21 Dpph Free Radical Scavenging Activity And Phenotypic Difference In Hepatoprotective Plant (silybum Marianum L.) kq2l

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DPPH free radical scavenging activity and phenotypic difference in hepatoprotective plant (Silybum marianum L.) Nisar Ahmad, Hina Fazal, Bilal Haider Abbasi, Shazma Anwar and Abdul Basir Toxicol Ind Health 2013 29: 460 originally published online 23 February 2012 DOI: 10.1177/0748233712436637 The online version of this article can be found at: http://tih.sagepub.com/content/29/5/460

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Article

DPPH free radical scavenging activity and phenotypic difference in hepatoprotective plant (Silybum marianum L.)

Toxicology and Industrial Health 29(5) 460–467 © The Author(s) 2012 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0748233712436637 tih.sagepub.com

Nisar Ahmad1, Hina Fazal2, Bilal Haider Abbasi1, Shazma Anwar3 and Abdul Basir4

Abstract Silybum marianum L. is medicinally important for its active principle component silymarin. Silymarin regenerates damaged hepatic tissues. On the basis of such regenerative properties, the radical scavenging activity (1,1-diphenyl-2-picrylhydrazyl (DPPH)) of different tissues and the phenotypic difference of the hepatoprotective species, S. marianum L. were evaluated. There was less phenotypic difference in purple and white varieties of S. marianum. Assay of the antioxidant potential of different parts of the plant revealed that significantly higher activity (78.2%) was observed in seeds of the purple flowering plant than seeds of white flowering plant (49%) after different time intervals. Young leaves collected from white flowering plant exhibit 64.8% activity, which is higher than the purple flowering plant (55.1%). Significantly, same activity was observed in mature leaves of white (52%) and purple flowering plants (50%). The main stem collected from both the varieties exhibits similar activity from 50 to 52%. A 67.2% activity was recorded for mature roots of white flowering plant followed by roots of the purple variety (65%). The present study revealed that seeds and roots of both the varieties scavenge and detoxify more DPPH free radicals than other plant parts and can be used as a source of natural antioxidants and food additives. Keywords Silybum marianum, DPPH, radical scavenging activity, phenotypic, tissues

Introduction Silybum marianum (L.) (milk thistle) is an important medicinal species in the family Asteraceae. S. marianum L. is a native of Southern Europe to Asia, specially cultivated for its seeds throughout the world. In Pakistan, it occurs as a weed in Punjab, Sindh and Khyber Pakhtunkhwa by the names of Unt katara, Poli and Karaiza, respectively. The medicinal part of the plant is its ripe seed that contains silymarin, consisting of silybin, silidianin and silichrystin. Silibinin, one the active components of silymarin, can regenerate the damaged hepatic tissues (Al-Anati et al., 2009; Jayaraj et al., 2007). Besides hepatoprotection, silibinin and its other components are also efficient as anticancerous against human breast carcinoma, cervical carcinoma, human adenocarcinoma and colon cancer cells (Bhatia et al., 1999; Hogan et al., 2007; Mokhtari,

2008; Sharma et al., 2003). Silymarin a combination of four active components influenced the production of superoxide dismutase, which is a potent antioxidant especially against toxic free radicals in hepatic tissues

1

Department of Biotechnology, Faculty of Biological Sciences, Quaid-i-Azam University, Pakistan 2 Medicinal Botanic Centre (MBC), Pakistan Council of Scientific and Industrial Research (PCSIR) Laboratories Complex, Pakistan 3 Department of Agronomy, Agriculture University Peshawar, Pakistan 4 Department of Agriculture, Abdul Wali Khan University, Ambar Campus, Pakistan Corresponding author: Nisar Ahmad, Department of Biotechnology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan. Email: [email protected]

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induced by alcohol. In addition, silymarin can efficiently reduce the leukotrienes action, which can damage hepatic tissues. (Chevallier, 1996; Nice, 2000; Weiss and Fintelmann, 2000). Recently, silymarin has shown its potential as anticholesterolaemic agent. Silymarin is also known to stimulate the enzyme RNA polymerase in the nucleus of liver cells, which has regenerative effect on liver. This results in enhancing ribosomal protein synthesis, which is helpful in regenerating hepatocytes. During stress conditions or incomplete body metabolism, toxic free radicals are produced. These toxic radicals are the products of oxidation reactions, which further start chain reactions and damage body cells and sometimes cause serious diseases like cancer (Ahmad et al., 2011b). Antioxidants (natural and synthetic) are those compounds that completely stop or reduce the speed of oxidation reactions. Different antioxidants can efficiently detoxify toxic free radicals and stop the chain reactions by undergoing oxidization themselves (Ahmad et al., 2011c). As a result, antioxidants like thiols, ascorbic acid or polyphenols are often used as reducing agents. Secondary metabolites are the main active ingredients of a medicinal plant, one of the groups are phenolics that act as an important antioxidant agent (Khanavi et al., 2009; Weiss and Fintelmann, 2000). These secondary substances are released naturally during different plant growth phases, some of the active agents that are produced against stress conditions are reactive oxygen species, hydroxyl radicals, superoxide anion radicals and nonfree radicals such as hydrogen peroxide (H2O2) and peroxide (Ahmad et al., 2011a; Ramarathnam et al., 1995). Natural antioxidants have become a major and interesting area of scientific research nowadays (Demo et al., 1998; Sanchez-Moreno et al., 1999). Natural antioxidants, especially present in medicinal plants, exhibit antiinflammatory, antimicrobial, antiviral, antiallergic and vasodilatory activities. Antioxidants of plants origin are also used as anticancer, antimutagenic and antiaging agents (Cook and Samman, 1996; Mandal and Gupta, 2001). The main objective of the current study is to evaluate the free radical scavenging activity (1,1-diphenyl-2-picrylhydrazyl (DPPH) activity) of different parts of S. marianum to find new potential sources of natural antioxidants, which were subjected to extraction using methanol as a solvent and to compare their highest antioxidant potential in different parts.

