In this work, hierarchical hafnium-containing multifunctional Linde type L (LTL) related zeotypes were prepared via top-down strategies, for the chemical valorization of LA, AnL and HMF via integrated catalytic transfer hydrogenation (CTH) and acid reactions in alcohol medium. This is the first report of CTH applications  of LTL related materials. The influence of the post-synthesis treatments/conditions  on the material properties and catalytic performances was studied. AnL and LA were converted to 2-butyl levulinate (2BL) and GVL in high total yields of up to ca. 100%, at 200°C, and GVL/2BL molar ratios up to 10. HMF conversion gave mainly the furanic ethers 5-(sec-butoxymethyl)furfural and 2,5-bis(sec-butoxymethyl)furan . Mechanistic, reaction kinetics and material characterization studies indicated that the catalytic results depend on a complex interplay of different factors . The recovered-reused solids performed steadily.The biomass-derived platform chemicals furfural and 5-(hydroxymethyl)furfural (HMF) may be converted to α-angelica lactone (AnL) and levulinic acid (LA). Presently, LA (synthesized from carbohydrates) has several multinational market players. Attractive biobased oxygenated fuel additives, solvents,  AMFs po hexoses ) or seco hexoses .On the other hand, HMF and FUR may be converted to α-angelica lactone (AnL) and levulinic acid (LA), and AnL may be an intermediate to LA . LA was e.g., butyl levulinate blends can improve fuel properties such as conductivity, combustion emission, freezing point  and γ-valerolactone (GVL) . LE and ng point . Furtherng point , and is ng point .2 (for hydrogenation) or organic H-donors such as secondary alcohols or acids ) (2 (mainly produced from fossil fuels) using (expensive) noble metal catalysts ) . High GVatalysts . Howeveratalysts  and, on ructures .i.e., not requiring external H2 supply and noble metals, specifically: hafnium supported on organic supports such as sulfonated sugar cane bagasse . Accord bagasse ; 2022b. Zeolites are (fully inorganic) crystalline microporous aluminosilicates used in the industry, and are particularly attractive for catalytic and adsorption processes . They po6Na3(H2O)21[Al9Si27O72] for Si/Al = 3, but higher ratios are possible) with LTL topology, which allows diffusion along 1D 12-membered ring (MR) channels that have ca. 0.71 nm × 0.71 nm apertures and cages with ca. 1.2 nm diameter  and conditions (leading to enhanced mesopore volume (Vmeso)), partly depending on the catalytic applications: alkane aromatization (Vmeso increased from 0.079 to 0.44 cm3 g−1) (meso increased from 0.01 to 0.29 cm3 g−1) (meso increased from 0.08 to 0.28 cm3 g−1) (d-xylose to xylitol (Vmeso increased from 0.010 to 0.14 cm3 g−1)  ; Knoevencm3 g−1) ; dealkylcm3 g−1) ; and hydcm3 g−1) . Al-Ani   and conditions  on the material properties and catalytic performances was studied. Mechanistic, reaction kinetics and material characterization studies indicated that the catalytic results depend on a complex interplay of different factors, and the relationships of material properties-catalytic performance may be different for different substrates.In this work, hierarchical multifunctional LTL zeotypes were prepared for integrated CTH and acid reactions of LA, AnL and HMF to useful bioproducts . The cat27Al MAS NMR spectroscopy (types of Al sites), attenuated total reflectance Fourier Transform Infrared (ATR FT-IR) spectroscopy (surface chemistry), elemental analysis for carbon , and FTIR of adsorbed pyridine (base probe) at 150 and 350°C (acid properties).All reagents and solvents were obtained from commercial sources and used as received  of hafnium acetylacetonate used as Hf precursor and finally calcination.Hafnium-containing hierarchical LTL zeotypes and a Zr-counterpart were prepared via post-synthesis partial dealumination using oxalic acid (oxac), desilication (D) using KOH treatments, followed by dealumination using H−1) in static air for 5 h, giving KL(oxac). Literature studies reported alkaline treatments of LTL type materials, using NaOH or KOH in the concentrations range 0.04–2.8 M  at 80°C for 3 h, with stirring. The solid was separated by filtration, thoroughly washed with hot Milli-Q water until neutral pH, dried overnight at 100°C, and finally calcined at 550°C  for 30 min at room temperature, with stirring. The solid was separated by filtration, thoroughly washed with hot Milli-Q water until neutral pH, and dried overnight at 100°C, giving D-HL(x), where x stands for the concentration of H2SO4 (0.10 ≤ x < 0.28 M).D-KL was simultaneously ion-exchanged and dealuminated using H for 1 h ; these c for 1 h . Hence, 2SO4 and then SSI of 0.28 mmolHf g−1 (the two treatments were carried out in a comparable fashion to that described above for the D-HL(x) materials). In a separate experiment, commercial KL was subjected to ion-exchange using 0.15 M aq. NH4NO3; the resultant material exhibited additional PXRD peaks, suggesting the presence of crystalline contaminations  where y is the hafnium concentration (in the range 0.28–1.68 mmolHf g−1). The SSI process involved gentle grinding of 1 g of the aluminosilicate support with the desired amount of Hf(acac)4 precursor for 30 min, using an agate pestle and mortar. The resultant material was calcined at 550°C (1°C min−1) under air flow (20 ml min−1) for 6 h. Zeolite 0.28Hf-HL was prepared in a similar fashion, albeit using the support HL and y = 0.28 mmolHf g−1. The Zr-counterpart, namely 0.28Zr-D-HL(0.15), was prepared by the same protocol, albeit using Zr(acac)4 as precursor (0.158 g Zr(acac)4 per g of D-HL(0.15)).The D-HL(x) precursors were subjected to SSI with Hf(acac)ca. 8 cm length, ca. 11 mm internal diameter) with a conic-shaped bottom, equipped with a PTFE-coated magnetic stirring bar (Supelco) and a PTFE valve (Normax) for purging. Each reactor was loaded with LA (in a concentration ranging from 0.14 to 0.45 M), catalyst (25.5 gcat L−1) and 2BuOH. The reactions of AnL and HMF were carried out using an initial substrate concentration of 0.45 M in 2BuOH. Bulk HfO2 was used in an equivalent molar amount of hafnium to that added together with the catalysts 1.40Hf-D-HL(x) to the reactor. Prior to control tests using D-HL(x), these materials were calcined under similar conditions to those described above for the Hf-containing zeotypes.The catalytic reactions were carried out using homemade tubular glass batch reactors  for quantification of the reaction products and remaining substrates.2SO4 at a flow rate of 0.8 ml min−1. The GC analyses were carried out using an Agilent 7820A GC equipped with a capillary column , spit injector (240°C) and a flame ionization detector (320°C), using H2 as carrier gas. The quantification of the substrates and reaction products was based on calibration curves using internal standards. Individual experiments were performed for a given reaction time, and the presented results are the mean values of at least two replicates with an error <6%.The HPLC analyses were carried out using a Knauer Smartline HPLC Pump 100 and a Shodex SH1011H + 300 mm × 8 mm (i.d.) ion exchange column , coupled to a Knauer Smartline 2520 UV detector (254 nm). The column temperature was set at 50°C, and the mobile phase was 0.005 M aq. Hsec-butoxymethyl)furfural (BMF) and 2,5-bis(sec-butoxymethyl)furan (BBMF) from HMF.The reaction products were identified using a Shimadzu QP2010 ultra-GC-MS  equipped with a split injector (240°C), ion source (200°C), interface (300°C), a Zebron ZB-5ms capillary GC column  and He as carrier gas (supporting databases: Wiley229 and NIST14). The identified products were 2-butyl levulinate (2BL) and GVL from LA; LA, 2BL and GVL from AnL; 5-methylfurfural (5MF), 5-—(molar concentration of Sub at reaction time t)/], and product (Prod) yield was calculated using formula 100 × [(molar concentration of Prod at time t)/]. Catalytic activities (mmol gcat h−1) were calculated based on the substrate conversion at 5 h reaction.The conversion (%) of the substrate (Sub) at a reaction time ca. 5 min), thoroughly washed with the reaction solvent (2BuOH) and dried at 85°C overnight. The used catalysts were thermally regenerated at 550°C (heating rate of 1°C min−1) for 5 h, under an air flow (20 ml min−1). Selected catalysts (1.40Hf-D-HL(0.15), 1.12Hf-D-HL(0.28)) were used for five consecutive 5 h-batch runs .The used catalysts were separated by centrifugation  consisted of contacting the fresh catalysts 1.40Hf-D-HL(0.15) and 1.12Hf-D-HL(0.28) with 2BuOH at 200°C for 5 h, under the same conditions as those used for a normal catalytic test, but without substrate; then, the solid was separated by centrifugation  and the supernatant liquid phase was passed through a 220 μm pore size PTFE membrane; the substrate (LA) was added to this solution  and left to react for 5 h at 200°C (giving LP-CT); finally, the homogeneous mixture was analyzed by GC and the results were compared to those without catalyst.2SO4 (giving D-HL(x), Si/Al in the range 5–41); solid-state impregnation (SSI) of hafnium and calcination. The influence of the acid concentration (x in the range 0.10–0.28 M H2SO4) and Hf loading (y in the range 0.28–1.68 mmolHf g−1) on the material properties was studied. The characterization studies of the zirconium counterpart 0.28Zr-D-HL(0.15) are discussed in the Supplementary Material ) were prepared from commercial KL (Si/Al = 3) via top-down strategies for the target reactions of LA, AnL and HMF. The strategies consisted of mild dealumination (giving KL(oxac), Si/Al = 5); desilication (D) to introduce mesoporosity ; dealumination using HMaterial ; the res2SO4, the latter possessed a higher Si/Al ratio, suggesting that the pretreatments facilitated dealumination. The SSI process did not significantly affect the Si/Al ratio, i.e., each pair of materials D-HL(x) and yHf-D-HL(x) with the same x possessed similar Si/Al ratio. Since no wash/extraction/filtration operations were performed after SSI, the amount of hafnium introduced in the SSI step remained in the solids, giving yHf-D-HL(x) with y in the range 0.28–1.68 mmolHf g−1 , increasing x led to increasing Si/Al ratio in the range 5-41, suggesting that partial removal of aluminum occurred (likely forming vacant sites) . AlthougolHf g−1 .Hf g−1 and of yHf-D-HL(0.28) with y < 1.40 mmolHf g−1 did not evidence the presence of other crystalline phases; peaks characteristic of hafnia monoclinic phase include ca. 28.6°, 31.8° and 34.7° (ICDD PDF-4+ 2020 reference code no. 04-005–4477) , the average crystallite size of the supported hafnium oxide particles  was very small, ca. 8–9 nm.The PXRD patterns of the materials KL, KL(oxac), HL, D-KL, D-HL(x) and yHf-D-HL(x) with x ≤ 0.15 indicated that the LTL topology was preserved , whereas05–4477) . These re.g., 73 and 71% RC for D-HL(0.15) and 0.28Hf-D-HL(0.15), respectively; 22 and 20% RC for D-HL(0.28) and 0.28Hf-D-HL (0.28), respectively. For the yHf-D-HL(0.15) materials, increasing y led to decreasing RC  . Increasasing RC , which m .The SEM images showed that, in general, the materials consisted of aggregates of small pseudo-spherical particles with sizes of up to 2 uptake as the relative pressure (p/p0) approached unity may be attributed to N2 sorption on the external surface of the relatively small crystallites. The desilicated materials D-KL and D-HL(x) exhibited type IV isotherms with an inflection point at p/p0 > 0.9  exhibited type I nitrogen sorption isotherms , charactinset of , which iinset of . Whereasy Vmicro . Consisty Vmicro .micro and Vmicro without affecting considerably SEM (130–148 m2 g−1) . Based on these results and PXRD, harsh dealumination seems to cause partial destruction and/or blockage of the micropores .For D-HL(x), increasing x up to 0.20 essentially led to decreasing S m2 g−1) . Materiaaterials . For x =micro and Vmicro, suggesting that Hf sites are at least partly located on the internal micropore surface (EM (117 and 74 m2 g−1 for D-HL(0.28) and 1.12Hf-D-HL(0.28), respectively), suggesting that Hf sites may be located on the mesopore surface.The introduction of Hf in D-HL(x) with x ≤ 0.20, led to a decrease in S surface . For x =ca. 0.55 and at 0.75 nm, assignable to the 8-MR (3D) and 12-MR (1D) channels. Especially the peak at ca. 0.55 nm was significantly attenuated for D-HL(x) with x ≤ 0.20 and negligible for D-HL(0.28)  . This waHL(0.28) .27Al MAS NMR spectra of KL, HL, Hf-HL, KL(oxac), D-KL, D-HL(x) and yHf-D-HL(x) with x ≤ 0.15 showed a prominent peak centered at ca. 63 ppm, assigned to Al sites in tetrahedral coordination (Altetra) . The materials D-HL(x) exhibited a weak resonance at ca. 0 ppm due to hexacoordinated  Al species (Alocta), which was not verified for D-KL, suggesting that Alocta was formed during the acid treatment. A comparison of the materials before and after the introduction of hafnium indicated no significant differences in the Altetra spectral region, but the Alocta peak disappeared. Hence, it seems that Alocta species were partially linked to the framework of D-HL(x) and converted to Altetra during SSI/calcination. According to the literature for zeolites, Alocta and Altetra sites may interconvert 4-x(OH)x) and non-hydroxylated (Al(OSi)4) aluminum sites.The Altetra) . Increasrconvert ; e.g., hrconvert . Possiblca. 