Plant material
A. jacquemontii was collected in the month of July from Lakary mountains, Shamshaki, District Karak, Khyber Pakhtunkhwa, Pakistan. The plant material was authenticated by Prof Dr. Mir Ajab Khan, Department of Plant Sciences, Quaid-i-Azam University Islamabad, Pakistan. A voucher specimen (PHM: 491) was deposited in the Herbarium of Medicinal Plants, Quaid-i-Azam University Islamabad, Pakistan.
Chemical and reagents
The study involved the use of analytical grade solvents which were purchased from Sigma (Sigma-Aldrich USA) i.e. n-hexane (n-Hex); Chloroform (CHCl3); Acetone (Ace); Ethyl acetate (EtOAc); Ethanol ((EtOH); Methanol (MeOH) and Dimethyl sulfoxide (DMSO). The reagents; Gallic acid (G7385); Quercetin (Q4951); 2,2-diphenyl-1-picryl-hydrazyl (Cat#757621; DPPH); Potassium acetate; Aluminum Chloride; Folin-ciocaltau (F-C) reagent -Sodium carbonate; Ascorbic acid (A5960); Ammonium molybdate; Sodium phosphate; Sulfuric acid; Ferric cyanide; Trichloroacetic acid and Potassium ferricyanide were procured from Merck (Merck KGaA, Germany). Roxithromycin (R4393); Cefixime (1097658); Terbinafine (T8826), Doxorubicin (D1515), Taxol (T1912) and Amphotericin-B (A2942) was obtained from Sigma (Sigma-Aldrich USA) were used Reference standards in the current study.
Preparation of extracts
Tubers of A. jacquemontii were washed with tap water then rinsed with distilled water. The material was air dried under shade at room temperature. After comminuting the plant material by using the commercial miller to coarse powder, 10 g of dried powder material was soaked separately in beakers, each containing 40 mL of extraction solvents of ranging polarity from highly non-polar to highly polar solvents including n-hexane (n-Hex), Chloroform (CHCl3), Acetone (Ace), Ethyl acetate (EtOAc), Ethanol (EtOH), Methanol (MeOH). Along with individual solvents, combinations of solvents at 1:1 were also used as follow: EtOH+CHCl3, MeOH+CHCl3, Ace+EtOAc, EtOH+EtOAc, and MeOH+EtOAc. The plant material was soaked for 48 h, thereafter filtered through Whatman No.1 filter paper. The residue was again dipped in respective solvent/s and this process was repeated thrice. The respective extracts were combined and concentrated by evaporating the solvent under reduced pressure in a rotary evaporator (Buchi, Switzerland) at 45 °C.
Phytochemical screening
Determination of total phenolic contents
The total phenolic contents of the extracts were determined by Folin–Ciocalteu method [15]. Briefly, stock solutions of extracts (4 mg/mL) were prepared in DMSO and 20 μL of each extract was transferred to each well of 96 well plate. The solutions were then mixed with 90 μL of F-C (Folin–Ciocalteu) reagent. After 5 min, the reaction mixture was mixed with 90 μL of Na2CO3 solution (7.5%). The reaction mixture was incubated for 1 h and absorbance was measured at 650 nm by using microplate reader (Bioteck). Blank (DMSO) and standard (gallic acid in DMSO) were run simultaneously. The resultant TPC is calculated as μg gallic acid equivalents per mg extract (μg GAE/mg extract).
Determination total flavonoid content (TFC)
The TFC of the extracts was determined by aluminum chloride method as reported previously [15]. Briefly, extract solution (20 μL, 4 mg/mL DMSO) was mixed with 10 μL of aluminum chloride (10%) and 10 μL of potassium acetate (1 M). Subsequently, distilled water was added to get a final volume of 200 μL. After 30 min of incubation (Incubator IC83 Yomato, Japan), absorbance was measured by using microplate reader (Bioteck) at 415 nm at 37 °C. Quercetin was used as standard flavonoid and results are expressed as μg quercetin equivalent per mg extract (μg QE/mg extract).
