Bioprospecting of pods of Moringa oleifera Lam. as novel antibacterial agent- Supplementary Data

In the treatment of bacterial diseases, increasing resistance to traditional chemotherapeutics has drawn the necessity for substitute remedies. In this context, here, we evaluated the bactericidal activity of pods of Moringa oleifera Lam., an ethno medicinal plant, against eight pathogenic bacterial strains, both Gram positive (Bacillus licheniformis, B. mycoides, B. subtilis and Staphylococcus aureus) and Gram negative (Escherichia coli, Pseudomonas aeruginosa, P. fluorescens and P. putida). Different organic solvent extracts, like ethyl acetate, acetone and alcohol, of pods of M. oleifera were examined for bactericidal activity against test microorganisms. Minimum inhibitory concentration, chromatographic analyses along with infrared spectroscopy, gas chromatography-mass spectroscopy and nuclear magnetic resonance spectroscopy was carried out for chemical characterization of active ingredient responsible for antibacterial activity. Both the Gram positive and Gram negative organisms showed variable sensitivity to different solvent extracts of M. oleifera pods. Ethyl acetate extracts showed maximum antibacterial activity with MIC value ranging from 1.30 to 4.10 mg/mL. IR analysis provided preliminary information about the amines, amides, aromatics and sulphur containing compounds of the active ingredient. GC-MS and NMR analyses indicated the presence of principal bioactive antibacterial compound 2-(benzoylsulfanyl)-1,3- thiazol,4-yl, benzoate with molecular formula C17H11NO3S2 from ethyl acetate extract of M. oleifera pods. The study concludes that the compound 2-(benzoylsulfanyl)-1,3-thiazol,4-yl, benzoate from ethyl acetate extract of pods of M. oleifera possess the antibacterial activity against the tested strains.

Bioprospecting of pods of Moringa oleifera Lam. as novel antibacterial agent

In the treatment of bacterial diseases, increasing resistance to traditional chemotherapeutics has drawn the necessity for substitute remedies. In this context, here, we evaluated the bactericidal activity of pods of Moringa oleifera Lam., an ethno medicinal plant, against eight pathogenic bacterial strains, both Gram positive (Bacillus licheniformis, B. mycoides, B. subtilis and Staphylococcus aureus) and Gram negative (Escherichia coli, Pseudomonas aeruginosa, P. fluorescens and P. putida). Different organic solvent extracts, like ethyl acetate, acetone and alcohol, of pods of M. oleifera were examined for bactericidal activity against test microorganisms. Minimum inhibitory concentration, chromatographic analyses along with infrared spectroscopy, gas chromatography-mass spectroscopy and nuclear magnetic resonance spectroscopy was carried out for chemical characterization of active ingredient responsible for antibacterial activity. Both the Gram positive and Gram negative organisms showed variable sensitivity to different solvent extracts of M. oleifera pods. Ethyl acetate extracts showed maximum antibacterial activity with MIC value ranging from 1.30 to 4.10 mg/mL. IR analysis provided preliminary information about the amines, amides, aromatics and sulphur containing compounds of the active ingredient. GC-MS and NMR analyses indicated the presence of principal bioactive antibacterial compound 2-(benzoylsulfanyl)-1,3- thiazol,4-yl, benzoate with molecular formula C17H11NO3S2 from ethyl acetate extract of M. oleifera pods. The study concludes that the compound 2-(benzoylsulfanyl)-1,3-thiazol,4-yl, benzoate from ethyl acetate extract of pods of M. oleifera possess the antibacterial activity against the tested strains.

Larvicidal efficacy of silver nanoparticles synthesized biologically using Swietenia mahagoni (L.) Jacq. leaf extract against Anopheles stephensi, Culex quinquefasciatus and Cx. vishnui group