1,620 cm−1 and 1,454 cm−1 (L); ca. 1,635 cm−1 and 1,540 cm−1 (B); and 1,490 cm−1 (L + B) , 1,540 cm−1 (B) and 1,635 cm−1 (B) decreased after acid treatment and were hardly distinguishable in the spectra of the materials with x = 0.28. The introduction of hafnium led to the appearance of new bands at ca. 1,608 and 1,448 cm−1 which became stronger with increasing y up to ca. 0.84 mmolHf g−1 . Hence, these two new bands may be attributed to Hf-containing acid sites. The appearance of these new bands after introducing Hf is in agreement with that reported by Tang et al. for Hf-USY  acidity  and Brønsted (B) acidity (pyridinium ions):  (L + B)  (Zhu et  (L + B) . A band r Hf-USY . For the−1, and B increased from 45 to 71 μmol g−1, respectively  led to decreasing amount of L and B acid sites . On the ectively ). These ectively .Hf g−1  and yHf-D-HL(0.28), a maximum amount of L acid sites was reached as a function of y; specifically, at y = 0.56 and 0.84 for materials with x = 0.15 and 0.28, respectively . For theHf g−1 .350/L150 up to ca. 0.33) and did not possess strong B acid sites (B350/B150 ≅ 0), i.e., Si/Al varied and Hf load was similar), the L acid strength increased with increasing x above 0.15 M H2SO4 (L350/L150 was similar for x = 0.10 and 0.15). On the other hand, the influence of y on the L acid strength was not straightforward: for yHf-D-HL(0.15), the L acid strength decreased with increasing y, whereas for yHf-D-HL(0.28) no straightforward relationship could be established. The acid properties may depend on several factors. The PXRD data indicated that condensed hafnium oxide species may be formed at higher y, which may impact on the amount and strength of accessible acid sites. The materials yHf-D-HL(x) may possess different metal  acid sites, chemical structures and coordination spheres, bond angles/lengths and binding energies , etc. For example, according to the literature, framework Lewis acid Hf sites may be hydrolyzed or non-hydrolyzed species, and the former may be stronger than the latter  materials possessed moderate to strong L acid sites . On the other hand, desilication and partial dealumination of the framework may form vacant sites for introducing hafnium via SSI, furnishing the zeolite with CTH activity. In this fashion, multifunctional LTL type catalysts may be produced, capable of promoting several paths that lead to target bioproducts.As mentioned in the Introduction, HMF, AnL and LA may be formed in carbohydrate conversion processes, and converted to GVL under acid and reduction conditions, and thus it is interesting to study their reactivities, envisaging future integrated reaction systems over multifunctional catalysts. The acid sites of the aluminosilicate LTL framework may promote the acid reactions (2SO4) on the LA reaction, keeping constant the amount of impregnated hafnium (y = 0.28 mmolHf g−1). Subsequently, the influence of the Hf loading (y in the range 0.28–1.68 mmolHf g−1) was studied for two sets of materials, namely  yHf-D-HL(0.15) and  yHf-D-HL(0.28). Kinetic, mechanistic, catalyst stability and finally substrate scope  were studied for selected catalysts.To study the influence of the post-synthesis conditions, the catalytic studies consisted of firstly studying the influence of the acid treatment  of D-HL(x)  materials gave GVL and 2BL in a high total yield (>90%) with molar ratios GVL/2BL in the range 0.2–3.5, at 200°C, 24 h. Without a catalyst, the reaction was much slower and GVL was not formed, under similar conditions  .2SO4), and the respective Hf-containing materials 0.28Hf-HL and 0.28Hf-D-HL(0.15), indicated that the aluminosilicates effectively converted LA to 2BL, but failed to give GVL , suggesting that the type of Hf sites may be determinant for GVL formation.Literature studies suggest that the conversion of LA to GVL in alcohol medium may be favoured by L and B acidity , such asgive GVL . The forThe zirconium catalyst 0.28Zr-D-HL(0.15) was less effective than its counterpart 0.28Hf-D-HL(0.15); the former led to less than half the GVL yield reached in the presence of 0.28Hf-D-HL(0.15)  . Hence, Hf g−1) and were subjected to similar acid treatment using x = 0.15 M H2SO4) indicated that the desilicated catalyst 0.28Hf-D-HL(0.15) was more active than 0.28Hf-HL , led to higher 2BL + GVL total yield  and higher GVL/2BL molar ratio  (A comparative study for (non-desilicated) 0.28Hf-HL and (desilicated) 0.28Hf-D-HL(0.15) (which possessed the same Hf loading (y = 0.28 mmolctively) , suggestHf g−1), i.e., comparing the 0.28Hf-D-HL(x) catalysts  increased with decreasing x in the order 1.55 (x = 0.28) < 1.68 (x = 0.20) < 1.74 (x = 0.15) < 1.91 (x = 0.10), which correlated with the increasing amounts of B and L acid sites  on the catalytic performance was studied keeping constant the Hf loading  decreased with increasing x, GVL yield and GVL/2BL ratios at 5 and 24 h increased . This maFurther studies were carried out to check the influence of the Hf loading (y), specifically for the two sets of materials yHf-D-HL(x) with x = 0.15 and 0.28. yHf-D-HL(0.15) led mainly to 2BL (51–71% yield), and GVL was formed in 22–38% yield at 200°C/24 h , whereasR2 = 0.9835)  > 0.33 (0.28Hf-D-HL(0.10)) > 0.32 (0.28Hf-D-HL(0.15)) > 0.17 (0.28Hf-D-HL(0.20)) > 0.15 (0.28Hf-D-HL(0.28)), and Smicro/SEM followed a similar order, 2.21 (0.28Hf-HL) > 1.37 (0.28Hf-D-HL(0.10)) > 0.94 (0.28Hf-D-HL(0.15)) > 0.72 (0.28Hf-D-HL(0.20)) > 0.07 (0.28Hf-D-HL(0.28)). Hence, for these materials with y = 0.28 mmolHf g−1, lower Smicro/SEM may be a main factor in favour of GVL formation. For the remaining materials with y > 0.28 mmolHf g−1, the L acid strength dependency on y (and thus on GVL yields) was not straightforward; higher y may lead to relevant differences in surface chemistry, impacting on catalytic performances.According to the literature, stronger L acidity may enhance CTH activity . However 0.9835) . These ricro/SEM ; specifii.e., as solvent and as reducing, etherification and esterification agent. The acid-catalyzed esterification of LA gives alkyl levulinates (LE), and the CTH of LA and LE may give 4-hydroxypentanoic acid (HPA) and alkyl 4-hydroxypentanoates (HPE), respectively, i.e., the carbonyl group in position C4 is reduced to an alcohol group. Literature studies reported that HPA/HPE may undergo etherification at the alcohol group in position C4, giving 4-alkoxypentanoic acid (APA) and alkyl 4-alkoxypentanoates (APE), respectively  plays multiple roles, ectively . Althougectively . Accordiectively .R2 ≥ 0.995) of ln ([LA]0/[LA]) on reaction time (considering the integrated rate law: ln ([LA]0/[LA]) = k t, where k is the kinetic constant and t is reaction time)  1.40Hf-D-HL(0.15) and  1.12Hf-D-HL(0.28), at 200°C . For theon time) . These rin 2BuOH .ca. 98% conversion , while 2BL concentration remained approximately constant (9–10% 2BL yield between 1 and 5 h)  using lower [LA]and 5 h) . Hence, micro/SEM than 1.40Hf-D-HL(0.15) , which may facilitate reaction pathways involving bulkier intermediates and transition states, e.g., LE/HPE/APE are bulkier than LA/HPA/APA, respectively. Luo et al. reported for methyl levulinate conversion to GVL over Hf-Beta, in 2BuOH, a dual-binding mechanism where the alkyl levulinate and the alcohol H-donor interacted (involving a hydride shift) with a single metal site forming a voluminous six-membered transition state  seems to effectively promote GVL formation via intermediate formation of 2BL. This catalyst possessed lower Son state . AccordiHf g−1 to 0.28Hf-D-HL(0.28), but higher L + B = 130 μmol g−1, L/B = 12 and mesopore specific surface area of ca. 660 m2 g−1  led to 29% GVL yield at 200°C/24 h  to hafnium silicates possessing high mesopore surface area, such as the mesoporous hafnium silicate Hf-TUD-1(50), previously described by our group . Hf-TUD-0 m2 g−1 ; for 0.20°C/24 h , entry 6To the best of our knowledge, this is the first study for LTL zeotype catalysts for LA conversion to GVL. ca. 1,460 cm−1 assignable to C-H vibrations of adsorbed organic matter   exhibited similar (white) colour, and the material balances closed in at least 91 mol%, considering GVL and 2BL as the useful bioproducts. ATR FT-IR spectroscopy indicated the presence of carbonaceous matter in the solids (exemplified for 0.28Hf-D-HL(0.15) in c matter , ca. 1,3e groups , ca. 1,7l groups , and ca.e groups . The thee groups .Elemental analysis of the washed-dried 0.28Hf-D-HL(x) catalysts (after a 5 h batch run) indicated that the carbon content (wt% C) increased with decreasing x, in the order 1.2 wt% (x = 0.28) < 1.7 wt% (x = 0.20) < 2.8 wt% (x = 0.15) < 3.7 w% (x = 0.10). While enhanced adsorption of carbonaceous matter did not seem to negatively affect LA conversion , it led to decreasing GVL yields and GVL/2BL ratios.The thermally regenerated solids 1.40Hf-D-HL(0.15) and 1.12Hf-D-HL(0.28) were reused and performed somewhat steadily for five consecutive 5 h-batch runs at 200 °C . For thee.g., hydration, CTH and lactonization) (The reaction of AnL to GVL involves acid and reduction chemistry (ization) , 2022b. ization) , but AnLization) , 2022a.ca. 100% conversion, 200 °C/24 h), but no GVL was formed (The reaction of AnL in the presence of HL and D-HL(0.15) gave LA and 2BL in high total yields , indicates a favourable effect of desilication on GVL yield; 9 and 29% for the non-desilicated and the desilicated materials, respectively, at 91–93% AnL conversion, 200°C/24 h.The introduction of hafnium was required to confer CTH activity for GVL formation . A compamicro/SEM (increasing x)  .i.e., 54% GVL yield and GVL/2BL = 1.26, at 100% AnL conversion, 200 °C/24 h.To check the influence of the Hf loading on the AnL reaction, comparative studies were carried out for yHf-D-HL(0.15) with y = 0.28 and 1.40, and, on the other hand, for yHf-D-HL(0.28) with y = 0.28 and 1.12 . For eacsec-butoxymethyl)furfural (BMF) were formed in total yields of 91 and 85%, at 95 and 90% HMF conversion, respectively, 200°C/24 h. The main product was BMF (73–75% yield) which may be formed via etherification of HMF. HMF to BMF may be enhanced by B acid sites  in 2BuOH at 180°C/6 h  were further explored for the reaction of HMF . The aluid sites . BMF may80°C/6 h . Elsayedd carbon . Hence, e.g., 35 and 54% yield for (non-desilicated) 0.28Hf-HL and (desilicated) 0.28Hf-D-HL(0.15), respectively). 