Streptomyces are characterized by metabolites with diverse structures. The genomic sequence of Streptomyces varsoviensis indicated that it possessed a biosynthetic gene cluster for DHB containing siderophores and metabolic profiling revealed metabolites correlated with catechol-type natural products. Here, we report the discovery of a series of catecholate siderophores produced by S. varsoviensis and a scale-up fermentation was performed to purify these compounds for structural elucidation. A biosynthetic route for the catecholate siderophores is also proposed. These new structural features enrich the structural diversity of the enterobactin family compounds. One of the new linear enterobactin congeners shows moderate activity against a food-borne pathogen Listeria monocytogenes. This work demonstrated that changing culture conditions is still a promising approach to explore unexplored chemical diversity. The availability of the biosynthetic machinery will enrich the genetic toolbox of catechol siderophores and facilitate such engineering efforts.Enterobactin is an archetypical catecholate siderophore that plays a key role in the acquisition of ferric iron by microorganisms. Catechol moieties have been shown to be promising siderophore cores. Variants of the conserved 2,3-dihydroxybenzoate (DHB) moiety with structural modifications expand the bioactivity.  Since t3+, due to the presence of the 2,3-dihydroxybenzamide motifs. Enterobactin is a remarkable tri-catechol siderophore with an exceptionally high affinity for Fe3+ (Ka = 1052), which is the highest affinity for iron reported to date -dependent pathway . EnterobE. coli . The NRPnd dimer . Since ervention . Enterobrvention .Streptomyces are a group of filamentous Gram-positive bacteria with the potential to produce bioactive natural products, including siderophores  was performed using antiSMASH (High performance liquid chromatography (HPLC) was conducted on a Waters HPLC system equipped with using an Agilent Eclipse XDB-C18 column  for analysis and Fisher X-bridge C18 column  for semi-preparation. Multi-wavelength monitoring was performed at 210, 254, 280 and 330 nm. High resolution electrospray ionization mass spectrometry (HR-ESI-MS) was performed on an Agilent Q-TOF HPLC mass spectrometer. NMR data were recorded on a Bruker 700 MHz system. The structures of enterobactin and congers were elucidated by a combination of HRMS and 1D and 2D NMR spectroscopic analyses. Size exclusion chromatography was performed on Sephadex LH-20 columns . Optical rotations were measured with the P-2000 high-accuracy multi-option digital polarimeter . Gene clusters prediction from ntiSMASH . SequencntiSMASH .2.2.S. varsoviensis CGMCC 4.1431 strain was obtained from the China General Microbiological Culture Collection Center. The strain was grown on medium B  at 30°C for 7 days for sporulation. The spores from one agar plate were harvested, and suspended in 1 mL 20% glycerol. 20 μl of spore suspension was transferred to 50 mL TSB (trypic soy broth) media at 30°C for 2 days as seed culture. Medium B and other two media  were used for fermentation and comparison . EtAC was removed by rotary evaporation and the resultant sample was dissolved in methanol and subjected for analysis on a Waters HPLC (10 μL injection). The mobile phase was a binary gradient of acetonitrile (ACN) and 0.1% formic acid (FA) in H2O, pumped at 0.6 mL/min through an Eclipse XDB-C18 column . Liquid chromatography for HPLC analysis was performed using a 30 min solvent gradient from 10–100% solvent B.The spores were harvested and the seed cultures were obtained by inoculating 10 μL of spore solution into 30 mL of TSB liquid medium and incubated at 30°C for 2 days, after which the resulting seed culture was transferred into production medium at 5% for 7 days fermentation. After the small-scale fermentation (50 mL) of S. varsoviensis strain, the cell mass was harvested by centrifugation and the supernatant was extracted with EtAC. The EtAC crude extract (10 g) was concentrated in vacuo, and the residue was applied to a silica gel column and separated using an increasing gradient of petroleum ether: EtAC from 5:1 to 1:5 as the mobile phase. Each 250 mL was collected as a fraction. A total of 17 fractions were collected and each fraction was analyzed by HPLC. Fraction D3 was concentrated (1.3 g) and further purified by a Sephadex LH-20 column using methanol as the mobile phase to give 45 subfractions (6 mL each subfraction) and analyzed by TLC and HPLC with UV detection at 254 nm, 300 nm, and 380 nm. Subfraction 25 and 26 and 41 have been chosen for further purification. Preparative HPLC was performed on a C18 X-bridge column  using a 0.1% formic acid in water/ACN as mobile phase, at a flow rate of 3 ml/min. An increasing elution gradient as follows 0–5 min (40% ACN), 5–6 min (40–45% ACN), 6–11 min, 45% ACN; 11–25 min, 45–100% ACN; 25–27 min, 100% ACN; 27–30 min, 100–40% ACN; 30–35 min, 40% ACN was used to afford compound 1 (5.0 mg), compound 3 (2.0 mg) with retention time at 19.6 min and 15.5 min. Compound 2 (4.9 mg), compound 4 (15.0 mg), compound 5 (23.1 mg) were purified from subfraction 41 with retention time at 20.0 min, 19.0 min and 20.7 min, respectively.For large-scale fermentation (25 L) of the 1: yellow oil; c = 0.42, MeOH). 1H NMR and 13C NMR data, see + ion at m/z 479.1299 .Compound 2: Yellow oil; c = 0.41, MeOH). 1H NMR and 13C NMR data, see + ion at m/z 702.1775 .Compound 3: 1H NMR : δH 7.36 , 6.97 , 6.77 , 4.76 , 4.03 , 3.95 , 3.79 .13C NMR : δC 172.4, 170.6, 149.6, 147.3, 119.8, 119.8, 119.8, 117.2, 62.8, 56.5, 52.9. HR-ESI-MS [M + H]+ ion at m/z 256.0818 .Compound 4: 1H NMR : δH 7.38 , 7.33 , 6.95 , 6.74 , 6.56 , 5.97 , 5.05 , 4.79 , 4.67 .13C NMR : δC 172.1, 170.7, 167.9, 164.9, 149.5, 147.8, 147.2, 147.1, 133.4, 121.1, 120.3, 120.3, 119.9, 119.9, 119.8, 119.0, 117.1, 111.3, 66.0, 53.1. HR-ESI-MS [M + H]+ 447.1038 .Compound 5: 1H NMR : δH 7.25 , 6.97 , 6.73 , 5.04 , 4.66 . 13C NMR : δC 170.7, 170.7, 149.5, 147.3, 120.1, 120.1, 119.7, 116.8, 65.8, 53.7. HR-ESI-MS [M + H]+ 670.1516 .Compound 2.4.1 and 2 was determined by using the Marfey’s assay with minor modifications  at 80°C overnight and then the hydrolysis solution was evaporated to dryness, respectively. The resulting residue was redissolved in 100 μl NaHCO3 solution (1 M), followed by the addition of 50 μL FDAA solution , and incubated at 55°C for 1 h. The derivatized solution was quenched by the addition of 100 μl HCl (2 M). Amino acid standards of L-Ser and D-Ser were derivatized in the same procedure. A 15 μL aliquot of such reaction mixture was subjected to HPLC analysis (340 nm). HPLC analyses were performed on an Agilent Eclipse XDB-C18 column  at a flow rate of 0.6 mL/min. Solvent A consisted of 0.1% formic acid with water, and solvent B was ACN. For the derivatized hydrolysate and the corresponding standards, the following gradient was used for HPLC analysis: 0–5 min, 20% B; 5–35 min, 20–55% B; 35–36 min, 55–100% B; 36–39 min, 100% B; 39–40 min, 100–20% B; 40–45 min, 20% B.The absolute configuration of compounds ications . Compoun2.5.Salmonella enterica ATCC 14028, Shigella dysenteriae CMCC 51335, Klebsiella pneumonia HS11286, Acinetobacter baumannii ATCC 19606, Pseudomonas aeruginosa PAO1) and three Gram-positive bacteria  were set for bioassay. All the tested strains were grown in LB broth to early stationary phase, diluted by LB broth to ~1.5 × 105 CFU/mL, and then inoculated into 96-well plate with 200 μL of LB in the well. The tested compounds with different concentrations in each wells using a serious dilution . No compound but methanol was used as negative control. The MIC (minimum inhibitory concentration) values were evaluated after incubation at 37°C for 16–18 h, using a plate reader at OD600.The antibacterial activities were determined in the 96-well plate with LB broth. Each compound for testing was prepared to a concentration of 1 mg/mL in methanol. Five Gram-negative bacteria  propanesulfonate (DDAPS), 20 μM FeCl3) was prepared in distilled Milli-Q water. Each compound was diluted to gradient concentration (approximately 0 to 100 μM), mixed with the CAS solution, and then added to a 96 well-microplate for 1 h incubation at room temperature. The chelating activity of each siderophore compound was calculated according to the absorption at OD630 nm using the Synergy H1 multimode reader (BioTek).Evaluation of the iron chelating activities of the different enterobactin-derived compounds was performed using the chrome azurol S (CAS) assay as previously described with some modifications . The CAS3.3.1.S. varsoviensis CGMCC 4.1431 allowed the identification of an NRPS gene cluster containing homologs of catechol siderophore biosynthetic genes (entA-entF). In silico analysis revealed that the NRPS cluster spans approximately ~26 kb, and functional assignments of individual ORFs were made by comparing of the deduced gene products with proteins of known or predicted function  are transcribed in the same orientation and may constitute an operon. Comparative analysis revealed that these genes are significantly identical to their counterparts in the enterobactin or bacillibactin gene cluster. EnsB1 showed 43% identity to an isochorismatase, EnsC is 40% identical to the isochorismate synthase, and EnsA resembles a 2,3-DHB dehydrogenase similar to EntA (45% identity) from E. coli. EnsB1, EnsC and EnsA are proposed to be responsible for the biosynthesis of the 2,3-DHB moiety from chorismic acid. EnsB2 encodes a carrier protein next to EnsB1. EnsB2 is proposed to function as a peptidyl-carrier protein (PCP) for tethering 2,3-DHB because it reveals similarity to the enterobactin synthetase component B EntB (43% identity). EnsE is a stand-alone adenylation (A) domain which is similar to DhbA involved in the catechol siderophore bacillibactin from Bacillus subtilis (67% identity/79% similarity) and EntA involved in enterobactin biosynthesis by E. coli (48% identity/65% similarity). EnsE catalyzes the formation of an adenylate from 2,3-DHB and tethers to the phosphopantetheine thiol of EnsB2. EnsF consists of a terminate NRPS module (C-A-PCP-TE) and is most similar to the NRPS of DhbF, which is involved in the biosynthesis of B. subtilis bacillibactin (46% identity/60% similarity). The featured 10 conserved residues in the amino acid binding pocket predict serine incorporation by EnsF. A TE domain at the C-terminal end of EnsF is likely responsible for the macrocyclization. EnsG is an esterase similar to enterobactin esterase that catalyzes the hydrolysis to convert cyclic enterobactin to linear form in E. coli , requiring 12 degrees of unsaturation. The molecular formula was established as C21H22N2O11. The UV spectrum displayed absorptions at λmax 207.5, 247.7, and 311.9 nm similar to catecholate metabolites , and the existence of two aromatic rings was supported by three aromatic protons in the downfield δH 7.33 , 6.96  and 6.74  with equal integrated area and. Analysis of the 13C NMR spectrum indicated the existence of 21 carbons attributable to four carbonyl carbons and 12 olefinic bond