Glutathione supplementation improves fat graft survival by inhibiting ferroptosis via the SLC7A11/GPX4 axis.

BACKGROUND: Autologous fat grafting is hampered by unpredictable graft survival, which is potentially regulated by ferroptosis. Glutathione (GSH), a powerful antioxidant used in tissue preservation, has ferroptosis-regulating activity; however, its effects on fat grafts are unclear. This study investigated the effects and mechanisms of GSH in fat graft survival. METHODS: Human lipoaspirates were transplanted subcutaneously into the backs of normal saline-treated (control) or GSH-treated nude mice. Graft survival was evaluated by magnetic resonance imaging and histology. RNA sequencing was performed to identify differentially expressed genes and enriched pathways. GSH activity was evaluated in vitro using an oxygen and glucose deprivation (OGD) model of adipose-derived stem cells. RESULTS: Compared with control group, GSH induced better outcomes, including superior graft retention, appearance, and histological structures. RNA sequencing suggested enhanced negative regulation of ferroptosis in the GSH-treated grafts, which showed reduced lipid peroxides, better mitochondrial ultrastructure, and SLC7A11/GPX4 axis activation. In vitro, OGD-induced ferroptosis was ameliorated by GSH, which restored cell proliferation, reduced oxidative stress, and upregulated ferroptosis defense factors. CONCLUSIONS: Our study confirms that ferroptosis participates in regulating fat graft survival and that GSH exerts a protective effect by inhibiting ferroptosis. GSH-assisted lipotransfer is a promising therapeutic strategy for future clinical application.

Exploration of the profiles of steroidal saponins from Rhizoma Paridis and their metabolites in rats by UPLC-Q-TOF-MS/MS.

INTRODUCTION: Steroidal saponins characterised by intricate chemical structures are the main active components of a well-known traditional Chinese medicine (TCM) Rhizoma Paridis. The metabolic profiles of steroidal saponins in vivo remain largely unexplored, despite their renowned antitumor, immunostimulating, and haemostatic activity. OBJECTIVE: To perform a comprehensive analysis of the chemical constituents of Rhizoma Paridis total saponins (RPTS) and their metabolites in rats after oral administration. METHOD: The chemical constituents of RPTS and their metabolites were analysed using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS). RESULTS: A reliable UPLC-Q-TOF-MS/MS method was established, and a total of 142 compounds were identified in RPTS. Specifically, diosgenin-type saponins showed the diagnostic ions at m/z 415.32, 397.31, 283.25, 271.21, and 253.20, whereas pennogenin-type saponins exhibited the diagnostic ions at m/z 413.31, 395.30, and 251.20. Based on the characteristic fragments and standard substances, 15 specific metabolites were further identified in the faeces, urine, plasma, and bile of rats. The metabolic pathways of RPTS, including phase I reactions (de-glycosylation and oxidation) and phase II reactions (glucuronidation), were explored and summarised, and the enrichment of metabolites was characterised by multivariate statistical analysis. CONCLUSION: The intricate RPTS could be transformed into relatively simple metabolites in rats through de-glycosylation, which provides a reference for further metabolic studies and screening of active ingredients for TCM.

Leucosceptrane Sesterterpenoids as a New Type of Natural Immunosuppressive Agents in Treating Sepsis.

Intragastric administration of the total sesterterpenoid extract (TSE) of medicinal plant Leucosceptrum canum at 2.5 g/kg dose protected mice from LPS-induced sepsis. Phytochemical investigation led to the isolation and identification of 47 leucosceptrane sesterterpenoids (1-47) including 30 new compounds (1-30) with complicated oxygenation patterns. Biological screening indicated their immunosuppressive activity via inhibiting IFN-γ secretion and/or proliferation of T cells with different potencies. Mechanism study of compounds 9, 25, and 32 revealed that they inhibited the activations of AKT-mTOR, JNK, p38 MAPK or ERK pathway in T cells and macrophages. In addition, compounds 9 and 25 induced G0/G1 cell arrest of T cells. The major component, leucosceptroid N (32), significantly lowered the levels of IL-6 and TNF-α in peripheral blood serum, and ameliorated the multiorgan damages of LPS-induced sepsis mice at 25 mg/kg dose. These findings suggest that leucosceptrane sesterterpenoids are a new type of potential immunosuppressive agents for sepsis treatment.

Unraveling the serial glycosylation in the biosynthesis of steroidal saponins in the medicinal plant Paris polyphylla and their antifungal action.

