Methanol extract from Vietnamese Caesalpinia sappan induces apoptosis in HeLa cells

Background

This study evaluated the cytotoxic activity of extracts from Caesalpinia sappan heartwood against multiple cancer cell lines using an MTT cell viability assay. The cell death though induction of apoptosis was as indicated by DNA fragmentation and caspase-3 enzyme activation.

Results

A methanol extract from C. sappan (MECS) showed cytotoxic activity against several of the cancer cell lines. The most potent activity exhibited by the MECS was against HeLa cells with an IC₅₀ value of 26.5 ± 3.2 μg/mL. Treatment of HeLa cells with various MECS concentrations resulted in growth inhibition and induction of apoptosis, as indicated by DNA fragmentation and caspase-3 enzyme activation.

Conclusion

This study is the first report of the anticancer properties of the heartwood of C. sappan native to Vietnam. Our findings demonstrate that C. sappan heartwood may have beneficial applications in the field of anticancer drug discovery.     

Extraction of saponins and toxicological profile of Teucrium stocksianum boiss extracts collected from District Swat,Pakistan

Background

The current era is facing challenges in the management of neoplasia and weeds control. The currently available anti-cancer and herbicidal drugs are associated with some serious side effects. Therefore numerous researchers are trying to discover and develop plant based alternative particularly for the rational management of cancer and weed control. Teucrium stocksianum possess antioxidant and analgesic activities. The current study was designed to evaluate crude saponins (CS), methanolic extract and sub-fractions of T. stocksianum for cytotoxic and phytotoxic potentials. CS, methanolic extract and sub-fractions were extracted from powdered plant material using different solvents. Cytotoxic potential of the extracts at a dose of 10, 100 and 1000 μg/ml were evaluated against Brine shrimp’s nauplii. Phytotoxic assay also performed at the same concentration against Lemna minor. Etoposide and Paraquat were used as positive controls in cytotoxic and phytotoxic assays respectively.

Results

The percent yield of crude saponins was (5%). CS demonstrated tremendous brine shrimp lethality showing < 10 μg/ml LC₅₀. The n-hexane (HF) and chloroform fractions (CF) demonstrated excellent cytotoxicity with 80 and 55 μg/ml LC₅₀ respectively. Whereas the methanolic extract (TSME), ethyl acetate (EAF) and aqueous fractions (AF) revealed moderate cytotoxicity showing 620, 860 and 1000 μg/ml LC₅₀ values respectively. In phytotoxic assay profound inhibition was displayed by HF (96.67%) and TSME (95.56%, 30 μg/ml LC₅₀) against the growth of Lemna minor at 1000 μg/ml respectively. Both CF and EAF demonstrated profound phytoxicity (93.33%) respectively at highest concentration (1000 μg/ml), while AF and CS demonstrated weak phytotoxicity with 1350 and 710 μg/ml LC₅₀ values respectively.

Conclusion

Cytotoxicity and phytotoxicity assays indicated that the crude saponins, n-hexane and chloroform fractions of T. stocksianum could play a vital role in the treatment of neoplasia and as potential natural herbicides. Therefore these sub-fractions are recommended for further investigation with the aim to isolate novel anti-cancer and herbicidal compounds.     

Cytotoxicity and hepatoprotective attributes of methanolic extract of Rumex vesicarius L.

Background

To evaluate the hepatoprotective potential and invitro cytotoxicity studies of whole plant methanol extract of Rumex vesicarius L. Methanol extract at a dose of 100 mg/kg bw and 200 mg/kg bw were assessed for its hepatoprotective potential against CCl₄-induced hepatotoxicity by monitoring activity levels of SGOT (Serum glutamic oxaloacetic transaminase), SGPT (Serum glutamic pyruvic transaminase), ALP (Alkaline phosphatase), TP (Total protein), TB (Total bilirubin) and SOD (Superoxide dismutase), CAT (Catalase), MDA (Malondialdehyde). The cytotoxicity of the same extract on HepG2 cell lines were also assessed using MTT assay method at the concentration of 62.5, 125, 250, 500 μg/ml.

Results

Pretreatment of animals with whole plant methanol extracts of Rumex vesicarius L. significantly reduced the liver damage and the symptoms of liver injury by restoration of architecture of liver. The biochemical parameters in serum also improved in treated groups compared to the control and standard (silymarin) groups. Histopathological investigation further corroborated these biochemical observations. The cytotoxicity results indicated that the plant extract which were inhibitory to the proliferation of HepG2 cell line with IC50 value of 563.33 ± 0.8 μg/ml were not cytotoxic and appears to be safe.

