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1.  Isorhamnetin 3-O-robinobioside from Nitraria retusa leaves enhance antioxidant and antigenotoxic activity in human chronic myelogenous leukemia cell line K562 
In this report, the isorhamnetin 3-o-robinobioside and its original extract, the ethyl acetate extract, from Nitraria retusa leaves, were evaluated for their ability to induce antioxidant and antigenotoxic effects in human chronic myelogenous leukemia cell line.
Nitraria retusa products properties were carried out by firstly evaluating their effects against lipid peroxidation induced by H2O2, using the thiobarbituric acid reactive substances species (TBARS) assay, and proceeding to the assay of cellular antioxidant activity, then doing the comet assay.
The isorhamnetin 3-o-robinobioside showed a protective effect against lipid peroxidation induced by H2O2. The same natural compound and ethyl acetate extract inhibited oxidation induced by 2,2′-azobis (2-amidinopropane) dihydrochloride in human chronic myelogenous leukemia cells with respectively 50% inhibitory concentration values of 0.225 mg/ml and 0.31 mg/ml, reflecting a significant antioxidant potential. The same two products inhibited the genotoxicity induced by hydroxyl radicals in the same human cell line (by 77.77% at a concentration of 800 μg/ml and by 80.55% at a concentration of 1000 μg/ml respectively).
The isorhamnetin 3- o-robinobioside and its original extract, the ethyl acetate extract, from Nitraria retusa leaves, have a great antioxidant and antigenotoxic potential on human chronic myelogenous leukemia cell line K562.
PMCID: PMC3439276  PMID: 22913434
2.  Antioxidant and antigenotoxic activities of Angelica keiskei, Oenanthe javanica and Brassica oleracea in the Salmonella mutagenicity assay and in HCT116 human colon cancer cells 
BioFactors (Oxford, England)  2006;26(4):231-244.
Epidemiological studies indicate that consumption of green-yellow vegetables rich in chlorophyll, vitamin C, vitamin E, and carotenoids reduce the risk of cancer. We sought to examine the antigenotoxic and antioxidant properties of chlorophyll-rich methanol extracts of Angelica keiskei, Oenanthe javanica, and Brassica oleracea (kale). In the Salmonella mutagenicity assay, A. keiskei caused dose-dependent inhibition against three heterocyclic amine mutagens in the presence of S9, O. javanica was antimutagenic only at the highest concentration in the assay (2 mg/plate), and B. oleracea showed no consistent inhibitory activity at non-toxic levels. None of the extracts were effective against three direct-acting mutagens in the absence of S9. Extracts of A. keiskei and, to a lesser extent O. javanica, inhibited two of the major enzymes that play a role in the metabolic activation of heterocyclic amines, based on ethoxyresorufin-O-deethylase and methoxyresorufin-O-demethylase assays in vitro. All three plant extracts were highly effective in assays which measured ferric reducing/antioxidant power, oxygen radical absorbance capacity, and Fe2+/H2O2-mediated DNA nicking. Finally, using the ‘comet’ assay, all three plant extracts protected against H2O2-induced genotoxic damage in human HCT116 colon cancer cells. These findings provide support for the antigenotoxic and antioxidant properties of chlorophyll-rich extracts of A. keiskei, O. javanica, and B. oleracea, through mechanisms that include inhibition of carcinogen activation and scavenging of reactive oxygen species.
PMCID: PMC2267880  PMID: 17119270
Antimutagen; antioxidant; heterocyclic amines; phytochemical; comet assay; D NA breaks
3.  Pharmacological, antioxidant, genotoxic studies and modulation of rat splenocyte functions by Cyperus rotundus extracts 
Cyperus rotundus Linn. (Cyperaceae) is a Tunisian medicinal plant used in folkloric (traditional) medicine to treat stomach disorders and inflammatory diseases. The present study explored the analgesic, anti-inflammatory and genotoxic activities of extracts from the aerial parts of C. rotundus. The antioxidant capacity and the modulation of splenocyte functions by these extracts were also investigated in mice. The phytochemical analysis was carried out using standard methods.
Aqueous, ethyl acetate, methanol and TOF-enriched extracts (300, 150, and 50 μg/ml) were evaluated for their analgesic and anti-inflammatory activities. 4, 2, and 1 mg/ml of each extract were tested to investigate their effect on lipid peroxidation. The genotoxic study was monitored by measuring the structural chromosome aberrations of mice treated with 300 mg/kg of extract. The proliferation of lymphocytes in the absence and presence of mitogens was assessed at a concentration range 1–1000 μg/ml.
The tested extracts were able to decrease the mouse ear oedema induced by xylene. Furthermore, it was shown that the same extracts reduced the number of abdominal contractions caused by acetic acid in mice, revealing the peripheral analgesic activity of these extracts. It is worth noting that mice treated with doses up to 300 mg/kg b.w. of Cyperus rotundus extracts did not exhibit any toxicity. The tested extracts significantly enhance lymphocyte proliferation at 1 mg/ml.
It appears that C. rotundus extracts contain potent components such as flavonoids that may potentially be useful for modulating the immune cell functions, provoking analgesic, anti-inflammatory and antioxidant effects.
PMCID: PMC3570401  PMID: 23388107
Cyperus rotundus; Analgesic activity; Anti-inflammatory activity; Lipid peroxidation effect; Chromosome aberrations; Immunomodulatory effect
4.  Antioxidant and antiproliferative activities of Abrus precatorius leaf extracts - an in vitro study 
The use of traditional medicine at the primary health care level is widespread and plant-based treatments are being recommended for curing various diseases by traditional medical practitioners all over the world. The phytochemicals present in the fruits, vegetables and medicinal plants are getting attention day-by-day for their active role in the prevention of several human diseases. Abrus precatorius is a widely distributed tropical medicinal plant with several therapeutic properties. Therefore in the present study, A. precatorius leaf extracts were examined for their antioxidant and cytotoxic properties in vitro in order to discover resources for new lead structures or to improve the traditional medicine.
In this study, antioxidant and antiproliferative properties of the different leaf extracts (hexane, ethyl acetate, ethanol and water) from A. precatorius were investigated along with the quantification of the polyphenol and flavonoid contents. The ability of deactivating free radicals was extensively investigated with in vitro biochemical methods like DPPH•, •OH, NO, SO2- scavenging assays and inhibition capability of Fe(II)-induced lipid peroxidation. Furthermore, antiproliferative activities using different human cancer cell lines and primary cell line was carried out by MTT method.