Sugar-sugar glycosyltransferases play important roles in constructing complex and bioactive saponins. Here, we characterized a series of UDP-glycosyltransferases responsible for biosynthesizing the branched sugar chain of bioactive steroidal saponins from a widely known medicinal plant Paris polyphylla var. yunnanensis. Among them, a 2'-O-rhamnosyltransferase and three 6'-O-glucosyltrasferases catalyzed a cascade of glycosylation to produce steroidal diglycosides and triglycosides, respectively. These UDP-glycosyltransferases showed astonishing substrate promiscuity, resulting in the generation of a panel of 24 terpenoid glycosides including 15 previously undescribed compounds. A mutant library containing 44 variants was constructed based on the identification of critical residues by molecular docking simulations and protein model alignments, and a mutant UGT91AH1Y187A with increased catalytic efficiency was obtained. The steroidal saponins exhibited remarkable antifungal activity against four widespread strains of human pathogenic fungi attributed to ergosterol-dependent damage of fungal cell membranes, and 2'-O-rhamnosylation appeared to correlate with strong antifungal effects. The findings elucidated the biosynthetic machinery for their production of steroidal saponins and revealed their potential as new antifungal agents.

Discovery of Unusual Sesterterpenoids from Colquhounia coccinea var. mollis and Their Metabolic Implications.

Three unusual sesterterpenoids featuring unprecedented rearranged colquhounane (C25) and tetranorcolquhounane (C21) frameworks, colquhounoids E (1) and F (3) and norcolquhounoid F (2), were isolated from a Lamiaceae medicinal plant Colquhounia coccinea var. mollis. Their structures were elucidated by spectroscopic analysis and quantum chemical calculations. A biomimetic inspired regioselective cyclopropane cleavage was achieved under acidic conditions. The immunosuppressive activities of these new sesterterpenoids were also evaluated.

A new sesquiterpene synthase catalyzing the formation of (R)-ß-bisabolene from medicinal plant Colquhounia coccinea var. mollis and its anti-adipogenic and antibacterial activities.

The sesquiterpene ß-bisabolene possessing R and S configurations is commonly found in plant essential oils with antimicrobial and antioxidant activities. Here, we report the cloning and functional characterization of a (R)-ß-bisabolene synthase gene (CcTPS2) from a Lamiaceae medicinal plant Colquhounia coccinea var. mollis. The biochemical function of CcTPS2 catalyzing the cyclization of farnesyl diphosphate to form a single product (R)-ß-bisabolene was characterized through an engineered Escherichia coli producing diverse polyprenyl diphosphate precursors and in vitro enzyme assay, indicating that CcTPS2 was a high-fidelity (R)-ß-bisabolene synthase. The production of (R)-ß-bisabolene in an engineered E. coli strain harboring the exogenous mevalonate pathway, farnesyl diphosphate synthase and CcTPS1 genes was 17 mg/L under shaking flask conditions. Ultimately, 120 mg of purified (R)-ß-bisabolene was obtained from the engineered E. coli, and its structure was elucidated by detailed spectroscopic analyses (including 1D and 2D NMR, and specific rotation). Four chimeric enzymes were constructed through domain swapping, which altered the product outcome, indicating the region important for substrate and product specificity. In addition, (R)-ß-bisabolene exhibited anti-adipogenic activity in the model organism Caenorhabditis elegans and antibacterial activity selectively against Gram-positive bacteria.

Secoiridoids from the traditional Chinese medicine Swertia pseudochinensis.

Detailed phytochemical investigation on the traditional Chinese medicine Swertia pseudochinensis Hara led to the isolation of ten undescribed secoiridoids and fifteen known analogs. Their structures were elucidated by extensive spectroscopic analysis (including 1D and 2D NMR, and HRESIMS). Selected isolates were assayed for their anti-inflammatory and antibacterial activities, and moderate anti-inflammatory activity via inhibiting the secretion of cytokines IL-6 and TNF-α in macrophages RAW264.7 induced by LPS were observed. Antibacterial activity against Staphylococcus aureus was not found at 100 µM.

Chemical investigation of Buddleja officinalis leaves and localization of defensive triterpenoids to its glandular trichomes.

