Collection of plant material
Stem bark of P.roxburghii was collected from local areas of Solan, HP, bark of P. wallichiana collected from Shimla, HP and bark of P.gerardiana collected from Rekongpeo, Kinnaur, HP. All plant drug samples were duly authenticated from Department of Forestry, YS Parmar University of Horticulture and Agriculture Sciences, Nauni, HP, India and samples were kept in institutional herbarium with voucher specimen Nos.13488, 13489, 13506. The plant part was dried in shade, powdered by the mechanical grinder and stored in air tight container till further use.
Preparation of extracts
The powdered plant material of stem bark was defatted using petroleum ether and extracted with soxhlet apparatus using 90 % v/v ethanol in water (hydro-alcoholic extraction). The solvent was recovered by evaporation under reduced pressure using rota evaporator. The semisolid mass was further freeze dried using lyophilizer at -80 °C for 24 h.
Chemicals
1,1-diphenyl-2-picrylhydrazyl (DPPH), rutin, naphthylethylenediamine dichloride, and standard markers for HPLC Gallic acid, tannic acid and quercetin were purchased from Sigma Chemicals. Ferric chloride, vanillin, trichloroacetic acid (TCA), Folin-Ciocalteu’s reagent, aluminium chloride (AlCl3) were purchased from Himedia Pvt Ltd. All other chemicals used in the present study were of analytical grade.
Phytochemical screening of plant extracts
The prepared hydroalcoholic extracts of all three plants were subjected to phytochemical screening tests to evaluate the presence of chemical constituents. The extracts was treated with Mayer’s reagent (Potassium mercuric iodide: formation of yellow coloured precipitate); Wagner’s reagent (Iodine in potassium iodide: formation of red brown/reddish precipitate); Dragendroff’s reagent (solution of potassium bismuth iodide: formation of red precipitate indicated the presence of alkaloids. The extract was boiled with 0.25 % w/v ninhydrin reagent; formation of blue colour indicated the presence of amino acids and proteins. A blackish red colour resulting from the addition of ferric chloride reagent to extracts filtrate indicated the presence of flavonoids. Occurrence of violet ring at the junction when extracts filtrate was treated with 2 drops of alcoholic α-naphthol solution was indicative of carbohydrates (Mollisch test). Fats and oils were detected with Sudan 3 treatment. 1 % gelatin solution containing sodium chloride was added to the extract, white precipitate showed the presence of tannins. Test solution was mixed with water and shaken; the formation of 1 cm froth was an indication of saponin glycoside. Salkowaski, sulphur powder test was done for steroids. Terpenoids were detected by formation of yellow precipitate when treated with lead acetate [25, 26].
Determination of total flavonoid content
Total flavonoid content (TFC) was determined by aluminium chloride assay using calorimetric estimation [27]. In different test tubes, 0.5 ml extract, 2 ml of distilled water, followed by 0.15 ml of sodium nitrite (5 % w/v) was added. After 5 min, 0.15 ml of aluminium trichloride (10 %) was added and incubated for 6 min. After incubation 2 ml of sodium hydroxide (4 % w/v) was added. After 15 min of incubation reaction mixture turns to pink and absorbance was measured against blank e.g. distilled water at 510 nm. A natural flavonoid rutin was used as standard. The TFC was expressed in mg of rutin equivalents per gram of extract.
Determination of total phenolic content
The total phenolic content was estimated according to Folin-ciocalteu phenol reagent method [28]. The solution of gallic acid was prepared in 80 % methanol for the standard curve. Folin-ciocalteu reagent was added to 100 μl of sample in ratio 1:10. The solution was mixed and incubated at room temperature for 1 min followed by the addition of 1.5 ml of 20 % sodium carbonate. Final mixture was shaken and incubated for 90 min in the dark at room temperature. The absorbance was taken at 725 nm and the phenolic content was expressed as Gallic acid equivalents GAE/g of sample.
Condensed tannin quantification
A volume (50 ml) of concentrations (100 mg/ml) of plant extract or standard solution of catechin (CE) was mixed with 3 ml of 4 % vanillin methanol solution. 1.5 ml of concentrated hydrochloric acid was added and 15 min after; the absorbance was measured against blank using distilled water at 510 nm. Tannin content was expressed as mg CE/g of sample, using a catechin calibration curve [29].
Estimation of β-carotene and lycopene
β- Carotene and Lycopene were determined according to the method of Nagata and Yamashita [30]. The dried extract was vigorously shaken with 10 ml of acetone-hexane mixture (4:6) for 1 min and filtered through Whatman No.4 filter paper. The absorbance of filtrate was measured at 453, 505, 645 and 663 nm. The content of β-carotene and lycopene were calculated using following equations:
$$ \begin{array}{l}\mathrm{Lycopene}\ \left(\mathrm{mg}/100\ \mathrm{ml}\right) = - 0.0458{\mathrm{A}}_{663} + 0.372{\mathrm{A}}_{505} + 0.0806{\mathrm{A}}_{453}\\ {}\upbeta \hbox{-}\ \mathrm{Carotene}\ \left(\mathrm{mg}/100\ \mathrm{ml}\right) = 0.216{\mathrm{A}}_{663} - 0.304{\mathrm{A}}_{505} + 0.452{\mathrm{A}}_{453.}\end{array} $$
The values are expressed as μg/g of extract.