2BL was formed in very low amounts, and thus the subsequent conversion of 2BL to GVL did not take place to a measurable extent (sec-butoxymethyl)furan (BBMF) was formed in up to 21% yield at 200°C/24 h, only in the presence of the Hf-containing catalysts. This path may involve integrated acid (etherification) and CTH (carbonyl group reduction) reactions.The introduction of hafnium in the materials led mainly to BMF  materials led to decreasing HMF conversion and increasing BBMF yield . Althougormation . Steric Hf g−1) and yHf-D-HL(0.28)   of Hf-zeolites for CTH reaction systems may depend on the type of substrate (The effect of the Hf loading was studied for yHf-D-HL(0.15)  . A higheubstrate . HMF is Hierarchical multifunctional LTL zeotypes were prepared for the chemical valorization of HMF, LA and AnL via integrated catalytic transfer hydrogenation (CTH) and acid reactions in 2-butanol (2BuOH) at 200°C. This is the first CTH application reported for LTL related materials.2SO4) and solid-state impregnation (SSI) of different amounts of hafnium (y), giving yHf-D-HL(x). The influence of the Hf loading (y) was studied for two groups of materials yHf-D-HL(x) with x = 0.15  and x = 0.28 .The catalysts were prepared via top-down strategies involving desilication, dealumination  may affect, to different extents, the textural properties, distributions and types (and strength) of acid sites, etc. Mechanistic and kinetic studies suggested that Al sites may promote esterification and etherification reactions , whereas Hf sites were required for CTH .Molecular level spectroscopic studies indicated: broader distributions of AlHf g−1 (lower Hf loading), GVL yields increased with decreasing Lewis acid strength and Smicro/SEM ratio. The decreasing L acid strength may avoid strongly adsorbed carbonaceous matter on the catalysts, and, on the other hand, lower Smicro/SEM may avoid steric hindrance and facilitate cyclization of intermediates , enhancing GVL formation. For materials with y > 0.28 mmolHf g−1, GVL formation may depend on an interplay of several factors, e.g., L acid strength, distribution and type of acid sites.For materials with y = 0.28 mmolAlthough 0.28Hf-D-HL(0.28) was mostly amorphous and mesoporous, it performed far superiorly to ordered mesoporous hafnium silicate Hf-TUD-1 possessing similar Hf loading. The top-down strategy and, on the other hand, having an LTL zeolite in its genesis, may result in unique surface properties of yHf-D-HL(0.28) type materials. The 1.12Hf-D-HL(0.28) catalyst led to 77% GVL yield at 200°C/24 h , and 91% GVL yield at 200°C/5 h .HMF was a more demanding substrate than LA and AnL for the formation of GVL. The main product was BMF, and GVL was not formed in measurable amounts at 200°C/24 h. HMF conversion to BMF and 2BL did not require Hf sites (HL was effective), whereas the diether BBMF was solely formed in the presence of Hf-containing catalysts (21% yield for 0.28Hf-D-HL(0.28)).Based on catalytic and characterization studies, the materials were relatively stable. The zirconium catalyst 0.28Zr-D-HL(0.15) possessed acid and CTH activity, but performed inferiorly to the analogue 0.28Hf-D-HL(0.15).Overall, post-synthesis modifications of commercial LTL zeolites may broaden their catalytic application profiles. The top-down strategies and conditions may be optimized to tune material properties and meet superior catalytic performances. A challenge may be to further enhance mesoporosity without considerable reduction of crystallinity.


Bacillus licheniformis-fermented products (SBLF) alleviates dextran sulfate sodium-induced colitis in mice by reducing inflammatory responses and the NLRP3 inflammasome activation in colon tissue.Quality of life is significantly impaired among individuals with inflammatory bowel disease (IBD), and the growth performance of animals is also affected by IBD. However, the side effects and poor therapeutic effects of medications for IBD limit the treatment effect. In this study, we demonstrated that oral administration of surfactin containing Bacillus licheniformis-fermented products (SBLF) and commercial surfactin (CS) on the treatment of dextran sulfate sodium (DSS)-induced colitis in a mouse model. We found that mice that received drinking water containing 3% DSS developed significant colitis symptoms, including increased disease activity index, body weight loss, shortening of the colon length, splenomegaly, colonic inflammation and colonic NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome activation. Notably, orally received SBLF, CS or clinical anti-inflammatory drug 5-aminosalicylic acid improved DSS-induced colitis symptoms in mice. These findings show that SBLF can improve IBD in mice by reducing colonic inflammation and inhibiting the NLRP3 inflammasome activation, suggesting that SBLF has the potential to be used as a nutraceutical in humans or a feed additive in economic and companion animals for preventing IBD.Inflammatory bowel disease (IBD) is a non-infectious disease characterized by chronic inflammation of the gastrointestinal tract. Currently, management of IBD is still a clinical challenge. The purpose of this study was to investigate the therapeutic potential of surfactin containing  The gut is the largest immune organ in the body and most immune cells can be found in the gut. The gut uses mucus and epithelial cells as a physical barrier to keep approximately 100 trillion microorganisms in the gastrointestinal tract . The twoThe treatment of IBD mainly relies on anti-inflammatory drugs , immunosuppressants and antibiotics . These tBacillus licheniformis is a facultative anaerobic Gram-positive bacterium that exists in soil or plants and forms endospores for its survival in difficult environments. Surfactin is a powerful lipopeptide biosurfactant that can be produced by B. licheniformis [B. licheniformis and demonstrated that the B. licheniformis-fermented products contain 5 × 109 CFU/g B. licheniformis spore and 14.3 mg/g surfactin, respectively [B. licheniformis-ferments (SBLF) has been shown to exert antiviral activity against the porcine epidemic diarrhea virus [niformis . We haveectively . The surBacillus subtilis was obtained from Sigma-Aldrich . DSS  was obtained from MP Biomedicals . ELISA kits for IL-1β, IL-6, tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MPC-1) were obtained from Thermo Fisher Scientific . NLRP3 antibody was obtained from Adipogen Life Science . IL-1β antibody was obtained from R&D Systems . Antibodies against Actin, ASC, cyclooxygenase-2 (COX-2) and 5-ASA were obtained from Santa Cruz Biotechnology . A myeloperoxidase (MPO) activity assay kit was obtained from Abcam .Commercial surfactin (CS) from B. licheniformis (ATCC 12713) was purchased from the Food Industry Research and Development Institute . The method used for SBLF preparation was described in our previous study [B. licheniformis were inoculated in sterile wheat bran and soybean meal-based substrates and fermented for 6 days at 30 °C in an anaerobic chamber with relative humidity above 80%. After fermentation, the solid-state fermented products were dried by incubation at 50 °C for 2 days. The dried fermented products were ground into a fine powder using a grinder to obtain SBLF. ATCC 127 was purc2O + vehicle group, received H2O with oral gavage of vehicle (0.5% methanol) daily, n = 3; DSS + vehicle group, received 3% (wt/vol) DSS in H2O with oral gavage of vehicle daily, n = 6; DSS + CS group, received 3% DSS in H2O with oral gavage of 20 mg/kg CS daily, n = 6; DSS + 20 mg/kg SBLF group, received 3% DSS in H2O with oral gavage of 20 mg/kg SBLF daily, n = 6; DSS + 5-ASA group, received 3% DSS in H2O with oral gavage of 40 mg/kg 5-ASA daily, n = 6; H2O + CS group, received H2O with oral gavage of 20 mg/kg CS daily, n = 3; H2O + SBLF group, received H2O with oral gavage of 20 mg/kg SBLF daily, n = 3. For DSS treatment, mice received 3% DSS in H2O and normal diet ad libitum for 5 days, followed by H2O and starvation for 1 day. CS, SBLF or 5-ASA were given daily by oral gavage during the DSS treatment. The body weight and disease activity index (DAI) of each mouse were recorded daily. On Day 7, following induction with DSS, mice were sacrificed for the collection of colonic tissue and spleen.C57BL/6JNal male mice (six-weeks old) were obtained from the National Laboratory Animal Center , and housed in the National Ilan University Animal Center at room temperature (23 ± 3 °C) and relative humidity (40–60%). The research design followed previous studies with minor modifications ,16. The The DAI score is a research tool used to quantify the symptoms of mice with IBD. The DAI score was calculated by combining the stool score and bleeding score. The stool scores included: 0 = well-formed stools; 1 = semi-formed stools that did not adhere to the anus; 2 = semi-formed stools that adhered to the anus. The bleeding scores included: 0 = no bleeding; 1 = slight bleeding; 2 = gross bleeding.The colonic tissues from mice were homogenized with lysis buffer followed by centrifugation at 13,000 rpm at 4 °C for 20 min. The protein concentration was determined by the BCA protein assay. The expression levels of IL-1β, IL-6, TNF-α, and MPC-1 in the colonic tissues were analyzed by ELISA. The expression levels of IL-1β in serum were analyzed by ELISA. The activity of MPO in colonic tissues was analyzed by the colorimetric activity assay kit.Colonic tissues were fixed in 10% formalin and embedded in paraffin. Sections were stained with H&E by Energenesis Biomedical Co., Ltd. . Histopathological scores were calculated by a pathologist by combining the inflammatory cell infiltration score and tissue damage score as described previously . The infp < 0.05, p < 0.01. and p < 0.001, respectively.The two-tailed t test was used to analyze two groups. For the analysis of three or more groups, ANOVA with Dunnett’s multiple comparisons test was used. The standard deviation of separate experiments is indicated by error bars. *, ** and *** represent p < 0.01), 1.67 ± 0.52 (p < 0.05) and 2.50 ± 0.84 (p < 0.05) on day 4, day 5 and day 6 post-DSS administration, respectively (p < 0.05), 1.83 ± 0.75 (p < 0.05) and 2.67 ± 0.52 (p < 0.05) on day 4, day 5 and day 6 post-DSS administration, respectively (p < 0.05), 1.50 ± 0.55 (p < 0.05) and 2.33 ± 1.03 (p < 0.05) on day 4, day 5 and day 6 post-DSS administration, respectively , 91.95 ± 3.01% (p < 0.001 compared to DSS) or 92.73 ± 3.63% (p < 0.001 compared to DSS) on day 7 compared to day 1 , 6.95 ± 0.19 cm (p < 0.001 compared to DSS) and 6.77 ± 0.22 cm (p < 0.001 compared to DSS), respectively , 0.086 ± 0.005 g (p < 0.001 compared to DSS) and 0.085 ± 0.004 g (p < 0.001 compared to DSS), respectively (p < 0.001 compared to DSS), CS  and 5-ASA  (p < 0.001 compared to DSS), 339.8 ± 91.3 pg/mL (p < 0.001 compared to DSS) and 226.3 ± 84.0 pg/mL (p < 0.001 compared to DSS) by SBLF, CS and 5-ASA, respectively (p < 0.01 compared to DSS), CS  and 5-ASA  (p < 0.001 compared to DSS), CS  and 5-ASA   C. DSS inectively D. The le to DSS) E. In add to DSS) F. These p < 0.001 compared to DSS) and 37.65 ± 13.09 pg/mL (p < 0.001 compared to DSS), respectively (p < 0.001 compared to DSS) and 34.93 ± 14.70 pg/mL (p < 0.001 compared to DSS), respectively (p < 0.001 compared to DSS) and 33.87 ± 21.53 pg/mL (p < 0.001 compared to DSS), respectively  is an infectious disease caused by ortality . Mycobac disease . Here, wB. licheniformis toxins has been reported, it has only happened in cases with inadequate food preparation conditions. In general, B. licheniformis is a safe bacterial strain and non-pathogenic to human and animals [In addition, although foodborne illness caused by  animals . The limWe demonstrated that SBLF is a natural substance that can alleviate DSS-induced colitis in mice. The beneficial effects of SBLF on colitis are mainly derived from its anti-inflammatory and anti-NLRP3 inflammasome effects. SBLF has the potential to be utilized as a nutraceutical or a feed additive for preventing IBD in humans or economic and companion animals.


