Abstract: The aim of the present study was to isolate the active constituents responsible for antioxidant activity. Radical scavenging activities of chromatographically isolated compounds from methanolic extracts of wood, bark, fruit and leaf were measured by the 1, 1-Diphenyl-2- Picrylhydrazyl (DPPH) method. The structures of isolated compounds were confirmed by spectroscopic techniques comprising of UV, IR, 13C NMR, P-NMR, Mass spectral and Co-TLC studies. The compound ANA 01, ANA 02 and ANA 04 were isolated from bark and confirmed as catechin, ellagic acid and gallic acid, respectively. The leaf extract resulted in separation of compounds ANA 03 (quercetin). The free radical scavenging activity of the different isolated compounds from methanolic extracts of Casuarina equisetifolia increased in a concentration dependent manner. ANA 04 (gallic acid) exhibited very strong antioxidant activity and when compared to ANA 01 (catechin), ANA02 (ellagic acid), ANA 03 (quercetin) and ANA05 (lupeol). This study suggests that the Casuarina equisetifolia could be pharmaceutically exploited for antioxidant properties.
INTRODUCTION
Casuarina equisetifolia (Casuarinaceae) is handsome tree with drooping branches, 10-50 m high (Mhaskar et al., 2000), it is found in dry hill sides and open forests of India, Sri Lanka and to Australia.
The following phytoconstituents have been isolated from the plant so far; kaempferol, quercetin (EL-Ansari et al., 1977), alicyclic acids (shikimic and quinic acid), amino acids (Madhusudanamma et al., 1978), taraxerol, lupenone, lupeol, gallic acid, sitosterol (Rastogi and Mehrotra, 1998), catechin and gallocatechin (Roux, 1957; Madhulata et al., 1985).
The plant is used as astringent (Mhaskar et al., 2000), diarrhea (Chopra et al., 1956), dysentery, cough, ulcers, toothache, lotion for swelling (Wealth of India, 1992) and diabetes (Prajapati et al., 2003). Pharmacological investigations of bark and wood showed significant anticancer and anthelmintic activities (Aher et al., 2006, 2008).
The biological activities, viz., anticancer, antibacterial (Wealth of India, 1992) and hypoglycemic, antifungal (Han, 1998) of the leaf has been reported.
The DPPH assay was used in the measurement of scavenging ability of isolated test compounds. The DPPH radical is reduced in the presence of an antioxidant molecule, the electron becomes paired off showing the color change from blue to uncolored methanol solutions stoichiometrical; depending on the number of electron taken up (Sundararajan et al., 2006; Choudhary, 2006).
The phenolic phytoconstituents isolated in earlier studies prompted to evaluate antioxidant potential of Casuarina equisetifolia.
MATERIALS AND METHODS
Authentification of Plant Material
The plant specimen was collected from Gangapur dam locality, Nashik (M.S.)
identified as Casuarina equisetifolia Linn. Family Casuarinaceae, Voucher
No. ANA1, Ref. No. BSI/WC/Tech./2005/867 dated 22.12.2005 by P.S.N. Rao, Joint
Director, Botanical Survey of India, Pune (M.S.).
Preparation of Plant Extracts
Coarsely powdered materials of leaf and bark (100 g each) were subjected
to reflux with 250 mL of methanol for 4 h coded as MEL and MEB followed by subsequent
filtration and evaporation to yield extract.
Separation and Isolation of Phytoconstituents by Chromatographic Methods
Gradient fractionation of acetone soluble part from methanolic bark extract
by column chromatography was performed by the using of toluene: ethyl acetate:
methanol (4:3:3) followed by methanol resulting in the isolation of compound
designated as ANA 01(catechin), ANA 04 (gallic acid) and ANA 02(ellagic acid).
Methanolic extract of leaf was chromatographed with toluene: ethyl acetate: formic acid (5:4:1), followed by spraying with ferric chloride. The presence of quercetin was evidenced in UV light. The methanolic extract of leaf was processed by PTLC to isolate compound coded as ANA 03 (quercetin).
Characterization of Isolated Compounds
The isolated compounds were characterized with help of physical properties
and the instrumental techniques.
Characterization of Compound ANA01
UV (MeOH) max: 219, 279 nm.
FT-IR: (KBr): 3820.10, 3391.85 (O-H), 2933.20, 2892.27, 1627.56 (C=O), 1522.37, 1471.52, 1289.96 (C-O-C), 1245.99, 1198.02 (C-O) and 868.70 cm-1.