Evaluation of free radical scavenging activity
1-1 Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay
The free radical scavenging activity of prepared samples was determined according to ability of extract to bleach to stable DPPH radicals. 0.5 ml of DPPH was added to 0.5 ml aliquots of standard or test solution in different concentrations: 10, 20, 40, 80, 160, 180, 200 μg/ml. Control test tubes were loaded with 0.5 mL of Dimethyl sulfoxide (DMSO) and 0.5 mL DPPH. After incubation at 37 °C for 30 min in dark, the absorbance was recorded at 517 nm. Ascorbic acid was used as a standard [31, 32]. The percentage scavenging by test sample at each concentration was calculated using following formula:
$$ \mathrm{Scavenging}\ \mathrm{DPPH}\ \left(\%\right) = \left[\left({\mathrm{Abs}}_{\mathrm{control}}\hbox{--} {\mathrm{Abs}}_{\mathrm{sample}}\right)\ /{\mathrm{Abs}}_{\mathrm{control}}\right] \times 100 $$
IC50 represents the level where 50 % of radicals scavenged by test or standard sample.
Nitric oxide scavenging assay
An inhibition of nitric oxide radicals was estimated using the Griess reaction method. Griess reagent was prepared by mixing 1 % sulphanilamide in 5 % v/v phosphoric acid and 0.01 % naphthylethylenediamine in distilled water in equal volumes. The solution of sodium nitroprusside (5 mM) in standard phosphate buffer (0.025 M, pH 7.4) was prepared and incubated with different concentrations of standard and test sample: 10, 20, 40, 80, 160, 180 and 200 μg/ml at 37 °C for 5 h. An equivalent amount of methanol was taken as control. After 5 h, 0.05 ml of incubated solution was removed and diluted with 0.5 ml of Griess reagent. The absorbance of chromophore formed during the digitization of nitrite with sulphanilamide and its subsequent coupling with naphthylethylenediamine was read at 546 nm. Ascorbic acid was used as a standard [33, 34]. The percentage scavenging by test fractions at each concentration was calculated using following formula:
$$ \mathrm{Scavenging}\ \mathrm{NO}\ \left(\%\right) = \left[\left({\mathrm{Abs}}_{\mathrm{control}}-{\mathrm{Abs}}_{\mathrm{sample}}\right)/{\mathrm{Abs}}_{\mathrm{control}}\right] \times 100 $$
IC50 represents the level where 50 % of radicals scavenged by test or standard sample.
Hydrogen peroxide (H2O2) scavenging assay
The ability of extract to scavenge H2O2 was determined according to method of Khaled-Khodjaa [35]. The solution of H2O2 (40 mM) was prepared in 50 mM phosphate buffer (pH 7.4). Different concentrations of sample and standard, 10, 20, 40, 80, 160, 180, 200 μg/ml (1.2 ml) were added to a H2O2 solution (0.6 ml). After 10 min, absorbance of H2O2 at 230 nm was determined against a blank solution containing phosphate buffer, ascorbic acid used as reference compound. The percentage of H2O2 scavenged by the sample was calculated using following formula:
$$ \mathrm{Scavenged}\ {\mathrm{H}}_2{\mathrm{O}}_2 = {\mathrm{Abs}}_{\mathrm{control}}\hbox{--}\ {\mathrm{Abs}}_{\mathrm{sample}}/{\mathrm{Abs}}_{\mathrm{control}} \times 100 $$
Reducing power assay
The reducing power of extracts was determined by the method of Oyaizu [36]. Briefly, 1 ml of sample was mixed with 2.5 ml of phosphate buffer (0.2 M, pH 6.6) and 2.5 ml of potassium Ferricyanide (1 %). The reaction mixture was incubated at 50 °C for 20 min. Then 2.5 ml of trichloroacetic acid (10 %) was added and centrifuged for 10 min. An aliquot 2.5 ml was mixed with 2.5 ml of distilled water and 0.5 ml of FeCl3 (0.1 %). The absorbance of all solutions was measured at 700 nm and expressed as mg of ascorbic acid equivalent per g of powder (mg A/g powder) and mg of quercetin equivalent per g of powder (mg QE/g powder).
Total antioxidant activity
The evaluation of total antioxidant activity of the extracts was done by a phosphomolybdenum method Ravishankar et al. [37]. 0.3 ml of extract was combined with 3 ml reagent solution (0.6 M sulfuric acid, 28 mm sodium phosphate and 4 mM ammonium molybdate). The reaction mixture was capped and incubated at 95 °C for 90 min. After cooling to room temperature, the absorbance was measured at 695 nm against blank (methanol 0.3 ml). Ascorbic acid was taken as the standard.
