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Byline: S. Khan, T. Iqbal, N. Ahmed and A. Jamil


Plants are one of the natural sources of bioactive molecules with potential abilities against different stress conditions. During pathogenic exposure, an array of biologically active molecules like reactive oxygen intermediates, phytoalexins and defense peptides are synthesized as measure of plant resistance to combat the intensifying conditions. Such molecules play a key role in advancement in potential therapeutics development. Psoralea corylifolia (P. corylifolia), a medicinal herb was evaluated under fungal stress and normal conditions for its inherent bioactive potential. Under fungal stress plant protein/peptide extracts showed greater antioxidant potential evaluated by DPPH radical scavenging activity assay. Mutagenicity of the extracts showed that the plant was safe to use. Hemolytic activity assay exhibited increased percentage in induced seedling extracts that instigates possible applications of extracts against cancer.

Keywords: Bioactive molecules, pathogenic, P. corylifolia, bioactive potential, proteins/peptides.


Recently, health concerns and toxicology of synthetic antioxidants have intensified the study for edible plants exhibiting effective, natural and non-toxic antioxidant compounds (Iqbal et al. (2012). Antioxidants act as repository of anti-inflammatory, antifungal, antibacterial and anti-carcinogenic (Servili et al. (2014). Reactive oxygen species (ROS), being one of the most rapid defense reactions induces production of antioxidant compounds. Consumption of these antioxidants from plants appears to be a healing realm against oxidative burst induced degenerative and pathological diseases like ageing and cancer (Pontiki et al. 2014; Tadhani et al. 2007).

Plants rely on proteomic pliability and bioactive molecules to amend themselves during pathogenic exposure. In response to microbial stress, crucial activities like induction and expression of genome-wide defensin genes, production of antioxidants and other bioactive molecules take place (Falak and Jamil, 2013). Pursuit for potential therapeutic agents especially under stress conditions is all muse on evaluating medicinal plants. Also mutagenicity and toxicity analysis at cellular level is necessary to develop safe measures of phyto- medicines.

In view of the above facts Psoralea corylifolia (leguminosae, Babchi) selected for evaluation of possible antioxidant, mutagenic and cytotoxic potential under biotic stress. Numerous studies report coumarins and flavonoids to be the major bioactive constituents of fructus psoralea (Chopra et al. (2013). Psoralea corylifolia has wide applications in ayurvedic and traditional Chinese medicine due to its antioxidant, antitumor, antibacterial, antifungal, immunomodulatory and anti-inflammatory activity (Gidwani et al. (2011); Qiao et al. (2006). Strong antioxidant activity in P. corylifolia fruit extract is reported in ABTS (2, 2V- azinobis[3-ethylbenzothiazoline-6-sulfonate] assay (Tang et al.(2004). Psoralen, isopsoralen, bakuchiol, corylifolin, corylin and especially psoralidin from powder, ether and chloroform extract of P. corylifolia showed strong antioxidant potential in comparison to butylated hydroxyl toluene and a-tocopherol (Jiangning et al. (2005).

In addition, this plant species possesses a wide range of biological activities like cytotoxicity, antimutagenic activity and bone calcification (Sah et al. (2006). Study on insecticidal and genotoxic activity of the plant is also reported while no literature was found on mutagenic and hemolytic activity of P. corylifolia.

In this study, fungal stress enhanced the antioxidant and hemolytic activity of Psoralea corylifolia seedlings while no mutagenic activity was observed in normal and fungal stress conditions.


Plant material: Seeds of Psoralea corylifolia were collected from local market in Faisalabad and authenticated from Department of Botany, University of Agriculture, Faisalabad, Pakistan. The seeds were sterilized and germinated in Petri plates (Falak and Jamil, 2013).

Crude protein extraction from Psoralea corylifolia seeds and seedlings: The seeds and seedlings of P. corylifolia were ground to a fine powder in pestle and mortar under liquid nitrogen and weighed upto 5 g each in sterilized centrifuge tube. The samples were homogenized in15 l of sodium phosphate buffer and 150 l of 1 m PMSF (phenylmethylsulfonyl fluoride) on polytrone for 15 minutes with 30 second intervals. The samples were centrifuged for 20 minutes at 10,000 rpm and 4 C. Supernatant having crude extract was collected and stored at -20 C for further study (Falak and Jamil, 2013).

