Plant collection
L. pinnatifidum whole plant was collected from Bagh, Azad Jammu and Kashmir, identified from Dr. Zafar, department of Plant sciences, Quaid i Azam university (QAU) Islamabad, and 175,701 accession number was assigned from herbarium of Pakistan, QAU Islamabad.
Preparation of extract and fractionation
The whole plant was wiped properly and dried under shade. Two kilo gram ground plant material was soaked in the methanol for 7 days and filtered, filtrate termed as LPM. Next was fractionation to separate plant’s compounds, from the crude extract, according to their polar contents. 50 g of crude extract was mixed in 200 m litre distilled water. n-hexane (C6H6), chloroform (CHCl3), ethyl-acetate (C4H8O2) and butanol (C4H9OH) were added correspondingly to obtain respective fractions. At the end, residues left in separating funnel, were termed as aqueous fraction. All the fractions were collected, evaporated, quantified and finally kept at 4 °C for further use.
Quantitative phytochemicals analysis
Total phenolic content
Total phenolics present in plant fractions were investigated by using Folin Ciocalteu methodology [9]. 1 ml of plant fraction dissolved in methanol was added in 1.5 ml of Folin ciocalteu reagent, diluted up to 10 folds. Then 1.2 ml of 7.5% Na2CO3 was mixed and mixture was placed at 27 °C for 90 min. The absorbance of mixture was checked by using ultraviolet–visible spectrophotometer, at wavelength of 760 nm. Using gallic acid as standard molecule, result articulation was done as milli gram equivalent of gallic acid.
Total flavonoids content
The content of total flavonoids was evaluated by following aluminium chloride (AlCl3) colorimetric method used by Baba and Malik [10]. One milli litre of plant fractions (one mg/ml ethanol) were assorted thoroughly with four milli litre distilled H2O and 0.3 ml of sodium carbonate (5%) solution respectively, then after 5 min of incubation, 0.3 ml aluminium chloride solution (10%) was put in the mixture and placed for 6 min. At last 1 mol/l sodium hydroxide solution was put in, and final volume was raised up to 10 ml by addition of distilled H2O. After 15 min, absorbance was reserved at 510 nm in Ultraviolet – Visible spectrophotometer. The TPC was measured by calibration curve using standard values of rutin. Results were articulated as mg rutin per gram equivalents dry weight.
In vitro antioxidant assessment
DPPH (1, 1-diphenyl-2-picryl-hydrazyl) radicals scavenging assay
Scavenging DPPH by plant fractions was measured by procedure used by Alam et al. [11] Stock solution was set by using 100 ml methanol and adding 24 mg of DPPH in it, this solution was placed at 20 °C. Optical density of this solution was measured and maintained at 0.908 (± 0.02), at 517 nm, by using methanol to dilute stock solution. 100 micro litre plant sample and 900 μl of DPPH solution were mixed thoroughly and incubated for about 15 min in dark, at 37 °C. In ultraviolet–visible spectrophotometer absorbance was taken at 517 nm. The ascorbic acid was positive control and antioxidant potential was calculated by formula given in eq. 1.
$$ \mathrm{Scavenging}\ \mathrm{effect}\ \left(\%\right)=\frac{\left[\ \mathrm{control}\ \mathrm{absorbance}-\mathrm{sample}\ \mathrm{absorbance}\right]}{\left[\mathrm{control}\ \mathrm{absorbance}\right]} \times 100 $$
Hydroxyl free radicals scavenging assay
Scavenging ability of free hydroxyl radical was examined by Choi et al. methodology [12]. To do so following procedure was carried out. 500 μl deoxyribose (2.8 mM) was mixed in phosphate buffer (50 mM) having pH value of 7.4, 200 μl of 100 mM FeCl3 and 100 μl EDTA (0.1 M) was put in reaction mixture. Next,100 μl hydrogen per oxide (200 mM) and plant sample (100 μl) are added.