Conclusions

Rumex vesicarius L. whole plant methanol extract exhibit hepatoprotective activity. However the cytotoxicity in HepG2 is inexplicable and warrants further study.     

Phytochemical and toxicological investigations of crude methanolic extracts,subsequent fractions and crude saponins of Isodon rugosus

Background

Isodon rugosus is used traditionally in the management of hypertension, rheumatism, tooth-ache and pyrexia. Present study was arranged to investigate I. rugosus for phytoconstituents, phytotoxic and cytotoxic activities to explore its toxicological, pharmacological potentials and to rationalize its ethnomedicinal uses. Briefly, qualitative phytochemical analysis of the plant extracts were carried out for the existence of alkaloids, flavonoids, saponins, oils, glycosides, anthraquinones, terpenoids, sterols and tannins. Plant crude methanolic extract (Ir.Cr), its subsequent fractions; n-hexane (Ir.Hex), chloroform (Ir.Chf), ethyl acetate (Ir.EtAc), aqueous (Ir.Aq) and saponins (Ir.Sp) in different concentrations were tested for phytotoxic and cytotoxic activities using radish seeds and brine shrimps (Artemia salina) respectively. The phytotoxic activity was determined by percent root length inhibition (RLI) and percent seeds germination inhibition (SGI) while the cytotoxicity was obtained with percent lethality of the brine shrimps.

Results

Ir.Cr was tested positive for the presence of alkaloids, glycosides, flavonoids, oils, terpenoids, saponins, tannins and anthraquinones. Among different fractions Ir.Sp, Ir.Chf, Ir.EtAc, and Ir.Cr were most effective causing 93.55, 89.32, 81.32 and 58.68% inhibition of seeds in phytotoxicity assay, with IC₅₀ values of 0.1, 0.1, 0.1 and 52 μg/ml respectively. Similarly, among all the tested samples, Ir.Sp exhibited the highest phytotoxic effect causing 91.33% root length inhibition with IC₅₀ of 0.1 μg/ml. Ir.Sp and Ir.Chf were most effective against brine shrimps showing 92.23 and 76.67% lethality with LC₅₀ values of 10 and 12 μg/ml respectively.

Conclusions

It may be inferred from the current investigations that I. rugosus contains different secondary metabolites and is a potential source for the isolation of natural anticancer and herbicidal drug molecules. Different fractions exhibited phytotoxic and cytotoxic activities, thus providing pharmacological basis for ethnomedicinal uses of this plant.     

Continuous,high-resolution biospeckle imaging reveals a discrete zone of activity at the root apex that responds to contact with obstacles

Background and Aims

Shining a laser onto biological material produces light speckles termed biospeckles. Patterns of biospeckle activity reflect changes in cell biochemistry, developmental processes and responses to the environment. The aim of this work was to develop methods to investigate the biospeckle activity in roots and to characterize the distribution of its intensity and response to thigmostimuli.

Methods

Biospeckle activity in roots of Zea mays, and also Jatropha curcas and Citrus limonia, was imaged live and in situ using a portable laser and a digital microscope with a spatial resolution of 10 μm per pixel and the ability to capture images every 0·080 s. A procedure incorporating a Fujii algorithm, image restoration using median and Gaussian filters, image segmentation using maximum-entropy threshold methods and the extraction of features using a tracing algorithm followed by spline fitting were developed to obtain quantitative information from images of biospeckle activity. A wavelet transform algorithm was used for spectral decomposition of biospeckle activity and generalized additive models were used to attribute statistical significance to changes in patterns of biospeckle activity.

Key Results

The intensity of biospeckle activity was greatest close to the root apex. Higher frequencies (3–6 Hz) contributed most to the total intensity of biospeckle activity. When a root encountered an obstacle, the intensity of biospeckle activity decreased abruptly throughout the root system. The response became attenuated with repeated thigmostimuli.

Conclusions

The data suggest that at least one component of root biospeckle activity resulted from a biological process, which is located in the zone of cell division and responds to thigmostimuli. However, neither individual cell division events nor root elongation is likely to be responsible for the patterns of biospeckle activity.     

