Fractions F8 Research Articles

Exploring the Antidiabetic and Antihypertensive Potential of Peptides Derived from Bitter Melon Seed Hydrolysate

Background/Objectives: Type 2 diabetes (T2D) has become a critical global health issue, with an increasing prevalence that contributes to significant morbidity and mortality. Inhibiting dipeptidyl peptidase-IV (DPP4) is a promising strategy for managing T2D. This study aimed to explore the DPP4 inhibitory peptide derived from bitter melon seed protein (BMSP) hydrolysate. Methods: Reversed-phase high-performance liquid chromatography (RP-HPLC) was utilized to fractionate the hydrolysate. Peptide in the highest activity fraction was analyzed using liquid chromatography-mass spectrometry (LC-MS/MS). Peptide synthetic was used for further characterizations, such as bioactivity exploration, inhibition mechanism, molecular docking, and peptide stability against in vitro simulated gastrointestinal (SGI) digestion. Results: The BMSP hydrolysate was digested with gastrointestinal proteases (GP) and assessed for DPP4 inhibitory activity, yielding an IC50 of 1448 ± 105 μg/mL. Following RP-HPLC fractionation, MPHW (MW4) and VPSGAPF (VF7) were identified from fraction F8 with DPP4 IC50 values of 128.0 ± 1.3 µM and 150.6 ± 3.4 µM, respectively. Additionally, MW4 exhibited potential antihypertensive effects through ACE inhibition with an IC50 of 172.2 ± 10.6 µM. The inhibitory kinetics and molecular docking simulations indicated that both MW4 and VF7 were competitive inhibitors of DPP4, while MW4 was also a competitive inhibitor of ACE. Importantly, both peptides remained stable during simulated gastrointestinal digestion, suggesting their resistance to human digestive processes and their capacity to maintain biological activity. Conclusions: The findings suggest that BMSP-GP hydrolysate may have potential in terms of the development of health foods or therapeutic agents. However, in vivo studies are also essential for further confirmation of efficacy.

Isolation of the active ingredients of antimalarial activity of the stem bark of Pseudocedrela kotschyi (Dry zone cedar)

Natural products, either as pure compounds or as standardized extracts provide unlimited opportunities for new drug discoveries because of the unmatched availability of chemical diversity. This study was carried out to isolate the active ingredients and determine the antimalarial activity of fractions of Pseudocedrela kotschyi stem bark following a scientific report of the antimalarial activity of crude extract of the plant material. Atotal of seven different fractions were obtained from 12g of the ethyl acetate crude extract using the flash chromatography method. GC-MS analysis of each of the seven ethyl acetate fractions was done and the identification of components present was based on a direct a comparison of the similarity index, retention times and mass spectral data with those for standard compounds. The compound prediction is based on NIST 05 Spectral library search programme. LD50 of 5 out of 7 fractions obtained was carried out using 45 mice of both sexes weighing 17-34g. The antimalarial (suppressive and curative) properties were evaluated in 40 mice in 3 different groups using NK 65 Plasmodium berghei berghei for each fraction. The plant treatments were compared against chloroquine and dimethyl sulfoxide. Results of GC-MS analysis of the seven ethyl acetate fractions of the plant extract showed the presence of forty-four different compounds, of which 3, 5-di-tert-butylphenol have been previously reported to have antimalarial properties. Acute toxicity (LD50 ) of fractions F1-4, F8, F9, F10-11, and F12-19 were 450mg/kg, 200mg/kg, 450mg/kg, 200mg/kg, and 450mg/kg respectively. All 7 fractions exhibited antimalarial activity. Remarkably, fractions F8 and F6-7 showed significant antimalarial activities for both suppressive and curative tests. These findings may benefit the antimalarial drug search in the current public health concerns of malaria disease.

