Partially-purified Extract Research Articles

Emulsion formulated using Hibiscus tiliaceus L. extract and flaxseed oil for topical application

The oil of Linum usitatissimum L., also known as flaxseed, is known for its many health benefits and has been widely used in food and cosmetic product. Hibiscus tiliaceus L. is a semi-mangrove whose wood and bark are used for fuel and fibre production. Preliminary studies have reported tyrosinase and melanogenesis inhibition activity in H. tiliaceus leaves dichloromethane extract (DE). Thus, it was believed that Hibiscus tiliaceus leaves could be used as an active ingredient in whitening products. The present study aimed to develop a stable formulation using flaxseed oil and H. tiliaceus partially purified extract (PE). Furthermore, this study aimed to evaluate the formulations’ physicochemical properties, microbial quality, storage stability, safety, and bioactivities. The presence of the active compound, friedelin, in partially purified extract (PE) obtained using Silica 60 column chromatography was identified using gas chromatography-mass spectrometry (GC-MS) analysis. Emulsions incorporated with 0.1% and 0.2% (w/w) PE, namely partially purified extract emulsion (PEE) 1 and 2 respectively, were found with satisfied physiochemical and safety evaluations. Both PEE 1 and 2 remarkably inhibited tyrosinase activity by 61% and 70%, respectively. They also exerted high efficacy in suppressing melanin content and tyrosinase activity in murine melanoma cells (B16F1). Furthermore, both PEE showed no antiproliferative activity on both melanoma and normal human dermal fibroblast (NHDF) cells and are unlikely to cause inflammation and affect the function of melanoma cells used in the melanogenesis assay. Interestingly, PEE 1 and 2 inhibited 34% and 32% extracellular melanogenesis at 0.5 mg/mL, which was higher than that of 1 and 2 mg/mL. No microbial growth was observed for in the microbial test when incorporating eugenol (0.4%) into the emulsions as an antimicrobial agent. PEE 1 yields better physicochemical properties, microbial quality and bioactivities upon storage at chilled, ambient and elevated temperatures. This study creates an approach to the development of natural whitening products using coastal plants.

Macroporous resin-assisted enrichment, characterizations, antioxidant and anticandidal potential of phytochemicals from Trachyspermum ammi.

Extract of de-oiled seeds of Trachyspermum ammi was purified using macroporous resins and the performance of three resins were evaluated to enrich major phytochemical component. A HPLC method has been developed to separate major phytochemical constituents in the crude (CTAE) and partially purified extracts (PTAE). Macroporous resin assisted enrichment and purification suggested XAD-16 as the most efficient (yield 29.8%) followed by XAD-7HP and Diaion HP-20. Concentrated PTAE was subjected to multiple preparative-TLC to afford three compounds, namely, rosmarinic acid-3-O-glucopyranoside (TA-1), kaempferol-(coumaroyl-glucosyl)-rhamnoside (TA-2) and quercetin-3-O-galactoside (TA-3). The structure of these compounds was elucidated from their corresponding spectroscopic characterizations in FT-IR, HR-MS, and partially by 1 H NMR. Total phenolic and flavonoid contents of the extracts were determined. Antioxidant activity by DPPH and ABTS radical scavenging, CUPRAC assays indicated the highest antioxidant potential of CTAE. Among the compounds, TA-1 exhibited the highest scavenging activity in ABTS (IC50 33.41µg/ml) and DPPH (IC50 69.23µg/ml), however, relatively lower than CTAE. In vitro anti-candidal activity against virulent strains of Candida spp. revealed C. albicans 4718 as the most susceptible (23.9µg/ml) to PTAE. PRACTICAL APPLICATIONS: Seeds of Trachyspermum ammi has been extensively investigated for volatile aromatic components of the essential oil. However, the de-oiled seeds have rarely been exploited for potential bioactive phenolics. The present investigation envisaged possible utilization of the de-oiled Trachyspermum seeds for its phenolic constituents, which could be used as natural antioxidant with additional benefits of anticandidal properties. Indeed, macroporous resin assisted enrichment and purification of extracts of T. ammi seeds generate valuable reference compounds, rosmarinic acid-3-O-glucopyranoside, kaempferol-(coumaroyl glucosyl)-rhamnoside, and quercetin-3-O-galactoside.

