compounds, including alkaloids, flavonoids, and glucosinolates. The chemical composition exhibited varietal differences across different Brassica species, with B. napus L. seeds showing higher concentrations of bioactive compounds. Furthermore, liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QToF-MS) analysis provided insights into the chemical composition, with sinapine isomers, feruloyl, and sinapoyl choline derivatives as major compounds in the seeds. This study contributes to a better understanding of the chemical diversity and quality control methods' approximations of B. napus L. seeds, highlighting their importance in functional food and nutraceutical applications.

Design, Synthesis, and Antifungal Activities of Phenylpyrrole Analogues Based on Alkaloid Lycogalic Acid.

Plant diseases caused by pathogenic fungi seriously affect the yield and quality of crops, cause huge economic losses, and pose a considerable threat to global food security. Phenylpyrrole analogues were designed and synthesized based on alkaloid lycogalic acid. All target compounds were characterized by 1H NMR, 13C NMR, and HRMS. Their antifungal activities against seven kinds of phytopathogenic fungi were evaluated. The results revealed that most compounds had broad-spectrum fungicidal activities at 50 µg/mL; 14 compounds displayed more than 60% fungicidal activities against Rhizoctonia cerealis and Sclerotinia sclerotiorum, and in particular, the fungicidal activities of compounds 8g and 8h against Rhizoctonia cerealis were more than 90%, which could be further developed as lead agents for water-soluble fungicides. The molecular docking results indicate that compounds 8g and 8h can interact with 14α-demethylase (RcCYP51) through hydrogen bonding with strong affinity.

Characterization and Dissolution Mechanism of Low-Molecular-Weight Organic Acids or Inorganic Mesoporous Particle-Based Piperine Amorphous Solid Dispersions.

The objective of the current study is to prepare amorphous solid dispersions (ASDs) containing piperine (PIP) by utilizing organic acid glycyrrhizic acid (GA) and inorganic disordered mesoporous silica 244FP (MSN/244FP) as carriers and to investigate their dissolution mechanism. The physicochemical properties of ASDs were characterized with scanning electron microscopy (SEM), powder X-ray diffraction (PXRD), and differential scanning calorimetry (DSC). Fourier transform infrared spectroscopy (FTIR) and one-dimensional proton nuclear magnetic resonance (1H NMR) studies collectively proved that strong hydrogen-bonding interactions formed between PIP and the carriers in ASDs. Additionally, molecular dynamic (MD) simulation was conducted to simulate and predict the physical stability and dissolution mechanisms of the ASDs. Interestingly, it revealed a significant increase in the dissolution of amorphous PIP in ASDs in in vitro dissolution studies. Rapid dissolution of GA in pH 6.8 medium resulted in the immediate release of PIP drugs into a supersaturated state, acting as a dissolution-control mechanism. This exhibited a high degree of fitting with the pseudo-second-order dynamic model, with an R2 value of 0.9996. Conversely, the silanol groups on the outer surface of the MSN and its porous nanostructures enabled PIP to display a unique two-step drug release curve, indicating a diffusion-controlled mechanism. This curve conformed to the Ritger-Peppas model, with an R2 > 0.9. The results obtained provide a clear evidence of the proposed transition of dissolution mechanism within the same ASD system, induced by changes in the properties of carriers in a solution medium of varying pH levels.

Structural characterization and screening of chemical markers of alkaloids in Aconiti lateralis radix Praeparata and its processed products by UHPLC/Q-TOF-MS/MS and GNPS combining multivariate statistical methods based on the clinic.