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Figure 1. Phenotypic difference between purple and white flowering Silybum marianum L. Values are means of triplicates. Purple and white colors indicating purple and white flowering in Silybum marianum. Means with common letters are not significantly different at P < 0.05.

Materials and method Plant material Test plants were collected from different areas of Districts Swat and Haripur of Khyber Pakhtunkhwa. Different parts including young, mature leaves, stem cutting, roots and seeds of purple and white varieties of S. marianum were collected and shade dried (28 + 2 C; Figure 12). The plants were authenticated with the help of taxonomic markers and deposited in herbarium (PES) of Medicinal Botanical Centre, PCSIR Laboratories Complex Peshawar, Pakistan.

Preparation of the extract Dried materials of different parts and seeds of S. marianum were ground and sieved to get fine powder from which the extracts were prepared. Methanolic extract of the plant was obtained by taking 10 g of powdered material in a separate container. Fifty milliliter of methanol was added to the powdered material and kept for 1 week with periodic shaking (the soaked material was stirred every 18 h using a sterilized glass rod), filtered, and the filtrate was collected. This procedure was repeated three times with fresh volume of methanol. The filtrates were pooled. The final extracts were ed through Whatman No. 1 filter paper (Whatman Ltd., England). The pooled methanol extracts were concentrated separately by rotary vacuum evaporator at 40 C and evaporated to dryness and stored at 4 C in an airtight bottle (Ahmad et al., 2010b). The extracts obtained

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from each part were dissolved in methanol independently to get stock solutions. The stock solution was prepared by dissolving pure extract of 5 mg in 20 ml of methanol independently.

DPPH scavenging activity in different parts of purple and white flowering varieties of S. marianum was measured using the stable radical (DPPH) in of hydrogen donating or radical scavenging ability. DPPH (1.25 mg) was dissolved in 40 ml (4) of methanol to make a stock solution. Plant extract of 5.0 mg was dissolved in 20 ml of methanol to make a stock solution. The procedure was repeated for each plant tissues. Sample solution of 0.5 ml was added to 1 ml of DPPH solution separately. These solutions were kept at room temperature in the dark for 30 min (incubation period). After an interval of 30 min, the absorbance was measured in a spectrophotometer at 517 nm. Lower absorbance of the reaction mixture indicated higher free radical scavenging activity using the equation (Ahmad et al., 2010a, 2010b): % DPPH scavenging activity ¼ 100 ½1 AE=AD where AE is the absorbance of the sample solution and AD is the absorbance of the DPPH solution with nothing added (blank, without extract).

Statistical analysis For statistical analysis, three replicates were conducted for each activity, and the experiments were repeated twice. The data were subjected to a oneway analysis of variance and Duncan’s Multiple Range Test (Statistix version 8.1, Analytical Software, Florida, USA); p < 0.05 value was regarded as significant.

Results and discussion The significance of natural antioxidants lies in the fact that these vital substances prevent the oxidation chain reactions and protect the body from induced oxidative stress of toxic free radicals. In fact, toxic free radicals attack on nucleic acid substances such as DNA and RNA, leading to mutational events. These toxic radicals also attacked enzymes, proteins and lipids causing degenerative diseases. Natural antioxidants from different tissues of medicinal plants function as freeradical scavengers and chain breakers, complexes of pro-oxidant metal ions and quenchers of singlet-

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Figure 2. Time dependent free radical scavenging activity in young leaves of white flowering Silybum marianum. Values are means of 3 replicates.

oxygen formation. S. marianum L. also contains the flavonoids called silymarin and is famous in curing a whole range of liver and gall bladder disorders (Weiss and Fintelmann, 2000). Similarly, various tissues of Silybum marianum L. exhibit higher antioxidant activities than vitamin C and E. Its extracts possess a significant scavenging activity against DPPH free radical and an inhibitory effect on H2O2. The thrust of the present research work was to evaluate the antioxidant activity in different parts and also to assess and compare the phenotypic difference between purple and white varieties of S. marianum. Percentage free radical scavenging activity in different parts of S. marianum was recorded in a timedependent manner. The phenotypic difference between purple and white flowering varieties of S. marianum is shown in Figure 2. There was no or less significant difference between different parts of both types of plants. The data were recorded in triplicates and the mean values are presented in Figure 1. Antioxidant activity was carried out to determine the antioxidant potential of different tissues and compare it with each other. Seeds of purple flowering variety of S. marianum had shown a significantly higher activity to detoxify DPPH free radicals when compared with other parts collected from wild plants (Figure 3). According to our data, the seeds of purple flowering variety of S. marianum have higher antioxidant activity (78.34%) than the seeds of white flowering variety of S. marianum (49.18%). The data were collected after 10, 20 and 30 min time intermittent to compare the highest antioxidant activity. From the