Phenolic composition by UHPLC-Q-TOF-MS/MS and stability of anthocyanins from Clitoria ternatea L. (butterfly pea) blue petals.

The aim of the present study was to evaluate the phenolic composition of crude lyophilized extracts (CLE) and partially purified (PPE) extracts of C. ternatea blue petals as well as the anthocyanin stability against pH, temperature and light in the presence and absence of fructooligosaccharides. Twelve compounds were tentatively identified by UHPLC-Q-TOF-MS/MS in CLE and PPE extracts. In direct/reverse spectrophotometric titration, anthocyanins showed colour changes between pH 2.25 to 10.20, and colour reversibility, maintaining antioxidant activity against the DPPH radical. The aqueous extracts at pH 3.6 and 5.4 exhibited thermal stability with the presence and absence of fructooligosaccharides with activation energy higher than 99kJ/mol. The addition of fructooligosaccharides in the extracts at pH 5.4 exposed to light provided a protective effect against anthocyanin photodegradation. The data show the technological potential of aqueous extract of C. ternatea blue petals as a natural colourant in a functional beverage model system.

Evaluation of Antimicrobial, Enzyme Inhibitory, Antioxidant and Cytotoxic Activities of Partially Purified Volatile Metabolites of Marine Streptomyces sp.S2A.

In the present study, marine actinobacteria Streptomyces sp.S2A was isolated from the Gulf of Mannar, India. Identification was carried out by 16S rRNA analysis. Bioactive metabolites were extracted by solvent extraction method. The metabolites were assayed for antagonistic activity against bacterial and fungal pathogens, inhibition of α-glucosidase and α-amylase enzymes, antioxidant activity and cytotoxic activity against various cell lines. The actinobacterial extract showed significant antagonistic activity against four gram-positive and two gram-negative pathogens. Excellent reduction in the growth of fungal pathogens was also observed. The minimum inhibitory concentration of the partially purified extract (PPE) was determined as 31.25 μg/mL against Klebsiella pneumoniae, 15.62 μg/mL against Staphylococcus epidermidis, Staphylococcus aureus and Bacillus cereus. The lowest MIC was observed against Micrococcus luteus as 7.8 μg/mL. MIC against fungal pathogens was determined as 62.5 μg/mL against Bipolaris maydis and 15.62 μg/mL against Fusarium moniliforme. The α-glucosidase and α-amylase inhibitory potential of the fractions were carried out by microtiter plate method. IC50 value of active fraction for α-glucosidase and α-amylase inhibition was found to be 21.17 μg/mL and 20.46 μg/mL respectively. The antioxidant activity of partially purified extract (PPE) (DPPH, ABTS, FRAP and Metal chelating activity) were observed and were also found to have significant cytotoxic activity against HT-29, MDA and U-87MG cell lines. The compound analysis was performed using gas chromatography-mass spectrometry (GC-MS) and resulted in three constituents; pyrrolo[1–a]pyrazine-1,4-dione,hexahydro-3-(2-methylpropyl)-, being the main component (80%). Overall, the strain possesses a wide spectrum of antimicrobial, enzyme inhibitory, antioxidant and cytotoxic activities which affords the production of significant bioactive metabolites as potential pharmacological agents.

An Antibiotic Complex from Lysobacter enzymogenes Strain C3: Antimicrobial Activity and Role in Plant Disease Control