Total phenolic content and total flavonoid content of the extracts were found in the range of 1.65 ± 0.22 to 25.48 ± 0.62 GAE mg/g dw and 6.20 ± 0.41 to 17.16 ± 1.04 QE mg/g dw respectively. The experimental results further revealed that A. precatorius extracts showed strong antiradical properties, capable to chelate Fe2+ and possess good inhibition ability of lipid peroxidation. In addition, as a first step towards the identification of phytoconstituents endowed with potent chemopreventive activities, we evaluated the inhibitory effects of A. precatorius extracts on the proliferation of four different human tumour cell lines such as human colon adenocarcinoma cells (Colo-205), human retinoblastoma cancer cells (Y79), human hepatocellular carcinoma cells (HepG2) and Leukemia cells (SupT1). Ethanol extract (APA) and ethyl acetate extract (APE) of A. precatorius had apparent capabilities of inhibiting the survival of tested human cancer cell lines. Moreover, it was observed that the A. precatorius extracts did not inhibit the growth of mice peritoneal macrophages, thus confirming that plants extracts are selective against the cancer cell lines.
This work provides a scientific support for the high antioxidant and antiproliferative activity of this plant and thus it may find potential applications in the treatment of the diseases caused by ROS. Further studies are needed to confirm in vivo anti-tumorgenicity and subsequent chemical characterization of the active molecule(s).
PMCID: PMC3600005  PMID: 23452983
5.  The influence of organic solvents on estimates of genotoxicity and antigenotoxicity in the SOS chromotest 
Genetics and Molecular Biology  2012;35(2):503-514.
In this work, the toxicity and genotoxicity of organic solvents (acetone, carbon tetrachloride, dichloromethane, dimethylsulfoxide, ethanol, ether and methanol) were studied using the SOS chromotest. The influence of these solvents on the direct genotoxicity induced by the mutagens mitomycin C (MMC) and 4-nitroquinoline-1-oxide (4-NQO) were also investigated. None of the solvents were genotoxic in Escherichia coli PQ37. However, based on the inhibition of protein synthesis assessed by constitutive alkaline phosphatase activity, some solvents (carbon tetrachloride, dimethylsulfoxide, ethanol and ether) were toxic and incompatible with the SOS chromotest. Solvents that were neither toxic nor genotoxic to E. coli (acetone, dichloromethane and methanol) significantly reduced the genotoxicity of MMC and 4-NQO. When these solvents were used to dissolve vitamin E they increased the antigenotoxic activity of this compound, possibly through additive or synergistic effects. The relevance of these results is discussed in relation to antigenotoxic studies. These data indicate the need for careful selection of an appropriate diluent for the SOS chromotest since some solvents can modulate genotoxicity and antigenotoxicity.
PMCID: PMC3389540  PMID: 22888301
antigenotoxicity; genotoxicity; interference; mitomycin C; 4-nitro-quinoline-1-oxide; SOS chromotest; solvents; vitamin E
6.  Effects of Payena dasyphylla (Miq.) on hyaluronidase enzyme activity and metalloproteinases protein expressions in interleukin-1β stimulated human chondrocytes cells 
Hyaluronidases have been found as the target enzymes in the development of osteoarthritis (OA) disease. While there is still no curative treatment for this disease, recent studies on the treatment of OA were focused on the effectiveness of natural products which are expected to improve the symptoms with minimal side effects. The aim of this study was to screen selected Malaysian plants on their anti-hyaluronidase activity as well as to evaluate the active plant and its derived fractions on its potential anti-arthritic and antioxidant activities.
A total of 20 methanolic crude extracts (bark and leaf) from ten different plants were screened using a colorimetric hyaluronidase enzymatic assay. The active plant extract (Payena dasyphylla) was then studied for its hyaluronidase inhibitory activity in the interleukin-1β (IL-1β) stimulated human chondrocytes cell line (NHAC-kn) using zymography method. The Payena dasyphylla methanolic bark extract was then fractionated into several fractions in where the ethyl acetate (EA) fraction was evaluated for its inhibitory effects on the HYAL1 and HYAL2 gene expressions using reverse transcription-polymerase chain reaction (RT-PCR) technique. While the MMP-3 and MMP-13 protein expressions were evaluated using western blot method. The phenolic and flavonoid contents of the three fractions as well as the antioxidant property of the EA fraction were also evaluated.
Bark extract of Payena dasyphylla (100 μg/ml) showed the highest inhibitory activity against bovine testicular hyaluronidase with 91.63%. The plant extract also inhibited hyaluronidase expression in the cultured human chondrocyte cells in response to IL-1β (100 ng/ml). Similarly, treatment with Payena dasyphylla ethyl acetate (EA) fraction (100 μg/ml) inhibited the HYAL1 and HYAL2 mRNA gene expressions as well as MMP-3 and MMP-13 protein expression in a dose dependent manner. Payena dasyphylla EA fraction has demonstrated the highest amount of phenolic and flavonoid content with 168.62 ± 10.93 mg GAE/g and 95.96 ± 2.96 mg RE/g respectively as compared to water and hexane fractions. In addition, the Payena dasyphylla EA fraction showed strong antioxidant activity with IC50 value of 11.64 ± 1.69 μg/mL.
These findings have shown that Payena dasyphylla might contained potential phenolic compounds that inhibiting the key enzyme in osteoarthritis development, which is the hyaluronidase enzyme through interruption of HYAL1 and HYAL1 gene expressions. The degradation of cartilage could also be inhibited by the plant through suppression of MMP-3 and MMP-13 protein expressions. We also reported that the inhibitory effect of Payena dasyphylla on hyaluronidase activity and expression might be due to its anti-oxidant property.
PMCID: PMC3848702  PMID: 23971790
Osteoarthritis; Hyaluronidase; MMP-3; MMP-13; Payena dasyphylla
7.  Piper betle shows antioxidant activities, inhibits MCF-7 cell proliferation and increases activities of catalase and superoxide dismutase 
Breast cancer is the most common form of cancer and the focus on finding chemotherapeutic agents have recently shifted to natural products. Piper betle is a medicinal plant with various biological activities. However, not much data is available on the anti-cancer effects of P. betle on breast cancer. Due to the current interest in the potential effects of antioxidants from natural products in breast cancer treatment, we investigated the antioxidant activities of the leaves of P. betle and its inhibitory effect on the proliferation of the breast cancer cell line, MCF-7.
