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The plant material consists of dried powdered seeds and fresh leaves of Raphanus sativus L. belonging to the family Brassicaceae and dried powdered seeds of Apium graveolens L. belonging to the family Apiaceae.
Plant collection and authentication
The seeds and leaves of Raphanus sativus Linn and seeds of Apium graveolens Linn were collected from Coimbatore and Ooty respectively, Tamilnadu, India during the month of June 2010. The plant was identified and authenticated by Mr. G.V.S. Murthy, Joint Director scientist, C-I/C, Botanical survey of India, TN Agricultural University Campus, Coimbatore bearing the reference no BSI/SC/5/08-09/Tech-730 and BSI/SC/5/08-09/Tech-766.
Chemicals and reagents
Angiotensin converting enzyme (EC.18.104.22.168) from rabbit lung, FAPGG [furnacryloyl-l-phenylalanylglycylglycine] and Lisinopril was purchased from Sigma - Aldrich, USA. 2-deoxy-2-ribose, (2-[4-(2-Hydroxyethyl)-1-piperazinyl] ethane sulphonic acid (HEPES) buffer, pyrocatechol and quercetin were purchased from Himedia Labs., Pvt. Ltd., Mumbai. Acetonitrile, (0.2μ filtered) was obtained from Sisco Research Laboratories Ltd. Mumbai. Thiobarbituric acid, Trifluroacetic acid, Trichloroacetic acid and Folin- ciocalteau reagent were purchased from SDF Chemicals Ltd., Mumbai. All other drugs and chemicals used for the work were purchased commercially.
Digital Balance (Sartorius Ltd, USA), Eppendorff minispin, Shimadzu-Jasco V-530 UV/Vis spectrophotometer, Elco L1/27 pH meter, Mechanical shaker, HPLC instrument with L-4000 UV detector, L-6200 Intelligent pump and LiChrosorb RP-18 column (50 mm - 7 mm) from Hitachi with Data Ace workstation was used for analysis (Hitachi Ltd., Tokyo, Japan).
Preparation of the extract
Preparation of ethanolic seed extract
The seeds of Raphanus sativus L. was collected and washed thoroughly with distilled water and dried in open air at room temperature for 24 hours. The seeds were powdered mechanically and sieved through No. 20 mesh sieve. About 500 g of powder was soaked with 2 litres of ethanol (95%) for 12 h and then macerated for 4 h at room temperature using a mechanical shaker. The extract was filtered with a porous cloth and the residue was again soaked with the same volume of 95% ethanol for 12 h and then further extracted for 4h and filtered. The filtrates were then combined together and concentrated under reduced pressure and evaporated at 40°C (Mirza, 2003). The R.sativus seeds ethanolic extract was designated as RSEE.
Preparation of aqueous leaf extract
Around 500g of leaves of Raphanus sativus L. was washed with tap water and then soaked in 2.75 L of distilled water for 3 days. The plant material was filtered off through a piece of permeable cloth and filter paper and the filtrate was collected. This procedure was repeated twice and the collective filtrate was concentrated in a rotary evaporator at 40°C to yield a thick, viscous extract (Gilani et al., 2004). The R.sativus leaf aqueous extract was designated as RLAE.
Preparation of methanolic seed extract
The seeds of Apium graveolens L. was collected, powdered mechanically, sieved through No. 20 mesh sieve and defatted with petroleum ether (60°-80°C) for 30 minutes. About 100g of defatted powdered seeds was extracted with 350 ml of methanol using a Soxhlet extractor. After thorough extraction, the methanolic extract was concentrated at 60°C (Singh et al., 2004). The A.graveolens seeds methanolic extract was designated as ASME.
The above extracts were used for the in vitro ACE inhibitory and in vitro anti-oxidant activities.
Chemical tests were carried out for the extract of Raphanus sativus L. and Apium graveolens L. for the presence of phytochemical constituents (Trease and Evans, 2002).
Test for tannins and phenolics
To the solution of the extract, 0.1% ferric chloride was added in drops and then observed for a formation of a bluish black or brownish green colouration.
Test for saponins
About 10 ml of the plant extract and 5 ml of water was shaken vigorously for a constant froth formation. About 3 drops of olive oil was further added to the frothing, mixed well and then observed for the formation of an emulsion.
Test for steroids
Around10 mg of extract was dissolved in 1ml of chloroform. 1ml of acetic anhydride was added following the addition of 2ml of concentrated sulphuric acid. Formation of reddish violet colour indicates the presence of steroids.
About one ml of concentrated sulphuric acid was added to 10mg of extract dissolved in 1 ml of chloroform. A reddish blue colour exhibited by chloroform layer and green fluorescence by the acid layer indicates that steroids are present in the test sample.
Test for flavonoids
To a portion of the extract, concentrated H2SO4 was added. A yellow colouration observed indicates the presence of flavonoids. The yellow coloration disappears on standing.
Few drops of 1% AlCl3 solution was added to a portion of extract. A yellow coloration indicates the presence of flavonoids.
