WORLD JOURNAL OF BIOLOGY AND BIOTECHNOLOGY Antifungal and antioxidant properties of chloroform soluble compounds of fennel seeds

Bio-pesticides are replacing chemicals being their potential alternatives. The present study was conducted to evaluate the antifungal effect of chloroform soluble fractions of Foeniculum vulgare seeds against seed rotting caused by Aspergillus flavus , A. welwitschia , A. minisclerotigenes, and A. oryzae . The effect was determined in terms of inhibition in fungal biomass production, the total content of cellular proteins, and the enzymatic rate of catalase and peroxidase. The maximum growth inhibition was recorded for A. flavus where the extract of F. vulgare reduced 97.29% fungal dry weight without significantly affecting the total cell protein content as well as the catalase and peroxidase enzymatic activities. Seed extract caused 92% inhibition in the mycelial growth of A. minisclerotigenes over the control treatment, reducing the cell protein content to 1.25 from 1.57 mg/g fresh weight. However, catalytic and peroxidase activities increased about three and two times, respectively. Under the stress of seed extract, A. welwitschia mycelial weight suppression was almost 72%, total protein content increased by two folds, and catalase and peroxidase activities decreased significantly. The effect of chloroform seed extract of F. vulgare was found to be insignificant on growth and selected physiological attributes of A. oryzae .

now cultivated in almost every country (Muckensturm et al., 1997).It contains protein, lipids, and carbohydrates along with minerals (Ca, Fe, Mg, Zn, P, K, and Na), dietary fiber, and vitamins (C and thiamin B1), relative to human needs.On account of its aroma, it is extensively utilized for culinary and medicinal purposes with diverse pharmacological (Rather et al., 2016).Essential oil and seeds extract of F. vulgare have been screened for their anti-fungal potential and found to be equally good with broad-spectrum antibiotics against Aspergillus niger, A. flavus, Fusarium graminearum and F. minliforme (Singh et al., 2006;Upadhyay, 2015).Likewise, aqueous and alcoholic seed extracts of F. vulgare inhibited the growth of Alternaria alternata, Mucor rouxii, and A. flavus (Thakur et al., 2013), and in some cases, antifungal activity of aqueous seed extract was greater compared to reference fungicidal agent ((Taie et al., 2013).Khan (2017) observed the substantial potential of methanolic and aqueous seed extract of F. vulgare against different species of Candida.Aamir et al. (2018) results indicated the significant antifungal potential of the aqueous, alcoholic, acetone, ethyl acetate, and chloroform extracts of fresh seeds of F. vulgare against A. niger.Natural agents displaying innovative chemistry and mode f action are always momentum as an alternative antimicrobial agent particularly on account of increasing resistance in the pathogen.F. vulgare may be a potential source for new antifungal agents against Aspergillus spp.OBJECTIVES: The purpose of this research work was to evaluate the in vitro antifungal potency of chloroform seed extract of F. vulgare against seed-borne Aspergilli.
th diverse pharmacological (Rather et al., 2016).Essential oil and seeds extract of F. vulgare have been screened for their anti-fungal potential and found to be equally good with broad-spectrum antibiotics against Aspergillus niger, A. flavus, Fusarium graminearum and F. minliforme (Singh et al., 2006;Upadhyay, 2015).Likewise, aqueous and alcoholic seed extracts of F. vulgare inhibited the growth of Alternaria alternata, Mucor rouxii, and A. flavus (Thakur et al., 2013), and in some cases, antifungal activity of aqueous seed extract was greater compared to reference fungicidal agent ((Taie et al., 2013).Khan (2017) observed the substantial potential of methanolic and aqueous seed extract of F. vulgare against different species of Candida.Aamir et al. (2018) results indicated the significant antifungal potential of the aqueous, alcoholic, acetone, ethyl acetate, and chloroform extracts of fresh seeds of F. vulgare against A. niger.Natural agents displaying innovative chemistry and mode f action are always momentum as an alternative antimicrobial agent particularly on account of increasing resistance in the pathogen.F. vulgare may be a potential source for new antifungal agents against Aspergillus spp.OBJECTIVES: The purpose of this research work was to evaluate the in vitro antifungal potency of chloroform seed extract of F. vulgare against seed-borne Aspergilli.