signals. Together with the DEPT135 and HSQC spectra, all the carbons were identified as one methyl (δC 53.2), two methylenes (δC 65.1 and 62.7), eight methines  and 10 quaternary carbons . The planar structure of 1 was assigned after careful analysis of the 2D NMR  spectra. Four spin coupling systems were confirmed by the 1H-1H COSY cross-peaks between H-5/H-6/H-7, H-9/H-10, H-5’/H-6’/H-7′, and H-9’/H-10′ (1. HMBC correlations from the α-CH proton (δ 4.75) to the C9’ and α-CH proton (δ 5.05) to the C9 carboxyl suggested that an amide linkage connected the three fragments. All the four spin-coupling fragments were connected together, and 1 was finally established as a new enterobactin derivative  and compared with L-and D-Ser amino acid standards under the same conditions. Comparison of the retention time of the 1 hydrolysate and amino acid standards using HPLC analysis revealed an L-configuration for the serine residue in 1. The absolute configuration at C9 of 1 was determined as 9S, 9’S. Thus, 1 was named as N,N′-bis-O-seryl methoxy serinate.The absolute configuration of 2 was obtained as yellow oil. It had a molecular formula of C31H31N3O16 based on the ion peak at m/z 702.1775 , indicating 18 degrees of unsaturation. The UV spectrum displayed absorption bands at λmax 210, 250, and 314 nm, suggesting the presence of the hydroxylated benzoyl chromophores similar to 1. The 1H NMR spectrum revealed one methyl proton in the upfield . The three aromatic rings were readily assigned based on the proton-splitting pattern in δH 7.30 (m), 6.95 (m), and 6.73 (m). NMR data of 2 showed 31 carbons contributing to four carbonyl carbons and 18 olefinic bond signals. These data were very similar to the planar structure of compound 1. In the 1H-1H COSY spectrum, six fragments including H-5’/H-6’/H-7′, H-9’/H-10′, H-5”/H-6”/H-7′, H-9”/H-10′, H-9/H-10, and H-5/H-6/H-7, were detected -O-seryl-O-seryl methoxy serinate.Compound detected , S23. Th3 was obtained as yellow and amorphous solid. HR-ESI-MS analysis of 3 afforded an [M + H]+ ion at m/z 256.0818 , establishing its molecular composition as C11H13NO6. According to the 1H and 13C NMR data . Compound 4 was identified as N,N′-bis-O-L-seryl-L-dehydroalanine, the derivative of enterobactin, based on the comparison of the NMR and MS data with previously published data , giving a molecular formula of C30H27N3O15. Based on the comparison of its NMR and MS data with previously published data, compound 5 was identified as known compound enterobactin  while 2 showed moderate activity against Listeria monocytogenes AB97021, with MIC at 25 μg/mL, but not against other tested Gram-negative or Gram-positive strains (MIC >100 μg/mL)  . The MICsoxazole .1–5. In the assay solution, the siderophore compounds seize the iron from the Fe-CAS-DDAPS complex to form the siderophore-CAS-DDAPS complex, causing the absorbance of CAS solution to decrease at 630 nm. The colorimetric shift is correlated with the iron chelating activity of each compound. Thus, compounds 1 and 2 showed higher iron chelating activity than enterobactin , which share an N-2,3-dihydroxybenzoyl-L-serine as a common structural unit, have been isolated and structurally elucidated. Of these compounds, 1–4 are 2,3-DHB monomer, dimer, or linear trimer congeners and 5 is enterobactin.In order to evaluate the siderophores produced by e moiety . All fivS. varsoviensis NRPS gene cluster ens is in accord with the biosynthetic logic. As shown in ensABC utilizing chorismite as a precursor, which is derived from the shikimate acid synthesis pathway. The synthesized 2,3-DHB is activated via EntE (A domain) and transferred to the EntB2 (PCP domain). The iterative NRPS EntF activates the L-serine and subsequently catalyzes the formation of each ester and amide bond. Finally, the TE domain finally catalyzes the macrocyclization of the trimer and release of the final enterobactin siderophore.Enterobactin is one of the well-known tri-catecholate siderophores and its metal-binding property has attracted considerable attention. Enterobactin consists of an L-serine trilactone linked by amide bonds to three 2,3-DHB moieties, and further sequence analysis showed that the Streptomyces species  can be found in the article/ZL, TH, SL, and ZD designed the experiments. ZL and QS conducted the experiments and analyzed the data. TH, ZL, and SL drafted, reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.This work was financially supported by grants from the National Key Research and Development Program of China (2018YFA0901900), National Natural Science Foundation of China (22207073), the Program of Shanghai Subject Chief Scientist (21XD1401300) and Major Project of Haihe Laboratory of Synthetic Biology (22HHSWSS00001).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Terfezia boudieri and T. claveryi, and the underlying mechanisms associated with their anti-inflammatory activities in RAW 264.7 macrophages stimulated with lipopolysaccharide/interferon-gamma (LPS/IFN-γ). Our results demonstrated that treatment with T. boudieri and T. claveryi extracts effectively suppressed the inflammatory response in LPS/IFN-γ-stimulated RAW 264.7 macrophages. Specifically, T. boudieri extract was found to reduce the production of nitric oxide and inhibit the expression of various pro-inflammatory markers, including inducible nitric oxide synthase, cyclooxygenase-2 (COX-2), tumor necrosis factor-α, and interleukin-6 (IL-6) at both the mRNA and protein levels. Similarly, T. claveryi extract exhibited comparable inhibitory effects, except for the expression of IL-6 and COX-2 at the protein level, where no significant effect was observed. Moreover, both studied extracts significantly downregulated the microRNA expression levels of miR-21, miR-146a, and miR-155, suggesting that T. boudieri and T. claveryi suppress the inflammatory response in LPS/IFN-γ-stimulated RAW 264.7 cells through an epigenetic mechanism. Furthermore, our study reveals a new mechanism for the anti-inflammatory properties of desert truffle extracts. We show for the first time that Terfezia extracts do not rely on the nuclear factor erythroid 2-related factor 2 pathway, previously linked to anti-inflammatory responses. This expands our understanding of natural product anti-inflammatory mechanisms and could have important implications for developing new therapies. To account for differences in truffle effects, extracts prepared were subjected to secondary metabolites profiling using UPLC-MS. UPLC-MS led to the annotation of 87 secondary metabolites belonging to various classes, including amino acids, carbohydrates, alkaloids, amides, fatty acids, sterols, and phenolic compounds. Therefore, these results indicate that T. boudieri and T. claveryi exhibit anti-inflammatory activities through suppressing multiple inflammatory mediators and cytokines and may be potential anti-inflammatory agents.Desert truffles have been used as traditional treatments for numerous inflammatory disorders. However, the molecular mechanisms underlying their anti-inflammatory effects in RAW 264.7 macrophages have yet to be fully elucidated. The present study investigated the anti-inflammatory activities of two main desert truffles,  However, the uncontrolled or persistent inflammatory response can contribute to the pathogenesis of several chronic inflammatory diseases, including arthritis, asthma, autoimmune diseases, atherosclerosis, cancer, and diabetes3. Inflammation is induced when innate immune cells, particularly macrophages, detect harmful stimuli like infection and tissue injury by activating germline-encoded pattern-recognition receptors (PRRs)5. These receptors can recognize pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharides (LPS), a component of the gram-negative bacterial cell wall, or damage-associated molecular patterns (DAMPs) produced from injured cells7. Notably, LPS can trigger inflammation-associated signaling pathways in macrophages through binding to the Toll-like receptors 4 (TLR4), which are then dimerized on the cell membrane. Receptor dimerization leads to the recruitment of myeloid differentiation primary response 88 (MyD88) and Toll/interleukin-1 receptor domain-containing adapter inducing interferon-β (TRIF) adaptor proteins to the intracellular domain of TLR4. Signaling via TLR4 activates an intracellular signaling cascade that results in activation of nuclear factor-kappa B (NF-κB), and mitogen-activated protein kinases (MAPKs), which eventually promote overproduction of the inflammatory mediators and cytokines11. The representative inflammatory mediators include nitric oxide (NO), histamine, prostaglandin E2 (PGE2), and pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6)14. Furthermore, inflammation is known to elevate the level of intracellular reactive oxygen species (ROS), resulting in oxidative stress16. In this context, the transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) has a pivotal role in protecting cells against inflammation and oxidative stress through modulating the expression of phase II detoxifying enzymes, such as heme oxygenase-1 (HO-1) and oxidative stress response genes, such as oxidative stress-induced growth inhibitor 1 (OSGIN1)19.Inflammation is a protective immune response that is crucial in eliminating pathogens and maintaining the tissue homeostasis21. Therefore, there is an imperative need for research on developing new natural-based anti-inflammatory compounds without side effects as alternatives to the current NSAIDs23. In this regard, truffles have a long history of use as traditional remedies to treat many human diseases, especially eye and skin diseases24.Currently, nonsteroidal anti-inflammatory drugs (NSAIDs) are the most widely prescribed drugs to treat both acute and chronic inflammatory disorders. However, their long-term use is associated with serious adverse effects, such as peptic ulcers, cardiovascular toxicity, hypertension, acute renal failure, and nephrotic syndrome25. Truffles have traditionally been utilized as functional foods and therapeutic agents due to their high content of amino acids, proteins, carbohydrates, fats, lipids, and minerals27. Moreover, they contain abundant bioactive compounds, such as phenolics, phytosterols, polysaccharides, flavonoids, and terpenoids, which are associated with their anti-inflammatory, immunomodulatory, antioxidant, anticancer, and antibacterial properties30. Edible truffles are taxonomically classified into two major types, tuber, and desert. Truffle species within the genus Tuber are recognized as "true" truffles, such as Tuber magnatum (white truffle) and Tuber melanosporum (black truffle), growing natively in Europe24. On the other hand, desert truffle species existing in arid and semi-arid areas of the Mediterranean belong to the genera Terfezia and Tirmania28. The genus Terfezia is composed of more than 20 species. T. boudieri and T. claveryi are among most popular desert truffle species31.Truffles are the hypogeous fruiting bodies of the ascomycetes fungi that thrive underground in depth between 5 and 10 centimetersT. boudieri and T. claveryi