LC-ESI-MS: m/e: 290 (5%, M+, C15H16O+6), 289 (12%, M+-1), 245 (32%, M+-CH3O2), 205 (100%, M+-C2H6O4), 203 (40%) and 179 (5%).
13C-NMR (DMSO, 300 MHZ): δ 156.59(C-7), 156.33(C-5), 155.51(C-3’), 144.99(C-4’), 130.75(C-8), 118.64(C-6), 115.26(C-6’), 114.64(C-9), 99.23(C-10), 95.27(C-5’), 94.02(C-2’), 81.12(C-3), 66.45(C-2) and 27.98(C-4).
1HNMR (DMSO, 300 MHZ): δ 9.01(s), 6.72(1H,H-6,8,s), 6.68(1H,H-2’,5’,s), 6.61(1H,H-6’,s), 5.88(s), 5.70(1H,H-5,7,s), 4.92(1H,H-3’,4’,s), 4.48(1H,H-3,s), 3.56(1H,H-3,s), 2.39(s),2.37(s) and 2.64(2H,H-4,d).
Characterization of Compound ANA0
UV (MeOH) max: 365.0, 254.0 nm.
FT-IR: (KBr): 3072.72, 3596.20(O-H), 1699.60(C=O), 1622.21(C=C), 1581.82, 1509.10, 1447.52, 1398.43(C-O), 1195.11 and 882.06 cm-1.
LC-ESI-MS: m/e: 301.8(10%, M+-C14H6O+8) 300.7(10%), 283.8(40%), 271.9(4%), 256.8 (50%, M+-H2O+O2), 228.9(100%, M+-CH3O4), 212.9(8%), 200.9(30%) and 184.9(60%).
13C-NMR (C6D5N, 300MHz): δ 164.23(C-7,7’), 142.09(C-2,3,2’,3’), 124.48(s), 123.11 (C-4,5,6,4’,5’,6’) and 111.92(C-1,1’).
1HNMR (C6D5N, 300 MHZ): δ 8.14(1H,H-1’,s) and 5.28(1H,H-2,3,2’,3’,s).
Characterization of Compound ANA03
UV (MeOH) max: 255, 372 nm.
FT-IR: (Kbr): 3405.97(O-H), 3100.53, 1667.12(C=O), 1627.56(C=C), 1522.37, 1471.52, 1319.97(C-O), 1262.97(C-O-C), 1198.94 and 868.70 cm-1.
LC-ESI-MS: m/e: 301.7 (0.5%, M+,C15H10O+6), 272.9 (6%),256.7 (15%), 229.2(8%), 192.8 (10%), 178.8(100%, M+-C6H6O2) and 150.9(65%, M+-C7H6O3).
13C-NMR (d6- acetone, 300 MHZ): δ 177.70 (C-4), 165.95 (C-7), 162.87 (C-5), 158.58 (C-3), 149.13 (C-3’), 148.35 (C-4’), 146.59 (C-2), 137.61 (C-1’), 124.51 (C-9), 122.04 (C-10), 116.98 (C-8), 116.36 (C-6), 104.88 (C-6’), 99.60 (C-2’) and 94.78 (C-5’).
1HNMR (d6- acetone, 300 MHZ): δ 7.74(1H,H-6,s), 7.73(1H,H-8,s), 7.65(1H,H-2’,s), 7.62 (1H,H-5’,s), 6.89(1H,H-6’,s), 6.86(1H,H-3,), 6.38(1H,H-7,s), 6.18(H-5,s) and 4.88(1H,H-3’,4’, s).
Characterization of Compound ANA04
UV (MeOH) λmax: 272.8 nm.
FT-IR: (KBr): 3285.28(O-H), 3072.72, 1703.34(C=O), 1615.63(C=C), 1501.90, 1446.96, 1339.41, 1245.99 and 867.96 cm-1.
LC-ESI-MS: m/e: 169.9 (M+, C6H6O+5,), 168.9 (100%, M+-1) and 125 (40%, M+-COOH).
13C-NMR (d6- acetone, 300 MHZ): δ 170.936 (C-7), 146.755 (C-3,C-5), 140.023 (C-4), 122.792 (C-1) and 110.79 (C-2,C-6).
1HNMR (d6-acetone, 300 MHZ): δ 9.136 (1H,H-7,s), 7.08 (1H,H-2,H-6,s) and 5.011 (1H,H-3, H-4,H-5,s).
Antioxidant Assay
DPPH Radical Scavenging Activity
Preparation of Test Solutions for Isolated Compounds
Quercetin from leaf, catechin, gallic acid and ellagic acid from bark were dissolved in methanol (500 μg mL-1 each).