English

Maerua pseudopetalosa (Gilg and Bened.) De Wolf tubers which are used traditionally as antitumor agent in Sudan were subjected to separation by column chromatography technique. Eight fractions were obtained for the ethyl acetate extract and twelve for the ethanolic extract. The ethanolic fractions F8, F9, F11 and F12, with high bioactivity were subjected to further investigations. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used for assessment of the cytotoxicity. Remarkable cytotoxicity against Michigan Cancer Foundation-7 (MCF-7) was shown, for the first time. Actually, the results revealed that F12 is a very promising one with remarkable activity against MCF-7 cell lines (43.51 µg/g at 72 h), high antioxidant activity (91.3% by 1,1-diphenyl-2-picryl-hydrazyl (DPPH) and 97% by ABTS) and flavonoid and phenolic contents (11.75 and 20.72 mg/g). Six compounds were detected in F9 (syrngic 11.65 µg/g, sinapic 8 µg/g) and F12 (gallic 88.12 µg/g, caffic 11.1 µg/g, sinapic 38.67 µg/g) which were not recorded in any previous work in the available literature by using high performance liquid chromatography (HPLC). Key words: Michigan Cancer Foundation-7 (MCF-7) breast cancer, Maerua pseudopetalosa, phenol and flavonoid contents, high performance liquid chromatography (HPLC) analysis, antioxidant activity.

Identificación y determinación estructural de un sesquiterpeno de las hojas de Tessaria integrifolia Ruiz & Pav. y evaluación de su actividad Leishmanicida

To identify and determine the phytoconstituent structure of Tessaria integrifolia Ruiz & Pav. leaves with leishmanicidal activity. Fluid extract of leaves was prepared, concentrated to soft extract, and used to evaluate leishmanicidal activity in Mesocricetus auratus with experimental leishmaniasis, at the dose of 250 mg/kg of soft extract by intramuscular route for 15 days. Extract was fractionated in 45 cm column chromatography with a 2.5 cm diameter, containing G-60 silica gel, and 70-230 mesh (Sigma-Aldrich®). Nine fractions were obtained and assessed on macrophages infected with Leishmania sp to determine the active fraction and isolate the active compound, by separation, purification, and crystallization, analyzed by nuclear magnetic resonance (NMR) of 1H, 13C, and liquid chromatography coupled to mass spectroscopy (LC/ MS). Fluid extract from the leaves of T. integrifolia presents leishmanicidal activity in M. auratus. Fraction F8 is active on infected macrophages at a dose of 14 μg/mL. An eudesman type sesquiterpene ((4aS, 5R, 6R, 8aR) -6-hydroxy-5, 8a-dimethyl-3- (1-methylethylidene) octahydronaphthalen-2 (1H) -one) was identified, by RMN 1 H, 13C, and LC / MS analysis. Fluid extract of leaves of Tessaria integrifolia Ruiz & Pav. presents leishmanicidal activity on Mesocricetus auratus with experimental leishmaniasis. Fraction F8 presents leishmanicidal activity on infected macrophages at a dose of 14 μg/mL. An eudesman type sesquiterpene was identified, according to 1 H, 13C, and LC / MS NMR analysis.

Recombinant Calponin of human filariid Brugia malayi : Secondary structure and immunoprophylactic potential

In the search for potential vaccine candidates for the control of human lymphatic filariasis, we recently identified calponin-like protein, that regulates actin/myosin interactions, in a proinflammatory fraction F8 (45.24-48.64kDa) of Brugia malayi adult worms. In the present study, the gene was cloned, expressed, and the recombinant Calponin of B. malayi (r-ClpBm) was prepared and characterized. r-ClpBm bears homology with OV9M of Onchocerca volvulus, a non-lymphatic filariid that causes loss of vision and cutaneous pathology. r-ClpBm was found to be a ∼45kDa protein that folds into a predominantly α-helix conformation. The protective efficacy of r-ClpBm against B. malayi infection in Mastomys coucha was investigated by assessing the course of microfilaraemia and adult worm burden in the host immunized with r-ClpBm and subsequently infected with infective third stage larvae (L3). Expression of the Calponin was detected in all life stages (microfilariae, L3, L4, L5 and adults) of the parasite and immunization with r-ClpBm partially protected M. coucha against establishment of infection as inferred by ∼42% inhibition in parasite burden. Upregulated cellular proliferation, TNF-α, IFN-γ, IL-1β, IL-4, nitric oxide (NO) release, expression of iNOS, and specific IgG, IgG1 and IgG2b in immunized animals correlated with parasitological findings. r-ClpBm immunization caused degranulation in majority of mast cells indicating possible involvement of mast cell products in reducing the parasite survival. It appears that complex mechanisms including Th1, Th2, NO and mast cells are involved in the clearance of infection. To the best of our knowledge this is the first report on cloning, expression of the gene and purification of r-ClpBm, determination of its secondary structure and its ability to partially prevent establishment of B. malayi infection. Thus, r-ClpBm may further be studied and developed in combination with other protective molecules of B. malayi as a component of potential filarial cocktail vaccine candidate.