MATERIALS AND METHODS: Fungal cultures: Four different

Aspergillus species [A.flavus (FCBP0051), A. welwitschia (FCBP0087), A. oryzae (FCBP1213) and A. minisclerotigenes (FCBP1353)] were procured from the First Fungal Culture Bank of Pakistan (FCBP), University of the Punjab, Lahore, Pakistan and maintained on Malt Extract Agar; MEA (Dhingra and Sinclair, 2017).Biological control assays: Growth assays were conducted in 250 mL conical flasks each containing 50 mL of malt extract broth (pH 6.5) amended with the respective concentration of chloroform extract of F. vulgare seeds.About 50 g of dry powdered F. vulgare seeds were soaked with 500 mL of methanol in airtight glass jars.After 7 days, undissolved material was removed by filtration through double-fold muslin cloth followed by Whatman filter paper.The crude methanolic soluble fraction of seeds was obtained by evaporation of filtrate in a hot air oven at 45 °C.The paste obtained was partitioned using chloroform in a separating funnel.The solvent was evaporated on a rotary evaporator and the weight of the solvent-soluble fraction was measured and then stored in preweighing sterilized glass bottles.Experimental setup for fungal growth assays: About 2 g crude chloroform extracts of seed were dissolved in 10 mL dimethyl sulfoxide (DMSO) to make a sticky solution.About 4 concentrations (w/v), 0, 0.02, 0.04, 0.08, and 0.12 % were prepared by dissoloving 0, 10, 20, 40, and 60 mg of stock solution per 50 mL of malt extract medium.Each experiment was replicated twice.Each treatment was inoculated with 6 mm agar plugs from actively growing 7 days old pure fungal culture and incubated at 25±2 °C for 10 days.Fungal dry weight was determined after overnight drying the filtered mycelia in a hot air oven at 40 °C.The effect of different concentrations of chloroform fractions of fennel seeds was determined in terms of percent increase/decrease in average fungal dry weight.Effect of pla

MATERIALS AND METHODS: Fungal cultures: Four different
Aspergillus species [A.flavus (FCBP0051), A. welwitschia (FCBP0087), A. oryzae (FCBP1213) and A. minisclerotigenes (FCBP1353)] were procured from the First Fungal Culture Bank of Pakistan (FCBP), University of the Punjab, Lahore, Pakistan and maintained on Malt Extract Agar; MEA (Dhingra and Sinclair, 2017).Biological control assays: Growth assays were conducted in 250 mL conical flasks each containing 50 mL of malt extract broth (pH 6.5) amended with the respective concentration of chloroform extract of F. vulgare seeds.About 50 g of dry powdered F. vulgare seeds were soaked with 500 mL of methanol in airtight glass jars.After 7 days, undissolved material was removed by filtration through double-fold muslin cloth followed by Whatman filter paper.The crude methanolic soluble fraction of seeds was obtained by evaporation of filtrate in a hot air oven at 45 °C.The paste obtained was partitioned using chloroform in a separating funnel.The solvent was evaporated on a rotary evaporator and the weight of the solvent-soluble fraction was measured and then stored in preweighing sterilized glass bottles.Experimental setup for fungal growth assays: About 2 g crude chloroform extracts of seed were dissolved in 10 mL dimethyl sulfoxide (DMSO) to make a sticky solution.About 4 concentrations (w/v), 0, 0.02, 0.04, 0.08, and 0.12 % were prepared by dissoloving 0, 10, 20, 40, and 60 mg of stock solution per 50 mL of malt extract medium.Each experiment was replicated twice.Each treatment was inoculated with 6 mm agar plugs from actively growing 7 days old pure fungal culture and incubated at 25±2 °C for 10 days.Fungal dry weight was determined after overnight drying the filtered mycelia in a hot air oven at 40 °C.The effect of different concentrations of chloroform fractions of fennel seeds was determined in terms of percent increase/decrease in average fungal dry weight.Effect of plant extracts on fungal physiology: Changes in total protein content and rate of peroxidase and catalase enzymes activities were determined and compared in extract-treated and non-treated strains.Fungal cells were grown and collected as described for growth assays.To remove extra moisture, filtered mycelia were blotted by pressing in folds of filter papers and used for physiological assays.Total cell protein content (mg g -1 fresh weight, FW) was calculated using the method of Lowry (1951).Peroxidase activity (mmol/min/mg protein) was determined by following the protocol of Kunwar and Khan (1982).Finally, catalytic activity (mmole H₂O₂ utilized/min/mg protein) in treated or non-treated fungal cells was assessed according to the method described by Glick (2009).

extracts on fungal ph
siology: Changes in total protein content and rate of peroxidase and catalase enzymes activities were determi ed and compared in extract-treated and non-treated strains.Fungal cells were grown and collected as described for growth assays.To remove extra moisture, filtered mycelia were blotted by pressing in folds of filter papers and used for physiological assays.Total cell protein content (mg g -1 fresh weight, FW) was calculated using the method of Lowry (1951).Peroxidase activity (mmol/min/mg protein) was determined by following the protocol of Kunwar and Khan (1982).Finally, catalytic activity (mmole H₂O₂ utilized/min/mg protein) in treated or non-treated fungal cells was assessed according to the method described by Glick (2009).