In vivo antipyretic,antiemetic, in vitro membrane stabilization,antimicrobial, and cytotoxic activities of different extracts from Spilanthes paniculata leaves

Background

The study was conducted to evaluate the in vitro antimicrobial activity, cytotoxic, and membrane stabilization activities, and in vivo antiemetic and antipyretic potentials of ethanolic extract, n-hexane and ethyl acetate soluble fractions of Spilanthes paniculata leaves for the first time widely used in the traditional treatments in Bangladesh.

Results

In antipyretic activity assay, a significant reduction (P < 0.05) was observed in the temperature in the mice tested. At dose 400 mg/kg-body weight, the n-hexane soluble fraction showed the effect (36.7 ± 0.63°C ) as like as the standard (dose 150 mg/kg-body weight) after 5 h of administration. Extracts showed significant (P < 0.001) potential when tested for the antiemetic activity compared to the standard, metoclopramide. At dose 50 mg/kg-body weight, the standard showed 67.23% inhibition, whereas n-hexane and ethyl acetate soluble fractions showed 37.53% and 24.93% inhibition of emesis respectively at dose 400 mg/kg-body weight. In antimicrobial activity assay, the n-hexane soluble fraction (400 μg/disc) showed salient activity against the tested organisms. It exerts highest activity against Salmonella typhi (16.9 mm zone of inhibition); besides, crude, and ethyl acetate extracts showed resistance to Bacillus cereus and Bacillus subtilis, and Vibrio cholera respectively. All the extracts were tested for lysis of the erythrocytes. At the concentration of 1mg/ml, ethanol extract, and n-hexane and ethyl acetate soluble fractions significantly inhibited hypotonic solution induced lysis of the human red blood cell (HRBC) (27.406 ± 3.57, 46.034 ± 3.251, and 30.72 ± 5.679% respectively); where standard drug acetylsalicylic acid (concentration 0.1 mg/ml) showed 77.276 ± 0.321% inhibition. In case of heat induced HRBC hemolysis, the plant extracts also showed significant activity (34.21 ± 4.72, 21.81 ± 3.08, and 27.62 ± 8.79% inhibition respectively). In the brine shrimp lethality bioassay, the n-hexane fraction showed potent (LC₅₀ value 48.978 μg/ml) activity, whereas ethyl acetate fraction showed mild (LC₅₀ value 216.77 μg/ml) cytotoxic activity.

Conclusions

Our results showed that the n-hexane extract has better effects than the other in all trials. In the context, it can be said that the leaves of S. paniculata possess remarkable pharmacological effects, and justify its folkloric use as antimicrobial, antipyretic, anti-inflammatory, and antiemetic agent. Therefore, further research may be suggested to find possible mode of action of the plant part.     

Investigations of anticholinestrase and antioxidant potentials of methanolic extract,subsequent fractions,crude saponins and flavonoids isolated from Isodon rugosus

Background

Based on the ethnomedicinal uses and the effective outcomes of natural products in various diseases, this study was designed to evaluate Isodon rugosus as possible remedy in oxidative stress, alzheimer’s and other neurodegenerative diseases. Acetylecholinestrase (AChE) and butyrylcholinesterase (BChE) inhibitory activities of crude methanolic extract (Ir.Cr), resultant fractions (n-hexane (Ir.Hex), chloroform (Ir.Cf), ethyl acetate (Ir.EtAc), aqueous (Ir.Aq)), flavonoids (Ir.Flv) and crude saponins (Ir.Sp) of I. rugosus were investigated using Ellman’s spectrophotometric method. Antioxidant potential of I. rugosus was determined using DPPH, H₂O₂ and ABTS free radicals scavenging assays. Total phenolic and flavonoids contents of plant extracts were determined and expressed in mg GAE/g dry weight and mg RTE/g of dry sample respectively.

Results

Among different fractions Ir.Flv and Ir.Cf exhibited highest inhibitory activity against AChE (87.44 ± 0.51, 83.73 ± 0.64%) and BChE (82.53 ± 0.71, 88.55 ± 0.77%) enzymes at 1 mg/ml with IC₅₀ values of 45, 50 for AChE and 40, 70 μg/ml for BChE respectively. Activity of these fractions were comparable to galanthamine causing 96.00 ± 0.30 and 88.61 ± 0.43% inhibition of AChE and BChE at 1 mg/ml concentration with IC₅₀ values of 20 and 47 μg/ml respectively. In antioxidant assays, Ir.Flv, Ir.Cf, and Ir.EtAc demonstrated highest radicals scavenging activities in DPPH and H₂O₂ assays which were comparable to ascorbic acid. Ir.Flv was found most potent with IC₅₀ of 19 and 24 μg/ml against DPPH and H₂O₂ radicals respectively. Whereas antioxidant activates of plant samples against ABTS free radicals was moderate. Ir.Cf, Ir.EtAc and Ir.Cr showed high phenolic and flavonoid contents and concentrations of these compounds in different fractions correlated well to their antioxidant and anticholinestrase activities.

