Evaluation of comparative free-radical scavenging capacity , and antioxidant activities of methanolic extracts of leaf , stem , and roots of Operculina turpethum

1 Department of Pharmacognosy, Trinity College of Pharmaceutical Sciences, Peddapally, Karimnagar, Andhra Pradesh, 505 172, India. 2 Department of Pharmacology, KVSR Siddhartha College of Pharmaceutical Sciences, Vijayawada, Andhra Pradesh, 520 010, India. 3 Department of Pharmacognosy, S. R. College of Pharmacy, Ananthasagar, Warangal, Andhra Pradesh, 506 371, India. 4 MakroCare Clinical Research Organization, Suite 1303, Newark, New jersey, 07102, USA.


INTRODUCTION
Reactive oxygen species (ROS) are responsible for various cellular anomalies like protein damage, deactivation of enzymes, alteration of DNA and lipid per oxidation which in turn leads to pathological condition like carcinogenesis, reperfusion injury, rheumatoid arthritis, diabetes, etc.The regular intake of antioxidants seems to limit or prevent the dangerous effects caused by ROS.Thus, to maintain cellular health, it is important to have a specific and effective antioxidant that scavenges multiple types of free radicals so that it can be used in multiple *Corresponding author.E-mail: shankar.pulipaka@gmail.com.Tel: +91-8121248937 or 9885589543.
diseases.Based on the efficiency of free radical scavenging, the compounds are classified into strong, moderate and weak antioxidants (Gramza et al., 2010).Naturally, there is a dynamic balance between the amount of free radicals produced in the body and antioxidants to scavenge or quench them to protect the body against deleterious effects.Therefore, it is obvious to enrich our diet with antioxidants to protect against harmful diseases.Hence, there has been an increased interest in the food industry and in preventive medicine in the development of "Natural antioxidants" from plant materials.That is why plants with antioxidant properties are becoming more and more popular all over the world (Indu and Thiraviam, 2010).To protect the cells and organ systems of the body against reactive oxygen species, humans have evolved a highly sophisticated and complex antioxidant protection system, that functions interactively and synergistically to neutralize free radicals (Pullaiah, 2007).
Operculina turpethum, which is the commonly known as trivit belonging to family Convolvulaceae, is a large stout perennial twinner with milky juice and fleshy branched roots (Shankaraiah et al., 2012).It also has anthelmintic, expectorant, antipyretic, hepatoprotective (Riaz et al., 2009), anti-inflammatory, purgative and antidiabetic properties (Shankaraiah et al., 2011(Shankaraiah et al., , 2012)).It contains a wide variety of phytoconstituents, which are useful in treatment of different ailments and includes glycosidic resin, coumarins, beta-sitosterol, and essential oils (John, 1988).It is reported to be highly medicinal and is cultivated occasionally in gardens as an ornamental plant.The aim of our study was to evaluate antioxidant activity of methanolic extracts of leaf, stem and roots of O. turpethum in vitro models.

Collection of plant material
O. turpethum (L.) Silva Manso plant material was collected from local areas of Vijayawada, Andhra Pradesh, India.Its parts were botanically authenticated by Prof. S. V. Raju, Taxonomist, Department of Botany, Kakatiya University, Warangal, Andhra Pradesh, India.The herbarium was maintained in the Department of Pharmacognosy and Phytochemistry, Vaagdevi College of Pharmacy, Hanamkonda.O. turpethum stem was washed under tap water and were efficiently dried under shade for about one week and protected from deterioration.The shade dried stem was grinded made into powder with the help of a laboratory mixer.These were efficiently dried under shade for about one week and protected from deterioration and then ground and made into powder.

Preparation of plant
O. turpethum leaves stem and roots were washed under tap water and were efficiently dried under shade for about one week and protected from deterioration.The shade dried leaves, stem and roots were ground made into powder with the help of a laboratory mixer.These were efficiently dried under shade for about one week and protected from deterioration and then ground and made into powder.

Soxhlation
The stem, root material was weighed (100 g) and using successive solvent extraction process (soxhlet apparatus) with methanol for 6 h.After completion of soxhlation process the liquid extract was collected and concentrated under reduced pressure below 50°C, until a soft mass was obtained in it and was dried and kept in a desiccators.

Maceration
The leaf material was weighed (250 g) and methanol was extracted at room temperature in a glass container for 3 days.The material was stirred from time to time to ensure proper extraction.After 3 days, the contents of the container were filtered through muslin cloth and the filtrate was concentrated under reduced pressure below 50°C, until a soft mass was obtained and then preserved in a desiccators.

