Influence Of Plant Extracts Biology Essay

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Abstract

Cardiovascular disease (Ide et al., 2001) and cancer (Valko et al., 2006) are ranked first and second leading causes of death in United States and many other industrialized countries. Consumption of fruits and vegetables regularly have shown to reduce the risk of cancer, cardiovascular disease, Alzheimer's disease, stroke, cataracts and some of the functional declines related to aging. These beneficial effects may be due to the presence of carotenoids and phytonutrients in fruits and vegetables. One proposed mechanism of antioxidant protection is the shielding of cellular DNA from oxidative damage and therefore mutations. The effect of fruit extracts in combating DNA damage was studied using whole blood DNA. The study was carried out in two parts- firstly; the DNA was isolated and was subjected to damage using H2O2 which results in the generation of free radicals that bring about damage (Athar et al.,1992). The extent of damage is monitored using Agarose Gel Electrophoresis (AGE). Secondly, along with the damaging agent, the plant extracts were added to the reaction mixture and incubated. Finally the damage control is recorded.Our results suggest that the plant extracts of Rauwolfia, Aesculus and Baptisia, protect genomic DNA against oxidative stress mediated damage.

Keywords: Phytonutrients, Oxidative damage, Agarose gel electrophoresis

Introduction

Reactive Oxygen Species (ROS) are produced by cellular metabolic processes and exogenous agents in the cell. Several oxygen free radicals such as superoxide anion (O2-), hydroxyl radicals (.OH) and hydrogen peroxide (H2O2) have been implicated in mediating various pathological processes, such as ischemia, inflammatory diseases and atherosclerosis. These reactive oxygen species may induce oxidative damage to biomolecules in cells such as carbohydrates, proteins, lipids and DNA which in turn leads to cardiovascular and neurodegenerative diseases, inflammation and others ( Ames 1983; Stadtnan, 1992; 1990). The eukaryotic cells have to always cope up with these free radicals through defense mechanisms such as antioxidant molecules and antioxidant enzymes. However, when the level of ROS increases beyond the capacity of these defense mechanisms, it may lead to oxidative damage of DNA.

At least two major human problems; aging and cancer involves ROS mediated DNA damage (Cerutte, 1994; Wiseman & Halliwell 1996). Oxidative damage can lead to mutations and is suspected to be major cause of cancer (Schwarz I 1984). Persistent oxidative damage alters the signaling cascade and gene expression which may result in cancer progression. Among the reactive oxygen species leading to cancer development, 80% of damage is caused by hydrogen peroxide H2O2, singlet oxygen (1O2) and hydroxyl radical (.OH) (Ghosal I 2003).

Plant food and products carry rich sources of biologically active compounds and many of these phytochemicals have been found to possess antioxidant potential (Craig 1999). Foods rich in antioxidants play an essential role in the prevention of cardiovascular diseases, cancer (Gerber et al. 2002; Kris Etherton et al. 2002; Serafini et al. 2002) and neurodegenerative diseases including Parkinson's and Alzheimer's disease (Di Matteo & Esposito, 2003). Essential oils and extracts of many plant species have become popular in recent years and research is going on to find out their biological properties for human welfare (Samarath et al. 2008; Cowan, 1999).

Our study aims to assess the potential of medicinal plant extracts in controlling DNA damage. The experiment was carried out by using three types of plant extracts- Rauwolfia serpentina, Aesculus hippocastanum, Baptisia tinctoria.

Rauwolfia serpentina or Sarpagandha, a member of family Apocynaceae is a native to South and East Asia and contain various bioactive chemicals like serpentinine, reserpine. The plant extract has been used to treat hypertension and certain neurodegenerative disorders like schizophrenia.

Aesculus hippocastanum also known as horse chestnut is the member of the family Sapindaceae and is used for vascular protection, anti-inflammatory and free radical scavenging properties. Baptisia tinctoria, also known as horse flyweed a native to Eastern North America belongs to the family Fabaceaeae. It has antimicrobial activity and promotes normal cellular metabolism that supports healthy tissues. These extracts were used in order to study their effect on the whole blood genomic DNA.

