Plant Materials
The plant materials viz. Mikania micrantha , Apluda mutica , Kyllinga nemoralis and Cleome rutidosperma were collected from various locations of Kolkata and Howrah, India and identifications were authenticated at Botanical Survey of India, Howrah. The voucher specimens of each plant materials were preserved at the Plant Chemistry department of our office under registry no. BSI/Chem/SD 006, BSI/Chem/SD 007, BSI/Chem/SD 008, and BSI/Chem/SD 009 separately. Plant materials were shade dried, pulverized and stored in an airtight container for further study.
Extraction of plant material
For preparation of plant extracts, one gram dry sample was extracted separately with 20 ml of methanol, 70% aq. ethanol, chloroform and benzene with continuous stirring, for 18–24 h at ambient temperature. The extracts were subsequently filtered with Whatmanno.1 filter paper and diluted to 25 ml with corresponding extracting solutions. These extracts were analyzed for their antioxidant profiling, anti-diabetic and anti-inflammatory study.
Antioxidant activity
Estimation of total phenolic content
Folin-Ciocalteu procedure was followed to estimate total phenolic content of the plant extracts (Datta et al. 2019), and was expressed as mg/g gallic acid equivalent (GAE). To 100 μl of different plant extract, 1.0 ml of Folin-Ciocalteu reagent and 0.8 ml of sodium carbonate (7.5%) were added and incubated for 30 mins after proper mixing. Absorption was measured at 765 nm (UV-visible spectrophotometer Shimadzu UV1800).
Estimation of total flavonoid content
Estimation of total flavonoid content was achieved following the standard method (Datta et al. 2019) and was expressed as mg/ g rutin equivalent (RE). To 0.5 ml of extract, 0.5ml of ethanolic aluminium chloride (2%) solution was added.Theabsorbance was measured at 420 nm(UV-visible spectrophotometer Shimadzu UV 1800) after 1 hr incubation.
Ferric Reducing Antioxidant Power (FRAP) Assay
Ferric Reducing Antioxidant Power (FRAP) was measured and expressed as trolox equivalent (TE) mg/ g dry extract (Datta et al. 2019). The FRAP working solution was prepared by mixing 300 mM sodium acetate buffer (pH 3.6), 10 mM TPTZ (2, 4, 6-tripyridyl triazine) solution in 40 mM HCl and 20 mM ferric chloride solution in a ratio of 10:1:1 (v/v/v). 1 ml of plant extract was then added to 2.85 ml of FRAP working solution and incubated at 37 °C for 30 min in dark. The increase in absorbance was measured at 593 nm in a UV–visible spectrophotometer (Shimadzu UV 1800).
Measurement of reducing power
The reducing power of the extracts wascompletedand calculated as ascorbic acid equivalent (AAE) in mg/ g of dry material (Datta et al. 2019). 100 μl of plant extracts were mixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and 2.5 ml potassium ferricyanide (1%). The mixture was incubated at 50°C for 20 min.2.5 ml aliquots was taken from mixture and added to the equal volume of 10% trichloroacetic acid, which was then centrifuged at 3000 rpm for 10 min. The upper layer of the resulting (2.5 ml) was added to the equal amount of distilled water and 0.5 ml freshly prepared ferric chloride solution (0.1%) and kept in dark for 5 min. The absorbance of resulting solution was estimated at 700 nm in a UV–visible spectrophotometer (Shimadzu UV 1800).
DPPH (2,2-Diphenyl-1-picryl-hydrazyl) assay
Free radical scavenging ability of the extracts was determined using the stable radical DPPH (1, 1-diphenyl-2-picrylhydrazyl) following the process of Dutta et al (Datta et al. 2019). 100 μl of the tested sample were mixed with 3.9 ml of methanolic DPPH solution (25 mg/L). After 30 mins incubation in dark, the absorbance was measured at 517 nm in a UV–visible spectrophotometer (Shimadzu UV1800). The capability of test sample to scavenge the DPPH radical was determined.
