Phytochemical Evaluation, Antioxidant and Antimicrobial Activities of Various Extracts from Leaves and Stems of Bryophyllum pinnatum

Antioxidant and antimicrobial activities of different extracts (methanol and ethyl acetate) of leaf and stem of Bryophyllum pinnatum were studied. The screening for the secondary metabolites was carried out using the standard methods. The antioxidant capacities of the different extracts were assessed using DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals and Ferric reducing antioxidant power (FRAP) while the antimicrobial activity of the extracts obtained were screened against Gram-positive, Gram-negative bacteria and fungi (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa Salmonella spp., Vibrio cholerae, Candida albicans and Aspergillus niger) using Agar well diffusion method. Both extracts obtained from leaf and stem of Bryophyllum pinnatum contained most of the phytochemical compounds tested for. However, anthocyanins and anthraquinone were not detected in leaf extracts while coumarin was absent in stem extracts. Quantification of bioactive compounds showed that both extracts contained the highest concentration of polyphenols (34.49 ±0.47 mg GAE/g and 32.32 ±1.2 mg GAE/g for methanol leaf and stem extracts respectively) while the least concentration was recorded for alkaloids (0.03±0.02 mg/g for methanol stem extract). Results revealed that the extracts showed dose-dependent scavenging of DPPH as well as the ability of the extracts to reduce FeCl3 solution, with methanol extracts exhibiting the highest scavenging and reducing capacity. However the leaves of Bryophyllum pinnatum had greater antioxidant activity than the stem by DPPH and ferric reducing assays, with IC50 values ranging from 3.147µg/ml to 3.80µg/ml for DPPH and 331.9 - 451 µg/ml for FRAP assays. The antimicrobial activity of various solvent extracts of leaf and stem reveal that microorganisms exhibited different sensitivities towards these extracts in a dose-dependent manner. Methanol leaf extract showed no activity against Escherichia coli while Pseudomonas aeruginosa was insensitive to ethyl acetate leaf extract. For stem extracts, Aspergillus niger, Vibrio cholerae and Pseudomonas aeruginosa were resistant to methanol extract while Aspergillus niger, Salmonella spp. and Pseudomonas aeruginosa was resistant towards ethyl acetate stem extract. The results obtained in this study showed that Bryophyllum pinnatum is a reservoir of bioactive compounds and both extracts exhibited significant antimicrobial and antioxidant activity

Phenolic Compounds from the Aerial Parts of Adenophora triphylla (Thunb.) A. DC. var. triphylla and their Free Radical Scavenging Activity

Adenophora triphylla (Thunb.) A. DC. var. triphylla (Family: Campanulaceae) is distributed in Japan, Korea, and China. It is locally known as “Saiyousyajin” in Japan and the roots are used in traditional medicine to treat chronic bronchitis and whooping cough, and also as anti-inflammatory and anti-tussive agents. Till now, there is no report on the chemical constituents of aerial parts. Thus, the main aim of this study was to isolate and identify major chemical constituents of aerial parts of A. triphylla var. triphylla, and to evaluate their free radical scavenging activity. The 70% methanol extract of the aerial parts was subjected to repeated column chromatography using MCI gel CHP-20P, Sephadex LH-20, ODS and silica gel columns to isolate the five phenolic components (1-5). Free radical scavenging activity of the extract and compounds was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity method. The structures of the isolated compounds were elucidated as luteolin (1), luteolin 4’-O-b-glucopyranoside (2), luteolin 7-O-b-glucopyranoside (3), luteolin 7-O-neohesperidoside (4) and chlorogenic acid (5) based on their nuclear magnetic resonance (NMR) spectral data and comparison with literature values. All these compounds were isolated for the first time from A. triphylla var. triphylla. Extract showed weak free radical scavenging activity. Among isolated compounds, luteolin (1), luteolin 7-O-b-glucopyranoside (3), luteolin 7-O-neohesperidoside (4) and chlorogenic acid (5) showed potent free radical scavenging activity. Results from this study suggest that the aerial parts of A. triphylla var. triphylla might be a potential plant source for the development of functional foods, however further detailed research is necessary

Selection of Pyramided Barley Advanced Lines for Stripe Rust, Leaf Rust and Crown Rust Diseases Using Molecular Markers

