Protective Effect of n-Butanol Extract from Viola yedoensis on Immunological Liver Injury.

Viola yedoensis Makino was used to treat inflammation, viral hepatitis, acute pyogenic infection, and ulcerative carbuncles. However, the protective effect on immunological liver injury (ILI) of V. yedoensis had been rarely reported. This study aimed to explore the protective effect of n-butanol extract (BE) from V. yedoensis on ILI in vitro and in vivo. In vitro, the BE significantly inhibited the secretions of Hepatitis B surface antigen (HBsAg) and Hepatitis B e antigen (HBeAg) in the HepG2.2.15 cells and the replication of HBV DNA. The research data in vivo revealed that the BE reduced the levels of alanine transaminase (ALT), aspartate transaminase (AST), and methane dicarboxylic aldehyde (MDA) in liver tissues of the ConA-induced mice, while increased the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and the effective contents of tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and the BE could ameliorate liver histological lesions. These results motivated a further investigation into the chemical constituents of BE. Four coumarins (esculetin, prionanthoside, cichoriin, and esculin) and one flavonoid (quercetin-3-O-galactoside) were isolated from the BE by silica gel column chromatography and recrystallization, of which structures were eventually confirmed by 1 H-NMR, 13 C-NMR, and MS.

The anthelmintic potentials of medicinal plant extracts and an isolated compound (rutin, C27H30O16) from Terminalia catappa L. against Gastrothylax crumenifer.

Paramphistomosis is a pathogenic disease that occurs frequently in tropical and subtropical countries including Thailand. This disease is affected in the parasites causing severe gastrointestinal disorders and death in infected animals. In the present study, we examined the anthelmintic efficacy of albendazole (ABZ) and crude plant extracts from barks of Bombax ceiba L., Diospyros rhodocalyx Kurz. and Vitex glabrata R.Br., and leaves of Terminalia catappa L. and Cassia alata L. against Gastrothylax crumenifer. The hightest anthelmintic activity on the parasites after 24 h incubation was observed in the n-butanol extract of T. catappa leaf. In this study, fractionation bioassay of n-butanol extract of T. catappa leaf was conducted to both separation and discrimination of rutin served as a new efficient compound (LC50 = 28.96; LC90 = 88.75 µg/mL) against G. crumenifer. This compound was confirmed by 1H nuclear magnetic resonance (1H NMR), 13C NMR, infrared (IR) and ultraviolet (UV) spectra as well as mass spectra data. The rutin-treated parasites with all dosages showed swift decrease of the motility and the relative motility (RM) and survival index (SI) were decreased obviously from 3 h until flukes were killed after 12 h of incubation. When observed with light microscopy, the parasites showed the earliest change in a limited region of the tegument. When observed by scanning electron microscopy, the parasites' tegument exhibited similar sequences of surface changes after treatments with rutin and ABZ, but less severity in ABZ treatment. The sequences of changes comprised swelling of folds and ridges, formation of blebbing, rupturing of blebs, erosions, lesions and the tegument demolition. Hence, rutin could be considered as the potential anthelmintic agent for treatment of paramphistomosis.

Phytochemicals of Periploca aphylla Dcne. ameliorated streptozotocin-induced diabetes in rat.

BACKGROUND: Periploca aphylla is used by local population and indigenous medicine practitioners as stomachic, tonic, antitumor, antiulcer, and for treatment of inflammatory disorders. The aim of this study was to evaluate antidiabetic effect of the extract of P. aphylla and to investigate antioxidant and hypolipidemic activity in streptozotocin (STZ)-induced diabetic rats. METHODS: The present research was conducted to evaluate the antihyperglycemic potential of methanol extract of P. aphylla (PAM) and subfractions n-hexane (PAH), chloroform (PAC), ethyl acetate (PAE), n-butanol (PAB), and aqueous (PAA) in glucose-overloaded hyperglycemic Sprague-Dawley rats. Based on the efficacy, PAB (200 mg/kg and 400 mg/kg) was tested for its antidiabetic activity in STZ-induced diabetic rats. Diabetes was induced via intraperitoneal injection of STZ (55 mg/kg) in rat. Blood glucose values were taken weekly. HPLC-DAD analysis of PAB was carried out for the presence of various polyphenols. RESULTS: HPLC-DAD analysis of PAB recorded the presence of rutin, catechin, caffeic acid, and myricetin. Oral administration of PAB at doses of 200 and 400 mg/kg for 21 days significantly restored (P < 0.01) body weight (%) and relative liver and relative kidney weight of diabetic rats. Diabetic control rats showed significant elevation (P < 0.01) of AST, ALT, ALP, LDH, total cholesterol, triglycerides, LDL, creatinine, total bilirubin, and BUN while reduced (P < 0.01) level of glucose, total protein, albumin, insulin, and HDL in serum. Count of blood cells and hematological parameters were altered in diabetic rats. Further, glutathione peroxidase, catalase, superoxide dismutase, glutathione reductase, and total soluble protein concentration decreased while concentration of thiobarbituric acid reactive substances and percent DNA damages increased (P < 0.01) in liver and renal tissues of diabetic rats. Histopathological damage scores increased in liver and kidney tissues of diabetic rats. Intake of PAB (400 mg/kg) resulted in significant improvement (P < 0.01) of above parameters, and results were comparable to that of standard drug glibenclamide. CONCLUSION: The result suggests the antihyperglycemic, antioxidant, and anti-inflammatory activities of PAB treatment in STZ-compelled diabetic rat. PAB might be used as new therapeutic agent in diabetic patients to manage diabetes and decrease the complications.

