Plant material: collection and identification
Plant materials were collected from the Nilgiri Hills of Tamil Nadu, India during the month of August, and dried under shade and powdered. Roots and aerial parts were powdered separately. The material was identified and authenticated by Dr. Prabhukumar K. M., Scientist and Head, Plant Systematics and Genetics Division, Centre for Medicinal Plants Research (CMPR), Kottakkal, India and the voucher specimen (CMPR 8670) was deposited at CMPR Raw Drug Museum.
Extraction
Roots and aerial parts were extracted with hexane, methanol, and water successively. All the extracts were concentrated and stored for further studies [20].
Qualitative phytochemical analysis
Test for alkaloids
Two tests were performed to confirm the presence of alkaloids in the samples. In the first test, (Hager’s) 2 mL of plant extract was mixed with 2 mL of Hager’s reagent (saturated aqueous solution of picric acid). An eye catching yellow precipitate pointed to a positive result [21].
In Wagner’s test for alkaloids, 2 mL of Wagner’s reagent (2 g of iodine and 6 g of potassium iodide in 100 mL distilled water) was added to 2 mL of plant extract. Reddish brown precipitate indicated positive result [22].
Test for flavonoids
Alkaline reagent test was used for the detection of flavonoids. To 2 mL of the test sample, a few drops of sodium hydroxide solution were added. An intense yellow color was developed and which faded on addition of dilute hydrochloric acid [21, 23].
Test for tannins
Presence of tannins in the extract was detected by treating 1 mL of plant extract with a few drops of 1% gelatin in 10% sodium chloride solution. Formation of a white precipitate indicates positive result [21].
Test for triterpenoids
Two milliliters of plant extract was mixed with 1 mL of chloroform and a few drops of concentrated sulfuric acid were added along the sides of the test tube. A reddish brown color at the interphase shows the presence of triterpenoids [22].
Test for glycosides
Acid hydrolysis of the plant extracts were done initially. To 2 mL of the hydrolysate, 3 mL chloroform was added and shaken well, kept undisturbed for the formation of two separate layers. To the chloroform layer, equal volume of 10% ammonia solution was added. Pinkish red color indicated the presence of anthraquinone glycosides (Borntrager’s test) [21, 22].
In the second test, 2 mL of plant extract was treated with 1 mL glacial acetic acid. A few drops of 5% ferric chloride and concentrated sulfuric acid were added by the sides of the test tube. Appearance of a reddish brown ring at the junction of two liquids indicated the presence of cardiac glycosides [21, 23].
Test for saponins
Foam test was used to detect the presence of saponins. Then, 2 mL of the extracts was mixed with 10 mL of distilled water and mixed vigorously. Observation of persistent foam indicated the positive result [22, 23].
Test for anthocyanins
One milliliter of plant extract and 1 mL of 2N sodium hydroxide solution were heated in water bath for 5 min at 100 °C. Formation of a bluish green color indicated the presence of anthocyanins [24].
Quantitative phytochemical analysis
Alkaloids
Alkaloids in a plant sample were quantitatively measured by Harborne methodology [20, 21]. Then, 2.5 g of plant sample taken in a 250 mL beaker was treated with 200 mL of 10% glacial acetic acid in ethanol and allowed to stand for 4 h for extraction. The extract was then concentrated in a water bath till it got reduced to 1/4th. Concentrated ammonium hydroxide solution was added drop wise till complete precipitation occurred. After 3 h of standing for sedimentation, the supernatant was discarded and the precipitate was washed with dilute ammonium hydroxide and then filtered. The residue was dried in an oven and weighed. Percentage of alkaloid content in a given amount of the sample was calculated by
$$ \%\mathrm{of}\ \mathrm{Alkaloid}=\left(\frac{\mathrm{Weight}\ \mathrm{of}\ \mathrm{alkaloid}}{\mathrm{Weight}\ \mathrm{of}\ \mathrm{sample}}\right)\times 100 $$
Flavonoids
Total flavonoid content of the sample was determined by aluminium chloride calorimetric method [25, 26]. In this reaction, aluminium chloride would form acid stable complexes with C-4 keto group and either C-3 or C-5 hydroxyl group of flavones and flavonols. Also, aluminium chloride formed acid labile complexes with ortho dihydroxyl groups in the A and B rings of flavonoids. Different concentrations of quercetin were used to generate calibration curve. Different concentrations of quercetin and the samples to be tested were prepared in methanol. Then, 0.5 mL of the test solutions and the standard solutions were mixed with 1.5 mL of methanol, 0.1 mL of 10% aluminium chloride, 0.1 mL of 1 M potassium acetate, and 2.8 mL of distilled water. For blank, 10% aluminium chloride was replaced by equal amount of distilled water. After 30-min incubation at room temperature, absorbance was measured at 415 nm in UV/visible spectrophotometer (Thermo Scientific Evolution 201). The results were obtained as microgram quercetin equivalent per milliliter of the sample.
