International Journal of Drug Development and Research

Keywords

Plumbagozeylanica , Phenolic, Flavonoids; DPPH,Antioxidant activity.

Introduction

Plumbagozeylanica (Family-Plumbaginaceae ) mainly called as “Chitrak” (Nquyen, et al., 2006) is a valuable Indian medicinal plant widely used in treatments of piles, diarrhea, leprosy and anasarca (Anonymous. 1989). Roots of plant have potential therapeutic properties like antianthrogenic, carditoxic, hepatoprotective, neuroprotective, anti-atherogenic, cardiotonic, hepatoprotective and neuroprotective properties (Tilak, et al., 2004). It is has been also reported that the plant have anticancer, antibacterial, antifungal and antitumor properties (Kavimani, et al., 1996). The leaves and roots of P.zeylanica contains an alkaloid called plumbagin (2- methoxy-5hydroxy-1, 4- napthoquinone), which externally is a strong irritant but a powerful germicide; stimulates muscular tissue in smaller doses and paralyzes in larger ones; stimulates the contraction of the muscular tissues of the heart and intestines; stimulates the secretion of sweat, urine and bile; and also has a stimulant action on the nervous system (Chopra, et al., 1996). Previously isolated constituents from P. zeylanica are Plumbagin, isoshinanolone, plumbagicacidbeta-sitosterol, 4- hydroxybenzaldehyde, Trans cinnamic acid, vanillic acid, 2, 5-dimethyl-7- hydroxychromone, indole-3-carboxaldehyde (Zhang, et al., 2003). The plant has great potential for various diseases and disorders along with great antioxidant activity.

Natural antioxidants such as phenols, flavonoids and tannins are increasingly attracting because they are natural disease preventing, health promoting and anti-ageing substances (Ozyurt Detal 2004),. Antioxidants may serve the task of reducing oxidative damage in humans induced by free radicals and reactive oxygen species under oxidative stress conditions. These conditions can cause DNA and protein damage, lipid peroxidation, cancer, ageing and inflammatory activity ( Blois MS et al., 1958). Phenolics are an important class of secondary plant metabolites possessing an impressive array of pharmacological activity. One of the more prominent properties of the phenolics is their excellent radical scavenging ability. Flavonoids are a group of polyphenolic compounds with known properties which include free radical scavenging, inhibition of hydrolytic and oxidative enzymes (Frankel E et al.,). Some evidence suggests that the biological actions of these compounds are related to their antioxidant activity (Gryglewski RJ, et al). An easy, rapid and sensitive method for the antioxidant screening of plant extracts is free radical scavenging assay using 1,1-diphenyl-2-picryl hydrazyl (DPPH) stable radical spectro photometrically. In the presence of an antioxidant, DPPH radical obtains one more electron and the absorbance decrease (Kolevaetal). In the present study we have attempted to investigate the antioxidant activity (free radical scavenging activity), determination of the content of total phenolics, total flavonoids in plant extracts and to explore relationship between phenolic content, flavonoid content and antioxidant activity.

Materials and Method

Chemicals:

DPPH (1, 1-Diphenyl-2-picrylhydrazyl), Ascorbic acid, FolinCiocatteu’sreagent, Gallic acid, anhydrous sodium carbonate,Catechin, Quercetin, EDTA (Ethylene diamine tetra acetic acid),Aluminum chloride, , Methanol . All chemicals were of AR grade .

Plant Material

The plant was obtained from Botanical garden of University of Rajasthan. The plant material washed with water to remove dirt and oven dried at 45°C.The dried plant material was pulverized using electric blender. Weighed portion of the sample was refrigerated to cold extraction.

Preparation of plant extract

Cold extraction method was employed for the extraction. 10gm of the powdered samples were weighed into conical flask ,90 ml of pure methanol was added and left for 72 hr .The mixture was filtered and this methanolic filtrate was concentrated under reduced pressure on rotary evaporator at 40 °C and then stored at 4 °C for further use. The filtrate was reconstituted in known amount of DMSO to obtain methanol extract of known concentration.