English

Ethyl acetate and ethanol extracts of the tuber parts of Maerua pseudopetalosa were subjected to further separation by column chromatography technique and eight fractions were obtained for the former and twelve for the latter one. The brine shrimp lethality assay was used for assessment of the toxicity. Remarkable cytotoxicity against brine shrimp larvae was shown, for the first time, by the ethanol extract. The fractions F8, F9, F11 and F12, with high cytotoxic values (1.25, 7.98, 0.185, 0.041 µg/ml, respectively), were subjected to gas chromatography/mass spectrometry analysis. Thirty three compounds were detected; which were not recorded in any previous work in the available literature. Fractions 8 and 9 were found to be cytotoxic due to the presence of oleate and linoleate compounds; with more cytotoxicity in fraction 8 as a result of the additional presence of decenoic acid. Also, fraction 12 was more cytotoxic than fraction 11 and this was attributed to the presence of a proline derivative (proline-N-methyl-butyl ester). This compound might be considered as the cause of the high toxicity of the fraction; since free proline was used as an inhibitor of breast cancer development. Surprisingly, M. pseudopetalosa tubers were used in the folkloric medicine by the natives of the South Blue Nile State for the treatment of breast cancer growth without any knowledge of its chemical constituents.   Key words: Capparaceae, brine shrimp larvae, bioactive compounds, column chromatography, GC/MS analysis, proline derivative. &nbsp

Protection against filarial infection by 45–49 kDa molecules of Brugia malayi via IFN-γ-mediated iNOS induction

Nitric oxide (NO) mediated mechanisms have been implicated in killing of some life-stages of Brugia malayi/Wuchereria bancrofti and protect the host through type 1 responses and IFN-γ stimulated toxic mediators’ release. However, the identity of NO stimulating molecules of the parasites is not known. Three predominantly NO-stimulating SDS-PAGE resolved fractions F8 (45.24–48.64kDa), F11 (33.44–38.44kDa) and F12 (28.44–33.44kDa) from B. malayi were identified and their proteins were analyzed by 2-DE and MALDI-TOF/TOF. Tropomyosin, calponin and de novo peptides were identified by 2-DE and MALDI-TOF/TOF in F8 and immunization with F8 conferred most significant protection against L3-initiated infection in Mastomys coucha. Immunized animals showed upregulated F8-induced NO, IFN-γ, TNF-α, IL-1β, IL-10, TGF-β release, cellular proliferative responses and specific IgG and IgG1. Anti-IFN-γ, anti-TNF-α, and anti-IL-1β significantly reduced F8-mediated NO generation and iNOS induction at protein levels. Anti-IFN-γ treated cells showed maximum reduction (>74%) in NO generation suggesting a predominant role of IFN-γ in iNOS induction. In conclusion, the findings suggest that F8 which contains tropomyosin, calponin and de novo peptides protects the host via IFN-γ mediated iNOS induction and may hold promise as vaccine candidate(s). This is also the first report of identification of tropomyosin and calponin in B. malayi.

Angiotensin I converting enzyme inhibitory peptides obtained after in vitro hydrolysis of pea (Pisum sativum var. Bajka) globulins.