A portion of the dried extract was heated for 3 min with 10 ml of ethyl acetate and filtered. To 4 ml of the filtrate around 1 ml of dilute NH3 solution was added and shaken vigorously. A yellow coloration indicates the presence of flavonoids.
Test for terpenoids
About 5 ml of the plant extract was first mixed with 2 ml of chloroform and then about 3 ml of concentrated H2SO4 was carefully added through the sides of the test tube to form a layer. A reddish brown coloration of the interface formed shows the presence of terpenoids.
Test for alkaloids
A small portion of the extract was stirred with few drops of
dil HCl and filtered.
To the filtrate, Dragendorff's reagent (potassium bismuth iodide
solution) was added and an orange brown precipitate indicates the presence of alkaloids.
To the filtrate, Mayer's reagent was added and a cream precipitate shows the presence of alkaloids.
Test for glycosides
To 1ml of the plant extract add equal volume of Fehling's solution A and B. Heat the above mixture in a water bath. Brick red colouration indicates presence of glycosides.
In vitro Angiotensin converting enzyme inhibitory activity
The Angiotensin converting enzyme inhibitory activity was carried out by using furnacryloyl-l-phenylalanylglycylglycine (FAPGG) as substrate (Holmquist et al., 1979). The extract and the standard drug Lisinopril (1 mg/ml) were prepared by dissolving in reaction buffer (HEPES 25mM, NaCl 293mM, pH 8.3). The assay mixture (750μl) consisting of 530μl of FAPGG (3mM in reaction buffer) and 200μl of extracts at different concentrations (100-800 µg/ml) is incubated for 3 min at 37°C. The reaction was initiated by adding 20 μl of ACE solution (0.05U/ml) to the test reaction and the samples were incubated for one hour at 37°C. The reaction was then stopped by adding 80 μl of 5% trifluoroacetic acid solution and samples were centrifuged at 9000 rpm for 5 minutes at room temperature.
The enzymatic activity is calculated by quantifying the decrease in FAPGG concentration by recording the decrease in absorbance at 345 nm using reversed-phase HPLC. Percentage enzyme inhibition was calculated by comparing the enzymatic activity with, and without inhibitor using the following formula,
ACE inhibition % = [1- ] x 100
The enzymatic activity was determined from the relative areas of the FAPGG peak of the assay with inhibitor and of the control sample without inhibitor. The assay was carried out in triplicate for each concentration of plant extract and Lisinopril (0.05-0.2ng/ml), which was used as the positive control (Lahogue et al., 2010; Ojeda et al., 2010).
High performance liquid chromatography (HPLC) analysis
Preparation of mobile phases for analysis
Acetonitrile and 1.1% Trifluoroacetic acid in water in the ratio of 75:25 v/v is used as mobile phase solution. The solution was sonicated for 15 min to dissolve completely and subjected to membrane filtration (Sartorius 0.45µ).
The isocratic reverse phase HPLC method was followed. The flow rate of the mobile phase was set to 1.5 ml/min with UV detection at 345nm.
HPLC analysis was carried out on a Hitachi L 4000 instrument, Using RP-18 column (50 mm - 7 mm, 3 µm pore size) with isocratic elution using Acetonitrile and 1.1% Trifluoroacetic acid in water in the ratio of 75:25 v/v; it was filtered through 0.45 µL filter (Sartorius, Germany) and using an ultrasonic bath was degassed before use. The column temperature was ambient and the total running time was 20 min at a flow rate of 1.5 ml/min, the injection volume was 20µL and the detection wavelength was set at 345 nm for analysis (Lahogue et al., 2010; Ojeda et al., 2010).
In vitro antioxidant activities
Reducing power ability
The reducing power ability was evaluated by the Fe3+-Fe2+ transformation in the presence of the plant extract. The development of Perl's Prussian blue at a wavelength of 700 nm gives the extent of transformation undergone by Fe3+ into Fe2+. The reaction was carried out by mixing 1ml of the extract at various concentrations (50-800 μg/ml) prepared in distilled water with 2.5 ml of K2Fe[CN]2 (1%) and 2.5 ml of PO4- buffer (0.25 M, pH 6.4). The solution was incubated for a period of 30 min at 50°C, and 2.5 ml of TCA (10%) was added to the mixture and centrifuged at 3000 g for 10 min. About 2.5 ml from the upper part of the centrifuged solution was diluted with 2.5 ml water and mixed with 0.5 ml of freshly prepared FeCl3 (0.1%). The absorbance of the above solution was measured at a wavelength of 700 nm using UV-spectrophotometer. The reference solution was prepared similarly, but contained water instead of the samples. All experiments were carried out in triplicate using butylated hydroxyltoluene (BHT) as positive control (Nagulendran et al., 2007).