Moreover, α-PGA/DOX NPs enhanced antitumor efficacy in vivo, the tumor inhibition rate was 67.4%, increasing 1.5 fold over DOX injection. α-PGA/DOX NPs also reduced the systemic toxicity and cardiotoxicity of DOX. In sum, α-PGA is a biosafe nanodrug delivery carrier with potential clinical application prospects.Poly(amino acids) have advanced characteristics, including unique secondary structure, enzyme degradability, good biocompatibility, and stimuli responsibility, and are suitable as drug delivery nanocarriers for tumor therapy. The isoform structure of poly(amino acids) plays an important role in their antitumor efficacy and should be researched in detail. In this study, two kinds of pH-sensitive isoforms, including α-poly(glutamic acid) (α-PGA) and γ-PGA, were selected and used as nanocarriers to prepare a nanodrug delivery system. According to the preparation results, α-PGA can be used as an ideal drug carrier. Selecting doxorubicin (DOX) as the model drug, an α-PGA/DOX nanoparticle (α-PGA/DOX NPs) with a particle size of 110.4 nm was prepared, and the drug-loading content was 66.2%. α-PGA/DOX NPs presented obvious sustained and pH-dependent release characteristics. The IC Cancer is a significant threat to humans, and the exploration of its treatment remains ongoing ,2,3. DruAs a component of the drug delivery system, nanocarriers play an important role ,12. As tPoly(glutamic acid) (PGA), as a typical poly(amino acid), has free carboxyl groups in its structure, which can provide a negative charge to interact with positively charged bioactive agents ,31. AccoAn effective method to enhance the DLC is to simplify the composition of the nanocarriers. In our previous study, hydrophilic polymers, such as polyethylene glycol and oligoethylene glycol derivatives, were utilized as nanocarriers to prepare drug-loaded NPs ,47. CompTo investigate the properties of PGA as nanocarrier, α-PGA and γ-PGA were utilized to construct DOX NPs via the antisolvent precipitation method in this work. After successful preparation, the physiochemical properties, morphology, and in vitro release characteristics of the PGA/DOX NPs (α-PGA/DOX NPs) were investigated. The in vitro antitumor efficacy against the 4T1 cell line and in vivo antitumor activity in 4T1 tumor-bearing mice were also estimated.α-PGA was synthesized according to a previous reference , γ-PGA w2.The mouse breast cancer (4T1) cell line was purchased from Peking Union Medical College Cell Center and cultured in RPMI-1640 medium containing 10% fetal bovine serum, 100 units of penicillin, and streptomycin under the condition of 37 °C and 5% COBALB/c female mice (20 ± 2 g) were purchased from Beijing HFK Bioscience Co., Ltd. . The mice were raised in an SPF-level laboratory animal room, kept on a 12 h light–dark cycle, and provided a standard diet of food and water ad libitum. All the experimental procedures complied with the Guidelines and Policies for Ethical and Regulatory for Animal Experiments and approved by the Animal Ethics Committee of Peking Union Medical College .The preparation method of drug-loaded PGA NPs was the reverse solvent precipitation and high-pressure homogenizer technique . Briefly−1 for 30 min and diluted with chromatographic methanol. The actual concentration of drug in the NPs was detected by HPLC  with the fluorescence/UV quantification method. The measurement was conducted according to previous reports [Briefly, 2 mL of NP solution was lyophilized and weighed precisely, then 1 mL chromatographic methanol was added. After dissolving completely, the methanol solution was centrifuged at 13,000 rpm min reports ,51. The −1 with deionized water, and the average hydrodynamic diameter (Dh), polydispersity index (PDI), and zeta potential were measured using a Malvern Zetasizer 3000 system . Measurements were conducted under a backscattering detection model (θ = 173°); a 4 mV He–Ne laser was utilized, and the wavelength was 633 nm. All samples were detected at 25 °C three times.These drug-loaded NPs were diluted to a concentration of 1 mg mL−1) was dropped on a clean silica gel sheet to dry, fixed with conductive adhesive, and then sprayed with gold for 6 min under negative pressure and a current of 30 mA, before the voltage was raised to 30 mV.The morphology of the α-PGA/DOX NPs was observed using a scanning electron microscope . The sample solution  and KBr powder (100 mg) were mixed to obtain KBr disks.FT-IR spectra of samples were analyzed with a spectrum 100 , spectra were recorded in the range of 400–4000 cmh and PDI. The storage stability of α-PGA/DOX NPs was measured at 4 °C. Dh and PDI of these NPs were recorded at 0, 2, 4, 6, 8, 10, 15, and 30 days. For the media stability, α-PGA/DOX NPs solution was mixed with 1.8% saline solution , 10% glucose solution , 2 × phosphate buffer saline , and plasma , then the mixture was incubated at 37 °C. After being incubated for 0, 2, 4, 6, and 8 h, the particle size and PDI were detected separately. All samples were measured in triplicate.The stability of α-PGA/DOX NPs was evaluated according to D−1. At the set time point, 1 mL of release medium was removed; meanwhile, the same volume of fresh media was added. The concentration of DOX was analyzed by HPLC, and each sample was repeated three times to calculate the cumulative percentage of drug release and draw the cumulative release curve.Cumulative drug release curves of NPs were plotted by the dialysis method. A total of 2 mL of α-PGA/DOX NP aqueous solution and DOX·HCl solution were transferred into dialysis bags; these dialysis bags were placed into 50 mL of PBS solution (pH = 7.4 and pH = 5.5) containing 0.5% sodium dodecyl sulfate at 37 °C, the concentration of DOX was 2 mg mL3 cells per well at 37 °C and 5% CO2. After a 24 h incubation, the DOX solution and α-PGA/DOX NPs were diluted to 0.01, 0.1, 0.5, 1, 2, 5, 10, 20, and 100 μg mL−1 with fresh RPMI-1640 and added into the well; the injected volume was 150 μL per well. The culture medium was replaced after incubation for 48 h before 10 µL CCK-8 solution was added to each well, then incubation was continued for 1.5 h. The optical density (OD) value was measured with a microplate reader at 450 nm wavelength to determine the cell viability. The half-maximal inhibitory concentration (IC50) was determined using the GraphPad Prism 5 software. The formula for calculating the cell inhibition rate is as follows:The 4T1 cells were cultured with different concentrations of α-PGA/DOX NPs, and the cytotoxicity of the NPs was investigated by the CCK-8 method according to the previous reports ,53. 4T1 7 mL−1 into the right axilla of BALB/c mice. When the tumor volume grew to about 150 mm3, the mice were randomly divided into four groups (n = 10): the negative control group, the positive control group, the blank control group, and the test group, which was treated with 5% glucose solution, DOX injection (3 mg Kg−1), α-PGA (1.5 mg Kg−1), and α-PGA/DOX NPs  via tail vein injection separately. The mice were treated every two days for seven consecutive times, their tumor volumes were measured, and their body weight was taken. After being sacrificed by cervical dislocation, the tumor tissue, heart, liver, and spleen were extracted and weighed, and the tumor inhibition rate (TIR), liver index, spleen index, and heart index in vivo were calculated.The 4T1 tumor-bearing mice model was established by subcutaneous injection of a 4T1 cell suspension (0.2 mL) with a concentration of 1 × 10After the blood collection, serum was prepared via centrifugation . LDH, CK, and CK-MB in the serum were measured using a biochemical autoanalyzer following the standard protocol for each Elisa plate.Hematoxylin and eosin (HE) staining was conducted according to previous paper . Tissuesp < 0.05 indicated statistical significance.All the experiments were conducted at least in triplicate (>3 independent experiments). The data are presented as the mean values ± standard deviation. Comparisons between groups were performed by one-way analysis of variance (ANOVA) , and Polymers possess a large number of isomers due to the chemical complexity, which should be studied in detail to illustrate structure–property relationships . For useh and PDI, α-PGA could be utilized as a nanocarrier to deliver IBU, OXC, DTX, and DOX in this experiment. Nanocarriers can encapsulate hydrophobic drugs via molecular interactions, including hydrophobic interaction, electrostatic interaction, hydrogen bond, and Van der Waals force. Therefore, these drugs could be entrapped to form nanoparticles successfully. Among these hydrophobic drug-loaded nanoparticles, PGA/DOX NPs showed the highest DLC and EE, which were 66.2 ± 4.3% and 72.7 ± 5.3%, correspondingly , and the PDI was decreased from 0.18 ± 0.02 to 0.12 ± 0.04 as storage time was prolonged (p > 0.05). These results indicate that α-PGA/DOX NPs showed good storage stability.The stability of α-PGA/DOX NPs including the storage stability and media stability was studied. The storage stability was assessed at 4 °C for 30 days, and the particle sizes of the α-PGA/DOX NPs were detected and are recorded in p > 0.05), while the particle size increased sharply after adding 0.9% normal saline and PBS buffer solution (>1000 nm). This suggested that α-PGA/DOX NPs presented good media stability in 5% glucose solution and mouse plasma, indicating that α-PGA/DOX NPs could be applied via intravenous administration with 5% glucose solution.The media stability of α-PGA/DOX NPs was investigated in 0.9% normal saline, 5% glucose solution, PBS buffer solution, and mouse plasma. The results are shown in p < 0.01) and 70.4 ± 5.5% within 144 h . Comparing with the neutral condition, DOX released fast in acidic condition. The reason for this pH-sensitive release was that α-PGA protonates in an acidic environment, and its affinity with DOX was weakened, resulting in a fast release. The above results indicated that α-PGA/DOX NPs have a pH-sensitive and sustained release profile, which means they could achieve a low release in blood circulation (pH 7.4) and fast release in tumor tissue (pH 5.5). This characteristic could be used for targeted drug delivery to the tumor microenvironment.The α-PGA/DOX NPs were constructed via electrostatic interaction, which may cause the release behavior to be affected by the pH value of the release medium. Hence, the release characteristics were analyzed in PBS solutions at pH 5.5 and pH 7.4 . There w50 value of free DOX and α-PGA/DOX NPs was 9.09 ± 0.92 and 1.06 ± 0.77 μg mL−1 separately, and the cytotoxicity of α-PGA/DOX NPs was enhanced approximately 8.5 fold after 48 h incubation. The antitumor efficacy of α-PGA/DOX NPs was promoted significantly in comparison with that of free DOX (p < 0.001). The