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Introduction: Toddalia asiatica (TA) is a classical traditional Chinese medicine used to treat rheumatoid arthritis and contusions. However, research regarding TA quality control is currently limited. Objective: We aimed to establish a strategy for identifying quality markers that can be used for the evaluation of the quality of TA. Method: A rapid and efficient ultra‐high‐performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UHPLC‐MS/MS) method was developed for the quantitative determination of 19 compounds in TA from different regions. Then, the extraction process of TA was successively optimized by single‐factor optimization and response surface methodology. Moreover, chemometrics was employed to confirm the correlation between quality and target compounds. Results: Utilizing the UHPLC‐MS/MS method, separation of the 19 bioactive compounds was achieved within 14 min. The method was validated in terms of linearity (r2 > 0.9982), precision (0.08%–3.70%), repeatability (0.50%–2.54%), stability (2.26%–5.46%), and recovery (95.8%–113%). The optimal extraction process (extraction solvent, 65% ethanol aqueous solution; solid–liquid ratio, 1:20; extraction time, 25 min) was determined with the total content of 19 bioactive compounds as indicator. Significant disparities were observed in the contents of target compounds across different batches of TA. Besides, all samples could be categorized into two distinct groups, and magnoflorine, (−)‐lyoniresinol, nitidine chloride, norbraylin, skimmianine, and decarine were identified as quality markers. Conclusion: In the present study, we developed a strategy to improve the quality control of TA. In consideration of the pharmacodynamic activity and statistical differences, six compounds are proposed as quality markers for TA. A rapid and efficient UHPLC‐MS/MS method was developed for the quantitative determination of 19 compounds in Toddalia asiatica from different regions. All samples could be categorized into two distinct groups, and magnoflorine, (−)‐lyoniresinol, nitidine chloride, norbraylin, skimmianine, and decarine were identified as quality markers. A new strategy of UHPLC‐MS/MS with chemometrics was developed to improve the quality control of Toddalia asiatica. [ABSTRACT FROM AUTHOR]

A new prenylated coumarin, 5, 7-dimethoxy-6-[(Z)-3'-methylbutan-1',3'-d-ienyl] coumarin (1), along with ten known compounds (2-11), was obtained from the roots of Toddalia asiatica (Linn.) Lam. Their structures were determined based on 1D and 2 D NMR spectroscopy as well as high-resolution mass spectrometry. Notably, the structure of (3S, 4R)-3,4-epoxypimpinellin was revised as its cis-epimer, compounds (2-6) were isolated from this genus for the first time. The cytotoxic activities of the isolated compounds 1-11 were evaluated against SW480, A549, MDA-MB-231, HepG 2, HEp-2, SGC7901 cancer cell lines. Compound 10 exhibited weak cytotoxicity against HEP 2 with IC50 value of 75.19 ± 1.17 µM. [ABSTRACT FROM AUTHOR]

Human hormone glucagon-like peptide-1 (GLP-1) lowers α-cell glucagon and increases β-cell insulin. GLP-1 is digested by dipeptidyl peptidase (DPP-4) enzyme; hence, the inhibition of DPP-4 enzyme prevents the digestion of GLP-1 and reduces the occurrence of type 2 diabetes condition. Present study developed a database consists of 1837 molecules structurally similar to selected query compounds applying PubChem screening and was further screened for drug-likeness and ADME/T behaviour using SwissADME tool. Total of six compounds, i.e. Toddalolactone, Ulopterol, 1-O-methyl 4-O-(2-oxo-1H-quinolin-4-yl) butanedioate, [2-oxo-3-(3-oxobutyl)-1H-quinolin-4-yl] acetate, N-(2,5-Dioxo-5,6-dihydro-2H-pyrano[3,2-c]quinolin-3-yl)acetamide, (2S)-2-amino-2-phenyl-1-[3-(trifluoromethyl)-6,8-dihydro-5H-[1,2,4] triazolo [4,3-a]1yrazine-7-yl] ethanone cleared screening and were tested for DPP-4 enzyme inhibitory activity comparing Alogliptin as standard drug applying AutoDock in silico approach (Argus Lab, Discovery Studio and PyMol). All the six compounds displayed RMSD values less than 2A0, and binding energies of − 8.5139 kcal/mol, − 9.438 kcal/mol, − 9.4783 kcal/mol, − 8.898 kcal/mol, − 9.022 kcal/mol, and − 7.604 kcal/mol, respectively. Study testified that Toddalolactone isolated from the plant Toddalia asiatica (− 8.5139 kcal/mol) confers 03 hydrogen bonds, 03 Pi-Pi stack bonds and RMSD values of 0.281 A0, phytocompound Ulopterol (− 9.438 kcal/mol) isolated from the plant Coleonema album, interacted with 04 amino acids of DPP-4 enzyme active site conferring stable hydrogen (02) and Pi-Pi bonds (02) with RMSD value 0.78 A0, represents increased DPP-4 enzyme inhibitory activity in comparison with standard drug Alogliptin binding energy of − 7.34 kcal/mol with only 02 interactive bonds. It is evidenced from the study that a higher number of bonds indicates the stability of the complex formed with increased enzyme inhibitory activity; hence, the phytocompound Toddalolactone derived from the plant Toddalia asiatica and Ulopterol from Coleonema album as a potential lead molecule shall be tested further resulting in in vitro and in vivo procedures in order to develop novel drugs in the treatment of Type 2 Diabetes mellitus condition. [ABSTRACT FROM AUTHOR]