Fungal induction: Fusarium solani was used to induce stress in P. corylifolia seedlings. Inoculum of Fusarium solani was prepared in Saboraud's medium (Oxoid) as described by Singariya et al. (2012) in 100 mL of distilled water and pH 5.7 was maintained. The inoculum was autoclaved for 15 minutes at 121C. On cooling it was inoculated with F. solani spores and incubated at 28C at 120 rpm for three days. Psoralea corylifolia seedlings were induced with 1 x 105 spores/ mL of the fungal culture and harvested along with their control samples 8 h post inoculation. The samples were preserved in liquid nitrogen and stored at -80C freezer till further use (Falak and Jamil, 2013).

Protein isolation: The fungus-induced seedlings were ground to a fine powder in liquid nitrogen without thawing and protein extraction was performed by following the TCA/acetone precipitation/phenol method described by Rastegari et al. (2011). These extracts were then further used for evaluation of antioxidant, hemolytic and mutagenic potential.

Antioxidant activity assay: DPPH % RSA method was followed after Noor et al. (2014). The protein samples (crude and induced protein extracts) were mixed with DPPH solution in 2:1 proportion followed by incubation for 15 min at room temperature. The absorbance was noted at 517 nm against ascorbic acid as standard. Each experiment was done in triplicate.

Hemolytic activity assay: Hemolytic activity of P. corylifolia extracts was conducted by following the method used by Shahid et al. (2013) and Zuber et al. (2014). After consent and counseling with volunteers 3 mL of freshly heparinized human blood sample was collected and centrifuged at 1000xg for 5 min. Plasma was discarded and 5 mL of autoclaved and chilled (4C) phosphate-buffered saline (PBS) with pH 7.4 was used thrice for washing cells. Each assay was performed with maintained erythrocytes concentration of 108 cells per mL.100L of each extract was mixed with 108cells/mL human blood cells separately following incubation for 35 min at 37 C and inverted after 10 min. The samples were immediately transferred on ice after incubation for 5 min and centrifuged at 1000xg for 5 min. 100 L of supernatant was collected from each tube and 10 times diluted with chilled PBS. PBS was taken as negative control whileTriton X-100 (0.1% v/v) was taken as positive control and same procedure was followed.

The absorbance was taken at 576 nm using Quant (Bioteck, USA). The % hemolysis for each sample was calculated.

Mutagenic activity test: Mutagenic activity was evaluated according to the procedure described by Razak et al. (2007). Mutagenic activity of P. corylifolia extracts was evaluated by the Muta-Chromplate kit (Environmental Bio-detection Products Incorporation (EBPI, Ontario, Canada)) according to manufacturer's protocol. This test kit works on the principle of Ames bacterial reverse-mutation test (Ames et al., 1975) performed in liquid culture (fluctuation test). Two mutant bacterial strains S. Typhimurium TA98 and S. typhimuriumTA100 were maintained on nutrient agar at 3 1C. The inoculum was prepared by inoculating bacterial strains into nutrient broth and incubated them for 18 - 24 h at 37 C.

Mutagenic activity assay: Plant extract, reagent mixture, deionized water, S9 activation mixture and standard mutagen were mixed in different bottles as shown in Table 1 and overnight culture of S. typhimurium test strains were used for inoculation. Each inoculated contents from bottles was added into 96-well microtitration plate and incubated for 4 days at 37 C.

Interpretation of results and statistical analysis: Positive results are shown by yellow color wells while negative results are indicated by purple color wells. Background plate was prepared for each test strain of salmonella to compare its natural rate of reverse mutation (fig 3). The extract with significantly higher number of yellow wells in test plate in contrast to yellow wells in the 'background' plate was considered mutagenic as shown in fig. 3. Positive controls were also prepared for comparison of results.