Reducing power method
About 2 ml of each sample and standard solutions were spiked with 2.5 ml of 1% potassium ferricyanide solution.This mixture was kept at 50°C in water bath for 20 min.After cooling, 2.5 ml of 10% trichloro acetic acid was added and centrifuged at 3000 rpm for 10 min.About 2.5 ml of supernant was mixed with 2.5 ml of distilled water and 1 ml of 0.1% ferric chloride and kept for 10 min.Control was prepared in similar manner without samples.The absorbance of resulting solution was measured at 700 nm (Carol et al., 2010).

DPPH free radical scavenging activity
About 150 ml of DPPH solution was added to 3 ml methanol and absorbance was taken immediately at 516 nm for control reading.Different volume levels of test sample (20, 40, 60, 80 and 100 μl) were screened and 100 μl of each dose level by dilution with methanol up to 3 ml was made.About 150 ml of DPPH solution was added to each test tube (Soni et al., 2006).Absorbance was measured at 516 nm in UV-visible spectrophotometer (Shimadzu, UV-1800, Japan) after 15 min using methanol as a blank.The percent reduction and IC50 were calculated and the free radical scavenging activity (FRSA) (% antiradical activity) was calculated using the following equation: Antiradical activity (%) = Control absorbance -Sample absorbance/Control Absorbance × 100 Each experiment was carried out in triplicate.

Nitric oxide free radical scavenging activity
Sodium nitroprusside (10 mg) in phosphate buffer saline was mixed with different volume levels of test sample (10, 20, 30, 40, 50 and 100 μl) and 100 μl of each dose level by dilution with methanol was made.The solution was incubated at room temperature for 150 min.The same reaction mixture without the extract but equivalent amount of methanol served as control.After the incubation period 5 ml of Griess reagent was added.The absorbance was taken in UVvisible spectrophotometer at 546 nm (Shreedhara et al., 2010).Ascorbic acid was used as positive control.The percent reduction and IC50 were calculated.Each experiment was carried out in triplicate.

Iron chelation activity
Iron chelation activity is a measure of antioxidant activity.Different concentrations of the extract and ascorbic acid solution each as 2 ml in 5% v/v methanol were incubated with methanolic Ophenanthroline solution (1 ml, 0.05% w/v) and ferric chloride solution (2 ml, 200 M) at ambient temperature for 10 min.After incubation, the absorbance of solutions was measured at 510 nm.The experiments were performed in triplicate.

Hydrogen peroxide (H2O2) scavenging activity
Hydrogen peroxide scavenging activity of plant extract was determined using a modification of the method.About 4 mM solution of H2O2 was prepared in phosphate-buffered saline (PBS, pH 7.4).H2O2 concentration was determined spectrophotometrically from absorbance at 230 nm.Plant extract corresponding to 50, 100, 150, 200, and 250 μl of 1 mg/ml plant extract stock solution in 4 ml distilled water were added to 0.6 ml hydrogen peroxide in PBS solution.Absorbance of H2O2 was determined at 230 nm (Muntasir, 2010).Absorbance was determined 10 min later against a blank solution similar to that mentioned earlier.

Determination of total phenol content
Total phenolic compound contents were determined by the Folin-Ciocalteau method.The extract samples (0.5 ml of different dilutions) were mixed with Folin-Ciocalteu reagent (5 ml, 1:10 diluted with distilled water) for 5 min and aqueous Na2CO3 (4 ml, 1 M) were then added.The mixture was allowed to stand for 15 min and the phenols were determined by colorimetric method at 765 nm.The standard curve was prepared by 0, 50, 100, 150, 200, 250, and 300 μg/ml solutions of gallic acid in methanol:water (50:50, v/v).Total phenol values are expressed in terms of gallic acid equivalent which is a common reference compound.

Data analysis
All statistics were calculated using Graph Pad Prism 5.0 software Pulipaka et al. 2027 (San Diego, CA, USA).Pharmacokinetic parameter values for groups were compared using analysis of variance with Tukey's and Dunnett's tests for multiple comparisons.The p value less than 0.05 were considered significant.

Reducing power method
The reducing power assay is a convenient and rapid screening method for measuring the anti oxidant potential.The reduction ability, that is, "Fe 3+ to Fe 2+ transformation" by measuring absorbance at 700 nm is as shown in Figure 1.