Conclusion

It may be inferred from the current investigations that the Ir.Sp, Ir.Flv and various fractions of I. rugosus are good sources of anticholinesterase and antioxidant compounds. Different fractions can be subjected to activity guided isolation of bioactive compounds effective in neurological disorders.     

Evaluation of phenolic profile,antioxidant and anticancer potential of two main representants of Zingiberaceae family against B164A5 murine melanoma cells

Background

Curcuma longa Linnaeus and Zingiber officinale Roscoe are two main representatives of Zingiberaceae family studied for a wide range of therapeutic properties, including: antioxidant, anti-inflammatory, anti-angiogenic, antibacterial, analgesic, immunomodulatory, proapoptotic, anti-human immunodeficiency virus properties and anticancer effects. This study was aimed to analyse the ethanolic extracts of Curcuma rhizome (Curcuma longa Linnaeus) and Zingiber rhizome (Zingiber officinale Roscoe) in terms of polyphenols, antioxidant activity and anti-melanoma potential employing the B164A5 murine melanoma cell line.

Results

In order to evaluate the total content of polyphenols we used Folin-Ciocâlteu method. The antioxidant activity of the two ethanolic extracts was determined by DPPH assay, and for the control of antiproliferative effect it was used MTT proliferation assay, DAPI staining and Annexin-FITC-7AAD double staining test. Results showed increased polyphenols amount and antioxidant activity for Curcuma rhizome ethanolic extract. Moreover, 100 μg/ml of ethanolic plant extract from both vegetal products presented in a different manner an antiproliferative, respectively a proapoptotic effect on the selected cell line.

Conclusions

The study concludes that Curcuma rhizome may be a promising natural source for active compounds against malignant melanoma.     

In vitro antioxidant capacity and free radical scavenging evaluation of active metabolite constituents of Newbouldia laevis ethanolic leaf extract

Background

The aim of the present study was to evaluate the in vitro antioxidant and free radical scavenging capacity of bioactive metabolites present in Newbouldia laevis leaf extract.

Results

Chromatographic and spectrophotometric methods were used in the study and modified where necessary in the study. Bioactivity of the extract was determined at 10 μg/ml, 50 μg/ml, 100 μg/ml, 200 μg/ml and 400 μg/ml concentrations expressed in % inhibition. The yield of the ethanolic leaf extract of N.laevis was 30.3 g (9.93%). Evaluation of bioactive metabolic constituents gave high levels of ascorbic acid (515.53 ± 12 IU/100 g [25.7 mg/100 g]), vitamin E (26.46 ± 1.08 IU/100 g), saponins (6.2 ± 0.10), alkaloids (2.20 ± 0.03), cardiac glycosides(1.48 ± 0.22), amino acids and steroids (8.01 ± 0.04) measured in mg/100 g dry weight; moderate levels of vitamin A (188.28 ± 6.19 IU/100 g), tannins (0.09 ± 0.30), terpenoids (3.42 ± 0.67); low level of flavonoids (1.01 ± 0.34 mg/100 g) and absence of cyanogenic glycosides, carboxylic acids and aldehydes/ketones. The extracts percentage inhibition of DPPH, hydroxyl radical (OH^.), superoxide anion (O₂^.-), iron chelating, nitric oxide radical (NO), peroxynitrite (ONOO⁻), singlet oxygen (¹O₂), hypochlorous acid (HOCl), lipid peroxidation (LPO) and FRAP showed a concentration-dependent antioxidant activity with no significant difference with the controls. Though, IC₅₀ of the extract showed significant difference only in singlet oxygen (¹O₂) and iron chelating activity when compared with the controls.

Conclusions

The extract is a potential source of antioxidants/free radical scavengers having important metabolites which maybe linked to its ethno-medicinal use.     