The leaves of P. betle were extracted with solvents of varying polarities (water, methanol, ethyl acetate and hexane) and their phenolic and flavonoid content were determined using colorimetric assays. Phenolic composition was characterized using HPLC. Antioxidant activities were measured using FRAP, DPPH, superoxide anion, nitric oxide and hyroxyl radical scavenging assays. Biological activities of the extracts were analysed using MTT assay and antioxidant enzyme (catalase, superoxide dismutase, glutathione peroxidase) assays in MCF-7 cells.
Overall, the ethyl acetate extract showed the highest ferric reducing activity and radical scavenging activities against DPPH, superoxide anion and nitric oxide radicals. This extract also contained the highest phenolic content implying the potential contribution of phenolics towards the antioxidant activities. HPLC analyses revealed the presence of catechin, morin and quercetin in the leaves. The ethyl acetate extract also showed the highest inhibitory effect against the proliferation of MCF-7 cells (IC50=65 μg/ml). Treatment of MCF-7 cells with the plant extract increased activities of catalase and superoxide dismutase.
Ethyl acetate is the optimal solvent for the extraction of compounds with antioxidant and anti-proliferative activities. The increased activities of catalase and superoxide dismutase in the treated cells could alter the antioxidant defense system, potentially contributing towards the anti-proliferative effect. There is great potential for the ethyl acetate extract of P. betle leaf as a source of natural antioxidants and to be developed as therapeutics in cancer treatment.
PMCID: PMC3533855  PMID: 23153283
Piper betle; Antioxidant; Phenolic; MCF-7; Cytotoxicity; Catalase; Superoxide dismutase; HPLC
8.  Phytochemical Analysis and Free Radical Scavenging Activity of Medicinal Plants Gnidia glauca and Dioscorea bulbifera 
PLoS ONE  2013;8(12):e82529.
Gnidia glauca and Dioscorea bulbifera are traditional medicinal plants that can be considered as sources of natural antioxidants. Herein we report the phytochemical analysis and free radical scavenging activity of their sequential extracts. Phenolic and flavonoid content were determined. Scavenging activity was checked against pulse radiolysis generated ABTS•+ and OH radical, in addition to DPPH, superoxide and hydroxyl radicals by biochemical methods followed by principal component analysis. G. glauca leaf extracts were rich in phenolic and flavonoid content. Ethyl acetate extract of D. bulbifera bulbs and methanol extract of G. glauca stem exhibited excellent scavenging of pulse radiolysis generated ABTS•+ radical with a second order rate constant of 2.33×106 and 1.72×106, respectively. Similarly, methanol extract of G. glauca flower and ethyl acetate extract of D. bulbifera bulb with second order rate constants of 4.48×106 and 4.46×106 were found to be potent scavengers of pulse radiolysis generated OH radical. G. glauca leaf and stem showed excellent reducing activity and free radical scavenging activity. HPTLC fingerprinting, carried out in mobile phase, chloroform: toluene: ethanol (4: 4: 1, v/v) showed presence of florescent compound at 366 nm as well as UV active compound at 254 nm. GC-TOF-MS analysis revealed the predominance of diphenyl sulfone as major compound in G. glauca. Significant levels of n-hexadecanoic acid and octadecanoic acid were also present. Diosgenin (C27H42O3) and diosgenin (3á,25R) acetate were present as major phytoconstituents in the extracts of D. bulbifera. G. glauca and D. bulbifera contain significant amounts of phytochemicals with antioxidative properties that can be exploited as a potential source for herbal remedy for oxidative stress induced diseases. These results rationalize further investigation in the potential discovery of new natural bioactive principles from these two important medicinal plants.
PMCID: PMC3867356  PMID: 24367520
9.  Evaluation of the genotoxic and antigenotoxic potential of Melissa officinalis in mice 
Genetics and Molecular Biology  2011;34(2):290-297.
Melissa officinalis (L.) (Lamiaceae), a plant known as the lemon balm, is native to the east Mediterranean region and west Asia. Also found in tropical countries, such as Brazil, where it is popularly known as “erva-cidreira” or “melissa”, it is widely used in aqueous- or alcoholic-extract form in the treatment of various disorders. The aim was to investigate in vivo its antigenotoxicity and antimutagenicity, as well as its genotoxic/mutagenic potential through comet and micronucleus assaying. CF-1 male mice were treated with ethanolic (Mo-EE) (250 or 500 mg/kg) or aqueous (Mo-AE) (100 mg/kg) solutions of an M. officinalis extract for 2 weeks, prior to treatment with saline or Methyl methanesulfonate (MMS) doses by intraperitoneal injection. Irrespective of the doses, no genotoxic or mutagenic effects were observed in blood and bone-marrow samples. Although Mo-EE exerted an antigenotoxic effect on the blood cells of mice treated with the alkylating agent (MMS) in all the doses, this was not so with Mo-AE. Micronucleus testing revealed the protector effect of Mo-EE, but only when administered at the highest dose. The implication that an ethanolic extract of M. officinalis has antigenotoxic/antimutagenic properties is an indication of its medicinal relevance.
PMCID: PMC3115325  PMID: 21734832
Melissa officinalis; comet assay; micronucleus test; genotoxicity; antigenotoxicity
10.  Larvicidal activity of medicinal plant extracts against Anopheles subpictus & Culex tritaeniorhynchus 
Background & objectives:
Mosquitoes transmit serious human diseases, causing millions of deaths every year and the development of resistance to chemical insecticides resulting in rebounding vectorial capacity. Plants may be alternative sources of mosquito control agents. The present study assessed the role of larvicidal activities of hexane, chloroform, ethyl acetate, acetone, and methanol dried leaf and bark extracts of Annona squamosa L., Chrysanthemum indicum L., and Tridax procumbens L. against the fourth instar larvae of malaria vector, Anopheles subpictus Grassi and Japanese encephalitis vector, Culex tritaeniorhynchus Giles (Diptera: Culicidae).
Larvicidal activities of three medicinal plant extracts were studied in the range of 4.69 to 1000 mg/l in the laboratory bioassays against early 4th instar larvae of An. subpictus and Cx. tritaeniorhynchus. The mortality data were subjected to probit analysis to determine the lethal concentrations (LC50 and LC90) to kill 50 and 90 per cent of the treated larvae of the respective species.
