Objective: To explore the pharmacological effects and molecular mechanism of quercetin 7-rhamnoside (Q7R) in the treatment of cholestatic hepatitis induced by alpha-naphthylisothiocyanate (ANIT).Methods: ANIT-induced cholestatic hepatitis rat model was used to investigate the hepatoprotective effects of three different doses of Q7R . Serum biochemical indices were detected using commercial kits. H&E and masson staining were used to observe hepatic tissue damage and collagen deposition in hepatocytes. The metabolism of bile acid-related substances was detected via HPLC-MS/MS by 5-(diisopropylamino) amylamine (DIAAA) derivative method. Hepatocyte injury, cholestasis, and inflammation were detected at the mRNA and protein levels using reverse transcription-polymerase chain reaction (RT-PCR) and western blotting, respectively.Results: Q7R can decrease the level of CYP7A1, and increase FXR, CYP27A1 so then improving abnormal bile acid secretion. Furthermore, Q7R can also ameliorating inflammation by reduce TNF-α, IL-1β, PTGS1, PTGS2, NCOA2, NF-κB level. Therefore, Q7R had an effective therapeutic effect on ANIT-induced cholestatic hepatitis, improving abnormal bile acid secretion, and inhibiting inflammatory responses.Conclusion: The results demonstrated that Q7R treat cholestatic hepatitis by regulating bile acid secretion and alleviating inflammation. The liver is the most important organ for the excretion of cholesterol from the body. Cholestasis is a common clinical disorder of intrahepatic cholesterol metabolism and bile acid metabolism that causes serious damage to the liver. A large amount of cholesterol is first converted into bile acids in the liver before being discharged from the body . Bile acHypericum japonicum is a dried whole plant of Hypericum japonicum Thunb., and has been widely applied to cure infectious hepatitis, acute and chronic hepatitis in traditional medicine. In addition, flavonoids, phloroglucinols, phenolic acids and xanthones and trimethylated acylphloroglucinol meroterpenoids isolated from Hypericum japonicum Thunb. have already been proved the hepatoprotective, anti-tumor, antibacterial, antiviral, and antioxidant activities  are used for treatment of cholestatic hepatitis with a long history. There have been many pharmacology and molecular mechanism researches on Chinese herbal medicine treat cholestatic hepatitis. Wang et al.  exploredtivities .Hypericum japonicum has the effect of improving cholestatic hepatitis . Kits for detecting albumin (ALB), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were purchased from Nanjing Jiancheng (Nanjing Jiancheng Bioengineering Institute). Ursodeoxycholic acid , Chenodeoxycholic acid , Cholic acid , Hyodeoxycholic acid , Deoxycholic acid  were purchased from meilunbio . d4-glycochenodeoxycholate  was purchased from ZZBIO . 5-(Diisopropylamino) amylamine , 1-[Bis (dimethylamino) methylene]-1H-1,2,3-triazolo  pyridinium 3-oxid hexafluorophosphate , 1-Hydroxybenzotriazole hydrate , Triethylamine  were purchased from Sigma (Sigma-Aldrich (Shanghai) Trading Co. Ltd.). Primary anti-FXR antibodies were purchased from Abcam (Abcam Plc.). anti-CYP7A1 antibodies were purchased from Boster , and anti-CYP27A1 antibodies were purchased from Affinity Biosciences . Secondary antibodies such as goat anti-mouse IgG (H + L) and goat anti-rabbit IgG (H + L) were purchased from CWBIO .Male Sprague-Dawley rats (SD) rats (200–220 g) were purchased from Guangzhou Ruige Biological Technology Co., Ltd. . All animals were acclimatized and kept in a well-ventilated environment at 22–25°C and 50%–60% relative humidity under a 12/12 h light/dark cycle for 1 week. Rats were allowed free access to food and water and were fasted for 1 day prior to the experiments. All animal care and experimental protocols were performed according to the guidelines for the Institutional Animal Care and Use of Laboratory Animals at Guangzhou Medical University .n = 6 rats each group): 1) control group and 2) model group: blank solvent, intragastrical administration (i.g.); (3) positive control group: ursodeoxycholic acid (UDCA) 50 mg/kg, i.g.; 4) high-dose group (Q7RH): 5 mg/kg Q7R, i.p.; 5) medium-dose group (Q7RM): 2.5 mg/kg Q7R, i.p.; and 6) low-dose group (Q7RL) 1.25 mg/kg, i.p. ANIT was administered 1 week later in all groups  and sacrificed, their body weights were recorded. Subsequently, blood was collected from the abdominal aorta for biochemical assessment, and livers were isolated and accurately weighed after the remaining blood on their surface was washed with PBS and wiped clean with filter paper. The formula for calculating the relative liver index was as follows: Liver index = Liver weight/Rat body weight × 100%.The isolated rat livers were fixed with 4% paraformaldehyde, embedded in paraffin, and sliced. Slices were stained with hematoxylin and eosin (H&E), and masson-stained. Histological changes were evaluated by randomly selecting visual fields using an upright microscope . The levels of alanine transaminase (ALT), aspartate transaminase (AST), gamma-glutamyl transferase (γ-GGT), total bilirubin (TBIL) and direct bilirubin (DBIL) were measured using a Chemray 240 Automatic Biochemical Analyzer . Albumin (ALB) was evaluated using commercial kits, according to the manufacturer’s instructions.PTGS1, PTGS2, NCOA2, IL-1β, NF-κB, FXR, and TNF-α mRNAs relative to GAPDH mRNA expression was detected using the 2−ΔΔCT method.Total RNA was extracted from liver tissue using an RNA extraction solution  according to the manufacturer’s instructions. The primer sequences used for PCR are listed in The proteins in liver tissue of rats in each group were extracted and quantified using the BCA method. The protein samples were then subjected to electrophoresis, membrane transfer, blocking, and incubation with anti-FXR (1:1000), anti-CYP7A1 (1:1000), anti-CYP27A1 (1:1000) antibodies. The membranes were washed three times with TBST and incubated with the corresponding horseradish peroxidase-labeled secondary antibodies (1:3000) for 1 h. After washing three times with TBST, the proteins were detected using an ECL chemiluminescence reagent. After exposure scanning , ImageJ software was used for analysis.Serum (100 μL) was added to 400 μL of cold methanol (100 ng/mL of D4-GCA), vortexing for 1 min, the precipitated protein was centrifuged  at 21380 g at 4°C for 15 min, and 400 μL supernatant was collected and repeated three times. The 1200 μL supernatant was combined and vacuum dried. 5 μL HOBt, 5 μL DIAAA-TEA, 5 μL HATU were successively added to the dried product, vortexed for 1 min, then incubated at room temperature for 2 min, add 35 μL acetonitrile, vortexed, centrifuged at 21,380 g for 15 min at 4°C, and collected 40 μL of the supernatant for analysis .Serum extracts were analyzed on a Sciex 4000 Triple Quadrupole HPLC-MS/MS system  using a Symmetry C18 analytical column . The mobile phase containing 1% formic acid aqueous solution (solvent A) and methanol solution (solvent B) was provided according to the following procedure: 0–8 min (5%–40% B); 8–15 min (40%–60% B); 15–18 min (60%–80% B); 18–20 min (80%–98% B); 20–25 min (98%–60% B); 25–30 min (60%–40% B). The injection volume and flow rate were 10 μL and 25 mL/min, respectively. The multiple reaction monitoring parameters were listed in The following MS parameters were used: curtain gas = 35 psi; ion spray voltage = +4500 V; temperature = 450°C; ion source gas 1 = 45 psi; ion source gas 2 = 45 psi; interface heater = on; collision gas = medium. The multiple reaction monitoring parameters are listed in t-test, and mean comparisons between three or more treatments were performed using one-way analysis of variance. p < .05 was considered statistically significant.All experimental data are expressed as the mean ± standard error (SEM). Statistical analyses were performed using GraphPad Prism 9.0 software. Mean comparisons between two data sets were performed using the p < .05) compared to that of the model group  (p < .05) in the positive control group and the different Q7R-treatment groups.Biochemical analys was conducted to evaluate the therapeutic effects of Q7R. Results showed that the levels of AST, ALT, ALP, γ-GGT, TBIL, and DBIL in the model group were significantly increased (p < .05) . In contThe results of H&E staining  showed tMasson staining  showed tQ7R treatment significantly attenuated the degree of hepatic putrescence, fibrosis, and inflammatory cell infiltration. These results revealed that Q7R protected against ANIT-induced liver fibrosis in cholestatic hepatitis rats.TNF-α, IL-1β, PTGS1, PTGS2, NCOA2, FXR, and NF-κB were measured. The results showed that the mRNA expression of TNF-α, IL-1β, PTGS1, PTGS2, NCOA2, and NF-κB were upregulated in the model group but were downregulated after administration Q7R treatment , a member of the nuclear receptor (NR) superfamily, and a major regulator of bile acid biosynthesis and enterohepatic circulation. Under normal physiological conditions, liver FXR activity regulates bile acid levels while limiting bile acid accumulation . When thlestasis .On the other hand, CYP27A1 is a key enzyme in the alternative pathway of bile acid synthesis. The inhibition of CYP7A1 leads to an increase in the expression of CYP27A1, which converts cholesterol into 27-hydroxycholesterol (27-HC). The molecule 27-HC is an important component of the negative feedback loop that regulates cholesterol biosynthesis. Thus, when CYP27A1 expression is elevated by pharmacological intervention, 27-HC production and cholesterol efflux are increased, and cholesterol accumulation is reduced, thereby alleviating the symptoms of the disease .Liver is the only organ that has all the enzymes required for bile acid synthesis. In the liver, CYP7A1 initiates the classical bile acid synthesis pathway by hydroxylating the steroid ring to form 7 α- Hydroxycholesterol. Then, CA and CDCA are generated by the catalyzed of CYP8B1, and CA continues to react to generate GCA and TCA. Next, GCA and TCA are transported out of the liver. In the intestine, GCA and TCA are hydrolyzed into CA by the bile salt hydrolase (BSH), CA at 7α- Dehydroxylase acts to biotransformed DCA. Meanwhile, in the bile acid alternative pathway, cholesterol is biotransformed to 27-HC by the action of CYP27A1 and then to CDCA by the action of CYP7B1. Similarly to the classical pathway, after a series of biotransformation, CDCA is transformed into tauro-β-Muricholic acid (T β-MCA), which is transported out of the liver and then hydrolyzed into β-MCA in the intestine. β-MCA biotransformation HDCA through 6 β-epimerization and additional 7 β-dehydroxylation . Accordivia the activated SHP pathway (An inflammatory response can be induced in the early stage of cholestasis, producing many inflammatory mediators, such as cytokines and chemokines, which induce the activation of neutrophils and their infiltration and aggregation in liver tissue . Bile ac pathway . Further pathway , whose tOn the other hand, TNF-α is a pro-inflammatory cytokine involved in normal inflammatory and immune responses and is mainly produced by activated monocytes and macrophages . TNF-α pPTGS2, also known as COX2, is an enzyme capable of initiating inflammation and promoting prostaglandin synthesis upon stimulation by various inflammatory factors, such as cytokines and bacteria. Prostaglandins can alter hepatic bile flow and may be related to the release and action of some cholelithogenic hormones. It is also involved in gallbladder contraction and water absorption and is closely related to the occurrence of cholecystitis and gallstones . SimilarIn conclusion, this study demonstrated that Q7R could activate FXR, CYP7A1, and CYP27A1, effectively regulate bile acid synthesis disorders, and ameliorate hepatocyte membrane damage. Moreover, Q7R can also reduce TNF-α, IL-1β, PTGS1, PTGS2, NCOA2, and NF-κB levels and alleviate the inflammatory response caused by cholestasis. Additionally, Q7R plays a hepatoprotective role. Taken together, the results of this study may provide support for further research and clinical application of Q7R in cholestatic hepatitis.