Lysobacter enzymogenes C3 is a bacterial biological control agent that exhibits antagonism against multiple fungal pathogens. Its antifungal activity was attributed in part to lytic enzymes. In this study, a heat-stable antifungal factor (HSAF), an antibiotic complex consisting of dihydromaltophilin and structurally related macrocyclic lactams, was found to be responsible for antagonism by C3 against fungi and oomycetes in culture. HSAF in purified form exhibited inhibitory activity against a wide range of fungal and oomycetes species in vitro, inhibiting spore germination, and disrupting hyphal polarity in sensitive fungi. When applied to tall fescue leaves as a partially-purified extract, HSAF at 25 mug/ml and higher inhibited germination of conidia of Bipolaris sorokiniana compared with the control. Although application of HSAF at 12.5 mug/ml did not reduce the incidence of conidial germination, it inhibited appressorium formation and suppressed Bipolaris leaf spot development. Two mutant strains of C3 (K19 and DeltaNRPS) that were disrupted in different domains in the hybrid polyketide synthase-nonribosomal peptide synthetase gene for HSAF biosynthesis and had lost the ability to produce HSAF were compared with the wild-type strain for biological control efficacy against Bipolaris leaf spot on tall fescue and Fusarium head blight, caused by Fusarium graminearum, on wheat. Both mutant strains exhibited decreased capacity to reduce the incidence and severity of Bipolaris leaf spot compared with C3. In contrast, the mutant strains were as efficacious as the wild-type strain in reducing the severity of Fusarium head blight. Thus, HSAF appears to be a mechanism for biological control by strain C3 against some, but not all, plant pathogenic fungi.

New alkaline protease from Nocardiopsis sp.: partial purification and characterization

A new alkaline protease from Nocardiopsis sp., isolated from a soil sample collected from the Northeast of Brazil is reported. The crude extract (CE) was designate as partially purified extract (PPE) after purification with ammonium sulphate (0–20%). Optimal pH and temperature for detection of protease activity were obtaining at pH 10.5 and 8.0, for CE and PPE, respectively, at 50 °C for both extracts. The enzyme was stable at alkaline pH and more than 75% activity was retained even after incubation for 120 min at pH's between 8.0 and 10.5, for both extracts. The use of specific inhibitors, made it possible to show that the enzyme belongs to the group of serine protease. Maximum protease activity was achieved at pH 8.0 (50 U/mg) for CE and at pH 10.0 for PPE (1260 U/mg). The CE was partial purified with Sephadex G-75 obtained 26-purification fold and 34% yield.

Plant growth regulators and phloem exudates modulate root nodulation of soybean.

A time-course study was conducted on the effect of synthetic ABA and phloem exudate on root nodulation, nitrogenase activity, and dry matter content of pouch-grown soybean [Glycine max (L.) Merr.]. A hypernodulating mutant (NOD1-3) derived from the cultivar Williams was evaluated. The normally-nodulating cultivar Williams 82 served as the control. All phases of nodulation (nodule initiation, development and function) were inhibited by 1-50 μM synthetic ABA treatment of roots of pouch-grown soybean. The ABA effect was more pronounced for the treatment made 24 h prior to inoculation. The magnitude of inhibition of nodulation by ABA and by phloem exudate from inoculated Williams 82 was decreased when application was delayed to 48 h post-inoculation. Synthetic indoleacetic acid and benzyladenine (1 and 5 μM) also inhibited the number of nodules per plant, and the magnitude of inhibition was greater in the normally-nodulating Williams 82 cultivar than in the NOD1-3 hypernodulating mutant. Changes in endogenous free ABA in cotyledons, leaves, and xylem sap of seedlings were monitored at 24-h intervals after inoculation in both lines, and appeared to respond to inoculation. Addition of crude phloem exudate from Williams 82 or hypernodulating NOD1-3 was inhibitory to root nodulation of NOD1-3 - this effect was concentration dependent. Partially-purified (acidic aqueous fraction) phloem exudate from Williams 82 suppressed nodulation in NOD1-3 by 54% (averaged over a 7-d time-course), while partially-purified phloem exudate (acidic aqueous fraction) from NOD1-3 was much less inhibitory (23%). ABA levels in the phloem fractions applied were nearly identical (15.6 vs 16.0 ng mL-1), which indicates that ABA was not directly affecting nodulation control. Partially-purified extract (acidic aqueous fraction) from leaves of nodulated NOD1-3 was much less inhibitory to nodulation of NOD1-3 seedlings than was the comparable fraction from Williams 82. Although free ABA applied exogenously was capable of altering nodulation, it does not appear that endogenous ABA levels directly account for the autoregulatory control of nodulation in soybean. This implies that there may be factor(s) moving in the phloem other than ABA that possibly impact expression of root nodulation. Alternatively, it may be that plant development is responsive to interactions among plant growth regulators mediated by subtle changes in individual hormone concentrations. The possibility remains that normally-nodulating and hypernodulating lines have differential sensitivity to ABA, and that this explains altered nodulation control at similar levels of endogenous ABA.