ABTS [2, 2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] assay
The 2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation (ABTS+) scavenging activity was measured according to the method described by Dutta et al (Datta et al. 2019). To aq. solution of ABTS (7mM), potassium persulphate solution (2.45 Mm, final concentration) was added and incubated in dark overnight for free radical generation. To determine the scavenging activity, 1 ml of diluted ABTS+ solution was added to 10 μl of plant extract, and the absorbance at 734 nm in a UV–visible spectrophotometer (Shimadzu UV1800).
Metal chelating property
For the estimation of metal chelating property of experimental plant extract, the process of Lin et al. was followed with slight modifications (Datta et al. 2019). 1 ml of plant extracts was added to a 200 µl ferrous chloride (2 mM) and 400µl ferrozine (5 mM). The mixture was incubated for 10 mins and absorbance was taken at 562 nm.
Anti-lipid peroxidation in linoleic acid system
Anti-lipid peroxidation was assayed as described by Dutta et al, with modifications (Datta et al. 2019). 1ml of plant extract was added 130 µl of linoleic acid solution followed by addition of 99.8% ethanol (10 ml) and 10 ml sodium phosphate buffer (pH 7, 0.2M). The mixture was made upto25 ml and incubated at 400 C upto 360 hours. Extent of oxidation was measured by thiocyanate method. 75% ethanol (10ml), 30% aq. Solution of ammonium thiocyanate (0.2 ml), sample solution (0.2 ml) and ferrous chloride (20mM in 3.5% HCl, 0.2 ml) added sequentially. The absorbance was measured at 500 nm after 3 mins incubation.
Quantitative profiling of phenolic acids and flavonoids by RP-HPLC
Preparation of standard solutions
Stock solution of 1mg/ml concentration of different phenolic acids and flavonoids viz. protocatechuic acid, gentisic acid, chlorogenic acid, p-hydroxy benzoic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, sinapic acid, salicylic acid, gallic acid and ellagic acid, catechin, rutin, myricetin, quercetin, naringin, apigenin and kaempferol were prepared by dissolving 10 mg of the respective phenolic acids in 1 ml HPLC-grade methanol and the and the resulting volume was made up to 10 ml with the solvent for the Mobile phase (methanol and 0.5% aq. acetic acid 1:9). A standard curve was obtained by further dilution at 20, 40, 60, 80 and 100 μg/ml with the mobile phase solvent system. The standard and working solutions were filtered through 0.45 μm PVDF-syringe filter prior injection.
Preparation of sample solution
1 g of each coarsely powdered plant samples were extracted using 5 ml 70% aq. ethanol with constant stirring for 24 h. The process was repeated for three times and the final volume was made upto 10 ml. The extracts were filtered through 0.45 μm PVDF-syringe filter prior injection.
Chromatograph analysis
Quantification of phenolic acids and flavonoids were performed following the method of Dutta et al (Datta et al. 2019). Separation was achieved by a reversed phase Acclaim C18 column (5 micron particle size, 250 x 4.6 mm). The mobile phase contains methanol (Solvent A) and 0.5% aq. acetic acid solution (Solvent B) and the column was thermostatically controlled at 280C and the injection volume was kept at 20 μl. The gradient elusion was 90 % solvent B and 10% solvent A and flow rate was settled from 1ml/min to 0.7 ml/min in 27 min, from 10 to 40 % solvent A with flow rate 0.7 ml/min for 23 min, 40% solvent A and 60% B with flow rate 0.7 ml/min primarily for 2 min and then flow rate altered from 0.7 to 0.3 ml/min in 65min, from 40 to 44% solvent A with flow rate 0.3 to 0.7ml/min in 70 min, 44% solvent A with flow rate 0.7 to 1ml/min for 10 min duration, solvent A changed from 44% to 58 % with flow rate 1ml/min for 5 min, 58 to 70% solvent A in 98 min at constant flow rate 1 ml/min. The mobile phase composition was taken to the original condition (solvent A: solvent B: 10: 90) in 101 min and allowed to run for another 4 min, before the injection of another sample. Total analysis time per sample was 105 min. The presence of phenolics were detected in HPLC chromatograms using a photo diode array UV detector at three different wavelengths (272, 280 and 310 nm) according to absorption maxima of investigated compounds. Each compound was identified by its retention time and by spiking with standards under the same conditions. The quantification of phenolic acids and flavonoids in the plant extracts were analysed by the measurement of the integrated peak area and the amounts were estimated using the calibration curve of the respective standard compound.