Drosera rotundifolia are traditionally used to treat cough symptoms during a common cold. The present study aimed to investigate the impact of extracts from D. rotundifolia and active compounds on the respiratory tract. Tracheal slices of C57BL/6N mice were used ex vivo to examine effects on airway smooth muscle (ASM) and ciliary beat frequency (CBF). Phosphodiesterase (PDE) inhibition assays were carried out to test whether PDE1 or PDE4 are targeted by the active compounds. An ethanol–water extract, as well as an aqueous fraction of this extract, exerted antispasmodic properties against acetylcholine-induced contractions. In addition, contractions induced by 60 mM K+ were abrogated by the aqueous fraction. Effects on ASM could be attributed to the flavonoids quercetin, 2″-O-galloylhyperoside and hyperoside. Moreover, the Drosera extract and the aqueous fraction increased the CBF of murine tracheal slices. Quercetin and 2″-O-galloylhyperoside were identified as active compounds involved in the elevation of CBF. Both compounds inhibited PDE1A and PDE4D. The elevation of CBF was mimicked by the subtype-selective PDE inhibitor rolipram (PDE4) and by 8-methoxymethyl-IBMX. In summary, our study shows, for the first time, that a Drosera extract and its flavonoid compounds increase the CBF of murine airways while antispasmodic effects were transferred to ASM.Extracts from  Drosera rotundifolia L. is a carnivorous plant that natively grows in peatland or other wet and oligotrophic regions of the world + ion and fragments in the respective MS2 spectra with the respective datasets from the literature  column  at 40 °C. Injection volume: 2 µL. Flow rate: 0.5 mL/min. Samples were dissolved in acetonitrile/water 1:1 (terature .v/v) was added, and the plant was extracted with an Ultra-Turrax rotor stator system under ice cooling . Then, the liquid extract was filtered, concentrated and lyophilized. Fast centrifugal partition chromatography FCPC  was used to fractionate the ethanol 90% extract from D. rotundifolia. For each fractionation procedure, 0.5 g of the dry extract was dissolved in 5 mL ethyl acetate/water 1:1 (v/v). Mobile phase: Ethyl acetate (saturated with water), stationary phase: water (saturated with ethyl acetate), ascending mode: 195 × g; 10 mL/min, fraction size: 5 mL. Selected fractions were purified via column chromatography on Sephadex® LH-20 (column size 130 × 29 mm i.d.) by isocratic elution with ethanol/water 1:1 (v/v) to isolate 2″-O-galloylhyperoside (yield: 51.4 mg).An amount of 230 g of fresh plant material was pulverized with liquid nitrogen. Ethanol 90%  at 600 MHz (1H) or 150 MHz (13C). The sample was dissolved in methanol-d4, and chemical shifts were referenced to the respective residual solvent signals .Identity was determined by mass spectrometry, followed by m/z 617.1160 [M + H]+ . 1H NMR  δ 7.64 , 7.50 , 7.13 , 6.78 , 6.33 , 6.16 , 5.68 , 5.45 , 3.93 , 3.82 , 3.69 , 3.59 . 13C NMR  δ 179.1 (C-4), 168.2 (C-7′″), 166.1 (C-7), 163.1 (C-5), 158.3 (C-8a), 158.0 (C-2), 149.8 (C-4′), 146.3 (C-3′″/5′″), 145.9 (C-3′), 139.9 (C-4′″), 135.0 (C-3), 123.1, 123.0 (C-1′), 121.6 (C-1′″), 117.1 (C-2′), 116.2 (C-5′), 110.6 (C-6′″/2′″), 105.7 (C-4a), 101.2 (C-1′’), 99.8 (C-8), 94.6 (C-6), 77.5 (C-5′’), 74.5 (C-2′’), 73.5 (C-3′’), 70.6 (C-4′’), 62.0 (C-6′’).Measured data were in accordance with those reported in the literature : ESI-qTODrosera dry extract was dissolved in acetonitrile/H2O, 1:1, v/v, (DE) or Krebs–Henseleit buffer (DFA) and analyzed at a concentration of 0.5 mg/mL via Waters Acquity UPLC system on an Acquity UPLC HSS T3  stationary phase . Mobile phase: (A) water with formic acid 0.1%; (B) acetonitrile with 0.1% formic acid; gradient: 0 to 2 min: isocratic at 10% B; 2 to 5 min: linear to 20% B; 5 to 11 min: linear to 45% B; 11 to 12.5 min: linear to 100% B; 12.5 to 14.5 min: isocratic at 100% B; 14.5 to 15 min: linear to 10% B. Column temperature: 40 °C. Flow rate: 0.5 mL/min. Injection volume: 2 µL. Compounds were detected using a Waters Acquity PDA eλ (λ = 200 to 800 nm) and a Waters Acquity QDa detector in positive ionization mode . For quantification, the respective UV maxima were used . For determination of the amount of 2″-O-galloylhyperosid, a four-point calibration, and for quercetin and hyperoside, a five-point calibration curve with standards (double injection) was used. Samples of DE and DFA were measured as independent triplicates (n = 3). Representative UV chromatograms of DE and DFA at λ = 360 nm are displayed in 2.Male and female C57BL/6N mice  were housed under standard conditions. Animal care followed the rules of German laws . The mice were sacrificed using CO2:CO2 95:5). Drosera dry extract (0.5 mg/mL) was solved in Krebs–Henseleit buffer containing 1 % DMSO (DE). DFA was obtained as described above. Hyperoside, 2″-O-galloylhyperoside and quercetin were dissolved in Krebs–Henseleit buffer; for quercetin, 0.5% DMSO was added to ensure solubility up to the highest concentration (100 µM). Control solutions contained equal amounts of DMSO depending on the respective test compound. Experiments were started after an equilibration time of 60 min. Tracheal tension was measured isometrically . The last ACh-induced contraction (100 µM) before adding test compounds was set as 100%. Test solutions were added 10 min before the next ACh bolus. When using a high potassium solution (60 mM K+), NaCl concentration was reduced to maintain osmolarity. At least 3 K+-induced contractions (each 20 min) were averaged and taken as control contraction. The maximal contraction represents the highest value, while the plateau (plat.) was calculated in minutes 19–20. IC50 values were calculated by the fitting of sigmoidal dose–response curves with a variable slope using GraphPad Prism version 3.00 for Windows .A Mayflower horizontal tissue bath  was used to measure contractions. Murine tracheal slices of 3 mm were placed in the 5 mL tissue bath at a preload of 0.2 cN. Krebs–Henseleit buffer (37 °C) was continuously gassed (O2. The medium was changed every 3 days. CBF was measured up to 7 days after dissection. Slices were placed in a 0.7 mL perfusion chamber at 37 °C and perfused with Krebs–Henseleit buffer (1 mL/min). The tissue bath was located at an inverted Nikon Eclipse Ti2 microscope  with a 100× oil objective. Drosera dry extract (0.2 mg/mL) was solved in Krebs–Henseleit buffer containing 0.3% DMSO (DE). The amount of DMSO was not exceeding 0.3%; as for this concentration, no effects on CBF were reported [A was prepared as described above. Quercetin, 8MmIBMX, and rolipram were solved in Krebs–Henseleit buffer containing 0.1% DMSO. 2″-O-Galloylhyperoside, ATP and salbutamol were solved without DMSO. In case DMSO was added, all test solutions of one experiment contained the same amount of DMSO. For calculation of the CBF, videos were recorded with a high-speed (200 frames per second) Basler aceA1300–200um camera  after an equilibration period of 20 min. Videos were captured and analyzed by the SAVA software  [basal), and effects on ciliary beating were expressed as a ratio (CBFRatio).Murine tracheal explants were placed in LHC-9 medium , cut into 0.5 mm slices and stored at 37 °C and an atmosphere of 5% COreported . DFA wasMI, USA) . CiliateMI, USA) . Data we50 values were calculated by a fitting of sigmoidal dose–response curves (variable slope) with GraphPad Prism version 3.00 for Windows .The activity of PDE4D and PDE1A was measured with assay kits #60345 and #60310 from Biomol . Assays were based on fluorescence polarization using fluorescein amidites-labeled cAMP and were performed according to the manufacturer’s instructions. Samples were recorded as duplicates in a 96-well plate containing equal amounts of DMSO (1%). The fluorescence polarization was measured at excitation wavelengths between 480 and 516 nm and emission wavelengths between 530 and 540 nm by using the plate reader CLARIOstar . The ICt-test, while ANOVA  followed by Student–Newman–Keul’s post hoc test was used for multiple comparisons. The null hypothesis of single experiments was that extracts or test solutions do not differ in the respective parameter. Statistical significance is considered if p-values are ≤0.05. Confidence intervals (CI) were used when IC50 values were calculated.Tissue bath experiments were performed using tracheal slices of 3 to 5 different mouse preparations. Measurements of CBF were carried out by using tracheal slices of at least 3 different preparations, while a single measurement represents an independent experiment. All results are expressed as the means ± SD. Different conditions were compared by a two-tailed paired Student’s


Caralluma tuberculata N. E. Brown. methanolic (CTME) and aqueous (CTAQ) extracts. Chemical characterization was done by high-performance liquid chromatography and gas chromatography–mass spectrometry analysis. The Complete Freund’s Adjuvant (CFA) was injected in left hind paw of rat at day 1 and dosing at 150, 300 and 600 mg/kg was started on the 8th day via oral gavage in all groups except normal and disease control rats (which were given distilled water), whereas methotrexate  was administered to standard control. The CTME and CTAQ exerted significant (p < 0.01–0.0001) in vitro anti-arthritic action. Both extracts notably reduced paw edema, and restored weight loss, immune organs weight, arthritic score, RBCs, ESR, platelet count, rheumatoid factor (RF), C-reactive protein, and WBCs in treated rats. The plant extracts showed significant (p < 0.05–0.0001) downregulation of tumor necrosis factor-α, Interleukin-6, -1β, NF-κB, and cyclooxygenase-2, while notably upregulated IL-4, IL-10, I-κBα in contrast to disease control rats. The plant extracts noticeably (p < 0.001–0.0001) restored the superoxide dismutase and catalase activities and MDA levels in treated rats. Both extracts exhibited significant anti-arthritic potential. The promising potential was exhibited by both extracts probably due to phenolic, and flavonoids compounds.Present research was planned to assess the in vitro and in vivo anti-arthritic potential of  Rheumatoid arthritis (RA), also known as rheumatism, is a chronic autoimmune disorder associated with tenacious inflammation, stiffness, pain and damage of synovial joints, which lead to physical disability. Symptoms of RA include fatigue, malaise, fever and pain in multiple joints, and prolonged morning stiffness along with various extra-articular manifestations . The worThe exact etiology of the disease is not known yet. There is a discharge of autoantibodies that contribute to the progression of disease apart from articular and systemic inflammation . The innThere is no distinct treatment for the RA; rather, different treatment strategies are adopted for the symptomatic relief and to delay the progression of disease . CommonlCaralluma tuberculata N. E. Brown. (CT), commonly known in Urdu as Chunga and in English as bittercress, is an ethanomedicinally imperious food plant belong to family Apocynaceae. It is a perennial leafless juicy herb, and its stem is consumed as a vegetable by local inhabitants [C. tuberculata (whole plant) using in vitro and in vivo tests that may aid in developing an easily available, cost-effective treatment strategy. Additionally, chemical characterization of it by high-performance liquid chromatography (HPLC) and gas chromatography–mass spectrometry (GC-MS) in order to explore its chemical profile. To now, GC-MS analysis of this plant has not been carried out.abitants ,14. It iabitants . Traditiabitants ,17. Prevabitants . Three fabitants ,19. Prevabitants . The proHealthy Wistar rats, weighing 120–170 g, were retained in the Animal House of the GC University, Faisalabad (GCUF) at temperature (25 ± 2 °C), humidity (60 ± 5%), for 12 h dark/light cycles with easy access to water and diet. The animals adapted for 2 weeks prior to the start of the study. The approval for animal study protocol was taken from Institutional Review Board, GCUF, (GCUF/ERC/2221). The guidelines of National Institute of Health which are concerned with the care and handling of rodents were followed.