EXPERIMENTAL STUDIES ON ANTIOXIDANT PROPERTIES OF CALOTROPIS GIGANTEA

Document Sample
EXPERIMENTAL STUDIES ON ANTIOXIDANT PROPERTIES OF CALOTROPIS GIGANTEA Powered By Docstoc
					  International Journal of Advanced Research OF ADVANCED RESEARCH IN
  INTERNATIONAL JOURNAL in Engineering and Technology (IJARET), ISSN
  0976 – 6480(Print), ISSN 0976 – 6499(Online) Volume 4, Issue 4, May – June (2013), © IAEME
             ENGINEERING AND TECHNOLOGY (IJARET)

ISSN 0976 - 6480 (Print)
ISSN 0976 - 6499 (Online)
                                                                       IJARET
Volume 4, Issue 4, May – June 2013, pp. 168-180
© IAEME: www.iaeme.com/ijaret.asp
Journal Impact Factor (2013): 5.8376 (Calculated by GISI)
                                                                       ©IAEME
www.jifactor.com




      EXPERIMENTAL STUDIES ON ANTIOXIDANT PROPERTIES OF
           CALOTROPIS GIGANTEA SOLVENT EXTRACTS

             David Mandepudi1,2, Bharath Kumar Ravuru3, Bhavani Mandepudi2
         1
          Faculty of Biotechnology, Jawaharlal Nehru Technological University-Hyderabad,
                             Hyderabad-500085, Andhra Pradesh, India
   2
     Department of Biotechnology, School of Engineering, Sir Padampat Singhania University,
                               Udaipur-313601, Rajasthan, India
     3
       School of Biotechnology, Vignan University, Guntur – 522213, Andhra Pradesh, India



  ABSTRACT

          The active metabolites from plant sources had been explored and extracted to serve
  the societies as part of the medicine either internally or externally for the treatment of
  ailments. In the similar lines, the Calotropis gigantea, has been investigated for antioxidant
  properties using ethanol, methanol, chloroform and aqueous solvent extracts of leaves, buds
  and flowers with the help of popular antioxidant testing methods of DPPH and hydrogen
  peroxide free radical scavenging activities and an additional reducing power test. In the
  results, many of the solvent extracts were reaching to variable saturation levels beyond the
  respective extract concentrations of 5µg/ml. Further, comparable free radical scavenging
  activities for some of the solvent extracts like, ethanol and methanol leaves, buds & flowers
  extracts in addition to methanol, chloroform and aqueous extracts of leaves, buds and
  flowers were observed with respect to free radical scavenging activities of the standard
  antioxidants, ascorbic acid and quercetin in the tests. However the reducing power of these
  solvent extracts of Calotropis gigantea leaves, buds and flowers were very low in comparison
  to that of the standard antioxidants, ascorbic acid and BHT. So, the observed free radical
  scavenging activities of the solvent extracts may be due to the presence of the detected active
  metabolites like, alkaloids, tannins, polyphenols etc. Though these results favor the use of
  Calotropisgigantea as part of the traditional medicine due to their auxiliary antioxidant
  properties, similar studies may be carried out for the better utilization and management of the
  Calotropis species.



                                               168
International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN
0976 – 6480(Print), ISSN 0976 – 6499(Online) Volume 4, Issue 4, May – June (2013), © IAEME

Keywords: Antioxidant, Ayurveda, drugs of abuse, Prodrug, Polyphenols, Calotropis
gigantean