Mosquito borne diseases are a global crisis, particularly in developing countries. Non-availability of apposite vaccines against these diseases has lead to sole dependence on the vector managerial steps for dropping the incidences. In the present study, we tried to evaluate the larvicidal potential of biologically synthesized silver nanoparticles (Ag NP) using aqueous leaf extracts of Swietenia mahagoni (L.) Jacq. against third instar larvae of Anopheles stephensi, Culex quinquefasciatus and Culex vishnui group. Aqueous extract of leaves reduced the aqueous silver ions to produce stable Ag NP. The characterization of synthesized nanoparticles was done through UV-Vis spectrum, Transmission electron microscope (TEM), X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscope. Third instar larvae of three mosquito species namely An. stephensi, Cx. quinquefasciatus and Cx. vishnui group were exposed to different concentrations of synthesized nanoparticles for 24, 48and 72 h. TEM measured the range of nanoparticle size as 8-9 nm whereas XRD measured as 6 nm. Cent percent mortality of larvae of An. stephensi was recorded at 80 ppm at 48 h. About 96 and 80% mortality of Cx. vishnui group and Cx. quinquefasciatus larvae respectively were noted at 80 ppm after 72 h of exposure. The result of regression analysis proved that the mortality rate (Y) was positively correlated with the period of exposure (X) and regression coefficients were close to one. Toxicity study on non-target species showed no injurious activity during experimental period. Results indicate, possibly a first report on mosquito larvicidal effect of Ag NP synthesized using S. mahagoni leaf extract which may be used to effectively control the larval forms of three important vector mosquitoes

Functional response analysis of Anisops sardea (Hemiptera: Notonectidae) against Culex quinquefasciatus in laboratory condition.

Background & objectives: Culex quinquefasciatus is the principal vector of lymphatic filariasis (LF). Application of alternative vector control methodologies are aimed at reduction of mosquito breeding sites and biting activity through the use of biological control methods. In the present study, functional response of aquatic Hemipteran backswimmer, Anisops sardea was assessed against Cx. quinquefasciatus larvae in laboratory bioassay. Methods: The functional respons of A. sardea was assessed against IIIrd instar larvae of Cx. quinquefasciatus. Respective handling times and coefficient of attack rates were determined by a non linear polynomeal regression equation. Results: The results of rates of predation in variable prey densities exhibited a ‘linear rise to plateau curve’, associated with ‘Type -II’ functional response. The logistic regression estimated a significant negative linear parameter (P1<0) which also supported the same observation. Associated ‘attack rates’ and ‘handling times’ were also calculated using the Holling Disc Equation. Interpretation & conclusions: The results of present experiments indicate that A. sardea can be used as a biocontrol agent against the larval forms of Cx. quinquefasciatus in temporarily available breeding places of mosquito with relatively clear water. However, a detailed field study has to be done to confirm these findings.

Laboratory evaluation of molluscicidal & mosquito larvicidal activities of leaves of Solanum nigrum L.

Background & objectives: Indiscriminate use of synthetic pesticides has created serious problem for the aquatic flora and fauna, and also resulted in appearance of pesticide resistance in vector population. This study was designed to evaluate the biocontrol efficacy of aqueous and solvent extracts of mature leaves of Solanum nigrum L., against fresh water snail Lymnaea acuminata f. rufescens (Gray) (an intermediate host of parasites causing fasciolopsiasis) and larvae of Culex vishnui group (Reuben) (vector of Japanese encephalitis). Methods: Aqueous and solvent extracts of fresh, mature, green/shed dried leaves of S. nigrum were tested against adult L. acuminata and larvae of Cx. vishnui group. The lethal concentration was determined and the appropriate lethal concentration at 24 h of benzene extract was also studied on non target organisms such as Daphnia sp, Diplonychus annulatum and Chironomus circumdatus. A qualitative phytochemical analysis was carried out in search of active ingredient and the chemical nature of the active substance was also evaluated by infrared (IR) analysis. Results: In a 72 h bioassay experiment with the aqueous extract, the highest mortality was recorded in 0.5 and 3 per cent extract against larvae of Cx. vishnui group and L. acuminata, respectivela. In the benzene solvent extract, the maximum mortality was recorded at a concentration of 150 ppm against L. acuminata and at 50 ppm against larvae of Cx. vishnui group with LC50 values of 55.45 and 11.59 ppm, respectively at 72 h. The log probit analysis (95% confidence level) recorded lowest value at 72 h of exposure. Qualitative phytochemical analysis reported the presence of some biochemical compounds, such as saponin, flavonoids, steroid and tannin. Among these, the toxic compound was detected by IR analysis having Rf = 0.87 (showed 66.70% and 76.70% mortality of L. acuminata and larvae of Cx. vishnui group, respectively). IR analysis provided preliminary information about the aliphatic amide nature of the active ingredient. Interpretation & conclusions: The study results provide considerable scope in exploiting local indigenous plant resources for molluscicidal and mosquito larvicidal activities.