have been investigated. Both species have been selected for the study based on the abundance of functional anti-inflammatory compounds in their chemical profile. Therefore, the aim of this study was to examine and compare the anti-inflammatory properties of T. boudieri and T. claveryi extracts in LPS/IFN-γ-stimulated RAW 264.7 macrophages, as well as to identify the underlying mechanisms related to these properties. Further, metabolites profiling using ultra-performance liquid chromatography-mass spectrometry was employed to provide a holistic overview of truffles metabolome.In the present study, the anti-inflammatory activities of ethanolic extracts from ® Green PCR Kit (Cat No. 218073), and miScript primer assays including Hs_RNU6-2_11 (Cat No. MS00033740), Mm_miR-21_2 (Cat. No. MS00011487), Mm-miR-146a*_1 (Cat. No. MS00024220), and Mm_miR-155_1 (Cat. No. MS00001701) were obtained from Qiagen . Dimethyl Sulfoxide , and Chloroform: Isoamyl alcohol 24:1  were purchased from SERVA . Rabbit polyclonal anti-COX-2 antibody (Cat. No. E-AB-70031), Mouse TNF-α (Cat. No. E-EL-M0049), and Mouse IL-6 (Cat. No. E-EL-M0044) Enzyme-Linked Immunosorbent Assay (ELISA) Kits were purchased from Elabscience . Acetonitrile and formic acid (LC–MS grade) were obtained from J. T. Baker (Netherlands). MilliQ water was utilized for UPLC-Orbitrap HRMS analysis. All other solvents employed in metabolites profiling were of HPLC–MS grade and were purchased from Sigma Aldrich .Dulbecco's Modified Eagle Medium (DMEM), high glucose (Cat. No. 41965-039), Fetal Bovine Serum , Penicillin-Streptomycin , -2, 5-diphenyltetrazolium bromide , Griess Reagent Kit (Cat. No. G7921), Isopropanol , Ethanol , RevertAid First Strand cDNA Synthesis Kit (Cat. No. K16.22), PowerUp™ SYBR™ Green Master Mix (Cat. No. A25741), mRNA primers , Pierce™ BCA Protein Assay Kit (Cat. No. 23225), NuPAGE™ LDS Sample Buffer , NuPAGE™ Sample Reducing Agent , NuPAGE™ MES SDS Running Buffer , NuPAGE™ 4 to 12%, Bis-Tris, 1.0 mm, Mini Protein Gel, 10-well (Cat. No. NP0321BOX), NuPAGE™ Transfer Buffer , Western Blotting Filter Paper, 0.83 mm thick, 8 × 13.5 cm (Cat. No. 84784), Pierce™ 20X TBS Tween™ 20 Buffer (Cat. No. 28360), Blocker™ BSA (10X) in PBS (Cat. No. 37525), Rabbit polyclonal anti-iNOS antibody (Cat. No. PA3-030A), Rabbit polyclonal anti-GAPDH antibody (Cat. No. PA1-987), Goat anti-Rabbit IgG (H + L) secondary antibody, horseradish peroxidase (HRP)-conjugated (Cat. No. 31460), and Pierce™ ECL Western Blotting Substrate (Cat. No. 32106) were all obtained from Thermo Fisher Scientific . HO-1 and OSGIN1 mRNA primers were purchased from Synbio Technologies . Cell Lysis Buffer , Protease Inhibitor Cocktail , and Prestained Protein Marker, Broad Range  were purchased from Cell Signaling . LPS from Escherichia coli 0111: B4 (Cat. No. L2630) was purchased from Sigma-Aldrich . Recombinant Murine IFN-γ (Cat. No. 315-05) was purchased from PeproTech . Phosphate Buffered Saline  was purchased from Lonza-Bioscience . Sulforaphane (Cat. No. 10496) was purchased from Cayman Europe Oü . QiAzol lysis reagent (Cat. No. 79306), Nuclease-Free Water (Cat. No. 129114), miScript II RT kit (Cat. No. 218161), miScript SYBRT. boudieri and T. claveryi, two desert truffles, were collected from Marsa Matruh, Egypt. Truffles were lyophilized and stored at − 20 °C for further analysis. To specify the concentration of the extracts used in this study, a stock solution was made by dissolving 100 mg of the ethanolic extract in 1 mL of DMSO, resulting in a 100 mg/mL concentration. The solution was then filtered using a 0.2 μm syringe filter to remove impurities. The 1 mg/mL solution was prepared by diluting the stock solution with DMEM medium. A working 100 μg/mL solution was produced by further diluting the 1 mg/mL solution with DMEM medium. The working solution was used to prepare all tested concentrations, ensuring the final DMSO concentration was kept at 0.05% v/v or lower.g for 30 min. 3 μL of supernatant were used for UPLC-Orbitrap HRMS analysis. Farag's exact parameters of chromatographic conditions and mass spectrometer parameters were followed32.Separately, the freeze-dried truffles were ground in a mortar with liquid nitrogen. The powder (150 mg) was then homogenized for five 20-s periods with 6 mL methanol in a Turrax mixer . To avoid heating, each mixing period was separated by 1 min. To remove plant debris, the extracts were vortexed vigorously and centrifuged at 3000× Terfezia species alongside searching the phytochemical dictionary database.Compounds were assigned by comparison of their retention times and MS spectral data  in both negative and positive ionization modes with those reported for RAW 264.7 cells  were maintained in DMEM, high glucose supplemented with 10% heat-inactivated FBS, and 1% Pen/Strep (100 units/mL penicillin and 100 µg/mL streptomycin) at 37 °C in a humidified 5% CO2 incubator.5 cells/mL in a 96-well plate and cultured in DMEM, high glucose supplemented with 10% heat-inactivated FBS and 1% Pen/Strep for 4 h at 37 °C in a humidified 5% CO2 incubator. After removing the medium, the cells were treated with increasing concentrations of T. boudieri and T. claveryi extracts  for 24 h to determine the non-cytotoxic concentrations to be used in the study. The cell viability was measured with the MTT colorimetric assay. After the incubation period, the medium was decanted, and the cells were incubated with serum-free DMEM containing 1 mg/mL of MTT for 2 h at 37 °C in a humidified 5% CO2 incubator. MTT was reduced by NAD(P)H-dependent cellular oxidoreductase enzymes of the metabolically active cells into formazan crystals, which were then dissolved in DMSO. The absorbance of each group was measured at 570 nm using SPECTROstar® Nano microplate reader , and the results were presented as the percentage of the control.RAW 264.7 cells were seeded at a density of 1 × 10T. boudieri and T. claveryi extracts in the presence of LPS/IFN-γ was also evaluated. RAW 264.7 cells were stimulated with LPS/IFN-γ (100 ng/10 U/mL), and co-incubated with the non-cytotoxic concentrations of T. boudieri and T. claveryi extracts 5, 10, and 20 µg/mL for 24 h in a 96-well plate. Additionally, the cells were treated with vehicle control, which was DMEM medium containing 0.05% v/v DMSO matching the maximum final concentration of DMSO in our treatments. Sulforaphane (SFN) (1 µM) was used as a positive control in all further experiments, so its cytotoxicity on RAW 264.7 cells with or without LPS/IFN-γ was also examined. The cell viability was then measured with MTT, as previously mentioned.In this study, RAW 264.7 cells were stimulated with LPS (100 ng/mL) plus IFN-γ (10 U/mL). Therefore, the cytotoxicity of 6 cells/mL in a 96-well plate and incubated for 4 h. The cells were then stimulated with LPS/IFN-γ (100 ng/10 U/mL) and co-incubated with T. boudieri and T. claveryi extracts at concentrations of 5, 10, and 20 µg/mL for 24 h at 37 °C in a humidified 5% CO2 incubator. SFN (1 µM) was used as a positive control. The nitrite accumulated in the culture medium was measured as an indicator of NO production using the Griess Reagent Kit by mixing 150 µL of the culture supernatant from each well with 130 µL of deionized water and 20 µL of Griess reagent and incubating at room temperature for 30 min in the dark according to the manufacturer’s instructions. The absorbance of the mixture was measured at 548 nm using a SPECTROstar® Nano microplate reader. The nitrite concentration in each sample was calculated based on a standard curve prepared with NaNO2.RAW 264.7 cells were seeded at a density of 1 × 106 cells/mL in a 6-well plate and incubated overnight. The cells were then stimulated with LPS/IFN-γ (100 ng/10 U/mL) and co-incubated with T. boudieri and T. claveryi extracts at concentrations of 5, 10, and 20 µg/mL for 6 h at 37 °C in a humidified 5% CO2 incubator. SFN (1 µM) was used as a positive control. Total RNA was extracted from the cells using QiAzol lysis reagent and used for cDNA synthesis. The cDNAs were synthesized from the total RNA using Thermo Scientific™ RevertAid™ First Strand cDNA Synthesis Kit for mRNA and miScript® II RT Kit for the microRNA (miRNA). The qPCR analyses were then performed using PowerUp™ SYBR™ Green Master Mix to quantify the mRNA expression of iNOS, COX-2, TNF-α, IL-6, HO-1, OSGIN1, and GAPDH (as a housekeeping gene). Furthermore, the miScript SYBR® Green PCR Kit was used to quantify the miRNA expression of miR-21, miR-146a, and miR-155, and RNU6-2 (as a housekeeping gene). The qPCR analyses were conducted using ABI Prism 7500 system (Applied Biosystems). The relative gene expression levels were determined by the 2−ΔΔCT method. The final results were expressed as the fold change of target gene expression in a target sample (treated sample) relative to a reference sample (untreated control), normalized to a reference gene: GAPDH (for mRNA) or RNU6-2 (for miRNA) as the following: fold change = 2−ΔΔCT, while ΔΔCT = ΔCT (treated sample) − ΔCT (untreated control), and ΔCT = CT (target gene) − CT (reference gene). The mRNA primers used in this study were designed using NCBI Primer-BLAST and are listed in Table ® Green PCR Kit. It was applied to all reactions allowing the detection of miRNAs in combination with miScript primer assays. Primer assays used in this study are listed in Table RAW 264.7 cells were seeded at a density of 1 × 106 cells/mL in a 6-well plate and incubated overnight. The cells were then stimulated with LPS/IFN-γ (100 ng/10 U/mL) and co-incubated with T. boudieri and T. claveryi extracts at concentrations of 5 and 20 µg/mL for 24 h at 37 °C in a humidified 5% CO2 incubator. SFN (1 µM) was used as a positive control. After incubation, the supernatants were collected, centrifuged at 1000× g for 20 min at 4 °C, and then stored at − 80 °C. The samples were subsequently analyzed for TNF-α and IL-6 proteins. The absorbance was measured at 450 nm using SPECTROstar® Nano microplate reader. The concentration of the mouse TNF-α and IL-6 proteins in each sample was calculated by comparing the absorbance of the samples to the standard curve.RAW 264.7 cells were seeded at a density of 1 × 106 cells/mL in a 6-well plate and incubated overnight. The cells were then stimulated with LPS/IFN-γ (100 ng/10 U/mL) and co-incubated with T. boudieri and T. claveryi extracts at concentrations of 20 µg/mL for 24 h at 37 °C in a humidified 5% CO2 incubator. SFN (1 µM) was used as a positive control. Total protein was extracted from the cells using an ice-cold cell lysis buffer containing a protease inhibitor cocktail. The protein concentration for each cell lysate was measured using Pierce™ BCA Protein Assay Kit. For western blotting, an equal amount of protein (10 μg) was loaded into each lane of the NuPAGE™ 4 to 12%, Bis–Tris Gel, and the Prestained Protein Marker. The gel ran into 1× NuPAGE™ MES SDS Running Buffer for 35 min at 200 Volts. The gel electrophoresis was conducted using the XCell SureLock™ Mini-Cell . After electrophoresis, the protein was transferred into a PVDF membrane through an electrotransfer process which was performed for 60 