Therapeutic potential of russelioside B as anti-arthritic agent in Freund's adjuvant-induced arthritis in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Caralluma species are traditional edible herbs used in folkloric medicine as antidiabetic, antioxidant, antipyretic, antirheumatic, anti-inflammatory and anthelmintic agents. C. quadrangula was selected in this study to document the traditional use of the genus as anti-rheumatic treatment and the possible mechanisms of action. AIM OF THE STUDY: The higher mortality rates and shorter survival among the patients suffering from rheumatoid arthritis (RA) led to the increased interest on searching for new treatments for RA. Russelioside B (RB), a major pregnane glycoside found in C. quadrangula, was evaluated as a new anti-rheumatic agent. MATERIALS AND METHODS: The n-butanol fraction of C. quadrangula was chromatographed on a silica gel column to isolate RB. The adjuvant-induced arthritis (AIA) model was established in rats by intradermal injection of complete Freund's adjuvant (CFA) to evaluate its anti-arthritic effect. Ibuprofen was used as a reference drug. Forty rats were randomly divided into 5 groups (n = 8): normal (NOR); CFA model (CFA); ibuprofen, 5 mg/kg; RB, 25 mg/kg and RB, 50 mg/kg. The treatments were initiated from day 16 when AIA model was established and continued up to day 40. Serum diagnostic rheumatoid markers, inflammatory cytokines, oxidative stress biomarkers, cartilage and bone degeneration enzymes were assessed. RESULTS: RB at 50 mg/kg b. wt., showed significant decreases in the activities of hyaluronidase and ß-glucouronidase enzymes as well significant decreases in the levels of proinflammatory cytokines as nuclear factor-kappa-B (NF-κB), tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1ß (IL-1ß) compared to the CFA group; 11.04 ± 0.61 pg/mg protein, 4.35 ± 0.25 pg/mg protein, 3.32 ± 0.13 pg/mg protein & 2.75 ± 0.14 pg/mg protein for RB, 50 mg/kg b. wt. group vs. 25.33 ± 2.13 pg/mg protein, 25.65 ± 2.1 pg/mg protein, 22.20 ± 1.34 pg/mg protein & 13.27 ± 1.40 pg/mg protein for the arthritic group, respectively. The total antioxidant capacity (TAC) was significantly restored to normal values in RB, 50 mg/kg treated rats (4.01 ± 0.09 nmol/mL vs. 3.71 ± 0.27 nmol/mL) and the levels of myeloperoxidase (MPO) reduced by 10-folds of the CFA arthritic group. Bone histomorphometry revealed that RB treatment significantly attenuated the CFA-induced bone loss in a dose-dependent manner. CONCLUSIONS: These findings suggested that the anti-arthritic effect of RB was mediated through the reduction of the rheumatoid markers, anti-inflammatory and antioxidant action, inhibition of cartilage and bone degenerative enzymes as well as attenuation of bone loss and osteoclastogenesis.

N-butanol extract of Hedyotis diffusa protects transgenic Caenorhabditis elegans from Aß-induced toxicity.

Hedyotis diffusa Willd (Rubiaceae) is a widely used and resourceful traditional Chinese medicine that exerts protection against aging and age-related diseases. However, the underlying mechanisms of the protective effects remain largely unclear. Alzheimer's disease (AD) is an age-related neurodegenerative disease, of which ß-amyloid (Aß)-induced toxicity has been suggested as a main cause. Herein, we use the transgenic Caenorhabditis elegans CL4176, CL2006, and CL2355 strains, which express human Aß1-42 peptide, to investigate the effects and the possible mechanisms of n-butanol extract of H.diffusa (HDB)-mediated protection against Aß toxicity in vivo. During the experiments, a method of quality control for HDB was established by HPLC. Additionally, we examined the effects of HBD on gene expression changes with qRT-PCR, aggregation of Aß plagues with thioflavin-S staining, and protein detection with GFP labeling. HDB improved lifespan, locomotion, and stress resistance. Further study showed that HDB decreased paralysis, the accumulation of ROS, and AChE activity. Moreover, HDB suppressed neuronal Aß-expression-induced defects in chemotaxis behavior and increased SOD activity. HDB also downregulated the Aß mRNA level and decreased the number of Aß deposits. Furthermore, HDB increased the expression levels of sod-3, daf-16, hsf-1, and hsp-16.2 gene and upregulated hsp-16.2::GFP and gst-4::GFP expression. Taken together, these results suggest that HDB may protect against Aß-induced toxicity in C. elegans via the insulin/insulin-like growth factor-1 (IGF-1) signaling pathway.