Biological evaluation
DPPH free radical scavenging assay
2, 2-diphenyl-1-picrylhydrazyl reagent was employed for the determination of free radical scavenging activity of the extracts [15]. Briefly, stock solutions of extracts (4 mg/mL) were prepared in DMSO. Aliquot of 10 μL of each extract was mixed with 190 μL of DPPH (0.004%). The reaction mixture was incubated in the dark for 1 h. The optical density was measured at 515 nm using microplate reader (Bioteck). Ascorbic acid was employed as positive standard while DMSO as negative control. The extracts were first screened at final concentration of 200 μg/mL and those exhibiting good quenching activity (≥ 50%) were tested at lower concentration to find IC50 values. Percent inhibition was calculated by the following formula:
$$ \mathrm{Percent}\ \mathrm{inhibition}\ \mathrm{of}\ \mathrm{the}\ \mathrm{test}\ \mathrm{sample}=\left[\%\mathbf{scavenging}\ \mathbf{activity}=\right(\mathbf{1}-{\mathbf{Ab}}_{\mathbf{s}}/{\mathbf{Ab}}_{\mathbf{c}\Big)}\ast \mathbf{100}\Big] $$
Where Abs is the absorbance of DPPH solution with sample, whereas Abc indicates the absorbance of negative control (containing the reagent except the sample). The IC50 was calculated by using Table curve software 2D version 4.
Determination of total antioxidant capacity (TAC)
Total antioxidant activity of extracts was evaluated following the methodology previously reported [15]. Mixing 100 μL of stock solution of each extract (4 mg/mL in DMSO) with 900 μL reagent solutions comprising of 0.6 M sulfuric acid, 4 mM ammonium molybdate and 28 mM sodium phosphate was done. The reaction mixtures were incubated at 95 °C for 90 min. After incubation the reaction mixtures were cooled down at room temperature and absorbance of each extract was measured at 695 nm by using micro plate reader. A typical blank containing DMSO (100 μL) was used as control. For the calibration curve, ascorbic acid was used at different concentrations with DMSO. The resultant TAC is expressed as μg ascorbic acid equivalent per mg extract (μg AAE/mg extract).
Reducing power assay
The reduction potential of the test samples were investigated according to the procedure described previously [15]. Briefly, 100 μL of each sample (4 mg/mL extract in DMSO) was mixed with 200 μL of phosphate buffer (0.2 M, pH 6.6) and 250 μL of 1% potassium ferricyanide solution. The resulting mixtures were incubated for 20 min at 50 °C. After incubation, the reaction mixtures were acidified with 200 μL of 10% trichloroacetic acid. The resultant mixtures were centrifuged at 3000 rpm for 10 min. Supernatant layer (150 μL) was mixed with 50 μL of 0.1% ferric chloride solution in a separate tube and optical density was measured at 630 nm using microplate reader. Ascorbic acid was maintained as positive control and results are expressed as μg ascorbic acid equivalent per mg extract (μg AAE/mg extract).
Brine shrimp lethality assay
The degree of lethality to brine shrimps was determined by following previously described protocol [16]. Stock solutions (100 mg/mL) of each sample were prepared in DMSO. Artemia salina (brine shrimp) eggs (Ocean Star, USA) were hatched in a bi-partitioned tank filled with artificial sea water (3.8% Sea salt supplemented with 6 mg/mL dried yeast, pH 7). The larger compartment was covered with aluminum foil while the smaller compartment was illuminated with a light source. After 24–48 h incubation period the phototropic nauplii were harvested by using micro pipette and transferred to 96 well plate. Various sub dilutions of 100 mg/mL DMSO stock solution of each extract were tested for lethality determination at final concentrations of 250, 125, and 62.5 μg/mL. The corresponding micro liter of each dilution was transferred to each well containing 10 nauplii and 300 μL sea water supplemented with dried yeast (6 mg/L). Negative control vial included DMSO, nauplii and sea water, but no sample, whereas positive control included 4 mg/ml standard drug doxorubicin, nauplii and sea water. After 24 h incubation period, dead nauplii were counted in each vial and LC50 was measured accordingly by comparing percentage mortality with standard drug using Table curve software 2D version 4.