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Figure 4. Time dependent free radical scavenging activity in seeds of white flowering Silybum marianum. Values are means of 3 replicates.

comparison, after 10-, 20- and 30-min intervals, it was concluded that at 30 min, the antioxidant activity was high. Seeds of S. marianum with purple flower showed 62.67, 74.02 and 78.34% activity after 10, 20 and 30 min, respectively, while white flowering seeds exhibit 37.79, 42.97 and 49.18% activity, respectively (Figures 2 and 4). From the experiment, it was concluded that the plant’s secondary metabolites scavenge more free radicals after 30 min than 10 min, as the time interval and incubation period increase, the plant extract scavenge more free radicals. Tawaha et al. (2007) documented the linear

Figure 5. Time dependent free radical scavenging activity in main stem of white flowering Silybum marianum. Values are means of 3 replicates.

relationship of antioxidant activity of alcoholic extracts of S. marianum with the total phenolics contents. Cai et al. (2004) also reported that the antioxidant activity of 112 Chinese wild herbs is correlated with the total phenolic contents. Present data are in line with the observation of many scientists who documented the relationship of antioxidant activity with total phenolic compounds (Cai et al., 2004; Zheng and Wang, 2001). The young leaves of white flowering variety of S. marianum possess higher antioxidant activity (64.8%) than purple flowering variety of S. marianum (55.25%; Figures 5 and 6). When the the incubation period is increased from 10 to 30 min, the young leaves of white flowering variety of S. marianum show 54.32, 55.61 and 64.8% activity, respectively (Figure 5); while young leaves of purple flowering plant show 50.22, 51.77 and 55.25% antioxidant activity, respectively (Figure 6). Studies conducted on free radical scavenging activities of medicinally important plants revealed that the efficiency of activities varies among different species. The activities are totally dependent on the methodologies that reflect the complexity of the mechanisms involved in total antioxidant capacity (Matkowski and Piotrowska, 2006). The complexity in the mechanisms of antioxidant activities even vary in related plant species. Mature leaves of purple flowering variety of S. marianum showed 45.99, 47.02 and 50.09% activity, respectively, at time intervals of 10, 20 and 30 min (Figure 7); however, the white flowering variety of

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Figure 7. Time dependent free radical scavenging activity in young leaves of purple flowering Silybum marianum. Values are means of 3 replicates.

S. marianum showed 48.70, 49.39 and 52.02% activity, respectively (Figure 4), and there is less significant difference between mature leaves of both purple and white flowering plants. In the overall experiment, the best results were observed in the seeds of purple variety. The antioxidant activity (DPPH) of S. marianum, essential components and different tissues was also reported by Hadaruga and Hadaruga (2009; Anderson et al., 1994; Carini et al., 1992; Chevallier, 1996; Huda-Faujan et al., 2009; Ka¨hko¨nen et al., 1999; Koch and Loffler, 1985; Pietrangelo et al., 1995; Weiss and Fintelmann,

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2000). After seeds of purple variety, the best antioxidant activities were also observed in the roots of both varieties. Roots of purple flowering variety of S. marianum represent 65% activity (Figure 8), while the roots of white variety exhibit 67.2% activity as shown in Figure 9. The main stem collected from both the varieties exhibits the activities in the range of 50– 52%, respectively (Figures 10 and 11).

Conclusion Different tissues of important medicinal plant S. marianum L. were subjected to extractions with solvent

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Figure 10. Time dependent free radical scavenging activity in main stem of purple flowering Silybum marianum. Values are means of 3 replicates.

Figure 11. Time dependent free radical scavenging activity in roots of purple flowering Silybum marianum. Values are means of 3 replicates.

methanol. Highest antioxidant potential was found in methanolic extract of purple seed variety followed by young leaves of white seed variety. From the experiment, it was observed that these plant parts have certain important constituents that are responsible for radical scavenging activity. S. marianum can regenerate the damaged hepatic tissues through scavenging

the harmful radicals by neutralizing them. Active components of silymarin also influence the production of superoxide dismutase enzymes, a powerful antioxidant especially active in destroying free radicals caused by alcohol in the liver. Radical scavenging activity was observed when discoloration occurred. Seeds were observed to have high

Figure 12. Plants, flowers and seeds of white and purple flowering varieties of Silybum marianum L.

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discoloration followed by roots and young and mature leaves. When the difference in the results was high between the DPPH solution and the sample, the percentage free radical activity is high or the sample was highly potential to scavenge the free radical of DPPH. This study reveals that the tested plant materials have significant free radical scavenging activity. The result of the present study suggests that these plant materials especially seeds can be used as a source of antioxidants for different diseases. Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

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