Studies on In Vitro Antioxidant Activities of Acetophenone Derivative of Helicanthus elasticus Linn. Stem (Loranthaceae)
K.A. Kedar*1, P.D. Chaudhari1, R.B. Jadhav2 and S.R. Chaudhari3
1Modern College of Pharmacy, Nigdi, Pune.
2 R.C. Patel College of Pharmacy, Shirpur, Maharastra. India.
3Amrutvahini College of Pharmacy, Tal- Sangamner, Dist-Ahmednagar, Maharastra, India
The present investigation evaluates the antioxidant activity of 7-methyl-1-phenyloctan-1-one of Helicanthus elasticus Linn. Stem (Loranthaceae) in various in vitro models. The n-hexane extract of Helicanthus elasticus Linn. (Loranthaceae) parasitic on Memecylon umbellatum Burm.f. (Melastomataceae) was subjected to column chromatography. The isolated compound was characterized by UV, IR, 1H NMR and Mass data. On comparing datas, it was confirmed that compound isolated from n-Hexane extract of Helicanthus elasticus was 7- methyl-1-phenyloctan-1-one. This is the first report stating the presence of such aromatic ketone from Helicanthus elasticus. The DPPH radical scavenging, nitric oxide scavenging and anti-lipid peroxidation activity of acetophenone derivative have IC50 value 10.23 µg/ml, 9.73 µg/ml, 16.45 µg/ml respectively. Thus results indicate that 7-methyl-1-phenyloctan-1-one showed potent antioxidant activity compare with standards, ascorbic acid and BHA.
The in vitro studies clearly indicate that the acetophenone derivative of Helicanthus elasticus Linn. Stem (Loranthaceae) has significant antioxidant activity.
Antioxidants are a type of complex compounds found in our diet that act as a protective layer for our body against certain diseases such as arterial, cardiac diseases, arthritis, cataracts and also premature aging along with several chronic diseases.1
A genus Loranthus is evergreen, shrubby, partial parasitic and stems are much branched, often joined. The different species of Loranthus genus mostly used in treatment of diseases of women as an antifertility agent, also in vesical calculi and kidney affection2. Bark of some species useful in wounds, mania and asthma3.
Helicanthus elasticus is a large bushy, usually glabrous plant. Barks are grey, smooth young parts glabrous or nearly so. Leaves are thick, coriaceous, usually opposite 3.8-10 by 2-4.5 cm, very variable in shape and, ovate elliptic, oblong-lanceolate. Midrib is prominent usually red. Flowers are sessile fascicled at the nodes; bracts are red and broadly ovate and about 1.2 mm. long2.
Angiosiospermic parasitic plant Helicanthus elasticus reported to contain biologically active substances such as flavonoid, quercetin and some proteins reported to have in-vitro Cytotoxicity4.
The purpose of this work was to study the antioxidant activity of acetophenone derivative of Helicanthus elasticus Linn.
MATERIAL AND METHODS:
The mistletoe, Helicanthus elasticus Linn. Parasitic on Memecylon umbellatum Burm.f. (Melastomataceae) were collected from Western Ghats (Latitude 17° 55' 0N and Longitude 73° 40' 0E and at altitude 1352 m) in February 2006.
A voucher specimen has been deposited in the herbarium of the Botanical Survey of India, under voucher no. LOT-1.
Preparation of plant Extract and fraction:
In brief, the powder sample (500gm) was extracted with n-hexane by cold maceration method. The extract was collected and combined and concentrated using rotary vacuum evaporator. The extract so obtained was then stored in desiccators before charging on column. The crude extract (3.5 g) obtained from above procedure was subjected to column chromatography (200g silica gel, #60-120) packed in hexane. Elution was started with hexane and polarity was gradually increased with ethyl acetate. The collected fractions (50ml each) was monitored by TLC using n- Hexane: Dichloroethane: Methanol (6.4: 6.4: 0.2) solvent system and visualization was done by Anisaldehyde-H2SO4. From all, fractions numbers (51-67) were combined and processes for recolumn chromatography. The Rf value of acetohenone derivative was found to be 0.8 and similar fractions were combined, evaporated and purified by crystallization with ethyl acetate.
Characterization of isolated compound:
The pure compound (70mg) obtained from column chromatography was subjected to UV, IR, Mass and NMR studies. The UV absorption spectrum of the compound in hexane was recorded in range of 200-500 nm on Shimadzu UV-1610 Spectrophotometer (with 10×10 mm quartz cuvettes). The IR Spectrum of the compound was recorded on FT-IR 8400S by press pellet technique with AR grade KBr.
A mass spectrum was recorded on LC-MS/MS Ion Trap Varian-500 MS. At electron energy of 70 V at source temperature 190oC. H NMR spectra was recorded on a Joel FT-NMR-300MHz in CDCl3 using TMS as an internal standard.
I) Determination of DPPH radical scavenging activity:5
1 ml of sample solution from all concentration range was taken in vials. To this, 5ml of methanolic solution of DPPH was added, shaken well and the mixture was incubated at 37 C for 20min. The absorbance was measured against methanol as a blank at 517nm. The absorbance of DPPH was taken as a control. The percent antiradical activity was calculated by using following formula6
% antiradical activity = (A Control. – A sample) / A Control X 100
Where A Control: Control absorbance (DPPH).