mechanism leading to this phenomenon could be that NPs transported more DOX into tumor cells through intracellular endocytosis and pinocytosis, compared with the passive diffusion of free DOX drugs into cells, thus avoiding the multidrug resistance of the cells to the drugs. These results revealed that α-PGA/DOX NPs significantly increased the antitumor effect of DOX.To research the antitumor effects of α-PGA/DOX NPs, the proliferation-inhibition effects of free DOX and α-PGA/DOX NPs against 4T1 cells were investigated by the CCK-8 method, and the results are shown in 3) showed no significant difference compared with that in the glucose solution group , indicating no tumor inhibition efficacy forα-PGA . Moreover, α-PGA/DOX NPs exhibited higher antitumor efficacy than free DOX injection; the tumor volume change was decreased 1.4 fold (781 vs. 1104 mm3), which was a significant difference (# p < 0.05).To further evaluate the antitumor efficacy of α-PGA/DOX NPs in vivo, the tumor volume changes, tumor inhibition rate based on the tumor weight, and apoptotic situation of the tumor tissue were recorded. The tumor volume of the α-PGA group .Furthermore, the antitumor activity was evaluated via the tumor weight, and the TIR was calculated b. The avAfter sacrifice, the apoptotic situation of the tumor tissue was observed via the HE staining method c,d. The All these results indicated that α-PGA/DOX NPs showed good antitumor efficacy; this phenomenon could be explained by the properties of α-PGA/DOX NPs. α-PGA/DOX NPs could be passively targeted into tumor tissues through the EPR effect. After entering tumor cells via endocytosis, DOX was released suddenly from the NPs owing to its pH sensitivity.p < 0.001). The α-PGA/DOX NP group also showed a significant difference compared with the DOX injection group (## p < 0.01). These results proved that α-PGA/DOX NPs could decrease the side effects of DOX.DOX, as the commercial chemotherapeutic agent, showed significant systemic toxicity and cardiotoxicity; hence, it was necessary to study the relative toxicity of α-PGA/DOX NPs. To estimate the systemic toxicity, body weight changes and the liver/spleen index were recorded. The body weight of the four groups showed two different tendencies a. The boThen, the liver and spleen indexes were calculated to evaluate systemic toxicity furthermore, and the results are shown in # p < 0.05). These results indicated that DOX caused severe heart injury and that α-PGA/DOX NPs could decrease the cardiotoxicity of DOX. Then, the relative heart index was calculated, which was 0.55 ± 0.01%, 0.56 ± 0.02%, 0.44 ± 0.03%, and 0.60 ± 0.05% for the glucose solution group, PGA group, DOX injection group, and α-PGA/DOX NP group, correspondingly. Similar to the result for heart weight, the heart index of the PGA group and the α-PGA/DOX NP group was increased and there existed no significant difference (p > 0.05) compared with the glucose solution group, while the heart index of the DOX injection group was decreased and showed a significant difference (*** p < 0.001). The difference was also presented between the DOX injection group and the α-PGA/DOX NP group (### p < 0.001), proving that the α-PGA/DOX NP group could decrease the cardiotoxicity of DOX.To research the cardiotoxicity of α-PGA/DOX NPs, the heart weight and the heart index were measured a. The hep > 0.05), indicating nontoxicity of PGA. The concentration of all enzymatic markers was increased in the DOX injection group significantly (*** p < 0.001), revealing that free DOX caused heart injury. Although the LDH of the α-PGA/DOX NP group increased from 57.5 ± 9.5 to 99.5 ± 7.5 U mL−1 , the CK and CK-MB were maintained, and no difference was shown (p > 0.05). Besides, all these markers of the α-PGA/DOX NP group decreased significantly compared with the DOX injection group (### p < 0.001). All these results suggested that α-PGA/DOX NPs could decrease the heart toxicity of DOX obviously.Moreover, the concentration of enzymatic markers, including LDH, CK, and CK-MB in serum, was enhanced significantly after myocardial injury and was measured using an Elisa plate b. ComparThe heart tissue was observed via the HE staining method, and the images are shown in These results revealed that DOX injection induced severe heart injury due to energy metabolism change, induction of apoptosis, intracellular calcium dysregulation, and oxidative stress ,63, whilTo research the structural influence of PGA, two isoforms, α-PGA and γ-PGA, were utilized as nanocarriers to encapsulate several hydrophobic drugs. It was confirmed that α-PGA could be applied to construct a nanodrug delivery system due to its appropriate structure and relatively low steric hindrance. α-PGA as an anionic polypeptide, interacted with cationic DOX via electrostatic interactions to assemble stable α-PGA/DOX NPs with high DLC and small particle diameter. α-PGA/DOX NPs presented good storage stability, media stability, and a pH-sensitive release profile. The in vitro antitumor activity of α-PGA/DOX NPs against 4T1 cells was enhanced approximately 8.5 fold compared with DOX injection. Animal experiments indicated that α-PGA/DOX NPs promoted the antitumor activity of DOX and reduced its toxicity. Compared with DOX injection, the tumor inhibition rate of α-PGA/DOX NPs increased by approximately 1.5 fold; the systemic toxicity and cardiotoxicity of DOX were decreased significantly, no body weight loss or abnormal enzymatic markers were detected, and normal heart tissue was observed. The advantage of α-PGA/DOX NPs in this study include simple preparation, high DLC, relative small particle size, and good antitumor efficacy. In summary, as a hydrophilic anionic polypeptide, α-PGA demonstrated promising as a nanocarrier to construct nanodrug delivery systems with potential applications in the clinic.


Cannabis sativa (C. sativa), phytocannabinoids have undergone extensive research on their structures, biosynthesis, and biological activities. Besides the phytocannabinoids, C. sativa is also rich in terpenes, alkaloids, and flavonoids, although little research has been conducted in this area.Flavonoids are necessary for plant growth and resistance to adversity and stress. They are also an essential nutrient for human diet and health. Among the metabolites produced in C. sativa, from their physical characteristics to their expression patterns. We screened the potentially step-by-step enzymes catalyzing the precursor phenylalanine to the end flavonoids using a conjoin analysis of gene expression with metabolomics from different tissues and chemovars. Flavonol synthase (FLS), belonging to the 2-oxoglutarate-dependent dioxygenase (2-ODD) superfamily, catalyzes the dihydroflavonols to flavonols. In vitro recombinant protein activity analysis revealed that CsFLS2 and CsFLS3 had a dual function in converting naringenin (Nar) to dihydrokaempferol (DHK), as well as dihydroflavonols to flavonols with different substrate preferences. Meanwhile, we found that CsFLS2 produced apigenin (Api) in addition to DHK and kaempferol when Nar was used as the substrate, indicating that CsFLS2 has an evolutionary relationship with Cannabis flavone synthase I.In this study, we identified 11 classes of key enzyme-encoding genes, including 56 members involved in the flavonoid biosynthesis in C. sativa and highlighted the key CsFLS genes that generate flavonols and their diversified functions in C. sativa flavonoid production. This study paves the way for reconstructing the entire pathway for C. sativa’s flavonols and cannflavins production in heterologous systems or plant culture, and provides a theoretical foundation for discovering new cannabis-specific flavonoids.Our study identified key enzyme-encoding genes involved in the biosynthesis of flavonoids in The online version contains supplementary material available at 10.1186/s12934-022-01933-y. Flavonoids are widely distributed throughout the plant kingdom . They fuCannabis sativa L. is an annual herb of the Cannabinaceae family, the genera Cannabis [C. sativa has been historically cultivated and utilized for 6000 years for food, textiles, and medicine [C. sativa also produces various non-cannabinoids constituents, including lignanamides, alkaloids, spiroindans, dihydrophenanthrenes, and flavonoids [C. sativa are currently understudied [C. sativa [C. sativa  [Cannabis . C. satimedicine . Much atavonoids . Flavonorstudied . Over 20. sativa , belongigelovii) , 22 {Reagelovii) , 23.Scutellaria baicalensis [p-coumaroyl: CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI). Subsequently, Nar is flowed to the biosynthesis of either flavonols such K, Q, and myricetin (M) by flavanone 3-hydroxylase (F3H) and flavonol synthase (FLS) or flavones such as Api, luteolin, and cannflavin A, B, and C in C. sativa via the catalytic action of flavone synthase (FNS) and other cytochrome P450 enzymes and transferases.Many studies have reported the biosynthesis of the core flavonoid skeleton in medicinal plants . For exacalensis  responsicalensis . The patC. sativa. However, the key enzyme-encoding genes involved in flavonol biosynthesis, have not been systematically identified in C. sativa. FLS, a member of the 2-oxoglutarate and Fe(II)-dependent dioxygenases (2-ODD) superfamily, converts dihydroflavonols to flavonols [C. sativa genome assembly. We also identified potentially encoding candidate genes by combining their expression patterns with flavonoid content in different tissues and chemovars of C. sativa. We eventually focused on the CsFLS2 and CsFLS3 genes, and found that they retained the conservative function of FLS and exhibited additional enzymatic properties in vitro. These results systematically constitute step-by-step genes involved in the biosynthesis of flavonoids in C. sativa and entitle a potential function of CsFLS different from FLS in other higher plants.Two gene-encoding enzymes in the early phenylpropanoid biosynthetic pathway, CsPAL and Cs4CL , as welllavonols . In thisC. sativa, we screened and explored 56 enzyme genes involved in flavonoid biosynthesis, including seven CsPALs, two CsC4Hs, six Cs4CLs, seven CsCHSs, four CsCHIs, eight CsFNSs, three CsF3'Hs, three CsF3Hs, five CsFLSs, three CsOMTs, and eight CsPTs using the BLASTP search and SwissProt database. The physical characteristics, including the amino acid (aa) length, molecular protein weight, isoelectric point, and their predicted subcellular localization, were also investigated  were highly expressed in root, flower, and bract. The CsCHS, CsCHI, CsFNS, CsF3'H, CsOMT, CsPT, and CsFLS gene families were mostly expressed in flowers, bracts, and leaves, while they showed relatively low transcriptional levels in roots, seeds, and stems, which were consistent with