®, Chelmsford, UK) at 40 °C for CTME and 50 °C for CTAQ under reduced pressure. It was further dried to a solid or semisolid mass through air-drying and stored in fridge at 8 °C till further use [The whole plant of CT (6 kg) was collected from Faisalabad in November 2020 and identified by a taxonomist of University of Agriculture, Faisalabad, against Voucher no. 1117-20-04. The plant was washed, dried under shade and processed through coarse grinding. To prepare methanolic (CTME) and aqueous extracts (CTAQ), the plant material was separately soaked in methanol and distilled water (DW) (1:5), extracted by a cold maceration process with daily shaking for up to seven days. Both extracts were filtered and passed through Whatman filter papers. The extraction residues were soaked in relevant solvent again and the process was repeated twice. The filtrates were pooled and concentrated by rotary evaporator  fitted with a UV-visible spectrophotometer. The absorbance was measured at 280 nm and then phenols and flavonoids were quantified by comparing retention time of the standards [HPLC analysis was executed by adopting an earlier method . The samtandards .Analysis was carried out using GC-MS . The column DB 5MS as stationary phase and 99.9% Helium was set to the rate of 1 mL/min. The oven temperature was set to 50 °C for 1 min then gradually raised at 25 °C/min to 120 °C for 5 min. The inlet was set to 275 °C and sample solution (1 µL) injected into the injector. The MS set to 230 °C for 51.133 min. The standard chromatogram was used to identify unknown compounds [The egg–albumin–protein denaturation assay was performed by following the earlier method . DiffereThe bovine serum albumin (BSA) denaturation procedure described earlier  was follv/v suspension was made with isosaline solution. The test solution comprised of PBS , 2 mL hypotonic saline (0.36% w/v NaCl), 0.5 mL extract  and 0.5 mL blood suspension. The solution test control consisted of 2 mL DDW, while the standard solution was comprised of piroxicam in place of the extract solution. The solutions were placed at 37 °C for 30 min and centrifuged for 15 min. The absorbance of supernatant was taken at 560 nm. The HRBC membrane stabilization assay was performed by the procedure described earlier . Brieflyn = 6). The group I served as a normal control (NCG) ; group II was the disease control (DCG) (negative control) and were given DW (1 mL). Group III was given standard drugs ; other groups were separately given CTME and CTAQ at 150, 300 and 600 mg/kg for 21 days [The male rats were indiscriminately distributed to nine groups  and were further determined on day 7, 12, 16, 20, 24 and 28. The arthritic scoring was also visually recorded and ranged from 0–4  at various days during the course of the study period. On the 29th day, blood samples were collected in plain and ethylenediamine tetraacetic acid (EDTA) tubes and subjected to hematologic and biochemical testing parameters through the chemical analyzer by using commercially available kits.All the rats were sacrificed after the 28th day following anesthesia with ether. The spleen and thymus were removed from each rat, clean and weighed. The left hind limb was removed from the rats for histopathological examination . The ankw/v liver homogenate was prepared by adopting previous methods [For this purpose, 10%  methods . Protein methods .To estimate superoxide dismutase (SOD) activity, pyrogallol method was adopted . To meas®, Waltham, MA, USA followed by synthesis of complementary DNA by adopting Thermo ScientificTM, Waltham, MA, USA kit method and kept at −20 °C.The pro- and anti-inflammatory cytokines  were quantified in blood after 28 days of CFA-induced arthritic study by qRT-PCR. For this purpose, RNA extraction was performed from blood by the TRIzol method using a kit from Invitrogen®, Seoul, Korea) method was used to run qRT-PCR . Briefly, a reaction mixture (20 µL) having 10 µL qPCR master mix, 1 µL cDNA, 1.5 µL each of forward and reverse primer and 6 µL of nuclease-free water was prepared and vortexed. The microplates were sealed with transparent film and given a short spin to avoid air bubbles. The programming of qPCR was set for 40 cycles at 90 °C for 2 min, 60 °C for 15 s and at 72 °C for 1 min. The relative expression (2−ΔΔCT) of biomarkers were calculated [For quantification and amplification, 2X Syber green qPCR master mix kit (GeneAlllculated . GAPDH wlculated .p < 0.05.All the data was expressed as mean ± standard deviation (S.D). The data of in vitro assays, paw diameter and body weight were analyzed by two-way analysis of variance (ANOVA) and that remaining by one-way ANOVA followed by Tukey’s test. The level of significance was The yields of CTME and CTAQ were 10.78% and 13.1% respectively. The phytochemical analysis indicated the presence of carbohydrates, flavonoids, phenols, tannins and alkaloids in CTME and CTAQ, whereas glycosides, steroids and terpenes were detected in CTME only. Saponins were detected in CTAQ only and proteins were not detected in any extract. Quantitative estimation of phytochemicals in CTME through HPLC showed that catechin, gallic acid, caffeic acid and ferulic acid were detected in it as mentioned in The GC-MS chromatogram of CTME confirmed the presence of various compounds as listed in p < 0.01–0.0001) inhibited protein denaturation as equated to standard, except CTME 800 and CTAQ 400 µg/mL, which were non-significant to the similar concentrations of standard control as shown in 50 (µg/mL) is given as piroxicam (437.6) > CTME (278.2) > CTAQ (271.5).Both extracts and piroxicam showed a concentration-reliant rise in percentage inhibition of egg albumin. The CTAQ exhibited highest percentage inhibition (76.53 ± 0.19%) as compared to CTME (73.96 ± 0.03%) and standard (74.62 ± 0.07%) at 1600 µg/mL. Both extracts considerably (p < 0.001) maximum percentage inhibition (83.41 ± 0.49%) at 1600 µg/mL in contrast to the standard control (81.59 ± 0.55%) and CTME (77.52 ± 0.38) as shown in p < 0.001–0.0001) inhibited BSA denaturation as compared to the standard except for CTAQ 100 µg/mL that was non-significant at similar concentration of standard control. Decreasing order of IC50 (µg/mL) is given as: CTME (753.4) > CTAQ (502.1) > piroxicam (279.4).The anti-denaturation assay using BSA revealed that both extracts inhibited BSA denaturation concentration dependently. The CTAQ showed notably (p < 0.0001) membrane stabilization dose-dependently and highest percentage protection was noticed with 1600 µg/mL. Whereas, the highest percentage protection was observed with CTAQ (67.64 ± 0.57%) at 1600 µg/mL as compared to standard drug (64.08 ± 0.53%) and CTME (61.32 ± 0.31%) as presented in 50 (µg/mL) is given as: CTME (386) > piroxicam (321.3) > CTAQ (217.6).Both extracts exhibited profound (p < 0.0001) rise in paw diameter was seen in the disease group at 8th day up to 28th day in comparison to normal control, and peak swelling was observed on 12th day. Treatment with both extracts prominently (p < 0.0001) decreased paw diameter in contrast to the DCG. Both extracts inhibited paw inflammation dose-dependently and significant percentage inhibition was observed at 600 mg/kg dosage. The maximum inhibitory effect on paw edema was noticed with CTAQ 600 mg/kg (4.18 ± 0.13 mm) on 28th day compared CTME (4.45 ± 0.13 mm) and the standard control group (4.26 ± 0.07 mm) as shown in The CFA administration resulted in inflammation in the injected paw in all rats and prominent swelling was saw at 8th day in all rats as compared to NCG. A noteworthy (p < 0.0001) decrease in body weight from day 8th until the end of study in comparison to normal rats. Whereas CTME, CTAQ (150–600 mg/kg) and methotrexate conspicuously (p < 0.05–0.0001) restored body weight as equated to DCG from 16th to 28th day. The loss of body weight was significantly ameliorated by CTAQ 300 (148.67 ± 4.93 g) followed in orders of effects by CTAQ 600 (148.5 ± 1.29 g), standard control (147.4 ± 3.21 g), and CTME 600 mg/kg (145 ± 1.83 g) in contrast to DCG (122.6 ± 3.05 g) at 28th day. The restoration of BW in CTME 600 and CTAQ 600 mg/kg-treated arthritic rats was non-significant as compared to standard control group on 28th day.There was decreased BW in arthritic rats as disease progressed in contrast to normal rats b. Animalp < 0.05–0.0001) in standard control (0.50 ± 0.54), CTAQ 600 (1.33 ± 0.51), CTAQ 300 (1.83 ± 0.41), CTAQ 150 (2.17 ± 0.75), CTME 600 (1.67 ± 0.52), CTME 300 (2.17 ± 0.41) and CTME 150 mg/kg (2.67 ± 0.52) treated groups in contrast to diseased rats as presented in A continuous increase in arthritic scoring was exhibited by DCG after CFA inoculation, in contrast to normal rats as shown in p < 0.0001) reduced in DCG in contrast to normal rats that notably (p < 0.05–0.0001) restored with CTME, CTAQ and methotrexate treated groups (p < 0.01) reduced values in the standard control (9.67 ± 1.53 mm/h) and CTAQ 600 (11.33 ± 1.53 mm/h)-treated arthritic rats as compared to DCG  were notably  restored CRP and RF levels in treated arthritic rats, in contrast to DCG.The systemic biomarkers like CRP (45.6 ± 2.25 mg/L) and RF (54.93 ± 1.72 IU/L) were increased significantly in the disease group in contrast to the normal group as shown in p < 0.0001) reduction in platelet count and ALP values in comparison to DCG (p < 0.05) in methotrexate and CTME 150-, 300- and CTAQ 150–600- mg/kg treated groups  which showed severe inflammation as shown in p < 0.05) in CTAQ, CTME at 600 mg/kg and standard therapy treated groups  as mentioned in After 28 days of studying CFA polyarthritis, it was found that the spleen (1.44 ± 0.11 g) and thymus (0.68 ± 0.04 g) weight elevated in the diseased control animals in comparison to the normal rats. The weight of organs restored considerably  in the CFA-induced arthritic study. The treatment with both plant extracts and standard drugs significantly augmented the SOD and CAT activities in arthritic rats as shown in p < 0.0001) reinstated the antioxidant enzyme activities as equated to DCG.It was noticed that there was a decreased activity (U/mg of protein) of SOD (6.56 ± 0.38) and CAT (8.25 ± 1.44) in 10% p < 0.0001) reinstated the MDA concentration in liver homogenate of treated arthritic rats as compared to diseased control rats (The concentration of MDA was profoundly upraised in the disease group (15.08 ± 0.19 µM/mg of protein) in comparison to the normal control (6.94 ± 0.14 µM/mg of protein). The treatment with CTME (9.15 ± 0.52 µM/mg of protein), CTAQ at 600 mg/kg (8.58 ± 0.18 µM/mg of protein) and standard control substantially (rol rats c.p < 0.0001) upregulation in the mRNA expression level of TNF-α, IL-6, -1β, and NF-κB was noticed in the diseased control group  in contrast to normal group. The level of those pro-inflammatory biomarkers were notably (p < 0.0001) reinstated in standard control, CTAQ 600 and CTME 600 mg/kg treated groups in contrast to DCG as presented in After 28 days of study, the mRNA expression of different inflammatory biomarkers was quantified by real-time PCR. A substantial (p < 0.0001) in diseased group (6.86 ± 0.11-fold) in contrast to normal while noticeably restored in standard group (2.09 ± 0.09-fold), CTAQ 600 (2.45 ± 0.09-fold) and CTME 600 mg/kg (2.75 ± 0.08-fold) treated animals in contrast to DCG (The levels of COX-2 were increased noticeably (t to DCG h.p < 0.0001) downregulation in IL-4,-10, and I-κBα expression in the diseased control group  in comparison to normal group in CFA induced polyarthritis. While a substantial (p < 0.0001) upregulation in the expression of these anti-inflammatory biomarkers in the standard control group , CTAQ 600  and CTME 600 mg/kg -treated groups was observed in contrast to diseased control animals as mentioned in A substantial  reduced in standard control group and CTAQ 600 mg/kg group as compared to DCG. The CRP production is aggravated by an increased level of pro-inflammatory cytokines such as IL-6 and TNF-α [Physical examination of disease progression is estimated by measuring arthritic score; this was notably reduced with treatment in contrast to DCG throughout the study period. The estimation of disease stage is done by measuring erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) and RF value in serum. These are also biomarkers for the estimation of systemic inflammation ,39. Incrnd TNF-α . The stup < 0.05–0.0001) downregulation of mRNA expression of pro-inflammatory cytokines and COX-2 in treated rats, while notably upregulating the expression of anti-inflammatory cytokines (IL-4 and IL-10) as compared to DCG. This showed the symptomatic and systemic relief by CT extract in CFA-induced polyarthritis as evident from histology slides of ankle joints of treated rats. The similar findings on arthritic parameters were exhibited in various previous studies [During an inflammatory process, pro-inflammatory cytokines  are released from activated monocytes and macrophages. TNF-α activates the release of two other pro-inflammatory cytokines that results in the release and infiltration of leukocytes resulting in vasodilation at the site of edema. TNF-α and IL-1β stimulate the production of MMPs inside the inflamed joint that exacerbated joint destruction. In order to cope with this, inhibition of pro-inflammatory cytokines is mandatory  to halt  studies ,43.The NF-κB is a member of group of transcription factors and responsible for different gene expressions that are involved in immunity and various inflammatory processes. The NF-κB have strong effect on synovial tissues in RA and is detected in activated form in human synovial tissues at early and later stages of inflammation . The I-κThe 2-Methoxy-4-vinylphenol exhibited antimicrobial, antioxidant, anti-inflammatory, analgesic . The γ-tC. tuberculata.Phytosterols such as stigmasterol, β-sitosterol, campesterol exhibited antioxidant, anti-inflammatory and antimicrobial activity. These exhibited anti-inflammatory potential via reducing COX-2, nitric oxide, and proinflammatory cytokines ,51,52. VC. tuberculata extracts exhibited anti-arthritic effects via stabilizing membrane, inhibiting protein denaturation, reducing edema, and restoring body and immune organ weights. Moreover, these treatments restored altered blood parameters in treated rats. Both extracts exhibited downregulation of TNF α, IL-6, -1β, NF-κB, and COX-2, while notably upregulating IL-4, -10, and I-κB as compared to DCG. Additionally, both extracts noticeably (p < 0.001–0.0001) restored the SOD, CAT activities and MDA level in treated rats in contrast to DCG. The CTAQ exerted profound effects in masking disease symptoms as compared to CTME at 600 mg/kg. There is an immense need for the isolation of required phytochemicals from CTME and CTAQ that are responsible for the anti-arthritic activity. Other anti-inflammatory mechanisms of CT should be found out. Clinical trials should be conducted on this substance prior to its use as a nutraceutical.It is concluded from the findings that