1. INTRODUCTION

        The molecular agents that prevent the oxidation of other molecules either by stopping
the transfer of electron or hydrogen are known as the antioxidants. But then, these antioxidant
molecules get themselves oxidized often acting as reducing agents [1]. While the oxidation
and reduction reactions are very important set of reactions in biological systems, the soldier
like antioxidants play a vital role in the sustenance of the life [2-3] on the planet, Earth. On
the other hand, the reactive species and free radicals were known to cause severe damage to
the cellular components of the life systems especially of human and may lead to various
ailments like early ageing, oxidative stress related with Alzheimer's disease,Parkinson's
disease,diabetes, rheumatoid arthritis, and neuro degeneration in motor neuron diseases
[2,3,4-13]etc. Since, these oxidation and reduction reactions were found to have the greater
significance in the realm of biological systems with antioxidant mechanism, plants and
animals established complex systems of antioxidants like glutathione, vitamins – C, D, A, K
& E; enzymes like catalase, superoxide dismutase, peroxidases, β-carotene, lutein, lycopene,
vitamin A, vitamin C, vitamin E, flavonoids, polyphenols, ascorbic acid [14-17] etc. to
regulate and check the unnecessary burst of any reactive species [11] like hydrogen peroxide
(H2O2), hypochlorous acid (HClO) and free radicals like hydroxyl radical (.OH) and
superoxide anion (O2-).           Owing to these facts, synthetic antioxidants like
butylatedhydroxytoluene (BHT) and buytlatedhydroxyanisole (BHA)were also developed or
derived in the form of health supplements or drug molecules to serve the purpose by various
pharmaceutical and health care companies [7,18]. However the natural antioxidants are the
best means for the human consumption and in the lines of acceptability for the environment
without the creation of any xenobiotic compounds [19] by the synthetic processes. So, these
facts elucidated the need to search for the novel and bioactive compounds [20] like
antioxidants directing the scientists to investigate the plant based solvent extracts to isolate
and identify the antioxidants that may be used as dietary supplements [21-23] or the drugs
that can save the life. In such scenario, many plants like, Azadiractaindica, Meliaazedarach,
Digitalis purpurea, Digitalis lanata, Daturastramonium, Atropa belladonna,
Papaversomniferum, Catharanthusroseus, Colchicum autumnale, erythroxylon coca etc.
were investigated for potential active metabolites likeazadiractin, cardenolides,
parasympatholytic agents, analgesics, anticancer agents, antigout agent, local anesthetics etc.
for the use of medicine and food supplements [20,24]. In the similar lines, the Calotropis sp.
known for its poisonous nature for centuries and for medicine in the traditional medical
practices [25-27] of several ethnic groups has been investigated for the possible active
metabolites using the residual solvent extracts of Calotropis gigantea leaves, buds and
flowers.

2. MATERIALS AND METHODS

2.1 Plant material & solvent extraction
       The Calotropis gigantia leaves, flowers and buds were collected [28] and processed
to obtain the respective dry powders and these dry powders were added to the solvents of
ethanol, methanol and chloroform and extracted the active metabolites by solvent extraction

                                              169
International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN
0976 – 6480(Print), ISSN 0976 – 6499(Online) Volume 4, Issue 4, May – June (2013), © IAEME

procedure and named them as the solvent extracts of ethanol leaves (EL), ethanol flowers
(EF), ethanol buds (EB); methanol leaves (ML), methanol flowers (MF), methanol buds
(MB); Chloroform leaves (CL), chloroform flowers (CF), chloroform buds (CB). The
aqueous extracts were also prepared by boiling the plant material (powder) individually and
processed [28] to obtain the final extracts of Aqueous leaves (AL), Aqueous flowers (AF)
and Aqueous buds (AB). These extracts were stored in the refrigerator until use in sterile
bottles.

2.2 Antioxidant Activity of solvent extracts
       The extracted solvent extracts of leaves, buds and flowers of Calotropis gigantea
were tested for the detection and estimation of antioxidant activity in terms of free radical
scavenging activity using DPPH [29-32] and Hydrogen peroxide [33-34] scavenging assay
methods.

2.3 The DPPH free radical scavenging activity
        The DPPH solution (3ml) of 0.02mg/ml in methanol was taken in three sets of clean
& dry test tubes and the solvent extracts of 1 ml were added respectively with an increasing
concentration in the range of (0-25µg/ml) in multiples of 5µg/ml. Similarly a standard
antioxidant, ascorbic acid of 1 ml was also added separately to the 3 ml DPPH solution in
three sets of test tubes and used as a reference for the assay. All those test tubes with the
solutions were vortexes and incubated at room temperature for 30 minutes under dark
conditions. The absorbance of these resulting solutions were recorded at 517nm using UV-
Visible double beam spectrophotometer. So the assay was performed in triplicates and the
results were expressed as mean values and the standard derivations were also calculated.
The DPPH free radical scavenging activity of the standard, ascorbic acid and the solvent
extracts were calculated using the equation (1).
          (% ) DPPH Free Radical Scavenging Activity (DFRSA) =             X100 (1)
        Where As is the absorbance of the DPPH in the absence of test solution (solvent
extract/ascorbic acid) and Ae is the absorbance of the test solution.