the distribution of flavonoids, including Nar, K, Q, Api, and cannflavins A and B. The candidate genes, including CsPAL7, CsC4H1, Cs4CL4, CsCHS9, CsCHI1, CsOMT21, CsPT3, and CsFLS2, were used to perform qRT-PCR , where CsFLS2 and CsFLS3 were located at Chr3 and Chr5, respectively, while CsFLS4 and CsFLS5 were presented on Chr7 in tandem duplication   and dihydroquercetin (K214N and F146A). We also established a model for CsFLS2  strain as N/C-terminal proteins fusion with two His-6 tags. Purified proteins were verified using Western blotting at approximately 45 kDa, which was consistent with the predicted molecular weights of 43.86 kDa for CsFLS2 and 45.65 kDa for CsFLS3  of the 2-ODD superfamily in 25 different plants with CsFLS2 and CsFLS3 [Both CsFLS2 and CsFLS3 converted dihydroflavonols (DHQ and DHK) to flavonols (Q and K), respectively  of DHK and DHQ for CsFLS2 was 16.28 µM and 5.49 µM, respectively, indicating that CsFLS2 had a higher affinity for DHQ than DHK  and Cs4CL (KC970301) in C. sativa, by searching expressed sequence tags against homologous sequences from other plants, which were named CsPAL4 and Cs4CL1 in this study. Nonetheless, both genes showed negative correlations with the content of detected flavonoids  and the Fe2+ binding site (His-Asp-His) [CsFLS1–3, which had both active sites, CsFLS4–5 retained the binding sites of 2-OG but lost those of Fe2+  rather than Gln (Q) at the same Gln 295 site in A. thaliana, explaining why CsFLS2 has less catalytic efficiency for DHQ than CsFLS3 , which belongs to the superfamily of 2-ODDs, lead to different expression pattern and subsequent subfunctionalization, where SlDMR6-1 exerted roles in pathogen infection, while SlDMR6-2 balanced salicylic acid levels in flowers and fruits [CsFLS promoters showed that potentially regulatory mechanism differed between them. The promoter of CsFLS2 had defense and stress responsiveness elements, while CsFLS3 possessed specific elements response for low-temperature, salicylic acid and Methyl jasmonate  . Unlike la rubra , G. bilo. biloba , and C. sinensis . CsFLS2 LS3 Fig. : Fig. S3d fruits . In hempC. sativa, suggesting the involvement of CsFLSs. Coincidentally, FLS and other flavonoid-related structural genes responded positively to different environmental variations [C. sativa plants and the biosynthesis of a substantial amount of bioactive flavonoids for downstream usage. Additionally, it was reported that flavonoids, synergistic with other non-phytocannabinoids compounds, exerts the entourage effect of boosting the bioactivities of phytocannabinoids [C. sativa.The accumulation of flavonols, such as quercetin and kaempferol, and flavanone varied in tissue specificity and chemovars of riations , 45 and riations . Hence, abinoids . TherefoC. sativa via a combination of transcriptomics and metabolomics of tissues of different chemovars , is a feminized plant, crossing Purple Kush and Dinamed Autoflowering CBD. DiKu and other commercial chemovars, Terra Italia, Swiss Dream, Pain killer, Gorilla Glue, and Red Pure, were grown in controlled growth chambers at 25 ℃ with 16:8 (light: dark) photoperiod in Yunnan Dali, China. The samples used in this study, including flowers, bracts, leaves, stems, and roots, were collected 20 days post-flowering (DPF). Once collected, samples were frozen in liquid nitrogen and stored at − 80 ℃ for further use.C. sativa (Accession number: GCA_900626175.1) were obtained from NCBI  [The reference genome and annotation files of rmation) . The traArabidopsis thaliana were downloaded from the Uniport [–5 and removing redundant sequences. Subsequently, the conserved domain (CD) search tool of NCBI was used to screen out those which did not have complete conserved domains to obtain the members of 11 classes in C. sativa. Molecular weights and isoelectric points were predicted using the ExPASy-ProSite website [Protein sequences of PAL, C4H, 4CL, CHS, CHI, FNS, F3′H, OMT, PT, F3H, and FLS from  Uniport  and homo website . Subcell website .Total RNA was extracted from the DiKu tissues of 20 DPF, with three biological replicates using RNAprep Pure  according to the manufacturer’s instruction. First-strand cDNA was subsequently synthesized using StarLighter Script RT all-in-one Mix .C. sativa genome sequence by employing HISAT2 tools, where 31,170 genes referred to 41,553 transcripts were annotated. Gene expression levels were estimated as fragments per kilobase of transcript per million fragments mapped (FPKM), of which log 2 (FPKM + 1) was assessed using TBtools [RNA sample sequencing was conducted via Illumina and PackBio sequencing platforms. After trimming the redundancy reads, clean reads were mapped to the reference  TBtools  to repre−(ΔΔCt) method [EF1α [qRT–PCR was performed on Rotor-Gene Q  using a StarLighter SYBR Green qPCR Mix kit . The program was set as 95 °C for five minutes, 95 ℃ for 30 s, 60 ℃ for 20 s, and 72 ℃ for 15 s for 40 cycles. Each sample was repeated at least thrice, and the data were analyzed using the 2) method  with EF1od [EF1α  as the rThe collected samples were lyophilized and then ground into power. Next, 100 mg of the ground sample was exposed to 1 ml of 70% methanol, sonicated at room temperature for 30 min, and placed at 4 °C overnight. The supernatant was retained after a 12,000 rpm centrifugation for 15 min. All samples were extracted twice following the steps above, and the mixed supernatant was filtered through a 0.22-μm organic membrane for total flavonoid content determination and LC–MS/MS analysis.3 (5%) were mixed and stayed for six minutes with an addition of 60-μl Al(NO3)3 (10%) with another six minutes stay. Then, 800-μl NaOH (4%) was added and fixed in 5-ml methanol with a thorough blending. After staying at RT for 15 min, the absorbance values were measured at 416 nm, and the standard curve was plotted using rutin standards to calculate the total flavonoid content of each sample. At least three biological replicates were determined.120 μl of diluted solution (in an appropriate proportion) and 60-ul NaNOThe Agilent UPLC 1290II-G6400 triple quadrupole mass spectrometer  was employed to determine the relative quantity of synthetic constituents. MS/MS spectra were obtained in negative ionization mode using a C18 column . The mobile phases were ammonium acetate (A) and acetonitrile (B) solutions with a linear gradient program: 0/5, 2/5, 2.5/18.5, 10.5/41, 11/59, 18/77, 22/95, 24/95, 24.1/5, and 26/5 (min/B%).CsFLS phylogenetic tree was constructed based on the NJ method by obtaining amino acid sequences from various plant FLSs from the GenBank database. Bootstrap tests with 1000 replicates were performed using the MEGA 6.0 software [The software .CsFLS genes and their distribution of exons and introns. Motif conserved motifs (motif parameter set to 10) were predicted using the MEME [TBtools software was used to investigate the chromosomal location of the MEME .A. thaliana (PDB ID: 1GP4) [Swiss-Model  was usedD: 1GP4)  as the tD: 1GP4) . In thisE. coli BL21 (DE3) strain and incubated at 37 ℃, 160 rpm for 2–3 h until OD 600 reached 0.6. The final concentration of 0.4-mM isopropylthio-β-galactoside was added and induced at 16 ℃, 130 rpm for 20 h. After sonication and centrifugation, it was purified through a nickel column and eluted with 250-mM imidazole. N/C-terminal fusion proteins with two His-6 tags were obtained from the condensed elution. After protein concentrations were determined using Bradford reagents , SDS–PAGE electrophoresis and subsequent Western blotting probed using mouse monoclonal antibody Anti-His  were performed. Enzyme reactions with recombinant FLS in a 500 μL system contained 20-mM Tris–Hcl (PH7.0), 1-mg ascorbic acid, 0.1-mg/ml bovine serum albumin, 50-μM ferrous sulfate, 1.5-mg/ml 2-ketoglutarate, 20-μg/ml substrate, and 30-μg recombinant protein reaction at 30 ℃ for 20 min followed by twice extraction using 500-μL ethyl acetate and evaporated dry at low temperature. The reactant was dissolved in 500-μL methanol and filtered for further Q-TOF analysis using the mobile phases of ammonium acetate (A) and acetonitrile (B) and a linear gradient program of 0/5, 2/5, 2.5/18.5, 20/30, 20.5/95, 23/95, 23.5/5, and 26/5 (min/B%). An Agilent 6460  triple quadrupole liquid mass spectrometer in the negative ion mode was used to perform the test as per the parameters set as follows: the scan range setting: 100–1000 m/z, atomization pressure: 35 psi, drying gas flow rate: 8 L min−1, protective gas flow rate: 11 L min−1, and protective gas temperature: 350 °C. At least three replicates were performed for each sample.CsFLS2 and CsFLS3 were cloned using the cDNA as the template and then constructed into a pET28a (+) expression vector. The primers are shown in Additional file The substrate concentration was changed to 50 μg/mL, and other conditions were unchanged. The catalytic products were quantified using HPLC with the corresponding standards, and at least three biological replicates were performed.The optimum pH was determined by conducting the enzymatic reaction at 30 ℃ for 30 min in three buffers  at 0.5 intervals. Meanwhile, the optimal temperature was determined by performing the catalytic reaction in pH 7.0 at a 15–50 ℃ gradient range with 5 ℃ interval for every 30 min. The reaction was conducted under the optimum conditions verified above with a substrate concentration range of 0–300 μM. The reactant was detected using HPLC with a linear gradient program of 0/5, 2/5, 2.5/18, 20/30, 20.5/95, 23/95, 23.5/5, and 26/5 (min/B%). The data was stimulated using. The corresponding enzyme kinetic parameters, such as Vmax and Kmax, were calculated by non-linearly fitting the Michaelis–Menten in the Graphpad software. The experiment was repeated at least thrice.Additional file 1: Table S1. Physical characteristics of the major enzyme-encoding genes of flavonoid metabolic pathway in C. sativa.Additional file 2: Table S2. Quantitative primers of the selected enzyme-encoding genes of flavonoid metabolic pathway in C. sativa.Additional file 3: Figure S1. Analysis of chromosomal location and gene structure of the CsFLS genes in C. sativa.Additional file 4:Table S3. Secondary structure predication of the CsFLS proteins in C. sativa.Additional file 5: Table S4. The list of flavonoid-related genes in the phylogenetic analysis from C. sativa and other plant species.Additional file 6: Figure S2. Western blotting of recombinant protein of CsFLS2 and CsFLS3.Additional file 7: Figure S3. Comparsion of CsFLS2 and CsFLS3 with other proteins belonging to DOXC 28/47 subgroup of 2-ODD superfamily.Additional file 8: Table S5. Cloning primers of CsFLS2 and CsFLS3 in C. sativa.Additional file 9: Figure S4. Cis-acting elements within the promoters of CsFLS2 and CsFLS3.