Synergistic activity and mechanism of action of Stephania suberosa Forman extract and ampicillin combination against ampicillin-resistant Staphylococcus aureus

Background

Ampicillin-resistant S. aureus (ARSA) now poses a serious problem for hospitalized patients, and their care providers. Plant-derived antibacterial that can reverse the resistance to well-tried agents which have lost their original effectiveness are the research objectives of far reaching importance. To this aim, the present study investigated antibacterial and synergistic activities of Stephania suberosa extracts (SSE) against ARSA when used singly and in combination with ampicillin.

Results

The majority chemical compounds of SSE were alkaloid (526.27 ± 47.27 mg/1 g of dried extract). The Minimum inhibitory concentration (MICs) for ampicillin and SSE against all ARSA strains were >512 μg/ml and 4 mg/ml, respectively. Checkerboard assay revealed synergistic activity in the combination of ampicillin (0.15 μg/ml) and SSE (2 mg/ml) at fractional inhibitory concentration index (FICI) <0.5. The killing curve assay had confirmed that the viability of ARSA was dramatically reduced from 5x10⁵ cfu/ml to 10³ cfu/ml within 6 h after exposure to SSE (2 mg/ml) plus ampicillin (0.15 μg/ml) combination. Electron microscopic study clearly revealed that these ARSA cells treated with this combination caused marked morphological damage, peptidoglycan and cytoplasmic membrane damage, and average cell areas significant smaller than control. Obviously, Immunofluorescence staining and confocal microscopic images confirmed that the peptidoglycan of these cells were undoubtedly disrupted by this combination. Furthermore, the CM permeability of ARSA was also increased by this combination. Enzyme assay demonstrated that SSE had an inhibitory activity against β-lactamase in concentrations manner.

Conclusions

So, these findings provide evidence that SSE has the high potential to reverse bacterial resistance to originate traditional drug susceptibility of it and may relate to three modes of actions of SSE: (1) inhibits peptidoglycan synthesis, resulting in morphological damage, (2) inhibits β-lactamases activity, and (3) increases CM permeability. It is widely recognized that many types of drugs are derived from alkaloids. So, this SSE offers the prominent potential to develop a novel adjunct phytopharmaceutical to ampicillin for the treatment of ARSA. Further active ingredients study, toxicity of it, and the synergistic effect on blood and tissue should be performed and confirmed in an animal test or in humans.     

Effect of root length on epicotyl dormancy release in seeds of Paeonia ludlowii,Tibetan peony

Background and Aims

Epicotyl dormancy break in seeds that have deep simple epicotyl morphophysiological dormancy (MPD) requires radicle emergence and even a certain root length in some species. However, the mechanisms by which root length affects epicotyl dormancy break are not clear at present. This study aims to explore the relationship between root length and epicotyl dormancy release in radicle-emerged seeds of Tibetan peony, Paeonia ludlowii, with discussion of the possible mechanisms.

Methods

Radicle-emerged seeds (radicle length 1·5, 3·0, 4·5 and 6·0 cm) were incubated at 5, 10 and 15 °C. During the stratification, some seeds were transferred to 15 °C and monitored for epicotyl–plumule growth. Hormone content was determined by ELISA, and the role of hormones in epicotyl dormancy release was tested by exogenous hormone and embryo culture.

Key Results

Cold stratification did not break the epicotyl dormancy until the root length was ≥6 cm. The indole-3-actic acid (IAA) and GA₃ contents of seeds having 6 cm roots were significantly higher than those of seeds with other root lengths, but the abscisic acid (ABA) content was lowest among radicle-emerged seeds. GA₃ (400 mg L⁻¹) could break epicotyl dormancy of all radicle-emerged seeds, while IAA (200 mg L⁻¹) had little or no effect. When grown on MS medium, radicles of naked embryos grew and cotyledons turned green, but epicotyls did not elongate. Naked embryos developed into seedlings on a mixed medium of MS + 100 mg L⁻¹ GA₃.

Conclusions

A root length of ≥6·0 cm is necessary for epicotyl dormancy release by cold stratification. The underlying reason for root length affecting epicotyl dormancy release is a difference in the GA₃/ABA ratio in the epicotyl within radicle-emerged seeds, which is mainly as a result of a difference in ABA accumulation before cold stratification.     