min at 30 Volts to transfer the protein from the gel to the PVDF membrane via 1× NuPAGE™ Transfer Buffer using the XCell II™ Blot Module . After blocking in 5% Blocker™ BSA for 1 h at room temperature, the membrane was incubated overnight at 4 °C with primary antibodies; Rabbit polyclonal anti-iNOS antibody (1: 2000), Rabbit polyclonal anti-COX-2 antibody (1: 1000), and Rabbit polyclonal anti-GAPDH antibody (1: 2500). The membrane was then incubated for 1 h at room temperature with Goat anti-Rabbit IgG (H + L) secondary antibody, horseradish peroxidase (HRP)-conjugated . Proteins were visualized with the Pierce™ ECL Western Blotting Substrate using ChemiDoc MP Imaging System . The density of western blotting bands was quantified using the ImageLab software (Bio-Rad Laboratories). The density of the protein bands was normalized to that of GAPDH. The density values were relatively expressed to the average value for the untreated control group, which was designated as 1.0.RAW 264.7 cells were seeded at a density of 1 × 10P value < 0.05 was considered statistically significant. All values obtained from the Griess assay, ELISA, and BCA assay were subjected to linear regression analysis.Data are expressed as mean ± standard error of the mean (SEM) for the indicated number of independently performed experiments. Statistical analysis was performed using SigmaPlot Version 14.0 . Statistical significance between multiple groups was calculated by one-way analysis of variance (ANOVA) followed by Student–Newman–Keuls post-hoc test, where T. boudieri and T. claveryi, via high-resolution UPLC-Orbitrap HRMS analysis. A total of 87 different metabolites belonging to various metabolite classes were annotated, including 21 amino acids and sugars, 11 alkaloids, 2 amides, 23 fatty acids/esters, 5 sterols, 4 phenolic, and 21 miscellaneous peaks, as listed in Table To assess for differences in the two desert truffles' metabolite composition, a non-targeted metabolite profiling of their ethanolic extracts was employed. The secondary metabolites were tentatively identified in the two desert truffles, +at m/z 280.1372 (C11H21NO7), peak 11 with [M − H]− at m/z 290.0881 (C11H17NO8), peak 12 with [M + H]+ at m/z 294.1547 (C12H23NO7) were annotated as fructosyl valine, N-fructosyl pyroglutamate and N-fructosyl isoleucine33, respectively.Truffles are typical edible fungi that are rich in nutrients, such as carbohydrates and amino acids, as revealed using LCMS analysis. Analysis showed the presence of several sugars and amino sugars like peak 10 with [M + H]T. boudieri containing more identified amino acids (16 peaks were annotated) and T. claveryi containing only 10 peaks.  were detected in both species. Identification of amino acids was mostly based on detection of nitrogen in their predicted formula and confirmed using tandem MS. For example, peak 5 showed [M + H]+ at m/z 337.1718 (C12H24N4O7) and yielded fragment ions at 319, 175 was annotated as N-Glucosyl arginine. Peak 6 showed [M + H]+ at m/z 175.1180 (C6H14N4O2) and yielded fragment ions at 158 was annotated as arginine. Peak 11 showed [M − H]− at m/z 290.0881 (C11H17NO8) and yielded fragment ions at m/z 128, 200, 272 was annotated as N-Fructosyl pyroglutamate. Peak 12 with an [M + H]+ at m/z 294.1547 (C12H23NO7) and fragment ions at m/z 276, 258, 230, 212, 86.0968; was annotated as N-fructosyl isoleucine33, while peak 15 with an [M + H]+ at m/z 328.1369 (C15H21NO7)+ and fragment ions at m/z 310, 292, 264, 178, 166; was annotated as N-Fructosyl phenylalanine34.Twenty-one amino acids and sugars were detected in the extracts of the two desert truffles, with 35, with the molecular formula (C14H16NO4)+. Furthermore, peak 32, (C25H42NO2)+ was annotated as dihydroxybuxadine-M. T. boudieri contains 7 alkaloids, whereas T. claveryi contains only 5 alkaloids, with Vincapyridine C being the only alkaloid present in both.MS spectral analysis also revealed the presence of nitrogenous compounds or alkaloids typically derived from amino acids. Based on their even high-resolution masses and improved response in positive ionization mode, 11 alkaloids (peaks 22–32) were annotated. Peak 24, for example, was annotated as puniceusine F+m/z at 200.2009 C12H26NO+ and 282.2785 C18H35NO+, annotated as dodecanamide and octadecenoic acid amide, respectively. The defining features of the monounsaturated fatty acid amide fragmentation patterns were based on losses of [M + H-17] and [M + H-35] product ion peaks observed in octadecenoic acid amide fragmentation, indicating the loss of ammonia and water from the carboxamide head group36.Truffles appeared to be enriched in nitrogenous compounds in addition to alkaloids, several nitrogenous compounds sub-classified as amides were identified. Peaks 33 and 34 showed [M + H]T. boudieri and T. claveryi revealed the presence of several fatty acids annotated by high-resolution masses and much higher response in negative ionization mode as major peaks. Octadecatrienoic acid  and octadecadienoic acid  were readily identified based on their high-resolution masses m/z 277.2175 and 279.2330 with predicted molecular formulas C18H30O2-, and C18H32O2-, respectively. A mass difference of 2 amu between the two peaks indicates the presence of an additional double bond.In the late elution region of the chromatogram, several peaks were observed. MS/MS spectra (Rt 10–18 m min) in the extracts of Terfezia species were annotated as hydroxy octadecanedioic acid (37), hydroxy-octadecadienoic acid (48), and hydroxy-octadecatrienoic acid (49) .MS/MS spectra also revealed several mono, di, and trihydroxy fatty acid conjugates assigned from their high-resolution masses and predicted formulas  + and yielded fragment ions at m/z 297, 233 was annotated as hydroxystigmast-4-en-3-one37. Peak 59 showed [M + H]+ at m/z 425.3044 (C28H40O3)+ and yielded fragment ions at m/z 407, 297, and 165, which was annotated as dankasterone A38. No sterols were detected in T. boudieri.In T. boudieri, peak 63 with [M-H]- at m/z 271.0812 (C12H15O7)−, peak 64 with [M − H]− at m/z 191.0343 (C10H8O4)−, peak 65 with [M − H]− at m/z 531.1538 (C26H28O12)− were annotated as hydroquinone-glucose-O-hexoside, scopoletin, and flavonol 3-O-petosyl hexoside, respectively. Only one peak 66 was identified in T. claveryi showed [M + H]+ at m/z 415.2115 (C24H30O6)+ was annotated as isomyrtucommulone B.The MS/MS spectra revealed a few phenolic compounds. Three peaks were detected in − at m/z 133.0144 (C4H6O5)− and yielded fragment ions at 115, 87,71, 87 was annotated as Malic acid. peak 69 showed [M − H]− at m/z 191.0198 (C6H8O7) and yielded fragment ions at 129, 111, and 87 annotated as Citric acid. Peak 70 showed [M − H]− at m/z 117.0190 (C4H6O4)− and yielded fragment ions at 117, 99, and 73 were annotated as Succinic acid. Few terpenes were detected in two types of truffles under study represented in peak 81 with [M + H]+ at m/z 291.1227 (C16H18O5)+, peak 84 with [M + H]+ at m/z 333.1697 (C19H25O5)+, peak 87 with [M + H]+ at m/z 307.2627 (C20H34O2)+ were annotated as unknown terpenoids.6 Annotated peaks belonged to organic acids identified in peaks 67, 69, 70, 71, 76, and 80. peak 67 showed [M − H]T. boudieri and T. claveryi extracts on LPS/IFN-γ-induced inflammation in RAW 264.7 cells, an MTT assay was performed to determine the optimal concentrations for use in the study. RAW 264.7 cells were incubated with increasing concentrations of T. boudieri and T. claveryi extracts (5–160 μg/mL) in the presence or absence of LPS/IFN-γ (100 ng/10 U/mL) for 24 h. SFN (1 µM) was used as a positive control. Both extracts did not significantly affect the cell viability at concentrations below 40 μg/mL compared to the untreated control. However, cell viability was decreased by approximately 63% with T. boudieri and 46% with T. claveryi when the concentration was increased to 160 μg/mL, revealing that T. boudieri has more cytotoxic effect on RAW 264.7 cells  for 24 h. SFN (1 µM) was used as a positive control. The nitrite levels in the cell culture medium were measured using the Griess Reagent Kit. As shown in Fig. 2, nitrite levels exhibited a substantial increase of 200% compared to the untreated control after stimulating cells with LPS/IFN-γ. However, treatment with both Terfezia extracts inhibited nitrite production in LPS/IFN-γ-stimulated cells in a dose-dependent manner. T. boudieri extract at concentrations of 5, 10, and 20 µg/mL significantly inhibited nitrite production by 11, 31, and 45%, respectively, and T. claveryi at concentrations of 10 and 20 µg/mL showed statistically significant inhibition of nitrite production by 38 and 41%, respectively. SFN (1 µM) also decreased nitrite levels by 29%.To investigate the anti-inflammatory effects of Terfezia extracts was due to suppression of iNOS gene expression, RAW 264.7 cells were treated with T. boudieri and T. claveryi extracts at concentrations of 5, 10, and 20 µg/mL in the presence of LPS/IFN-γ (100 ng/10 U/mL) for 6 h. SFN (1 µM) was used as a positive control. iNOS mRNA expression was then determined using qPCR. As shown in Fig. Terfezia extracts, T. boudieri at concentrations of 5, 10, and 20 µg/mL dose-dependently suppressed the upregulation of iNOS mRNA expression by 23, 28, and 30%, respectively, and T. claveryi extract at concentrations of 5, 10, and 20 µg/mL downregulated iNOS mRNA expression by 24, 34, and 42%, respectively, in a dose-dependent manner. SFN (1 µM) also inhibited the iNOS mRNA expression by 38%. These results indicate that Terfezia extracts decreased nitrite production in LPS/IFN-γ-stimulated macrophages by downregulating iNOS transcription.To investigate whether the reduction of nitrite production following treatment with Terfezia extracts on the COX-2 mRNA expression, RAW 264.7 cells were treated with T. boudieri and T. claveryi extracts at concentrations of 5, 10, and 20 µg/mL in the presence of LPS/IFN-γ (100 ng/10 U/mL) for 6 h. SFN (1 µM) was used as a positive control. COX-2 mRNA expression was then determined using qPCR. Figure Terfezia extracts, T. boudieri strongly downregulated the mRNA expression of COX-2 by approximately 60% (5–20 µg/mL), and T. claveryi extract at only 20 µg/mL significantly downregulated the mRNA expression of COX-2 by 35%. Significant differences were also observed in the inhibitory effects of both Terfezia extracts on the COX-2 mRNA expression, with T. boudieri being statistically significant compared to T. claveryi at 5, 10, and 20 µg/mL. SFN (1 µM) also inhibited the COX-2 mRNA expression by 31%.To examine the suppressive effect of Terfezia extracts on the mRNA expression of pro-inflammatory cytokines TNF-α and IL-6, RAW 264.7 cells were treated with T. boudieri and T. claveryi extracts at concentrations of 5, 10, and 20 µg/mL in the presence of LPS/IFN-γ (100 ng/10 U/mL) for 6 h. SFN (1 µM) was used as a positive control. The mRNA expression of TNF-α and IL-6 was then determined using qPCR. Figure Terfezia extracts, T. boudieri and T. claveryi at only 20 µg/mL significantly reduced the mRNA expression of TNF-α by 23, and 30%, respectively  by 23, 25, and 30%, respectively, and also