The screened LN-SDS is featured with suppressed water reactivity and high ionic conductivity at temperatures ranging from room temperature to −15 °C. Additionally, NaTi2(PO4)3‖LiMn2O4 batteries operating with LN-SDS manifest impressive electrochemical performance at both room temperature and −15 °C, especially the cycling stability and low-temperature performance.Low-cost and ecofriendly electrolytes with suppressed water reactivity and raised ionic conductivity are desirable for aqueous rechargeable batteries because it is a dilemma to decrease the water reactivity and increase the ionic conductivity at the same time. In this paper, Li A. Aη) ploeasing ω f. Accordeasing ω e, due toη* increase as n increases  show that both zero shear viscosity and ncreases d,f. The irection g. Meanwh+/Na+ ions resulting from the added inorganic salts contribute to lower the melting point. For example, LN-SDS-90 exhibits a melting point of −3.71 °C, much lower than that of −0.35 °C for SDS-90 . The weak peak position is centered at 7.6, 8.9, 10.9, and 11.3 °C as n equals 30, 60, 90, and 120, respectively a. The weperature c. As n etal ions ,66. Liketal ions ,40. The olutions ,40. Note3 decreased to only 0.016 mS cm−1 at 10 °C from 73.8 mS cm−1 at room temperature [−1 at 0 °C  [+ electrolytes (4.5–10.7 mS cm−1) [The conductivity for conventional WIS-based electrolytes dramatically declined as the temperature deceased ,28. For  at 0 °C . As for  at 0 °C . For examS cm−1) ,18,25 anmS cm−1) .σ is the ionic conductivity, σ0 is the pre-exponential factor, Ea is the activation energy, k is the Boltzmann’s constant, and T is the absolute temperature.The relatively high ionic conductivity of LN-SDS-n (n ≥ 30) at low temperatures can be deeply understood by the Arrhenius equation, expressed as ,67:σ = σTK above which the ionic conductivity abruptly increases, we analyzed the plots in the temperature range of −15–5 °C. A linear relationship exists between log σ and T−1 for LN-SDS-n (n = 30–120), indicating that the electrolyte conductivities obey the Arrhenius law at the measured temperatures [eratures . The Arreratures . In otheEa decreased as n increased from 0 to 90, and then increased with increasing n to 120 , the n to 120 . The Ea ctrolyte . Therefo−1 at −40 °C [+ through the channel of zwitterion groups, which function as dissociation enhancers for lithium salts [+, Na+, SO42−, and SDS were randomly distributed at the temperatures ≤ 0 °C (273.15–258.15 K). Such a situation also occurred in LN-SDS-0 , the Li+ MSD values for LN-SDS-90 were not improved in the applied voltage range of 0.5–2.0 V nm−1, as compared to LN-SDS-0. However, the Na+ and SO42− MSD values for LN-SDS-90 greatly improved, especially for the case with applied voltages of more than 1.0 V nm−1. The MSD results indicate that the presence of SDS provides great contributions to improve the Na+ and SO42− migration rates, but has a minor effect on the Li+ migration rate. In other words, the improved ionic conductivity for LN-SDS-90 at the temperatures ≤ 0 °C is mainly attributed to the improved Na+ and SO42− migration rates.The SDS micelle-assisted formation of directional ion transport channels facilitated the improvement of the ionic conductivity, as reflected by the MSD plots a–f. The 2SO4, and Na2SO4, resulting in a decrease in the freezable water concentration and, hence, lowered electrolyte melting points  is attributed to the formation of directional ion transport channels without (at 298.15 K) and with (at 273.15–258.15 K) applying an electric field. When working, the cations and anions form quasi-continuous ion flows in the channels, enabling the high ionic conductivities. In addition, the decreased HBNs of water, reduced freezable water concentration, and improved NaCdis) of NTP retained 56 mA h g−1 after 1000 cycles at 1 A g−1, presenting a capacity retention of 64.6% relative to the initial Cdis  delivered Cdis values of 59.4, 67.1, 62.8, 60.9, 65.1, and 55.6 mA h g−1 in sequence , however, the rate capability and cycling stability of NTP‖LMO were greatly improved. Among the electrolytes, LN-SDS-90 was the top performer. The Cdis of NTP‖LMO operating with LN-SDS-90 retained 47.1 mA h g−1 after 2000 cycles with varying C rates, presenting a capacity retention of 70.4%. When working at 5 °C for 2000 cycles, a Cdis of 30.8 mA h g−1 was retained, presenting a 76.8% capacity retention and a nearly 100% CE (Ro) represented by the intercept at the x-axis was 0.84 Ω before cycling and increased by only 0.58 Ω after 2000 cycles at 5 °C. In addition, the charge transfer resistance (Rct) represented by the semicircle in the high- to medium-frequency region increased by only 6.85 Ω after cycling, according to the fitting results with Zsimpwin software. It should be noted that the most rigorous proof for ARMB stability is the performance at low C rates, rather than the high-rate performance [Cdis retained 41.0 mA h g−1, presenting a 61.8% capacity retention and a nearly 100% CE  render NTP‖LMO with high-capacity retention b, their  100% CE . The ohmformance ,17. Ther 100% CE e. The slEbattery, W h kg−1) and specific power  of a battery can be obtained according to the GCD curves  of ARMBs without and with cycles of less than 300  finding the synergistic effect of Li+, Na+, SO42−, and SDS that endows LN-SDS-n (n ≥ 1) with a lowered melting point, reduced freezable water concentration, suppressed water reactivity, and high ionic conductivity in a wide temperature range, and (ii) proposing a micelle-assisted formation mechanism of directional ion transport channels for the SDS-containing electrolytes with high ionic conductivity, based on the theoretical computation results. Using NTP‖LMO as a probe, the electrolytes were screened. LN-SDS-90 was found to be a top performer that can significantly improve the electrochemical properties of NTP‖LMO ARBs. As a low-cost and ecofriendly electrolyte, LN-SDS-90 is a promising candidate for application in ARBs with an output potential smaller than 2 V.In summary, a new type of electrolyte, i.e., LN-SDS-n, has been designed that is composed of Li


Sulfides including H2S, a gasotransmitter, generated from l-cysteine by some enzymes including cystathionine-γ-lyase (CSE), are pro-nociceptive, since they enhance Cav3.2 T-type Ca2+ channel activity expressed in the primary afferents, most probably by canceling the channel inhibition by Zn2+ linked via coordinate bonding to His191 of Cav3.2. Given that germanium is reactive to sulfur, we tested whether THGP would directly trap sulfide, and inhibit sulfide-induced enhancement of Cav3.2 activity and sulfide-dependent pain in mice. Using mass spectrometry and 1H NMR techniques, we demonstrated that THGP directly reacted with sulfides including Na2S and NaSH, and formed a sulfur-containing reaction product, which decreased in the presence of ZnCl2. In Cav3.2-transfected HEK293 cells, THGP inhibited the sulfide-induced enhancement of T-type Ca2+ channel-dependent membrane currents. In mice, THGP, administered systemically or locally, inhibited the mechanical allodynia caused by intraplantar Na2S. In the mice with cyclophosphamide-induced cystitis and cerulein-induced pancreatitis, which exhibited upregulation of CSE in the bladder and pancreas, respectively, systemic administration of THGP as well as a selective T-type Ca2+ channel inhibitor suppressed the cystitis-related and pancreatitis-related visceral pain. These data suggest that THGP traps sulfide and inhibits sulfide-induced enhancement of Cav3.2 activity, leading to suppression of Cav3.2-dependent pain caused by sulfide applied exogenously and generated endogenously.Poly-  •v3.2 activity by interacting with Zn2+ linked to Cav3.2.Sulfide enhances Ca•THGP, an organogermanium compound hydrolysate, directly reacts with sulfide.•v3.2 activity.THGP inhibits the sulfide-induced enhancement of Ca•v3.2-depedent pain caused by sulfide in mice.THGP suppresses the Ca•The organogermanium is useful to treat sulfide-induced symptoms including pain. Cyclophosphamide (CPA) and sodium sulfide (Na2S) were purchased from Sigma-Aldrich . Zinc chloride (ZnCl2), sodium hydrogen sulfide (NaSH), TTA-A2 and cerulein were purchased from Kishida Chemicals Co., Ltd. , Strem Chemicals, Inc. , Alomone Labs.  and Bachem , respectively. THGP, NaSH and Na2S were dissolved in ultra-pure water for the MS/MS and whole-cell patch clamp experiments, and in phosphate buffer  or Tris-HCl buffer  prepared with D2O instead of H2O for 1H NMR analysis. For administration to mice, THGP, NaSH, Na2S, CPA and cerulein were dissolved in saline, and TTA-A2 was in 0.5% methyl cellulose containing 1.3% DMSO and 10% Tween-80.Poly-2.2m/z 100 to 250. The ion at m/z 195 in the negative ion full-scan mass spectra obtained by MS analysis of the mixture of THGP and NaSH was selected as a precursor for MS/MS analysis, since the relative signal at m/z 195 in the mixture of THGP and NaSH was greater than that in THGP alone. Product ion spectra were measured at a scan range from m/z 50 to 250 with the collision energy of −20 and −35 V. Data were acquired and analyzed using LabSolutions LCMS software (Shimadzu). The values of calculated m/z were determined using ChemBioDraw Ultra 11.0 .NaSH at 1 mM and THGP at 1 mM were mixed and incubated for 10 min at room temperature, and the structure of the reaction products was analyzed by tandem mass spectrometer, LCMS-8060 , equipped with heated ESI probe. The pH of the mixture was adjusted to 7.0 with NaOH. Mass spectrometry (MS) and tandem mass spectrometry (MS/MS) were carried out in the negative ion mode. The samples were analyzed by flow injection analysis. The mobile phase consisted of water/methanol  and it was pumped at a flow rate of 0.2 mL/min. The overall run time was 1 min. The injection volume was 2 μL. Probe voltage was set at −3000 V, DL temperature was 150 °C and 250 °C, heat block temperature was 400 °C, and interface temperature was 100 °C. Nebulizer gas flow was 3 L/min, drying gas flow was 10 L/min, and heating gas flow was 10 L/min. ESI mass spectra for reaction products were measured at a scan range from 2.31H NMR) analysis, a phosphate buffer solution  was prepared with D2O and used for preparing samples. The solution of THGP at a final concentration of 10 mM was mixed with NaSH at 10 mM or Na2S at 1.25–10 mM, 10 min (or 5 min in some experiments) before beginning the NMR analysis. In the experiments to study the reaction between THGP and Na2S, each at 10 mM, in the presence of ZnCl2 at 0.1–10 mM, a Tris-HCl buffer solution  instead of the phosphate buffer were prepared using D2O and deuterium chloride solution, in order to avoid the formation of Zn3(PO4)2 precipitation. The deposited ZnS in the reaction mixture was filtered off immediately before NMR analysis. 1H NMR spectrum of the reaction mixtures or its filtrates was measured at 25 °C by a JEOL JNM-ECA 800 .In proton nuclear magnetic resonance (2.4v3.2 T-channels (hCav3.2-HEK293) , 105  and 71  , respectively, of the expected collision-induced dissociation product ions of a possible sulfur-containing reactant in the mixture of THGP and NaSH. The MS/MS analysis of the mixture of THGP and NaSH with a greater collision energy, −35 V, provided a collision-induced dissociation product ion at m/z 123 corresponding to the calculated m/z 122.8960 (74Ge) of the dissociation product ion of the sulfur-containing reactant, in addition to the signals at m/z 105 and 71 detected in MS/MS analysis of THGP alone  D. The MS5 and 71 , being cs report . On the 5 and 71 E. It is GP alone . CollectGP alone F.1H NMR technique. In the NMR spectrum of THGP at 10 mM alone, two proton triplet signals were observed at δH 2.48 (a) and 1.57 (b), indicating two methylene groups adjoining a carbonyl group and a Ge atom, respectively (δH 2.40 (a’) and 1.42 (b’), respectively, indicating that a large proportion (83%) of THGP changed to the sulfur-containing reaction product through the reaction with sulfide  and (b), shifted to the higher magnetic fields, (a’) and (b’), respectively, in a manner depending on concentrations of Na2S  and (b) signals decreased 2 precipitation in this experiment. Under this condition, 82–83% of 10 mM THGP, once mixed with 10 mM Na2S in the absence of ZnCl2, changed to the sulfur-containing reaction product, as estimated from changes in % peak area of triplet signals from (a) and (b) to (a’) and (b’), respectively (2 in a concentration-dependent manner (2 at 3 and 10 mM decreased the % peak areas of (a’) and (b’) signals of the 10 mM THGP/10 mM Na2S mixture from 82-83% to 46–47% and 0%, respectively, and conversely, (a) and (b) signals increased , the cells were stimulated with Na2S at 10 μM almost doubled T-currents, an effect persisting for at least 10 min  in a dose-dependent manner  at a peak time, 3–6 h after oral administration of Ge-132 at 100 mg/kg in rats [2+ and THGP would react with sulfide in a competitive manner at least in a solution state, which is also associated with solubility equilibrium of ZnS in a biological fluid.The acceleration of Careatitis , as showreatitis ,27,28. InCl2 see  suggest ian body , it is e2S is now known as a multifunctional gasotransmitter along with NO and CO, and exhibits a variety of biological activity through its interaction with metals binding to proteins including heme proteins, antioxidant activity, sulfhydration of cysteine residues present in proteins, and others [2S is also inhibited by THGP. Sulfide/H2S interacts with several metals, such as zinc, iron, copper and nickel [Hd others . It is ld nickel , whereas5v3.2-dependent pain caused by sulfide applied exogenously or generated endogenously. Therefore, Ge-132 may serve as a medicine for treatment of pathological pain. H2S, a toxic gas, at higher concentrations binds to cytochrome c oxidase in the electron transport chain, leading to cell death [2S may also be protected by THGP. Endogenous sulfide/H2S appears to play a dual role, i.e. being pro-inflammatory and anti-inflammatory (protective), and long-term complete inhibition of H2S-generating enzymes may not necessarily be beneficial, particularly in the cardiovascular system [2S generated in pathological conditions and maintain appropriate blood sulfide/H2S levels.Our study demonstrates that THGP, a hydrolysate of Ge-132, directly reacts with sulfide, resulting in suppression of Call death , and neell death . Such a r system ,52. In tFS, YS, TN and AK designed this project. FS, NK, YS, KS, HM, HY, GT, SM, YK and MT performed the experiments. FS, NK, YS, KS, HM, TN, HY, GT, SM, YK, MT and AK analyzed data and discussed results. FS, NK and AK wrote the manuscript. TO and SY contributed materials and technical guiding.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.