RATIONAL: Aconiti Lateralis Radix Praeparata (AC) is a traditional Chinese medicine with a long history of use. However, the current research on the material basis of AC and its processed products is still not comprehensive, especially the changes in lipo-diterpenoid alkaloids (LDAs) that can be hydrolyzed into diester-diterpenoid alkaloids in AC before and after processing. This study aimed to provide material basis guidance for the clinical use of AC and its processed products by comprehensively analyzing the changes in substances between AC and its processed products. METHODS: An ultra-high-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF-MS/MS) approach was optimized to chemical profiling. The MS data were processed using molecular networking combined with the in-house library database to fast characterize the compounds. Multivariate statistical methods were adopted to determine the dissimilarities of components in AC and its processed products. RESULTS: A total of 310 compounds were tentatively identified from AC, including 109 potential new alkaloids, of which 98 were potential novel LPAs. A metabolomics approach was applied to find the characteristic marker components. As a result, 52 potential chemical markers were selected to distinguish the AC samples of different extraction methods and 42 potential chemical markers for differentiating between AC and its processed products were selected. CONCLUSION: The results indicate that UHPLC/Q-TOF-MS/MS and Global Natural Products Social Molecular Networking coupled with multivariate analysis strategies was a powerful tool to rapidly identify and screen the chemical markers of alkaloids between the AC samples and its processed products. These results also indicate that the toxicity of water extracts of AC and its processed products were decreased. This research not only guides the clinical safe use of AC and its processed products, but also extends the application of the molecular networking strategy in traditional herbal medicine.

Anti-inflammatory potential of aconitine produced by endophytic fungus Acremonium alternatum.

Argemone mexicana belonging to family Papaveraceae is a traditional medicinal plant widely utilized by tribal people in India for treating various ailments like skin infections, wounds and inflammation. This plant is very rich in alkaloidal content, which has a great potential in the treatment of anti-inflammatory disorders. Therapeutically promising bioactive molecules are often produced by endophytic fungi associated with medicinal plants. In this investigation, endophytic fungi were isolated from various parts of A. mexicana and screened for alkaloidal content. Among these, one of the fungal isolate, Acremonium alternatum AMEF-5 producing maximum alkaloids showed significant anti-inflammatory activity. Fractionation of this crude fungal extract through column chromatography yielded eight fractions, which were further screened for anti-inflammatory activities. Fraction 3 exhibited significant anti-inflammatory activity by the inhibition of lipoxygenase enzyme (IC50 15.2 ± 0.09 µg/ml), scavenging of the nitric oxide radicals (IC50 11.38 ± 0.35 µg/ml), protein denaturation (IC50 14.93 ± 0.4 µg/ml), trypsin inhibition (IC50 12.06 ± 0.64 µg/ml) and HRBC stabilization (IC50 11.9 ± 0.22 µg/ml). The bioactive alkaloid in fraction 3 was identified as aconitine which was confirmed by UV, FTIR, HPLC, HRMS, 1H NMR, and 13C NMR analysis. This study demonstrates that endophytic fungi serve a potential source for sustainable production of therapeutically important alkaloids.

Analysis of glycoalkaloid distribution in the tissues of mealworm larvae (Tenebrio molitor).

Solanine (SOL) and chaconine (CHA) are glycoalkaloids (GAs) produced mainly by Solanum plants. These plant secondary metabolites affect insect metabolism; thus, they have the potential to be applied as natural plant protection products. However, it is not known which GA concentration induces physiological changes in animals. Therefore, the aim of this study was to perform a quantitative analysis of SOL and CHA in the larvae of Tenebrio molitor using LC‒MS to assess how quickly they are eliminated or metabolised. In this experiment, the beetles were injected with 2 µL of 10-5 M SOL or CHA solution, which corresponds to a dosage range of 0.12-0.14 ng/mg body mass. Then, 0.5, 1.5, 8, and 24 h after GA application, the haemolymph (H), gut (G), and the remainder of the larval body (FB) were isolated. GAs were detected in all samples tested for 24 h, with the highest percentage of the amount applied in the FB, while the highest concentration was measured in the H sample. The SOL and CHA concentrations decreased in the haemolymph over time, while they did not change in other tissues. CHA had the highest elimination rate immediately after injection, while SOL slightly later. None of the GA hydrolysis products were detected in the tested samples. One possible mechanism of the detoxification of GAs may be oxidation and/or sequestration. They may be excreted by Malpighian tubules, with faeces or with cuticles during moulting. The results presented are significant because they facilitate the interpretation of studies related to the effects of toxic substances on insect metabolism.

