Plant material: collection and identification
Leaves and bark of the S. palghatense were collected from Nelliyampathy region of Nemmara forest division, Palakkad, Kerala, India. The material was identified and authenticated by Dr. P. Sujanapal, Scientist, Silviculture Department, Kerala Forest Research Institute, Peechi, Thrissur and Dr. A.K. Pradeep, Assistant Professor, Department of Botany, University of Calicut. Collected samples of leaves and bark were dried under shade and powdered separately. The voucher specimen was deposited in the Calicut University Herbarium, Department of Botany, with accession number 7054.
Morpho-anatomical characterization
The shape of leaf, margin, color, and nature of bark were examined. For studying, stomatal morphology and venation pattern, paradermal sections, as well as clearing of leaf with 5% sodium hydroxide or epidermal peeling by partial maceration employing Jeffrey’s maceration fluid [3] were prepared. A rotary microtome has been used to section the paraffin-embedded leaf and bark of the plant sample. The sections were 10–12 μm thick. Toluidine blue has been used to stain the sections [4].
Pharmacognostic characterization
Powder microscopy
Cell structure and behavior of the leaf and bark powders were observed using standard procedures. The powder form of leaf and bark sieved, stained with safranin, mounted in glycerin, and observed under microscope [5, 6].
Fluorescence analysis
The powders of both leaves and bark of S. palghatense was treated with several reagents such as acetone, acetonitrile, methanol, and distilled water. It was further observed under UV and visible light (254 nm and 366 nm) to note the color produced after treatment and fluorescence character were studied [7, 8].
Physicochemical characterization
Various physicochemical parameters (water-soluble extractive, alcohol-soluble extractive, petroleum ether extractive, ethyl acetate-soluble extractive, crude fiber, vitamin A, vitamin C, minerals, heavy metals, total ash, acid insoluble ash, water-soluble ash, and sulphated ash) were investigated on the powdered sample of both leaves and bark of S. palghatense [9,10,11,12,13,14,15].
Phytochemical characterization
Extraction
The collected leaves and bark of S. palghatense were separately shade dried and powdered. Soxhlet extraction method was used to extract leaves and bark of the plant sample and were performed using different solvents including, chloroform, ethyl acetate, methanol, and distilled water. After the extraction, the solvent was evaporated using a rotary evaporator [16]. The total extractive values were calculated on dry weight basis by the following formula:
$$ \mathrm{Percentage}\kern0.5em \mathrm{extract}\mathrm{ive}\kern0.5em \mathrm{value}\kern0.5em \left(\mathrm{yield}\%\right)=\frac{\mathrm{Weight}\kern0.5em \mathrm{of}\kern0.5em \mathrm{dry}\kern0.5em \mathrm{extract}}{\mathrm{Weight}\kern0.5em \mathrm{of}\kern0.5em \mathrm{the}\kern0.5em \mathrm{plant}\kern0.5em \mathrm{sample}\kern0.5em \mathrm{taken}\kern0.5em \mathrm{for}\kern0.5em \mathrm{extract}\mathrm{ion}}\times 100 $$
The dried extracts were stored in refrigerator for further studies.
Qualitative phytochemical analysis
Preliminary phytochemical investigations for secondary metabolites were carried out using standard procedures. The metabolites tested were alkaloids, flavonoids, glycosides, saponins, carbohydrate, tannins, reducing sugar, starch, phenolics, anthraquinone, fats and oils, essential oils, proteins, aminoacids, steroids, and coumarin [16,17,18,19,20,21,22].
Quantitative phytochemical analysis
Alkaloids
Alkaloids in a plant sample were measured quantitatively [17, 23]. In a 250 ml beaker, 2.5 g of the plant sample 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 until it was reduced to 1/4th of its original volume. Concentrated ammonium hydroxide solution was added drop wise till complete precipitation was achieved. The supernatant was discarded after 3 h of sedimentation, and the precipitate was washed with dilute ammonium hydroxide and filtered. In an oven, the residue was dried and measured. The percentage of alkaloid content in a given amount of sample was determined using the following formula,
$$ \mathrm{Percentage}\kern0.5em \mathrm{of}\kern0.5em \mathrm{alkaloid}=\frac{\mathrm{Weight}\kern0.5em \mathrm{of}\kern0.5em \mathrm{alkaloid}}{\mathrm{Weight}\kern0.5em \mathrm{of}\kern0.5em \mathrm{sample}}\times 100 $$
Flavonoids
The aluminum chloride colorimetric assay was used to determine total flavonoid content. In a 10 ml volumetric flask, a reaction mixture of 1 mg/ml extract and 4 ml distilled water was prepared. To the flask, 0.30 ml of 5% sodium nitrite was treated and after 5 min, 0.3 ml of 10% aluminum chloride was mixed. Two milliliters of 1 M sodium hydroxide was treated and diluted to 10 ml with distilled water after 5 min. A set of quercetin reference standard solutions (20, 40, 60, 80, and 100 g) were prepared. An UV/Visible spectrophotometer was used to measure the absorbance of the test and standard solutions against the reagent blank at 510 nm. The total flavonoid content of the extract was calculated as μg/mg [24].