Potential microbial resistance and fewer side effects of herbal extracts have led to focus on novel therapeutic agents from plants. Advancement in scientific techniques have revealed the complex chemical composition of plants and revaluated the phytomedicine industrial applications. Psoralea corylifolia being part of Chinese pharmacopoeia and ayurvedic medicine has been used to treat different diseases and skin disorders (Chopra et al. (2013). However, to the best of our knowledge no studies to date have been conducted to compare protein extracts in aqueous phosphate buffer under fungal stress.

In our study, the bioactive potential of crude protein extracts of control and Fusarium solani induced seeds and seedlings of Psoralea corylifolia were evaluated.

Antioxidant activity: Antioxidant activity is the ability to scavenge free radicals in organisms. Isoflavones from plants are reported to possess antioxidant activity with protective effect against oxidative damage (Devi et al. (2009).

Antioxidant potential of P. corylifolia was studied in crude extracts of control and Fusarium solani induced seedling extracts. All experimental samples showed significant antioxidant activity (Figure 1). Seed crude extract possessed more activity than seedling crude while 8 h induced extract showed greater activity than all protein extracts. Seed and seedling crude extracts showed less antioxidant activity than 8 h induced extract as compared to ascorbic acid (positive control). Maximum antioxidant activity of 62% was possessed by 8 h induced protein extract.

Many phytochemicals including psoralen, isopsoralen, psoralidin, corylifol and bavachinin were reported to possess antioxidant activity form P. corylifolia. Bhawya and Anilakumar (2011) studied different solvent extracts of P. corylifolia and reported to possess strong % DPPH radical scavenging activity. Water extract showed 40.42% which is in agreement to our results while maximum activity was shown by ethanol extract 91.0%. Methanolic, ethyl acetate, chloroform, hexane and acetone extracts showed 76.42%, 61.27%, 46.24%, 65.93% and 31.05%, respectively. Xiao et al. (2010) isolated antioxidant components from P. corylifolia seeds by using a combination of thin layer chromatography (TLC) and high-speed counter-current chromatography (HSCCC). Separation by two phases HSSC produced 3.19 mg psoralidin, 5.91 mg psoralen, 6.26 mg isopsoralen, 2.43 mg bavachinin and 0.92 mg corylifol A with maximum purity.

Also aqueous and solvent extracts of P. corylifolia seeds were also reported to possess superoxide anion quenching. Contrary to our results, maximum superoxide quenching activity (89%) was shown by alcohol and water (1:1) extract (AWEP) (Kiran and Raveesha (2010). Jiangning et al. (2005) also studied antioxidant activity of powder and extracts of P. corylifolia. Bakuchiol, corylin and corylifolin were found responsible for strong antioxidant activity and psoralidin showed stronger antioxidant potential than BHT, while psoralen and isopsoralen possessed no antioxidant activity at 0.02% and 0.04%.

It is concluded from the results in Figure 1 that 8 h induced protein extract of P. corylifolia showed significant antioxidant activity which might be due to induction and synthesis of some proteins for defense purpose in addition to antioxidant compounds like bakuchiol, corylin, corylifolin and psoralidin. Therefore, fungal stress enhanced antioxidant activity of the seedlings as compared to other plant extracts (Figure. 1) which assures efficient use of plant in the treatment of oxidative stress and related diseases.

Hemolytic activity: Hemolytic activity of seeds and seedling crude extracts were analyzed against human erythrocytes. The results showed that hemolytic activity of seed crude and seedling crude extracts was non- significant (Figure 2). In comparison, seed crude extract showed greater activity (8.51%) than the seedling crude extract (5.69%). However, according to results, 8 h induced protein extracts showed greater hemolytic activity of 9.33% among all extracts.

Radiation-induced hemolysis of human erythrocytes by IBG-RA-26 extract of Psoralea corylifolia was studied with efficacy in 25-1000 g/mL concentration range. Significant protection against radiation was observed in 25-50 g/mL range (Arora et al., 2011). Other plants like Achyranthes aspera are reported to possess very low haemolytic activity to human erythrocytes (1.3%) (Priya et al., 2010). Moderate hemolytic activity was found in aqueous extract of Lantana camara human erythrocytes with 7.93% at 1000 g/mL concentration (Kalita et al., 2011).