2.4 Hydrogen peroxide free radical scavenging activity
        Estimation of antioxidant activity of enzyme, catalase [35] was adopted to detect and
evaluate the hydrogen peroxide scavenging activity of solvent extracts of Calotropis gigantia
leaves, buds and flowers. The solvent extracts were taken as test solutions of 0.4ml with
varying concentrations in multiples of 5µg/ml in the concentration range of (5-25) µg/ml
separately in three sets of clean and dry test tubes and then added 0.6ml of 40mM hydrogen
peroxide solution (in 0.1M phosphate buffer of pH 7.4) to each test sample and incubated
them for 15 min under dark conditions. Similarly, a standard antioxidant solution of quercetin
was prepared and used for the assay following the above standard procedure with varying
concentration in multiples of 5µg/ml in the concentration range of (5-25)µg/ml. After the
incubation period, 1ml of dichromate solution in acetic acid (5% potassium dichromate
aqueous solution mixed with glacial acetic acid in the ratio of 1:3) was added to each solvent
extract test solution and standard quercetin solution separately and mixed them well. These
test solutions were further incubated in boiling water bath for 10min, where the characteristic
green color appeared in the test tubes. 2ml of deionized water (MilliQ water) was added to
each test solution and the resultant solution was used for the measurement of absorbance at

                                             170
International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN
0976 – 6480(Print), ISSN 0976 – 6499(Online) Volume 4, Issue 4, May – June (2013), © IAEME

620nm using UV-Visible double beam spectrophotometer. The control sample was prepared
without any extract or standard antioxidant, containing only deionized water and the
hydrogen peroxide as well as dichromate solutions in parallel with test sample& standard
samples of quercetin. Hence the test was performed in triplicates and the measured
absorbance at 620nm for each sample was taken as mean value and used this data for further
analysis.
       The (%) hydrogen peroxide free radical scavenging activity of solvent extracts & the
standard, quercetin were calculated as per the equation (2).
(% ) Hydrogen peroxide Free Radical Scavenging Activity (HFRSA) =            X 100(2)
       Where AR is the absorbance of control sample, and Ae is the absorbance of extract or
standard at 620nm.

2.5 Reducing power assay
        The solvent extracts of Calotropis gigantea leaves, buds and flowers were evaluated
[36], where 1ml of the variable concentration of residual solvent extracts (0-1 mg/ml) were
mixed with 2.5ml of 0.2M phosphate buffer (pH 6.6) and 2.5ml of 1% potassium ferricyanide
(K3Fe CN6) solution respectively and then incubated the resulting solutions at 50oC for 20
minutes. Added 2.5ml of 10% trichloroacitic acid to each reaction mixture in the test tubes
and then centrifuged at 3000g for 10 min where the supernatant of 1ml was withdrawn
carefully and mixed with 2.5ml of distilled water and further added 0.5ml of 0.1% ferric
chloride solution to get the final solution. The absorbance of the resulting solution for each
test sample was measured using UV-Visible double beam spectrophotometer at 700nm. The
ascorbic acid and BHT were taken as the standard antioxidants for positive controls following
the above procedure and the experiments were performed in triplicates. The mean absorbance
for each test sample was calculated and used the data for further analysis.

3. RESULTS

        The free radical scavenging activity of Calotropis gigantea leaves, buds and flowers
were calculated using DPPH and hydrogen peroxide methods as the (%) reduction in the
absorbance and used them to plot the free radical scavenging activity profiles of Calotropis
gigantea leaves extracts, buds extracts and flowers extracts against their respective
concentrations as represented in the Figures 1-6. These profiles in the figures also include the
standard or reference antioxidant (Ascorbic acid for DPPH method and quercetin for H2O2
Method) profile, providing a means for the comparison of the solvent extract antioxidant
profiles of Calotropis gigantea leaves, buds and flowers. The saturation free radical
scavenging activities and the corresponding Effective Dosage for the 50% free radical
scavenging activities (ED50) were noted for all the solvent extracts and represented in the
Figures 7 & 8 with respect to DPPH and hydrogen peroxide methods. Similarly the reducing
power of the residual solvent extracts of Calotropis gigantea leaves, buds and flowers were
represented as the absorbance profiles against their respective variable concentrations as
shown in the Figures 9-11 along with the absorbance profiles of standards, ascorbic acid and
BHT. The reducing power of the standards were so high that the ascorbic acid test results
were diluted to 5% and the BHT results were diluted to 12% for the convenience. However
the data represented in the profiles and the figures were taken as the average of the triplicates.