Mosquito larvicidal potential of four common medicinal plants of India.

Background & objectives: Mosquitoes transmit serious human health diseases, causing millions of deaths every year. Plants may be sources of alternative mosquito control agents. The present study was carried out to assess the role of larvicidal activities of the crude extracts of four plants viz. Alternanthera sessilis L. (Amaranthaceae), Trema orientalis L. (Cannabaceae), Gardenia carinata Smith. (Rubiaceae) and Ruellia tuberosa L. (Acanthaceae) against Culex quinquefasciatus Say in laboratory bioassay. Methods: Selective concentrations (0.5, 1 and 1.5%) of crude extract of all four plant leaves were tested against Ist to IVth instar larvae of Cx. quinquefasciatus. Log probit analysis (at 95% confidence level) revealed the LC50 values. Preliminary qualitative phytochemical analyses of crude extracts were also done. The lethal concentrations (%) of crude extracts at 24 h against IIIrd instar larvae were also studied on non-target organisms. Result: In a 72 h bioassay experiment with crude extract, the highest mortality was recorded in 1.5 per cent extract. A. sessilis showed the highest mortality (76.7 %) at 1.5 per cent crude extract against IInd instar larvae having LC50 value of 0.35 per cent, followed by R. tuberosa (LC50 =1.84%), G. carinata (LC50 = 2.11) and T. orientalis (LC50 = 2.95%). The regression equation showed a dose-dependent mortality, as the rate of mortality (Y) was positively correlated with the concentration (X). Phytochemical analysis of the crude extract showed the presence of many bioactive phytochemicals such as steroids, alkaloids, terpenes, saponins, etc. No changes in the swimming behaviour and survivality of non-target organism were noticed at the studied concentrations. Interpretation & conclusions: Crude extract of the four selected plants showed larvicidal activity against Cx. quinquefasciatus. The extracts at the studied concentrations did not produce any harmful effect on non-target organisms.

Mosquito larvicidal activity of Solanum nigrum berry extracts.

Phytochemicals are widely used as biocontrol agent against vector mosquitoes. The present study was undertaken to isolate and evaluate the mosquitocidal activity of various extracts of berries of S. nigrum against Culex quinquefasciatus. Crude and chloroform: methanol (1:1, v/v) extracts of fresh, mature, green berries of S. nigrum were tested against Cx. quinquefasciatus. The lethal concentration was determined and the chemical nature of the active substance was evaluated. A qualitative phytochemical analysis of chloroform: methanol (1:1, v/v) extract was performed in search of the active ingredient. The appropriate lethal concentrations at 24 h for chloroform: methanol (1:1, v/v) extract was also studied on non-target organisms. In a 72 h bioassay experiment with crude extract, the highest mortality was recorded in 3 per cent extract. In the chloroform: methanol (1:1, v/v) solvent extract, the maximum mortality was recorded at a concentration of 120 μg/ml. The log probit analysis (95% confidence level) recorded lowest LC50 value at 72 h of exposure. Both crude and chloroform: methanol (1:1, v/v) extracts showed good larvicidal activity against Cx. quinquefasciatus. The isolated active ingredient may be tested as a potential larvicide after determination of its structure.

Efficacy of extracts of six medicinal plants of India against some pathogenic bacteria.