All plant extracts showed moderate effects after 24 h of exposure; however, the highest toxic effect of bark methanol extract of A. squamosa, leaf ethyl acetate extract of C. indicum and leaf acetone extract of T. procumbens against the larvae of An. subpictus (LC50 = 93.80, 39.98 and 51.57 mg/l) and bark methanol extract of A. squamosa, leaf methanol extract of C. indicum and leaf ethyl acetate extract of T. procumbens against the larvae of Cx. tritaeniorhynchus (LC50 =104.94, 42.29 and 69.16 mg/l) respectively.
Interpretation & Conclusions:
Our data suggest that the bark ethyl acetate and methanol extract of A. squamosa, leaf ethyl acetate and methanol extract of C. indicum, acetone and ethyl acetate extract of T. procumbens have the potential to be used as an ecofriendly approach for the control of the An. subpictus, and Cx. tritaeniorhynchus.
PMCID: PMC3171902  PMID: 21808141
Anopheles subpictus; Culex tritaeniorhynchus; medicinal plant extracts; larvicide
11.  Phytochemical analysis, antimicrobial and radical-scavenging properties of Acalypha manniana leaves 
SpringerPlus  2013;2:503.
Acalypha manniana (Euphorbiaceae) is a plant popularly used in Cameroon and in several parts of Africa for the treatment of various microbial diseases like diarrhea and skin infections.
The present study was designed to evaluate the phytochemical composition, antimicrobial and radical-scavenging activities of A. manniana methanol leaf extract and its fractions.
The methanol extract was partitioned into hexane, ethyl acetate and residual fractions and phytochemical analysis was conducted using standard methods. The broth microdilution method was used to evaluate the antimicrobial activity against nine bacterial species and four dermatophyte species. The free radical scavenging activities of the methanol extract and its fractions were evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay.
The results obtained showed that A. manniana contains alkaloids, tannins, anthocyanins, flavonoids, phenols and steroids. The methanol extract as well as the hexane, ethyl acetate and residual fractions exhibited both antibacterial and antidermatophytic activities that varied between the microbial species (MIC = 0.12 – 2.04 mg/mL). These tested samples also showed high radical-scavenging activities (RaS50 = 3.34 – 4.80 μg/mL) when compared with vitamin C used as reference antioxidant (RaS50 = 1.74 μg/mL).
These findings provide evidence that the studied plant possesses antimicrobial and antioxidant properties and may act as potential antioxidant for biological systems susceptible to free radical-mediated reactions.
PMCID: PMC3795206  PMID: 24130962
Acalypha manniana; Euphorbiaceae; Extracts; Phytochemicals; Antimicrobials; Free radical scavenging
12.  Investigation on Antibacterial and Antioxidant Activities, Phenolic and Flavonoid Contents of Some Thai Edible Plants as an Alternative for Antibiotics 
This study was aimed to examine the antibacterial and antioxidative properties of seven edible plants from Thailand to develop alternative antibiotics as feed additives. The plants include Citrus aurantifolia Swingle (Lime) fruits and its leaves, Sesbania grandiflora L. (Agati sesbania) leaves, Piper sarmentosum Roxb (Wild betal) leaves, Curcuma domestica Valeton (Turmeric) roots, Morinda citrifolia L. (Beach mulberry) leaves, Cassia siamea britt (Siamea cassia) leaves, and Cocos nucifera L. (Coconut) peels. The plants were extracted by methanol, n-hexane, chloroform, ethyl acetate, butanol and water. Antibacterial activities with minimum inhibitory concentration (MIC) were determined by agar diffusion assay against Escherichia coli, Burkholderia sp., Haemopilus somnus, Haemopilus parasuis, and Clostridium perfringens that were considered pathogenic strains in livestock infection. Methanol extracts of C. aurantifolia Swingle fruits and leaves showed the broadest spectrum of antibacterial activities except for C. perfringens. Butanol extract of S. grandiflora L. leaves showed the strongest activity against Burkholderia sp. with MIC, 135 μg/mL. P. sarmentosum Roxb leaves showed antibacterial activities against E. coli, Burkholderia sp. and H. parasuis. Ethyl acetate and water extracts from C. domesitca Valeton roots showed MIC of 306 μg/mL and 183 μg/mL, respectively against only C. perfringens. Antioxidative activity was determined by 2-diphenyl-2-picryl hydrazyl photometric assay. The methanol extracts of C. aurantifolia Swingle fruits and P. sarmentosum Roxb leaves showed the highest antioxidant activity among all the extracts with 3.46 mg/mL and 2.70 mg/mL effective concentration 50% (EC50) values, respectively. Total contents of phenolics and flavonoids were measured from the plant extracts. Methanol extracts of S. grandiflora L. and chloroform extracts of C. domestica Valeton were found to have the highest amount of total phenolics, 41.7 and 47.8 μg/mL, respectively. Flavonoid content of methanol extracts in S. grandiflora L. T was 22.5 μg/mL and the highest among plant extracts tested. These results indicated that C. aurantifolia Swingle, S. grandiflora L., P. sarmentosum Roxb, and C. domestica Valeton have antibacterial and antioxidant activities and can be used as alternative antibiotics or potential feed additives for the control of animal pathogenic bacteria.
PMCID: PMC4150179  PMID: 25178298
Plant Extract; Antibacterial Activity; Antioxidant Activity; Polyphenol; Flavonoid
13.  In vitro antimicrobial activity on clinical microbial strains and antioxidant properties of Artemisia parviflora 
Artemisia parviflora leaf extracts were evaluated for potential antimicrobial and antioxidant properties. Antimicrobial susceptibility assay was performed against ten standard reference bacterial strains. Antioxidant activity was analyzed using the ferric thiocyanate and 2, 2-Diphenyl-1-Picrylhydrazyl (DPPH) assays. Radical scavenging activity and total phenolic content were compared. Phytochemical analyses were performed to identify the major bioactive constitution of the plant extract.
Hexane, methanol and ethyl acetate extracts of A. parviflora leaves exhibited good activity against the microorganisms tested. The n-hexane extract of A. parviflora showed high inhibition of the growth of Pseudomonas aeruginosa, Escherichia coli and Shigella flexneri. Methanol extract showed strong radical scavenging and antioxidant activity, other extracts showed moderate antioxidant activity. The major derivatives present in the extracts are of terpenes, steroids, phenols, flavonoids, tannins and volatile oil.
