Plant material
EG is a prostrate (stretched out with face on the ground in adoration), annual plant with branches up to 15 cm long; the whole plant appearance is short-hairy or sparsely furry. The plant is typically collected from the wild for native use. The official name of the plant is Euphorbia granulata Forssk. Common names include Euphorbia forsskaolii, Euphorbia turcomanica, lubaina, and spurge. Its local name is lubaina. In English, it is called as desert spurge. Whole plant EG was collected during the month of July–August (2017) from the peripheral areas of Khanewal city in Punjab province, Pakistan. Botanical identification was done from Government College University, Lahore, and a sample was retained there in the herbarium with a voucher number 3820/Bot. Moreover, the plant name has been checked with http://www.theplantlist.org for the accepted name in accordance with the International Plant Names Index (IPNI).
Preparation of plant extract
The non-essential elements from the collected material were detached mechanically and the whole plant was washed three times using distilled water. Subsequently, plant material was shade dried at temperature ranging between (21–30 °C) for 30 days. The dehydrated plant material was mechanically condensed to granular powder and stored in an air-tight vessel. From the obtained plant powder, approximately 500 g of material was taken in a large beaker thereafter; 2 liters of dichloromethane (DCM) was then added to it, and the mixture was then macerated for 72 h and then again macerated with methanol for 72 h with intermittent shaking and stirring. The filtrate was extracted three times with the fresh solvent and all the extract was then combined. Multi-layered muslin cloth was used for coarse filtration of the macerated plant material. Once the coarse filtration is completed, the plant extract was filtered through Whatman no. 1 filter paper for fine filtration. The acquired filtrate was then resolved under reduced pressure (760 mmHg) at 40 °C in a rotary evaporator (Heidolph Laborota 4000, USA). The semisolid mass assimilates indicated percentage yield of 29.6%, and later it was desiccated in an oven at 40 °C. To speed up the process of extraction, grinding was done and later size reduction was performed with a pestle and mortar. After size reduction, plant material was macerated in (70:30) water and methanol. The grinded material was macerated in aqueous methanol mixture three times, then filtered and evaporated on rotary evaporator [18, 19].
Solvents and chemicals
The standard drug indomethacin was locally acquired from Chiesi Pharmaceuticals, Pakistan; tramadol (Tonoflex injection) and paracetamol (Provas injection) were purchased from SAMI Pharmaceuticals Pvt. Ltd. Acetic acid and brewer’s yeast was purchased from Punjab Chemicals Pvt. Ltd. Lahore, Pakistan. Carrageenan was purchased from Sigma Aldrich, USA. Sterile normal saline (NS) (PAKSOL pharmaceuticals pvt Ltd) and sterile water for injection was purchased from local distributer, Muller and Phipps (M&P), Lahore, Pakistan. Dimethyl sulfoxide (DMSO) from Sigma Aldrich, USA; Dulbecco’s modified Eagle medium (DMEM); Roswell Park Memorial Institute (RPMI 1640) and L15 complete medium from (Hyclone, USA); penicillin/streptomycin solution (Hyclone, USA); phosphate buffer saline (PBS; Hyclone, USA); fetal bovine serum (FBS; Hyclone, USA); MTT reagent (Bio world, Dublin); and trypsin (Hyclone, USA). The doses of the plant extract were prepared in DMSO and diluted in sterile normal saline under aseptic laboratory conditions in a clean room for all the experiments.
Experimental animals
With prior approval from the ethical committee for the animal experiments (Institutional Review Committee for Biomedical Research, University of Veterinary and Animal Sciences, Lahore, Pakistan), approval number is IRCBR/886-E-17/PCOL/UVAS. Wistar rats (female) aging 1 to 2 months of Rattus norvegicus species weighing 100–150 g were purchased from University of Health Sciences (UHS), Lahore. The experimental rats, (ER), now onwards, were retained under routine laboratory conditions of 22–25 °C with alternate light/dark 12/12-h period. Pellet form feed was given to ER and water ad libitum. For every experiment, 20 ER were used and distributed randomly in five groups and each group comprises of four ER.