downregulated by treatment with T. claveryi  by 20, 27, and 40%, respectively, as shown in Fig. To examine the potential anti-inflammatory effects of Terfezia extracts were related to modulation of the Nrf2 signaling pathway, RAW 264.7 cells were firstly treated with T. boudieri and T. claveryi extracts at concentrations of 5, 10, and 20 µg/mL in the presence of LPS/IFN-γ (100 ng/10 U/mL) for 6 h. SFN (1 µM) was used as a positive control. qPCR was then performed to measure the mRNA expression of Nrf2 targets (HO-1 and OSGIN1). The results depicted in Fig. T. boudieri, T. claveryi, and SFN did not lead to a significant activation of the mRNA expression of HO-1 and OSGIN1. Consequently, the concentration of T. boudieri and T. claveryi was increased to a non-cytotoxic concentration of 40 µg/mL. SFN was also increased up to 5 µM. As shown in Fig. T. boudieri and T. claveryi (40 µg/mL) did not exhibit any significant effect on either HO-1 or OSGIN1 mRNA expression.To determine whether the anti-inflammatory effects of Terfezia extracts were associated with epigenetic modulation of gene expression, RAW 264.7 cells were treated with T. boudieri and T. claveryi extracts at concentrations of 5, 10, and 20 µg/mL in the presence of LPS/IFN-γ (100 ng/10 U/mL) for 6 h. SFN (1 µM) was used as a positive control. qPCR was then performed to measure the miRNA expression of miR-21, miR-146a, and miR-155. As shown in Fig. 7, miR-21, miR-146a, and miR-155 were significantly upregulated by 206, 263, and 247%, respectively, in LPS/IFN-γ-induced cells compared to the untreated controls and significantly reduced by treatment with Terfezia extracts in a dose-dependent manner. Significant differences were also observed in the inhibitory effects of Terfezia extracts on the miR-21, and miR-146a, with T. claveryi (20 µg/mL) being statistically significant compared to T. boudieri (20 µg/mL). Figure T. boudieri (10 and 20 µg/mL) caused a significant reduction in miR-21 upregulation by approximately 35%, whereas T. claveryi  showed significant inhibition of miR-21 upregulation by 21, 50, and 64%, respectively. Similarly, in Fig.  T. boudieri  significantly reduced the upregulation of miR-146a by 28, 54, and 58%, respectively, while T. claveryi  demonstrated highly significant inhibition of the upregulated miR-146a by 46, 70, and 85%, respectively. In Fig.  T. boudieri and T. claveryi at only 20 µg/mL significantly decreased miR-155 upregulation by 28 and 35%, respectively. Additionally, SFN (1 µM) led to downregulation of miR-21, miR-146a, and miR-155 expression by 55, 72, and 36%, respectively.To investigate whether the anti-inflammatory effects of Terfezia on TNF-α and IL-6 mRNA expression and their corresponding secretion levels, RAW 264.7 cells were treated with T. boudieri and T. claveryi extracts at concentrations of 5 and 20 µg/mL in the presence of LPS/IFN-γ (100 ng/10 U/mL) for 24 h. SFN (1 µM) was used as a positive control. The concentration of TNF-α and IL-6 proteins secreted into the cell culture supernatant was measured using ELISA. As shown in Fig. Terfezia extracts, T. boudieri at 20 µg/mL significantly reduced TNF-α and IL-6 production by 24, and 14%, respectively. It was also found that T. claveryi at 20 µg/mL and SFN (1 µM) displayed a significant reduction in TNF-α production by 27 and 34%, respectively, but they did not exhibit any significant inhibition of IL-6 production  for 24 h. SFN (1 µM) was used as a positive control. The expression of iNOS and COX-2 proteins were examined using western blotting. As shown in Fig. Terfezia extracts, T. boudieri (20 µg/mL) significantly inhibited the iNOS and COX-2 expression by 46 and 36%, respectively. Similarly, T. claveryi (20 µg/mL) suppressed the expression of iNOS by 40%, but it did not demonstrate any significant effect on the COX-2 expression. SFN (1 µM) also inhibited the iNOS and COX-2 expression by 28 and 33%, respectively. The expression of the GAPDH, which was used as a loading control was not changed  and piperlongumine alkaloids in the chemical profile of T. claveryi. In an in vitro study to evaluate the anti-inflammatory activities of tempol, it was determined that tempol treatment inhibited NO production in LPS-induced macrophages as well as suppressed the expression of cytokines  and inflammatory enzymes  in IL-1β-stimulated chondrocytes42. Similarly, in activated macrophages, Sun et al. demonstrated that piperlongumine or its analog inhibited LPS-induced production of NO and PGE2 by downregulating the expression of iNOS and COX-2, respectively. They also reported that piperlongumine analog reduced LPS-dependent induction of NF-κB and MAPKs43. Phenolics are among the most essential classes of secondary metabolites, present in the fungal fruiting bodies with proven anti-inflammatory activities44. From our analysis, T. boudieri was characterized by the presence of phenolic compounds, particularly scopoletin, which exerted anti-inflammatory activities in croton oil-treated mouse ears by reducing the overproduction of TNF-α and PGE245. The anti-inflammatory actions of truffles can be attributed to their amino acid contents, which are associated with prostaglandin metabolism46. Pyroglutamic acid was identified in T. claveryi, and its derivatives significantly inhibited the secretion of NO, TNF-α, and IL-6 in LPS-treated RAW 264.7 macrophages47. On the other hand, T. boudieri was rich in stachydrine, which exhibited anti-inflammatory properties by suppressing the levels of NO, PGE2, iNOS, COX-2, TNF-α, and IL-6 as well as blocking the IL-1β-mediated potentiation of NF-κB pathway in IL-1β-induced chondrocytes48. Furthermore, T. boudieri contained N-acyl homoserine lactone, and analogues of this compound have been shown to inhibit cytokine release of IL-6 and IL-8 in different in vitro-stimulated cell lines49. Fatty acids present in truffles potentially support their anti-inflammatory characteristics because of their high contents of polyunsaturated fatty acids (PUFAs), which are the precursors of the eicosanoids46. The metabolome of both Terfezia species was distinguished by the abundance of PUFAs, especially linoleic acid. Saiki et al. indicated that linoleic acid extracted from Agaricus brasiliensis reduced NO secretion and inhibited the expression of TNF-α, IL-6, IL-1β, and iNOS in RAW 264.7 macrophages. Linoleic acid also suppressed the activation of the NF-κB pathway50. The metabolite profiling of T. boudieri and T. claveryi contained phenylacetic acid and cinnamic acid, respectively, two active aromatic compounds with anti-inflammatory activities52.Our metabolomic analysis of 53. Additionally, Heiss et al. indicated that SFN reduced the production of the inflammatory mediators, including NO, PGE2, and TNF-α, accompanied by downregulation of iNOS and COX-2 proteins in LPS-induced RAW 264.7 cells54. SFN-mediated anti-inflammatory activity has been attributed in part to the activation of the Nrf2, as reported by Lin et al.55. In addition to the previous studies, Saleh et al. demonstrated that SFN modulated the TLR-associated miRNAs, such as miR-146a and miR-155, in LPS/IFN-γ-induced RAW 264.7 cells56. As a result, SFN can be considered as a suitable benchmark for assessing the anti-inflammatory properties of other medications.SFN was used as a positive control in this study because it possesses a potent anti-inflammatory activity, mediated through suppressing the TLR4 oligomerization57. Therefore, in vitro models of macrophages are important tools in evaluating the efficacy of the anti-inflammatory drugs through assessing the inflammatory response and cytotoxicity. RAW 264.7 macrophage cells are generally used to investigate the anti-inflammatory properties of drugs, and significantly activated by LPS and/or IFN-γ59. IFN-γ is included in combination with LPS in the macrophage polarization, and the induction of macrophages with either one of them, leads to the secretion of several inflammatory mediators60. In the current study, to examine the effects of T. boudieri and T. claveryi extracts on the inflammatory pathways, RAW264.7 cells were stimulated with LPS (100 ng/mL) plus IFN-γ (10 U/mL). The rationale for selecting LPS/IFN-γ instead of LPS alone was based on our optimization experiments. We found that RAW 264.7 cells treated with LPS alone exhibited weak immune responses, and the addition of IFN-γ was able to potentiate the immune response by activating signaling pathways that lead to the production of inflammatory cytokines and NO.Macrophages play pivotal roles during the inflammatory response, such as phagocytosis of microbes, antigen presentation, and secretion of inflammatory mediators. Moreover, macrophages are essential for maintaining homeostasis and tissue regeneration after injury56. Initially, we determined the non-cytotoxic concentrations of T. boudieri, and T. claveryi extracts with or without LPS/IFN-γ in RAW 264.7 cells using an MTT assay. Our data showed that treatment of RAW 264.7 cells with T. boudieri and T. claveryi extracts in the presence or absence of LPS/IFN-γ was not associated with a decrease in the cellular viability at concentrations between 5–20 μg/mL. Therefore, 5, 10, and 20 μg/mL concentrations were selected for both extracts in all further experiments.The increased production of NO, as measured by the Griess assay, supported this finding. Our results align with the previously published study by Saleh et al., which has shown that LPS/IFN-γ produces a more robust immune response than LPS alone2. Once LPS/IFN-γ activates macrophages, the production of inflammatory mediators, including cytokines , eicosanoids , and NO is increased62. NO is a key signaling molecule that is vital to the inflammatory response. NO is released as a cellular signaling molecule to increase the vasodilation in blood vessels by activating iNOS, which subsequently leads to an apparent increase in the blood flow and recruitment of leukocytes to the region of inflammation63. Therefore, to investigate the anti-inflammatory effects of T. boudieri and T. claveryi extracts, we analyzed the NO production and the iNOS expression at the mRNA and protein levels in LPS/IFN-γ-stimulated RAW 264.7 cells. Since NO is rapidly oxidized to nitrite, the nitrite level in the culture medium was measured as an indicator of the NO production64. Here, we found that T. boudieri and T. claveryi extracts inhibited NO production in a dose-dependent manner, accompanied by a simultaneous reduction in the iNOS mRNA expression. These data were consistent with our Western blotting analysis, in which both extracts exhibited significant inhibition of the expression of iNOS protein. Our results indicate that Terfezia extracts effectively improve inflammatory conditions by inhibiting NO's overproduction and the expression of iNOS in LPS/IFN-γ-stimulated RAW 264.7 cells.The inflammatory response is mediated by a wide range of mediators forming complex regulatory networks that prevent further tissue damage and restore the normal physiology of the inflamed tissues65. As a consequence, a plethora of pharmacological agents has been developed to suppress COX-2 