RESULTS AND DISCUSSION: Effect on growth and antioxidant properties of A. oryzae: Seeds of F. vulgare have been used as a traditional medicine to cure m RESULTS AND DISCUSSION: Effect on growth and antioxidant properties of A. oryzae: Seeds of F. vulgare have been used as a traditional medicine to cure many microbial diseases (Dua et al., 2013).The production of fungal biomass of A. oryzae was inhibited with the increase in the concentration of chloroform soluble compounds of F. vulgare seeds (figure 1A-E).In the control treatment, the dry weight of A. oryzae was 4.39 g which increased to 6.33 g in 10 mg treatment.However, under the effect of 20 and 40 mg extract concentration, an insignificant difference was observed in the dried fungal matt of A. oryzae markedly declined to 3.17 g when the fungal strain was grown in the medium amended with 60 mg extract concentration (figure 1A).The higher concentration of extract inhibit the fungal biomass production probably due to the increase in quantity of bioactive metabolites (e.g.oxygenated monoterpenes) in the seed extract (Badgujar et al., 2014).The antifungal compounds might cause denaturation of the enzymes responsible for growth and reproduction in the fungi.Proteins play a major role in fungal growth metabolism.A concentration-dependent increase in A. oryzae total cell protein content was recorded (figure 1B) in the fungal cells is directly correlated with nitrogen metabolism (Akhtar et al., 2022).Proteolysis or denaturation is usually the main reason for lower cellular protein content (Lubaina and Murugan, 2013).Activities of antioxidant enzymes can be used as indicators of stress physiology in cells.Enhanced activity of antioxidant enzymes activity helps the organisms to grow under stress and is a good sign of stress tolerance which in the present case is the chloroform extract of F. vulgare seeds.Catalytic activity in A. oryzae cells rose with the addition of F. vulgare seed extract.In the control treatment, the rate of catalytic activity was 1.94 mmol/min/mg protein that enhanced to 2.57, 3.67, 3.70, and 3.74 mmol/min/mg protein due to the extract concentrations 10, 20, 40, and 60 mg (figure 1C).However, A. oryzae peroxidase enzyme activity declined to 0.07 mmol/min/mg protein from 0.20 mmol/min/mg protein (control treatment) as the extract concentration increased to 60 mg (figure 1D).

y microbial diseases (Dua et al., 2013).The production of fungal biomass of A. ory
ae was inhibited with the increase in the concentration of chloroform soluble compounds of F. vulgare seeds (figure 1A-E).In the control treatment, the dry weight of A. oryzae was 4.39 g which increased to 6.33 g in 10 mg treatment.However, under the effect of 20 and 40 mg extract concentration, an insignificant difference was observed in the dried fungal matt of A. oryzae markedly declined to 3.17 g when the fungal strain was grown in the medium amended with 60 mg extract concentration (figure 1A).The higher concentration of extract inhibit the fungal biomass production probably due to the increase in quantity of bioactive metabolites (e.g.oxygenated monoterpenes) in the seed extract (Badgujar et al., 2014).The antifungal compounds might cause denaturation of the enzymes responsible for growth and reproduction in the fungi.Proteins play a major role in fungal growth metabolism.A concentration-dependent increase in A. oryzae total cell protein content was recorded (figure 1B) in the fungal cells is directly correlated with nitrogen metabolism (Akhtar et al., 2022).Proteolysis or denaturation is usually the main reason for lower cellular protein content (Lubaina and Murugan, 2013).Activities of antioxidant enzymes can be used as indicators of stress physiology in cells.Enhanced activity of antioxidant enzymes activity helps the organisms to grow under stress and is a good sign of stress tolerance which in the present case is the chloroform extract of F. vulgare seeds.Catalytic activity in A. oryzae cells rose with the addition of F. vulgare seed extract.In the control treatment, the rate of catalytic activity was 1.94 mmol/min/mg protein that enhanced to 2.57, 3.67, 3.70, and 3.74 mmol/min/mg protein due to the extract concentrations 10, 20, 40, and 60 mg (figure 1C).However, A. oryzae peroxidase enzyme activity declined to 0.07 mmol/min/mg protein from 0.20 mmol/min/mg protein (control treatment) as the extract concentration increased to 60 mg (figure 1D).