Anti-inflammatory activity
The anti-inflammatory strength of aqueous methanolic extract of whole plant was assessed against using carrageenan-induced paw edema model. ER were randomly divided into five groups counting four ER in each group. ER were deprived of feed 1 h before experiment. Group I was administered sterile normal saline as blank or negative control 10 ml/kg; group II was given standard drug indomethacin 10 mg/kg, as positive control; group III, IV, and V were given plant extract 50, 100, and 200 mg/kg, respectively. After 1 h of intra-peritoneal (i.p.) administration of standard drug and plant extract, 1% carrageenan solution approximately 100 μl was injected into the left hind paw of each ER as an inflammatory mediator. The paw volume of each ER was then determined immediately at 0 h and after 3 h of carrageenan injection using liquid immersion model [20]. To measure the volume of the paw edema, a glass beaker was filled with distilled water and positioned on a weighing balance; the weight of the beaker was tared. The paw volume of each ER was measured by immersion of the inflamed paw into water. A force F is applied to the balance against the movement of liquid inside the beaker and that was equal to weight of the paw. The paw volume was measured using Eq. (1).
$$ \mathrm{Volume}=\left(\mathrm{Weight}\right)/\left(\mathrm{Specific}\ \mathrm{gravity}\ \mathrm{of}\ \mathrm{liquid}\right) $$
(1)
As the specific gravity of water at room temperature is taken as 1 so each 1 g increase in weight is equal to the 1 cm3 increase in volume.
The mean paw edema was calculated for all groups and compared, with that of negative control group. Percentage inhibition of the inflammation was then measured using Eq. (2) given as:
$$ \mathrm{percentage}\ \mathrm{inhibition}=\left(\left(\mathrm{Vt}-\mathrm{Vo}\right)\mathrm{control}-\left(\mathrm{Vt}-\mathrm{Vo}\right)\ \mathrm{treated}\right)/\left(\mathrm{Vt}-\mathrm{Vo}\right)\ \mathrm{control}\times 100 $$
(2)
where Vt is paw volume at time t = 3 h and Vo is volume at 0 h time.
Analgesic activity
Eddy’s hot plate model
The ER were randomly distributed into five groups comprising four ER in each group. All animals were introverted from feed 2 h before the beginning of experiment. Pre-testing of ER on Eddy’s hot plate kept at 55 °C ± 0.1 °C was then performed. ER displaying latency time greater than 15 s were omitted from the experiment. In our study, none of the ER displayed latency time greater than 15 s in pre-testing examination so there was no exclusion. The groups were injected with the following: group I was injected sterile NS 10 ml/kg; group II was given tramadol 20 mg/kg; group III, IV, and V were given EG 50, 100, and 200 mg/kg via i.p. route of administration, respectively. Thirty minutes after administration of corresponding treatment, the ER were placed on Eddy’s hot plate and latency time (time for which ER remained on the hot plate without licking or flicking of hind limb or jumping) was then measured for 1 min and recorded [21]. The percentage analgesia was calculated by Eq. (3).
$$ \mathrm{percentage}\ \mathrm{Analgesia}\ \left(\%\right)=\frac{\mathrm{Test}\ \mathrm{latency}-\mathrm{Control}\ \mathrm{latency}}{\mathrm{Cut}\ \mathrm{off}\ \mathrm{time}-\mathrm{Control}\ \mathrm{latency}}\times 100 $$
(3)
Writhing model
ER were divided into five groups comprising four ER in each group. The peripheral analgesic potential was assessed by acetic acid-induced abdominal writhing. The groups were given with following: group I was administered sterile NS 10 ml/kg; group II was administered indomethacin 10 mg/kg; group III, IV, and V were administered plant extract 50, 100, and 200 mg/kg, respectively. After group treatment, approximately 50 μl of 1% acetic acid solution in sterile NS was given to each ER through i.p. route of administration. Twenty minutes later, the abdominal constrictions or writhing was observed and counted for 1 min and recorded [22].