The compounds form lamellar self-assemblies in crystals. The energetic landscapes of single crystals of a series of studied amphiphilic gelators have been analyzed to explore the gelling properties. The presented results may be used as model systems to understand which supramolecular interactions observed in the solid state and what energy contributions are desired in the designing of new low-molecular-weight organic gelators. Lauric acid was used to synthesize the low-molecular-weight organogelators (LMOGs), derivatives of two endogenous (L)-alanine, (L)-leucine, and three exogenous (L)-valine, (L)-phenylalanine, and (L)-proline amino acids. The nature of processes responsible for the gel formation both in polar and in apolar solvents of such compounds is still under investigation. Knowing that the organization of surfactant molecules affects the properties of nano scale materials and gels, we decided to elucidate this problem using crystallographic diffraction and energy frameworks analysis. The single crystals of the mentioned compounds were produced successfully from heptane/ Pharmaceutical gels are willingly applied in pharmaceutical formulations where active pharmaceutical ingredients (API) are combined to yield a final medicinal product of well-defined properties. The stability of the obtained formulation is a crucial issue in the pharmaceutical and cosmetics industries . StabiliSmall particles are unstable with respect to agglomeration to the bulk caused by van der Waals forces, in the absence of any repulsive forces. This repulsive interaction can be facilitated by two methods: electrostatic and steric stabilization . ElectroIn many cases, the shape , size, aAnother pharmaceutical application of surfactants leads in their ability to form organogels . The orgIn both nanoparticle and organogel drug delivery systems, the biocompatibility of the surfactants used is a crucial issue. Herein, we decided to overcome the natural toxicity of the common surfactants by condensation of naturally occurred molecules: fatty (lauric) acid and amino acids. Indeed, several studies already showed that such amides can act as efficient and biodegradable gelators ,28,29,30N-dodecanoyl-L-amino acid derivatives. The energy framework approach was previously successfully applied in explaining many problems regarding polymorphism, co-crystals, supramolecular synthons, solid state reactions and sol-to-gel transition process [The role of the interactions between such surfactants during gelation is still under investigation. The self-assembled fibrillary network (SAFiN) formation was proved to be related to the hydrogen bond’s formation ability ,33,34,35 process ,42,43,44Amides based on amino acids linked to long alkyl chains (>C4) do not crystallize easily to give single crystals of good quality. In the Cambridge Structural Database (version 5.42 with updates)  there arN-dodecanoyl-L-amino acid derivatives for which we managed to obtain single crystals by recrystallization from heptane/tBuOMe mixture to explore if the solid state supramolecular synthons and the energy frameworks may be an indicator for desirable gelling properties. Obtained gelators were proved to be suitable for organogels and hydrogels formation.Here, we present the study of crystal packing and energy frameworks of a series of amphiphilic gelators based on 1H NMR (500 MHz) and 13C NMR (125 MHz) spectra were taken on a Bruker AVANCE 500 MHz spectrometer using CDCl3 as a solvent  temperature gels in toluene, water, t-BuOMe and petroleum ether were formed as presented in α radiation (λ = 1.54184 Å). The structural models were obtained by direct methods using the SHELXS-97 program [F2 using the SHELXL-97 program implemented on Olex2.refine package [R1 = 10.96% [The crystal structures of the analyzed compounds 1-Ala, 2-Pro, 3-Phe, 4-Leu, and 5-Val were determined based on the X-ray diffraction experiment performed on single crystals at 120 K on SuperNova X-ray diffractometer, CuK program  and refi package . Crystal= 10.96% . The pro15H29NO3 (M = 271.39 g/mol): triclinic, space group P1 (no. 1), a = 4.8852(2) Å, b = 5.5958(2) Å, c = 30.2891(13) Å, α = 90.765(3), β = 91.034(4), γ = 105.561(4), V = 797.37(6) Å3, Z = 2, T = 120.10(10) K, μ(CuKα) = 0.615 mm−1, Dcalc = 1.1303 g/cm3, 15,118 reflections measured (5.84 ≤ 2Θ ≤ 144.06), 5297 unique  which were used in all calculations. The final R1 was 0.0448 (I ≥ 2σ(I)) and wR2 was 0.1537 . CCDC No. 2226607.Crystal Data for 1-Ala, C17H31NO3 (M = 297.43 g/mol): monoclinic, space group P21 (no. 4), a = 6.65959(13) Å, b = 8.08116(17) Å, c = 15.9905(3) Å, β = 90.4021(17)°, V = 860.54(3) Å3, Z = 2, T = 120.10(10) K, μ(CuKα) = 0.612 mm−1, Dcalc = 1.1478 g/cm3, 4825 reflections measured (5.52° ≤ 2Θ ≤ 143.54°), 2282 unique  which were used in all calculations. The final R1 was 0.0297 (I ≥ 2σ(I)) and wR2 was 0.0900 . CCDC No. 2226609.Crystal Data for 2-Pro, C21H33NO3 (M = 347.48 g/mol): orthorhombic, space group P212121 (no. 19), a = 5.3354(2) Å, b = 12.7541(6) Å, c = 28.9824(14) Å, V = 1972.19(16) Å3, Z = 4, T = 120.10(10) K, μ(CuKα) = 0.607 mm−1, Dcalc = 1.1703 g/cm3, 5142 reflections measured (6.1° ≤ 2Θ ≤ 140.02°), 3303 unique  which were used in all calculations. The final R1 was 0.0487 (I ≥ 2σ(I)) and wR2 was 0.1494 . CCDC No. 2231565—redetermination of CCDC No. 1959468 CSD Refcode WUWJUW [Crystal Data for 3-Phe, C18H35NO3 (M = 313.47 g/mol): orthorhombic, space group P22121 (no. 18), a = 5.32892(18) Å, b = 13.4149(4) Å, c = 27.0501(8) Å, V = 1933.73(10) Å3, Z = 4, T = 120.10(10) K, μ(CuKα) = 0.564 mm−1, Dcalc = 1.0767 g/cm3, 6310 reflections measured (6.54° ≤ 2Θ ≤ 144.2°), 3157 unique  which were used in all calculations. The final R1 was 0.0387 (I ≥ 2σ(I)) and wR2 was 0.1046 . CCDC No. 2226608.Crystal Data for 4-Leu, C17H33NO3 (M = 299.44 g/mol): orthorhombic, space group P212121 (no. 19), a = 32.7663(7) Å, b = 9.9958(2) Å, c = 5.68440(11) Å, V = 1861.79(7) Å3, Z = 4, T = 120.10(10) K, μ(CuKα) = 0.566 mm−1, Dcalc = 1.0677 g/cm3, 8144 reflections measured (9.24° ≤ 2Θ ≤ 143.98°), 3259 unique  which were used in all calculations. The final R1 was 0.0639 (I ≥ 2σ(I)) and wR2 was 0.2052 . CCDC No. 2226610.Crystal Data for 5-Val, Chttp://www.ccdc.cam.ac.uk/conts/retrieving.html  (accessed on 13 December 2022).CCDC No. 2226607–2226610 and 2231565 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via Hirshfeld analysis is a well-known method for exploring crystal data to gain deeper insight into the characteristics of intermolecular interactions in a solid state ,53,54,55N-dodecanoyl-L-amino acid derivatives was based on the optimization of well-defined approaches which usually allow obtaining the amino acids of high purity in acceptable yields [tBuOMe mixture. The alternative approaches tested to prepare the targeted fatty acid amides were less efficient [The synthesis of the five e yields . Thus, tfficient ,29,60,61N-lauroyl-L-alanine (1-Ala)-based hydrogel was tested as a supramolecular drug carrier [3 melts above 19 °C. The gelation of alcohols was not successful; however, gel obtained with 40% v/v aqueous ethyl alcohol was stable for a few days at ambient temperature, and it changed slowly from nanofiber (gel) to a crystalline form by dissolving-crystallisation process. The last observation proved that gels could be obtained in solvent mixtures, where the solubility of the gelator in at least one of the solvents used is low. The stability of gels is higher in the cases, where a possibility of dissolving of nanofiber of gelators and its recrystallization is limited. On the other hand, solvents in which the gelator is well soluble  might not be gelated in a pure form.The enriched with gold nanoparticles  carrier . The gel carrier . The exaP1, P21, P212121 and P22121, As pure enantiomers all studied compounds crystallized in chiral space groups  seems not to determine the crystal packing mode. The 3-Phe, 4-Leu and 5-Val which form similar folded layers show the conformations of the aliphatic chain from synclinal  through anticlinal (3-Phe) up to antiperiplanar (4-Leu) ones . The separation of hydrophilic and lipophilic regions  which maa and c directions. Various colors represent various energy terms: red—coulombic, yellow—polarization, green—dispersion, and blue—total energy. The scale for tube size was 150, the energy cut-off 5 kJ/mol. The energy frameworks indicate the dominance of coulombic and dispersion energies in the crystal nets. In crystal packing, there is an evident division for hydrophilic and lipophilic sections . Within 22(8) motif resulting in high total energy of ca. −116 kJ/mol. At the higher level of the graph set theory both of the mentioned motifs link into a R44(24) ring. The final patterning in 1-Ala may be considered to be parallel tapes between which dispersion forces are present. Such arrangement does not seem to ease a possible formation of gel, since the hydrophilic parts are strongly bonded and the rearrangement of the molecules to accommodate solvent would demand a large energy input.In the case of 1-Ala, the supramolecular primary level motif is a C(4) chain formed between amide groups (E_tot = −55.6 kJ/mol), which is assisted by dispersion interactions between parallelly arranged amphiphilic molecules. However, it is not the strongest interaction in this crystal. The carboxylic acid fragments preserve the usual cyclic R2-Pro exhibits only a simple C(7) herring-bone pattern via O–H…O_amide contact (E_tot = −42 kJ/mol). The maximum total energy of pairwise interaction is smaller than in the other structures (−46 kJ/mol) and comes mainly from dispersion term. This may indicate possible formation of polymorphs. However, the blocked N atom limits the prospects of this compound to interact freely with solvent molecules.44(20) ring with four donors and four hydrogen bond acceptors, in which the same functional groups are involved. These crystals are not isostructural, since the alkyl parts of each are arranged differently. However, some degree of similarity among these lamellar structures is observed, which is also in accordance with the energy frameworks.Comparing the crystal packing of 3-Phe, 4-Leu, and 5-Val, the main noticeable feature is the common second level graph set RThe 3-Phe structure is dominated by two interactions of very similar total energies (−68.7 and −62.7 kJ/mol); however, their origin is entirely different. One of the interactions is a strong O–H…O_amide hydrogen bond with the highest contribution of coulombic factor (−87.1 kJ/mol). The other one is N–H…O_acid with moderate coulombic (−36.6 kJ/mol) and quite high dispersion (−60.3 kJ/mol) inputs.The strongest interaction in 4-Leu comes from O–H…O_amide hydrogen bond with high coulombic contribution (−77 kJ/mol) has a total energy of −54.5 kJ/mol. However, another pairwise total energy nearly equally strong is an effect of a combination of a weaker N–H…O_acid hydrogen bond (E_Coul = −31.5 kJ/mol) supported by high dispersion term (−52.6 kJ/mol) associated with a parallel alignment of the alkyl chains.22(8) ring. Additionally, the amide N–H…O_acid interaction is assisted by a weaker C–H…O_amide one (motif R22(11)) giving the contribution to the electrostatic energy.Structure of 5-Val is stabilized by similar interactions, but the much higher coulombic energetic term of ca. −92 kJ/mol in the case of O–H…O_amide h-bond is a consequence of an additional contribution of an accompanying C–H…O_acid contact which closes the RN-dodecanoyl-L-amino acid derivatives have been successfully determined. Energy frameworks analysis was applied for the first time to study the gelling behavior of a series of low-molecular-weight gelators, suitable for the formation of both organo- and hydrogels. The presence of carboxylic O–H and amide N–H groups impacted the most the crystal net energetics giving high coulombic contributions to the total energy in each of the studied structures. Three of the analyzed crystals  had the same cyclic supramolecular synthon R44(20), stabilized by hydrogen bonds. The different alignment of the aliphatic chains had a minor contribution to the total energy in the crystals, but the alkyl chains exhibited wide range of conformations from synclinal  through anticlinal (3-Phe) up to antiperiplanar (4-Leu) ones. This information may be useful when predicting the tangling ability of the lipophilic parts of the gelator molecules.The crystal structures of a series of Spontaneous formation of self-assembled fibrillary network (SAFiN), at least in the case of apolar solvents, seems to have its origin in two phenomena: (1) the hydrophilic parts of amphiphilic organogelators interact to each other influencing the stability of the gels as well as the energy necessary to initiate the gelling process; (2) the hydrophobic fibers may tangle up trapping in voids lipophilic solvent molecules. The combination of crystallographic studies and energy frameworks analysis may be a useful help in understanding the gelling behavior and in designing new low-molecular-weight organogelator (LMOG) materials.