Effect on growth and antioxidant properties of A. flavus:

Increasing concentration of F. vulgare seed extract caused significant growth inhibition of A. flavus (figure2A-E).The antifungal potential of Cymbopogon nervatus has been reported for its inhibitory effect against Aspergillus flavus for aflatoxins production.Similarly, durin

Effect on growth and antioxidant properties of A. flavus:
Increasing concentration of F. vulgare seed extract caused significant growth inhibition of A. flavus (figure2A-E).The antifungal potential of Cymbopogon nervatus has been reported for its inhibitory effect against Aspergillus flavus for aflatoxins production.Similarly, during the present study, antifungal potential of F. vulgare has been proven very effective against A. flavus biomass production.The weight of the mycelium fungal cells grown in a non-treated growth medium was 6.16 g which was reduced to 0.16 g when cells were grown in 60 mg extract  The transcriptome profile of any organism under stress is needed to adjust continuously.That may result in the overexpression of some genes (Gupta et al., 2013).A similar pattern of protein content was observed in cells of A. flavus that improved with a rising concentration of extract (figure 2B).

the present study, antifungal potential of F. vulgare has
een proven very effective against A. flavus biomass production.The weight of the mycelium fungal cells grown in a non-treated growth medium was 6.16 g which was reduced to 0.16 g when cells were grown in 60 mg extract  The transcriptome profile of any organism under stress is needed to adjust continuously.That may result in the overexpression of some genes (Gupta et al., 2013).A similar pattern of protein content was observed in cells of A. flavus that improved with a rising concentration of extract (figure 2B).

Catalase and peroxidase activities showed decreasing pattern with the addition of F. vulgare chloroform extract (figure 2C &  D).Cellular response to a particular stress is highly hooked on the tolerance or susceptibility level which is generally under the effect of genes or proteins expression (Nouri et al., 2015) Effect on growth and antioxidant properties of A. minisclerotigenes: Results of present assays showed that chloroform extract of F. vulgare seeds significantly arrested the growth of A. minisclerotigenes fungus even at the lowest concentration (figure 3A & E).Almost complete cessation in the growth of A. minisclerotigenes was achieved which might be due to the impact of antifungal compound in extract on mitosis and cell division (McCarroll et al., 2002).

In contrast to A. oryzae and A. flavus, the total cell protein content of A. minisclerotigenes which is an important parameter to study the changes in cellular metabolism (Shoaib et al., 2022) decreased with the increase in the concentration of seed extract (figure 3B).

Catalase and peroxidase activities of fungal cells increased with the in Catalase and peroxidase activities showed decreasing pattern with the addition of F. vulgare chloroform extract (figure 2C &  D).Cellular response to a particular stress is highly hooked on the tolerance or susceptibility level which is generally under the effect of genes or proteins expression (Nouri et al., 2015) Effect on growth and antioxidant properties of A. minisclerotigenes: Results of present assays showed that chloroform extract of F. vulgare seeds significantly arrested the growth of A. minisclerotigenes fungus even at the lowest concentration (figure 3A & E).Almost complete cessation in the growth of A. minisclerotigenes was achieved which might be due to the impact of antifungal compound in extract on mitosis and cell division (McCarroll et al., 2002).
In contrast to A. oryzae and A. flavus, the total cell protein content of A. minisclerotigenes which is an important parameter to study the changes in cellular metabolism (Shoaib et al., 2022) decreased with the increase in the concentration of seed extract (figure 3B).
Catalase and peroxidase activities of fungal cells increased with the increasing concentration of F. vulgare extract (figure 3C & D).Undergrowth stress conditions, reactive oxygen species (ROS) are produced which are generated as the result of oxygen metabolism.Catalase and peroxidase are antioxidant enzymes that help the cell to maintain the hydrogen peroxide to a harmless level and save the cells from damage by these ROS (Mustapha et al., 2009).
reasing concentration of F. vulgare extract (figure 3C & D).Undergrowth stress conditions, reactive oxygen species (ROS) are produced which are generated as the result of oxygen metabolism.Catalase and peroxidase are antioxidant enzymes that help the cell to maintain the hydrogen peroxide to a harmless level and save the cells from damage by these ROS (Mustapha et al., 2009).