142 ESTER FORMATION IN RELATION TO HARVEST MATURITY AND CONTROLLED-ATMOSPHERE STORAGE OF `GALA' APPLES

Volatile esters from acids and alcohols are important components of flavor and odor perception in apples (Malus domestica Borkh.). We are interested in understanding the biochemical basis for ester synthesis/flavor retention in `Gala' apples held in controlled atmosphere storage. The relationship between acetyl CoA alcohol transferase (AAT) acetate ester-formin activity, non-ethylene volatile emission, and flesh volatile content of `Gala' apples during the maturation period and after removal from CA storage was investigated. At the appropriate times, apples were sampled for volatile compounds in the headspace and flesh using solid sorbent along with purge-and-trap capillary gas chromatography. Subsequently, acetate ester forming activity was assayed on partially-purified extracts of cortical tissue. During storage, the accumulation of the major flavor notes butyl acetate and 2-methyl butyl acetate in the flesh was decreased as oxygen levels in storage atmospheres were lowered. AAT activity is closely linked to the onset of climacteric ripening and is sensitive to atmospheres having low oxygen contents.

Ester biosynthesis in “Rome” apples subjected to low-oxygen atmospheres

Abstract The relationship between acetate ester-forming activity of acetyl CoA alcohol transferase (ACAT), non-ethylene volatile emission, and flesh volatile content of “Rome” apples ( Malus domestica Borkh.) after removal from 9 months storage in low-oxygen environments was investigated. Apple samples held at 0–1°C were removed from refrigerated air (RA) or controlled-atmosphere (CA) storage at 1.0 or 0.5% v/v O 2 /1% CO 2 and placed in ambient laboratory conditions. Every three days fruit flesh and headspace were analyzed for volatile compounds using capillary gas chromatography. Acetate ester forming activity was assayed spectrophotometrically on partially-purified extracts of cortical tissue. Patterns of acetate ester formation depended upon storage environment and alcohol moiety precursor. Ethyl acetate content was always higher in the headspace and flesh of RA fruit, regardless of time after storage. Propyl acetate concentrations in CA stored apple flesh were higher until post-storage day 12 when they decreased. Headspace propyl acetate levels were higher in RA controls until days 9–15 when more emanated from CA fruit. Butyl acetate concentrations were lower in flesh and headspace of CA fruit until day 15, when RA fruit headspace levels decreased. Headspace and flesh concentrations of 2-methyl-1-butyl acetate were higher in CA apples. Acetate ester-forming activity was detectable at day 0 in 1.0% oxygen-stored fruits, but not in 0.5% oxygen-stored fruits. By post-storage day 9, ACAT activity in CA apples reached maximal levels, only to decrease by day 15. RA-stored apples had more ACAT activity at day 0, but did not substantially increase in activity like the 0.5% oxygen-stored apples removed from CA storage. It is unknown whether the ACAT protein is reactivated by exposure to ambient oxygen or synthesized de novo.

Purification to homogeneity and partial amino acid sequence of a fragment which includes the methyl acceptor site of the human DNA repair protein for O6-methylguanine