                                               171
International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN
0976 – 6480(Print), ISSN 0976 – 6499(Online) Volume 4, Issue 4, May – June (2013), © IAEME




 Fig.1 DPPH free radical scavenging activity profiles of Calotropis gigantea leaves extracts
                                             p




  Fig.2 DPPH free radical scavenging activity profiles of Calotropis gigantea buds extracts
                                              p




Fig.3 DPPH free radical scavenging activity profiles of Calotropis gigantea flowers extracts
                                            p


                                            172
International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN
0976 – 6480(Print), ISSN 0976 – 6499(Online) Volume 4, Issue 4, May – June (2013), © IAEME




   Fig.4 Hydrogen peroxide free radical scavenging activity of Calotropis gigantea leaves
                                          extracts




Fig.5Hydrogen peroxide free radical scavenging activity of Calotropis gigantea buds extracts
     Hydrogen




  Fig.6 Hydrogen peroxide free radical scavenging activity of Calotropis gigantea flowers
                                         extracts

                                            173
International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN
0976 – 6480(Print), ISSN 0976 – 6499(Online) Volume 4, Issue 4, May – June (2013), © IAEME




   Fig.7 Variations in the saturation of DFRSA of Calotropis gigantea extracts on DPPH
                    radicals and the corresponding ED50 concentrations




  Fig.8 Variations in the saturation of HFRSA of Calotropis gigantea extracts on hydrogen
                peroxide radicals and the corresponding ED50 concentrations




  Fig. 9 Comparative reducing power of Calotropis gigantea leaves extracts along with the
                       standard antioxidants, ascorbic acid and BHT

                                           174
International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN
0976 – 6480(Print), ISSN 0976 – 6499(Online) Volume 4, Issue 4, May – June (2013), © IAEME




  Fig. 10 Comparative reducing power of Calotropis gigantea buds extracts along with the
                   standard antioxidants, ascorbic acid (AA) and BHT




 Fig. 11 Comparative reducing power of Calotropis gigantea flowers extracts along with the
                       standard antioxidants, ascorbic acid and BHT


4. DISCUSSION

        The free radical scavenging activity of many plant based extracts and the standard
                                                                                    [37
antioxidants like ascorbic acid and quercetin were dependent on their concentration [37-41],
however such free radical scavenging antioxidant properties of Calotropis gigantea solvent
                                                                           gigant
extracts on free radicals like that of DPPH were found to be saturating (for concentrations
                                           1-6.
beyond 5µg/ml) as shown in the Figures 1 6. The details of the results were discussed as
follows.

                                          gigantea
4.1 Antioxidant properties of Calotropis giganteaLeaves extracts
        The leaves extracts of Calotropis gigantea (Figures 1 & 4) reached the saturation
values of free radical scavenging activity by 5µg/ml concentration whereas the standard
antioxidant, ascorbic acid followed a gradually increasing free radical scavenging activity to
the saturation level at around 15µg/ml concentration. Conversely, the quercetin, the other
standard antioxidant was found (Figure 4) to reach the saturation levels of free radical