Buddleja officinalis is a traditional Chinese medicinal plant covered with glandular and non-glandular trichomes on leaves. Phytochemical investigation of its leaves led to the identification of one undescribed tetranorcycloartane 3-oxo-25,26,27,29-tetranorcycloartan-24-oic acid (1) and one first identified natural product tetranorcycloartane 3-oxo-25,26,27,29-tetranorcycloartan-24-oic methyl ester (2), along with an undescribed megastigmane glucoside (3) and 14 known constituents (4-17). Structures of undescribed chemicals were elucidated by comprehensive 1D and 2D NMR, MS and CD analysis. Further chemical investigation resulted in six triterpenoids (4-9) being localized to the trichomes of B. officinalis. The major trichome components cycloeucalenone (4) and 24-oxo-29-norcycloartan-3-one (5) showed potent antifeedant activity against a generalist insect cotton bollworm (Helicoverpa armigera), but no obvious activity against the specialist herbivore Hyphasis inconstans. Compounds 4 and 7 also displayed inhibitory effects on seed germination of Arabidopsis thaliana. In addition, 1 and 4 exhibited moderate antibacterial activity toward three gram-positive bacteria.

Secondary metabolites from the Mongolian medicine Lomatogonium carinthiacum.

Systematic phytochemical investigation on the Mongolian medicinal herb Lomatogonium carinthiacum led to the isolation of 12 monoterpenoids including three new secoiridoids (1, 2 and 4) and one new iridoid glycoside (13), one new monoterpenoid alkaloid (3), and three new sesquiterpenoids (14-16). Comprehensive spectroscopic analysis (including 1D and 2D NMR, and HRESIMS) and quantum chemistry computations (including ECD and NMR calculations) were applied to elucidate their structures. Weak immunosuppressive activities were observed for the new isolates via inhibiting T cell proliferation and cytokine IFN-γ secretion in vitro.

Efficacy and cerebral mechanism of acupuncture and moxibustion for treating primary dysmenorrhea: study protocol for a randomized controlled clinical trial.

BACKGROUND: Acupuncture or moxibustion has been proven to be effective for patients with primary dysmenorrhea (PDM). However, the respective advantages and potential central mechanism of acupuncture and moxibustion are worthy of investigating to promote their further application. METHODS: In this randomized controlled neuroimaging trial, 72 patients with PDM will be randomly assigned to three groups: acupuncture treatment group, moxibustion treatment group, and waiting list group. The acupuncture treatment group and moxibustion treatment group will receive acupuncture or moxibustion, respectively, for a total of 3 sessions over 3 consecutive menstrual cycles, and the waiting list group will not take acupuncture or moxibustion during these 3 menstrual cycles. The COX Menstrual Symptom Scale (CMSS), visual analog scale (VAS), and Pain Catastrophizing Scale (PCS) will be used to evaluate the clinical efficacy. The Self-rating Depression Scale (SDS), Self-rating Anxiety Scale (SAS), and 36-Item Short Form Health Survey (SF-36) will be used to assess the mental state and quality of life at baseline and at the end of treatment. Functional magnetic resonance imaging (fMRI) will be performed for detecting the cerebral activity changes at baseline and at the end of the treatment. The clinical data and imaging data will be analyzed among the groups. Correlation analysis will be conducted to investigate the relationship between brain functional changes and symptom improvement. DISCUSSION: The application of the randomized controlled neuroimaging trial will provide objective and valid evidence about how acupuncture and moxibustion treatment relieve menstrual pain. The results of this study would be useful to confirm the potential similarities and differences between acupuncture and moxibustion in clinical efficacy and central mechanism for patients with PDM. TRIAL REGISTRATION: Chinese Clinical Trial Registry ChiCTR2100043732 . Registered on 27 February 2021.

Acupuncture Modulates the Spontaneous Activity and Functional Connectivity of Calcarine in Patients With Chronic Stable Angina Pectoris.