AIM: Observation of neuroprotective effects of Toddalia asiatica (TA) on cerebral ischemia-reperfusion injury (CIRI) in rats by investigating the effects and mechanisms of drugs on the polarization of microglia M1/M2 subtype and the Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor-κB (NF-κB) pathway. METHODS: The modified thread occlusion method was used to establish a rat model of CIRI, and the rats were randomly divided into the model group, Toddalia asiatica (1.08 g/kg) group, donepezil hydrochloride (0.45 mg/kg) group, and sham group, with 16 rats in each group. Based on the assessment of neurofunctional changes in each group of rats, HE and Nissl staining were used to observe the pathological changes in brain tissue. TUNEL staining was performed to detect neuronal apoptosis, immunohistochemistry was used to detect the expression of M1 microglia marker ionized calcium-binding adapter molecule 1 (Ibal), M2 microglia marker arginase 1 (Argl), and TLR4 localization. Western blot was used to detect the expression of microglia polarization proteins and proteins related to TLR4/MyD88/NF-κB pathway in the hippocampus region. RESULTS: Compared with sham group, the model group rats had higher neurological function scores (P<0.01), and neuronal arrangement in the hippocampus and cortex was loose and disordered, Nissl bodies decreased, and neuronal apoptosis increased. The numbers of M1 microglia marker Iba1-, M2 microglia marker Arg1-, and TLR4-positive cells were significantly increased. In addition, the protein levels of TLR4, MyD88, p-NF-κB p65, NF-κB p65, p-NF-κB inhibitory factor (p-IκB), Ibal, interleukin-6 (IL-6), and Arg1 in the hippocampus were elevated (P< 0.05, while IL-4 and IL-10 expression were decreased (P<0.01). Compared with model group, the Toddalia asiatica group and Donepezil hydrochloride group showed increased protein expression of Arg1, IL-4 and IL-10 (P<0.05), while the other indicators were decreased (P<0.05, P<0.01). CONCLUSION: Toddalia asiatica possesses neuroprotective effects on CIRI rats, which may be attributed to its ability to regulate M1/M2 polarization and inhibit the TLR4/MyD88/NF-κB-mediated inflammatory pathway. [ABSTRACT FROM AUTHOR]

Two new coumarins with a rare, oxidized geranyl group, named toddalins I (1) and J (2), were isolated from the roots of Toddalia asiatica , a traditional Chinese herb used to treat many diseases, especially athletic injuries. Their structures were determined using extensive spectroscopic methods, including 1D NMR, 2D NMR and HRESIMS. Toddalins I (1) and J (2) exhibited moderate in vitro anti-inflammatory activities by the inhibition of lipopolysaccharide (LPS)-induced NO production in RAW 264.7 macrophages with IC 50 values of 12.6 and 15.6 μ M, respectively. [Display omitted] • Two new coumarins (1 and 2) were isolated from the roots of Toddalia asiatica. • Their structures were elucidated by means of extensive spectroscopic analyses. • Toddalins I (1) and J (2) exhibited moderate in vitro anti-inflammatory activities. [ABSTRACT FROM AUTHOR]

We reported the discovery of six novel coumarins, toddasirins A–F (1 – 6), each endowed with modified isoprenyl or geranyl side chains, derived from the roots of Toddalia asiatica. Comprehensive structural elucidation was achieved through multispectroscopic analyses, single-crystal X-ray diffraction experiments, and advanced quantum mechanical electronic circular dichroism (ECD) calculations. Furthermore, the anti-inflammatory activity of these compounds was assessed. Notably, compounds 1 – 3 and 6 demonstrated notable inhibitory effects on nitric oxide (NO) production in lipopolysaccharide (LPS)-induced RAW 264.7 cells, with 50% inhibitory concentration (IC 50) values of 3.22, 4.78, 8.90, and 4.31 μmol·L−1, respectively. [ABSTRACT FROM AUTHOR]