Effect on growth and antioxidant properties of A. welwitschia:

An even decline in A. welwitschia biomass production was seen with the addition of F. vulgare seed extract in the growth medium.

In the highest concentration, approximately 75% reduction was observed (figure 4A & E).These results suggested that this extract ca

Effect on growth and antioxidant properties of A. welwitschia:
An even decline in A. welwitschia biomass production was seen with the addition of F. vulgare seed extract in the growth medium.
In the highest concentration, approximately 75% reduction was observed (figure 4A & E).These results suggested that this extract can be an alternative to synthetic fungicides for the treatment of A. welwitschia infections.Results depicted an increase in A. welwitschia total cell protein content with the rising concentration of seed extract and at the highest concentration of plant extract cell proteins became almost double (figure 4B).The increasing concentration of chloroform extract exhibited an inhibitory effect on both catalase and peroxidase enzymatic rates as compared to the control (figure 4C).Peroxidase enzyme lost its activity from 0.56 to 0.14 mmol/min/mg protein at 60 mg concentrations (figure 4D).

be an alternative to synthetic fungicides for the treatment of
. welwitschia infections.Results depicted an increase in A. welwitschia total cell protein content with the rising concentration f seed extract and at the highest concentration of plant extract cell proteins became almost double (figure 4B).The increasing concentration of chloroform extract exhibited an inhibitory effect on both catalase and peroxidase enzymatic rates as compared to the control (figure 4C).Peroxidase enzyme lost its activity from 0.56 to 0.14 mmol/min/mg protein at 60 mg concentrations (figure 4D).

The decline in antioxidant enzyme rate is an indication that due to the F. vulgare extract, the transcript level of these enzyme coding genes is suppressed.It has been widely accepted that response of each organism to a particular stress is different for other organisms (Caverzan et al., 2012).

Figure 1 :
1
Figure1: Effect of different concentration of chloroform soluble fraction (0.02-0.08%) of Foeniculum vulgare seed on growth, total protein content, and antioxidant properties of Aspergillus oryzae.(About 10, 20, 40, and 60 mg of stock solution of chloroform fraction per 50 mL of growth medium is equivalent to 0.02, 0.06 and 0.08% concentration, respectively).monoterpenes) in the seed extract(Badgujar et al., 2014).The antifungal compounds might cause denaturation of the enzymes responsible for growth and reproduction in the fungi.Proteins play a major role The decline in antioxidant enzyme rate is an indication that due to the F. vulgare extract, the transcript level of these enzyme coding genes is suppressed.It has been widely accepted that response of each organism to a particular stress is different for other organisms (Caverzan et al., 2012).

Figure 1 :
Figure1: Effect of different concentration of chloroform soluble fraction (0.02-0.08%) of Foeniculum vulgare seed on growth, total protein content, and antioxidant properties of Aspergillus oryzae.(About 10, 20, 40, and 60 mg of stock solution of chloroform fraction per 50 mL of growth medium is equivalent to 0.02, 0.06 and 0.08% concentration, respectively).monoterpenes) in the seed extract(Badgujar et al., 2014).The antifungal compounds might cause denaturation of the enzymes responsible for growth and reproduction in the fungi.Proteins play a major role in fungal growth metabolism.A concentration-dependent increase in A. oryzae total cell protein content was recorded (figure1B) in the fungal cells is directly correlated with nitrogen metabolism(Akhtar et al., 2022).Proteolysis or denaturation is usually the main reason for lower cellular protein content(Lubaina and Murugan, 2013).Activities of antioxidant enzymes can be used as indicators of stress physiology in cells.Enhanced activity of antioxidant enzymes activity helps the organisms to grow under stress and is a good sign of stress tolerance which in the present case is the chloroform extract of F. vulgare seeds.Catalytic activity in A. oryzae cells rose with the addition of F. vulgare seed extract.In the control treatment, the rate of catalytic activity was 1.94 mmol/min/mg protein that enhanced to 2.57, 3.67, 3.70, and 3.74 mmol/min/mg protein due to the extract concentrations 10, 20, 40, and 60 mg (figure1C).However, A. oryzae peroxidase enzyme activity declined to 0.07 mmol/min/mg protein from 0.20 mmol/min/mg protein (control treatment) as the extract concentration increased to 60 mg (figure1D).Effect on growth and antioxidant properties of A. flavus:Increasing concentration of F. vulgare seed extract caused significant growth inhibition of A. flavus (figure2A-E).The antifungal potential of Cymbopogon nervatus has been reported for its inhibitory effect against Aspergillus flavus for aflatoxins production.Similarly, during the present study, antifungal potential of F. vulgare has been proven very effective against A. flavus biomass production.The weight of the mycelium fungal cells grown in a non-treated growth medium was 6.16 g which was reduced to 0.16 g when cells were grown in 60 mg extract