Barley diseases are the major yield limiting factors for barley cultivation in Nepal. Stripe/Yellow rust (P. striformis f.sp. hordei and P. striformis f.sp. tritici), leaf rust (Puccinia hordei), and crown rust (P. coronata) are the major rust diseases in Nepal. Pyramiding resistance genes against all these rust diseases are possible through molecular marker assisted breeding. Sweden originated barley variety ‘Bonus’ is found resistant to stripe rust and having linked microsatellite markers for stripe rust and crown rust resistance. Similarly, Nepalese hull-less barley variety ‘Solu Uwa’ and Nepalese awn-less barley landrace NPGR Acc# 2478 have linked microsatellite markers for leaf rust resistance. Therefore, one polymorphic sequence tagged sites (STS) marker (ABG054) for stripe rust resistance, two polymorphic simple sequence repeats (SSR) markers (Bmac0144h and HVM049) for leaf rust and one polymorphic SSR marker (Bmag0006) for crown rust resistance were used to select the advanced barley lines (at F8 stage) from above parents. Field screening of stripe rust resistance was also conducted. Among 51 advanced and field disease resistance lines from Bonus/Solu Uwa cross, we have selected 10 pyramided lines for all three types of barley rust resistance. Similarly, among 39 advanced and field disease resistance lines from Bonus/NPGR Acc#2478 cross we have selected three pyramided lines and advanced for further yield testing for general cultivation purpose. The chances of losing the desired gene are high in late generation selection using molecular marker assisted selection (MAS); but the chances of getting agronomically superior varietal output will also increase

Screening of Actinomycetes from Soil for Antibacterial Activity

Actinomycetes are Gram positive, free living saprophytes which are distributed in soil as one of the major populations and are primary source of antibiotics. This study was carried out with a quest to isolate actinomycetes from soil samples of different places and assess their antibacterial activity. Isolation of actinomycetes was carried out by serial dilution of soil sample followed by spread plate method. The antimicrobial extract was extracted using ethyl acetate. Assessment of antimicrobial activity was performed by using Agar cup plate assay against test organisms (Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Salmonella typhi, Salmonella paratyphi, Bacillus subtilis, Staphylococcus aureus). Antibacterial activity was tested against Methicillin Sensitive Staphylococcus aureus and Methicillin Resistant Staphylococcus aureus in the isolates having effective inhibitory activity against Staphylococcus aureus. From 15 soil samples of 12 different locations, 121 actinomycetes isolates were isolated. Among them, 58 (47.9%) isolates were inhibitory against at least 1 test organism in primary screening, of which 22 isolates effective against more than 1 test organism was chosen for secondary screening. Out of them, 8 were inhibitory against 2 test organisms while 14 were inhibitory against 3 test organisms. Staphylococcus aureus was found to be the most susceptible test organism with its susceptibility against 12 actinomycetes isolates. Among 12 isolates effective against Staphylococcus aureus, 10 were found to have an inhibitory effect against Methicillin Susceptible Staphylococcus aureus while 6 were found to have inhibitory effect against Methicillin Resistant Staphylococcus aureus strain. The findings of this study highlight the inhibitory potential of actinomycetes and the need for further investigation for obtaining novel antimicrobial agents from actinomycetes from various unexplored areas

Antibiogram of Escherichia coli and Staphylococcus aureus Isolated from Milk Sold in Kathmandu District

The emergence of antibiotic resistance in microorganisms and the presence of such isolates in milk pose a great risk to public health. Therefore, this study aims to determine the antibiotic susceptibility pattern of Escherichia coli and Staphylococcus aureus isolated from milk and assess the microbial quality of milk. For this, a total of 70 milk samples were collected and the total bacterial count (TBC) was determined. E. coli and S. aureus were isolated using their respective selective media while antibiotic susceptibility testing was carried out by Kirby Bauer Disc Diffusion method. The TBC showed that the raw milk samples contained two-fold higher microbial load while the pasteurized milk samples contained four-fold higher microbial loads than the standard guidelines. A total of 62 isolates were identified from culture-positive milk samples of which 32 were E. coli and 30 were S. aureus. A significant correlation was observed between microbial load and the organism isolated (r = 0.339, p<0.01). All S. aureus isolates were susceptible to Chloramphenicol while 40% were resistant to Cefoxitin, indicating the presence of Methicillin resistant S. aureus (MRSA). Also, 12 multidrug resistant (MDR) S. aureus were identified. While for E. coli, all were susceptible to Chloramphenicol but resistant to Ampicillin. Also, 9 MDR E. coli were detected. Higher resistance was observed among isolates from the raw milk samples than the pasteurized milk. It can be concluded that the milk produced by small-scale farms and dairy industries of Kathmandu district are of poor quality. Hence, routine microbial quality assessment and antimicrobial resistance monitoring should be followed to safeguard public health