Protective effects of n-Butanol extract and iridoid glycosides of Veronica ciliata Fisch. Against ANIT-induced cholestatic liver injury in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Veronica ciliata Fisch. is a traditional medical herb that present in more than 100 types of Tibetan medicine prescriptions, most of which are used for liver disease therapy. Iridoid glycosides have been identified as the major active components of V.ciliata with a variety of biological activities. AIMS OF THE STUDY: The aim of this study is to explore the protective effect and potential mechanism of n-Butanol extract (BE) and iridoid glycosides (IG) from V.ciliata against ɑ-naphthyl isothiocyanate (ANIT)-induced hepatotoxicity and cholestasis in mice. MATERIALS AND METHODS: Mice were intragastrically (i.g.) given BE and IG at different dose or positive control ursodeoxycholic acid (UCDA) once a day for 14 consecutive days, and were treated with ANIT to cause liver injury on day 12th. Serum levels of hepatic injury markers and cholestasis indicators, liver index and liver histopathology were measured to evaluate the effect of BE and IG on liver injury caused by ANIT. The protein levels of tumor necrosis factor-α (TNF-α), nuclear factor kappa B(NF-κB), interleukin-6 (IL-6), Na+/taurocholate cotransporting polypeptide (NTCP), bile salt export pump (BSEP), multidrug resistance-associated protein 2 (MRP2), and the levels of oxidative stress indicators in liver tissue were investigated to reveal the underlying protective mechanisms of BE and IG against ANIT-induced hepatotoxicity and cholestasis. RESULTS: The n-Butanol extract (BE) and iridoid glycosides (IG) isolated from V.ciliata significantly decreased serum level of cholestatic liver injury markers aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), γ-glutamyl transferase (GGT), total bile acid (TBA), total bilirubin (TBIL), and direct bilirubin (DBIL) in ANIT-treated mice. Histopathology of the liver tissue showed that pathological damages were relieved upon BE and IG treatment. Meanwhile, the results indicated BE and IG notably restored relative liver weights, inhibited oxidative stress induced by ANIT through increasing hepatic level of superoxide dismutase (SOD), reduced glutathione (GSH), catalase (CAT) and decreasing hepatic content of malondialdehyde (MDA). Western blot revealed that BE and IG inhibited the expression of pro-inflammatory factors TGF-α, IL-6 and NF-κB. Furthermore, the decreased protein expression of bile acid transporters NTCP, BSEP, MRP2 were upregulated by BE and IG in a dose-dependent manner. CONCLUSION: The results have demonstrated that BE and IG exhibited a dose-dependently protective effect against ANIT-induced liver injury with acute intrahepatic cholestasis in mice, which might be related to the regulation of oxidative stress, inflammatory response and bile acid transport. In addition, these findings pointed out that iridoid glycosides as main active components of V.ciliata play a critical role in hepatoprotective effect of V.ciliata.

Anti-inflammatory activity of ethyl acetate and n-butanol extracts from Ranunculus macrophyllus Desf. and their phenolic profile.

ETHNOPHARMACOLOGICAL RELEVANCE: The members of the genus Ranunculus have counter-irritating properties and thus, they are traditionally used for treating anti-inflammatory disorders and other skin conditions. Ranunculus macrophyllus Desf. is a wild medicinal plant growing in Algeria and traditionally used to treat some cutaneous skin disorders. AIM: The aim of this study was to characterize the composition of the ethyl acetate and n-butanol extracts from Ranunculus macrophyllus Desf. as well as to elucidate and to compare their effect against acute skin inflammation. Moreover, both the antioxidant activity and the acute toxicity of the plant extracts were also studied. MATERIALS AND METHODS: Spectrophotometric and chromatographic methods were employed to identify and quantify phenolic compounds and triterpenoids from R. macrophyllus Desf. fractions. The antioxidant activity was estimated using the phosphomolebdenum, DPPH, reducing power and ß-carotene bleaching assays. The ethyl acetate and n-butanol extracts were screened for their anti-inflammatory activities using ex-vivo membrane stabilizing assays and in-vivo acute skin inflammation model. RESULTS: Ethyl acetate fraction showed the highest amounts of total phenolic compounds (413 ± 4 µg GAE/mg extract) and triterpenoids (70.4 ± 1.8 µg UAE/mg extract). Rutin, hesperidin, myricetin and kaempferol were the major compounds identified in the different fractions. Ethyl acetate fraction exhibited strong DPPH• radical scavenging ability (IC50 1.6 ± 0.2 µg/mL), high total antioxidant capacity (447 ± 7 µg AAE/mg extract) and reducing power (514 ± 8 µg AAE/mg extract). Ethyl acetate fraction inhibited (73.4 ± 0.3) % of linoleic acid peroxidation. Ethyl acetate and n-butanol fractions did not have any visible toxicity at 2000 mg/kg and presented excellent membrane stabilizing ability. The inhibition of xylene induced ear inflammation was (38 ± 4) % and (46 ± 1) % for RM-B and RM-EA, respectively. CONCLUSIONS: The high content of both phenolic compounds and triterpenoids combined with the remarkable anti-inflammatory effect and antioxidant activity of ethyl acetate and n-butanol extracts from R. macrophyllus Desf. support the wide spread use of this traditional plant on some skin disorders (inflammatory skin disorders).