Investigation of the effects of curcumin and piperine on cyclophosphamide-induced brain injury in rats.

Cyclophosphamide (CP) is an antineoplastic drug widely used in chemotherapy. Curcumin (CUR) and piperine (PP) show a protective effect on neurodegenerative and neurological diseases. This research was designed to measure several biochemical parameters in the brain tissue of CP-applied rats to investigate the impact of combined CUR-PP administration. The study evaluated six groups of eight rats: Group 1 was the control; Groups 2 and 3 were administered 200 or 300 mg/kg CUR-PP via oral gavage; Group 4 received only 200 mg/kg CP on day 1; Groups 5 and 6 received CP + CUR-PP for 7 days. Data from all parameters indicated that CP caused brain damage. Phosphorylated TAU (pTAU), amyloid-beta peptide 1-42 (Aß1-42), glutamate (GLU), and gamma amino butyric acid (GABA) parameters were the same in Groups 4, 5, and 6. On the other hand, 8-hydroxy-2-deoxyguanosine (8-OHdG), nitric oxide (NO), interleukin-6 (IL-6), nuclear factor kappa beta (NF-kß), malondialdehyde (MDA), and tumor necrosis factor-alpha (TNF-α) levels in the CP + CUR-PP groups were lower than those in the CP group (p < 0.05). However, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and reduced glutathione (GSH) parameters were higher in the CP + CUR-PP groups compared to the CP group (p < 0.05). It is thought that the similarity of Groups 5 and 6 with Group 4 in Aß1-42, pTAU, GLU, and GABA parameters hinder the determination of treatment protection however, they might have a therapeutic effect if the applied dose or study duration were changed. This study attempted to evaluate the effects of a CUR-PP combination on CP-induced brain damage in rats by measuring biochemical parameters and performing histopathological examinations. Based on the findings, this CUR-PP combination could be considered an alternative medicine option in cases with conditions similar to those evaluated in this study.

Spatial distribution of metabolites in processing Ziziphi Spinosae Semen as revealed by matrix-assisted laser desorption/ionization mass spectrometry imaging.

Ziziphi Spinosae Semen (ZSS) is the first choice for the treatment of insomnia. This research aimed to reveal the spatial distribution of identifying quality markers of ZSS and to illustrate the metabolite quality characteristics of this herbal medicine. Here, we performed a matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) in situ to detect and image 33 metabolites in ZSS, including three saponins, six flavonoids, four alkaloids, eight fatty acids, and 12 amino acids. The MALDI images of the metabolites clearly showed the heterogeneous spatial distribution in different regions of ZSS tissues, such as the cotyledon, endosperm, and radicle. The distribution area of two saponins, six flavonoids, and three alkaloids increased significantly after the fried processing of ZSS. Based on the ion images, samples with different processing technologies were distinguished unambiguously by the pattern recognition method of orthogonal partial least squares discrimination analysis (OPLS-DA). Simultaneously, 23 major influencing components exerting higher ion intensities were identified as the potential quality markers of ZSS. Results obtained in the current research demonstrate that the processing of ZSS changes its content and distribution of the medicinal components. The analysis of MALDI-MSI provides a novel MS-based molecular imaging approach to investigate and monitor traditional medicinal plants.

Fatty acid and nutrient profiles, diosgenin and trigonelline contents, mineral composition, and antioxidant activity of the seed of some Iranian Trigonella L. species.