The results obtained with n-hexane extract were particularly significant as it strongly inhibited the growth of P. aeruginosa, E. coli and S. flexneri. The major constituent of the n-hexane extract was identified as terpenes. Strong antioxidant activity could be observed with all the individual extracts. The antimicrobial and antioxidant property of the extracts were attributed to the secondary metabolites, terpenes and phenolic compounds present in A. parviflora and could be of considerable interest in the development of new drugs.
PMCID: PMC3582536  PMID: 23171441
Artemisia; Terpenoids; Antimicrobial; Antioxidant; Radical scavenging activity
14.  Gas chromatography-mass spectroscopy analysis of bioactive petalostigma extracts: Toxicity, antibacterial and antiviral activities 
Pharmacognosy Magazine  2014;10(Suppl 1):S37-S49.
Petalostigma pubescens and Petalostigma triloculare were common components of pharmacopeia's of multiple Australian Aboriginal tribal groupings which traditionally inhabited the areas in which they grow. Among these groups, they had a myriad of medicinal uses in treating a wide variety of bacterial, fungal and viral infections. This study was undertaken to test P. pubescens and P. triloculare leaf and fruit extracts for the ability to inhibit bacterial and viral growth and thus validate Australian Aboriginal usage of these plants in treating bacterial and fungal diseases.
Materials and Methods:
P. pubescens, and P. triloculare leaves and fruit were extracted and tested for antimicrobial, antiviral activity and toxicity. The bioactive extracts were further examined by RP-HPLC and GC-MS to identify the component compounds.
The methanol, water and ethyl acetate leaf and fruit extracts of displayed potent antibacterial activity. The methanol and ethyl acetate extracts displayed the broadest specificity, inhibiting the growth of 10 of the 14 bacteria tested (71%) for the leaf extract and 9 of the 14 bacteria tested (64%) for the fruit extracts. The water extracts also had broad spectrum antibacterial activity, inhibiting the growth of 8 (57%) and 7 (50%) of the 14 bacteria tested, respectively. All antibacterial extracts were approximately equally effective against Gram-positive and Gram-negative bacteria, inhibiting the growth of 50-75% of the bacteria tested. The methanol, water and ethyl acetate extracts also displayed antiviral activity in the MS2 plaque reduction assay. The methanol and water extracts inhibited 26.6-49.0% and 85.4-97.2% of MS2 plaque formation, respectively, with the fruit extracts being more potent inhibitors. All ethyl acetate extracts inhibited 100% of MS2 plaque formation. All extracts were also non-toxic or of low toxicity. Analysis of these extracts by RP-HPLC showed that the P. triloculare ethyl acetate fruit extract was the least complex of the bioactive extracts. Subsequent analysis of this extract by GC-MS revealed that it contained 9 main compounds: acetic acid; 2,2-dimethoxybutane; 4-methyl-1,3-dioxane; decane; unadecane; 2-furanmethanol; 1,2-benzenediol; 1,2,3-benzenetriol; and benzoic acid.
These studies validate Australian Aboriginal therapeutic usage of Petalostigma species and indicate their medicinal potential.
PMCID: PMC4047571  PMID: 24914307
Antibacterial; antiviral; Australian medicinal plants; euphorbiaceae; Petalostigma pubescens; Petalostigma triloculare; quinine bush
15.  Analysis and comparison of the active components and antioxidant activities of extracts from Abelmoschus esculentus L 
Pharmacognosy Magazine  2012;8(30):156-161.
Abelmoschus esculentus L. is a healthy vegetable belonging to the family Malvaceae. This article reports the contents of total phenolics (TP) and total flavonoids (TF) in 80% methanol extracts of the flower (FL), fruit (FR), leaf (L), and seed (S) of A. esculentus, and in 0, 10, 30, 50, and 70% methanol eluates (ME), through the HP-20 column chromatography of 80% of the methanol fruit extract after it is defatted with petroleum and extracted with ethyl acetate. All the names of the samples are shortened for AEE-FL, AEE-FR, AEE-L, AEE-S and 0% MEF-WE, 10% MEF-WE, 30% MEF-WE, 50% MEF-WE, 70% MEF-WE respectively. In addition, the effects of the aforementioned extracts on 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) radical-scavenging and on ferric reducing antioxidant power (FRAP) have been evaluated.
Materials and Methods:
The antioxidant activity of the extracts and the enrichment fraction of A. esculentus were also evaluated by two assays, the DPPH radical-scavenging and ferric reducing antioxidant power (FRAP). The content measurement of TF and TP adopts the UV-2102 PCS method, and the measurement of the antioxidant activity adopts the Infinite M 200 method.
The experiment results show that all the different parts and different enrichment fractions of the water extracts of A. esculentus contain phenolics and flavonoids. Through the research of antioxidant activity we know that all the parts of the methanol extracts and different enrichment fractions of water extracts in the A. esculentus have the effect of scavenging free radicals, among which the antioxidant activity in the 50% MEF-WE part is the strongest. Here, the main components of antioxidant activity must be the flavonoids and phenolics, and furthermore, we know that there is a direct relationship between the contents of flavonoids and phenolics and the antioxidant activity.
The study suggests that A. esculentus may be the potential rich source of natural antioxidant. The experiment result provided a scientific basis for the further research and development of A. esculentus.
PMCID: PMC3371438  PMID: 22701290
Abelmoschus esculentus L; antioxidant activity; 1,1-Diphenyl-2-picryl-hydrazyl; ferric reducing antioxidant power; the total flavonoid; the total phenolic acid
16.  Estimation of total phenol and in vitro antioxidant activity of Albizia procera leaves 
BMC Research Notes  2013;6:121.
Research on natural products has gained a wide popularity due to the potential of discovering active compounds. The antioxidant properties contained in plants have been proposed as one of the mechanisms for the observed beneficial effect. Therefore, the present study investigated the antioxidant activity and total phenolic contents of various solvent extracts of Albizia procera leaves.
Antioxidant activity of the methanol extract and its derived fractions petroleum ether (APP), carbon tetrachloride (APC), dichloromethane (APD), ethyl acetate (APE), and residual aqueous fraction (APA) of the leaves of Albizia procera was performed by in vitro chemical analyses. Total phenolic content of the APM and other five fractions were also determined. APM and its derived fractions were also subjected to preliminary phytochemical screening test for various constituents.