The antifungal and antiaflatoxigenic effects of four distinct plant species against Aspergillus flavus and Aspergillus parasiticus were investigated. Essential oils and methanolic extracts were prepared from aerial parts of Lippia javanica, Ocimum gratissimum, Satureja punctata, and stem barks of Toddalia asiatica by hydro-distillation and maceration, respectively. The poisoned food method was used to confirm the antifungal activity of essential oils and methanolic extracts from four different plant species against Aspergillus flavus and Aspergillus parasiticus, and high-performance liquid chromatography was used to quantify the antiaflatoxigenic activity. The essential oils of Satureja punctata and Lippia javanica showed the highest antiaflatoxigenic activity against the fungi strains tested at concentrations of 1.25, 2.5, and 5 µL/mL, followed by Ocimum gratissimum essential oil while Toddalia asiatica essential oil exerted moderate antiaflatoxigenic activity. Meanwhile, the methanolic extracts showed a wide spectrum of low to high antifungal and antiaflatoxigenic activities at concentrations of 125, 250, and 500 µg/mL against A. flavus and A. parasiticus. This study has indicated that the essential oils of Satureja punctate, Lippia javanica, and Ocimum gratissimum had substantial antifungal and antiaflatoxigenic activities compared to their methanolic extracts, while Toddalia asiatica methanolic extract had a moderate antifungal activity compared to its essential oil. [ABSTRACT FROM AUTHOR]

Medicinal plants are rich sources of several diverse bioactive constituents with a wide range of biological effects. The screening of bioactive compounds plays a vital role in the protection of human health. Toddalia asiatica (L.) Lam., belonging to Toddalia genus of Rutaceae family, is a folk medicine in China used for hundreds of years. The purpose of the current study is to monitor the phytochemical compounds in the root of Toddalia asiatica using gas chromatography and mass spectrometry (GC–MS). Totally 27 compounds were identified and the chromatograph showed peaks with individual compounds. The major constituents identified in root methanolic extract were anliinosoquinoline (20.18%), pimpinellin (20.18%), pyrrolidine (18.92%), 1H-pyrrole (18.41%) and 2-phenyl-6-vinylindolizine (15.53%) and many other compounds were identified as low level. Many of these phytocomponents have antibacterial, antifungal, antifouling, immunomodulatory and antitumor activities, which justify the folklore use of root extract in traditional system to cure various ailments. [ABSTRACT FROM AUTHOR]

A new sesquiterpene, named todasinoid A (1), together with eight known compounds (2−9) were isolated from the roots of Toddalia asiatica. The gross structure of todasinoid A were established by analyses of the NMR and HRESIMS data. A comparison of the experimental ECD spectrum of 1 with the calculated ECD spectra for a model compound (1a) resolved the absolute configuration of 1. The known compounds were identified to be bullatantriol (2), aculeatin (3), 6-(3-ethoxy-2-hydroxy-3-methylbutyl)-5,7-dimethoxy-2H-1- benzopyran-2-one (4), trans-N-p-coumaroyltyramine (5), feruloyltyramine (6), −sitosterol (7), sitosterol Dglucoside(8), 7α-hydroxysitosterol (9) by comparing the NMR data and specific rotations with reported data in literature. Compound 1 is an eremophilane-type sesquiterpenoid containing a mercaptolactate side-chain that is rarely found in nature. Compounds 2, 5, and 9 were isolated from this plant for the first time. Bioassay study revealed that compound 5 exhibited inhibitory effects against -glucosidase with an IC50 of 320 M, being more active than the positive control acarbose. [ABSTRACT FROM AUTHOR]