Background: Acupuncture is an effective adjunctive therapy for chronic stable angina pectoris (CSAP), while the underlying mechanism is unclear. This study aimed to investigate the central pathophysiology of CSAP and explore the mechanism of different acupoint prescriptions for CSAP from the perspective of brain-heart interaction. Methods: Thirty-seven CSAP patients and sixty-five healthy subjects (HS) were enrolled, and thirty CSAP patients were divided into two acupoint prescriptions groups (Group A: acupoints on the meridian directly related to the Heart; Group B: acupoints on the meridian indirectly related to the Heart). The Magnetic Resonance Imaging data and clinical data were collected at baseline and after treatment. The comparisons of brain spontaneous activity patterns were performed between CSAP patients and HS, as well as between baseline and after treatment in CSAP patients. Then, the changes in resting-state functional connectivity before and after treatment were compared between the two acupoint prescriptions. Results: Chronic stable angina pectoris patients manifested higher spontaneous activity on the bilateral calcarine, left middle occipital gyrus, right superior temporal gyrus, and right postcentral gyrus. After acupuncture treatment, the spontaneous activity of the left calcarine, left cuneus, and right orbitofrontal gyrus was decreased. The left calcarine was identified as region-of-interest for functional connectivity analysis. Compared with group B, CSAP patients in group A had significantly increased functional connectivity between left calcarine and the left inferior temporal gyrus/cerebellum crus 1, left hippocampus, left thalamus, and left middle cingulate cortex after treatment. Thresholds for all comparisons were p < 0.05, Gaussian Random Field corrected. Conclusion: Regulating the aberrant spontaneous activity of the calcarine might be an underlying mechanism of acupuncture for CSAP. The multi-threaded modulation of functional connectivity between calcarine and multiple pain-related brain regions might be a potential mechanism for better efficacy of acupuncture at points on the meridian directly related to the Heart.

Secoiridoids and triterpenoids from the traditional Tibetan medicine Gentiana veitchiorum and their immunosuppressive activity.

Two undescribed secoiridoids (dehydroxyl-swerimilegenin H and 9-oxo-swerimuslactone A) with eight known ones, and two undescribed triterpenoids [28-O-(3,4-dihydroxyl-benzyl)-lupeol and 17-hydroperoxide-28-norurs-12-en-3-one] with seven known ones, were isolated from the aerial parts of Gentiana veitchiorum, a traditional Tibetan medicine. The structures of these compounds were elucidated by detailed spectroscopic analyses (including 1D and 2D NMR, HRMS, IR, and specific rotation) and comparison with structurally related known compounds. The isolates were selected to evaluate for their immunosuppressive activity via inhibiting the proliferation of T cells and cytokine IFN-γ production in T cells. Among them, 28-O-(3,4-dihydroxyl-benzyl)-lupeol exhibited significant effect by inhibiting the proliferation of T cells (IC50, 20.08 µM) and T cell IFN-γ production (IC50, 7.29 µM).

Cloning and Functional Characterization of a Squalene Synthase from Paris polyphylla var. yunnanensis.

Paris polyphylla Smith var. yunnanensis (Franch.) Hand. - Mazz. is a precious traditional Chinese medicine, and steroidal saponins are its major bioactive constituents possessing extensive biological activities. Squalene synthase (SQS) catalyzes the first dedicated step converting two molecular of farnesyl diphosphate (FDP) into squalene, a key intermediate in the biosynthetic pathway of steroidal saponins. In this study, a squalene synthase gene (PpSQS1) was cloned and functionally characterized from P. polyphylla var. yunnanensis, representing the first identified SQS from the genus Paris. The open reading frame of PpSQS1 is 1239 bp, which encodes a protein of 412 amino acids showing high similarity to those of other plant SQSs. Expression of PpSQS1 in Escherichia coli resulted in production of soluble recombinant proteins. Gas chromatography-mass spectrometry analysis showed that the purified recombinant PpSQS1 protein could produce squalene using FDP as a substrate in the in vitro enzymatic assay. qRT-PCR analysis indicated that PpSQS1 was highly expressed in rhizomes, consistent with the dominant accumulation of steroidal saponins there, suggesting that PpSQS1 is likely involved in the biosynthesis of steroidal saponins in the plant. The findings lay a foundation for further investigation on the biosynthesis and regulation of steroidal saponins, and also provide an alternative gene for manipulation of steroid production using synthetic biology.

Carbon Arc Lamp Therapy Enhances the Repair Potential of Chronic Soft Tissue Injury in Rats Compared with the Ability of Photobiomodulation Therapy.