Phytochemical screening revealed the presence of saponins, steroids, tannins, glycosides and flavonoids in the extracts. Amongst the extracts, APE showed the highest total phenolic content (449.18 ± 18.41mg of gallic acid equivalent/g of extract). In DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging test, the IC50 value of APM, APP, APC, APD, APE and APA was 43.43, 63.60, 166.18, 41.15, 11.79, and 63.06 μg/mL, respectively. Therefore, among the APM and its derived fractions, APE showed the highest antioxidant activity which is comparable to that of standard ascorbic acid (AA) (IC50 10.12 μg/mL). The total antioxidant capacity was found to be varied in different fractions. The reducing activity on ferrous ion was ranked as APE > APD > APM > APA > APC.
The above evidences suggest that APE of A. procera leaf is a potential source of natural antioxidant and can be used to prevent diseases associated with free radicals.
PMCID: PMC3621163  PMID: 23531304
Antioxidants; Free radical scavenging; Phytochemical constituents; Total phenolic content
17.  Antibacterial and antioxidant properties of the methanol extracts of the leaves and stems of Calpurnia aurea 
In South Africa, Calpurnia aurea (Ait.) Benth is used to destroy lice and to relieve itches, to destroy maggots and to treat allergic rashes, particularly those caused by caterpillars. Antioxidants play an important role protecting against damage by reactive oxygen species. Plants containing flavonoids have been reported to possess strong antioxidant properties.
The antibacterial, antioxidant activities and phenolic contents of the methanol extracts of the leaves and stems of Calpurnia aurea were evaluated using in vitro standard methods. Spectrophotometry was the basis for the determinations of total phenol, total flavonoids, flavonols, and proanthocyanidins. Tannins, quercetin and catechin equivalents were used for these parameters. The antioxidant activities of the stem extract of Calpurnia aurea were determined by ABTS, DPPH, and ferrous reducing antioxidant property (FRAP) methods. Laboratory isolates of 10 bacteria species which included five Gram-positive and five Gram-negative strains were used to assay for antibacterial activity of this plant.
The results from this study showed that the antioxidant activities of the stem extract of Calpurnia aurea as determined by the total phenol, flavonoids, and FRAP methods were higher than that of the leaves. On the other hand, the leaf extract of the plant has higher level of total flavonols and proanthocyanidins. The leaf extract also has higher radical scavenging activity as shown in 1, 1-Diphenyl-2-picrylhydrazyl (DPPH), and 2,2¿-azinobis-3- ethylbenzothiazoline-6-sulfonic acid (ABTS) assay. The leaf extract showed activity against seven of the bacterial organisms.
The results from this study indicate that the leaves and stem extracts of Calpurnia aurea possess antioxidant properties and could serve as free radical inhibitors or scavenger or, acting possibly as primary antioxidants. Although, the antibacterial properties of Calpurnia aurea are not as effective as the standard drugs- Chloramphenicol and Streptomycin, they still possess some activity against bacterial strains used in this study. Calpurnia aurea may therefore be a good candidate for functional foods as well as pharmaceutical plant-based products.
PMCID: PMC2556645  PMID: 18803865
18.  Antioxidant and anti-dermatophytic properties leaf and stem bark of Xylosma longifolium clos 
The present study was carried out to assess the phytochemical and anti-dermatophytic effect of the leaf and bark extracts of Xylosma longifolium Clos. The leaf and stem bark are used by the indigenous people of Manipur, India for treatment of skin diseases.
The leaves and stem barks of Xylosma longifolium were extracted using petroleum ether, chloroform and methanol respectively. The different extracts of each plant parts were tested for antioxidant activity using DPPH assay. The phenolic content was assayed using Folin-Ciocalteu colorimetric method. Each extracts was further analysed by RP-HPLC to quantify some individual flavonoid components. The anti-dermatophytic activity was evaluated both by agar diffusion method and micro wells dilution method against the Microsporum boullardii MTCC 6059, M. canis (MTCC 2820 and MTCC 32700), M. gypseum MTCC 2819, Trichophyton ajelloi MTCC 4878, T. rubrum (MTCC 296 and MTCC 3272).
The free radical scavenging activity values were ranged from 0.7 to 1.41 mg/ml and 0.6 to 1.23 mg/ml, respectively for leaf and stem bark extracts. The amount of total phenolic contents of the extracts occurred in both leaf and bark in the range of 12 to 56.6 mg GAE/100 g and 16 to 58 mg GAE/100 g respectively. RP-HPLC analysis for flavonoids revealed the presence of two major flavonoid compounds, rutin and catechin. Kaempferol was in trace or absent. Methanol leaf extract showed significant low inhibitory effect against tested fungus Trichophyton ajelloi MTCC 4878 (0.140625 mg/ml) as the most sensitive. These finding suggest that the methanol leaf extract tested contain compounds with antimicrobial properties.
The results of our study may partially justify the folkloric uses on the plant studied and further provide an evidence that the leaf extract of Xylosma longifolium might be indeed a potential sources of antimicrobial agents.
PMCID: PMC3718714  PMID: 23819459
Xylosma Longifolium; Phenolic; Flavonoid; Antioxidant; RP-HPLC; Anti-Dermatophytic
19.  In vitro analysis on bactericidal screening and antioxidant potentiality of leaf and root extracts of Thottea siliquosa (Lam.) Ding Hou. An ethnobotanical plant 
Natural products of plant origin are potential source of novel antimicrobial and antioxidative agents. Thottea siliquosa (Lam.) Ding Hou. (T. siliquosa). A medicinal herb used by local tribals for treating various ailments. The present study aims at the phytochemical screening, GC-MS analysis, in vitro antibacterial activity and antioxidant potentiality of root and leaf extracts of T. siliquosa.
Hot continuous Soxhlet extraction, GC-MS analysis, antibacterial analysis by disc diffusion, microdilution assay and antioxidant potentialities by hydroxyl radical and nitric oxide radical scavenging. The data was statistically analyzed.