Ethnomedicinal documentation and anti-inflammatory effects of n-butanol extract and of four compounds isolated from the stems of Pituranthos scoparius: An in vitro and in vivo investigation.

ETHNOPHARMACOLOGICAL RELEVANCE: Pituranthos scoparius is a medicinal plant belonging to the Apiaceae family. It thrives in North Africa, and is widely distributed in the high plateau of most parts of the Sahara in Algeria. This plant is widely used in the Algerian traditional medicine to treat numerous infectious diseases, dermatoses, nervous breakdowns, digestive disorders, and calm abdominal pain. AIM OF THE STUDY: The aim of the present work was to examine the ethnomedicinal uses of Pituranthos scoparius in Setif region, Algeria, isolate and identify the chemical constituents of the n-butanol stem extract of P. scoparius (BEPS), and to determine the toxicity and anti-inflammatory effects of these compounds in addition to the extract. MATERIALS AND METHODS: The anti-inflammatory effects of BEPS and the four compounds isolated from the extract were evaluated using the in vitro protein denaturation assay, whereas the topical anti-inflammatory activity was assessed using the croton oil-induced ear edema in mice. Toxicity was determined based on assessment of in vitro cytotoxicity using hemolytic activity against human red blood cells (RBCs). RESULTS: Four compounds, identified as the rare isorhamnetin-3-O-ß-apiofuranosyl (1 â†’ 2)-ß glucopyranoside (2), in addition to three known compounds, namely isorhamnetin-3-O-ß-glucoside (1), D-mannitol (3), and isorhamnetin-3-O-ß-glucopyranosyl-(1 â†’ 6)-ß-glucopyranoside (4) were isolated from BEPS. These compounds were characterized by means of NMR and high-resolution mass spectral (HRMS) data. These four compounds were isolated for the first time from this traditional Algerian medicinal plant. Screening of the extract indicated the presence of alkaloids, polyphenols, flavonoids, free quinones, coumarins and tannins. Topical anti-inflammatory effect showed that the four isolated compounds, as well as BEPS, exhibit a significant (p < 0.05) dose-dependent (0.5 and 1 mg/ear) anti-inflammatory effect. At a dose of 1 mg/ear, compounds 1, 2, and 4, exhibited remarkable anti-inflammatory effect with a percentage inhibition of 85.50 ± 2.78, 79.78 ± 4.68, and 75.78 ± 2.98%, respectively. Results from in vitro cytotoxicity showed that the % lysis of the extract, along with isolated compounds was found to be virtually nontoxic. CONCLUSIONS: These results suggest that BEPS and isolated compounds are safe, nontoxic, and exert remarkable anti-inflammatory effects, and can be new sources of natural anti-inflammatory agents.

A new glucoside with a potent α-glucosidase inhibitory activity from Lycium schweinfurthii.

A new glucoside, 3-methoxy-4-O-ß-D-glucopyranosyl-methyl benzoate, has been isolated from Lycium schweinfurthii along with five known compounds through bioactivity guided fractionation of the total plant methanolic extract towards α-glucosidase inhibitory activity. All the isolated compounds were tested for their inhibitory effect on α-glucosidase enzyme. As a result, four of them showed a potent inhibitory activity and thus constitute a therapeutic approach to decrease postprandial hyperglycemia in diabetic patients.