Tannins
Tannin content was measured by a modified Folin-Ciocalteu method [27, 28]. Then, 0.5 mL of the sample was mixed with 3.75 mL of distilled water. To this mixture, 0.25 mL of Folin-Ciocalteu reagent and 0.5 mL of 35% sodium carbonate solution were added and incubated at room temperature for 30 min. Then, absorbance was measured at 725 nm. Total tannin content was expressed as tannic acid equivalents in μg/mL of plant extract.
Phenols
Total polyphenols in a sample was determined by spectrophotometric method [28, 29]. Polyphenols formed a blue complex with some specific redox reagents that could be quantified spectrophotometrically. This blue complex is phosphotungstic acid-phosphomolybdenum complex and its maximum absorption depends on the alkaline solution and the concentration of phenolic compounds [30]. One milliliter of the sample extract was added to tubes containing 5 mL of 1:10 dilution of Folin–Ciocalteu reagent in water and 4 mL of 7.5% sodium carbonate solution. The tubes were then incubated for 1 h at room temperature. The absorbance was finally measured at 765 nm. Total phenol content was expressed as gallic acid equivalents in μg/mL of plant extract.
In vitro anti-oxidant assays
DPPH radical scavenging assay
A stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH), having a free electron, showed high absorbance at 517 nm. DPPH solution has a characteristic deep purple color which gets faded and converted to golden yellow upon accepting hydrogen from corresponding donors. This discoloration was proportional to the concentration and the scavenging activity of the compounds present in the plant extracts. Then, 2.8 mL of 100 μM DPPH in methanol was mixed with different concentrations (12.5, 25, 37.5, and 50 mg/mL) of plant extracts in 0.2 mL of methanol. Dosages for all the assays were planned according to the inhibition shown by the extracts at minimum concentration. The mixture was then incubated at room temperature for 30 min. Absorbance was measured at 517 nm after shaking well. DPPH and methanol solution without the extract served as control and methanol solution alone as blank. Same test was repeated with different concentrations of ascorbic acid to get a standard graph [23, 31].
$$ \mathrm{Percentage}\ \mathrm{of}\ \mathrm{DPPH}\ \mathrm{scavenging}\ \mathrm{activity}=\left(\frac{\mathrm{Absorbance}\ \mathrm{of}\ \mathrm{control}-\mathrm{Absorbance}\ \mathrm{of}\ \mathrm{test}}{\mathrm{Absorbance}\ \mathrm{of}\ \mathrm{control}}\right)\times 100 $$
ABTS radical scavenging assay
ABTS (2,2′-azinobis (3-ethylbenzothiazoline)-6-sulfonic acid) radical is generated when a strong oxidizing agent like potassium permanganate or potassium per sulfate reacts with ABTS salt. The ability of the anti-oxidants present in the extract to scavenge ABTS radical generated in the aqueous phase is assayed comparing to a positive standard ascorbic acid. Blue-green color of the ABTS radical solution gets reduced by the hydrogen donated by the anti-oxidants and this can be measured by suppression in the absorption spectrum of wavelength 734 nm [32]. Equal quantities of 7.4 mM ABTS solution and 2.6 mM potassium per sulfate solution were mixed and incubated at room temperature for 12 h in dark to prepare the working solution. One milliliter of the working solution was mixed with 60 mL of methanol and its absorbance was measured at 734 nm. Absorbance value was adjusted to 1.17 by adding methanol or working solution. When the absorbance reached 1.17, absorbance of the samples was measured. Different concentrations (0.25, 0.5, 0.75, and 1 mg/mL) of the extracts were taken as samples. Samples were prepared by mixing 150 μL of the extract with 2850 μL of the working solution and incubated at 2 h in dark. Methanol was taken as the blank and working solution in methanol was taken as control. Standard graph was prepared by repeating the experiment with different concentrations of ascorbic acid.
