Gastrodin and parishin compounds alcohol derivatives, the major bioactive components in Gastrodia elata, have attracted significant attention due to their promising potential health benefits. The simultaneous separation of these compounds from crude extracts poses significant challenges due to their structural homology. In this study, a separation method based on modified multiple dual-mode (MDM) high-speed counter-current chromatography was established for the efficient preparation and separation of four polar similar compounds of G.elata. Tert-butyl methyl ether/n-butyl alcohol/acetonitrile/water (acidified with 0.7% trifluoroacetic acid) (2:3:1:5, v/v/v/v) was used as two-phase polar solvent system, four target compounds were simultaneously separated by two cycles in modified MDM mode. Parishin A, gastrodin, parishin B and parishin E were obtained with purities of 90.2%, 98.3%, 87.8% and 93.5%, respectively. The structure of the target compounds was confirmed by ¹H NMR and 13C NMR analysis of the isolated material. Moreover, it provides a novel and scalable technical platform for the efficient isolation of structurally similar compounds from complex natural product matrices.
The major histocompatibility complex (MHC) is an essential part of the adaptive immune systems that present antigenic peptides at the cell surface for recognition and activation of circulating T lymphocytes, which is vital for our body to recognize and respond to foreign pathogens, including viruses, bacteria, and cancerous cells. Human leukocyte antigen (HLA) genes encode proteins located on the surface of most cells in the body, and studies have shown that protein glycosylation plays a significant role in the folding of human leukocyte antigen (HLA) proteins, loading of peptides, and forming of HLA-peptide complex. In the meantime, some of those peptides presented by the MHC system are also glycosylated. Thus, glycosylation has a huge impact on cellular uptake, proteolytic processing, presentation by MHC, and subsequent T-cell priming. However, glycosylation characterization of the whole MHC system has often been neglected during conventional immunopeptidomics analysis due to the lower abundance of glycosylated peptides and increased complexity of the mass spectrum. To tackle this problem, we utilized hydrophilic interaction chromatography to enrich glycopeptides from both peptides bound to HLA and digest of HLA proteins that obtained from immunoprecipitation of MHC complex from cell lysate. Enriched glycopeptides are analyzed by mass spectrometry that enables the characterization of glycosylation of MHC by database search.
To develop a rapid analytical method based on cold-induced liquid-liquid extraction coupled with ultra-performance liquid chromatography-tandem mass spectrometry (CI-LLE-UPLC-MS/MS) for the determination of perchlorate in human urine. After spiking urine samples with stable isotope-labeled internal standards, the samples were centrifuged with a mixture of acetonitrile and water (60∶40, V/V) and then kept at -40 ℃ for 30 min. The supernatant was diluted tenfold with acetonitrile and separated on a Poroshell 120 PFP column (50 mm×2.1 mm, 1.9 μm) with a gradient elution of methanol and 1% acetic acid aqueous solution. Perchlorate was quantified by UPLC-MS/MS using the internal standard method. The method exhibited good linearity (coefficient of determination R2>0.999), with a detection limit of 0.3 μg/L and a quantification limit of 1.0 μg/L. The average recoveries at low, medium, and high spiked levels were 101.2%-106.7%, and the intra-day and inter-day relative standard deviations were both less than 5%(n=6). The determination result of perchlorate in the human urine standard reference material (SRM 3668) were (2.75±0.11) and (13.67±0.34) μg/L, both within the reference ranges. Analysis of 243 real urine samples from China revealed a 100% detection rate for perchlorate, with concentrations ranging from 1.91 to 87.41 μg/L and a median value of 18.74 μg/L. The method features simple pretreatment, rapid detection, and high sensitivity, precision, and accuracy, making it suitable for large-scale detection of perchlorate in human urine.
An analytical method for the determination of 32 pesticide residues in human urine was established using amino multi-walled carbon nanotubes (NH2-MWCNTs)-based dispersive solid-phase extraction combined with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). 25% saturated sodium chloride solution was added to precipitate protein in the urine. The pH was adjusted to 5 using 20 mmol/L ammonium acetate solution. The target analytes were adsorbed onto amino multi-walled carbon nanotubes, followed by desorption with acetonitrile. Finally, the extract was analyzed by UPLC-MS/MS. Good linearity for the 32 pesticides was observed within the concentration range of 1-100 μg/L, with correlation coefficients (r) all above 0.999. Fortified at three concentration levels (4, 40, and 200 μg/L, n=6), average recoveries were obtained in the range of 91.2%-110.0%, with relative standard deviations ranging from 1.1%-4.6%. The limits of detection and quantification were determined to be 0.003-0.03 μg/L and 0.01-0.1 μg/L, respectively. The adsorption process of the 32 pesticides onto the NH2-MWCNTs was found to be well described by the pseudo-second-order kinetic model and the Langmuir isotherm model. This method is simple and rapid to operate, accurate, and highly sensitive, enabling the rapid detection of 32 pesticide residues in human urine.