Phytochemicals of Periploca aphylla Dcne. ameliorated streptozotocin-induced diabetes in rat

BACKGROUND@#Periploca aphylla is used by local population and indigenous medicine practitioners as stomachic, tonic, antitumor, antiulcer, and for treatment of inflammatory disorders. The aim of this study was to evaluate antidiabetic effect of the extract of P. aphylla and to investigate antioxidant and hypolipidemic activity in streptozotocin (STZ)-induced diabetic rats.@*METHODS@#The present research was conducted to evaluate the antihyperglycemic potential of methanol extract of P. aphylla (PAM) and subfractions n-hexane (PAH), chloroform (PAC), ethyl acetate (PAE), n-butanol (PAB), and aqueous (PAA) in glucose-overloaded hyperglycemic Sprague-Dawley rats. Based on the efficacy, PAB (200 mg/kg and 400 mg/kg) was tested for its antidiabetic activity in STZ-induced diabetic rats. Diabetes was induced via intraperitoneal injection of STZ (55 mg/kg) in rat. Blood glucose values were taken weekly. HPLC-DAD analysis of PAB was carried out for the presence of various polyphenols.@*RESULTS@#HPLC-DAD analysis of PAB recorded the presence of rutin, catechin, caffeic acid, and myricetin. Oral administration of PAB at doses of 200 and 400 mg/kg for 21 days significantly restored (P < 0.01) body weight (%) and relative liver and relative kidney weight of diabetic rats. Diabetic control rats showed significant elevation (P < 0.01) of AST, ALT, ALP, LDH, total cholesterol, triglycerides, LDL, creatinine, total bilirubin, and BUN while reduced (P < 0.01) level of glucose, total protein, albumin, insulin, and HDL in serum. Count of blood cells and hematological parameters were altered in diabetic rats. Further, glutathione peroxidase, catalase, superoxide dismutase, glutathione reductase, and total soluble protein concentration decreased while concentration of thiobarbituric acid reactive substances and percent DNA damages increased (P < 0.01) in liver and renal tissues of diabetic rats. Histopathological damage scores increased in liver and kidney tissues of diabetic rats. Intake of PAB (400 mg/kg) resulted in significant improvement (P < 0.01) of above parameters, and results were comparable to that of standard drug glibenclamide.@*CONCLUSION@#The result suggests the antihyperglycemic, antioxidant, and anti-inflammatory activities of PAB treatment in STZ-compelled diabetic rat. PAB might be used as new therapeutic agent in diabetic patients to manage diabetes and decrease the complications.

Chemical composition and anti-inflammatory activity of n-butanol extract of Piper sarmentosum Roxb. In the intestinal porcine epithelial cells (IPEC-J2).

ETHNOPHARMACOLOGICAL RELEVANCE: Piper sarmentosum Roxb. (PS) is a terrestrial herb primarily distributed in tropical and subtropical regions of Asia. It is widely used in folk medicine in certain countries of Southeast Asia for the treatment of fever, toothache, coughing and pleurisy, which showed the anti-inflammatory activity of PS. AIM OF THE STUDY: This study aimed to investigate the chemical constituents and the molecular mechanism and related metabolic pathway by which n-butanol extract of PS (PSE-NB) exerts its anti-inflammatory effects. MATERIALS AND METHODS: Chemical constituents of PSE-NB was analyzed using ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) technique. Anti-inflammatory effects of PSE-NB were investigated in lipopolysaccharide (LPS)-induced IPEC-J2 cells. RESULTS: In total, 218 compounds, including 94 alkaloids and 26 phenolics were tentatively identified, which indicating alkaloids and phenolics were the main constituents of PSE-NB. In addition, the current cell experiment in vitro showed that PSE-NB (10-500 µg/mL) pre-treatment before LPS stimulation significantly decreased mRNA expression of IL-1ß, IL-6 and TNF-α in IPEC-J2 cells compared with LPS treatment (p < 0.05). PSE-NB improved mRNA expression of tight junction proteins (ZO-1 and Occludin) and NHE3, which were reduced by LPS stimulation (p < 0.05). Moreover, PSE-NB (10 µg/mL) alleviated LPS-induced protein expression of p65 and p-p65 (p < 0.05), and reduced p65 translocation into the nucleus induced by LPS. At the same time, metabolic pathway analysis indicated that PSE-NB exerts anti-inflammatory effects mainly via augmentation of methionine metabolism in IPEC-J2 cells. CONCLUSIONS: Taken together, the results suggested that alkaloids and phenolics were the main constituents in PSE-NB. PSE-NB might attenuate LPS-induced inflammatory responses in IPEC-J2 cells by regulating NF-κB signaling pathway and intracellular metabolic pattern.

Photocatalytic degradation of nevirapine with a heterostructure of few-layer black phosphorus coupled with niobium (V) oxide nanoflowers (FL-BP@Nb2O5).

The degradation and removal of antiviral drugs in water has emerged remains a major challenge. This work presents, the photodegradation of nevirapine (NVP) with a novel p-n heterostructure of FL-BP@Nb2O5 nanoparticles synthesized via hydrothermal method. Several characterization techniques revealed a successful formation of the heterostructure with well aligned band positions that promoted excellent separation of charge carriers. A systematic study was conducted on the effect of initial pH, initial catalyst loading and initial concentration on the degradation kinetics of NVP. Degradation efficiency of 68% was achieved with the FL-BP@Nb2O5 after 3 h with 5 ppm initial concentration solution of NVP, at a working pH of 3 and 15 mg of photocatalyst. The stable fragment resulting from the degradation of NVP was n-butanol as evidenced by LC/MS. The successful degradation of NVP transpired with synergistic effect exhibited by the heterostructure that led to accelerated formation of reactive species that were responsible for the breaking down of NVP into smaller fragments. A TOC removal percentage of 19.03% after the photodegradation of NVP was observed, suggesting a successful break down of NVP to simpler non-toxic carbon-containing compounds.