Antipyretic activity
Brewer’s yeast-induced pyrexia model
Antipyretic strength of the plant extract was appraised against using methods previously described. ER were randomly allocated into five groups comprising four ER in each group. The standard body temperatures of the ER were examined by inserting a digital thermometer into their anal cavities for about 1 min. The rectal temperature readings observed were recorded as pre-treatment or normal body temperature. Thereafter, pathogenic fever was induced in ER by injecting 15% brewer’s yeast (Saccharomyces cerevisiae) suspension at dose adjustment of 1 ml/kg, subcutaneously under the nape of the neck and 24 h later the rectal temperature of all the ER was measured again. ER that did not show a baseline increase of 0.3 °C temperature was excluded from the study. In our study, none of the ER was omitted due to the aforesaid criteria. Later on, group I was given sterile NS 10 ml/kg; group II received standard drug paracetamol 150 mg/kg; and group III, IV, and V were given plant extract 50, 100, and 200 mg/kg, via i.p. route of administration, respectively. Rectal temperature was then noted directly at 0 h and after 1, 2, 3, and 4 h post drug treatment [23, 24].
Sterility test
Preparations were intended to be injected through intra-peritoneal route of administration, so they needed to comply with sterility protocol. Following the standards of the International Pharmacopoeia, we cross-checked by carrying out sterility test using fluid thioglycolate medium for anaerobic microbes and soya bean-casein medium for aerobic microbes, incubated for 14 days with preparations separately under the same conditions in which original tests were performed. The intended preparations were inoculated with equal volumes of mentioned medium. Thereafter, we have checked the appearance of any visible growth of microbes at 3rd, 5th, 7th, 9th, 11th, and 14th day after inoculation. In macroscopic examination, no visible growth or turbidity were observed in any of the culture plate. Hence, it was ascertained that plant extract and standard drugs used earlier were all sterile by replicating the similar conditions as were used during experiments [23].
In vitro anti-proliferation test
Cell culture
Human breast cancer cell lines MCF-7, Skbr3, and MDA-MB-231 were provided by the Centre of Excellence in Molecular Biology (CEMB), PU Lahore. Receptor-based classification categorizes these cells into estrogen positive (ER+), e.g., MCF-7, Human epidermal growth factor receptor-2 positive (HER2), e.g., Skbr3 and triple-negative breast cancer (TNBC) type cancer cells, which do not possess any of the classical receptors of breast cells, e.g., MDA-MB-231 cell line. The MCF-7 cells were cultured in DMEM and Skbr3 cells were cultured in RPMI 1640, whereas MDA-MB-231 cells were grown in L15 complete medium; all the medium were supplemented with 10% FBS (v/v) and 1% penicillin-streptomycin solution (v/v) at 37 °C, 5%CO2, and 95% relative humidity except for MDA-MB-231 cell line, which was incubated in CO2-free environment.
MTT assay
The cytotoxic potential of EG whole plant extract was appraised as described before with slight modifications. Briefly, the cells were seeded into 96-well plate at a density of 2 × 104 cells/well and incubated for 24 h, followed by the treatment with different dilutions of plant extract (0.78,1.56,3.12, 6.25,12.5,25,50,100 µg/ml) for 48 h. After 48 h, MTT reagent (5 mg/ml) was added in each well with serum-free medium (200 μl) and subjected to further incubation for 4–6 h; thereafter, the contents were removed from each well. In order to dissolve the formed crystals of purple formazan, 150 μl of DMSO was added to each well and kept for 20 min at 37 °C on an orbital shaker for 15 min. The absorbance was then calculated using an ELISA plate reader at 490 nm. Absorbance of control (without any treatment) was used as reference for calculation of cytotoxicity and cell viability [25].
Statistical analysis
All the data were expressed in terms of mean ± S.D. The experimental results were analyzed for statistical significance by one-way ANOVA followed by post hoc Dunnett’s test for multiple comparisons. p < 0.05* was considered significant.