The present study reports the enhanced antifungal, aflatoxin B 1 (AFB 1) inhibitory activities and mode of action of chitosan-based nanoencapsulated Toddalia asiatica essential oil (neTAEO). Twenty-seven different chemical components were recognized from T. asiatica essential oil (TAEO) using gas chromatography–mass spectrometry (GC–MS). The caryophyllene oxide (CO) (25.4%), and 1,3-hexadiene, 3-ethyl-2,5-dimethyl- (HED) (24.08%) were documented as significant compounds. The Z -average particles diameter (Z-APD) of the neTAEO ranged between 18.41 and 131.8 nm. The neTAEO showed enhanced and most promising antifungal and AFB 1 inhibitory activity than TAEO. In viable maize model assay, neTAEO effectively preserved the maize from fungal invade and AFB 1 biosynthesis. The neTAEO significantly disturbs membrane integrities of Aspergillus flavus by inhibiting ergosterol biosynthesis followed by the extreme release of ions (Mg2+ and K+) and UV-absorbing (260 and 280 nm) cellular constituents. The in-silico molecular docking showed that the major active components of TAEO viz. , CO and HED were active against AFB 1 synthesizing leading genes Ver-1 and Omt-A with docking scores ranging from −4.8 to −7.7. The obtained results confirm that neTAEO showed promising antifungal and AFB 1 inhibitory activities; hence, it could be used as an alternative green strategy to protect food grains from fungal invade and AFB 1 production during storage. [ABSTRACT FROM AUTHOR]

Foods contaminated with fungi and their toxins cause many infections in developing and underdeveloped countries. Owing to the development of resistance by fungal pathogens to existing chemical antifungal agents, there is an urgent need to look for new eco-friendly antimicrobial agents. There is an increasing demand from agro-food industries for new environmentally compatible fungicides. Plant essential oils are an excellent source of antifungal volatile substances which are appropriate substitutes for food protection. Keeping this in mind we evaluated the efficacy of selected essential oils in suppressing the growth of Alternaria alternata isolated from stored wheat grains using in vitro and in vivo models. Preliminary antifungal activities varied among the tested oils with the zones of inhibition ranging from 10.66±1.15 mm to 19±1.00 mm. Among the tested oils T. asiatica oil showed significant inhibitory effects on A. alternata at 2.5 µL/mL. Further, its mechanistic action was related to the impairment of the squalene gene (SqSy) which was involved in the biosynthesis of sterol and ergosterol membrane factors. This was further supported by altered sterol profile of A. alternata with 29.4±2.40% reduction of total ergosterol content at 30 µL/mL. Moreover, prolonged exposure to T. asiatica oil as a fumigant controlled the total fungal count up to 68.75% compared to the control for a period of 12 months. T. asiatica oil proved to be a promising candidate to control A. alternata and its mycotoxin residues in stored wheat. It can be used as a sustainable and effective alternative to develop new fungicide. [ABSTRACT FROM AUTHOR]

Objective: To investigate the therapeutic mechanism of Tujia medicine Toddalia asiatica alcohol extract (TAAE) for synovial pannus formation in rats with college-induced arthritis (CIA)., Methods: Sixty male SD rats were randomized into normal control group, CIA model group, TGT group, 3 TAAE treatment groups at low, medium and high doses ( n =10). Except for those in the normal control group, all the rats were subjected to CIA modeling using a secondary immunization method and treatment with saline, TGT or TAAE by gavage once daily for 35 days. The severity of arthritis was assessed using arthritis index (AI) score, and knee joint synovium pathologies were examined with HE staining. Serum levels of TNF-α, IL-6, and IL-1β were detected with ELISA; the protein expressions of PI3K, Akt, p-PI3K, p-Akt, VEGF, endostatin, HIF-1α, MMP1, MMP3, and MMP9 in knee joint synovial tissues were determined using Western blotting, and the mRNA expressions of TNF‑α, IL-6, IL-1β, VEGF, HIF-1α, PI3K, and Akt were detected with RT-PCR., Results: Treatment of CIA rat models with TAAE and TGT significantly alleviated paw swelling, lowered AI scores, and reduced knee joint pathology, neoangiogenesis, and serum levels of inflammatory factors. TAAE treatment obviously increased endostatin protein expression, downregulated p-PI3K, p-Akt, MMP1, MMP3, MMP9, VEGF, and HIF-1α proteins, and reduced TNF‑α, IL-6, IL-1β, PI3K, Akt, VEGF, and HIF-1α mRNA levels in the synovial tissues, and these changes were comparable between high-dose TAAE group and TGT group., Conclusion: TAAE can improve joint symptoms and inhibit synovial pannus formation in CIA rats by regulating the expressions of HIF-1α, VEGF, endostatin, MMP1, MMP3, and MMP9 via the PI3K/Akt signalling pathway.