Materials and Methods

Isolation of DNA from human whole blood

Blood samples obtained from the healthy donor was used to obtain DNA using phenol-chloroform method (Sambrook et al. 2001) and the concentration of DNA was measured using spectrophotometric analysis. The obtained DNA was refrigerated for further use.

Extract preparation

Extracts were obtained from a West Bengal based homeopathic medicinal shop. The dry weight per ml of the extract was measured and 1mg/ml of stock solution was prepared for carrying out the experiment.

Studying the impact of plant extracts on DNA

DNA concentration of 473μg was subjected to 10mM of H2O2 in order to induce damage and the mixture was incubated along with varying concentrations of the plant extracts (50μg- 400μg) at 37°C followed by UV treatment for 30 minutes. After incubation, the samples were analyzed using 1.2% Agarose gel electrophoresis to monitor the effect of the extracts on damaged DNA. The gel was viewed under a gel documentation unit for comparison.

The DNA samples were also analyzed by using the extracts in order to examine their DNA damaging capability in the absence of H2O2.

Results and Discussion

Lane 1 2 3 4 5 Lane 1 2 3 4 5

1(a) 1(b)

Fig 1(a) shows the effect of only Rauwolfia extract on DNA :

Lane 1: only DNA; 2: DNA+ 50µg of extract; 3: DNA+100µg of extract; 4: DNA+200µg of extract; 5: DNA+400µg of extract

1(b) shows the effect of Rauwolfia on DNA in presence of H2O2 at different concentration of the plant extracts:

Lane 1: only DNA; 2: DNA+10mM H2O2; 3: DNA+10mM H2O2 + 50µg of extract; 4: DNA+10mM H2O2 + 100µg of extract; 4: DNA+10mM H2O2 + 200µg of extract

Lane 1 2 3 4 5 Lane 1 2 3 4 5

Fig 2(a) Fig 2(b)

Fig 2(a) shows the effect of only Aesculus extract on DNA:

Lane 1: only DNA; 2: DNA+ 50µg of extract; 3: DNA+100µg of extract; 4: DNA+200µg of extract; 5:

DNA+400µg of extract

2(b) shows the effect of Aesculus on DNA in presence of H2O2 at different concentration of the plant extracts:

Lane 1: only DNA+ 10mMH2O2; 2: DNA+10mM H2O2 + 50µg of extract; 3: DNA+10mM H2O2 + 100µg of extract; 4: DNA+10mM H2O2 + 200µg of extract 5: only DNA

Lane 1 2 3 4 5 Lane 1 2 3 4 5

Fig 3(a) Fig 3(b)

Fig 3(a) shows the effect of only Baptisia extract on DNA:

Lane 1: only DNA; 2: DNA+ 50µg of extract; 3: DNA+100µg of extract; 4: DNA+200µg of extract; 5: DNA+400µg of extract

3(b) shows the effect of Baptisia on DNA in presence of H2O2 at different concentration of the plant extracts:

Lane 1: only DNA; 2: DNA+10mM H2O2; 3: DNA+10mM H2O2 + 50µg of extract; 4: DNA+10mM H2O2 + 100µg of extract; 4: DNA+10mM H2O2 + 200µg of extract

From Fig 1(a) and (b) we can infer that I extract acts as a DNA protector even in the presence of H2O2. Fig 2(a) and (b) shows that Aesculus though shields the DNA from damage but is not that much effective at lower concentrations (i.e, at 50µg/ml and 100 µg/ml damage is observed in presence of H2O2). Fig 3(a) and (b) depicts that Baptisia is capable of masking DNA in presence of H2O2 thereby showing its inhibitory effect on any DNA damage brought about due to strand breaks induced by H2O2 (Zhu et al.2001).

Conclusion

Fruits, vegetables and plant extracts are new candidates for prevention of oxidative stress (Osawa et al. 1994, Noda et al. 1997). The present study was based on three plant extracts namely, Rauwolfia, Aesculus and Baptisia. The results obtained demonstrated that all the three extracts act as potential candidate for prevention of oxidative damage generated by H2O2 Among the three, Aesculus is less effective in action as compared to other two extracts.

Therefore, it can be concluded that these extracts exhibit interesting antioxidant properties thereby controlling DNA damage and reducing deleterious consequences of free radical generation.