Pesticidal and pest repellency activities of rhizomes of Drynaria quercifolia (J. Smith) against Tribolium castaneum (Herbst)

Background

Tribolium castaneum (Herbst) is a harmful pest of stored grain and flour-based products in tropical and subtropical region. In the present study, rhizome of Drynaria quercifolia (J. Smith) was evaluated for pesticidal and pest repellency activities against T. castaneum, using surface film method and filter paper disc method, respectively. In addition, activity of the isolated compound 3,4-dihydroxybenzoic acid was evaluated against the pest.

Results

Chloroform soluble fraction of ethanol extract of rhizome of D. quercifolia showed significant pesticidal activity at doses 0.88 to 1.77 mg/cm² and significant pest repellency activity at doses 0.94 to 0.23 mg/cm². No pesticidal and pest repellency activity was found for petroleum ether, ethyl acetate and methanol soluble fractions of ethanol extract as well as for 3,4-dihydroxybenzoic acid.

Conclusion

Considering our findings it can be concluded that chloroform soluble fraction of rhizome of D. quercifolia is useful in controlling T. castaneum of stored grain and flour-based products.

Electronic supplementary material

The online version of this article (doi:10.1186/0717-6287-47-51) contains supplementary material, which is available to authorized users.     

Anti-inflammatory components of the Vietnamese starfish Protoreaster nodosus

Background

In the present study, we examined the inhibitory effects of a methanolic extract, dichloromethane fraction, water layer, and polyhydroxylated sterols (1–4) isolated from the Vietnamese starfish Protoreaster nodosus on pro-inflammatory cytokine (IL-12 p40, IL-6, and TNF-α) production in LPS-stimulated bone marrow-derived dendritic cells (BMDCs) using enzyme-linked immunosorbent assays (ELISA).

Results

The methanolic extract and dichloromethane fraction exerted potent inhibitory effects on the production of all three pro-inflammatory cytokines, with IC₅₀ values ranging from 0.60 ± 0.01 to 26.19 ± 0.64 μg/mL. Four highly pure steroid derivatives (1–4) were isolated from the dichloromethane fraction and water layer of P. nodosus. Potent inhibitory activities were also observed for (25S) 5α-cholestane-3β,4β,6α,7α,8β,15α,16β,26-octol (3) on the production of IL-12 p40 and IL-6 (IC_50s = 3.11 ± 0.08 and 1.35 ± 0.03 μM), and for (25S) 5α-cholestane-3β,6α,8β,15α,16β,26-hexol (1) and (25S) 5α-cholestane-3β,6α,7α,8β,15α,16β,26-heptol (2) on the production of IL-12 p40 (IC_50s = 0.01 ± 0.00 and 1.02 ± 0.01 μM). Moreover, nodososide (4) exhibited moderate inhibitory effects on IL-12 p40 and IL-6 production.

Conclusion

This is the first report of the anti-inflammatory activity from the starfish P. nodosus. The main finding of this study is the identification oxygenated steroid derivatives from P. nodosus with potent anti-inflammatory activities that may be developed as therapeutic agents for inflammatory diseases.     

Chloroplast localization of Cry1Ac and Cry2A protein- an alternative way of insect control in cotton

Background

Insects have developed resistance against Bt-transgenic plants. A multi-barrier defense system to weaken their resistance development is now necessary. One such approach is to use fusion protein genes to increase resistance in plants by introducing more Bt genes in combination. The locating the target protein at the point of insect attack will be more effective. It will not mean that the non-green parts of the plants are free of toxic proteins, but it will inflict more damage on the insects because they are at maximum activity in the green parts of plants.

Results

Successful cloning was achieved by the amplification of Cry2A, Cry1Ac, and a transit peptide. The appropriate polymerase chain reaction amplification and digested products confirmed that Cry1Ac and Cry2A were successfully cloned in the correct orientation. The appearance of a blue color in sections of infiltrated leaves after 72 hours confirmed the successful expression of the construct in the plant expression system. The overall transformation efficiency was calculated to be 0.7%. The amplification of Cry1Ac-Cry2A and Tp2 showed the successful integration of target genes into the genome of cotton plants. A maximum of 0.673 μg/g tissue of Cry1Ac and 0.568 μg/g tissue of Cry2A was observed in transgenic plants. We obtained 100% mortality in the target insect after 72 hours of feeding the 2^nd instar larvae with transgenic plants. The appearance of a yellow color in transgenic cross sections, while absent in the control, through phase contrast microscopy indicated chloroplast localization of the target protein.