                                             175
International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN
0976 – 6480(Print), ISSN 0976 – 6499(Online) Volume 4, Issue 4, May – June (2013), © IAEME

scavenging activity on hydrogen peroxide by 5µg/ml concentration, but then its saturation
free radical scavenging activity was only of about half the free radical scavenging activity
saturation values of ascorbic acid (Figure 1). Similarly the Calotropis gigantea leaves
extracts recorded the higher (twice) free radical scavenging saturation activities in case of
ethanol, methanol & chloroform leaves extracts on DPPHradicals compared to that of
hydrogen peroxide radicals; whereas the aqueous leaves extracts exhibited higher free radical
scavenging activity on hydrogen peroxide compared to that of DPPH radicals (Figures 1, 4, 7
& 8). These results were also supported by the increasing reducing power of Calotropis
gigantea leaves extracts as shown in Figure 9 where the leaves extracts reducing power
profiles against the concentration were gradually increasing but then these profiles were very
low in reducing power in comparison to that of the standard antioxidants, ascorbic acid and
BHT. The effective dosage for 50% saturating free radical scavenging activity (ED50) as
shown in the Figures 7 and 8 for various Calotropis gigantea solvent extracts, brought out
ethanol leaves extract with lowest ED50 concentration of 2µg/ml on DPPHfreeradicals and
aqueous leaves extract with highest ED50 concentration of 4µg/ml on hydrogen peroxide free
radicals.

4.2 Antioxidant properties of Calotropis giganteaBuds extracts
        The free radical scavenging activity of Calotropis gigantea buds extracts were shown
in the Figures 2, 7 & 4, 8 with respect to DPPH and hydrogen peroxide free radicals where
the methanol buds extract recorded higher percentage of free radical scavenging activity on
DPPH radicals (80%) and the least ED50 concentration was found as 2.2µg/ml with respect
to hydrogen peroxide radicals. However the ethanol buds extract has shown decreasing
activity on DPPH radicals beyond 5 µg/ml concentration whereas its converse profile was
observed on hydrogen peroxide radicals, otherwise all other buds extracts exhibited the
saturating free radical scavenging activity for the respective concentrations (>5µg/ml). These
results were also supported by the moderate and decreasing reducing power of the Calotropis
gigantea solvent extracts of leaves, buds and flowers as shown in the Figure 10.

4.3 Antioxidant properties of Calotropis giganteaFlowers extracts
        The Calotropis gigantea flowers extract free radical scavenging activity with respect
to variation in their respective concentrations in the solvents were shown in the figures 3 & 6
where ethanol flowers extract exhibited higher and lower scavenging activities on DPPH
(76%) and hydrogen peroxide (15%) radicals respectively. On the other hand aqueous
flowers extracts had the least scavenging activity on DPPH (10%) free radicals and the higher
scavenging activity on hydrogen peroxide (41%) free radicals. Conversely, the chloroform
flowers extracts recorded moderately saturating free radical scavenging activity on both
DPPH and hydrogen peroxide free radicals whereas these chloroform flower extracts
reducing power profiles were found to exhibit lowest saturating reducing powers as shown in
the Figure 11. In another interesting finding, the methanol flowers extracts recorded
increasing free radical scavenging activity on hydrogen peroxide free radicals beyond the
concentration of 5 µg/ml. However the reducing power of methanol flowers extract was
found to be decreasing with increasing concentration whereas the reverse effects were found
in case of aqueous flowers extracts. These observed free radical scavenging activities of the
solvent extracts of Calotropis gigantea leaves, buds and flowers may be due to the bioactive
compounds like tannins, polyphenols and alkaloids [42-43] etc. present as the residual
components in these solvent extracts.

                                             176
International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN
0976 – 6480(Print), ISSN 0976 – 6499(Online) Volume 4, Issue 4, May – June (2013), © IAEME

5. CONCLUSIONS

        The study has provided an evidence for the antioxidant free radical scavenging
activities of Calotropis gigantea leaves, buds and flowers extracts of ethanol, methanol,
chloroform and aqueous solvent extracts. These results also demonstrated the comparable
free radical scavenging activities for some of the solvent extracts like, ethanol leaves extract,
methanol leaves extract, ethanol buds extract, methanol buds extract, ethanol flowers extract
and methanol flowers extract on DPPH radicals with that of the standard antioxidant, ascorbic
acid. Similarly, methanol, chloroform and aqueous extracts of leaves, buds and flowers also
exhibited the comparable free radical scavenging activity on hydrogen peroxide radicals with
that of the standard antioxidant, quercetin. However the reducing power of these solvent
extracts of Calotropis gigantea leaves, buds and flowers were very low in comparison to that
of the standard antioxidants like ascorbic acid and BHT. So these results support the use of
Calotropis as part of the traditional medicine to a considerable extent as these extracts
recorded the moderate antibiotic and wound healing properties [44-48] that may aid as part of
the treatment to terminate the pathogens and their effects on animals and humans.