n fungal grow
h metabolism.A concentration-dependent increase in A. oryzae total cell protein content was recorded (figure1B) in the fungal cells is directly correlated with nitrogen metabolism(Akhtar et al., 2022).Proteolysis or denaturati

y, during the present study, antifungal po
Figure 3.14: Effect of F. vulgare on growth, total protein content and activities of antioxidant enzymes of A. oryzae.Treatments are chloroform soluble fraction of F. vulgare ground seeds.

Figure 2 :
Figure 2: Effect of different concentration of chloroform soluble fraction (0.02-0.08%) of Foeniculum vulgare seed on growth, total protein content, and antioxidant properties of Aspergillus flavus.(About 10, 20, 40, and 60 mg of stock solution of chloroform fraction per 50 mL of growth medium is equivalent to 0.02, 0.06 and 0.08% concentration, respectively).The transcriptome profile of any organism under stress is needed to adjust continuously.That may result in the overexpression of some genes(Gupta et al., 2013).A similar pattern of protein content was observed in cells of A. flavus that improved with a rising concentration of extract (figure2B).Catalase and peroxidase activities showed decreasing pattern with the addition of F. vulgare chloroform extract (figure2C & D).Cellular response to a particular stress is highly hooked on the tolerance or susceptibility level which is generally under the effect of genes or proteins expression(Nouri et al., 2015) Effect on growth and antioxidant properties of A. minisclerotigenes: Results of present assays showed that chloroform extract of F. vulgare seeds significantly arrested the growth of A. minisclerotigenes fungus even at the lowest concentration (figure3A & E).Almost complete cessation in the growth of A. minisclerotigenes was achieved which might be due to the impact of antifungal compound in extract on mitosis and cell division(McCarroll et al., 2002).In contrast to A. oryzae and A. flavus, the total cell protein content of A. minisclerotigenes which is an important parameter to study the changes in cellular metabolism(Shoaib et al., 2022) decreased with the increase in the concentration of seed extract (figure3B).

Figure 3 :
Figure 3: Effect of different concentration of chloroform soluble fraction (0.02-0.08%) of Foeniculum vulgare seed on growth, total protein content, and antioxidant properties of Aspergillus minisclerotigenes.(About 10, 20, 40, and 60 mg of stock solution of chloroform fraction per 50 mL of growth medium is equivalent to 0.02, 0.06 and 0.08% concentration, respectively).Catalase and peroxidase are antioxidant enzymes that help the cell to maintain the hydrogen peroxide to a harmless level and save the cells from damage by these ROS(Mustapha et al., 2009).Effect on growth and antioxidant properties of A. welwitschia:An even decline in A. welwitschia biomass production was seen with the addition of F. vulgare seed extract in the growth medium.In the highest concentration, approximately 75% reduction was observed (figure4A & E).These results suggested that this extract can be an alternative to synthetic fungicides for the treatment of A. welwitschia infections.Results depicted an increase in A. welwitschia total cell protein content with the rising concentration of seed extract and at the highest concentration of plant extract cell proteins became almost double (figure4B).The increasing concentration of chloroform extract exhibited an inhibitory effect on both catalase and peroxidase enzymatic rates as compared to the control (figure4C).Peroxidase enzyme lost its activity from 0.56 to 0.14 mmol/min/mg protein at 60 mg concentrations (figure4D).The decline in antioxidant enzyme rate is an indication that due to the F. vulgare extract, the transcript level of these enzyme coding genes is suppressed.It has been widely accepted that response of each organism to a particular stress is different for other organisms(Caverzan et al., 2012).

Figure 4 :
Figure 3.6: Effect of F.vulgare on growth, total protein content and activities of antioxidant enzymes of A. flavus.Treatments are chloroform soluble fraction of F. vurlgare ground seeds.