$$ \mathrm{Percentage}\ \mathrm{of}\ \mathrm{ABTS}\ \mathrm{scavenging}\ \mathrm{activity}=\left(\frac{\mathrm{Absorbance}\ \mathrm{of}\ \mathrm{control}-\mathrm{Absorbance}\ \mathrm{of}\ \mathrm{test}}{\mathrm{Absorbance}\ \mathrm{of}\ \mathrm{control}}\right)\times 100 $$
Hydroxyl radical scavenging assay
Oxidative stress developed by the reactive oxygen species (ROS) are commonly believed to cause damage to the biomolecules [33, 34]. Hydroxyl radicals are one of the potent ROS developed in the cells. The ability of the anti-oxidant compounds to compete with salicylic acid for scavenging the hydroxyl radicals was assayed. Reaction mixture contained 1 mL of 1.5 mM ferrous sulfate, 0.7 mL of 6 mM hydrogen peroxide, 0.3 mL of 20 mM sodium salicylate, and 1 mL of various concentrations (0.25, 0.5, 0.75, and 1 mg/mL) of the extracts. The reactants were mixed and incubated at 37 °C for 1 h and then the absorbance was measured at 562 nm. The absorbance measured was due to the hydroxylated salicylate complex. Percentage of scavenging was calculated by the equation:
$$ \%\mathrm{of}\ \mathrm{scavenging}=1-\left(\frac{A_1-{A}_2}{A_0}\right)\times 100 $$
A0 is the absorbance of control (reactants without extract), A1 is the absorbance of reactants in the presence of extract, and A2 is the absorbance of reactants except sodium salicylate in the presence of extracts. Ascorbic acid was the standard used.
In vitro anti-bacterial assay
Anti-bacterial activity of the roots and aerial parts extracts of the plant was tested with different bacterial strains by agar gel well-diffusion method [35]. Clinical strains of Staphylococcus aureus, Pseudomonas sp., Klebsiella sp., Escherichia coli, and Salmonella typhi were collected from a local clinical lab. Some other strains of the same species were also used for the study. Muller Hinton agar plates were prepared and the wells were cut with a micro tip. The extracts were prepared at a concentration of 1 mg/mL. Further, 100 μL extracts (hexane, methanol, and water) in dimethyl sulfoxide (DMSO) were added to the wells and incubated at 37 °C for 24 h. Then, 100 μL of DMSO was used as the blank. Inhibition zones were measured to know the anti-bacterial potential. The positive samples were further analyzed by applying increasing concentrations of extracts (0.25, 0.50, 1.0, 2.0 mg/mL).
In vitro anti-inflammatory assays
Trypsin inhibition assay
Continuous-rate spectrophotometric method developed by Sigma Aldrich [36] with slight modifications was used for the assay. The activity of the inhibitor was expressed as the inhibition of active fraction mass units. The substrate used was 0.25 mM Nα-benzoyl-l-arginine ethyl ester (BAEE) in phosphate buffer at pH 7.6. One unit of BAEE will show an absorbance of 0.001 per minute at 7.6 pH. Enzyme (0.05 mM) was prepared in ice-cold 0.001 M hydrochloric acid and the extract fractions in DMSO. The reaction mixture contains a total volume of 3.4 mL with 3 mL substrate, 0.2 mL enzyme, and 0.2 mL inhibitor. Enzyme and the inhibitor fractions were incubated for 10 min, then the substrate was added and the readings were taken at 253 nm by time scanning for 10 min. Phenyl methyl sulfonyl fluoride (PMSF) was used as the standard inhibitor.
Blank → 0.2 mL DMSO, 0.2 mL HCl, and 3 mL substrate
Activity of the native enzyme (N) → 0.2 mL DMSO, 0.2 mL enzyme, and 3 mL substrate
Activity of enzyme in the presence of inhibitor (I) → 0.2 mL inhibitor, 0.2 mL enzyme, and 3 mL substrate
$$ \%\mathrm{of}\ \mathrm{Inhibition}=\left(\frac{N-I}{N}\right)\mathrm{x}\ 100 $$
Lipoxygenase inhibition assay
Lipoxygenase (LOX) inhibition assay developed by Arthon and Barrett [26] with slight modifications were applied for activity analysis. LOX (EC 1.13.11.12) type I-B (soya bean) was the enzyme used and linoleic acid was the substrate used. Further, 0.93 μM solution of enzyme was prepared in 0.2 mM borate buffer at pH 8.6. Then, 0.32 mM substrate was also prepared in same buffer at the same pH. The assay mixture contains 50 μL enzyme, 360 μL substrate, and 1.59 mL borate buffer making the final volume of 2 mL. While adding inhibitor to the reaction mixture, 50 μL of inhibitor was added and the buffer volume was reduced to 1.54 mL to maintain the total volume at 2 mL. Formation of hydroperoxy-octadecadienoic acid was the indicator of LOX activity, which could be measured in a spectrophotometer at 234 nm. Vanillin was used as the standard inhibitor. The blank solution contained 50 μL DMSO, 1590 μL buffer, and 360 μL substrate.
$$ \mathrm{Inhibition}=\left(\frac{N-I}{N}\right)\mathrm{x}\ 100 $$
where N is the activity of the native enzyme and I is the activity of the enzyme in the presence of inhibitor.