Biofuel Production from Waste Cooking Oils and its Physicochemical Properties in Comparison to Petrodiesel

Haphazard mining and consumption of fossil fuels have reduced petroleum reserves causing fossil fuel depletion and environmental degradation; thus, reflecting the need for the cheaper, renewable and eco-friendly alternative source of petroleum to meet the fuel demand. A million liters of edible oil used for cooking foods and date expired oils from oil manufacturers are discarded into sewage. This study primarily intends to study the feasibility of biodiesel production using such waste oils. In this work, biodiesel was prepared from waste cooking oils by a process called transesterification with NaOH as a catalyst. Our results showed that methyl ester (biodiesel) (92.67±0.90%), soap materials (1.33±0.224%), and glycerol (6±0.68%) were obtained after the transesterification of waste cooking oil. The physicochemical properties of biodiesel such as density, viscosity, volatility, surface tension, and flashpoint were analyzed, which were found to be 0.862±0.006 g/cm3, 2.23±0.021 cP, 0.327×10-3±4.5×10-6 g/s, 32.03±0.138 dyne/cm, 169.67±0.810°C, respectively. These properties were compared with that of commercial diesel as well as with the values specified by the American Society for Testing and Materials (ASTM) D6751. The density and the surface tension of the biodiesel were found similar to that of petrodiesel but its volatility was 3 times lower. Fourier-transform infrared spectroscopy (FTIR) spectra of the biodiesel showed methyl ester functional group at 1436 cm-1. Based on the cost of the materials used for production, the cost of biodiesel was estimated to be about 81 Nepalese rupees (0.67 USD) per liter. The properties of biodiesel also met the standard values of ASTM D6751. These findings indicate that waste oil is one of the feasible biodiesel sources and it can be used as a suitable alternative to petrodiesel

Comparative Assessment of Antibiotic Resistance in Lactic Acid Bacteria Isolated from Healthy Human Adult and Infant Feces

Lactic acid bacteria are normal inhabitants of the gastrointestinal tract of humans. Their occurrence in infant and adult feces is abundant. The current study assesses and compares the antibiotic resistance in lactic acid bacteria isolated from healthy human adult and healthy infant fecal samples. A total of 255 lactic acid bacteria isolates (126 from adult feces and 129 from infant feces) were isolated and characterized from 60 fecal samples. Lactobacillus spp., Pediococcus spp. and Enterococcus spp. were included in the study. The study was done using the WHONET software for the analysis of antibiotic susceptibility data of lactic acid bacteria. Most of the Lactobacillus and Pediococcus strains were sensitive to vancomycin. Enterococcus strains showed resistance against vancomycin. Ampicillin, ciprofloxacin and cefuroxime resistance were significantly (p<0.05) higher in Lactobacillus strains isolated from adult fecal samples than those isolated from infant fecal samples. A similar pattern was observed in Enterococcus strains with erythromycin, gentamycin and tobramycin resistance. Pediococcal isolates from adult feces showed significantly higher resistance against tobramycin, ciprofloxacin, gentamycin, cefotaxime and cefuroxime in comparison with infant fecal isolates. Antibiotic resistance was exhibited by lactic acid bacteria against most commonly used antibiotics and it was higher in strains isolated from adult fecal samples than in the strains isolated from infant fecal samples. The increasing trend in antibiotic resistance from infant to adult might be due to food habits and antibiotic intakes. Thus, the widespread antibiotic resistance in different lactic acid bacteriamay pose a food safety concern as well

Biofuel Production from Waste Cooking Oils and its Physicochemical Properties in Comparison to Petrodiesel