Phytochemical screening of the ethyl acetate and methanolic extract of leaf and root revealed the presence of phenols, alkaloids, tannins and saponin. The extract revealed a pool of phytochemicals by comparison with authentic standards from spectral library. Both the extracts has shown their broad spectrum of inhibition against the selected bacteria Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumonia compared with standard antibiotic drug streptomycin. The extracts showed antioxidant activity by scavenging of free radicals such as hydroxyl and nitric oxide. The IC50 values of the ethyl acetate extracts leaf and root and standard in this assay were 167.5±0.67, 99.4±1.2, 192±2.5 µg/mL respectively. Similarly those methanolic extracts of leaf and root were 269.5±0.89 and 289.1±2.66 µg/mL respectively. Similarly, ethyl acetate and methanolic extracts also caused a moderate dose-dependent inhibition of nitric oxide with an IC50 range 65.5±1.55 to 148 ±3.09 µg/mL. The inhibitory activities were found to be dose dependent.
The present study provides evidence that ethyl acetate and methanol extract of leaf and root of T. siliquosa are potential source of natural antioxidants and bactericidal nature. It is essential that research should continue to isolate and purify the bio active components of this natural plant and use in drug discovery and development.
PMCID: PMC3793157
Antibacterial activity; Antioxidant potentiality; Phytochemicals; Soxhlet extraction; Thottea siliquosa.
20.  Effects of Carvacrol on Sister Chromatid Exchanges in Human Lymphocyte Cultures 
Cytotechnology  2003;43(1-3):145-148.
Carvacrol is a predominant aromatic compound in oil of oregano. It has naturally remarkable antibacterial, antiviral, antifungal and antiparasital effects. In this study, genotoxic and antigenotoxic activities of carvacrol were investigated by the in vitro sister chromatid exchange (SCE) assay on human peripheral blood lymphocytes. The genotoxicity test was performed with carvacrol in two donors. On the other hand, inhibitory effect of carvacrol was tested in the presence of mitomycin C (MMC) in the same assay. According to data, all doses of carvacrol did not increase the formation of SCE, whereas it inhibited the rate of SCE induced by MMC. In conclusion, carvacrol exhibited a significant antigenotoxic activity in mammalian cells, indicating its potential for use as an antigenotoxic agent.
PMCID: PMC3449596  PMID: 19003219
carvacrol; lymphocyte culture; sister chromatid exchange
21.  Evaluation of the Bronchorelaxant, Genotoxic, and Antigenotoxic Effects of Cassia alata L. 
Aqueous-ethanolic extract of Cassia alata (AECal) and its derived fractions obtained through liquid-liquid fractionation were evaluated for their bronchorelaxant, genotoxic, and antigenotoxic effects. Contractile activity of rats' tracheas in the presence of tested materials, as well as its modifications with different inhibitors and blockers, was isometrically recorded. The antigenotoxic potential of AECal was evaluated on cyclophosphamide- (CP-) induced genotoxicity in the rat. Animals were pretreated with the extract, then liver comet assay was performed. AECal and its chloroformic fractions (CF-AECal) relaxed the contraction induced by Ach, but both were significantly less potent in inhibiting contraction induced by KCl (30 mM; 80 mM). Propranolol, indomethacin, L-NAME, methylene blue, and glibenclamide did not modify the relaxant effect of CF-AECal. TEA altered the response of trachea to CF-AECal. CF-AECal caused a rightward shift without affecting the Emax in cumulative concentration-response curves of Ach only at low concentrations. In animals pretreated with the extract, the percentage of CP-induced DNA damage decreased. Our results suggest that (1) muscarinic receptors contribute at least in part to the relaxant effects of CF-AECal; (2) CF-AECal interferes with membrane polarization; and (3) AECal is not genotoxic in vivo and contains chemopreventive phytoconstituents offering protection against CP-induced genotoxicity.
PMCID: PMC3655621  PMID: 23710211
22.  Antioxidant Effects of Spinach (Spinacia oleracea L.) Supplementation in Hyperlipidemic Rats 
Increased consumption of fresh vegetables that are high in polyphenols has been associated with a reduced risk of oxidative stress-induced disease. The present study aimed to evaluate the antioxidant effects of spinach in vitro and in vivo in hyperlipidemic rats. For measurement of in vitro antioxidant activity, spinach was subjected to hot water extraction (WE) or ethanol extraction (EE) and examined for total polyphenol content (TPC), oxygen radical absorbance capacity (ORAC), cellular antioxidant activity (CAA), and antigenotoxic activity. The in vivo antioxidant activity of spinach was assessed using blood and liver lipid profiles and antioxidant status in rats fed a high fat-cholesterol diet (HFCD) for 6 weeks. The TPC of WE and EE were shown as 1.5±0.0 and 0.5±0.0 mg GAE/g, respectively. Increasing the concentration of the extracts resulted in increased ORAC value, CAA, and antigenotoxic activity for all extracts tested. HFCD-fed rats displayed hyperlipidemia and increased oxidative stress, as indicated by a significant rise in blood and liver lipid profiles, an increase in plasma conjugated diene concentration, an increase in liver thiobarbituric acid reactive substances (TBARS) level, and a significant decrease in manganese superoxide dismutase (Mn-SOD) activity compared with rats fed normal diet. However, administration of 5% spinach showed a beneficial effect in HFCD rats, as indicated by decreased liver TBARS level and DNA damage in leukocyte and increased plasma conjugated dienes and Mn-SOD activity. Thus, the antioxidant activity of spinach may be an effective way to ameliorate high fat and cholesterol diet-induced oxidative stress.