BACKGROUND: Fenugreeks (Trigonella L. spp.), belonging to the legume family (Fabaceae), are well-known multipurpose crops that their materials are currently received much attention in the pharmaceutical and food industries for the production of healthy and functional foods all over the world. Iran is one of the main diversity origins of this valuable plant. Therefore, the aim of the present study was to explore vitamins, minerals, and fatty acids profile, proximate composition, content of diosgenin, trigonelline, phenolic acids, total carotenoids, saponins, phenols, flavonoids, and tannins, mucilage and bitterness value, and antioxidant activity of the seed of thirty populations belonging to the ten different Iranian Trigonella species. RESULTS: We accordingly identified notable differences in the nutrient and bioactive compounds of each population. The highest content (mg/100 g DW) of ascorbic acid (18.67 ± 0.85‒22.48 ± 0.60) and α-tocopherol (31.61 ± 0.15‒38.78 ± 0.67) were found in the populations of T. filipes and T. coerulescens, respectively. Maximum content of catechin was found in the populations of T. teheranica (52.67 ± 0.05‒63.50 ± 0.72 mg/l). Linoleic acid (> 39.11% ± 0.61%) and linolenic acid (> 48.78 ± 0.39%) were the main polyunsaturated fatty acids, with the majority in the populations of T. stellata (54.81 ± 1.39‒63.46 ± 1.21%). The populations of T. stellata were also rich in trigonelline (4.95 ± 0.03‒7.66 ± 0.16 mg/g DW) and diosgenin (9.06 ± 0.06‒11.03 ± 0.17 mg/g DW). CONCLUSIONS: The obtained data provides baseline information to expand the inventory of wild and cultivated Iranian Trigonella species for further exploitation of rich chemotypes in the new foods and specific applications.

Cytotoxicity of alkaloids isolated from Peganum harmala seeds on HCT116 human colon cancer cells.

BACKGROUND: The present study aimed to elucidate the potential anticancer activity and mechanism of P. harmala's alkaloid extract, harmine (HAR), and harmaline (HAL) in HCT-116 colorectal cancer cells. METHODS AND RESULTS: P. harmala's alkaloid was extracted from harmala seeds. HCT-116 cells were treated with P. harmala's alkaloid extract, HAR and HAL. Cytotoxicity was determined by MTT assay, apoptotic activity detected via flow cytometry and acridine orange (AO)/ethidium bromide (EB) dual staining, and cell cycle distribution analyzed with flow cytometry. The mRNA expression of Bcl-2-associated X protein (Bax) and glycogen synthase kinase-3 beta (GSK3ß) was measured by real-time PCR. Furthermore, the expression of Bax, Bcl-2, GSK3ß and p53 proteins, were determined by western blotting. The findings indicated that, P. harmala's alkaloids extract, HAR and HAL were significantly cytotoxic toward HCT116 cells after 24 and 48 h of treatment. We showed that P. harmala's alkaloid extract induce apoptosis and cell cycle arrest at G2 phase in the HCT116 cell line. Downregulation of GSK3ß and Bcl-2 and upregulation of Bax and p53 were observed. CONCLUSION: The findings of this study indicate that the P. harmala's alkaloid extract has anticancer activity and may be further investigated to develop future anticancer chemotherapeutic agents.

Trigonelline hydrochloride attenuates silica-induced pulmonary fibrosis by orchestrating fibroblast to myofibroblast differentiation.

BACKGROUND: Silicosis represents a paramount occupational health hazard globally, with its incidence, morbidity, and mortality on an upward trajectory, posing substantial clinical dilemmas due to limited effective treatment options available. Trigonelline (Trig), a plant alkaloid extracted mainly from coffee and fenugreek, have diverse biological properties such as protecting dermal fibroblasts against ultraviolet radiation and has the potential to inhibit collagen synthesis. However, it's unclear whether Trig inhibits fibroblast activation to attenuate silicosis-induced pulmonary fibrosis is unclear. METHODS: To evaluate the therapeutic efficacy of Trig in the context of silicosis-related pulmonary fibrosis, a mouse model of silicosis was utilized. The investigation seeks to elucidated Trig's impact on the progression of silica-induced pulmonary fibrosis by evaluating protein expression, mRNA levels and employing Hematoxylin and Eosin (H&E), Masson's trichrome, and Sirius Red staining. Subsequently, we explored the mechanism underlying of its functions. RESULTS: In vivo experiment, Trig has been demonstrated the significant efficacy in mitigating SiO2-induced silicosis and BLM-induced pulmonary fibrosis, as evidenced by improved histochemical staining and reduced fibrotic marker expressions. Additionally, we showed that the differentiation of fibroblast to myofibroblast was imped in Trig + SiO2 group. In terms of mechanism, we obtained in vitro evidence that Trig inhibited fibroblast-to-myofibroblast differentiation by repressing TGF-ß/Smad signaling according to the in vitro evidence. Notably, our finding indicated that Trig seemed to be safe in mice and fibroblasts. CONCLUSION: In summary, Trig attenuated the severity of silicosis-related pulmonary fibrosis by alleviating the differentiation of myofibroblasts, indicating the development of novel therapeutic approaches for silicosis fibrosis.