DNA repair by O6-methylguanine-DNA methyltransferase (O6-MT) is accomplished by removal by the enzyme of the methyl group from premutagenic O6-methylguanine-DNA, thereby restoring native guanine in DNA. The methyl group is transferred to an acceptor site cysteine thiol group in the enzyme, which causes the irreversible inactivation of O6-MT. We detected a variety of different forms of the methylated, inactivated enzyme in crude extracts of human spleen of molecular weights higher and lower than the usually observed 21-24kDa for the human O6-MT. Several apparent fragments of the methylated form of the protein were purified to homogeneity following reaction of partially-purified extract enzyme with O6-[3H-CH3]methylguanine-DNA substrate. One of these fragments yielded amino acid sequence information spanning fifteen residues, which was identified as probably belonging to human methyltransferase by virtue of both its significant sequence homology to three procaryote forms of O6-MT encoded by the ada, ogt (both from E. coli) and dat (B. subtilis) genes, and sequence position of the radiolabelled methyl group which matched the position of the conserved procaryote methyl acceptor site cysteine residue. Statistical prediction of secondary structure indicated good homologies between the human fragment and corresponding regions of the constitutive form of O6-MT in procaryotes (ogt and dat gene products), but not with the inducible ada protein, indicating the possibility that we had obtained partial amino acid sequence for a non-inducible form of the human enzyme. The identity of the fragment sequence as belonging to human methyltransferase was more recently confirmed by comparison with cDNA-derived amino acid sequence from the cloned human O6-MT gene from HeLa cells (1). The two sequences compared well, with only three out of fifteen amino acids being different (and two of them by only one nucleotide in each codon).

Settlement and recruitment of sea urchins (Strongylocentrotus spp.) in a sea-urchin barren ground and a kelp bed: are populations regulated by settlement or post-settlement processes?

I sampled recruitment of very small sea urchins (Strongylocentrotus spp.) by using the anesthetic magnesium chloride to remove individuals from substrata collected in sea-urchin barren grounds (barrens) and kelp beds at Naples Reef near Santa Barbara, California, USA. Preliminary sampling found low numbers of newly settled individuals(<0.6 mm test diam) from April–July in 1984 and 1985, and in April, 1986. In early May, 1986, I found many newly settled seaurchins (0.3 to 0.6 mm, 5 to 17 d old), and I compared the densities of the cohort on several types of natural substrata in barrens and kelp-bed habitats. Newly settled individuals of both purple sea urchins (S. purpuratus) and red sea urchins (S. franciscanus) were present in similar, high densities (1 000 S. purpuratus m-2) on foliose red algal turf, a dominant substratum ofthe kelp bed, and on crustose coralline algae, the dominant substratum of an adjacent barrens. Larvae of S. purpuratus reared and tested in the laboratory showed high rates of settlement on both red algal turf and on crustose coralline algae, but significantly lower rates on rock. Larvae also settled in response to a partiallypurified extract of coralline algae. The reduced settlement on natural rock surfaces relative to either algal treatment and the significant settlement in response to the extract of coralline algae indicate that larvae discriminate between natural substrata and probably respond to a settlement cue other than, or in addition to, a simple microbial (bacterial) film. The similar densities of young recruits of S. purpuratus on dominant substrata of barrens and kelp bed show that, at least in this case, differential settlement cannot explain the high densities of sea urchins in the barrens habitat. Movement between barrens and kelp bed is unlikely given the small sizes of the newly recruited sea urchins relative to the large distances often involved. Reduced post-settlement mortality of newly settled individuals in the barrens remains the most likely mechanism leading to the higher densities of sea urchins in barrens relative to kelp-bed habitats.

A critical look at the importance, prevalence and control of toxocariasis and the possibilities of immunological control

The visceral infection of humans with Toxocara canis is particularly prevalent in children and may cause a variety of symptoms that commonly persist for 6–24 months. The ocular infection usually causes permanent loss of visual acuity. Human infection is acquired by ingestion of embryonated T. canis eggs with contaminated dirt. Review of recent reports indicates that patent T. canis infection is widely prevalent in the general population of dogs all over the world (3–81%) and results in a substantial contamination of the ground (0.3–87%). The results of sensitive and specific serological tests suggests that about 7% of the clinically healthy human population of the United States, about 5% of that of Canada, and about 4% of that in Great Britain is infected with the parasite. Control of transmission of the parasite to man is often attempted by eliminating the infection in dogs, reducing the population of dogs and the environmental contamination with their feces, and educating the public about the zoonotic potential of toxocariasis. The evidence reviewed indicates that these methods are only marginally effective. Because T. canis relies on congenital and lactogenic transmission to persist in nature, only a procedure that effects the sustained killing of the reservoir larvae in the tissues of the bitch, or of newly-acquired parasites, is expected to be successful. Research with mice, rabbits and dogs demonstrated that prior infections of the host induce the development of protective immunity to reinfections. This procedure, however, leaves remnant populations of larvae from the immunizing infections that are resistant to anthelmintics and to the effect of prior irradiation. Hyperimmunization with partially-purified extracts of T. canis larvae induced 37% resistance to a challenge in mice when the extract was administered alone, and 76% resistance when administered with lipopolysaccharide adjuvant. Production of complete resistance, however, will probably require the prior control of the immunosuppression induced by the parasite. T. canis infections inhibit the production of homologous protective immunity and antibody responses to heterologous antigens, probably by interfering with the activity of helper T-cells, competing with protective antigens, and suppressing antibody synthesis. The evidence indicates, however, that an anti- T. canis vaccine to eliminate the parasite in dogs is feasible.