Ethyl acetate fraction of Toddalia asiatica was fractionated to yield fifteen previously undescribed prenylated coumarins, asiaticasics A-O (1 – 15) along with nine (16 – 24) known derivatives. The structures of these undescribed coumarins were established by spectroscopic analysis and reference data. Biological activity evaluation showed that compound 3 with the IC 50 value of 2.830 μM and compound 12 with the IC 50 value of 0.682 μM owned anti-inflammatory activity by detecting the rate of lactate dehydrogenase release in pyroptosis J774A.1 cells. The results showed that the expression of Caspase-1 and IL-1 β was decreased in a dose-dependent manner in the compound 12 treatment group, suggesting that compound 12 may reduce pyroptosis by inhibiting NLRP3 inflammasome. To further determine that compound 12 treatment can inhibit macrophage pyroptosis, morphological observation was performed and the results were consistent with the bioactivity evaluation. [Display omitted] • Twenty-four coumarins were isolated from the ethyl acetate fraction of Toddalia asiatica. • Fifteen coumarins that have not been described before. • Two undescribed compounds showed low IC 50 values and could exert effective anti-inflammatory effects. • Compound 12 inhibited pyroptosis in a dose-dependent manner. [ABSTRACT FROM AUTHOR]

• ODCE, a CMC expansion strategy, was developed. • CMC was extended unidirectionally. • ODCE was compatible with the concurrent determination of multiple compounds. • Improvement of ULOQ reached 50-fold. To facilitate the safety, efficacy and rationality of clinical application of traditional Chinese medicines (TCMs), pharmacokinetic research played an indispensable role. The key challenge during pharmacokinetic investigation lied at the substantial fluctuation of compound concentrations in the plasma over the course of absorption. Taking the pharmacokinetics of six compounds after administration of Toddalia asiatica (TA) as an example, an efficient strategy was established by introducing the online double collision energy (ODCE) into the quantification process applying ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). During the analytical program, double collision energy (DCE) was optimized to establish the dual calibration curve (DCC) with large concentration monitoring coverage (CMC) for meeting the wide content range of certain target compounds. Method validation test was performed in terms of linearity, precision, sensitivity, matrix effect, recovery, etc. The results displayed that the CMC of todarolactone with high exposure in plasma was extended from 1.25–2,500 ng/mL to 1.25–125,000 ng/mL. Furthermore, a rapid UHPLC-MS/MS method integrated with ODCE was successfully applied to the determination of six compounds in rat plasma, revealing an extremely high plasma concentration of todarolactone (16,662 ng/mL). This strategy could expand the range of quantification while retaining extraordinary sensitivity. Consequently, it could be a fit-for-purpose strategy to quantify compounds over a wide concentration range for in vivo process monitoring. [ABSTRACT FROM AUTHOR]

Toddalia asiatica L., a significant medicinal plant in the family Rutaceae, has been applied to treat rheumatoid diseases for decades. Its pharmacological activities are mainly attributed to the existence of generous coumarins and alkaloids; however, the pharmacokinetics of Toddalia asiatica L. remain unclear. A high-performance liquid chromatography-tandem mass spectrometry (HPLC–MS/MS) method was established for the simultaneous determination of four coumarins, three alkaloids and one flavonoid (hesperidin, nitidine chloride, chelerythrine, toddalolactone, isopimpinellin, pimpinellin, bergapten and dictamnine) in rat feces as well as four coumarins and one alkaloid (toddalolactone, isopimpinellin, pimpinellin, bergapten and dictamnine) in rat plasma and urine. Chromatographic separation was accomplished on an Agilent ZORBAX SB-C18 column (2.1 × 150 mm, 5 μm) with acetonitrile (containing 0.1% formic acid) and 5 mmol/L aqueous ammonium formate for gradient elution. A correlation coefficient greater than 0.9925 reflected the excellent linearity of the analytical response. The lower limits of quantification were 30.0, 10.0, 10.0, 30.0, 5.0, 10.0, 2.5 and 2.5 ng/mL for hesperidin, nitidine chloride, chelerythrine, toddalolactone, isopimpinellin, pimpinellin, bergapten and dictamnine, respectively. The intra- and inter-day precision were less than 12.7%, and the accuracy was between −11.8% and 12.9%. In summary, this study is the first to study the pharmacokinetics and excretion of T. asiatica extract after oral administration, which may provide a scientific basis for its clinical applications. [Display omitted] • The pharmacokinetics and excretion of T.asiatica extract after oral administration was revealed for the first time. • Established and validated for simultaneous analysis in rat feces, urine and plasma of eight components by the LC-MS/MS method. • Pimpinellin, isopimpinellin, bergapten and dictamnine may excreted in the form of bile or metabolites. [ABSTRACT FROM AUTHOR]