Conclusion

Locating the target protein at the point of insect attack increases insect mortality when compared with that of other transgenic plants. The results of this study will also be of great value from a biosafety point of view.     

Improving the large scale purification of the HIV microbicide,griffithsin

Background

Griffithsin is a broad spectrum antiviral lectin that inhibits viral entry and maturation processes through binding clusters of oligomannose glycans on viral envelope glycoproteins. An efficient, scaleable manufacturing process for griffithsin active pharmaceutical ingredient (API) is essential for particularly cost-sensitive products such as griffithsin -based topical microbicides for HIV-1 prevention in resource poor settings. Our previously published purification method used ceramic filtration followed by two chromatography steps, resulting in a protein recovery of 30%. Our objective was to develop a scalable purification method for griffithsin expressed in Nicotiana benthamiana plants that would increase yield, reduce production costs, and simplify manufacturing techniques. Considering the future need to transfer griffithsin manufacturing technology to resource poor areas, we chose to focus modifying the purification process, paying particular attention to introducing simple, low-cost, and scalable procedures such as use of temperature, pH, ion concentration, and filtration to enhance product recovery.

Results

We achieved >99% pure griffithsin API by generating the initial green juice extract in pH 4 buffer, heating the extract to 55°C, incubating overnight with a bentonite MgCl₂ mixture, and final purification with Capto™ multimodal chromatography. Griffithsin extracted with this protocol maintains activity comparable to griffithsin purified by the previously published method and we are able to recover a substantially higher yield: 88 ± 5% of griffithsin from the initial extract. The method was scaled to produce gram quantities of griffithsin with high yields, low endotoxin levels, and low purification costs maintained.

Conclusions

The methodology developed to purify griffithsin introduces and develops multiple tools for purification of recombinant proteins from plants at an industrial scale. These tools allow for robust cost-effective production and purification of griffithsin. The methodology can be readily scaled to the bench top or industry and process components can be used for purification of additional proteins based on biophysical characteristics.     

In vivo analgesic,antipyretic, and anti-inflammatory potential in Swiss albino mice and in vitro thrombolytic activity of hydroalcoholic extract from Litsea glutinosa leaves

Background

The study was conducted to evaluate the in vitro thrombolytic activity, and in vivo analgesic, anti-inflammatory and antipyretic potentials of different hydrocarbon soluble extracts of Litsea glutinosa leaves for the first time widely used in the folkloric treatments in Bangladesh. This work aimed to create new insights on the fundamental mechanisms of the plant extracts involved in these activities.

Results

In thrombolytic activity assay, a significant clot disruption was observed at dose of 1 mg/mL for each of the extracts (volume 100 μL) when compared to the standard drug streptokinase. The n-hexane, ethyl acetate, chloroform, and crude methanolic extracts showed 32.23 ± 0.26, 37.67 ± 1.31, 43.13 ± 0.85, and 46.78 ± 0.9% clot lysis, respectively, whereas the positive control streptokinase showed 93.35 ± 0.35% disruption at the dose of 30,000 I.U. In hot plate method, the highest pain inhibitory activity was found at a dose of 500 mg/kg of crude extract (15.54 ± 0.37 sec) which differed significantly (P <0.01 and P <0.001) with that of the standard drug ketorolac (16.38 ± 0.27 sec). In acetic acid induced writhing test, the crude methanolic extract showed significant (P <0.01 and P <0.001) analgesic potential at doses 250 and 500 mg/kg body weight (45.98 and 56.32% inhibition, respectively), where ketorolac showed 64.36% inhibition. In anti-inflammatory activity test, the crude methanolic extract showed significant (P <0.001) potential at doses 250 and 500 mg/kg body weight (1.51 ± 0.04 and 1.47 ± 0.03 mm paw edema, respectively), where ketorolac showed 1.64 ± 0.05 mm edema after 3 h of carrageenan injection. In antipyretic activity assay, the crude extract showed notable reduction in body temperature (32.78 ± 0.46°C) at dose of 500 mg/kg-body weight, when the standard (at dose 150 mg/kg-body weight) exerted 33.32 ± 0.67°C temperature after 3 h of administration.

Conclusions

Our results yield that the crude hydroalcoholic extract has better effects than the other in all trials. In the context, it can be said that the leaves of L. glutinosa possess remarkable pharmacological effects, and justify its traditional use as analgesic, antipyretic, anti-inflammatory, and thrombolytic agent.     