Improved Butanol Production Using FASII Pathway in E. coli.

n-Butanol is often considered a potential substitute for gasoline due to its physicochemical properties being closely related to those of gasoline. In this study, we extend our earlier work to convert endogenously producing butyrate via the FASII pathway using thioesterase TesBT to its corresponding alcohol, i.e., butanol. We first assembled pathway genes, i.e., car encoding carboxylic acid reductase from Mycobacterium marinum, sfp encoding phosphopantetheinyl transferase from Bacillus subtilis, and adh2 encoding alcohol dehydrogenase from S. cerevisiae, responsible for bioconversion of butyrate to butanol in three different configurations (Operon, Pseudo-Operon, and Monocistronic) to achieve optimum expression of each gene and compared with the clostridial solventogenic pathway for in vivo conversion of butyrate to butanol under aerobic conditions. An E. coli strain harboring car, sfp, and adh2 in pseudo-operon configuration was able to convert butyrate to butanol with 100% bioconversion efficiency when supplemented with 1 g/L of butyrate. Further, co-cultivation of an upstream strain (butyrate-producing) with a downstream strain (butyrate to butanol converting) at different inoculation ratios was investigated, and an optimized ratio of 1:4 (upstream strain: downstream strain) was found to produce ∼2 g/L butanol under fed-batch fermentation. Further, a mono-cultivation approach was applied by transforming a plasmid harboring tesBT gene into the downstream strain. This approach produced 0.42 g/L in a test tube and ∼2.9 g/L butanol under fed-batch fermentation. This is the first report where both mono- and co-cultivation approaches were tested and compared for butanol production, and butanol titers achieved using both strategies are the highest reported values in recombinant E. coli utilizing FASII pathway.

Bioassay-Guided Separation of Centipeda minima Using Comprehensive Linear Gradient Centrifugal Partition Chromatography.

A comprehensive linear gradient solvent system for centrifugal partition chromatography (CPC) was developed for the bioassay-guided isolation of natural compounds. The gradient solvent system consisted of three different ternary biphasic solvents types: n-hexane-acetonitrile-water (10:2:8, v/v), ethyl acetate-acetonitrile-water (10:2:8, v/v), and water-saturated n-butanol-acetonitrile-water (10:2:8, v/v). The lower phase of the n-hexane-acetonitrile-water (10:2:8, v/v) was used as the stationary phase, while its upper phase, as well as ethyl acetate-acetonitrile-water (10:2:8), and water-saturated n-butanol-acetonitrile-water (10:2:8, v/v) were pumped to generate a linear gradient elution, increasing the mobile phase polarity. We used the gradient CPC to identify antioxidant response elements (AREs), inducing compounds from Centipeda minima, using an ARE-luciferase assay in HepG2 cells, which led to the purification of the active molecules 3-methoxyquercetin and brevilin A. The developed CPC solvent systems allow the separation and isolation of compounds with a wide polarity range, allowing active molecule identification in the complex crude extract of natural products.

Protective effects of Amygdalus mongolica on rats with renal fibrosis based on serum metabolomics.

ETHNOPHARMACOLOGICAL RELEVANCE: Renal fibrosis (RF) is a common outcome of various progressive chronic kidney diseases (CKDs) and, thus, seriously endangers human health. As the active ingredient of Amygdalus mongolica, amygdalin inhibits RF. Furthermore, our previous studies demonstrated that n-butanol extract (BUT) and petroleum ether extract (PET), which are effective components of A. mongolica, have an anti-renal fibrosis effect. However, their potential mechanisms of action are unclear and need further verification. AIMS OF THE STUDY: The aims of this study were to further investigate the effects and potential mechanisms of A. mongolica extracts in the treatment of RF. MATERIALS AND METHODS: The animals were divided into the control group, RF model group, PET group and BUT group. The RF rat model was established through unilateral ureteral obstruction (UUO). Biochemical indicators, including blood urea nitrogen (BUN), serum creatinine (Scr), and hydroxyproline (HYP, a routine marker of fibrosis), and the antioxidant index (including superoxide dismutase (SOD) and malondialdehyde (MDA)) were measured to evaluate the anti-RF effects of the extracts of A. mongolica. The histomorphology of renal tissue was observed and scored by HE and Masson staining. A serum metabonomic analysis based on UPLC-Q-TOF/MS was performed to assess the changes in the metabolic profile among the different groups. RESULTS: The results showed that PET and BUT significantly improved tubulointerstitial damage and fibrosis by reducing the levels of Scr, BUN, HYP, and MDA and increasing the level of SOD. Moreover, no significant differences in efficacy were observed between the BUT and PET groups. According to the metabolomics analysis, seventy-four potential biomarkers were identified, and eight crucial biomarkers were further selected. These key biomarkers significantly contributed to RF progression by participating in six metabolic pathways, including pathways involved in arginine and proline metabolism, histidine metabolism, nicotinamide metabolism, pentose and glucuronate interconversion, ascorbate and aldarate metabolism, and amino sugar and nucleotide sugar metabolism. In addition, eight key biomarkers and six crucial biomarkers were restored to levels similar to those observed in controls following the treatment with PET and BUT, respectively. CONCLUSIONS: The outcomes of these studies demonstrate the renoprotective effects of A. mongolica extracts in rats with RF and revealed the mechanism underlying these antifibrotic effects on metabolic activity for the first time.