Pea seeds represent a valuable source of active compounds that may positively influence health. In this study, the pea globulins were digested in vitro under gastrointestinal condition and potentially bioaccessible angiotensin I converting enzyme (ACE) inhibitory peptides were identified. The degree of hydrolysis after pepsin, 14.42%, and pancreatin, 30.65%, were noted. The peptides with the highest ACE inhibitory properties were separated using ion exchange chromatography on DEAE-cellulose. Thirteen peptides fractions were obtained but only four showed potential antihypertensive properties. The highest inhibitory activity was determined for the fraction F8 (IC50 = 0.0014 mg/mL). This fraction was separated on Sephadex G10 and two peptide fractions were obtained. The peptides fraction (B) with the highest ACE inhibitory activity (IC50 = 0.073 mg/mL) was identified by ESI-MS/MS. The sequences of ACE inhibitory peptides were GGSGNY, DLKLP, GSSDNR, MRDLK, and HNTPSR. Based on Lineweaver-Burk plots for the fraction B, the kinetic parameters as K m, Vmax, and K i and mode of inhibition were determined. This fraction belongs to uncompetitive inhibitor of ACE activity. The seeds of pea are the source of precursor protein, which releases the ACE inhibitory peptides as a result of enzymatic hydrolysis.

Comparative study chromatographic fractions activities from Terminalia ivorensis and Ketoconazole as standard antifungal on Trichophyton mentagrophytes var. interdigitale in vitro growth.

The present study was undertaken to evaluate in-vitro antimicrobial activity of Ketoconazole and extracts from trunk’s barks of Terminalia ivorensis A. Chev (Combretaceae). In vitro antimicrobial activity of all the extracts was done by agar slant. Extracts were incorporated in Sabouraud medium culture and extract has been inserted to this medium according to the Agar slant method and Ketoconazole were used as standards for antifungal assay. Antimicrobial activity was determined by diminution of mushroom in the assay tubes. For each extract five tests were done. The antifungal activity was more pronounced against Trichophyton mentagrophytes var. interdigitale. The fraction F8 showed best antifungal activity. T. ivorensis barks extracts showed better antifungal activity than commercially used antibiotics. Demonstration of antimicrobial activity of T. ivorensis provides the scientific basis for the use of this plant in the traditional treatment of diseases and may help to discover new chemical classes of antibiotic substances that could serve as selective agents for infectious and cutaneous diseases. This investigation has opened up the possibility of the use of this plant in drug development for human consumption possibly for the treatment of various infections caused by opportunists’ mushrooms.

Bioactivity of venom extracted from the sea anemone Anthopleura asiatica (Cnidaria: Anthozoa): Toxicity and Histopathological studies

The bioactivity of the venom from a locally available sea anemone, Anthopleura asiaticacollected from the Mumbai coast was studied. The crude venom from sixty sea anemones (Sixty numbers) was extracted in aqueous medium. The protein content of the crude venom was 4.3459±0.027 mg/ml. The crude venom was found to be lethal at 1ml when injected intra-peritoneal to kasuali strain male albino mice (20±2 g). The crude venom was partially purified by anion exchange chromatography using a step-wise gradient of 0.1-1.0M NaCl and 10 fractions each of 15 ml, F1 – F10 were collected. Fractions F8, F9 and F10 exhibited lethality to mice, upon envenomation. The symptoms of toxicity observed in the mice indicated that the venom affected the central nervous, cardiovascular and urinary systems. Histopathological study revealed accumulation of polymorphic nuclear cells with necrosis in brain, hemolysis in heart, occlusion with hemolysed blood in kidney and necrosis, vacuolation with pleomorphic nuclear material and hemolysis in liver. Key words: Sea anemone, venom, toxicity, histopathology.

Antisecretory actions of Baccharis trimera (Less.) DC aqueous extract and isolated compounds: Analysis of underlying mechanisms