PMCID: PMC3999804  PMID: 24772405
spinach; comet assay; liver TBARS; tail DNA; hyperlipidemic rat
23.  Evaluation of Antimicrobial and Wound Healing Potential of Justicia flava and Lannea welwitschii 
Microbial infections of various types of wounds are a challenge to the treatment of wounds and wound healing. The aim of the study is to determine the antimicrobial, antioxidant, and in vivo wound healing properties of methanol leaf extracts of Justicia flava and Lannea welwitschii. The antimicrobial activity was investigated using agar well diffusion and microdilution methods. The free radical scavenging activity of the methanol leaf extracts was performed using 1,1-diphenyl-2-picryl-hydrazyl (DPPH). The rate of wound contraction was determined using excision model. The test organisms used were Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 4853, Bacillus subtilis NTCC 10073, Staphylococcus aureus ATCC 25923, and clinical strains of Candida albicans. The MICs of methanol leaf extract of J. flava against test organisms were E. coli (7.5 mg/mL); P. aeruginosa (7.5 mg/mL); S. aureus (5 mg/mL); B. subtilis (7.5 mg/mL); and C. albicans (5 mg/mL). The MICs of methanol leaf extract of L. welwitschii against test organisms were E. coli (5 mg/mL); P. aeruginosa (10 mg/mL); S. aureus (5 mg/mL); B. subtilis (2.5 mg/mL); and C. albicans (2.5 mg/mL). The MBC/MFC of the extract was between 10 and 50 mg/mL. The IC50 of the reference antioxidant, α-tocopherol, was 1.5 μg/mL and the methanol leaf extracts of J. flava and L. welwitschii had IC50 of 65.3 μg/mL and 81.8 μg/mL, respectively. The methanol leaf extracts of J. flava and L. welwitschii gave a significant reduction in wound size as compared to the untreated. The rates of wound closure after the application of the extracts (7.5% w/w) were compared to the untreated wounds. On the 9th day, J. flava extract had a percentage wound closure of 99% (P < 0.01) and that of L. welwitschii exhibited wound closure of 95% (P < 0.05) on the 13th day compared to the untreated wounds. The two extracts significantly (P < 0.01) increased the tensile strength of wounds compared to the untreated wounds. The extracts treated wound tissues showed improved angiogenesis, collagenation, and reepithelialization compared to the untreated wound tissues. The preliminary phytochemical screening of J. flava and L. welwitschii leaf extracts revealed the presence of tannins, alkaloids, flavonoids, and glycosides. The above results indicate that methanol leaf extracts of J. flava and L. welwitschii possess antimicrobial and wound healing properties which may justify the traditional uses of J. flava and L. welwitschii in the treatment of wounds and infections.
PMCID: PMC3789403  PMID: 24159350
24.  Antimicrobial, Antioxidant, and Wound Healing Properties of Kigelia africana (Lam.) Beneth. and Strophanthus hispidus DC. 
Microbial infections of various types of wounds are a challenge to the treatment of wounds and wound healing. The study was to investigate antimicrobial and antioxidant properties of methanol leaf and stem bark extracts of Kigelia africana and methanol leaf and root extracts of Strophanthus hispidus and also to determine wound healing properties of the extracts. The antimicrobial activities of the methanol extracts were determined against two Gram-positive and two Gram-negative bacteria and a fungus using agar diffusion and micro-dilution methods. The antioxidant activity was determined using 1,1-diphenyl-2-picryl–hydrazyl (DPPH) method. The influence of the extracts on rate of wound closure was investigated using the excision wound model and histopathological investigation of treated and untreated wound tissues performed. The MICs of leaf extract of K. africana against test organisms were 2.5–7.5 mg/mL and stem bark extract were 2.25–7.5 mg/mL. The leaf extract of S. hispidus had MIC range of 2.5–7.5 mg/mL and 2.5–10 mg/mL for root extract. The IC50 of leaf and stem bark extracts of K. africana were 56.9 and 13.7 μg/mL, respectively and leaf and root of S. hispidus were 49.8 and 45.1 μg/mL, respectively. K. africana extracts (7.5% w/w) showed significant (P < 0.05) wound contraction at day 7 with 72% of wound closure whiles significant (P < 0.05) wound contractions were observed on day 11 for stem bark of K. africana, leaf and root extracts of S. hispidus. Wound tissues treated with the extracts showed improved collagenation, re-epitheliazition and rapid granulation formation compared with untreated wound tissues. The extracts were found to contain alkaloids, saponins, tannins, flavonoids, carbohydrates, and sapogenetic glycosides. The HPLC finger-printing of the extracts were developed. The leaf, stem bark and root extracts of K. africana and S. hispidus exhibited antimicrobial, antioxidant, and enhanced wound healing properties and these may justify the medicinal uses of the plants for treatment of microbial infections and wounds.
PMCID: PMC3639673  PMID: 23662099
25.  Efficacy of indigenous plant extracts on the malaria vector Anopheles subpictus Grassi (Diptera: Culicidae) 
Background & objectives:
Mosquito control is facing a threat due to the emergence of resistance to synthetic insecticides. Insecticides of plant origin may serve as suitable alternative biocontrol techniques in the future. The purpose of the present study was to assess the ethyl acetate, acetone and methanol extracts of Andrographis paniculata, Eclipta prostrata and Tagetes erecta leaves tested for oviposition-deterrent, ovicidal and repellent activities against malaria vector, Anopheles subpictus Grassi (Diptera: Culicidae).
The dried leaves of the three plants were powdered mechanically and extracted with ethyl acetate, acetone and methanol. One gram of crude extract was first dissolved in 100 ml of acetone (stock solution). From the stock solution, test solution concentrations of 31.21- 499.42 mg/l for oviposition- deterrence assay and repellency and 15.60 - 998.85 mg/l were used in ovicidal assay. The percentage oviposition- deterrence, hatching rate of eggs and protection time were calculated. One-way analysis of variance was used for the multiple concentration tests and for per cent mortality to determine significant treatment differences.
The percentage of effective oviposition repellency was highest at 499.42 mg/l and the lowest at 31.21 mg/l in ethyl acetate, acetone and methanol extracts of A. paniculata, E. prostrata and T. erecta. The oviposition activity index (OAI) value of ethyl acetate, acetone and methanol extracts of A. paniculata, E. prostrata and T. erecta at 499.42 mg/l were -0.91, -0.93, -0.84, -0.84, -0.87, -0.82, -0.87, -0.89 and -0.87, respectively. Mortality (no egg hatchability) was 100 per cent with ethyl acetate and methanol extracts of A. paniculata, E. prostrata and T. erecta at 998.85 mg/l. The maximum adult repellent activity was observed at 499.42 mg/l in ethyl acetate extracts of A. paniculata, E. prostrata and methanol extracts of T. erecta, and the mean complete protection time ranged from 120 to 150 min with the different extracts tested.
Interpretation & conclusions:
The acetone extract of A. paniculata, methanol extract of E. prostrata and T. erecta showed good oviposition-deterrent, ovicidal and repellent activities respectively. These results suggest that the leaf extracts of A. paniculata, E. prostrata and T. erecta may have the potential to be used as an ideal eco-friendly approach for the control of the An. subpictus.
PMCID: PMC3193720  PMID: 21985822
Anopheles subpictus; ovicidal; oviposition-deterrent; plant extracts; repellent activities

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