Hydroxyl radical scavenging assay
Hydroxyl radical scavenging activity of the extract is determined by its ability to scavenge the hydroxyl radicals generated by the Fe3+- EDTA-H2O2-ascorbic acid system by a reaction known as Fenton reaction (Halliwell et al., 1994). The reaction mixture amounts to a final volume of 1.0 ml which contains 100 μl of 2-deoxy2-ribose (28 mM) in phosphate buffer solution (20 mM, pH 7.4), 500 μl of the extracts at various concentrations (5-80 μg/ml) in buffer solution, 200 μl of 1.04 mM EDTA and 200 μM FeCl3 (1:1v/v), 100 μl of H2O2 (1.0 mM) and 100 μl of ascorbic acid (1.0 mM). Test samples were incubated at 37°C for 1 h. The free radical damage inflicted on the substrate, deoxyribose was assessed with the thiobarbituric acid test. A mixture of 1ml of thiobarbituric acid (1%, TBA) and 1ml trichloroacetic acid (2.8%, TCA) are added to the test tubes and heated for 20 min at 100°C. After cooling, the absorbance at a wavelength of 532 nm was measured against a blank solution containing deoxyribose and buffer. The positive control used for this assay was quercetin (5-80 µg/ml) (Nagulendran et al., 2007).
Super oxide anion radical scavenging activity
The super oxide anion radical scavenging activity of the plant extracts can be measured by assessing the ability of the extracts to scavenge superoxide anions produced in a non enzymatic phenazine methosulfate-nicotinamide adenine dinucleotide (PMS-NADH) system by the interaction between NADH, oxygen and PMS. The extent of scavenging is determined from the reduction of nitro blue tetrazolium (NBT). In the assay the superoxide anion was generated in a final volume of 3 ml of 100 mM Tris-Hydrochloric acid buffer (pH 7.4) containing 0.75 ml of 300µM NBT solution, 0.75 ml of 936 µM NADH solution and 0.3 ml of various concentrations (25-400 μg/ml) of the extract. The reaction was started by adding 0.75 ml of PMS (120 μM) to the mixture. The mixture after incubation for 5 min at 25° C, absorbance was measured at 560 nm in UV spectrophotometer (Nagulendran et al., 2007).
Nitric oxide radical scavenging assay
Various concentrations of the extract (50-800 μg/ml) and sodium nitroprusside (5mM) in phosphate buffer saline (0.025 M, pH 7.4) in a total volume of 3 ml was incubated at room temperature for a period of 150 min. After which, 0.5 ml of the incubated solution and 0.5 ml Griess' reagent (1% sulphanilamide, 2% O-Phosphoric acid and 0.1% naphthyethylene diamine dihydrochloride) were added togather and allowed to react for 30 min. Control samples without the test compounds but with equal volume of buffer was prepared in a similar manner as done for the test. The absorbance of the chromophore formed during diazotisation of nitrite with sulphanilamide and successive coupling with naphthyethylene diamine dihydrochloride was measured at 546 nm. The percentage inhibition of the extracts and standard was calculated. The experiment was carried out in triplicate using curcumin (50-800 μg/ml) as positive control (Rao et al., 2008).
Ferrous chelating ability
In the Fe2+ chelating assay, Fe2+ level in the assay mixture is monitored by measuring the formation of the Fe2+ -ferrozine complex. The reaction mixture containing various concentrations (50-800 μg/ml) of the extract was added to 2mM FeCl3(0.1 ml) and 5mM ferrozine (0.2 ml) to begin the reaction and the resulting mixture was shaken and left to stand for 10 min at 25° C. The absorbance of the assay solution was measured at 562nm. The experiment was carried out in triplicate using ascorbic acid (50-800µg/ml) as positive control. The percentage chelating effect of ferrozine-Fe2+ complex formation was calculated (Kumaran and Joel, 2006).
Estimation of total phenolic content
Total phenolics present in the extract can be estimated using folin-ciocalteu reagent and pyrocatechol as the standard. About 0.1 ml of the extract (1 mg/ml) in water was accurately transferred to an Erlenmeyer flask (100 ml) and the total volume was made up to 46 ml by distilled water. To this mixture 1 ml of folin -ciocalteu reagent, followed by 3ml of 2% Na2CO3 after 3 min was added. The mixture was vigorously shaken for 2 h at room temperature and the absorbance measured at a wavelength of 760 nm. The total phenolic compounds present in the extract were expressed as µg pyrocatechol equivalent (PCE) with the use of the standard pyrocatechol graph (Hsu, 2006).
Total flavanoid content
Total flavonoids present in the extract can be determined by treating it with aluminium nitrate and then comparing it to that of the standard quercetin. One mg of the extract was first added to 1ml of ethanol (80%) and from which 0.5 ml was added to the test tubes containing 0.1 ml of Al2(NO)3 (10%), 0.1 ml of 1 M CH3COOK and 4.3 ml of ethanol (80%). After a period of 40 min the absorbance of the supernatant solution was measured at 415 nm using UV spectrophotometer (Hsu, 2006).
Calculation of percentage inhibition (%I)
The extracts were evaluated for its percentage scavenging abilities at five different concentrations (µg/ml) and the concentration of extracts required to scavenge the radicals was calculated. The percentage inhibition (I%) of individual samples can be found out by using the following formula:
% I = X 100
Where Ac denotes the absorbance of the control and As the absorbance of the sample. The concentration of the extract required to produce 50% inhibition of free radicals was calculated.
All data were expressed as mean ± standard error of mean (S.E.M) and statistical analysis was performed using one way analysis of variance (ANOVA).