Kinetic plots were used to assess the theoretical resolving power of sub-1 μm non-porous particle-packed columns in UHPLC conditions. The use of sub-750 nm particle-packed columns enables substantially faster (> 2x) separations for high molecular-weight molecules such as peptides and proteins in the high-velocity regime compared to state-of-the-art 1.5 μm packed columns. This gain, however, is fundamentally constrained by intrinsic limitations: the markedly reduced permeability restricts attainable column lengths under current pressure limits (P = 1500 bar), while mitigation of viscous heating requires small internal diameters, thereby increasing susceptibility to extra-column dispersion. Furthermore, only analytes with sufficiently low diffusion coefficients can achieve meaningful efficiencies, positioning nanoparticle columns primarily for (bio)macromolecular separations. Self-assembly-based column packing can produce highly ordered sphere arrangements (i.e., face-centered cubic structures) that markedly reduce eddy dispersion. Such column technology, based on 750 nm particles, would enable separations that are 20-35 times faster than conventional randomly packed columns using 1.5 µm particles. However, due to the markedly reduced permeability and higher column performance per unit length, such improvement in speed would be limited by the extremely short optimal column length of face-centered cubic packed columns (< 1.5 cm), rendering these columns impractical when used in current LC systems.
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The chemical constituents of Cinnamomum longepaniculatum leaves were isolated and purified using separation techniques such as silica gel column chromatography, macroporous adsorption resin, gel column chromatography, and preparative high-performance liquid chromatography. The isolated compounds were identified by combining spectroscopic techniques including high-resolution electrospray ionization mass spectrometry(HR-ESI-MS), nuclear magnetic resonance(NMR), ultraviolet spectroscopy(UV), and infrared spectroscopy(IR) and literature data. A total of nine compounds were identified: cinlongeligan A(1), salicifoneoliganol(2),(-)-syringaresinol-4-O-β-D-apiofuranosyl-(1→2)-β-D-glucopyranoside(3), syringaresinol mono-β-D-glucoside(4), episyringaresinol-4″-O-β-D-glucopyranoside(5),(+)-medioresinol 4'-O-β-D-glucopyranoside(6), lyoniside(7), lantibetin(8),(+)-isolariciresinol-9-O-β-D-glucoside(9). Compound 1 was a novel lignan derivative. Except for compounds 7 and 9, all others were isolated from the genus Cinnamomum for the first time. The results of antioxidant activity showed that compounds 1 and 3 had significant scavenging capacity against 2,2-diphenyl-1-picrylhydrazyl(DPPH) free radical with IC_(50) values of(10.35±0.46) and(9.73±0.62) μg·mL~(-1), respectively.
A total of 9 constituents(catalpol, rehmannioside A, rehmannioside D, ajugol, aucubin, regaloside A, regaloside B, regaloside C, and regaloside E) from Baihe Dihuang Decoction in the plasma samples of normal and depression rats were simultaneously determined by ultra-performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS). Normal and depression model rats were used to study the pharmacokinetics and elimination patterns of active constituents from Baihe Dihuang Decoction. A depression model was established in rats via the chronic unpredictable mild stress(CUMS) protocol. After gavage with Baihe Dihuang Decoction, blood samples were collected from orbital venous plexus of both normal and model rats at designated time points. Ultra-high performance liquid chromatography-triple quadrupole-mass spectrometry(UHPLC-QqQ-MS) was employed to investigate the pharmacokinetics of 9 major compounds in the multiple reaction monitoring(MRM) mode. Key pharmacokinetic parameters were determined through non-compartmental mode analysis in Phoenix 64. The results revealed significant differences in the pharmacokinetic profiles of the constituents of Baihe Dihuang Decoction between the normal and model groups. Compared with the normal group, the model group exhibited significant increases in the C_(max) values and decreases in T_(max) of catalpol, aucubin, and regaloside A. For regaloside B, regaloside C, and regaloside E, the AUC, MRT, t_(1/2), and T_(max) were higher in the model group than in the normal group. Rehmannioside A and rehmannioside D showed similar trends, with lower C_(max), AUC, t_(1/2), and T_(max) in the model group than in the normal group, and their plasma concentration-time curves shifted from a double-peak profile in the normal group to a single-peak profile in the model group. These marked differences in the pharmacokinetic characteristics of Baihe Dihuang Decoction between normal and model groups indicate that the physiological state can substantially alter the absorption and elimination of the decoction, providing a scientific basis for its administration.