From our results, significant increase in hemolytic activity of 8 h induced protein extract as compared to 8 h control might be due to increased synthesis of some protein against pathogens possessing hemolytic property. Less hemolytic potential of seed and seedling crude extract as shown in figure 2 makes it safe for optimization of the extracts in pharmacological uses. Overall, all extracts possess less to moderate hemolytic activity which supports the use of the herb in safe measures.

Mutagenic activity: Mutagenic activity investigation of seed and seedling crude extracts was performed against two test strains of Salmonella TA98 and TA100 for evaluation of their potential ability to cause cancer. All test samples were found to be non-mutagenic as shown in Table 2.

Mutagenic activity was evaluated by Fluctuation method entirely performed in liquid culture is based on Ames test used for detection of carcinogenicity and gene mutation level (Lewis et al., 1993). The mechanism involved in this test requires mutant strains of Salmonella typhimurium which require histidine for growth. The chemical substances like carcinogens have ability to revert to state in which bacterium does not require histidine for growth. The frequency of mutation was measured by pH indicator that cause color changes from purple to yellow. The changes in color of sample are compared to natural background rate activity. This is first time to study the mutagenic potential of P. corylifolia with and without stress (fungal).

To the best of our knowledge no studies have been found regarding mutagenic ability of Psoralea corylifolia while antimutagenic activity is reported in the respective plant. Quinone reductase (QR) enzyme induced by chemical substances possesses protective effect against mutagens and carcinogens. Lee et al. (2009) studied the effect of seed extracts of P. croylifolia on induced QR enzyme in Hepa 1c1c7 hepatoma cells. Methanolic extract of P. croylifolia seeds showed 1.5 fold (1.5 FIC) increased QR induction at 1.2 g/mL concentrations while psoralidin isolated further from active fraction showed 1.5 FIC at 0.5 g/mL concentrations. Also essential oil from plant is reported to cause genotoxic effects of 6.713% and 8.864% in adult Cx. quinquefasciatus at 0.034 mg/cm2 and 0.069 mg/cm2 revealed by comet assay method (Dua et al., 2013).

From mutagenic screening of P. corylifolia extracts during the current study, it was found that they are non-mutagenic which assures their safety to use in pharmacological formulations.

Table. 1. Fluctuation assay set-up.

###Treatments###Constituents (ml)

###Standards###Plant###Reagent###S9 mix###Deionized###Test

###K2Cr2O2and H2N3###extract###mixture###Water###strain



###Test sample###0.005###2.5###2.0###15.5###0.005

Standard mutagen###0.1###2.5###17.4###0.005

Table. 2. Mutagenic activity of protein extracts of Psoralea corylifolia. Each reading represents mean of three readings. K2Cr2O2 and H2N3 are positive controls.

###Samples###Mutant count for TA98/96###Mutant count for TA100/96###Interpretation

###Seed crude###0###8###Non-Mutagenic

###Seedling crude###9###8###Non-Mutagenic

###8 hpi C###7###5###Non-Mutagenic

###8 hpi###7###7###Non-Mutagenic



###H 2N 3###93###91###Mutagenic

Conclusion: In our study potent biological activities of P. corylifolia were evaluated under normal and stressed conditions. Significant antioxidant activity was shown by all extracts (crude and protein extracts) estimated by % DPPH radical scavenging activity assay, a maximum of 62% was observed in induced seedling extract. While the extracts possessed low to moderate hemolytic activity and no mutagenic activity was observed in all these extracts. Increase in induced extracts activities as compared to crude extracts might be due to synthesis of some defensive proteins/peptides expressed in response to fusarium solani stress could be used in pesticides or fungicide development while non-mutagenic nature of plant assures its safety for human drug development.

Acknowledgements: The research was conducted under a research project No. 280 funded by Pakistan-US Science and Technology Support Program of Higher Education Commission, and indigenous PhD fellowship awarded to the first author.


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Publication:Journal of Animal and Plant Sciences
Date:Oct 31, 2015

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