A sample: Sample/standard absorbance.
Nitric oxide radicals were generated from sodium nitroprusside solution at physiological pH. Sodium nitroprosside (1 ml, 10 mM) was mixed with 1ml solution of compound (10-80µg/ml) in phosphate buffer (pH 7.4). The mixture was incubated at 25 C for 150 min. To 1 ml of incubated solution, 1ml of Griess reagent (a-naphthyl-ethylenediamine dihydrochloride 0.1% in water and sulfanilamide 5% in H3PO4) was added and absorbance was read at 546 nm. The same reaction mixture without the sample but equivalent amount of distilled water was served as control.
III) Determination of Reducing Power Assay:8
To 1ml of different concentrations of sample (10-80µg/ml), 2.5ml of phosphate buffer (0.25 M) and 2.5 ml of potassium ferricyanide (1% w/v) were added. The mixture was incubated at 50 C for 20min. After incubation 2.5ml of TCA was added to the reaction mixture, which was then centrifuged at 3000 rpm for 10 min. Then 2.5ml of supernatant was taken and to it add 2.5ml of distilled water and 0.5ml of ferric chloride solution (0.1% w/v) and the absorbance was measured at 700 nm.
The mixture containing 0.5 ml of homogenate, 1ml of 0.15 ml KCl and 0.5 ml of different conc. of drug extract were prepared. Lipid peroxidation was initiated by adding 100 µl of 1mM ferric chloride .The reaction mixtures were incubated for 30 min. at 37 C. After incubation the reaction mixture was stopped by adding 2ml of ice-cold 0.25N HCl containing 15% TCA and 0.38% TBA and 0.2 ml of 0.05% BHT. These reactions mixtures were heated for 60 min at 80ºC. Cooled and centrifuged at 5000 rpm for 15 min. The absorbance of the supernatant was measured at 532nm against a blank with contained all reagent except liver homogenate and drug. Identical experiments were performed to determine the normal (without drug and ferric chloride) and induced (without drug) lipid peroxidation leveling the tissue. The % of anti-lipid peroxidation effect (%ALP) was calculated by following formula.
Ferric chloride O. D. - Sample O. D.
%ALP = ------------------------------------------- ×100
Ferric chloride O. D. - Normal O. D
RESULT AND DISCUSSION:
The UV absorption spectrum of the compound in hexane was recorded in range of 200-500 nm on Shimadzu UV-1610 Spectrophotometer (with 10×10 mm quartz cuvettes). Light absorption of isolated compound in hexane in range of 200-500 nm. UV (hexane) λ max (log Є) 256 (0.016), 277 (0.012), 280(0.010), 310 (0.012) (Fig.1).
The IR spectrum of the compound was recorded on FT-IR 8400S by press pellet technique with AR grade KBr. IR (KBr), nmax 2925.81, 2858.31, 1757.03, 1718.46, 1654.81, 1560.3, 1508.23, 1458.23, 1458.08, 1388.65 cm-1 (Fig.2).
A mass spectrum was recorded on LC-MS/MS Ion Trap Varian-500 MS. From spectral data, molecular ion peak and base observed at m/z 218 (M+ H+) and 147 (M+ H+) respectively (Fig.3). So from above data fragmentation pattern of compound given below.
Interpretation of Mass spectra of compound
An H NMR spectrum was recorded on a Joel FT-NMR-300MHz in CDCl3 using TMS as an internal standard. From NMR data compound contain aromatic ring (7.3 d), CH2-CH2-CH2 (0.5-2 d) (Fig.4).
The compound isolated was white flakes like in nature, UV (n- Hexane) lmax (log e) 371(0.008), 310(0.012), 280 (0.0100), 277(0.012), 256 (0.016). lmax above 200nm means it may contain conjugated diene or α β unsaturated ketone. IR (KBr), nmax 2925.81, 2858.31, 1757.03, 1718.46, 1654.81, 1560.3, 1508.23, 1458.23, 1458.08, 1388.65 cm-1.The Rf = 0.8, The turbo spray MS shown molecular ion peak at m/z 218 (M+H+). From NMR data compound contain aromatic ring (7.3 d), CH2-CH2-CH2 (0.5-2 d).
On comparing datas, it was confirmed that compound isolated from n-Hexane extract of Helicanthus elasticus was 7- methyl-1-phenyloctan-1-one.
The isolated compound was further investigated for its in vitro antioxidant activity using DPPH (Fig. 5) and nitric oxide radicals (Fig. 6), lipid-peroxidation (Fig. 8) and reducing power (Fig. 7). The IC50 values were 10.23 µg/ml, 9.73 µg/ml, 16.45 µg/ml respectively. Thus these results indicate that compound was equipotent to reference standards, ascorbic acid and BHA.
Several acetophenone derivatives have been reported to have potent anti-oxidant activity10, anti-inflammatory activity11. It was also found that, photo-antioxidant effects of such compounds can be improved by enlarging the conjugation system of group bonded to 2- hydroxyl benzoyl carbonyl group12.
Authors are thankful to Dr. Milind Sardesai and Dr. C. B. Salunke for taxonomical identification of the plant material.
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