activity and thereby mitigate the inflammatory response. Thus, to examine the suppressive effect of T. boudieri and T. claveryi extracts, we measured the mRNA and protein expression of COX-2 in LPS/IFN-γ-stimulated RAW 264.7 cells. Our study findings demonstrated that both extracts effectively reduced the mRNA expression of COX-2. Notably, T. boudieri exhibited more potent inhibitory action on both mRNA and protein levels. Taken together, these data suggest that the anti-inflammatory properties of Terfezia extracts are associated with the suppression of COX-2 expression in LPS/IFN-γ-stimulated macrophages.Prostaglandins are eicosanoid-derived molecules that participate in modulating numerous physiological processes, particularly during immune responses. Prostaglandins are produced through arachidonic acid metabolism by cyclooxygenases, which exist in two isoforms: COX-1 and COX-2. COX-1 is produced constitutively in most cells, whereas COX-2 is induced in response to inflammatory stimuli12. Among cytokines, TNF-α is the primary mediator of inflammation with several effects, including stimulating other cytokine secretion, activating cell adhesion molecules, and promoting cell growth and proliferation12. IL-6 is another important cytokine released during inflammation, and its dysregulation causes a variety of inflammatory disorders66. Hence, the dysregulation in the production of inflammatory cytokines is commonly related to inflammatory diseases, making them potential therapeutic targets. In the current study, we assessed the expression of pro-inflammatory cytokines TNF-α and IL-6 at the mRNA and protein levels to identify the possible effects of T. boudieri and T. claveryi extracts on the inflammation mediated by LPS/IFN-γ in RAW 264.7 cells.Cytokines are essential signaling proteins regulating the crosstalk between different cell types involved in the immune and inflammatory responseT. boudieri extract decreased the expression of TNF-α and IL-6 in a dose-dependent manner at the mRNA and protein levels. In contrast, T. claveryi was found to decrease the mRNA expression of TNF-α and IL-6, along with the inhibition of TNF-α protein production, while no significant effect was detected on IL-6 protein levels. Consistent with our results, Darwish et al. revealed that T. claveryi displayed anti-inflammatory activity by reducing TNF-α, IL-1β, and IFN-γ in LPS-stimulated WBCs67. These results reveal that Terfezia extracts possess anti-inflammatory properties via suppressing the pro-inflammatory cytokines involved in the inflammatory process.Interestingly, T. claveryi treatment decreased the mRNA expression of COX-2 and IL-6 but did not show the same effect on the protein levels. Several possibilities could explain this discrepancy, including differences in the time frame of our measurements, post-transcriptional modifications, protein stability, and other regulatory factors68. Firstly, the time frame of our measurements could be a contributing factor. We measured the mRNA expression levels at 6 h after stimulation with LPS/IFN-γ, while protein expression levels were measured at 24 h. Therefore, it is possible that T. claveryi treatment had a transient effect on the mRNA levels that were not reflected at the protein expression levels at 24 h. Secondly, post-transcriptional modifications can affect mRNA's stability and translation efficiency, which may not always be reflected at the protein expression levels. After transcription, mRNA undergoes several post-transcriptional modifications such as splicing, polyadenylation, and transport from the nucleus to the cytoplasm, affecting its stability and translation efficiency69. Thirdly, protein stability can also contribute to the discrepancy between mRNA and protein expression levels. Even if the mRNA levels are decreased, the protein levels may remain stable due to post-translational modifications or other factors68.Following the earlier findings, it can be observed that T. boudieri and T. claveryi extracts were related to activation of the Nrf2 signaling pathway in RAW 264.7 cells induced by LPS/IFN-γ through assessing the gene expression of Nrf2 target genes, HO-1 and OSGIN1. Inflammatory cells produce numerous inflammatory mediators, which subsequently attract more inflammatory cells to the site of injury, leading to increased oxidative stress levels. Meanwhile, persistent oxidative stress is associated with chronic inflammation. Nrf2 signaling pathway is also essential in reducing inflammation-related disorders, including atherosclerosis, asthma, autoimmune diseases, and rheumatoid arthritis15. Several studies have reported that the activation of the Nrf2 signaling pathway suppressed cytokines, chemokine, iNOS, and COX-2 secretion, which modulate the NF-kB and other inflammatory cascades that regulate the transcription and activity of downstream target proteins during the inflammation process19. Our preliminary results demonstrated that exposure to various concentrations  of T. boudieri, T. claveryi, as well as SFN at a concentration of 1 µM, did not result in a significant upregulation of the mRNA expression of HO-1 and OSGIN1. Since SFN is known to be a potent activator of Nrf2, we initially expected it to upregulate the expression of Nrf2 target genes. However, we did not observe any significant effects on the expression of these genes at a concentration of 1 µM. Therefore, we increased the concentration of SFN to 5 µM to determine if this would lead to changes in Nrf2 target genes expression. The concentration of T. boudieri and T. claveryi was also increased to a non-cytotoxic concentration of 40 µg/mL, as assessed by MTT assay. As opposed to our initial results, we observed that SFN at a concentration of 5 µM activated the gene expression of HO-1 and OSGIN1 in our study. This is consistent with previous research by Doss et al., who reported a significant increase in HO-1 mRNA levels in erythroid cells treated with 5 µM SFN, whereas lower concentrations (100 nM-1 µM) did not produce the same effect70. On the other hand, treatment with T. boudieri and T. claveryi at concentration of 40 µg/mL did not show any significant induction on the gene expression of HO-1 and OSGIN. These results imply that the anti-inflammatory effects of Terfezia extracts are not mediated through Nrf2 activation.Nrf2 is a key transcription factor, playing a central role in the inflammation signaling pathways and oxidative stress responses. Therefore, the present study examined whether the anti-inflammatory effects of Terfezia extracts, this study explored the effects of T. boudieri and T. claveryi extracts on the expression of inflammatory miRNAs, including miR-21, miR-146a, and miR-155 in RAW 264.7 cells activated with LPS/IFN-γ. The selection of these miRNAs in the study was based on their relevance to the TLR4 signaling pathway71. miRNAs play significant roles in regulating immune cell functions in both innate and adaptive immune systems through targeting inflammation-related pathways, such as TLR472. miR-21 is a negative regulator of the TLR4-induced immune response that suppresses the NF-κB activity and promotes the production of the anti-inflammatory cytokine IL-10 by targeting the programmed cell death protein 4 (PDCD4)73. Furthermore, miR-146a was identified to be an NF-κB-dependent gene that targets interleukin-1 receptor-associated kinases1 (IRAK1) and TNF receptor-associated factor 6 (TRAF6) in the TLR4 signaling pathway, suggesting it as a negative regulator of the innate immune response74. Moreover, miR-155 is known to play a significant role in inflammation by targeting multiple proteins involved in the TLR4 signaling pathway75. Ceppi et al. reported that miR-155 negatively regulated the inflammatory response to LPS in monocyte-derived dendritic cells. This negative regulation by miR-155 was related to its ability to target TA76. The results of the present study demonstrated upregulation of miR-21, miR-146a, and miR-155 in response to LPS/IFN-γ stimulation, consistent with previous studies79. Interestingly, treatment with T. boudieri and T. claveryi extracts was observed to inhibit the LPS/IFN-γ-induced upregulation of these miRNAs in a concentration-dependent manner, suggesting that both Terfezia extracts suppress the inflammatory response in activated macrophages through regulating the expression of miR-21, miR-146a, and miR-155.In order to gain a deeper understanding of the anti-inflammatory mechanisms of 80. However, our results demonstrate that the anti-inflammatory properties of T. boudieri and T. claveryi are independent of Nrf2 activation. As such, the effects observed at the miR-21, miR-146a, and miR-155 are equally independent of any effects at the Nrf2 level. Although the specific mechanisms involved are not yet fully understood, our results suggest that these compounds may utilize alternative pathways to regulate inflammation. One possible mechanism is the inhibition of NF-κB signaling, a central inflammation regulator. While some natural compounds have been shown to inhibit NF-κB signaling, we did not investigate this pathway in our study.Nrf2 activation is known to regulate inflammation by activating miRNAs, such as miR-21, miR-146a, and miR-155T. boudieri and T. claveryi metabolome suggesting that NF-κB signaling may indeed play a role in the observed effects50. In addition, previous research from our laboratory has also suggested a role for Glycogen synthase kinase 3 (GSK3) in mediating anti-inflammatory response that is also Nrf2 independent81. As such, further research is necessary to determine the involvement of these pathways in the anti-inflammatory effects of T. boudieri and T. claveryi. Overall, our study suggests that these compounds may have the potential as alternative anti-inflammatory agents that act through non-traditional pathways.Nevertheless, our findings are supported by similar results regarding the effects of some biologically active substances in T. boudieri and T. claveryi extracts in regulating the inflammatory response in LPS/IFN-γ-stimulated RAW 264.7 macrophages through modulation of the TLR4 activation. The results of our experiments showed that treatment with these extracts led to a concentration-dependent reduction in the production of NO that coincided with the downregulation of iNOS at both the mRNA and protein levels. Both extracts downregulated the COX-2 mRNA expression, with T. boudieri further reducing the expression of COX-2 protein. Furthermore, T. boudieri extract dose-dependently downregulated TNF-α and IL-6 mRNA and protein levels, while T. claveryi extract showed significant inhibition of TNF-α and IL-6 mRNA expression without affecting IL-6 and COX-2 protein levels. This study also offers novel insights into the epigenetic suppressive properties of T. boudieri, and T. claveryi extracts on miR-21, miR-146a, and miR-155 expression in LPS/IFN-γ-induced RAW 264.7 macrophages, proposing that Terfezia extracts could exert anti-inflammatory activities by suppressing the miRNAs involved in the TLR4 signaling pathway. Additionally, our findings showed that T. boudieri and T. claveryi exhibited anti-inflammatory effects through an Nrf2-independent manner. Furthermore, our study demonstrated differences in the chemical composition and anti-inflammatory effects of T. boudieri and T. claveryi extracts, despite both species being desert truffles. These differences can be attributed to the distinct chemical compositions of the two species. These differences highlight the importance of identifying and characterizing the specific bioactive compounds in each species, which could have different pharmacological properties and therapeutic potential. Finally, the present study suggests that T. boudieri and T. claveryi could serve as promising alternative agents for treating inflammation. However, the efficacy and safety of these extracts in vivo should be evaluated using appropriate animal models in future studies.In conclusion, our results suggest that targeting specific inflammatory mediators associated with TLR4-mediated signaling could be an effective therapeutic approach for mitigating inflammation induced by LPS/IFN-γ in RAW 264.7 macrophages. The current study highlights the potential anti-inflammatory properties of Supplementary Information.