Anti-inflammatory activity
Albumin denaturation assay
A solution of 0.2 % w/v of Bovine serum albumin (BSA) was prepared in Tris buffer (pH 6.8). Both extract and standard drugs (diclofenac sodium) were diluted in concentrations: 500, 1000, 1500, 2000 and 2500 μg/ml). 5 ml of 0.2 % w/v BSA was transferred to tube containing 50 μg/mL of extract/standard. The control tube consists of 5 mL 0.2 % w/v BSA solution with 50 μl methanol. The samples was heated at 72 °C for 5 min and cooled at room temperature for 15 min [38, 39]. The optical density of the solution was read at 660 nm and percentage inhibition of precipitation (denaturation of proteins) was determined as compared to control using following formula: % Inhibition = (Abs control - Abs sample) /Abscontrol × 100.
Membrane stabilization assay
Human red blood cells (HRBC) membrane stabilization method was used to study the anti-inflammatory activity. Blood was collected from healthy volunteers who was not taken any analgesic medication for two weeks and mixed with equal volume of sterilized Alsever solution (2 % dextrose, 0.8 % sodium citrate, 0.5 % citric acid and 0.42 % sodium chloride in water). Blood was centrifuged at 3000 RPM for 15 min. Packed cells were washed with isosaline (0.85 %, pH 7.2) and a suspension was made with isosaline (10 %). Different concentrations of extract: 50, 100, 250, 500 and 1000 ug/ml were prepared in isosaline. The assay mixture contained 0.5 ml of HRBC suspension, phosphate buffer (0.15 M pH 7.2), 2 ml hyposaline (0.36 %) and 1 ml of various concentrations of extract and incubated at 37 °C for 30 min. Then, the mixture was centrifuged at 3000 RPM for 20 min. Diclofenac sodium was used as reference standard [40, 41]. The absorbance of supernatant solution was estimated using spectrophotometer at 560 nm.
$$ \begin{array}{l}\%\ \mathrm{Hemolysis}\ \mathrm{was}\ \mathrm{calculated}\ \mathrm{b}\mathrm{y}:\ \mathrm{O}\mathrm{D}\ \mathrm{of}\ \mathrm{test}/\mathrm{O}\mathrm{D}\ \mathrm{of}\ \mathrm{control} \times 100\hfill \\ {}\mathrm{Percentage}\ \mathrm{protection} = 100 - \mathrm{O}\mathrm{D}\ \mathrm{of}\ \mathrm{the}\ \mathrm{test}/\mathrm{O}\mathrm{D}\ \mathrm{of}\ \mathrm{control} \times 100\hfill \end{array} $$
Antimicrobial activity
Procurement of microorganisms
The bacterial strains were obtained from Institute of Microbial Technology, Chandigarh. The bacterial species: gram-positive Staphylococus aureus (S. aureus) (MTCC 737), gram-negative Pseudomonas aeruginosa (P. aeruginosa) (MTCC 741) and Escherichia coli (E. coli) (MTCC 739), Klebsiella pneumoniae (K. pneumoniae) MTCC 1427), and yeast represented by Candida albicans (MTCC 3958) Saccharomyces cerevisiae (MTCC 827) were used for evaluating antimicrobial activity.
Determination of antibacterial and antifungal activities
Antibacterial activity
Muller Hinton agar plates with 4 % NaCl supplementation were prepared. Sterilized swabs were dipped in standardized bacterial suspension with an inoculum size of 1.5 × 108 cfu/ml prepared above and excess culture was removed by turning the swab against the side of the tube. Inoculum was spread evenly over the entire surface of Muller Hinton Agar plates. These plates were allowed to dry for at least 15 min and then well (7 mm diameter) were made on petri dish using sterile cork borer. About 25 μl extracts were introduced into bore agar wells using a sterile dropping pipette. These plates were kept inside the refrigerator at 4 °C for 6 h to allow proper diffusion of extracts into the medium. The plates were then examined for antibacterial activities of extracts after 24 h of incubation at 37 °C [42, 43]. Antimicrobial activity was determined by measuring the diameter zone of inhibition in mm.
Antifungal activity
Sabouraud dextrose agar (SDA) plates were prepared and sterilized swabs were dipped in standardized fungal suspension with an inoculum size of 1.5 × 107 cfu/ml prepared above and excess culture was removed by turning the swab against the side of the tube. Inoculum was spread evenly over the entire surface of SDA plates. These plates were allowed to dry for at least 15 min and then well (7 mm diameter) were made on petri dish using sterile cork borer. About 25 μl extracts were introduced into bore agar wells using a sterile dropping pipette. These plates were kept inside the refrigerator at 4 °C for 6 h to allow proper diffusion of extracts into the medium. The plates were then examined for antifungal activities of extracts after 72 h of incubation at 25 °C. The antimicrobial activity was determined by measuring the diameter zone of inhibition in mm [42, 43].
Statistical analysis
Results were expressed as mean ± standard deviation (SD). Statistical analysis was performed by one-way ANOVA followed by Bonferroni’s multicomparison test as post hoc. The software GraphPad Prism (version 6.0) was used and a probability (p) value < 0.05 was considered to be statistically significant.