Dekkera anomala YAE-1 strain separated from “airag” (Mongolian fermented mare’s milk) produces β-glucosidase, which can convert ginsenoside Rb1 from Panax ginseng. Ginseng-derived bioactive components such as ginsenoside Rb1 have various immunological and anticancer activities. Airag was collected from five different mare milk farms located near Ulaanbaatar, Mongolia. YAE-1 strains were isolated from airag to examine the hydrolytic activities of β-glucosidase on Korean Panax ginseng using an API ZYM kit. Supernatants of selected cultures having β-glucosidase activity were examined for hydrolysis of the major ginsenoside Rb1 at 40°C, pH 5.0. The YAE-1 strain was found to be nearly identical at 99.9% homology with Dekkera anomala DB-7B, and was thus named Dekkera anomala YAE-1. This strain exerted higher β–glucosidase activity than other enzymes. Reaction mixtures from Dekkera anomala YAE-1 showed great capacity for converting ginsenoside Rb1 to ginsenoside Rd. The β-glucosidase produced by Dekkera anomala YAE-1 was able to hydrolyze ginsenoside Rb1 and convert it to Rd during fermentation of the ginseng. The amount of ginsenoside Rd was highly increased from 0 to 1.404 mg/ml in fermented 20% ginseng root at 7 days. In 2018, health functional food sales totaled US$2.1 billion and red ginseng products accounted for 44%, or US$927 million in sales [ng roots , 4. Amonng roots  and anting roots . GinsenoAirag is the local name for fermented mare’s milk in Mongolia. The traditional beverage is fermented by a co-culture of yeasts and lactic acid bacteria and is produced by churning mare’s milk in a leather sack with a wooden masher.Airag does not use an inoculated starter and is instead fermented by mixing the lactic acid bacteria and yeast in mare’s milk. Therefore, the diversity of yeast and lactic acid bacteria is very high.Dekkera anomala is one of several spoilage yeasts found in the genus Dekkera. D. anomala was differentiated from other species of the genus morphologically by the formation of blastese, single filamentous pseudohyphae, and physiologically by the fermentation of lactose [ lactose .D. anomala YAE-1 could be used to hydrolyze the ginsenoside Rb1. The objectives of this study are to separate and characterize D. anomala YAE-1 having β-glucosidase activity from airag, and to investigate the enzymatic capacity of D. anomala YAE-1 strain on the hydrolysis of a major ginsenoside (Rb1) in Korean ginseng.The conversion of ginsenosides by LAB enzymes has great potential importance for human health due to biological, immunological and medicinal functions. We have hypothesized that the β-glucosidase activity of the Airag was prepared according to the method . Airag sThe experimental strains were isolated from airag, a Mongolian fermented milk product. Samples were plated according to the method -14. Escu18S rRNA gene sequencing of selected strains was conducted according to the method . PCR seqTo determine the optimum pH (3.0-7.0) and temperature (20-40°C) for growth of strain YAE-1, 3% (v/v) of the yeast strain was inoculated in YM broth for 72 h in an incubator, and the specimen was checked for viable cell count.Enzyme activity was detected using the API ZYM Kit  in accordance with the manufacturer's instructions. Selected colonies from the surface agar plates were suspended in medium  to a turbidity adjusted to a McFarland No. 5.0-6.0 standard (BioMérieux). Each microcupule of the API ZYM gallery containing 19 dehydrated chromogenic enzyme substrates was inoculated with 50 μl of the suspension, and the strip was incubated 4 h at 37°C. (BioMérieux).Carbohydrate fermentation tests were performed according to the method . Selecteβ-Glucosidase activity of the supernatant was determined by the rate of hydrolysis of 5 mM p-nitrophenyl-β-D-glucopyranoside  at 40°C and pH 7.0 (50 mM potassium phosphate buffer). The optimum pH and temperature on β-glucosidase activity were measured over a range of pH from 3.0 to 9.0 and temperature from 25°C to 70°C, respectively. β-Glucosidase activity of the supernatant was determined according to the method .1 under optimum condition  of β-glucosidase. The reaction condition was cultured at 40°C for 48 hours. Conversion of ginsenoside Rb1 was checked by TLC and HPLC analysis [Selected colonies were inoculated in YM broth (pH 4.0) (HiMedia Laboratories) for 3 days at 30°C. Supernatant activities were tested for hydrolysis of the major ginsenoside Rbanalysis .Ginseng roots were sliced and sterilized at 75°C for 10 min. A 20% (w/v) ginseng root solution was incubated with 3% (v/v) of yeast strain YAE1 at 30°C for 7 days. The fermentation characteristics of ginseng root by the strain YAE-1 were analyzed by TLC and HPLC, and viable cell counts were performed according to the method .Hydrolyzed ginsenosides obtained from filtrate samples showing β-glucosidase activity were analyzed using the reversed-phase HPLC system Shimadzu LC-6AD  and an ACE-5-C18 column (4.6 × 250 mm) equilibrated with solvent A (H2O). HPLC analysis was conducted according to the method .All analyses were repeated at least 3 times and are expressed as means ± standard deviation (SD) .1, Rb2, Rd, Re, and Rg1 were hydrolyzed by the strains, strain YAE-1 can hydrolyze ginsenoside Rb1 converted to Rd in minor quantity, while no conversions occurred for ginsenosides Rb2, Rd, Re and Rg1 , Airag B (strain YAB1-YAB8), Airag C (strain YAC1-YAC6), Airag D (strain YAD1-YAD8), Airag E (strain YAE1-YAE8), and they were examined for β-glucosidase activity. Fifteen strains showed esculin-positive reaction. The YAE-1 strain was found to be the strongest esculin positive. When the major ginsenosides Rb and Rg1 . β-Glucompound K -18.Penicillium dipodomyicola strain isolated from the soil of wild ginseng also biotransformed major ginsenosides into compound K. The optimum transforming conditions for this fungus are 40°C, medium pH of 4.0 - 6.0 and incubation time of 7 days [Aspergillus usamii KCTC6954 converted ginsenoside Rb1 to compound K and the incubation time was 15 days. The optimum temperature and pH of β-glucosidase produced by this mold is 60°C and 6.0 [Candida allociferrii JNO301 isolated from meju (fermented soybean) converted ginsenosides of red ginseng extract, and the optimum temperature and pH were 20-30°C and 5-8, respectively. C. allociferrii JNO301 converted ginsenoside Rb1 to Rd → F2, Rb2 → Compound O, Rc → Mc1, Rf → Rh1 [Cladosporium fulvum was hydrolyzed with Rb1 to Rd. The enzyme has an optimal pH of 5.5 and an optimal temperature of 45°C [f 7 days . Aspergi and 6.0 . On the Rf → Rh1 . The β-g of 45°C .D. anomala DB-7B  than α-galactosidase (≥ 5 nmol) and α–glucosidase (0 nmol). On the other hand, fungi (P. dipodomyicola and A. usamii KCTC6954), bacteria (Cl. fulvum) and yeast  had β–glucosidase activity [D. anomala YAE-1 fully consumed carbohydrates such as glucose, D-galactose, N-acetyl-D-glucosamine, D-cellulose, D-lactose, while glycerol and raffinose were consumed 90% and 50%, respectively. C. allociferrii JNO301 can utilize carbohydrates such as glucose, galactose, N-acetyl-D-glucosamine, glycerol and raffinose, but lactose is not utilized [Enzymatic activities of activity -18. D. autilized .Cl. fulvum was 5.5 and 45°C, respectively [P. dipodomyicola isolated from soil of wild ginseng best converted ginsenoside Rb1 to minor ginsenosides under a pH value from 4.0 to 6.0 and the best fermentation temperature was 40°C [The β-glucosidase of the supernatant exhibited maximum enzyme activity at 40°C , but graectively . Furtherwas 40°C . Thus, t1 was tested under optimum condition for β-glucosidase . After 48 h, 3.095 mg/ml of ginsenoside Rb1 was fully hydrolyzed by supernatant with β-glucosidase activity, and released 3.0 mg/ml of ginsenoside Rd  for 48 h at optimum condition of 30°C. Hydrolysis of the major ginsenoside Rboside Rd , (Table oside Rd . Our resar mass. . Many reentation , 20, 21.us foods . Most mius foods . Jo et aus foods  reported1 to ginsenoside Rd in 20% fermented ginseng root. The peak size of ginsenoside Rb1 was small and that of ginsenoside Rd was significantly large after 7 days, as revealed in HPLC chromatograms (1 was converted to Rd, reducing ginsenoside Rb1 and increasing Rd. The factor that absolutely affects ginsenoside conversion is fermentation time. Rd was increased as the main ginsenoside in the final fermentation product of ginseng roots, confirming that Rb1 was decreased continuously during the 7-day fermentation. The quantity of minor ginsenoside Rd was elevated from 0 to 0.060 ± 0.011 mg/ml at 7 days in Paenibacillus sp. MBT213, which converts ginsenoside Rb1 into ginsenoside Rd from Panax ginseng [Paenibacillus sp. MBT213 was absolutely affected by fermentation time as was the ginsenoside conversion by D. anomala YAE-1. The initial viable cell count of D. anomala YAE-1 was 4.85 log CFU/ml, and maximum cell count was 7.39 log CFU/ml after 7 days (D. anomala YAE-1 has strong β-glucosidase activity, which can be used to convert major ginsenoside Rb1 to Rd during the fermentation of ginseng. It is also believed that the converted ginsenoside Rd has the potential to be used in a variety of health functional foods and pharmaceutical products.D. anomala YAE-1 hydrolyzed ginsenoside Rbatograms . Furtheratograms . In ferm ginseng . The ginr 7 days . D. anom