ACKNOWLEDGEMENT

       Dr. M Lakshmi Narasu, Professor & Dr. ArchanaGiri, Assistant Professor, Faculty of
Biotechnology, Jawaharlal Nehru Technological University, Hyderabad, Dr.
ArchanaGajbhiye, Head, Department of Biotechnology, Sir Padampat Singhania University,
Udaipur were acknowledged for their suggestions and critical comments on the research
work. Mr. Ashok Ghosh, President, Ms. Rinu Ghosh, Vice President, Prof. P.C. Deka, Vice
Chancellor and Prof. AchintyaChaudary, Dean, School of Engineering of Sir Padampat
Singhania University, Udaipur were also acknowledged and thanked for their financial
support and approvals for the research work.

REFERENCES

[1]     J Chaudiere, and R. Ferrari-Iliou, Intracellular antioxidants: From chemical to
biochemical mechanisms, Food and Chemical Toxicology, 37, 1999, 949–962.
[2]     T Finkel, and N.J. Holbrook, Oxidants, oxidative stress and the biology of ageing,
Nature,408, 2000, 239–247.
[3]     M Valko, D. Leibfritz, J. Moncol, M.T. Cronin, M. Mazur, and J. Telser, Free
radicals and antioxidants in normal physiological functions and human disease, The
International Journal of Biochemistry and Cell Biology,39, 2007, 44–84.
[4]     Y Christen, Oxidative stress and Alzheimer disease, The Americal journal of clinical
nutrition, 71, 2000, 621S–629S.
[5]     L.A del Río, L.M. Sandalio, J.M. Palma, P. Bueno, and F.J. Corpas, Metabolism of
oxygen radicals in peroxisomes and cellular implications, Free Radical Biology &
Medicine,13, 1992, 557–580.
[6]     D.C Hooper, G.S. Scott, A. Zborek, T. Mikheeva, R.B. Kean, H. Koprowski, and
S.V. Spitsin, Uric acid, a peroxynitrite scavenger, inhibits CNS inflammation, blood-CNS
barrier permeability changes, and tissue damage in a mouse model of multiple sclerosis,The
FASEB Journal,14, 2000, 691–698.


                                              177
International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN
0976 – 6480(Print), ISSN 0976 – 6499(Online) Volume 4, Issue 4, May – June (2013), © IAEME