Determination of total phenolics

The determination of total phenolics based on Folin-Ciocalteu reagent assay (Singleton and Rossi., 1965). An aliquot (1ml) of extracts and standard solution of Gallic acid (100 mg/ml) was added to 25 ml volumetric flask, containing 9 ml distilled water. The distilled water itself was used as blank. One ml of Folin-Ciocalteu reagent was added to the mixture and shaken. After 5 min, 10 ml of 7% Na2CO3 solution was added to the mixture. The solution was diluted to volume (25 ml) with distilled water and mixed. After incubation for 90 min at room temperature, the absorbance against prepared reagent blank was determined at 750 nm with an UV-Vis Spectrophotometer. The total phenolic content of root extracts expressed as mg Gallic acid equivalents (GAE)/100 G fresh weights. All samples were analyzed in triplicates.

Determination total flavonoids

Total flavonoid content was measured by the aluminum chloride colorimetric assay (Zhishen, et al., 1999). An aliquot (1 ml) of extracts and standard solution of catechin (100 mg/ml) was added to 10 ml volumetric flask containing 4 ml of distilled water. To this 0.3 ml 5 % NaNO2 were added. After 5 min, 0.3 ml 10 % AlCl3 was added. Then after 1 min, 2ml of 1 M NaOH was added and the total volume was made up to 10 ml with distilled water. The solution was mixed well and the absorbance was measured against prepared reagent blank at 510 nm. Total flavonoid content of root extracts expressed as mg catechin equivalents (CE)/100 G fresh weights. All samples were analyzed in triplicates.

DPPH radical scavenging assay: The free radical scavenging activities of the plant extracts were measured employing the modified method of Blois (1958). 1 ml each of the different concentrations (1000, 500, 250, 125 and 62.5 μg/ml) of extracts or standard (ascorbic acid) in a test tube was added 1 ml of 0.3 mM DPPH in methanol. The mixture was vortexed and then incubated in a dark chamber for 30 min after which the absorbance was measured at 517 nm against a DPPH control containing only 1 ml of methanol in place of the extract. Percentage scavenging activity was calculated using the expression (Gulcin, 2009): % scavenging activity = [Absorbance of control - Absorbance of sample/Absorbance of control] ×100

Results

The results of the phenolic and flavonoid contents are shown in Table1. Experiments were done in triplicates and mean values and standard deviation were calculated.

Total Phenolic content

The phenolic content of the studied plant parts was found to be maximum in leaf (28.25±0.01 mg/g.d.wt.) followed by stem (24.95±0.02 mg/g.d.wt.) and root (14.26±0.01mg/g.d.wt.).

Total Flavonoid content

The total flavonoid content of the studied plant parts was found to be maximum in leaf (2.41±002 mg/g.d.wt.) followed by stem (2.025±0.001 mg/g.d.wt.) and root (1.20±0.015 mg/g.d.wt.).

DPPH Radical Scavenging Activity

The results of DPPH free radical scavenging activity on the methanolic extracts isolated from root, stem and leaves of the plant are shown in Table 2. IC50 values and Regression equation were calculated for each extract by statistical analysis. Plant extracts were evaluated at different concentration ranging from50 μg/ml to 400 μg/ml.

The methanolic extract of root exhibited maximum free radicle scavenging activity ranging from 56.42% to 96. 27% with an IC50 value of 24.67 μg/ml, followed by methanolic extract of leaf (51.86 to 90.13 %) with an IC50 value of 32.43 μg/ml and methanolic extract of stem (58.47 to 91.57 %) with an IC50 value of 72.31 μg/ml