Lavandula angustifolia, is one of the main lavender essential oil production crops in Xinjiang Province, China. Several cases of local usage indicated that ‘Taikong blue’ lavender essential oil (TLEO) had excellent anti-inflammatory and antioxidant properties for skin problems. However, to date, substantial data on these functions are lacking. In this study, we aimed to investigate the composition and bioactivities of TLEO and the potential underlying mechanisms through LPS-induced inflammatory models of HaCaT and RAW264.7 cells.‘Taikong blue’ lavender, a space-bred cultivar of The composition of TLEO was determined by GC‒MS. To study the anti-inflammatory and antioxidative properties of TLEO, we induced HaCaT and RAW264.7 cells by LPS. TLEO  was used to treat inflamed cells with dexamethasone  as the standard drug. A variety of tests were carried out, including biochemical assays, ELISA, RT‒PCR, and western blotting. Docking of components was performed to predict potential ligands.The GC‒MS analysis revealed that 53 compounds (> 0.01%) represented 99.76% of the TLEO, and the majority of them were esters. TLEO not only reduced the levels of oxidative stress indicators  but also protected the SOD and CAT activities. According to the RT‒PCR, ELISA, and Western blot results, TLEO decreased inflammation by inhibiting the expression of TNF-α, IL-1β, IL-6, and key proteins  in MAPK-NF-кB signaling. Molecular docking results showed that all of the components (> 1% in TLEO) were potent candidate ligands for further research.The theoretical evidence for TLEO in this study supported its use in skin care as a functional ingredient for cosmetics and pharmaceutics.The online version contains supplementary material available at 10.1186/s12906-022-03800-0. Skin is the largest organ of the body as well as the first defensive line to protect the organism against external stimulation, while macrophages are immune cells that protect host homeostasis by phagocytosis, cytokine production, and antigen presentation , 2. HoweTherefore, managing uncontrolled inflammation could be a good way to regulate inflammatory responses. Currently, there are two types of anti-inflammatory drugs used in therapeutics: steroidal anti-inflammatory drugs (SAIDs) and nonsteroidal anti-inflammatory drugs (NSAIDs) . NeverthLavandula angustifolia Mill was brought to Xinjiang, China, in the 1950s. 'Taikong blue' lavender is a new variety of L. angustifolia Mill produced by mutagenesis in space breeding techniques. From September 27th to October 15th, 2004, 100 g L. angustifolia Mill seeds were released into space on a Long March 2-D carrier rocket as the 20th recoverable science and technology experimental satellite. After a year of cultivation, the researchers discovered that the first space seeds of 'Taikong blue' lavender had numerous benefits over L. angustifolia Mill cultured in the same place, such as longer blooming periods and higher oil yield, and it has become a significant cultivated species in Xinjiang  assays with HaCaT and RAW264.7 cells. HaCaT and RAW264.7 cell suspensions were added to each well of a 96-well plate, and the cell number in each well was approximately 2 × 10For analysis of the roles of TLEO in LPS-induced protective effects, HaCaT and RAW264.7 cells were grown and treated in 96-well plates as described above. After 12 h of culture, the cells were processed for MTT assays as described previously . Cell cy3 to determine ROS and NO levels. ROS production was identified using the ROS Detection Kit (Beyotime Biotechnology). In brief, the supernatant was discarded, and 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA)  diluted with DMEM was added to the plate. The cells were then cultured for 20 min in the incubator. Cells were washed three times using DMEM without FBS to remove DCFH-DA from the plate. The ROS level was measured using a microplate reader infinite with excitation at 488 nm and emission at 525 nm.HaCaT and RAW264.7 cell suspensions were added to each well of a 96-well plate. Finally, the cell number in each well was approximately 2 × 10After the treatments described above, the NO production assay was performed in triplicate by using the Griess assay according to the instructions given by the kit. The absorbance values of the colored solution were measured using a microplate reader at 540 nm. These values were converted to micromoles per liter (μmol/L) with a standard curve obtained by adding 0–80 μmol/L sodium nitrate to fresh culture media. This experiment was performed in triplicate individually .6 cells/dish and 1.8 × 106 cells/dish, respectively. After the treatments described in 2.3, measurement of MDA levels in cells was performed using a Lipid Peroxidation MDA Assay Kit. MDA is an end-product of fatty acid peroxidation and can be measured via a chromogenic reaction between MDA and thiobarbituric acid (TBA). Briefly, 100 μL of cell lysate was mixed with 200 μL of MDA working solution followed by incubation in a 100 °C water bath for 15 min and then cooled to 25 °C. The mixture was centrifuged at 1000 × g for 10 min, and 200 μL of supernatant was used to measure absorbance at 532 nm. The TBARS concentration was calculated from an MDA standard curve.Three milliliters of HaCaT and RAW264.7 cells at the logarithmic growth stage were inoculated in 35 mm dishes at a density of 1.5 × 10For the CAT activity assay, cells were treated as described in 2.3. The cell lysates were centrifuged at 1600 rpm at 4 °C for 20 min, the supernatant was diluted to the proper concentration, and the protein concentrations were determined by a BCA protein assay kit (Beyotime Institute of Biotechnology). The catalase activity was measured following the kit’s instructions by a microplate reader at a wavelength of 520 nm.SOD activity was measured by a total superoxide dismutase assay kit with WST-8. After the same treatments described in 2.3, the protein concentrations of the cell lysate supernatant were determined by a BCA protein assay kit (Beyotime Biotechnology), and SOD activity was measured by an infinite microplate reader at a wavelength of 450 nm according to the manufacturer’s instructions , 16.6 cells/dish (HaCaT) and 1.8 × 106 cells/dish (RAW264.7 cells). Briefly, after the treatments of the cells as described in 2.3, total RNA was extracted from the cells with TRIzol. Complementary DNA (cDNA) synthesis was conducted with a ReverTra Ace qPCR RT kit. Quantitative PCR was performed on a CFX96™ real-time PCR machine. The mRNA level was calculated using the 2−∆∆CT method [Quantitative real-time polymerase chain reaction (qRT‒PCR) was used to detect the mRNA levels of proinflammatory cytokines. Three milliliters of cells were plated in a petri dish (35 mm) at a density of 1.5 × 10T method . The seq(1.5 × 106 cells/dish) and RAW264.7 cells (1.8 × 106 cells/dish) were inoculated in 35 mm dishes and incubated for 24 h at 37 °C in 5% CO2. After the same treatments of the cells as described in 2.3, a brief centrifugation step at 2000 × g was applied to remove dead cells and cell debris, and the supernatants were subjected to ELISAs. ELISAs were performed following the manufacturer’s instructions for TNF-α, IL-1β, and IL-6 levels.HaCaT cells 6 cells/dish HaCaT cells and 1.8 × 106 cells/dish RAW264.7 cells (each 3 mL) were seeded in dishes (35 mm). The cells were treated in the same way as in 2.3. Then, the cells were washed with cold PBS three times, and the proteins were extracted with a Total Protein Extraction kit. Protein concentrations were determined by a BCA protein assay kit. Equal amounts of proteins were analyzed in polyacrylamide gels by SDS‒PAGE and subsequently transferred to PVDF membranes. PVDF membranes were blocked with 5% nonfat milk in PBST and incubated with primary antibody overnight at 4 °C. Subsequently, the PVDF membranes were washed with PBST and incubated with secondary antibody for 1 h at room temperature. Bands were analyzed by chemiluminescence  with horseradish peroxidase-conjugated IgG [A total of 1.5 × 10ated IgG .The ADME properties of all TLEO components were predicted using Swiss-ADME. The 3D conformations of all 11 components (< 1% from GC‒MS result) were downloaded from the PubChem database . Analysis of variance (ANOVA) was performed using SPSS software . One-way ANOVA and Scheffe’s method were used to analyze the differences between the means. A significant difference was recognized at the level of For TLEO, 53 compounds were identified and are listed in Table Different doses of TLEO were added to cell cultures with or without LPS treatments to determine their effects on cell viability. As shown in Fig. p < 0.05) alleviated inflammatory mediator and oxidative factor production and protected antioxidative enzyme activities, similar to the control or even better. Notably, macrophages are more sensitive to LPS treatment. TLEO (0.1%) inhibited NO and ROS production to a lesser extent than DEX treatment. Compared with LPS-treated HaCaT and RAW264.7, the NO yield in 0.1% TLEO treatment was reduced by about 16.36 μmol/L and 111.83 μmol/L, respectively, which was lower than the NO reduction after DEX treatment (9.54 μmol/L and 27.37 μmol/L respectively). However, for the inhibition of iNOS protein expression, the best results were found with 0.01% TLEO (p < 0.05) in the two cell lines. The inhibition rates of iNOS in the 0.01% TLEO treatment group were approximately 49.49% and 32.49%. 