Characterization of ethyl acetate and n-butanol extracts of Cymbopogon schoenanthus and Helianthemum lippii and their effect on the smooth muscle of the rat distal colon.

ETHNOPHARMACOLOGY RELEVANCE: Cymbopogon schoenanthus (C. schoenanthus) and Helianthemum lippii (H. lippii) are Saharan species found in the South West of Algeria, in the region of Bechar. Both plants are used in traditional medicine to treat gastrointestinal disorders. OBJECTIVE: The aim of our study was to characterize the composition of the ethyl acetate (EtOAc) and n-Butanol (n-BuOH) extracts of C. schoenanthus and H. lippii, and to elucidate and compare their effect on the reactivity of the rat distal colon. MAIN METHODS: The plants were macerated in a hydroalcoholic solution. After concentration, the aqueous solutions of the residues were submitted to liquid-liquid extractions to obtain EtOAc and n-BuOH extracts. The phenolic and flavonoid content of the extracts was determined by high performance liquid chromatography coupled with mass spectrometry with a time of flight analyzer (HPLC-TOF/MS). The effect of the extracts was tested on the rat distal colon, namely on the basal tone and on KCl- and Ach-induced precontracted preparations. RESULTS: HPLC-TOF/MS identified 32 phenols and flavonoids in the extracts. The four extracts relaxed the rat distal colon, the effect being noticed on the basal tone and on the KCl- and Ach-induced precontractions. The EtOAc and the n-BuOH extracts of H. lippii decreased the basal tone of the rat distal colon more markedly than the correspondent extracts of C. schoenanthus. Moreover, the n-BuOH extract of C. schoenanthus decreased the basal tone more markedly than the EtOAc extract of this plant but there was no difference between extracts of H. lippii. The EtOAc extracts of both C. schoenanthus and H. lippii totally reverted both the KCl- and the Ach-induced precontraction of the rat distal colon. However, the n-BuOH extracts of the two plants reverted the Ach-precontracted colon but not the colon that has been precontracted with KCl. CONCLUSION: Extracts of H. lippii contain a higher level of phenols compared to the extracts of C. schoenanthus. All extracts of C. schoenanthus and H. lippii caused marked relaxation of the isolated rat distal colon, either when applied directly or when tested over KCl- and Ach-induced precontraction. These results give support to the use of C. shoenanthus and H. lippii in traditional medicine, namely for gastrointestinal diseases.

Antineuroinflammation activity of n-butanol fraction of Marsilea crenata Presl. in microglia HMC3 cell line.

Background Neuroinflammation is one of the main causes of neurodegenerative events. Phytoestrogen is a group compounds that have an estrogen-like structure or function. Phytoestrogen has a high potential to overcome neuroinflammation caused by estrogen deficiency in postmenopausal women. Marsilea crenata Presl. is a plant known to contain phytoestrogens. This research aimed to analyze the activity of an n-butanol fraction of M. crenata leaves in inhibiting the classical pathway activation of microglia HMC3 cell line to M1 polarity, which has proinflammatory characteristics. Methods Microglia HMC3 cell line was cultured in Eagle's minimum essential medium and induced with IFN-γ for 24 h to activate the cell to M1 polarity in 24-well microplates. The n-butanol fraction was added with various doses of 62.5, 125, and 250 ppm and genistein 50 µM as a positive control. The expression of major histocompatibility complex II (MHC II) as a marker was tested using a confocal laser scanning microscope. Results The result of MHC II measurement shows a significant difference in the MHC II expression in the microglia HMC3 cell line between the negative control and all treatment groups at p<0.05, indicating a non-monotonic dose-response profile. Conclusions The best dosage to inhibit MHC II expression was 250 ppm with the value of 200.983 AU. It is then concluded that n-butanol fraction of M. crenata leaves has antineuroinflammation activity due to its phytoestrogens.

Therapeutic effect of n-butanol fraction of Huang-lian-Jie-du Decoction on ulcerative colitis and its regulation on intestinal flora in colitis mice.