Volume of 100 micro litre ascorbic acid 300 mM was put in reaction mixture and allowed to incubate at room temperature for 60 min. Then one ml, 2.8% TCA and one ml, 10% w/v TBA prepared in sodium hydroxide (50 mM) was mixed and placed in water bath for 15 min. On cooling, 532 nm wavelength was used to measure optical density. Radical neutralizing power was quantified using given formula.
$$ \mathrm{Scavenging}\ \mathrm{Activity}\ \left(\%\right)=\frac{\left[1-\mathrm{Sample}\ \mathrm{Absorbance}\right]\ }{\left[\mathrm{Control}\ \mathrm{Absorbance}\right]} \times 100 $$
Nitric oxide scavenging assay
Scavenging potential of each fraction was assessed by procedure used by Anu and Usha [13]. It was done by taking sodium nitroprusside (100 μl, 10 mM) in saline phosphate buffer and intermixed with plant sample (100 μl). Sodium-nitroprusside generate nitric oxide radicals that interact with oxygen and give rise to nitrite ion specie, which can be detected by Griess reagent. After 3 h of incubation 1 ml of Griess reagent was added. Griess reagent is made by taking equal volume of sulfanil-amide (1%) in phosphoric acid (5%) and naphthylethylene diamine di-hydrochloride (0.1%) in distilled water. The absorbance was taken at 546 nm in UV – Visible spectrophotometer. Scavenging power was measured by formula given in equation one.
Chelating power assay
Aptitude of chelating iron (II) of plant fractions was assessed by following Karatoprak et al. [14] Plant samples were mixed in methanol and serial dilutions were made. 200 μl of plant aliquot was blended with methanol (900 μl) plus FeCl2.2H2O (100 μl, 2 mM) and incubated for 5 min. 400 μl ferrozine (5 mM) was put in reaction solution and left to incubate for 10 min. By using UV – Visible spectrophotometer absorbance was read at 562 nm wavelength Chelating potency was quantified by using formula given in equation one.
Reducing power assay
Protocol of Phatak and Hendre was trailed to determine reducing activity of plant fractions [15]. About 2 ml plant sample is mixed in 2 ml phosphate buffer (0.2 M) of pH 6.5. Volume of 2 m litre potassium ferricyanide (10 mg/l) is added in mixture. After 20 min incubating at fifty degree centigrade, trichloroacetic acid (2 ml, 10%) was mixed in it. Then centrifugation was done at 3000 rpm speed for 10 min. After centrifugation 2 m litre supernatant was taken gently and diluted by adding two milli litre D. W and 0.5 ml (0.1%) FeCl3. After 10 min, UV–Visible spectrophotometer was used to take absorbance at 700 nm. And gallic acid was standard in this assay.
Phosphomolybdenum assay
Antioxidant potency of plant fractions was evaluated by phospho-molybdenum assay by Hossain and Shah [16]. In eppendorf 100 μl of plant aliquots were allowed to mix with 1000 μl of reagent containing sodium phosphate (28 mM), 4 mM of ammonium molybdate [(NH4)2MoO4] and sulfuric acid (0.6 M). After mixing eppendorfs were incubated in water bath at 90 °C for 90 min, to prevent direct exposure of light eppendorfs were covered by aluminium foil. After incubation, the reaction mixture is allowed to cool at normal temperature and absorbance was taken at 765 nano meters. Ascorbic acid was proceeded as a standard.
ß-carotene bleaching assay
Antioxidant dimension of plant’s fractions was determined by betacarotene bleaching methodology used by Hatami et al. [17] Amount of two milli gram ß carotene was added in chloroform (10 ml) to make ß carotene solution. 200 mg of Tween 80 and linoleic acid were added in this solution and chloroform was evaporated from it. Volume of 50 ml of D. W was mixed in reacting mixture and vortexed strongly to have a uniformed emulsion made by ß carotene linoleate. Volume of 250 μl of that emulsion was taken and mixed with 30 μl plant sample (30 μl). Immediately optical density was checked at 470 nm. For 2 h mixtures were placed at 45 °C in water bath and absorbance was read again. In this assay catechin served as a standard.
$$ \mathrm{Bleaching}\ \mathrm{inhibition}\%=\left[\left\{\mathrm{AA}(120)-\mathrm{AC}\ (120)\right\}/\left\{\mathrm{AC}(0)-\mathrm{AC}(120)\right\}\right]\times 100 $$
Here, AA (120); sample absorbance on 120 min, AC (120) and AC (0); control absorbance on 120 and 0 min respectively.