Baccharis trimera (Less.) DC. (Asteraceae) is a species native to South America used in Brazilian folk medicine to treat gastrointestinal and liver diseases, kidney disorders and diabetes. Previous studies from this laboratory confirmed the antacid and antiulcer activities of the plant aqueous extract (AE) in rat and mouse models. To investigate the mechanisms involved in the antacid action of AE and isolated compounds from Baccharis trimera. AE was assayed in vivo in cold-restraint stress gastric ulcers and in pylorus-ligated mice. Nine fractions (F2-F10) previously isolated from AE were assayed in vitro on acid secretion measured as [(14)C]-aminopyrine ([(14)C]-AP) accumulation in rabbit gastric glands, and on gastric microsomal H(+), K(+)-ATPase preparations. Chlorogenic acids (F2, F3, F6, F7), flavonoids (F9), an ent-clerodane diterpene (F8) and a dilactonic neo-clerodane diterpene (F10) have been identified in these fractions. Intraduodenal injection of AE (1.0 and 2.0 g/kg) in 4h pylorus-ligated mice decreased the volume (20 and 50%) and total acidity (34 and 50%) of acid secretion compared to control values. Administered orally at the same doses AE protected against gastric mucosal lesions induced in mice by restraint at 4°C. Exposure of isolated rabbit gastric glands to fractions F8 (10-100 μM) and F9 (10-300 μg/ml) decreased the basal [(14)C]-AP uptake by 50 and 60% of control (Ratio=6.2±1.1), whereas the remaining fractions were inactive. In the presence of the secretagogues F2 and F4 (30-300 μg/ml) decreased the [(14)C]-AP uptake induced by histamine (His) with a 100-fold lower potency than that of ranitidine. F5 and F6 reduced the [(14)C]-AP uptake stimulated by carbachol (CCh), but they were 10 to 20-fold less potent than atropine. F8 (diterpene 2) and F9 (flavonoids) decreased both the His- and CCh-induced [(14)C]-AP uptake, whereas F10 (diterpene 1) was inactive against the [(14)C]-AP uptake stimulated by secretagogues. Diterpene 2 was the most active of all tested compounds being 7-fold less potent than ranitidine and equipotent to atropine in reducing acid secretion in vitro. This compound also reduced the gastric H(+), K(+)-ATPase activity by 20% of control, while the remaining fractions were inactive on the proton pump in vitro. The results indicate that Baccharis trimera presents constituents that inhibit gastric acid secretion by acting mainly on the cholinergic regulatory pathway. The plant extract also contains compounds that exert moderate inhibition of the histaminergic regulatory pathway of acid secretion and the gastric proton pump. Altogether these active constituents appear to provide effective inhibition of acid secretion in vivo, which may explain the reputed antiulcer activity of the plant extract.

Amélioration par fractionnement chromatographique de l’activité anticandidosique d’un extrait hexanique de Mitracarpus scaber Zucc. sur la croissance in vitro de Candida albicans et Candida tropicalis

In the goal to bring our contribution to the struggle against opportunistic candidosis, we tested seventeen chromatographic fraction gotten from an hexanic extract of a Mitracarpus scaber Misca codified on the in vitro growth of Candida albicans and Candida tropicalis. Antifungal tests have been made on Sabouraud medium culture. Extract has been inserted to this medium according to the Agar slant method. The range of concentrations has been fixed between 3.125 μg/ml and 12 μg/ml. Results showed that in this interval of values, alone fractions F6, F7, F8 permitted a clean inhibition to 3 125 μg/ml of the growth of the 2 strains of Candida. Among them, the fraction F8 has possessed a better anticandidosic activity with IC50 = 16 μg/ml on Candida albicans against IC50 = 45 μg/mls on Candida tropicalis which is the least sensible. So, the chromatographic fraction F8 obtained from a hexanic extract is the one that permits a better concentration of active principles of Misca.

Characterization of the TE-NORM waste associated with oil and natural gas production in Abu Rudeis, Egypt