0.1% TLEO reduced the ROS production of HaCaT and RAW264.7 stimulated by LPS, 74.09%, and 46.75%, respectively, which were higher than those in the DEX treatment group (12.22% and 32.80%). While for the inhibition of MDA production, 0.01% TLEO was used (p < 0.05) in HaCaT cells, which reduced the LPS-induced MDA of 52.42 μmol/mg to 29.70 μmol/mg. And 0.001–0.1% TLEO resulted in the same significance (p < 0.05) downregulation in the RAW264.7 cell line, the content of MDA was reduced by 5.88 μmol/mg, respectively, 10.38 μmol/mg and 8.37 μmol/mg. The SOD activity of the two LPS-stimulated cell lines under 0.01% TLEO treatment recovered to 13.45 U/mg and 30.00 U/mg, respectively, and the effect was similar to that of the DEX treatment group (13.77 U/mg and 28.88 U/mg), and both reached the normal level (12.58 U/mg and 30.27 U/mg). The same phenomenon was found with CAT only under treatment with 0.1% TLEO in LPS-treated HaCaT cells (p < 0.05), 0.1% TLEO increased CAT activity by 15.58 U/mg.As shown in Fig. p < 0.05) at the mRNA and protein levels in the LPS-treated RAW264.7 cells. At the mRNA expression level, TLEO inhibition rates for TNF-α, IL-1β, and IL-6 ranged from 40 to 50%, 12%-72%, and 40%-85%, respectively. While at the protein level, 0.001%-0.1% TLEO reduced the production of TNF-α treated with LPS by 462.79 pg/mL, 484.50 pg/mL, and 497.73 pg/mL, respectively, which was similar to that of the DEX treatment group (476.11 pg/mL). 0.001%-0.1% TLEO reduced the production of IL-1β in LPS treatment to 20.39 pg/mL, 20.26 pg/mL, and 17.52 pg/mL, respectively, similar to the DEX treatment group (20.37 pg/mL). 0.001%-0.1% TLEO reduced the production of IL-6 treated with LPS by 272.87 pg/mL, 432.60 pg/mL, and 774.83 pg/mL, respectively, with certain dose dependence. However, in the case of LPS-activated HaCaT cells, DEX showed the best inhibition of cytokine expression, the inhibition rates of TNF-α, IL-1β, and IL-6 reached 76.98%, 60.03%, and 62.65%, respectively .TLEO inhibited the gene expression of TNF-α, IL-1β, and IL-6 (p < 0.05). In the LPS-treated HaCaT model, the inhibition rates of 0.01% and 0.1% TLEO on the phosphorylation expression level of p38 were 36.35% and 43.46%, respectively. The inhibition rate of the phosphorylation expression level of p38 in the DEX treatment group was not significant. While TLEO of 0.001% and 0.01% significantly reduced the phosphorylation level of JNK, by 37.28% and 21.35% (p < 0.05), respectively, and the inhibition effect of 0.001% TLEO on JNK phosphorylation level was better than that of DEX (inhibition rate 23.82%). In the inflammatory model of LPS-stimulated RAW264.7, the inhibition rates of 0.001%-0.1% TLEO on p38 phosphorylation levels were 20.15%, 34.23%, and 23.82%, respectively, all slightly lower than those in the DEX treatment group (41.89%). Similarly, TLEO of 0.001%-0.1% showed a significant inhibition effect on JNK phosphorylation levels, with inhibition rates of 39.45%, 26.82%, and 25.61%, respectively (p < 0.05). DEX also slightly reduced the expression compared to that of the LPS group, although its effect was not as strong as that of TLEO.To investigate whether TLEO inhibits NF-κB activation via the MAPK pathway, we used Western blotting to examine the effects of TLEO on the LPS-stimulated phosphorylation of JNK and p38 MAPKs in HaCaT and RAW264.7 cells. As shown in Fig. p < 0.05). However, in the LPS-treated RAW264.7 cells, all TLEOs had almost the same inhibitory effects on the phosphorylation of these three NF-κB proteins. The inhibition rates of 0.001%-0.1% TLEO on IκBα, p65, and p50 phosphorylation levels ranged from 25%-50%, 15%-25%, and 10%-20%, respectively. The inhibition rate of the phosphorylation level of IκBα in the 0.001% TLEO treatment group (48.05%) was significantly higher than that in the DEX treatment group (25.05%) (p < 0.05). The inhibition rates of the phosphorylation level of p65 in the 0.001%-0.1% TLEO treatment group were 24.80%, 23.30%, and 18.95%, respectively, which was similar to that of the DEX treatment group (22.75%). The inhibition rates of the phosphorylation level of p50 in the 0.001%-0.1% TLEO treatment group were 18.59%, 14.29%, and 14.57%, respectively, significantly lower than the DEX treatment group (27.79%) (p < 0.05). However, in both LPS-stimulated cell lines, DEX had slight or no effects on the downregulation of NF-κB pathway proteins  in other LEOs were found to be significantly higher in TLEO, such as lavandulol acetate (4.77%), cis-β-ocimene (3.04%), 1-octen-3-yl acetate (2.62%), caryophyllene (2.49%), terpinen-4-ol (2.44%), -alloocimene (1.23%), β-ocimene (1.2%), geranyl acetate (1.11%), and β-myrcene (1.1%) [The major components in TLEO are linalool and linalyl acetate , similar to other e (1.1%) , 22. Spacimene 1.%, β-ocime (1.1%) . The maie (1.1%) .Dexamethasone (DEX), a standard anti-inflammatory drug, was used as a positive control in this study. DEX not only reduces the production of inflammatory cytokines, such as IL-6, TNF-α, and NO, by LPS-induced inflammation –27 but aE.coli. Through the toll-like receptor 4 (TLR4), LPS enters the body and activates macrophages, neutrophils, and endothelial cells like keratinocytes, which results in the production of numerous inflammatory factors, and oxidative stress markers and leads to organ dysfunction . So, the downregulation of the TLR4 pathway is one of the strategies to reduce inflammation in cells. We studied the most prominent pathway of inflammation, which is MAPK-NF-κB signaling. In our present study, TLEO showed good inhibition of the excessive production of NO and ROS in the LPS-treated HaCaT cells and RAW264.7 murine macrophages. This ROS-alleviating effect was also observed by other researchers. Kozics et al. (2017) found that pretreatment with EO from L. angustifolia could inhibit the oxidant stress induced by H2O2 in human hepatoma cells (HepG2) [L. stoechas could also significantly decrease the production of MDA and increase the activities of SOD and CAT to inhibit alloxan-induced oxidative stress in rats [We induced inflammation in our experiment with lipopolysaccharide (LPS), an endotoxin produced by  (HepG2) , 27, 29. in rats .L. angustifolia EO significantly reduced TNF-α and COX-2 expression by inhibiting NF-кB activity [L. x intermedia essential oil against C. rodentium-induced colitis in C57BL/6 mice, and the expression of typical inflammatory factors, such as TNF-α, IFN-γ, IL-22, and iNOS, was remarkably downregulated [The ROS alleviating effect of TLEO may be due to the downregulation of key proteins in MAPK-NF-κB signaling Fig. . In the activity . Baker eegulated .Our preliminary experiments showed that TLEO exhibited a better anti-inflammatory effect than essential oils from ‘French blue’ lavender and 701 lavender (types available in Xinjiang), two commonly used varieties in Xinjiang’s industry Fig. S. The comConsidering all 11 ligands, IKB-α protein from both humans and mice exerted the weakest effect, and JNK showed the highest effect. Caryophyllene, lavandulol acetate, and geranyl acetate were the top 3 ligands that bound to proteins in the MAPK-NF-κB signaling pathway with the lowest affinities. These 3 compounds are relatively abundant in TLEO and are present in trace amounts in other common LEOs. In addition to these compounds, linalyl acetate, as a major component in almost all lavender essential oils, also maintained a relatively high binding affinity , while 1-octen-3-yl-acetate had the weakest interaction.Caryophyllene interacted with JNK and p38 in both humans and mice with the lowest affinities > -7.0 kcal/mol. The human JNK protein interacted with caryophyllene by five alkyl bonds at Arg 72, Leu 76–77, Ile 86, and Ile 147 and four van der Waals bonds. The ligand shared eight alkyl bonds as well as four van der Waals bonds with human p38 amino acids ranging from 38 to 171 and was enriched with leucine residues. These properties make p38-caryophyllene more stable than JNK-caryophyllene in humans. However, in mice, JNK and p38 showed the same number of alkyl bonds, varying in the quantity of van der Waals bonds. As p50 and p65 work as dimers to activate the pathway, interacting with one of them could cause changes in the signal. Caryophyllene interacted with both human and mouse p50 with an affinity of -6.0 kcal/mol. These molecules shared alkyl and van der Waals bonds with five common residues: Lys 92, Ile 94, Gly 162, Tyr 163, and Phe 217  and JNK(B) protein from swiss model. Fig. S3. Molecular docking of TLEO components with Human and Mice IkB-αprotein. Fig. S4. Moleculardocking of TLEO components with Human and Mice JNK protein.Fig. S5. Molecular docking of TLEO components withHuman and Mice p50 protein. Fig. S6. Molecular docking of TLEO components with Human and Mice p65 protein. Fig. S7. Moleculardocking of TLEO components with Human and Mice p38 protein.Fig. S8. Effects of TLEO on mRNA andprotein level of pro-inflammatory cytokines IL-6 by LPS-induced RAW264.7cells. The data are the means ± S.D (n = 3). Statistical analysis was performedby one-way ANOVA with a scheffe’s test. “*” and “#”  indicate significant difference (p <0.05) compared with the LPS-treated group. Fig. S9. Total ionchromatography from GC-MS analysis of TLEO. Table S1. ADME analysis of TLEOcomponents. Table S2. Validation of mice protein IKB-α and JNK. Table S3. Molecular docking of major TLEO componentswith protein IKB-α. Table S4. Molecular docking of major TLEO componentswith protein JNK. Table S5. Molecular docking of major TLEO components withprotein p50. Table S6. Molecular docking of major TLEO components with protein p65. Table S7.  Molecular docking of major TLEO components with protein p38.