A strategy combining non-targeted metabolomics with targeted quantification of characteristic components was employed to systematically analyze the chemical constituents of Rubia cordifolia from multiple production regions. Non-targeted metabolomic analysis was performed on 28 samples of R. cordifolia from Shanxi, Shaanxi, and Henan provinces using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UHPLC-Q-TOF-MS). Targeted quantification was conducted using ultra-high-performance liquid chromatography-triple quadrupole tandem mass spectrometry(UPLC-QqQ-MS/MS) to determine the contents of three characteristic cyclopeptides(RA-Ⅴ, RA-Ⅶ, and RA-Ⅻ) and two representative quinone components(purpurin and mollugin) in 33 samples from Shanxi, Shaanxi, Henan, Hebei, and Yunnan provinces. A total of 1 604 metabolites were identified by non-targeted analysis, and clear intergroup differences were observed among samples from Shanxi, Shaanxi, and Henan provinces. KEGG pathway enrichment analysis of differential metabolites revealed metabolic pathways related to amino acids, terpenoids, and flavonoids. Targeted quantitative analysis showed that the average contents of RA-Ⅴ, RA-Ⅶ, total cyclopeptides, and purpurin were highest in samples from Yunnan, the average content of RA-Ⅻ was highest in samples from Henan, and the average contents of mollugin and total quinones were highest in samples from Hebei. This study revealed that R. cordifolia from different production regions exhibited certain differences in chemical composition, with differential metabolic pathways mainly involving amino acid, terpenoid, and flavonoid metabolism. The targeted quantitative results indicate that Yunnan and Hebei are the dominant production regions for R. cordifolia rich in cyclopeptides and quinones, respectively. This study adopted a research strategy combining non-targeted metabolomics with targeted quantification of characteristic components, providing data support and methodological references for the origin tracing, quality control, and clinical application of R. cordifolia.
Understanding how nutritional management and genetic background influence hepatic metabolism is important for improving the nutritional quality of bovine products in tropical production systems. This study evaluated breed-associated differences under a common dietary regimen and diet-related differences within the Nelore breed with respect to hepatic cholesterol, lipid-soluble antioxidant vitamins, fatty acid composition and lipid quality indices. Thirty-six intact male cattle were allocated to three experimental groups: Nelore fed a silage-concentrate diet, crossbred (½ Nelore × ½ Angus) fed the same diet, and Nelore fed a high-grain diet. Hepatic cholesterol and antioxidant vitamins were determined by high-performance liquid chromatography, whereas fatty acid composition was analysed by gas chromatography with flame ionisation detection. No significant differences were observed in hepatic cholesterol concentration for either the breed-associated or diet-related contrasts (p > 0.05). Breed-associated differences were detected for β-carotene, β-tocopherol, γ-tocopherol and docosahexaenoic acid (22:6n-3) (p < 0.05). Diet-related differences significantly affected β-carotene, tocopherols, tocotrienols, several individual fatty acids and lipid quality indices, including the n-6/n-3 ratio and hypocholesterolaemic and hypercholesterolaemic fatty acid indices (p < 0.05). Principal component analysis showed clear separation of animals by dietary treatment, with β-carotene, α-tocopherol and tocotrienols contributing strongly to sample discrimination. Overall, diet-related differences within the Nelore breed were associated with significant changes in a broader range of hepatic lipid and antioxidant variables, whereas breed-associated differences under a common dietary regimen were observed for a more limited set of variables. These findings highlight the importance of nutritional management in modulating hepatic composition and support the use of liver biomarkers to assess metabolic responses in beef cattle.