Objective: The effects of photobiomodulation therapy (PBMT) and carbon arc lamp therapy (CALT) on the repair of chronic soft tissue injury were compared. Background data: PBMT improves soft tissue repair of chronic injury. However, there has been no research on the effect of CALT. Methods: Human umbilical vein endothelial cells (HUVECs) were irradiated using PBMT and CALT at 2 J/cm2 to observe their effects on cell proliferation and migration. The effects of PBMT and CALT on soft tissue injury repair were assessed using a chronic gastrocnemius injury model of the posterior limb in rats. The malondialdehyde (MDA), superoxide dismutase (SOD), and prostaglandin E2 (PGE2) were examined by biochemical analyses. The degree of tissue damage repair was evaluated by the immunohistochemical method [CD45, CD34, vascular endothelial growth factor (VEGF), and actin] and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method. Results: Treatment by PBMT and CALT significantly accelerated the proliferation and migration of HUVECs. Moreover, significant decreases in the contents of MDA and PGE2 were observed in the PBMT and CALT groups, while SOD activity was increased. The histological assessment shows that the content of inflammatory cells and apoptotic cells significantly decreased in the CALT group. However, the microvascular density, VEGF content, and actin content were increased in the CALT group. Conclusions: The results demonstrate that CALT has a stronger effect on promoting chronic soft tissue injury repair in comparison with PBMT.

Immunosuppressive gentianellane-type sesterterpenoids from the traditional Uighur medicine Gentianella turkestanorum.

Whole plants of Gentianella turkestanorum are commonly used as a traditional Uighur medicine. A phytochemical investigation led to the isolation of eight undescribed gentianellane-type sesterterpenoids (18-epi-nitidasin, gentianelloids D-F, and 18-epi-gentianelloids C-F), one undescribed 11,12-seco-gentianellane (18-epi-alborosin), and three known analogs (nitidasin, gentianelloid C and alborosin) among which gentianelloid C was found for the first time from a natural source. The structures of these compounds were elucidated by extensive spectroscopic analyses (including 1D and 2D NMR, HRMS, IR, and specific rotation) and in the case of 18-epi-gentianelloid C by the single-crystal X-ray diffraction analysis. A putative biosynthetic route for these sesterterpenoids was proposed. The immunosuppressive activity of the isolated compounds was also evaluated by their ability to inhibit the proliferation of T cells and T cell cytokine IFN-γ production. Nitidasin suppressed IFN-γ production with an IC50 value of 16.50 µM, while gentianelloid F and alborosin inhibited the proliferation of and IFN-γ production in T cells with IC50 values of 12.40-14.66 µM.

Antifeedant, cytotoxic, and anti-inflammatory neo-clerodane diterpenoids in the peltate glandular trichomes and fresh leaves of Ajuga forrestii.

The Lamiaceae plant Ajuga forrestii Diels is a traditional Chinese herbal medicine with abundant glandular trichomes (GTs), but their chemistry and biological functions remain uninvestigated. Here, a panel of six highly functionalized neo-clerodane diterpenoids was localized to the peltate GTs of A. forrestii using laser microdissection coupled with HPLC analysis, indicating that the GTs of A. forrestii are an excellent material for the elucidation of the yet unclear biosynthetic pathway of natural neo-clerodane diterpenoids. In addition, four undescribed neo-clerodane diterpenoids with an acyclic C-9 side chain including two pairs of 1:1 mixture of inseparable diastereomers, ajuforrestins D-G, were isolated from the fresh leaves of A. forrestii together with six known compounds. The structures of the undescribed compounds were elucidated by spectroscopic (including 1D and 2D NMR and HR-ESI-MS) analyses. Biological assays indicated that the major GT compound ajugacumbin B and undescribed ajuforrestins D/E showed antifeedant activity against Helicoverpa armigera, suggesting that neo-clerodanes in A. forrestii should be involved in plant defence against insects. Moreover, the abietane diterpenoid ajuforrestin B exhibited significant anti-inflammatory activity on the secretion of interleukin-2 (IL-2) and cytotoxicity against three cancer cell lines, NCI-H1975, HepG2 and MCF-7, suggesting that ajuforrestin B could positively contribute to the therapeutic effects of this traditional Chinese medicine.

Characterization of defensive cadinenes and a novel sesquiterpene synthase responsible for their biosynthesis from the invasive Eupatorium adenophorum.