Soybean oil body (SOB) emulsions were prepared using OBs extracted at pH 11.0 and pH 7.0. The pH 11.0-SOB comprised oleosins, whereas pH 7.0-SOB comprised extrinsic proteins and oleosins. All SOB emulsions were heated at 60–100 °C for 15 min. Heating may lead to the release of extrinsic proteins from the surface of pH 7.0-SOB due to heat-induced denaturation. The total proportion of α-helix and β-sheets gradually decreased from 77 (unheated) to 36.2% (100 °C). During stomach digestion, the extrinsic protein hydrolysis of heated pH 7.0-SOB emulsions was fast between 60 and 80 °C, and it then slowed between 90 and 100 °C; heating inhibited the oleosin hydrolysis of pH 7.0- and 11.0-SOBs. Heat treatment promoted aggregation and coalescence, and it resulted in increased particle sizes for all emulsions. Larger aggregates were found in heated pH 7.0-SOB emulsions, and larger oil droplets were found in heated pH 11.0-SOB emulsions. After intestinal digestion, the droplets of all SOB emulsions gradually dispersed, and particle sizes decreased. Different heating temperatures had lesser effects on particle sizes and microstructures. Lipolysis was affected by the extraction pH and heating. For pH 11.0-SOB emulsions, the FFA release tendency was greatly affected by the heating temperature, and heating to 80 °C resulted in the highest FFA release (74%). However, all pH 7.0-SOB emulsions had similar total FFA releases. In addition, the droplet charges of heated pH 7.0-SOB emulsions were lower than those of unheated pH 7.0-SOB emulsions in both the intestine and stomach phases; however, the charge changes in different pH 11.0-SOB emulsions showed the opposite tendency. This study will offer guidance regarding the application of SOB emulsions in food. Soybeans are widely cultivated as oil crops across the world. The protein and lipid contents of soybeans are 37.7–44.5% and 17.2–21.6%, respectively , which aDue to the need for food processing and storage, heat treatment is one of the most common techniques used in the field of food. Furthermore, heating is a common method used to increase the digestibility of proteins in various foods . HoweverOBs could be considered as special protein-stabilized oil droplets because the entire surface was covered by proteins . We consIn this study, pH 7.0- and pH 11.0-SOB emulsions were each treated at 60 °C, 70 °C, 80 °C, 90 °C, and 100 °C. The digestive regularities of pH 7.0- and pH 11.0-SOB emulsions were researched in the gastrointestinal stage, including protein hydrolysis, zeta potentials, particle sizes, microstructures, and FFA releases. This study will offer theoretical guidance for the application of SOB emulsions in food, such as the delivery of functional nutrients and dietary lipid.We bought soybean seeds (Zhonghuang 37) from the local market . Protein markers (14.4–97.4 kDa) were bought from Solarbio . Pancreatin, pepsin, Fast Green and Nile Red, and bile salts were bought from Sigma-Aldrich . All chemical reagents were analytical grade.w/w), and the mixture was ground via a crushing machine  at 22,000 r/min for 2 min. Then, four layers of gauze were used to filter the resulting slurry. Sucrose (62.5 g) was added to the filtrate (312.5 g) and mixed well. The mixture was divided into two parts, and their pHs were adjusted to 11.0 and 7.0, respectively, using 0.2–2 mol/L KOH and HCl solutions. The mixtures were centrifuged at 43,667× g and 4 °C for 30 min. The floats were collected from the upper layer and mixed again with fresh ID water. The pHs of the mixtures were adjusted to 11.0 and 7.0, respectively, and they were centrifuged under the same conditions. The above steps were repeated twice. The obtained SOBs were called pH 11.0-SOB and pH 7.0-SOB, respectively.The SOBs were extracted using the modified methods described by Han et al. (2020) . Soybeang) again, and the floats were analyzed using Tricine-SDS-PAGE. NaN3  was added into the other SOB emulsions, which were immediately used for further analysis.SOBs (8.0 g) were dispersed into the ID water to prepare the SOB emulsions . All emulsions were sheared  using a dispersion machine . The pH 7.0- and pH 11.0-SOB emulsions were heated at 60 °C, 70 °C, 80 °C, 90 °C, and 100 °C for 15 min and immediately cooled. An unheated SOB (4 °C) was used as the control. Some samples were centrifuged  . Each SOWhen simulated stomach digestion was completed, the pH of each mixture was adjusted to 7.0 with KOH (0.2–2 mol/L). The simulated intestinal fluid (SIF) was composed of CaCl2 (0.3 mol/L), KCl (25 mmol/L), and NaCl (0.75 mol/L). Bile salts (0.62 g) were added into the prepared SIF (57.6 mL). Then, pancreatin was added into the solution, and the final concentration was 6.35 mg/mL. This solution simulated intestinal digestion at pH 7.0 and 37 °C, which lasted for 3 h.Samples were collected at 120 min (termed G120) in the stomach phase, cooled, and diluted 400 times using citric acid solution . In addition, samples were collected at 180 min (termed I180) in the intestine phase and cooled. The initial emulsions and all samples were also diluted for the number of times described above using phosphate solution . Zeta potentials were carried out using a Laser Nano-ZS Particle Analyzer  at 25 °C ,20.Samples from stomach and intestinal digestion were diluted 1000 times using a citric acid solution  and a phosphate solution , respectively. The particle sizes were determined using a Laser Nano-ZS Particle Analyzer  at 25 °C.v/v) was added to all samples, and the mixed solutions were heated at 100 °C for 5 min. Then, 8 μL of each sample was loaded into the sample well, and samples were electrophoresed at a constant voltage of 30–35 mV until all samples entered into the stacking gel, and the constant voltage was then changed to 100 mV until complete. The gel was stained using Coomassie Brilliant Blue G-250. The intensities of different protein bands were analyzed via Image Lab 3.0 Software .Tricine-SDS-PAGE was conducted according to the method of Zhao et al. (2016) , albeit w/v) Nile Red dissolved in 10% (v/v) acetone and 0.1% (w/v) Fast Green aqueous solution, respectively. Samples (200 μL) were stained with Nile Red (20 μL) and Fast Green (25 μL) and stored with restricted lighting for 10 min [Microstructures were observed using a Confocal Laser Scanning Microscope (CLSM) 880 . The oils and proteins were stained using 0.02%  ,25. FFA KOHV and KOHm represent the consumed volume and content of KOH, respectively; M represents the molecular weight of SOBs, i.e., 876.56 g/moL; and lipidM represents the weight of TAGs.p < 0.05 was considered to be statistically significant.Experiments were performed on 3 separate occasions in triplicate using freshly prepared samples. The data were presented as the means with standard deviations. Statistical analysis was performed using SPSS statistics version 25 following an ANOVA model . A confidence level of The protein compositions of unheated and heated pH 7.0- and pH 11.0-SOB emulsions are shown in −1) and amide III (1230–1300 cm−1). In order to reveal the protein structure of the pH 7.0-SOB, we quantitatively fitted amides I and III via the Raman spectrum  had no obvious influence on the zeta potentials of pH 11.0-SOB emulsions.Before digestion, the average particle size of the unheated pH 7.0-SOB emulsion was 658 nm, and the particle sizes of heated pH 7.0-SOB emulsions first decreased to 459 nm (60 °C), before remaining constant at about 327 nm (70–100 °C). This result occurred due to extrinsic proteins released from the pH 7.0-SOB surface via heating a,b. ConvIt was shown that the particle sizes of pH 7.0- and pH 11.0-SOB emulsions increased significantly in the stomach phase . The reaThe droplets were observed using CLSM, and oil phases and proteins were marked with red and green fluorescent dyes . Before During intestinal digestion, TAGs were hydrolyzed using pancreatin, and the total FFA release increased gradually with increasing digestive time . The FFAIn this study, we prepared emulsions using SOBs extracted at pHs of 7.0 and 11.0 and discussed the effects of heat treatment (60–100 °C) on their digestive characteristics. It was found that the pH 11.0-SOB had only oleosins , while the pH 7.0-SOB had extrinsic proteins (7S and 11S) and oleosins. Obviously, extrinsic proteins were released from pH 7.0-SOBs heated at 60–100 °C due to heat-induced denaturation. The total proportion of α-helix and β-sheet in the unheated pH 7.0-SOB was 77%, while the total proportion of α-helix and β-sheet decreased from 61.1 (60 °C) to 36.2% (100 °C). The heating temperatures had no significant effects on particle sizes and microstructures, and they had a greater effect on the zeta potentials of pH 7.0-SOBs than those of pH 11.0-SOBs before digestion. In stomach digestion, (1) the extrinsic protein hydrolysis of pH 7.0-SOB emulsions gradually accelerated from 60 to 80 °C, before slowing from 90 to 100 °C, and heat treatment inhibited oleosin hydrolysis for both the pH 7.0- and 11.0-SOB. Next, (2) the particle sizes increased for all emulsions, and heat treatment resulted in larger oil droplets in pH 11.0-SOB emulsions and larger aggregates in pH 7.0-SOB emulsions. Finally, (3) heat treatment decreased and increased the positive charges of SOB emulsions extracted at pH 7.0 and 11.0, respectively. After intestinal digestion, (1) the droplets gradually dispersed, and the particle sizes of all SOB emulsions decreased. Next, (2) heated pH 7.0–SOB emulsions had more negative charges than unheated emulsions, while the charge changes in pH 11.0-SOB emulsions showed the opposite trend. Finally, (3) for all pH 7.0-SOB emulsions, the total FFA releases were similar (about 70%) at 180 min, but different temperatures greatly affected the FFA releases of pH 11.0-SOB emulsions, and heating at 80 °C resulted in the highest FFA releases (74%). FFA release was improved in tandem with the increases in the heating temperatures during the initial digestion stage. Therefore, extraction pH and heat treatment had important effects on the digestive characteristics of SOB emulsions, which will provide guidance for selecting proper extraction pHs and heating temperatures for the application of SOB emulsions in the field of food.