However, the energetic performance of cyclo-N5− compounds has certain limitations and cannot go beyond that of CL-20. In order to reach the higher energy, in this work, we presented two kinds of polynitrogen species, N4 and N8. Two isomers of N4 and four isomers of N8 were fully calculated by using density functional theory (DFT). Theoretical results show that all these polynitrogen materials exhibit excellent heats of formation (7.92–16.60 kJ g−1), desirable detonation performance , as well as the remarkable specific impulses (330.1–436.2 s), which are much superior to CL-20. Among them, N4-2 (tetraazahedrane)  and cube N8-4  have the highest energetic properties, which are expected to become promising high-energy-density-materials. Moreover, electrostatic surface potentials, Frontier molecular orbitals, infrared spectra, natural bond orbital charges, and weak interactions were also investigated to further understand their relationship between structure and performance.All-nitrogen materials, as a unique branch of energetic materials, have gained huge attentions, of which  With th0.0 GPa) . Unfortu−1), N = N (418.2 kJ mol−1) bonds in structure and forming N≡N (946 kJ mol−1) (3−), pentanitrogen cation (N5+) and pentazolate anion (cyclo-N5-) are successfully synthesized 6(H3O)3(NH4)4Cl, was reported in 2017  to RDX, and could not surpass CL-20  in the structures, which affect the whole performance  . Howeverthesized . N5+ waeratures . Hence,  in 2017 . Subsequstimated . It is dss CL-20 . Main reformance . Therefoformance .x, x from 3 to 60) are particularly attractive due to the extremely high heats of formation arising from abundant N-N and N = N high-energy bonds  calculations are helpful methods and widely used by many researches . So, an equation suggested by For covalent compounds, the intermolecular interactions within the crystals were considered to improve the accuracy of the density of the molecule  was performed with K-J equation proposed by Q is heat of detonation, N is the number of moles of the gas generated per gram, M̅ is the average molecular weight of gaseous product, and ρ is the calculated density.In the cases of this study, all the molecules have the characteristic of c ≥ 2a + b/2, so the calculation method of Q, N, and M̅ are:a, b, c and d stand for the number of C, H, O, N atoms in the explosive molecule, respectively; ΔHf is the standard heat of formation of the explosive. In addition, the detonation performances of these compounds were also evaluated by the EXPLO5 V6.05.04 program  thermochemical code at 6.86 MPa with an expansion ratio 70:1, and the supersonic section ratio is considered to be Ae/At = 10, whereas the initial temperature is 298.15 K  and detonation pressure  and 1.536 Å (N1-N2), and the bond angles are uniform 90°. N4-1 is a flat molecule, which can be evident from the dihedral angles of N4-N1-N2-N3, 0.021 and N1-N2-N3-N4, -0.017. Compound N4-2 exhibits a regular tetrahedron structure, with the bond length of 1.452 Å and bond angle of 60, and therefore is known as tetraazahedrane (TdN4). As for four N8 allotropes, N8-1shows a shape similar to a windmill, of which the bond lengths of double bonds (N1 = N2) are 1.220 Å and those of single bonds (N2-N3) are 1.509 Å. While N8-2 and N8-3 exhibit good planarity, with the dihedral angles of -179.9 , -0.02  and 0.06 . The N-N bonds distance in N8-2 range from 1.133 to 1.388 Å, where N2-N3 of azido group has the shortest value of 1.133 Å. The bond lengths of pentazole ring are 1.345 Å (N7-N8) and 1.304 Å (N6-N7), which are close to those of N8-3 . Similarly, the bond angles of pentazole ring in N8-2 and N8-3 are both in the range of 103.7°–114.1°. N8-4 shows a special cube structure, possessing the bond length of 1.521 Å and bond angle of 90.The optimized structures of two N4 isomers, tetraazahedrane (N4-2) has the shorter N-N bonds than rectangle N4-1, suggesting that the three-dimensional tetrahedral structure is conducive to enhancing the molecular stability. In addition to chain N4, tetrahedral form of N4 has already received growing concerns  has the longest bond distance of 1.521 Å. The structure of this material is the analogue of cubane (CH)8, which is the skeleton of high-energy-density material ONC. The internuclear N–N–N angles of 90° are far from the standard angle, which is considered to store powerful energy. ONC with remarkable energetic performance is a representative example. However, the longer bond lengths and the unusual bond angles of N8-4 may generate the possibility of instability. During subsequent calculations, we found that N8-4 has the highest total energy (-437.44 au) among four N8 isomers . As for N8 compounds, N8-3 and N8-4 exhibit the largest maximum surface of ESP with the values of 48.46 kcal mol−1 and 41.42 kcal mol−1, respectively. The surface minima and maximum of ESP also illustrate the main electrophilic and nucleophile reaction sites, respectively. Meanwhile, molecular surface area in different electrostatic potential intervals was obtained to help us to better understand quantitative distribution of ESP  of six N program . As shown of ESP . Among s4 isomers show only one typical peak, as well as the cube N8, of which the peaks are at 561, 980, 792 cm−1, respectively. For other N8 isomers, the spectra are more complex. The strong peak at 1527 cm−1 of N8-1 is assigned to stretching vibration modes of the three N=N bonds in structure, while other two weak peaks at 467 and 1093 cm−1 arise from the stretching vibrations of single bonds. As for N8-2, the peaks at 980, 1204, 1408 cm−1 result from the stretching vibration modes of pentazole ring, and the strongest absorption at 2262 cm−1 arises from the stretching vibrations of N1–N2 and N2–N3 bonds in azide group. Another weak absorption at 784 cm−1 is assigned to stretching vibration of N1-N4 bond connecting pentazole and -N3. The peak at 235 cm−1 in the spectrum of N8-3 arises from the torsion vibration of fused N4-N5 bond. And others at 965, 1027, 1316 cm−1 belong to the stretching vibrations of two five-membered rings, of which the strongest absorption at 1027 cm−1 is stretching vibrations of N1-N2, N7-N8.The IR spectra of the all-nitrogen compounds were calculated and the results are presented in 2)ρ of all compounds are also obtained. Several spikes in the range of -0.4–-0.25 au of all compounds correspond to the chemical bonding interactions, while the spikes ranging in 0–0.1 au mean the repulsive forces. It can be seen that there is no spike in the scatter plot of two N4 isomers, suggesting their weaker repulsions than N8 isomers. Among the N8 allotropes, the plot of cube N8 (N8-4) has two spikes with the value of 0.030 and 0.075 au, and others only respectively have one spike. According to the above analysis, cube N8 seems to exhibit the stronger steric effects and thus the poorer stability than other N8 conformations.Exploring the noncovalent interactions of a molecule is beneficial to help us understand the structural characteristics and stabilities of energetic materials. In this work, interaction region indicator (IRI) analysis as an effective method were adopted and the results are shown in 4 and N8 isomers were explored. The HOMO and LUMO orbitals and the energy gaps are presented in N8-3, whose HOMOs and LUMOs mainly occupy the unfused N atoms. The HOMOs-LUMOs distributions of these N4 and N8 materials exhibit good symmetrical features, apart from N8-2. The energy gaps for all materials are 2.74, 11.30, 5.36, 5.24, 6.25, 5.06 eV, respectively. Previous researches have demonstrated that a molecule with better stability may possess higher energy gap  and lowest unoccupied molecular orbital (LUMO) of all Nergy gap . Hence, 8 isomers were calculated to understand their charge, bond order, and bond type, and the results are presented in 8 isomers exhibit positive charges, whereas the other six N atoms exhibit negative charges. N8-2 shows the most positive charge with the value of 1.519 (N2). For N8-1 and N8-3, their distributions of NBO charges are axisymmetric, which are consistent with the characteristics of their structures. And the N atoms in N8-3 has the more negative charges than those in N8-1. The negative charges in N8-4 are -0.005, which is the lowest among the four N8 isomers.Natural bond orbital charges  of four 4 and N8 isomers, the density and the heat of formation as two important factors should be obtained in advanced. In this study, the densities of all materials were calculated using fH298K(g)) were estimated by the atomization approach  and cube N8 (N8-4) are highest, with the values of 1.502 g cm−3 and 1.749 g cm−3. The densities of other three N8 molecules are relatively similar in the range of 1.664–1.697 g cm−3. Unfortunately, the densities of these all-nitrogen materials seem no advantages compared to traditional CHON energetic materials, like RDX (1.80 g cm−3), HMX (1.91 g cm−3) and CL-20 (2.04 g cm−3).As presented in 4 and N8 allotropes has very excellent heats of formation of 7.92–16.60 kJ g−1, of which cube N8 has the highest value of 16.60 kJ g−1. This may arise from the numerous N-N and N=N bonds in the structure. The excellent enthalpies of these all-nitrogen materials are much superior to representative CHON explosives, such as RDX (0.36 kJ g−1), HMX (0.35 kJ g−1) and CL-20 (0.90 kJ g−1). In addition, the six poly-nitrogen materials exhibit prominent detonation performance. The values of detonation velocities fall in the range of 9747–11620 m s−1, while the detonation pressures are in the range of 36.80–61.10 GPa . Among them, N8 isomers show higher detonation performance than N4 ones due to the higher density. There is no doute that N8-4 has the best detonation performance . All the all-nitrogen materials presented in this work has much higher energy density than CL-20 . And compared to N5+N3− in our previous work  exhibits higher density and higher detonation performance based on the same structural composition of pentazole ring and azide . The above discussion not only confirms that all-nitrogen materials have the great prospects because of the huge energy, but also that covalent poly-nitrogen compounds would be more powerful than all-nitrogen salts.The heats of formation (details in Supporting Information) and detonation performance of all compounds are shown in ous work , N8-4 and N8 isomers also possess exceptional theoretical specific impulse (Isp). The values of Isp for two N4 isomers are 410.3 s and 409.7 s, respectively, which are higher than those of N8-1 (356.9 s), N8-2 (330.1 s), N8-3 (339.4 s), but poorer than N8-4 (436.2 s). Cube N8 in this work combines the best detonation performance and best specific impulse, suggesting the unique advantages of cage structure. And the specific impulses (330.1–436.2 s) of six poly-nitrogen molecules are much superior to that of CL-20 (272.6 s). It is believed that these ploy-nitrogen materials are expected to become the promising candidates as solid rocket propellants.In addition to the excellent detonation performance, N4  and four structures of cyclo-N8  were designed and calculated through density functional theory methods. The optimized structures, molecular electrostatic potentials, Frontier molecular orbitals, non-covalent interaction, natural bond orbital, and most importantly, the energetic properties of six poly-nitrogen materials were fully investigated. According to the theoretical calculation, N4-2 with tetrahedron structure possesses shorter N-N bond lengths than rectangle-shaped N4-1, and thus the higher molecular stability. Cube N8-4 has the longest bond lengths and unusual bond angle among N8 isomers, which may lead to the poor stability. The results from Frontier molecular orbitals and non-covalent interaction are also consistent with structure analysis. The densities of six compounds are 1.441–1.749 g cm−3, and the heats of formation are 7.92–16.60 kJ g−1. With such remarkable enthalpies, these N4 and N8 isomers exhibit excellent detonation performance  and high specific impulses (Isp: 330.1–436.2 s), which are superior to CL-20 . In particular, N4-2 and N8-4 have highest energy density among N4 and N8 species, respectively, revealing their huge development potential as next-generation HEDMs beyond CL-20.In this study, two structures of cyclo-N