Discussion

The results of the DPPH radical scavenging activities of the extracts revealed that the plant extracts contain free radical scavenging activity which could exert a beneficial action against pathological alterations caused by generation of free radicals. The IC50 value (the concentration required to inhibit radical formation) for stem, root and leaf extract is found to be72.3 μg/ml, 24.6μg/ml and 32.43μg/ml respectively. Polyphenols of plant kingdom are one of the most effective antioxidative constiuents . It is important to estimate phenolic contents of plant extracts so as to justify their controls with to antioxidant activity ( Choi et al ., 2007) . In the present study we estimate total phenolic content of stem, root, leaf extracts by F.CR method. FCR method is one of the oldest and commonly used colorimetric techniques for estimating total phenolic content of a range of substances including plant extracts. The phenolic compounds react with FCR only under basic conditions to form blue complex having maximum absorption near 750 nm. Though the chemical nature of FCR is undefined, the total phenols assay by FCR is convenient, simple, and reproducible. A large data has been accumulated, and it has become a routine assay in studying the phenolic antioxidants (Dasgupta and De, 2004; Huang et al., 2005; Chung et al., 2006; Harish and Shivanandappa, 2006; Coruh et al., 2007; Ardestani and Yazdanparast, 2007; Kekuda et al., 2011; Rekha et al., 2012; Junaid et al., 2013). Total phenolic contents in stem, root, leaf was found to be 24.95 ± 0.021 mg, 14.26 ± 0.015 mg and 28.25 ± 0.01 mg respectively. Flavonoids as one of the most diverse and widespread group of natural compounds are probably the most important natural phenols. These compounds possess a broad spectrum of chemical and biological activities including radical scavenging properties. Using the standard plot of quercetin (y = 0.0148x, R2 =0.975), the flavonoid contents of Plumbago zeylanica leaves, stem and root were found to be 2.41±0.021 mg, 2.025±0.001mg and 1.20±0.015 mg quercetin equivalent/g of dry sample respectively.

Conclusion

The present investigation revealed that the leaves, stem and root of Plumbago zeylanica contain significant amount of phenols and flavonoids and possess antioxidant property. The objective of this study was to get information of the amount of phenolics and flavonoids in the parts of Plumbagozeylanica. Further intention of this study is to correlate relationship between antioxidant activity and total phenolic content, total flavonoid content and evaluate Plumbago zeylanica as a potential source of natural bioactive chemicals.

Tables at a glance

< Table 1 Table 2

Figures at a glance

Figure 1 Figure 2

References

1. Anonymous., (1989): In: The Wealth of India: A Dictionary of Indian Raw Materials and industrial Products. CSIR New Delhi India, 2:163-164.
2. Chopra, R.N., Nayar, S.L., Chopra, I.C., (1996): Glossary of Indian medicinal plants, NISCOM Publishers New Delhi , 55-60.
3. Kavimani, S., Ilango, R., Madheswaran, M., Jayakar, B., Gupta, M., Majumdar, U.K., (1996): Antitumor activity of plumbagin against dalton’sascitic lymphoma. Ind. J. Pharm. Sci ., 58: 194-196.
4. Nguyena, A.T. ,Malonne, H., Duez, P., Vanhaelen- Fastre, R., Vanhaelen, M., Fontaine, J., (2004): Cytotoxic constituents from Plumbagozeylanica . Fitoterapia , 75:500-504.
5. Singleton, V. L., Rossi, J. A., (1965): Colorimetry of total phenolics with phosphomolybdicphosphotungsticacid reagents. Am J. Enolo.Viticul. , 16:144-158.
6. Tilak, J.C., Adhikari, S., Devasagayam, T. P., ( 2004): Antioxidant properties of Plumbagozeylanica , an Indian medicinal plant and its active ingredient, plumbagin, Redox Rep ., 9: 219-227.
7. Zhang, Q.R., Mei, Z.N., Yang, G.Z., Xiao, Y.X., (2007): Chemical constituents from aerial parts of PlumbagozeylanicaLinn .Zhong Yao Cai ., 30:558-560.
8. Zhishen, J., Mengcheng ,T., Jianming, W., (1999): The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals Food Chem , 64: 555-559.
9. Blois MS. Antioxidant determinations by the use of a stable free radical.Nature.1958; 181, 1199-1150.
10. Ozyurt D, Ozturk BD,Apak R. Determination of totalflavonoid content of Urticadioica L. by a new method.Adnan Menderes University, 4th AACD Congress, Turkey 2004.
11. Frankel E. Nutritional benefits of flavonoids. International conference on food factors: Chemistry and Cancer Prevention, Hamamatsu, Japan.1995; 6, 2-4. 7.
12. Gryglewski RJ, Korbut R, Robak J. On the mechanism of antithrombotic action of flavonoids.BiochePharmacology.1987; 36, 317- 321.
13. Koleva II, Van Beek TA, Linssen JPH, Groot A,EvstatievaL. Screening of plant extracts for antioxidant activity: a comparative study on three testing methods. Phytoche Analysis. 2002; 12, 138-147.