Estimation of anti-diabetic property
Extraction of plant material
1g of powdered plant sample was extracted with 5 ml 70% ethanol on constant stirring for 24 h at the ambient temperature. The resulting extracts were filtered using Whatman No.1 filter paper and the filtrate was concentrated under vacuum at room temperature. Dried extracts were weighed and further dissolved in double distilled water to yield a stock solution concentration ranging from 5μg/ml to 50μg/ml.
α- Amylase inhibition property
This assay was carried out using a modified procedure of McCue and Shetty (McCue, K. Shetty 2004). Different concentration (5–50μg/ ml) of plant extract was mixed with 250 µl of sodium phosphate buffer (0.02M, pH 6.9) containing α-amylase solution (0.5 mg/ml). This solution was incubated at 250C for 10 min, followed by addition of 250 µl of starch solution (1%) in 0.02M sodium phosphate buffer (pH 6.9) then further incubated at 250C for 10min. The reaction was completed by addition 500 µl of dinitrosalicylic acid (DNS) reagent. The reaction mixture was incubated for 5min in boiling water. The absorbance was measured at 540 nm using spectrophotometer after cooling to room temperature and further dilution with 5ml distilled water. Acarbose (Acarbose, 25mg from Orchid Chemicals & Pharmaceuticals Ltd.) was used as standard. The α-amylase inhibitory activity was calculated as percentage inhibition (%).
α- Glucosidase inhibition property
The inhibition of α-glucosidase assay of the extracts was completed according to the method described by Kim et al.(Kim et al. 2005), using α-glucosidase from Saccharomyces cerevisiae. p-nitrophenylglucopyranoside (pNPG) solution prepared in phosphate buffer (20mM , pH 6.9) was used as substrate. 100 µl of α-glucosidase (1.0U/cc) was pre-incubated with in different concentration (5 – 50μg/ml) of the extracts for 10min, followed by addition of 50 µl of pNPG (3mM) to initiate reaction. The reaction mixture was incubated at 370C for 20min and terminated by adding 2ml of sodium carbonate (0.1M). The absorbance was measured at 405 nm using spectrophotometer. Acarbose was used as standard andα-glucosidase inhibitory activity was calculated as percentage inhibition (%).
Anti-inflammatory activity
The plant extract was prepared following the same procedure that used for anti-diabetic activity. The anti-inflammatory activity was studied in terms of inhibition of albumin denaturation which was studied according to Vallabh et. al.(Vallabh et. al. 2009) with minor modifications. The reaction mixture (0.5 ml) consisted of 0.45 ml of bovine serumalbumin (5% aqueous solution) and 0.05 ml of plant extracts (50 and 200 μg/cc of final volume) which was incubated in boiling water bath for 10 mins.After cooling the turbidity was measured at 660nm. Diclofenac (Dicloran 50 mg, JB Chemicals & Pharma Ltd.) was used as standard and protein denaturation activity was calculated as percentage inhibition (%).
Statistical analysis
All the experiments were done using triplicate samples. Results were represented as value ± standard error mean (SEM).Experimental results were subjected to univariate analysis of variance (ANOVA), followed by Tukey test (p ≤ 0.05) using the statistical package for the social sciences SPSS version17.0 (SPSS Inc., Chicago,Illinois, USA).