Immunization of dogs with a Leishmania infantum-derived vaccine

A partially-purified extract of Leishmania infantum has been administered to healthy dogs. Post-immunization sera were found to neutralize the infectivity of L. infantum and to abate the development of L. major. Muramyl dipeptide and one of its derivates, murabutide, were the best adjuvants.

M-associated protein antibodies in patients with rheumatic fever.

Sequential serum samples obtained from 50 rheumatic fever subjects and from control individuals matched for time, age and geographical location were tested for antibodies against the M-associated protein antigens, MAP I and MAP II. Antibody titres were determined by the complement fixation test with a partially-purified extract of Streptococcus pyogenes serotype M30 as the MAP I antigen and an acid extract of serotype M48 as the MAP II antigen. Titres of MAP I antibody exceeded those of MAP II antibody in all but six rheumatic fever subjects. Anti-MAP I titres in excess of 40 were significantly more common in rheumatic fever subjects than in matched controls (p less than 0.001) or matched subjects with a diagnosis of acute post-streptococcal glomerulonephritis (p less than 0.01). Peak MAP I titres were present at the time of admission to hospital in the sera of 40 of the 50 rheumatic fever subjects. In the remainder peak titres occurred within 10 days. Antibody titres were maintained for a mean of 10.3 weeks before declining. Changes in MAP antibody titres were independent of changes in antistreptolysin O and anti-DNAase B titres. Normal children aged between 6 and 15 had higher MAP antibody titres than 2-5-year-old children. Rheumatic fever subjects had significantly higher mean titres of MAP I antibody than matched controls in each age group.

レンサ球菌感染症の血清学的診断法とその意義に関する研究

The antibody titers were measured against extracellular products of group A streptococci, deoxyribonuclease-B (DNase-B), nicotine-amide adenine dinucleotidase (NADase), streptolysin-O SLO), cellular components of group A streptococci, partially purified extracts (PPE), (C-polysaccharide (C-poly) and peptidoglycan (PG), in the sera of patients infected with streptococci or its sequelae.The results obtained were as follows.1) Antibody titers against DNase-B and NADase were high in the sera of patients with acute glomerulonephritis or with acute rheumatic fever.2) There were no significant differences in antibody titers against PPE between healthy group and patient group, of children. In each group of patient high antibody titers against PG occurred at high frequency. Antibody titers against C-poly were high in the sera of acute glomerulonephritis, acute rheumatic fever and anaphylactoid purpura.3) No correlation was observed between ASO titers and anti DNase-B titers, as well as between ASO titers and anti NADase titers. In the patients with acute glomerulonephritis antibody titers against DNase-B and NADase were high at early stage, but had a tendency to become lower as time went on.There were almost no changes in the antibody titers of the sera of the patients with mucocutaneous lymphoide syndrome (MCLS).

Insulin-like hypoglycemic and immunological activities in honeybee royal jelly

An aqueous extract of royal jelly from Apis mellifera produced hypoglycemia when injected into larvae of Manduca sexta. Application of specific radioimmunoassay to the partially-purified extract showed that royal jelly contains several insulin-like peptides, the major immunoreactive component of which has an apparent mol. wt similar to that of bovine insulin. These results suggest the existence of a peptide in the honeybee having both biological and structural similarities to vertebrate insulin.