The sensitivity of the pathogenic multi-drug resistant bacteria (Aeromonas hydrophila, Bacillus licheniformis, Bacillus mycoides, Bacillus niacini, Bacillus subtilis, Escherichia coli, Geobacillus thermodenitrificans, Klebsiella pneumoniae, Paenibacillus koreensis, Paenibacillus larvae larvae, Proteus vulgaris, Pseudomonas aeruginosa, Pseudomonas flourescens, Pseudomonas putida and Staphylocccus aureus) was tested against aqueous, acetone and ethanol extracts of mature leaves of Mimosa pudica Linn. (Mimosaceae) and Moringa oleifera Lam. (Moringaceae), stems of Michelia champaca Linn. (Magnoliaceae) and Musa paradisiaca Linn.(Musaceae), roots of Momordica charantia Linn. (Cucurbitaceae) and Murraya koenigii Linn. (Rutaceae) by agar well diffusion method. Gatifloxacin was the most effective antibiotic against all the reference bacteria. Though all the extracts were found effective, the ethanol extract showed maximum inhibition against the test microorganisms followed by acetone and aqueous extract. Bacillus niacini is the most resistant bacteria and Klebsiella pneumoniae is the most sensitive bacteria against all the extracts used. MIC values of each bacterium were also determined.

Plant extracts as potential mosquito larvicides.

Mosquitoes act as a vector for most of the life threatening diseases like malaria, yellow fever, dengue fever, chikungunya ferver, filariasis, encephalitis, West Nile Virus infection, etc. Under the Integrated Mosquito Management (IMM), emphasis was given on the application of alternative strategies in mosquito control. The continuous application of synthetic insecticides causes development of resistance in vector species, biological magnification of toxic substances through the food chain and adverse effects on environmental quality and non target organisms including human health. Application of active toxic agents from plant extracts as an alternative mosquito control strategy was available from ancient times. These are non-toxic, easily available at affordable prices, biodegradable and show broad-spectrum target-specific activities against different species of vector mosquitoes. In this article, the current state of knowledge on phytochemical sources and mosquitocidal activity, their mechanism of action on target population, variation of their larvicidal activity according to mosquito species, instar specificity, polarity of solvents used during extraction, nature of active ingredient and promising advances made in biological control of mosquitoes by plant derived secondary metabolites have been reviewed.

Functional responses of Laccotrephes griseus (Hemiptera: Nepidae) against Culex quinquefasciatus (Diptera: Culicidae) in laboratory bioassay.

Background and objectives: In integrated vector management programmes, major emphasis is given on the application of biocontrol agents. In the present study, the hemipteran water bug, Laccotrephes griseus (Hemiptera: Nepidae) was evaluated to establish its functional response against the immature Culex quinquefasciatus Say in the laboratory. Methods: Adult water bugs (L. griseus) were collected from different ponds in Bankura, West Bengal, India. Predation rate was determined in semi-field conditions. Functional response, regression equation analysis and rate of predation were also determined in the presence of an alternative prey. Results: The long-term predation experiment (up to 30 days) revealed that L. griseus is a very good predator of mosquito larvae. The functional curve analysis of L. griseus exhibited a Type II functional response. Their handling times and coefficient of attack rates were 29.37 min and 2.17 respectively. Regression equations showed that consumption rate was directly proportional to prey and predator densities and inversely proportional to the search area. The selectivity index, niche width and food breadth were also determined. Interpretation and conclusion: The present study revealed that L. griseus can consume a good number of mosquito larvae in the laboratory conditions. The long-term bioassay also indicates their predatory response against mosquito larvae in the semi-field conditions. The result of selectivity index, niche width and food breadth were also highest for Cx. quinquefasciatus. In these respects, the water bugs L. griseus are indicative of their release in the aquatic habitat where Cx. quinquefasciatus is the most dominant mosquito species.

Antibacterial activities of some plant extracts used in Indian traditional folk medicine

Objective:To evaluate the antibacterial activity of the leaf extracts of Cestrum diurnum, Ocimum sanctum, Carcica papaya, Solanum villosum, Vitex negundo, and Clerodendron inerme against two gram positive bacteria (Staphylococcus aureus MTCC 2940 and Bacillus subtilis MTCC 441) and two gram negative bacteria (Escherichia coli MTCC 739 and Pseudomonas aeruginosa MTCC 2453). Methods: The sensitivity of two gram positive and two gram negative pathogenic multi-drug resistant bacteria to extracts of leaves of six medicinal plants used as popular medicine in India was studied in vitro by the disk diffusion method and minimal inhibitory concentration (MIC). Results: All the bacterial strains were found to be sensitive to aqueous, n-hexane and ethanol extracts. But, it is evident that the organic extracts were comparatively more effective than aqueous extracts. Conclusions: It can be concluded that the leaf extracts of the six medicinal plants possess antibacterial activity against human pathogens.