[7]     P Jha, M. Flather, E. Lonn, M. Farkouh, and S. Yusuf, The Antioxidant Vitamins
and Cardiovascular Disease: A Critical Review of Epidemiologic and Clinical Trial Data,
Annals of Internal Medicine,123, 1995, 860–872.
[8]     K.T Khaw, and P. Woodhouse, Interrelation of vitamin C, infection, haemostatic
factors, and cardiovascular disease, BMJ,310, 1995, 1559–1563.
[9]     A Krieger-Liszkay, Singlet oxygen production in photosynthesis, Journal of
Experimental Botany,56, 2004, 337–346.
[10] A Nunomura, R. Castellani, X. Zhu, P. Moreira, G. Perry, and M. Smith,.
Involvement of oxidative stress in Alzheimer disease, Journal of Neuropathology and
Experimental Neurology,65, 2006, 631–641.
[11] S Raha, and B.H. Robinson, Mitochondria, oxygen free radicals, disease and ageing,
Trends in Biochemical Sciences,25, 2000, 502–508.
[12] M Valko, M. Izakovic, M. Mazur, C.J. Rhodes, and J. Telser, Role of oxygen radicals
in DNA damage and cancer incidence, Molecular and Cellular Biochemistry,266, 2004, 37–
56.
[13] A Wood-Kaczmar, S. Gandhi, and N.W. Wood, Understanding the molecular causes
of Parkinson's disease, Trends in Molecular Medicine,12, 2006, 521–528.
[14] R A. Miller, and B.E. Britigan, Role of oxidants in microbial
pathophysiology,Clinical Microbiology Reviews,10, 1997, 1–18.
[15] N Smirnoff, L-Ascorbic acid biosynthesis, Vitamins and Hormones,61, 2001, 241–
266.
[16] A.L Sowell, D.L. Huff, P.R.Yeager, S.P.Caudill, and E.W. Gunter, Retinol, alpha-
tocopherol, lutein/zeaxanthin, beta-cryptoxanthin, lycopene, alpha-carotene, trans-beta-
carotene, and four retinyl esters in serum determined simultaneously by reversed-phase
HPLC with multiwave length detection, Clinical Chemistry,40, 1994, 411–416.
[17]     S Vertuani, A. Angusti, S. Manfredini, The Antioxidants and pro-antioxidants
network: An overview, Current Pharmaceutical Design,10, 2004, 1677–1694.
[18] H Sies, Strategies of antioxidant defense, Europian Journal of Biochemistry,215,
1993, 213–219.
[19] S Debajyothi, and T. Ankit,            Xenobiotics, oxidative stress, free radicals Vs.
Antioxidants: Dance of death to heaven’s life. Asian Journal of Research in Pharmaceutical
Sciences. 1(2), 2011, 36-38.
[20] M.F Balandrin, J.A. Klocke, E.S. Wurtele, and W.H. Bollinger, Natural plant
chemicals: Sources of industrial and medicinal materials. Science, 228,1985, 1154-1160.
[21] S.J Ahmad, K.K. Pillai, M. Ali, V. Nair, D. Anand, and S. Singh, Anti-inflammatory
and Antinociceptive Activity of Lawsoniainermis Linn Alcoholic Extract in Rats,
International Journal of Biological Sciences and Technology, 2, 2010, 16-21.
[22] I.F Benzie, 2003. Evolution of dietary antioxidants, Comparative Biochemistry and
Physiology Part A: Molecular & Integrative Physiology,136, 113–126.
[23] P.H Canter, H. Thomas, and E. Ernst, Bringing medicinal plants into cultivation:
opportunities and challenges for biotechnology, Trends in Biotechnology, 23, 2005, 180–185.
[24] S Karuppusamy, A review on trends in production of secondary metabolites from
higher plants by in vitro tissue, organ and cell cultures, Journal of Med Plants Res, 3, 2009,
1222–1239.
[25] A. Hamilton, and G.J. Martin, The People and Plants Initiative in Ethnobotany:
Methods and Manual (WWF International, London: Chapman& Hall, 1995).


                                             178
International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN
0976 – 6480(Print), ISSN 0976 – 6499(Online) Volume 4, Issue 4, May – June (2013), © IAEME

[26] V.L Kumar, and N.Basu, Anti-inflammatory activity of Calotropis procera, Journal
of Ethnopharmacology, 44, 1994, 123-125.
[27] A Mitra, S. Chakraborty, B. Auddy, P. Tripathi, , S. Sen, A.V. Saha, and B.
Mukherjee, Evaluation of chemical constituents and free-radical scavenging activity of
Swarnabhasma (gold ash) an ayurvedic drug, Journal of Ethnopharmacology, 80, 2002,147–
153.
[28] M David, K.R. Bharath, and M. Bhavani, Study of Calotropis gigantea R. Br. extracts
on growth and survival dynamics of selected pathogenic microorganisms, International
Journal of Biological Engineering, 1, 2011,1-5.
[29] SA Adesegun, A. Fajana,             C.I. Orabueze, and H.A.B. Coker, Evaluation of
Antioxidant Properties of PhaulopsisfascisepalaC.B.Cl. (Acanthaceae), eCAM, 6, 2009,
227–231.
[30] W Brand-Williams, M.E. Cuvelier, and C. Berset, Use of a free radical method to
evaluate antioxidant activity, LWT - Food Science and Technology, 28, 1995, 25-30.
[31] K Marxen, K.H. Vanselow, S. Lippemeier, R. Hintze, A. Ruser, and U.P. Hansen,
Determination of DPPH radical oxidation caused by methanolic extracts of some microalgal
species by linear regression analysis of spectrophotometric measurements. Sensors, 7, 2007,
2080-2095.
[32] P Molyneux, The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for
estimating antioxidant activity, Songklanakarin Journal of Science and Technology, 26, 2004,
211-219.
[33] M P. Czochra, and A.J. Widensk, Spectrophotometric determination of H2O2
activity, AnalyticaChemicaActa, 452, 2002, 177–184.
[34] C S. Moreno, Review: Methods used to evaluate the free radical scavenging activity
in foods and biological systems, Food Science and Technology International,8, 2002, 121-
137.
[35] A K. Sinha, Colorimetric assay of catalase, Analytical Biochemistry, 47,1972, 389–
394.
[36] G C. Yen, and H.Y Chen, Antioxidant activity of various tea extracts in relation to
their antimutagenicity, Journal of Agricultural Food Chemistry, 43, 1995, 27–32.
[37] H Meena, H.K. Pandey, P. Pandey, M.C. Arya, and Z. Ahmed, Evaluation of
antioxidant activity of two important memory enhancing medicinal plants Baccopamonnieri
and Centellaasiatica, Indian Journal of Pharmacology, 44, 2012, 114-117.
[38] A M. Pisoschi, and G.P. Negulescu, Methods for total antioxidant activity
determination: A review, Biochemistry and Analytical Biochemistry, 1, 2011, 106.
[39] C A. Rice-Evans, N.J. Miller, and G. Paganga, Structure antioxidant activity
relationships of flavonoids and phenolic acids, Free Radical Biology and Medicine, 20,1996,
933-956.
[40] S Y. Wang, and H.S. Lin, Antioxidant activity in fruits and leaves of blackberry,
raspberry, and strawberry varies with cultivar and developmental stage, Journal of
Agricultural Food Chemistry, 48, 2000, 140–146.
[41] H Zielinski, and H. Kozlowska, Antioxidant activity and total phenolics in selected
cereal grains and their different morphological fractions, Journal of Agricultural Food
Chemistry,48, 2000, 2008–2016.