Assessment of the phytochemical constituents and antioxidant activity of a bloom forming microalgae Euglena tuba

Background

Unstable generation of free radicals in the body are responsible for many degenerative diseases. A bloom forming algae Euglena tuba growing abundantly in the aquatic habitats of Cachar district in the state of Assam in North-East India was analysed for its phytochemical contents, antioxidant activity as well as free radical scavenging potentials.

Results

Based on the ability of the extract in ABTS^•+ radical cation inhibition and Fe³⁺ reducing power, the obtained results revealed the prominent antioxidant activity of the algae, with high correlation coefficient of its TEAC values to the respective phenolic and flavonoid contents. The extract had shown its scavenging activity for different free radicals and 41.89 ± 0.41 μg/ml, 5.83 ± 0.07 μg/ml, 278.46 ± 15.02 μg/ml and 223.25 ± 4.19 μg/ml were determined as the IC₅₀ values for hydroxyl, superoxide, nitric oxide and hypochlorous acid respectively, which are lower than that of the corresponding reference standards. The phytochemical analysis also revealed that the phenolics, flavonoids, alkaloids, tannins and carbohydrates are present in adequate amount in the extract which was confirmed by HPLC analysis.

Conclusions

The results showed that 70% methanol extract of the algae possesses excellent antioxidant and free radical scavenging properties.     

A rapid,controlled-environment seedling root screen for wheat correlates well with rooting depths at vegetative,but not reproductive,stages at two field sites

Background and Aims

Root length and depth determine capture of water and nutrients by plants, and are targets for crop improvement. Here we assess a controlled-environment wheat seedling screen to determine speed, repeatability and relatedness to performance of young and adult plants in the field.

Methods

Recombinant inbred lines (RILs) and diverse genotypes were grown in rolled, moist germination paper in growth cabinets, and primary root number and length were measured when leaf 1 or 2 were fully expanded. For comparison, plants were grown in the field and root systems were harvested at the two-leaf stage with either a shovel or a soil core. From about the four-leaf stage, roots were extracted with a steel coring tube only, placed directly over the plant and pushed to the required depth with a hydraulic ram attached to a tractor.

Key Results

In growth cabinets, repeatability was greatest (r = 0·8, P < 0·01) when the paper was maintained moist and seed weight, pathogens and germination times were controlled. Scanned total root length (slow) was strongly correlated (r = 0·7, P < 0·01) with length of the two longest seminal axile roots measured with a ruler (fast), such that 100–200 genotypes were measured per day. Correlation to field-grown roots at two sites at two leaves was positive and significant within the RILs and cultivars (r = 0·6, P = 0·01), and at one of the two sites at the five-leaf stage within the RILs (r = 0·8, P = 0·05). Measurements made in the field with a shovel or extracted soil cores were fast (5 min per core) and had significant positive correlations to scanner measurements after root washing and cleaning (>2 h per core). Field measurements at two- and five-leaf stages did not correlate with root depth at flowering.

Conclusions

The seedling screen was fast, repeatable and reliable for selecting lines with greater total root length in the young vegetative phase in the field. Lack of significant correlation with reproductive stage root system depth at the field sites used in this study reflected factors not captured in the screen such as time, soil properties, climate variation and plant phenology.     

Antimicrobial and Antimycobacterial Activities of Methyl Caffeate Isolated from Solanum torvum Swartz. Fruit

Abstract

Solanum torvum Swartz. (Solanaceae) fruit is traditionally used for the treatment of bacterial and fungal infections. The methanolic extract was subjected to activity guided fractionation by column chromatography over silica gel. The structure of the compound was elucidated using physical and spectroscopic data. The antimicrobial activity was screened using five Gram-positive bacteria, six Gram-negative bacteria, seven clinical isolates and four fungi. Antimycobacterial activity was screened against two Mycobacterium strains. The zone of inhibition by methyl caffeate ranged from 0 to 22 mm. The lowest minimum inhibitory concentration (MIC) values of methyl caffeate were: 50 μg/ml against P. vulgaris, 25 μg/ml against K. pneumoniae (ESBL-3971), 8 μg/ml against M. tuberculosis (H³⁷Rv) and 8 μg/ml against M. tuberculosis (Rif^R). Methyl caffeate showed moderate antimicrobial and prominent antimycobacterial activities. Methyl caffeate can be evaluated further for drug development.