DPPH Radical Scavenging Activity
DPPH scavenging activity was measured by spectrophotometric method (Choudhary,
2006; Ravishankara et al., 2002). Methanolic solution of DPPH 100 μL
(1.3 mg mL-1) was added in 4 mL of methanol to give initial absorbance
of 0.9 at 516 nm. To methanolic solution of DPPH (100 μL), test extracts
dissolved in methanol were added at different concentrations (25-100 μg
mL-1). The decrease in absorbance of test mixtures was read at 516
nm against test blank and the percentage inhibition (Table 1)
was calculated by using the formula:
Statistical Analysis
Results were expressed as Mean±SD of three replicates and average
value was considered. The IC50 value was calculated using linear
regression analysis of the percent inhibition obtained using different concentrations.
The regression equation was obtained and the concentration required to produce
50% inhibition (IC50) was calculated.
RESULTS AND DISCUSSION
IC50 values showed by isolated polyphenolic compounds i.e., gallic
acid, ellagic acid and catechin from bark justifies anti oxidant potential {IC50
μg mL-1 2.23, 3.67 and 12.14>14.10 (ascorbic acid)} supports
the anticancer activity of bark (Acharya et al., 2004; Aher et al.,
2008; Velavan et al., 2007; Wealth of India, 1992).
Isolation of Phytoconstituents
The isolated constituents were characterized with help of the instrumental
techniques as UV spectroscopy, IR spectroscopy, Mass spectrometry and NMR spectroscopy.
Co-TLC of the isolated constituents with authentic compounds was also used as
criteria for their identification. Methanolic extract of bark offered three
compounds. These were identified as ANA01 (catechin), ANA02 (ellagic acid) and
ANA04 (gallic acid), respectively. Methanolic extract of leaf showed the presence
of ANA03 (quercetin).
Radical Scavenging Activity
The DPPH racial scavenging activity of the isolates increases with increasing
concentration. The isolated compounds show more scavenging activity in comparison
to ascorbic acid (Table 1).
The gallic acid from bark was found to most effective (IC50 2.23 μg mL-1) than catechin, ellagic acid from bark and quercetin from leaf (IC50 12.14, 3.67 and 3.67 μg mL-1) when compared with standard ascorbic acid (IC50 14.10 μg mL-1).
Oxidation is involved in decomposition of pharmaceutical preparation containing steroids, vitamins and antibiotics. Reactive Oxygen Species (ROS) are produced continuously in the cells as accidental bye-products of metabolism which are cytotoxic and are important factors for several pathological conditions such as cardiovascular diseases, diabetes, inflammation and cancer etc. (Acharya et al., 2004).
Flavonoids and phenolic compounds widely distributed in plants which have been reported to exert multiple biological effect, including antioxidant, free radical scavenging abilities, anti-inflammatory, anticarcinogenic etc. (Velavan et al., 2007).
DPPH is one of the free radicals, stable at room temperature generally used for testing preliminary radical scavenging activity of a compound or a plant extract. The decrease in the concentration of DPPH radical due to scavenging ability of compounds isolated from Casuarina equisetifolia showed better activity.
Therefore, in this study, the antioxidant properties of the isolated compounds
from methanol extracts of bark and leaf of Casuarina equisetifolia were
examined for DPPH radical scavenging activity according to the method described
and the results of the screening are shown in Fig. 1 as comparable
with known antioxidant ascorbic acid. In terms of antioxidant activity, all
the compounds investigated exhibited a rather high degree of activity.
| Table 1: | Antiradical activity for isolated compounds from various plant parts observed with DPPH |
| Values are Mean of three replicates and expressed as Mean±SEM | |
| Fig. 1: | DPPH scavenging activity of isolated compounds |
In particular, compound gallic acid isolated from bark displayed the highest activities as antioxidant activity as removal of the stable radical DPPH (Fig. 1) and the lowest activity were found in catechin (from bark). As expected, the overall activity of the isolated compounds were higher than that of commercial antioxidant ascorbic acid, the reference antioxidant (Fig. 1).
In conclusion, the screening of antioxidant activity performed on Casuarina equisetifolia, which traditionally used as anti cancer and for swelling (Aher et al., 2008; Wealth of India, 1992) shows that they are endowed with potentially exploitable antioxidant activities.
ACKNOWLEDGMENTS
Authors are thankful to the Dr. V.R. Gudsoorkar, Principal, NDMVPS College of Pharmacy, Nashik for providing necessary facilities. The authors are also thankful to I.I.T., Powai, Bombay for permitting in spectral analysis and assisting in structural elucidation of the isolated compounds.