The chemical constituents of the ethyl acetate extract from Achyrocline satureioides were rapidly analyzed using ultra performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry(UPLC-Q-TOF-MS/MS) combined with the Global Natural Products Social Molecular Networking(GNPS) molecular network. The mass spectrometric characteristics of the phloroglucinol compounds were clarified. Guided by these features, systematic isolation was performed using ODS-C_(18), Sephadex LH-20, and semi-preparative high performance liquid chromatography(HPLC). The structures were elucidated using modern spectroscopic techniques such as nuclear magnetic resonance(NMR) spectroscopy and high-resolution mass spectrometry(HR-MS). A total of 38 compounds were identified from the ethyl acetate fraction by mass spectrometry, including 23 flavonoids. Additionally, 18 compounds were isolated from this fraction. Among them, compound 1 was a new unsaturated fatty acid, and compounds 2, 3, and 7-10 were phloroglucinols. Compounds 2, 3, 7, and 8 were obtained from this plant for the first time. The in vitro anti-proliferative activities of compounds 2, 3, and 7-10 against the non-small cell lung cancer cell lines A549 and H1975 were evaluated using the CCK-8 assay. Compound 8 exhibited significant anti-lung cancer activity. This integrated approach using LC-MS/MS combined with GNPS enables efficient analysis of the chemical components in the ethyl acetate fraction of A. satureioides and facilitates the guided isolation of phloroglucinol compounds, thereby enriching the understanding of its material basis.
One new cassane diterpenoid(1) and six known compounds(2-7) were isolated from the 95% ethanol extract of fresh Caesalpinia decapetala pods by silica gel column chromatography, gel filtration chromatography, and semi-preparative HPLC. Compound 1 was identified as a highly oxidized cassane diterpenoid, and its absolute configuration was determined by comprehensive spectroscopic analysis and electronic circular dichroism(ECD) calculations, being named caesalpin L. The known compounds were characterized as caesalsappanin K(2), bonducellin(3), 4-hydroxy-3,5-dimethoxybenzaldehyde(4), thellungianin C(5), p-hydroxybenzoyl coumaric acid anhydride(6), and gallic acid(7). Compounds 2 and 4-7 were obtained from this plant species for the first time. The anti-inflammatory activity of compounds 1 and 2 was comparable to that of the positive control.
This study presents a preliminary investigation into the antimicrobial, antibiofilm, and antiproliferative potential of Eremothecium coryli, an endophytic fungus isolated from the seeds of Basella alba L. The biological activities were evaluated using a crude extract-based screening study, employing the lyophilized cell-free supernatant (LCFS) of the fungal culture. Antibacterial and antibiofilm activities were assessed against selected multidrug-resistant bacterial pathogens, while antiproliferative activity was evaluated against breast cancer cell lines and a normal breast epithelial cell line. The LCFS demonstrated measurable antibacterial activity and significant biofilm-inhibition in a concentration-dependent manner. In cytotoxicity assays, selective growth inhibition of breast cancer cell lines was observed when compared with normal cells. Chemical profiling using Liquid Chromatography-Mass Spectrometry and Gas Chromatography-Mass Spectrometry enabled putative metabolite identification, revealing the presence of compounds previously reported to exhibit antimicrobial and anticancer activities. However, these identifications are tentative and based on spectral library matching. Overall, this preliminary investigation highlights the biological potential of E. coryli-derived metabolites. Further purification and validation are necessary to confirm compound identities, elucidate mechanisms of action, and assess in vivo relevance.
This study aims to reveal the differences of polysaccharides in Hedysari Radix before and after honey processing. After separation and purification of Hedysari Radix polysaccharides(HPS) and honey-fried Hedysari Radix polysaccharides(H-HPS) by gradient elution, UV spectrophotometry was employed to determine the content of polysaccharides and uronic acids in different eluate fractions. The molecular weights of different eluate fractions were determined by high-performance gel permeation chromatography(HPGFC). Ion chromatography(IC) was employed to detect the monosaccharide composition and content differences among eluate fractions. The results displayed that the gradient elution of HPS and H-HPS yielded three distinct fractions, which were the fractions from elution with distilled water, 0.1 mol·L~(-1) NaCl, and 0.3 mol·L~(-1) NaCl, respectively. The total polysaccharide and uronic acid content in all eluate fractions of H-HPS were higher than those in the eluate fractions of HPS. Both HPS and H-HPS showed a distribution pattern where the distilled water eluate had the highest total polysaccharide and uronic acid content, followed by the 0.1 mol·L~(-1) NaCl eluate, and the 0.3 mol·L~(-1) NaCl eluate had the lowest. The molecular weight distribution profiles demonstrated that all elution fractions were uniform in structure. After honey processing, the peak symmetry was improved and the structural uniformity was enhanced. During elution under different ionic strengths, with the increase in ionic strength, the polysaccharide distribution tended to be more uniform, with increased molecular size and enhanced stability. In addition, honey processing led to a reduction in the overall molecular weight and a narrower distribution range of polysaccharides, rendering them more stable under the same elution conditions. In particular, the fraction eluted with 0.3 mol·L~(-1) NaCl exhibited high uniformity and a large molecular size. In terms of monosaccharide composition, honey processing induced characteristic changes across fractions. Specifically, the distilled water eluate contained the fewest monosaccharides; the elution with 0.1 mol·L~(-1)NaCl introduced fucose while increasing glucose, xylose, and mannose; the elution with 0.3 mol·L~(-1) NaCl doubled galacturonic acid content, making it a unique component, while glucuronic acid completely disappeared. The study found that honey processing, through reactions such as hydrolysis and decarboxylation, significantly increases polysaccharide and uronic acid content while restructuring their molecular architecture. This provides a theoretical foundation for elucidating the effect-enhancing mechanism of honey processing for Hedysari Radix and for evaluating and developing the polysaccharides in Hedysari Radix and its processed products.