Protective Effect of the Total Alkaloid Extract from Bulbus Fritillariae pallidiflorae in a Mouse Model of Cigarette Smoke-Induced Chronic Obstructive Pulmonary Disease.

Purpose: In recent years, the incidence of chronic obstructive pulmonary disease (COPD) has been increasing year by year, but therapeutic drugs has no breakthrough. The total alkaloid extract from Bulbus Fritillariae pallidiflorae (BFP-TA) is widely used in treating lung diseases. Therefore, this study aimed to investigate the protective effect and mechanism of BFP-TA in COPD mice. Methods: BFP-TA was prepared by macroporous adsorbent resin, and the material basis of BFP-TA was analyzed by HPLC-ELSD and UHPLC-MS/MS. Then, the COPD mouse model was induced by cigarette smoke (CS) for 12 weeks, administered at weeks 9-12. Subsequently, the body weight, lung-body ratio, pulmonary function, histopathology, and the levels of pro-inflammatory cytokines, matrix metalloproteinases (MMPs) and oxidative stress markers in the serum of mice were determined. The expressions of related protein of EMT and MAPK signaling pathways in the lung tissues of mice were detected by Western blot. Results: The alkaloid relative content of BFP-TA is 64.28%, and nine alkaloids in BFP-TA were identified and quantified by UHPLC-MS/MS. Subsequently, the animal experiment showed that BFP-TA could improve pulmonary function, and alleviate inflammatory cell infiltration, pulmonary emphysema, and collagen fiber deposition in the lung of COPD mice. Furthermore, BFP-TA could decrease the levels of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1ß), MMPs (MMP-9 and MMP-12) and MDA, while increase the levels of TIMP-1 and SOD. Moreover, BFP-TA could decrease the protein expressions of collagen I, vimentin, α-SMA, MMP-9, MMP-9/TIMP-1, Bax, p-JNK/JNK, p-P38/P38, and p-ERK/ERK, while increase the level of E-cadherin. Conclusion: This study is the first to demonstrate the protective effect of BFP-TA in CS-induced COPD mouse model. Furthermore, BFP-TA may improve airway remodeling by inhibiting the EMT process and potentially exert anti-inflammatory effect by inhibiting the MAPK signaling pathway.

Functionalized magnetic particles coupled with LC-MS strategy facilitated discovery of trace thioalkaloids with potent immunosuppressive activity.

Trace natural products (TNPs) are still the vital source of drug development. However, the mining of novel TNPs is becoming increasingly challenging due to their low abundance and complex interference. A comprehensive strategy was proposed in which the functionalized magnetic particles integrated with LC-MS for TNPs discovery. Under the guidance of the approach, fifteen trace Nuphar alkaloids including seven new ones, cyanopumiline A sulfoxide (1), cyanopumiline C sulfoxide (8) and cyanopumilines A-E (4-5, 10, 12-13) featuring an undescribed nitrile-containing 6/6/5/6/6 pentacyclic ring system were isolated from the rhizomes of Nuphar pumila. Their structures and absolute configurations were determined on the basis of detailed spectroscopic data analysis and single-crystal X-ray diffraction analysis. Notably, a concise method based on 13C NMR spectroscopy was established to determine the relative configurations of spiroatoms. Biologically, compounds 1-12 exhibited potent immunosuppressive activities with IC50 values ranging from 0.1-12.1 µM against anti-CD3/CD28 induced human peripheral T cell proliferation. Mechanistic studies revealed that 4 could dose-dependently decrease pro-inflammatory cytokines and the expression levels of CD25 and CD71.