Eupatorium adenophorum is a malignant invasive plant possessing extraordinary defense potency, but its chemical weaponry and formation mechanism have not yet been extensively investigated. We identified six cadinene sesquiterpenes, including two volatiles (amorpha-4,7(11)-diene and (-)-amorph-4-en-7-ol) and four nonvolatiles (9-oxo-10,11-dehydroageraphorone, muurol-4-en-3,8-dione, 9-oxo-ageraphorone and 9ß-hydroxy-ageraphorone), as the major constitutive and inducible chemicals of E. adenophorum. All cadinenes showed potent antifeedant activity against a generalist insect Spodoptera exigua, indicating that they have significant defensive roles. We cloned and functionally characterized a sesquiterpene synthase from E. adenophorum (EaTPS1), catalyzing the conversion of farnesyl diphosphate to amorpha-4,7(11)-diene and (-)-amorph-4-en-7-ol, which were purified from engineered Escherichia coli and identified by extensive nuclear magnetic resonance (NMR) spectroscopy. EaTPS1 was highly expressed in the aboveground organs, which was congruent with the dominant distribution of cadinenes, suggesting that EaTPS1 is likely involved in cadinene biosynthesis. Mechanical wounding and methyl jasmonate negatively regulated EaTPS1 expression but caused the release of amorpha-4,7(11)-diene and (-)-amorph-4-en-7-ol. Nicotiana benthamiana transiently expressing EaTPS1 also produced amorpha-4,7(11)-diene and (-)-amorph-4-en-7-ol, and showed enhanced defense function. The findings presented here uncover the role and formation of the chemical defense mechanism of E. adenophorum - which probably contributes to the invasive success of this plant - and provide a tool for manipulating the biosynthesis of biologically active cadinene natural products.

Production of the Inaccessible Sesquiterpene (-)-5-Epieremophilene by Metabolically Engineered Escherichia coli.

(-)-5-Epieremophilene, an epimer of the versatile sesquiterpene (+)-valencene, is an inaccessible natural product catalyzed by three sesquiterpene synthases (SmSTPSs1-3) of the Chinese medicinal herb Salvia miltiorrhiza, and its biological activity remains less explored. In this study, three metabolically engineered Escherichia coli strains were constructed for (-)-5-epieremophilene production with yields of 42.4-76.0 mg/L in shake-flask culture. Introducing an additional copy of farnesyl diphosphate synthase (FDPS) gene through fusion expression of SmSTPS1-FDPS or dividing the FDP synthetic pathway into two modules resulted in significantly improved production, and ultimately 250 mg of (-)-5-epieremophilene were achieved. Biological assay indicated that (-)-5-epieremophilene showed significant antifeedant activity against Helicoverpa armigera (EC50 =1.25 µg/cm2 ), a common pest of S. miltiorrhiza, implying its potential defensive role in the plant. The results provided an ideal material supply for studying other potential biological activities of (-)-5-epieremophilene, and also a strategy for manipulating terpene production in engineered E. coli using synthetic biology.

Diterpenoids and Flavonoids from the Twigs of Cephalotaxus fortunei var. alpina.

Three new diterpenoids (a cephalotane, an abietane and a 9(10→20)-abeo-abietane) and one new flavonoid, together with 11 known compounds, were isolated from the twigs of Cephalotaxus fortunei var. alpina. The new compounds were identified by comprehensive spectroscopic (including 1D and 2D-NMR and HR-ESI-MS) analysis. Anti-inflammatory, immunosuppressive and cytotoxic activities of three new compounds were evaluated. 3ß,20-epoxyabieta-8,11,13-triene-3α,12-diol showed weak cytotoxicity against tumor cell lines NCI-H1975, HepG2, MCF-7, while fortalpinoid R and 3-acetonyl-3,5,7,4'-tetrahydroxy-2-methoxyflavanone were not active at 80 µM. None of these compounds showed anti-inflammatory and immunosuppressive activities.

Aqueous extraction followed by dispersive solid phase extraction with in situ derivatization for the determination of aflatoxins in traditional Chinese medicines.

A green sample preparation method based on aqueous extraction followed by dispersive solid phase extraction (d-SPE) with in situ derivatization (ISD) was developed for the determination of aflatoxins (AFs) in traditional Chinese medicines (TCMs). AFs in TCMs were extracted by alkaline aqueous solution and converted to substituted coumaric acids. Then, mixed-mode anion exchange (MAX) sorbent was used to isolate and enrich the substituted coumaric acids. During the elution by acetonitrile/trifluoroacetic acid solution, AFs were reconstructed and in situ derivatized. Several parameters affecting the procedure were evaluated. The developed preparation method coupled with high performance liquid chromatography-fluorescence detection was successfully applied for AFs determination in TCMs. The limit of detection (LOD) reached 10 pg/mL for AFs. Good linearity was obtained in three orders of magnitude with correlation coefficients ranging from 0.9996 to 0.9999. The relative recoveries of the method were between 72.7% and 114.5% with intra- and inter-day relative standard deviations (RSDs) less than 9.5% and 10.1%, respectively. The method was successfully applied to determine AFs in 15 kinds of TCMs in China, with the results verified by IAC standard method.