Glycosides
One milliliter extract and 1 ml Baljet’s reagent (freshly prepared 95 ml 1% picric acid and 5 ml 10% NaOH, blended immediately before use and filtered through a sintered glass funnel) are combined and allowed to stand for 1 h. The solution was diluted with 2 ml distilled water and mixed thoroughly. A spectrophotometer was used to measure the color intensity against a blank at 495 nm [25].
Saponins
Ten milligrams diosgenin was dissolved in 16 ml methanol and 4 ml distilled water to make a standard saponin solution. Vanillin reagent (8%, 0.25 ml) was applied to the aliquots for each tube, and sulfuric acid (72% v/v, 2.5 ml) was progressively added on the inner side of the tube wall. The solutions were thoroughly combined before being moved to a 60 °C water bath. After 10 min of incubation, the tubes were cooled for 3–4 min in an ice cold water bath. Against a reagent blank, the absorbance was measured at 544 nm. In aqueous methanol (80%, 0.1 ml), 0.1 g of freeze-dried sample was dissolved. At 544 nm, 0.25 ml aliquot was taken for spectrophotometric determination of total saponins [26].
Total carbohydrate
One hundred milligrams of the sample was taken in a boiling tube and hydrolyzed for 2–3 h with 5 ml of 2.5 N HCl in a boiling water bath before being cooled to room temperature. Until the effervescence ceased, neutralized it with solid sodium carbonate. The volume was made up to 100 ml, centrifuged, and the supernatant was collected, and 0.2 to 1 ml was taken for analysis. The standards were prepared by taking 0.2–1 ml from the working standard of glucose (10 mg/100 ml) with distilled water to make up to 1 ml. One milliliter water serves as a blank; fill the tubes with distilled water to 1 ml, then add 4 ml anthrone reagent and heat in a boiling water bath for 8–10 min. The glucose concentration in the sample was determined [27].
Tannin
The Folin-Ciocalteu method was used to determine the amount of tannin in the sample. The measurement of blue color produced by the reduction of phosphotungstic molybdic acid by tannin-like compounds in an alkaline medium is used to colorimetrically estimate tannins. With distilled water, 1 ml of extract and a standard solution of tannic acid (20–100 μg) were made up to 7.5 ml. After that, 0.5 ml Folin-Ciocalteu reagent and 1 ml sodium carbonate solution (35%) were added. The volume was made up to 10 ml with distilled water and the absorbance was assessed at 700 nm [28].
Reducing sugar
Homogenized sample (100–500 mg) was transferred to a beaker. Then, 5 ml warm ethyl alcohol was added and mixed for 15–20 min with a glass rod. In a centrifuge tube, the supernatant was decanted. The residue was re-extracted for two times with 3 ml portion of ethyl alcohol. The supernatant was combined in a centrifuge tube, and the ethanol was evaporated on a water bath (70–80 °C) until the residue was dry. The sugar was dissolved in 10 ml distilled water. Centrifuged for 15–20 min at 1000–1200 rpm. Pipette the standard solutions of 0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 ml into clean numbered test tubes. In two numbered test tubes, a 1 ml alcohol extract of the sample was taken. Further, 3.0 ml solution volume prepared by adding 3.0, 2.5, 2.0, 1.5, 1.0, and 0.5 ml distilled water to the appropriate numbered tubes. Three milliliters of alkaline dinitrosalicylic acid reagent was added. For 5 min, all test tubes are placed in a boiling water bath. One milliliter sodium potassium tartrate solution was added to all tubes while they were still hot. The absorbance of orange red color was measured at 510 nm after proper mixing and cooling [29].
Phenol
The aliquot of sample was pipetted out and the volume in tube was increased to 3 ml with distilled water. Then, 0.5 ml Folin-Ciocalteau reagent and 2 ml sodium bicarbonate solution (20%) were added; the tubes were immersed in boiling water bath for 1 min. In a spectrophotometer, the absorbance was measured at 750 nm after the tubes were cooled [30].
In vitro antioxidant activity
DPPH radical scavenging assay
Different solvent extracts (chloroform, ethyl acetate, methanol, and distilled water) of leaves and bark of S. palghatense were tested for its scavenging activity against the stable free radical DPPH (2,2-diphenyl-1-picrylhydrazyl). When DPPH is in its radical form, it has a 517 nm absorption band that disappears when it is reduced. Different concentrations of the sample were added to 0.375 ml of freshly prepared DPPH solution in methanol. The volume was made up to 2 ml with respective solvents. The absorbance of the reaction mixture was measured at 517 nm after 20 min of incubation in the dark. The percentage inhibition was calculated and concentration needed for IC50 was estimated [31].