Evaluation of target specific larvicidal activity of the leaf extract of Typhonium trilobatum against Culex quinquefasciatus Say

Objective: To investigate the target specific larvicidal potential of an edible herb Typhonium trilobatum (T. trilobatum) (L.) Schott against mosquito Culex quinquefasciatus (Cx. quinquefasciatus) Say. Methods: Different concentrations of crude and methanol extract of T. trilobatum mature leaves were treated against Cx. quinquefasciatus larvae. LC50 concentration of crude extract on mosquito 3rd instar larvae was tested on Chironomus circumdatus and Diplonychus annulatum larvae. Preliminary phytochemical analysis was performed in search of plant’s secondary metabolites. Results:100%mortality of 1st instar mosquito larvae was recorded at 0.4%concentration after 72 h of exposure of crude extract. At 72 h 0.5%concentration produced 100%, 89.99%and 79.99%mortality of 2nd, 3rd and 4th instar larvae respectively. 50 ppm methanol extract showed 73.67%mortality of 3rd instar mosquito larvae at 72 h. 400 ppm concentration was responsible for 100%mortality in 24 h. Application of LC50 concentration (of 3rd instar mosquito larva) against non target organisms like C. circumdatus and D. annulatum larvae produced no significant mortality among them. Secondary metabolites like terpenoids and free glycoside bound anthraquinones were found. Conclusions:This experimental study was a pioneer attempt to establish T. trilobatum as a valuable resource of effective target specific mosquito larvicide.

In vitro synergistic effect of doxycycline & ofloxacin in combination with ethanolic leaf extract of Vangueria spinosa against four pathogenic bacteria.

Background & objectives: The in vitro antibacterial activity of ethanolic leaf extract of Vangueria spinosa Roxb. (Rubiaceae) alone and in combination with antibiotics (doxycycline and ofloxacin) by means of fractional inhibitory concentration indices (FICI) as well as by the use of time-kill assays against one Gram-positive bacterium (Staphylococcus aureus) and three Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa) was studied. Methods: Antibacterial activity was assayed by using the microdilution method. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined for the ethanolic leaf extract of V. spinosa alone and also in combination with antibiotics using the fractional inhibitory concentration (FIC) and time-kill assay method. Synergism was also tested using checker board dilution method. Results: MIC/MBC values for ethanolic leaf extract of V. spinosa against all the tested bacteria ranged between 25.5 - 52.6/22.4 - 60.5 μg/ml, for doxycycline 4.0/4.0 - 4.5 μg/ml and for ofloxacin 0.625 - 2.5/1. 25 - 5.0 μg/ml respectively. The average log reduction in viable cell count in time-kill assay ranged between 2.4 log10 - 4.5 log10 cfu/ml after 1 h of interaction and between 3.9 log10 -5.0 log10 cfu/ml after 3 h interaction in 1 × MIC to 4 × MIC. When leaf extract and antibiotics were combined, the average log reduction in viable cell count for doxycycline from 1.5 log10 - 5.18 log10 cfu/ml and for ofloxacin 3.06 log10- 5.39 log10 cfu/ml. Synergistic actions were observed in all the cases except against P. aeruginosa which showed an additive effect for ofloxacin and plant extract combination. Interpretation & conclusions: This study provides a preliminary report of synergistic activity of V. spinosa Roxb, ethanolic leaf extract with antibiotics.

Antibacterial potentiality of Argemone mexicana solvent extracts against some pathogenic bacteria

The sensitivity of two Gram positive (Staphylococcus aureus and Bacillus subtilis) and two Gram negative (Escherichia coli and Pseudomonas aeruginosa) pathogenic multi-drug resistant bacteria was tested against the crude extracts (cold aqueous, hot aqueous, and methanol extracts) of leaves and seeds of Argemone mexicana L. (Papaveraceae) by agar well diffusion method. Though all the extracts were found effective, yet the methanol extract showed maximum inhibition against the test microorganisms followed by hot aqueous extract and cold aqueous extract.