In vitro anti-inflammatory assay
Anti-inflammatory potency was calculated in accordance to the protocol of Kulkarni et al. [8]. Reaction blend comprising of test sample and aqueous soln. of bovine albumin (1%) was incubated at 37 °C (20 min) and then at 51 °C (20 min). Absorbance was measured at 660 nm using spectrophotometer. Loprin was standard drug used and test was performed in triplicate. Following formula used to determine inhibition percentage of protein denaturing.
$$ \mathrm{Inhibition}\ \mathrm{of}\%\mathrm{denaturation}=\left[\mathrm{Abs}\ \mathrm{of}\ \mathrm{Control}-\mathrm{Abs}\ \mathrm{of}\ \mathrm{sample}/\mathrm{Abs}\ \mathrm{of}\ \mathrm{control}\right]\ \mathsf{x}100 $$
In vivo studies
Sprague-Dawley rats of both sexes were of almost 6 weeks old rats about 150–200 g weight maintained at standardized laboratory conditions at primate facility of the QAU, Islamabad. Rats had ad libitum access to water and basal chow. Ethical Committee of QAU, Islamabad permitted study design, for animal’s care and experimentation.
Acute-toxic studies
Female Sprague Dawley rats (Rattus novergicus) weighted 150 g - 180 g were separated in 3 groups, randomly, 3 rats in each. Rats were administered orally with LPM, LPH, LPC, LPE, LPB and LPA at varied doses (250 mg/kg, 500 mg/kg, 1000 mg/kg, 2000 mg/ kg, 3000 mg/kg and 4000 mg/kg) in the morning. The animals were observed for any change in physical appearance, irregular behaviour and mortality after 30 min for 6 h then after 24 h for 15 days. Given doses did not produce any behavioural irregularity or mortality.
Anti inflammatory activity
To find out the anti inflammatory potency of plant, carrageenan mediated hind paw edema was trailed in this study [18]. Male Sprague-Dawley rats (150–200 g) were separated randomly in to 15 groups; each containing 7 animals. Volume of normal paw was measured before experimentation. Group I was given saline (1%) and diclofenac potassium (10 mg/kg) was given to Group-II. Animals of remaining groups were treated with plant fraction dosage of 10, 200, and 400 mg/kg at fasting. Carrageenan 1 ml/kg (1% in saline w/v) was injected in hind paw, about 30 min earlier to dose administration. Digital plethysmometer was used to measure paw volume instantly after injecting carrageenan (0 h) and repeated after every 1 h up to 4 h. Paw edema volume of each rat was calculated and percentage inhibition of every groups was measured.
$$ \mathrm{EV}=\mathrm{PVA}-\mathrm{PVI} $$
EV; edema volume, PVI; initial paw volume, PVA; paw volume after injecting carrageenan
$$ \mathrm{Edema}\ \mathrm{inhibition}\%=\frac{\mathrm{EVc}-\mathrm{Et}}{\mathrm{EVc}}\times 100 $$
EVc; Control group edema volume, EVt; Sample group edema volume.
Statistical analysis
Whole data is presented as mean ± SD. In vitro analysis contained triplicate evaluation while seven animals were used for each in vivo group. The Graph Pad Prism 5 was used for in vitro activities, for assessing correlation and IC50. Statistix 8.1 (1-way analysis of variance) was used for in vivo investigation. Observed significance level was p ≤ 0.05 for in vitro and p ≤ 0.01 for the in vivo analysis.