Haphazard mining and consumption of fossil fuels have reduced petroleum reserves causing fossil fuel depletion and environmental degradation; thus, reflecting the need for the cheaper, renewable and eco-friendly alternative source of petroleum to meet the fuel demand. A million liters of edible oil used for cooking foods and date expired oils from oil manufacturers are discarded into sewage. This study primarily intends to study the feasibility of biodiesel production using such waste oils. In this work, biodiesel was prepared from waste cooking oils by a process called transesterification with NaOH as a catalyst. Our results showed that methyl ester (biodiesel) (92.67±0.90%), soap materials (1.33±0.224%), and glycerol (6±0.68%) were obtained after the transesterification of waste cooking oil. The physicochemical properties of biodiesel such as density, viscosity, volatility, surface tension, and flashpoint were analyzed, which were found to be 0.862±0.006 g/cm3, 2.23±0.021 cP, 0.327×10-3±4.5×10-6 g/s, 32.03±0.138 dyne/cm, 169.67±0.810°C, respectively. These properties were compared with that of commercial diesel as well as with the values specified by the American Society for Testing and Materials (ASTM) D6751. The density and the surface tension of the biodiesel were found similar to that of petrodiesel but its volatility was 3 times lower. Fourier-transform infrared spectroscopy (FTIR) spectra of the biodiesel showed methyl ester functional group at 1436 cm-1. Based on the cost of the materials used for production, the cost of biodiesel was estimated to be about 81 Nepalese rupees (0.67 USD) per liter. The properties of biodiesel also met the standard values of ASTM D6751. These findings indicate that waste oil is one of the feasible biodiesel sources and it can be used as a suitable alternative to petrodiesel

Antibiogram of Escherichia coli and Staphylococcus aureus Isolated from Milk Sold in Kathmandu District

The emergence of antibiotic resistance in microorganisms and the presence of such isolates in milk pose a great risk to public health. Therefore, this study aims to determine the antibiotic susceptibility pattern of Escherichia coli and Staphylococcus aureus isolated from milk and assess the microbial quality of milk. For this, a total of 70 milk samples were collected and the total bacterial count (TBC) was determined. E. coli and S. aureus were isolated using their respective selective media while antibiotic susceptibility testing was carried out by Kirby Bauer Disc Diffusion method. The TBC showed that the raw milk samples contained two-fold higher microbial load while the pasteurized milk samples contained four-fold higher microbial loads than the standard guidelines. A total of 62 isolates were identified from culture-positive milk samples of which 32 were E. coli and 30 were S. aureus. A significant correlation was observed between microbial load and the organism isolated (r = 0.339, p<0.01). All S. aureus isolates were susceptible to Chloramphenicol while 40% were resistant to Cefoxitin, indicating the presence of Methicillin resistant S. aureus (MRSA). Also, 12 multidrug resistant (MDR) S. aureus were identified. While for E. coli, all were susceptible to Chloramphenicol but resistant to Ampicillin. Also, 9 MDR E. coli were detected. Higher resistance was observed among isolates from the raw milk samples than the pasteurized milk. It can be concluded that the milk produced by small-scale farms and dairy industries of Kathmandu district are of poor quality. Hence, routine microbial quality assessment and antimicrobial resistance monitoring should be followed to safeguard public health

Phenolic Compounds from the Aerial Parts of Adenophora triphylla (Thunb.) A. DC. var. triphylla and their Free Radical Scavenging Activity

Adenophora triphylla (Thunb.) A. DC. var. triphylla (Family: Campanulaceae) is distributed in Japan, Korea, and China. It is locally known as “Saiyousyajin” in Japan and the roots are used in traditional medicine to treat chronic bronchitis and whooping cough, and also as anti-inflammatory and anti-tussive agents. Till now, there is no report on the chemical constituents of aerial parts. Thus, the main aim of this study was to isolate and identify major chemical constituents of aerial parts of A. triphylla var. triphylla, and to evaluate their free radical scavenging activity. The 70% methanol extract of the aerial parts was subjected to repeated column chromatography using MCI gel CHP-20P, Sephadex LH-20, ODS and silica gel columns to isolate the five phenolic components (1-5). Free radical scavenging activity of the extract and compounds was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity method. The structures of the isolated compounds were elucidated as luteolin (1), luteolin 4’-O-b-glucopyranoside (2), luteolin 7-O-b-glucopyranoside (3), luteolin 7-O-neohesperidoside (4) and chlorogenic acid (5) based on their nuclear magnetic resonance (NMR) spectral data and comparison with literature values. All these compounds were isolated for the first time from A. triphylla var. triphylla. Extract showed weak free radical scavenging activity. Among isolated compounds, luteolin (1), luteolin 7-O-b-glucopyranoside (3), luteolin 7-O-neohesperidoside (4) and chlorogenic acid (5) showed potent free radical scavenging activity. Results from this study suggest that the aerial parts of A. triphylla var. triphylla might be a potential plant source for the development of functional foods, however further detailed research is necessary