Enterobacter cloacae and Klebsiella oxytoca.2,3-butanediol is an important platform compound which has a wide range of applications, involving in medicine, chemical industry, food and other fields. Especially the optically pure -2,3-butanediol can be employed as an antifreeze agent and as the precursor for producing chiral compounds. However, some -2,3-butanediol overproducing strains are pathogenic such as C. glutamicum strain, CGS9, was engineered to produce optically pure -2,3-butanediol efficiently. Firstly, the gene bdhA from B. subtilis 168 was integrated into strain CGS9 and its expression level was further enhanced by using a strong promoter Psod and ribosome binding site (RBS) with high translation initiation rate, and the -2,3-butanediol titer of the resulting strain was increased by 33.9%. Then the transhydrogenase gene udhA from E. coli was expressed to provide more NADH for 2,3-butanediol synthesis, which reduced the accumulation of the main byproduct acetoin by 57.2%. Next, a mutant atpG was integrated into strain CGK3, which increased the glucose consumption rate by 10.5% and the 2,3-butanediol productivity by 10.9% in shake-flask fermentation. Through fermentation engineering, the most promising strain CGK4 produced a titer of 144.9 g/L -2,3-butanediol with a yield of 0.429 g/g glucose and a productivity of 1.10 g/L/h in fed-batch fermentation. The optical purity of the resulting -2,3-butanediol surpassed 98%.In this study, a (3R)-acetoin overproducing C. glutamicum is a competitive candidate for -2,3-butanediol production.To the best of our knowledge, this is the highest titer of optically pure -2,3-butanediol achieved by GRAS strains, and the result has demonstrated that The online version contains supplementary material available at 10.1186/s12934-022-01875-5. It is employed as a fuel additive due to its high combustion value(27.2 kJ/g) [The increasing prices and environmental impacts of fossil fuels had made sustainably produced synthetic fuels and chemicals get more attention, which would allow us to decrease net CO.2 kJ/g) . It is a.2 kJ/g) –6. And ton value2.2 kJ/g [2 emissions [Bacillus subtilis [Bacillus amyloliquefaciens [Bacillus licheniformis [Klebsiella oxytoca [Klebsiella pneumoniae [Paenibacillus polymyxa [Serratia marcescens [Enterobacter cloacae [Enterobacter aerogenes [Escherichia coli [Zymomonas mobilis [Pichia pastoris [K. pneumoniae SDM isolated from orchard soil accumulated 150 g/L 2,3-butanediol at 38 h in a 5-L bioreactor, representing the highest titer achieved by the wild type [E. cloacae SDM 09 utilizing a mixture of glucose and xylose within 44 h [B. licheniformis MW3 (ΔbudC), which accumulated 123.7 g/L -2,3-butanediol with 99% optical purity at 42 h. However, some over-producing strains are conditionally pathogenic and require higher requirements for production processes which limited their potential in industrial application.The synthesis of 2,3-butanediol by traditional petrochemical-based processes results in gargantuan energy consumption and COmissions . Construsubtilis , 10, Bacefaciens , 12, Bacniformis , 14, Kle oxytoca , 16, Kleeumoniae , 18, Paepolymyxa , 20, Serrcescens , 22, Ent cloacae , 24, Enterogenes , 26, Eschia coli , Zymomon mobilis  and Pichpastoris , etc. Siild type . Howeverild type , which lhin 44 h . Ge et aCorynebacterium glutamicum, a GRAS microorganism with good biotransformation properties and clear genetic backgrounds, is a very mature strain for industrial scale amino acid production [L. lactis into C. glutamicum ATCC13032 and deleted genes of competing pathway. The final strain produced 6.3 g/L 2,3-butanediol with a yield of 0.33 g/g·glucose and a productivity of 0.2 g/L/h. Yang et al. [C. glutamicum ATCC 13032, which could produce 18.9 g/L 2,3-butanediol from 80 g/L glucose in CGXII medium. Indeed, both the titer and yield of 2,3-butanediol achieved by C. glutamicum are not ideal compared with other over-producing strains. However, metabolic engineered C. glutamicum has exhibited excellent performance in (3R)-acetoin production with a titer up to about 100 g/L [C. glutamicum has great potential to produce 2,3-butanediol with high titer and yield.oduction . Radoš eoduction  introduc 100 g/L . Given tC. glutamicum CGS9, a (3R)-acetoin over-producing strain, was used to be further engineered for -2,3-butanediol production. It can produce15.70 g/L (3R)-acetoin in flask culture with a yield of 0.408 g/g glucose, which was 83.4% of the theoretical yield [In this study, al yield . A seriealsSD operon in the genome, in which biosynthesis pathways of the major by-products were disrupted and the TCA cycle was weakened by downregulating the expression of the gltA gene. In order to convert (3R)-acetoin to -2,3-butanediol, the gene bdhA encoding 2,3-butanediol dehydrogenase from B. subtilis 168, under control of the constitutive promoter Ptrc (without lacO sequence), was inserted into the genome of CGS9 at the ∆ldh locus, generating the strain CGK1. Strains were cultivated in CGXIIP medium containing 40 g/L glucose. As shown in Fig. alsSD (3 copies) and the relative lower gene dosage of bdhA (1 copy). Consequently, the enzyme activity of BDH could be not sufficient for converting all the (3R)-acetoin into -2,3-butanediol. It was found that -2,3-butanediol was converted to (3R)-acetoin when glucose is depleted, which due to that the NADH produced from glycolysis was not enough and 2,3-butanediol can be converted to acetoin to regenerate NADH to maintain a constant oxidation-reduction state [CGS9  is a . The resulting strain CGK2 showed a significant increase in BDH enzyme activity and -2,3-butanediol production. As shown in Fig. P < 0.05, t-test). These results suggested that carbon flux was further directed toward -2,3-butanediol synthesis by improving the expression level of bdhA.The expression level of heterologous genes was affected by several factors, including gene dosage, promoter strength, secondary structure of mRNA and RBS sequence . To impry and 2R,R-2,3-butudhA gene from E. coli W1485 encodes a transhydrogenase that partially converts NADPH to NADH, and the latter is beneficial to the conversion of (3R)-acetoin to -2,3-butanediol [Psod, consisting of the genes bdhA and udhA with RBS-10, was introduced into the chromosome of CGS9 at the ∆ldh locus to generate strain CGK3.The conversion between -2,3-butanediol and (3R)-acetoin could regulate the redox balance . The udhtanediol . The art+ ratio significantly reduced from 1.19 to 0.74, and the NADH/NAD+ ratio increased from 0.72 to 0.94 , which might due to that the growth of CGK3 was inhibited by the change of reducing power. Thus, the glucose consumption rate needs to be improved, which can lead to increase in -2,3-butanediol productivity.As expected, comparing the reducing power in strains CGK2 and CGK3 at 12 h, the NADPH/NADPatp operon structure consisted of the gene atpI, atpB, atpE, atpF, atpH, atpA, atpG, atpD and atpC [+-ATPase encoded by the gene atpG worked as the main shaft for the rotation of the H+-ATPase rotor [+-ATPase was reduced to 70% of the original by replacing T to C at 817 bp and C to T at 818 bp of atpG in C. glutamicum ATCC14067, which resulted in an increase of 24% in specific glucose consumption during the exponential phase [It was observed that microorganisms could increase the glycolytic flux to compensate for the lack of ATP, which improved the consumption of glucose . Therefoand atpC , and these rotor . Accordial phase . Thus, tAs shown in Fig. bdhA and udhA can further enhance the production and yield of -2,3-butanediol, an episomal plamid pECK1 with additional copy of bdhA-udhA was constructed and introduced into strain CGK4, generating strain CGK5. As shown in Fig. bdhA and udhA in CGK4 was sufficient to convert (3R)-acetoin to -2,3-butanediol. Considering the stability of the production strain and bio-safety, strain CGK4 without plasmid and marker gene (antibiotic resistance gene) was chosen to further produce -2,3-butanediol in fed-batch fermentation.To test if the increase of the expression level of ld of 2R,R-2,3-but600 of 70, and dissolved oxygen also dropped to less than 2%, and then fluctuated between 1 and 2%. The dissolved oxygen gradually increased to 70% from 72 to 130 h. Meanwhile, the (3R)-acetoin production increased significantly during this stage and reached a final concentration of 24.50 g/L at 130 h. It can be inferred that the increased dissolved oxygen during 72–130 h resulted in an increase in NADH consumption by respiration, and a shortage in NADH supply for the conversion of acetoin to 2,3-butanediol.To evaluate potential of CGK4 for further industrial application, strain CGK4 was cultured in LBRC medium  in a 5-LIt is well known that control of oxygen supply is the most critical factor for efficient production of 2,3-butanediol . The anaC. glutamicum compared with other over-producing strains -2,3-butanediol productivity of R,3R-2,3-utamicum .In summary, a series of promising -2,3-butanediol producers were constructed by metabolic engineering of a (3R)-acetoin overproducing strain. The best engineered strain CGK4 produced 144.9 g/L -2,3-butanediol with a yield of 0.429 g/g glucose at a rate of 1.10 g/L/h in a 5-L fermenter and the optical purity was over 98%, which is the highest level of -2,3-butanediol production in GRAS strains at present. This study provided the possibility of microbial economically viable production of -2,3-butanediol by GRAS microorganism at industrial level.Primers were synthesized by GENEWIZ . Plasmids were extracted using the Axyprep™ Plasmid Miniprep Kit  and DNA was perfied using the SanPrep Column Plasmid Mini-Prep Kit . BHI broth was purchased from Hopebio . Yeast extract was purchased from Angel . 2,3-butanediol and acetoin standards were purchased from Sigma . Other reagents were purchased from Sangon Biotech .C. glutamicum ATCC 13,032 ∆pta∆ack∆ldh∆buta∆nagD∆ppc, ∆ackA::Ptuf -alsSD, ∆butA::Ptuf-alsSD, ∆nagD::Ptuf-alsSD, P1-gltA) [Escherichia coli DH5α was used for plasmid construction and was grown in LB medium. BHI broth (74 g/L) was used for the tube culture and transformation of C. glutamicum. CGIII medium was used for pre-cultures. Batch fermentation of 2,3-butanediol was conducted in CGXIIP medium [C. glutamicum, kanamycin 25 mg/L, for E. coli, kanamycin 40 mg/L.The original strain was CGS9 (P1-gltA) . StrainsP medium . LBRC meP medium  with theP medium . AntibioC. glutamicum via two-step homologous recombination [All the primers used in this study are listed in Additional file bination .Ptrc-bdhA into the chromosome, the plasmid pD-ldh-A1 was constructed as follows: promoter Ptrc was amplified from the plasmid pEC-XK99E using the primer pair trc-F/R, and bdhA1-F/R was used to amplify the bdhA gene from B. subtilis 168, after which these fragments were fused using trc-F/bdhA1-R. The resulting fragment was digested with SdaI/SalI and ligated between the corresponding sites of pD-sacB-ldh. AAAGGAGGACAACC was used as RBS sequence in plasmid pD-ldh-A1, which was used in our previous studies [ldh-A2 and pD-ldh-AU were constructed analogously to integrate the Psod-bdhA and Psod-bdhA-udhA into the chromosome respectively. The RBS sequence of these two plasmids was RBS-10 (ACGAGAAAAAAATTCGAACCCGAGAAAGGAGGTATT) designed by using the RBS Calculator (design mode) of De Novo DNA for the bdhA with the highest target translation initiation rate (TIR).To integrate the  studies . The plaatpG, the plasmid pD-sacB-atpGT817C, C818T was constructed as follows: the flanking regions of the atpG gene with relevant modifications were amplified from genomic DNA of C. glutamicum using the primer pairs atpGU-F/R and atpGD-F/R. The corresponding flanking fragments were fused using atpGU-F/atpGD-R. The fused product was digested with XbaI/SphI and ligated between the corresponding sites of pD-sacB to construct pD-sacB-atpGT817C, C818T.To introduce mutations into the endogenous gene Psod-bdhA-udhA from the plasmid pD-ldh-AU and pec-F/R was used to amplify the pEC-XK99E, then the plasmid pECK1 was constructed by the method of CPEC.To construct the plasmid pECK1, the primer pair AU1-F/R was used to amplify the artificial operon C. glutamicum pre-cultures, and then 1 mL of the resulting seed culture was transferred into 50 mL CGIII medium with 20 g/L glucose and grown at 220 rpm for 12 h. For batch fermentation, the seed culture was used to inoculate a 250-mL shake flask containing 50 mL CGXIIP medium to an initial OD600 of 1 and grown at 180 rpm on a rotary shaker. All fermentations were performed at 30 °C.Single colonies were used to inoculate 5 mL BHI medium and grown at 220 rpm overnight to prepare For fed-batch fermentation, 200 mL of CGIII seed culture was used to inoculate a 5-L fermenter  containing 1.8 L LBRC medium. The agitation speed was maintained at 350 rpm. All cultivations were carried out at 30 °C with an aeration rate of 1 vvm. The initial pH of the medium was 7.0. During the fermentation process, the pH value was not controlled. Fed-batch fermentation was conducted by an interim feeding method. The initial glucose concentration was 50 g/L, and an appropriate amount of feeding medium was added to maintain its concentration between 20 and 60 g/L.600) using a UV–Vis spectrophotometer. Glucose was measured using an SBA Bio-analyzer . Metabolite concentrations were determined by HPLC using an HPX-87 H (300 mm × 7.8 mm) organic acid, as described previously [− 1, followed by a ramp of 1 °C min− 1 to 80 °C (2 min), and finally 15 °C min− 1 to 230 °C (4 min). Nitrogen was used as the carrier gas. The temperature of injector and detector were set at 230 °C.Cell growth was determined by measuring the optical density at 600 nm . The intracellular NADH and NAD+ contents were determined using the Coenzyme I NAD(H) Content Assay Kit , and the intracellular NADPH and NADP+ contents were determined using the Coenzyme II NADP(H) Content Assay Kit .NADH, NADThe crude enzyme solution was extracted when the growth of the engineered strain to the exponential growth phase, and the method was as described previously . Total pAdditional file 1: Figure S1. Identification of2,3-butanediol enantiomers by GC-FID. A: The optically pure standards of-2,3-butanediol, -2,3-butanediol and meso-2,3-butanediol hadretention times of 22.957, 23.098 and 23.973 min; B: Fermentation products ofCGK1 in CGXIIP medium; C: Fed-batch fermentation products of CGK4 in LBRCmedium at 132 h; D: The standard of 2,3-butanediol -2,3-butanediol:-2,3-butanediol: meso-2,3-butanediol= 0: 98: 2). E: Fermentationproducts of CGK4 in CGXIIP medium. 1: -2,3-butanediol, 2:-2,3-butanediol, 3: meso-2,3-butanediol. Table S1. Primers used in this study.