                                            179
International Journal of Advanced Research in Engineering and Technology (IJARET), ISSN
0976 – 6480(Print), ISSN 0976 – 6499(Online) Volume 4, Issue 4, May – June (2013), © IAEME

[42] T Hatano, R. Edamatsu, M. Hiramatsu, A. Mori, Y. Fujita, and A. Yasuhara, Effects
of interaction of tannins with co-existing substances VI. Effects of tannins and related
polyphenols on superoxide anion radical and on DPPH radical, Chemical and Pharmaceutical
Bulletin, 37,1989, 2016-2021.
[43] M Tanaka, C.W. Kuei, Y. Nagashima, and T. Taguchi, Application of
antioxidativemaillrad reaction products from histidine and glucose to sardine products,
Nippon Suisan Gakkaishi, 54, 1998, 1409-1414.
[44] A M. Ashraful, M.R. Habib, F. Nikkon, M. Rahman, and M.R. Karim, Antimicrobial
activity of Akanda (Calotropis gigantea L.) on some pathogenic bacteria, Bangladesh journal
of scientific and industrial research, 43(3), 2008, 397-404.
[45] P Chiranjibi, C.S. Reddy, and N.K. Dhal, Phytomedicinal study of coastal sand dune
species of Orissa, Indian journal of traditional knowledge. 7(2), 2008, 263-268.
[46] V P. Kumar, N. S. Chauhan, H. Padh and M. Rajani, Search for antibacterial and
antifungal agents from selected indian medicinal plants. Journal of Ethnopharmacology, 107,
2006, 182-188.
[47] V Kurucheve, J. G. Ezhilan, and J. Jayaraj, Screening of higher plants for
fungitoxicity against Rhizoctoniasolani in vitro, Indian phytopathology, 50(2), 1997, 235-
241.
[48] Z S. Nudrat, R. Deo, and U. Mukundan, Herbal remedies used by Warlis of Dahanu to
induce lactation in nursing mothers, Indian Journal of Traditional Knowledge, 6(4), 2007,
602-605.
[49] Ramesh U and Kurian Joseph, “The Holistic Approach of Ayurveda Based Wellness
Tourism in Kerala”, International Journal of Advanced Research in Management (IJARM),
Volume 3, Issue 2, 2013, pp. 29 - 39. ISSN Print: 0976 – 6324, ISSN Online: 0976 – 6332.




                                           180

				
DOCUMENT INFO
Shared By:
Categories:
Tags:
Stats:
views:3
posted:6/21/2013
language:
pages:13