The chemical constituents from Syneilesis aconitifolia were investigated by comprehensive column chromatography techniques, including silica gel, ODS, Sephadex LH-20, and semi-preparative high performance liquid chromatography, and six compounds were isolated from the methanol extract of S. aconitifolia. Their structures were identified by NMR, HR-ESI-MS, and single crystal X-ray diffraction and the compounds were named saconitifolia C(1), saconitifolia D(2), 3β-angeloyloxy-6β-hydroxy-8-epi-eremophilenolide(3), 6β-hydroxy-8β-methoxyeremophil-7(11)-12,8α-olide(4), 8βH-eremophil-3,7(11)-dien-12,8α(14,6α)-diolide(5), 8β-hydroxyeremophil-3,7(11)-diene-8α,12(6α,15)-diolide(6), 8β-methoxyeremophil-3,7(11)-diene-8α,12(6α,15)-diolide(7), and 3β-angeloyloxy-6α,15-epoxy-10βH-eremophil-7(11)-en-12,8β-olide(8). Compounds 1-8, including two new compouds(1 and 2), were sesquiterpene lactones, and compounds 3-8 were isolated from S. aconitifolia for the first time. All of the compounds showed no cytotoxic activity on HeLa or GL261 cells.
Leishmania panamensis, a protozoan parasite of the Viannia subgenus, causes American tegumentary leishmaniasis (ATL) throughout Central and South America, with clinical manifestations ranging from cutaneous lesions to mucosal involvement. Despite the availability of a reference genome for L. panamensis MHOM/COL/81/L13 strain, comprising 30.69 megabases across 35 chromosomes and 8,665 predicted protein-coding genes, comprehensive proteogenomic analyses to refine these annotations have remained limited. This study utilised a proteogenomic approach to enhance the genome annotation of the L. panamensis MHOM/COL/81/L13 strain by integrating publicly available liquid chromatography-tandem mass spectrometry data with a custom six-frame translated genome database. Through systematic peptide mapping and validation, we identified 50 novel protein-coding genes previously absent from the reference annotation and corrected 50 existing genes. The newly identified genes encode proteins containing functional domains, including thioredoxin, glucosyltransferases, and myotubularin, which likely contribute to parasite metabolism, host-pathogen interactions, and intracellular survival mechanisms. These findings substantially refine the genomic resource available for L. panamensis research and provide potential targets for diagnostic and therapeutic development. Future studies will incorporate orthogonal validation methods such as RNA-seq and Ribosome profiling to distinguish between functional genes and translational noise. The refined annotation enables more accurate functional genomic studies and enhances our understanding of the molecular basis underlying L. panamensis pathogenicity and immune evasion strategies. Moreover, the peptide-supported novel and corrected protein-coding genes identified in this study represent a valuable resource for the future exploration of candidate biomarkers and may be provide the molecular targets for improved diagnosis, prognosis, and therapeutic intervention in American tegumentary leishmaniasis.
Even moderate exposures to pesticides and plasticizers have been associated with neurotoxicity and autism characteristics; however, most studies of urine and blood provide a short-term exposure window compared to teeth, which capture long-term exposures from 13-14 weeks in-utero through early childhood. We investigated cumulative pesticide and plasticizer exposures in shed baby teeth from children with and without autism using a comprehensive targeted and non-targeted analysis with two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GCxGC-TOFMS). Compound identification was confirmed with standards and high-fidelity spectral library matches. Sixty compounds were detected in more than 50% of participants (n = 38). Of these, 95% (57/60) were higher in cases compared to controls, and 19/60 were statistically significantly elevated. Thirty-nine of the 60 compounds detected are known developmental toxicants or endocrine-disrupting compounds. To date, 21/60 compounds lack Environmental Protection Agency (EPA) developmental toxicity data. Compounds exhibiting higher levels in cases than in controls include benzyl benzoate (fragrance and food preservative) and organophosphate esters, which are frequently found in flame retardants and other baby products, PEX tubing, food contact materials, and products applied to the skin. Interestingly, five of the top nine compounds that differentiated cases from controls migrate from baby bottles. Elevated levels of toxicants in autism cases in our pilot study highlight a need for larger case-control studies to understand the effects of long-term exposure on autism.