The present study was conducted to characterize the Technically Enhanced Naturally Occurring Radioactive Materials (TE-NORM) waste generated from oil and gas production. The waste was characterized by means of dry screening solid fractionation, X-ray analysis (XRF and XRD) and γ-ray spectrometry. Sediment of the TE-NORM waste was fractionated into ten fractions with particle sizes varying from less than 100 μm to more than 3 mm. The results showed that the TE-NORM waste contains mainly radionuclides of the 238U, 235U and 232Th series. The mean activity concentrations of 226Ra (of U-series), 228Ra (of Th-series) and 40K in the waste samples before fractionation (i.e. 3 mm) were found to amount to 68.9, 24 and 1.3 Bq/g (dry weight), respectively. After dry fractionation, the activity concentrations were widely distributed and enriched in certain fractions. This represented a 1.48 and 1.82-fold enrichment of 226Ra and 228Ra, respectively, in fraction F8 (2.0–2.5 mm) over those in bulk TE-NORM waste samples. The activity ratios of 238U/ 226Ra, 210Pb/ 226Ra, 223Ra/ 226Ra and 228Ra/ 224Ra were calculated and evaluated. Activity of the most hazardous radionuclide 226Ra was found to be higher than the exemption levels established by IAEA [International Atomic Energy Agency, 1994. International Basic Safety Standards for the Protection against Ionizing Radiation and for the Safety of Radiation Sources. GOV/2715/94, Vienna]. The radium equivalent activity (Ra-eq), radon ( 222Rn) emanation coefficient (EC) and absorbed dose rate ( D γr) were estimated and these are further discussed.

Specific Isoforms of Actin-binding Proteins on Distinct Populations of Golgi-derived Vesicles

Golgi membranes and Golgi-derived vesicles are associated with multiple cytoskeletal proteins and motors, the diversity and distribution of which have not yet been defined. Carrier vesicles were separated from Golgi membranes, using an in vitro budding assay, and different populations of vesicles were separated using sucrose density gradients. Three main populations of vesicles labeled with beta-COP, gamma-adaptin, or p200/myosin II were separated and analyzed for the presence of actin/actin-binding proteins. beta-Actin was bound to Golgi cisternae and to all populations of newly budded vesicles. Centractin was selectively associated with vesicles co-distributing with beta-COP-vesicles, while p200/myosin II (non-muscle myosin IIA) and non-muscle myosin IIB were found on different vesicle populations. Isoforms of the Tm5 tropomyosins were found on selected Golgi-derived vesicles, while other Tm isoforms did not colocalize with Tm5 indicating the association of specialized actin filaments with Golgi-derived vesicles. Golgi-derived vesicles were shown to bind to F-actin polymerized from cytosol with Jasplakinolide. Thus, newly budded, coated vesicles derived from Golgi membranes can bind to actin and are customized for differential interactions with microfilaments by the presence of selective arrays of actin-binding proteins.

Cellular Mechanisms of Action of a Ouabain-Like Factor in Vascular Smooth-Muscle Cells

Ouabain-like factor (OLF) has been implicated to play an important role in certain forms of hypertension. We isolated OLF from the urine of salt-loaded healthy subjects by stepwise chromatographic procedures. The post-salt fraction (F IV) eluted from Sephadex G-25 was rechromatographed on Sephadex G-10. A late small-molecular-weight fraction F8 inhibited Na-K-ATPase in vitro (OLF activity). The effects of OLF on intracellular Ca2+ concentrations ([Ca2+]i) and pH (pHi) were examined in cultures of vascular smooth-muscle cells using the fluorescent probes fura-2 and 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), respectively. Preincubation with OLF increased basal [Ca2+]i from 87 +/- 6 to 160 +/- 8 nM (p < 0.001) and enhanced arginine vasopressin-stimulated maximal [Ca2+]i (418 +/- 11 vs. 523 +/- 14 nM, p < 0.01). This effect was similar to that of ouabain. OLF also induced a rapid transient increase of [Ca2+]i (82 +/- 9 vs. 253 +/- 23 nM, p < 0.01); [Ca2+]i returned to levels slightly above baseline within approximately 4 min. OLF-stimulated [Ca2+]i was attenuated by verapamil (126 +/- 5 nM, p < 0.01) and was also reduced in Ca(2+)-free medium (104 +/- 9 nM, p < 0.01). As opposed to OLF, ouabain did not exhibit this fast transient effect on [Ca2+]i. Amiloride (10(-3) M) blocked the sustained effect of OLF on [Ca2+]i (77 +/- 11 vs. 86 +/- 12 nM, NS). OLF induced an increase of pHi from 7.09 +/- 0.03 to 7.28 +/- 0.04 (p < 0.002).(ABSTRACT TRUNCATED AT 250 WORDS)