DPPH free radical scavenging activity
Free radical scavenging activity of ascorbic acid, methanolic extracts of Operculina turpethum leaves (MEOTL), stem (MEOTS), and roots (MEOTR) were performed by DPPH method.The reduction capability of the DPPH radical is determined by the decrease in its absorbance.

Nitric oxide scavenging activity
The nitric oxide scavenging of ascorbic acid, MEOTL, MEOTS, and MEOTR were carried out after incubation for 150 min with different concentrations.The methanolic and stem and root extracts exhibited scavenging activity lower than ascorbic acid at similar concentrations (Figure 3).Here, methanolic stem extract scavenging activity is higher when compared with other extracts as shown in Table 3.The IC 50 values of the ascorbic acid, MEOTL, MEOTS, and MEOTR were found to be 16.10 ± 1.57, 16.45 ± 1.89, 16.10 ± 1.24 and 16.90 ± 1.66 µg, respectively.

Iron chelation scavenging activity
The scavenging activity of iron chelation by ascorbic acid,  MEOTL, MEOTS, and MEOTR were carried out.The methanolic and stem and root extracts exhibited scavenging activity lower than ascorbic acid at similar concentrations (Figure 4).The methanolic stem extract

Hydrogen peroxide scavenging activity
The scavenging activity of hydrogen peroxide by ascorbic acid, MEOTL, MEOTS, and MEOTR exhibited scavenging activity higher than ascorbic acid at similar   concentrations.The results suggested that methanolic stem extract scavenging activity was higher (Figure 5) when compared with other extracts depicted in Table 5.

Total phenolic activity
The total phenolic content of MEOTL, MEOTS, and MEOTR were performed.The methanolic extracts of stem and root exhibited scavenging activity lower than gallic acid at similar concentrations as shown in Figure 6.
Based on the results obtained from the method followed, the methanolic stem extract scavenging activity was higher when compared with extracts as sown in Table 6.

DISCUSSION
For many centuries, plants have been used throughout the world as drugs and remedies for treatment of various diseases as they have great potential for producing new drugs of great benefit to humankind.There are many approaches to search for new biologically active principles in higher plants.Natural flora has gained its attention in the treatment of common cold to dreadful diseases, namely, AIDS, cancer, etc, such plants are called medicinal plants which have curative properties due to the presence of various complex chemical substances of different composition, namely, grouped as alkaloids, glycosides, corticosteroids, terpenoids, isoflavanoids, steroids, etc (Shankaraiah et al., 2012).
O. turpethum is a perennial plant with milky juice belongs to family Convolvulaceae.This plant is widespread in old tropics from East Africa to North Australia, and is common in Godavari, Andhra Pradesh, India (John et al., 2005).It is widely distributed in tropical Africa and Asia.In India, it is found in damp and it occurs almost throughout India up to an altitude of about 1000 m.It is sometimes grown in gardens for its beautiful flowers.It is rare on open sandy soils.It is occasionally cultivated in India.Traditionally, O. turpethum root is prescribed in the treatment of snake bite and scorpion sting, but it is not an antidote to either snake-venom or scorpion-venom.In constipation, it is an effective laxative.It is used in periodic fevers and in the treatment of anemia accompanied by spenomegaly.It is also used to  et al., 2006).Several studies reported the antioxidant activity of these plants including Zingiber officinale (Atef et al., 2013), Cinnamomum longepaniculatum (Cui et al., 2013), and Boerhaavia diffusa (Jitender et al., 2013).
In the present study, the MEOTL, MEOTS, and MEOTR were evaluated for antioxidant activity.The percent reduction and IC 50 values were calculated using standard methods.The percent reduction values are summarized in Tables 1 to 6. Finally, the present study results suggested that among all extracts, MEOTS has higher antioxidant activity.

Conclusion
The findings reported by the comparison of antioxidant activity of MEOTL, MEOTS, and MEOTR.The stem methanolic extract has higher antioxidant activity than other extracts.Although, promising results have been obtained, more concentrated efforts are still needed for the characterization of the comparison of antioxidant activity with MEOTL, MEOTS, and MEOTR.Further investigations are needed to identify the lead molecule and to elucidate the structure and exact mechanism of action for their antioxidant activity.

Table 6 .
Total phenolic content of ascorbic acid, MEOTL, MEOTS, and MEOTR by total phenolic activity method.flatulence and colic and in the treatment of obesity to decrease fat.It is used to treat dropsy, dyspepsia with constipation and flatulence, gout and rheumatism, and other inflammations (Suresh relieve