An analytical method for determining pesticide residues in fruit juices, vegetable juices, and wine was developed and validated for 32 active compounds, including 11 fluorinated ones. Sample extraction was carried out using a mixture of acetone, dichloromethane, and petroleum ether. The analysis was performed with gas chromatography-tandem mass spectrometry (GC-MS/MS). The method was applied to the analysis of 81 commercial samples. A total of 81 samples were analysed: 34 fruit juices, 6 vegetable juices, and 41 wine samples. After calculating concentrations in raw fruit or vegetables, maximum residue level (MRL) exceedances were found in 4 samples. Calculation of the chronic risk assessment confirmed that for the analysed samples consumer exposure was within acceptable limits. However, due to three active substances, acute exposure could pose a risk.
Chemoselective bioconjugation techniques are essential tools for precise modification and functional analysis of proteins. Here, we describe a detailed protocol for histidine-specific labeling using thiophosphorodichloridate reagents, forming stable thiophosphoramidate linkages. The procedure includes synthesis of the labeling reagents, protein modification, purification by ion-exchange chromatography, and subsequent copper-catalyzed azide-alkyne cycloaddition (CuAAC) for further conjugation.
This paper aims to evaluate the feasibility of clinical application of high-pressure Schisandrae Chinensis Fructus decoction pieces(HPSCH) compared with that of traditional Schisandrae Chinensis Fructus decoction pieces(SCH), based on the efficacy of mice with nonalcoholic fatty liver disease(NAFLD) induced by a high-fat diet. The optimal preparation process of HPSCH was investigated by using schisandrin content and total extractive yield as indices. After one week of adaptive feeding, C57BL/6 mice were assigned to a normal control group, while the remaining mice were fed an HFD for eight weeks to establish the NAFLD mouse model. After being successfully modeled, the mice were divided into a blank control group, blank administration group, simple model group, fenofibrate group, SCH group, and HPSCH group. All groups were administered by intragastric administration for four weeks. Body weight changes of mice were recorded, and liver indices were calculated. The levels of blood lipid and liver function index in the serum of mice were measured. Hepatic histopathological changes were observed by using hematoxylin-eosin(HE) staining and oil red O staining. Mice's feces were collected, and the 16S rDNA high-throughput sequencing method was employed to analyze changes in the gut microbiota of mice. Untargeted lipidomic analysis of the liver was conducted by using liquid chromatography-mass spectrometry(LC-MS), and principal component analysis(PCA) and orthogonal partial least squares discriminant analysis(OPLS-DA) were adopted to screen and identify differential metabolites. Kyoto Encyclopedia of Genes and Genomes(KEGG) database was used to perform relevant pathway analysis. Integrated analyses of gut microbiota and differential metabolites were performed to compare the similarities and differences in efficacy of HPSCH and SCH. The results show that both the HPSCH group and SCH group exert similar effects on biochemical indices in serum and hepatic pathological changes, lipid metabolism can be regulated, and liver injury in NAFLD mice can be improved. The two groups show similar changes in microbial abundance, trending toward the blank control group and maintaining intestinal homeostasis. Compared with those of the simple model group, the differential metabolites and metabolic pathways of both the HPSCH group and SCH group show similarity, and the differential metabolites are mainly triglycerides, diglycerides, phosphatidylcholines, phosphatidylglycerols, and so on. Metabolic pathways include glycerophospholipid metabolism, choline metabolism, retrograde endocannabinoid signaling, thermogenic lipolysis, and so on. This indicates that HPSCH and SCH share common advantages in improving hepatic function in mice and achieve comparable effects. HPSCH can improve blood lipid and liver function indices in NAFLD mice, attaining the therapeutic effect through the regulation of gut microbiota and lipid metabolism. Notably, HPSCH achieved the same efficacy as SCH at half the conventional dose, demonstrating its feasibility for clinical application. Moreover, the application of HPSCH may contribute to the conservation of medicinal resources.