Superoxide radical scavenging assay
Superoxide scavenging activity of the different solvent extract of the plant sample was determined by nitroblue tetrazolium (NBT) reduction method [32]. It depends on the light-induced superoxide generation by riboflavin and the corresponding reduction of NBT. Various concentrations of the plant extracts was added to the reaction mixture consisting of 0.1 M ethylene diamine tetra acetic acid (EDTA) containing 0.3 mM NaCN, 0.12 mM riboflavin, 1.5 mM NBT, and 0.067 M phosphate buffer making up the volume to a total of 3 ml. The tubes were uniformly illuminated with an incandescent lamp for 15 min and the optical density was measured at 560 nm before and after the illumination. The percentage inhibition was evaluated by comparing the absorbance values of the control and the experimental tubes [32].
Hydroxyl radical scavenging activity
Hydroxyl radical scavenging activity was measured by the competition between deoxy 2-ribose and plant extracts. The reaction mixture contained ferric chloride (100 μM), EDTA (100 μM), deoxy-2-ribose (2800 μM), phosphate buffer (100 μM, pH 7.4), hydrogen peroxide (100 μM), ascorbic acid (100 μM), and various volumes of sample (2–12 μl) in a final volume of 1 ml. Incubated reaction mixture at 37 °C for 1 h. At the end of incubation, 1.5 ml of acetic acid (20%, pH 3.5), 0.2 ml of sodium dodecyl sulfate (SDS, 8.1%), and 1.5 ml of tertiary butyl alcohol (TBA, 0.8%) were added to 0.4 ml of reaction mixture. After thorough mixing, the reaction mixture was incubated at 100 °C for 1 h, and the formation of thiobarbituric acid reactive substances (TBARs) was determined using a spectrophotometric method at 532 nm. All of the experiments were performed in triplicate, and the percentage of inhibition was determined using the following formula: Percentage of inhibition = [Ac − As]/Ac × 100; Ac and As is the absorbance of control and sample respectively [33].
Ferric-reducing antioxidant power assay
The antioxidant capacity of different extracts was estimated. The method measures the ferric reducing ability (ferric-reducing antioxidant power, FRAP). When a ferric tripyridyl triazine (Fe III-TPTZ) complex is reduced to ferrous (Fe II) form at low pH, an intense blue color with an absorption maximum at 595 nm is developed. FRAP reagent (25 ml 300 mmol/L acetate buffer, pH 3.6; 2.5 ml 10 mmol/L TPTZ (2,4,6-tripyridyl-s-triazine) in 40 mmol/L HCl and 2.5 ml 20 mmol/L Fecl3.6H2O solution), prepared freshly and 900 μl was mixed with different concentration of plant extract and made the volume up to 1 ml using distilled water. The reaction system was incubated at 37 °C for 20 min. Reading was taken against distilled water blank at the absorption maximum 595 nm in spectrophotometer. The percentage increase in the ferric reducing activity was then calculated [34].
Anti-diabetic activity
Alpha amylase inhibitory assay
One percent phosphate buffer and the starch solution was prepared and incubated with 500 μl enzyme (α-amylase) for 10 min at 37 °C. Then, 1 ml of (20, 40, 60, 80, and 100 μg/ml) isolated pooled fractions from methanol extract of leaves and bark of S. palghatense was added to the enzyme solution. Further, 2 M of NaOH is applied to stop the reaction process. One milliliter of dinitro salicylic acid is mixed and the reaction is maintained in the hot water bath for 5 min. After completion of incubation, test tubes were cooled by running tap water, and the final volume of test solution was to make up to 10 ml using sterile distilled water and absorbance was measured at 540 nm. Acarbose was used as a reference substance [35].
Alpha glucosidase inhibitory assay
One milligram of the α-glucosidase enzyme (isolated from Saccharomyces cerevisiae) was suspended with 100 ml neutral phosphate-buffered saline buffer which contains the 200 mg of bovine serum albumin [36]. The various concentrations (20, 40, 60, 80, and 100 μg/ml) of isolated pooled fractions from methanol extract of leaves and bark of S. palghatense were added with reaction mixture (10 μl of pH 6.8 phosphate buffer; 490 μl of 5 mM p-nitro phenyl α-d glucopyranoside (p-NPG)). The reaction mixture was incubated at 37 °C for 5 min then added 250 μl of α-glucosidase (0.15 unit/ml) and again incubated at 37 °C for 15 min. Then reaction mixture was allowed to cool and added 2 ml of sodium carbonate (200 mM) to stop the reaction. The activity of enzyme inhibition was measured at 405 nm and acarbose was utilized as a reference compound.
Percentage calculation
Percentage of free radical scavenging and α-amylase and α-glucosidase inhibition was measured using the following formula: Percentage Inhibition/scavenging activity = [(Acontrol – Asample)/(Acontrol)] × 100. Where A = absorbance.