Sapovirus (SaV) infection is increasing globally. Concurrently, several SaV-outbreaks were observed in children of Zhejiang province, China, in recent years, In this study, the genotypes of Sapovirus from seven outbreaks in the Zhejiang province were analysed.A total of 105 faecal samples were collected from children aged between 4 and 17 years from the Zhejiang Provincial Center for Disease Control and Prevention between October 2021 and February 2023. Genotypes were processed using reverse transcription polymerase chain reaction and Sanger sequencing, while next-generation sequencing was used to generate a complete viral genome. Deduced amino acid sequences were analysed to detect VP1 gene mutations.In total, 60 SaV-positive patients were detected at a 57.14% (60/105) positivity rate. Positive rates in the seven outbreaks were: 22.22% (2/9), 15.00% (3/20), 93.10% (27/29), 84.21% (16/19), 28.57% (2/7), 53.33% (8/15) and 33.33% (2/6), respectively. Four genotypes were identified in the seven outbreaks, of which, GI.1 accounted for 14.29% (1/7), GI.2 accounted for 14.29% (1/7), GI.6 and GII.5 accounted for 14.29% (1/7), and GI.6 accounted for 57.14% (4/7). All patients were children and outbreaks predominantly occurred in primary schools and during cold seasons. Additionally, the complete sequence from the GI.6 outbreak strain showed high homology (identity: 99.99%) with few common substitutions  in VP1 protein.SaV genotype diversity was observed in the seven outbreaks, with GI.6 being the main SaV genotype in Zhejiang province. It demonstrated high homology and may provide a platform for SaV prevention and control measures.The online version contains supplementary material available at 10.1186/s12985-023-02202-z. Sapovirus genus of the Caliciviridae family and is a causative agent of acute gastroenteritis. SaV infects individuals of all age groups, and is implicated in both sporadic acute gastroenteritis cases and outbreaks [Sapovirus (SaV) belongs to the utbreaks . SaV is utbreaks , with prutbreaks .The SaV genome is a positive single-strand RNA, approximately 7.3–7.5 kb in size, and encodes two structural VP1–VP2) and seven non-structural proteins (NS1–NS7). SaV is also highly diverse in terms of genes and antigens. The VP1 gene sequence is the main classification criterion-based on gene sequences, SaV is divided into 19 genogroups (GI–GXIX), with GI, GII, GIV, and GV primarily infecting humans, and the remaining genogroups primarily infecting animals  and seve. VP1 is Acute gastroenteritis is a global public health issue, particularly in low- and middle-income countries . DespiteSince its first discovery in Japan in 1976, SaV sporadic cases and outbreaks in humans have been reported globally  in SwedeResearch involving biological samples from humans was approved by the ethics committee of the Zhejiang Provincial Center for Disease Control and Prevention .Refer to the Guidelines on Outbreak Investigation, Prevention and Control of Norovirus Infection (2015), within three days, the same gathering setting presented over five patients diagnosed acute gastroenteritis, with the epidemiological association, manifesting over three diarrhoea episodes or two vomiting episodes in 24 h, and at least two patients detected SaV positivity on laboratory diagnosis. Patients meeting these criteria were included. An epidemiological survey was performed by the Center for Disease Control and Prevention in Prefecture-level cities.In total, 105 faecal samples were collected from seven SaV acute gastroenteritis outbreaks  from October 2021 to February 2023.Escherichia coli and Salmonella).Faecal samples were prepared in 10% faecal phosphate-buffered saline (PBS) , centrifuged at 4500 rpm at 4 °C for 30 min, and supernatants stored at − 80 °C. In nucleic acid assays, samples were negative for other viruses and bacteria associated with acute gastroenteritis  and the manufacturer’s instructions, viral nucleic acid was extracted from 200 μL of faecal supernatant, eluted in 50 μL elution buffer, and stored at − 80 °C.Taq, 0.5 μL of RT enzyme, 0.6 μL of forward primer (20 μM), 0.6 μL of reverse primer (20 μM), 0.3 μL of probe (20 μM), 5 μL of template RNA, and 5 μL of RNase-free water. Amplification conditions were as follows: reverse transcription at 50 °C for 30 min, initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s, and annealing and extension steps at 62 °C for 1 min. Primer and probe sequences are listed  in a final volume of 25 μL, containing 12.5 μL of buffer, 0.5 μL of A partial SaV VP1 (800 bp) sequence was amplified using the PrimeScript™ One-Step RT-PCR Kit Ver.2 (TaKaRa 055RA). The final reaction volume was 50 μL, containing 25 μL of buffer, 2 μL of enzyme mix, 1 μL of forward primer, 1 μL of reverse primer, 13 μL of RNase-free water, and 8 μL of RNA template. Forward primers were SVF13 and SVF14 and reverse primers were SVR13 and SVR14 as listed in Additional File Taq™ Hot Start Version (TaKaRa 007RA). The final volume was 50 μL and contained 5 μL of buffer, 4 μL of dNTPs, 0.25 μL of Taq enzyme, 0.5 μL of forward primer, 0.5 μL of reverse primer, 38.75 μL of RNase-free water, and 1 μL of first the PCR product. The forward primer was SV1245Rfwd and the reverse primer was SVR2  was used to synthesise double-stranded cDNA using random primers. cDNA was purified using Agencourt® AMPure® XP beads. Libraries were constructed using the MATRIDX Metagenomic DNA Library Construction Kit  and purified using Agencourt® AMPure® XP beads. The KAPA Library Quantification Kit for the Illumina® platform was used to determine library concentrations, after which, the library was sequenced using the Illumina NextSeq 2000 Reagent Kit P2 (100 cycles and single end) on an Illumina NextSeq 2000 platform.Raw sequencing data were processed using quality control, alignment, and consensus sequence generation steps. Firstly, Fastq data were used to trim primer sequences and filter out low-quality reads. Secondly, all cleaned reads were mapped to the SaV reference sequence (Genbank: MN509083) using the Burrows-Wheeler Aligner. To eliminate duplicate sequences and non-unique mapped reads, used SAMtools to ulteriorly filter data. The sequencing depth was obtained using BCFtools. Finally, the remaining mapped reads were assembled to obtain whole sequences.DNA sequences were assembled using DNAMAN software, MEGA 7.0 was used to align sequences using the Clustal W method and generate a phylogenetic tree, which was constructed using the neighbour-joining algorithm and maximum composite likelihood model with 1000 bootstraps. The Geneious Prime (Ver. 2022.1.1) was used to align DNA and amino acid sequences using the MAFFT alignment method, and sites were confirmed according to the SaV strain . All reference sequences were obtained from the National Centre for Biotechnology Information (NCBI) GenBank database. All sequences obtained were submitted to GenBank (GenBank accession numbers: OR351085-OR351108).In total, 105 cases form seven outbreaks were included; 9 patients from the SX-2021-1 outbreak, 20 from the HuZ-2022-1 outbreak, 29 from the HuZ-2022-2 outbreak, 19 from the WZ-2022-3 outbreak, 7 from the JX-2022-4 outbreak, 15 from the TZ-2022-5 outbreak, and 6 from the JX-2023-1 outbreak. All outbreaks were well distributed across the province; SX-2021-1, HuZ-2022-1, HuZ-2022-2, JX-2022-4 and JX-2023-1 occurred in the north of Zhejiang province, while TZ-2022-5 and WZ-2022-3 occurred in the midlands and south, respectively. Patients in outbreaks manifested with mild stomachache, diarrhoea, vomit, nausea and fever symptoms. All outbreaks lasted for 3–5 days.As indicated Table , 60 SaV-Median patient ages across outbreaks  were 7, 5, 11, 16, 5, 10 and 10 years old, respectively, and all were children < 18 years old  and the USA  in NCBI, SaV strain identities were 97.8% (Japan-LC380411), 97.8% (USA-MG012443) and 97.8% (USA-MN509083). Thus, all strains demonstrated high homology.VP1 measured 1698 bp and contained 565 amino acids. Hu/Nichinan-2–1-day3/2005/JP as the reference to confirm the site of amino acid. Sapovirus/Hu/GI.6/Nichinan/FP05284/2005/JPN, Hu/US/2015/GI.6/Nashville9367 and Hu/US/2016/GI.6/CA-RGPS-1084 as the references to compare the mutations of VP1 protein. Four highly variable regions  were identified in P2 of VP1. Amino acid sequence comparison analyses of these four regions showed that the GI.6 sequence contained two amino acid substitutions (Y300S and N302S) in HVR1. One amino acid substitution (A415G) in HVR4 compared with Sapovirus/Hu/GI.6/Nichinan/FP05284/2005/JPN, but when compared with the other two strains (Hu/US/2015/GI.6/Nashville9367 and Hu/US/2016/GI.6/CA-RGPS-1084), HVR4 exhibited an amino acid substitution (S415G). Additionally, it exhibited other amino acid mutations in VP1. When compared with Sapovirus/Hu/GI.6/Nichinan/FP05284/2005/JPN, three amino acid mutations  were identified. When compared with Hu/US/2015/GI.6/Nashville9367, three amino acid mutations  were identified. When compared with Hu/US/2016/GI.6/CA-RGPS-1084, three amino acid mutations  were identified.Amino acid sequences of the complete VP1 region of GI.6 were analysed Fig. . CompletZhejiang province is located in southeast China, covers 105,500 square kilometres and contains 11 cities. Approximately 65.77 million people live in the province, of which 9.07 million are children. The province is economically prosperous with considerable transportation hubs and good internal and external communication links. Thus, viral transfer is easily facilitated. SaV is an important virus as it causes acute gastroenteritis in children, often causing outbreaks and leading to public health issues. To understand more about the seven recent SaV outbreaks in Zhejiang province, we investigated these outbreaks in depth, reported genotypes, and identified VP1 amino acid mutations.SaV mainly infects individuals with low immunity, such as children and the elderly, but children under 5 years old are more often targeted  as indicSaV is genotypically diverse, however, GI and GII genogroups are globally dominant , 26. FroIn our study, GI.1 and GI.2 were detected, but GI.6 was dominant across the seven outbreaks. GI.6 was also the main genotype in the SaV outbreak in Osaka, Japan, from 2004 to 2005 . HoweverVP1 gene variability is the main SaV classification criterion [The VP1 protein is one of the main structural proteins in the SaV virion and riterion . As viruriterion . In our riterion . Two amiGI.6 was the dominant genotype in seven SaV outbreaks in Zhejiang province, China. Highly homology and genotype diversity was also identified in outbreaks. Children and schools were the primary targets. Therefore, more studies and surveillance programs are required to investigate the molecular epidemiology underpinning human SaV.Additional file 1. Primer sequences used in the study