Huang-lian-Jie-du Decoction (HLJDD) is a classical prescription for clearing away heat and detoxification. In order to screen the effective fraction of HLJDD in the treatment of ulcerative colitis (UC) in mice and explore its effects on intestinal flora in UC mice, we prepared different polar fractions of HLJDD by system solvent extraction method. Subsequently, the contents of 13 active compounds in different polar fractions of HLJDD were determined by HPLC. Further, the UC model induced by dextran sodium sulfate was used to evaluate the therapeutic effects of different polar fractions of HLJDD. Finally, cecal contents were used for sequencing and analysis of bacterial 16S rRNA genes. The results showed that the yield of HLJDD-n-butanol (HLJDD-NBA) fraction was the highest, and the content or proportion of 13 active compounds in HLJDD-NBA fraction were the most similar to HLJDD. In addition, in vivo pharmacodynamic experiments showed that HLJDD-NBA intervention not only significantly alleviated the clinical symptoms of UC mice and ameliorated the pathological damage of colon tissue, but also showed significant anti-inflammatory and antioxidative effects (p < 0.05), which were comparable to HLJDD (p > 0.05). Moreover, both HLDD and HLJDD-NBA treatments can restore the intestinal flora homeostasis of UC mice by inhibiting the growth of intestinal pathogens and preventing the decrease of beneficial bacteria. Meanwhile, they can also significantly correct the dysfunction of intestinal flora in UC mice. In conclusion, we proved that HLJDD-NBA fraction is an effective fraction of HLJDD in treating UC in mice, and it can maintain the intestinal flora homeostasis of UC mice, which increases our understanding of the mechanism of HLJDD in treating UC and lays a foundation for the development of new anti-ulcer drugs.

Effect of tea root-derived proanthocyanidin fractions on protection of dentin collagen.

OBJECTIVES: Proanthocyanidins (PAs) have been widely used as effective agents for dentin collagen cross-linking to enhance the biomechanics and biostability of dentin in vitro. However, the effects and protective mechanisms of various tea root-derived PA components on dentin remain undefined. This study evaluated the effects of these tea root-derived PA components on dentin biomechanics and biostability. METHODS: In this study, ethyl acetate and n-butyl alcohol were used to extract PAs with different degrees of polymerization from tea roots; the effects of these PA extracts on dentin were evaluated. RESULTS: Dentin was treated with glutaraldehyde, ethyl acetate, n-butyl alcohol, or water. PAs with a high degree of polymerization, extracted using n-butyl alcohol, were able to more effectively improve dentin collagen cross-linking, increase resistance to bacterial collagenase digestion, and enhance dentin elasticity, relative to treatment with glutaraldehyde or PAs with a low degree of polymerization (extracted using ethyl acetate). Additionally, treatment with aqueous extract of tea roots was detrimental to dentin stability and function. CONCLUSIONS: PAs with a high degree of polymerization were effective for dentin protection and restoration in vitro, suggesting clinical treatment potential for tea root-derived PAs.

In vitro assessment of antibacterial, antifungal, enzyme inhibition and hemolytic activities of various fractions of Rhynchosia pseudo-cajan.

The aims of the present investigation were to assess the antibacterial, antifungal, enzyme inhibition and hemolytic activities of various fractions of Rhynchosia pseudo-cajan Cambess. The methanolic extract of the plant was dissolved in the water (distilled) and then partitioned with the n-hexane, chloroform, EtOAc and n-BuOH sequentially. Antibacterial activity was checked against Escherichia coli, Pasturella multocida, Bacillus subtilis and Staphylococcus aureus by the disc diffusion method using streptomycin sulphate, a standard antibiotic, as positive control. Chloroform and ethyl acetate soluble fractions showed good activity against Escherichia coli, Bacillus subtilis and Staphylococcus aureus. These fractions also showed good MIC values. The n-butanol soluble and remaining aqueous fraction also showed good activity against some strains. Antifungal activity was studied against four fungi i.e. Aspergillus niger, Aspergillus flavus, Ganoderma lucidum and Alternaria alternata by the disc diffusion method using fluconazole, a standard antifungal drug, as positive control. Chloroform, n-butanol and ethyl acetate soluble fraction showed good activity only against G. lucidum. Enzyme inhibition studies were done against four enzymes i.e. α-glucosidase, butyrylcholinesterase, acetyl cholinesterase and lipoxygenase. Aqueous fraction possessed very good activity against α-glucosidase, even greater than acarbose, a reference standard drug. Its IC50 value was found as 29.81±0.12 µg/ml as compared to acarbose having IC50 38.62±0.04 µg/ml. Chlroform and ethyl acetate soluble fractions also showed good activity against α-glucosidase. Ethyl acetate soluble and remaining aqueous fractions showed good activity against lipoxygenase. All the studied fractions showed very less toxicity i.e. <2.5%.