Protein kinase inhibition assay
Streptomyces 85E strain was used for protein kinase inhibition assays following the published protocol [17]. The microorganism was refreshed in sterile Trypton Soy broth (Merck, Germany) for 24–48 h. The cultured was inoculated ISP4 mineral medium in petri plates. 5 mm Whattman filter paper discs impregnated with 5 μL of 20 mg/mL extracts were placed on seeded plates. The plates were incubated at 28 °C for 72 h. The bald zones of hyphae formation inhibition were measured. Surfactin was used as positive control while DMSO impregnated discs were included as negative control in order to confirm the non toxic effect of DMSO.
In vitro antileishmanial activity
In vitro antileishmanial activity was performed with Leishmania tropica khw23 strain [18]. The Leishmanial parasite was kindly provided by Dr. Gul Shahnaz and protocol was followed as described [18]. The parasite was cultured in M199 media supplemented with 10% Fetal Bovine Serum at 24 °C.The culture (promastigotes) was harvested at concentration of 1 × 106 cells/mL. Stock solution of each test samples was prepared in DMSO (20 mg/mL) and serially diluted in 96 well plate. The anti leishmanial activity of each sample was determined at concentrations ranging from 62.5–250 μg/mL. The cultured plates, inoculated with parasite and test samples, were incubated at 24 °C for 72 h. Amphotericin-B was employed as positive control while distilled water as a negative control. The live promastigotes were counted under light microscope using improved Neubauer chamber. The data obtained was then statistically analyzed and LC50 was estimated using table curve 2D Ver.4 software.
Antibacterial activity
Antibacterial assay was performed against five bacterial strains (two Gram positive (Micrococcus luteus ATCC# 10240 and Staphylococcus aureus ATCC# 6538) and three Gram negative (Bordetella bronchiseptica ATCC# 4617, Salmonella typhimurium ATCC# 14028 and Enterobacter aerogenes ATCC# 13048) following the standard protocol [15]. The strains were purchased from ATCC culture bank and maintained in the lab. The strains were recultured by inoculating them from stored slants onto nutrient broth media and incubated for 18–24 h. The refreshed inoculum was then swabbed onto petri plates filled with nutrient agar. Sterilized deionized water was used to adjust the turbidity to 104 CFU/mL by comparing with McFarland 0.5 BaSO4 turbidity standard. The extract (5 μL of 20 mg/ml DMSO) infused filter paper discs (6 mm) were placed on swabbed nutrient agar plate. Roxithromycin (4 mg/ml), Cefixime-USP (4 mg/ml) and DMSO impregnated discs were included as positive and negative control, respectively. After 24 h incubation period, clear zone of growth inhibition around sample and standard treated discs was measured by using vernier caliper. The samples having ≥11.0 mm zone of inhibition were tested at lower concentrations (100–50 and 25 μg/ml) by using microtiter plate broth dilution method to find minimum inhibitory concentration (MIC).
Antifungal activity
Antifungal activity of each sample was evaluated following the method described previously [17]. The fungal strains were kindly provided by First Culture Bank of Pakistan Punjab University Lahore Pakistan. Fungal cultures (Aspergillus fumigatus FCBP# 66, Fusarium solani FCBP# 0291, Mucor specie FCBP# 0300, Aspergillus flavus FCBP# 0064 and Aspergillus niger FCBP# 0198) were cultured on Sabouraud Dextrose Agar medium (SDA; Merck Germany). Prior to the sensitivity determination, the spores of fungal strains were harvested in 0.02% between 20 solution and their turbidity was adjusted according to McFarland 0.5 turbidity standard. Then 100 μL of each harvested spores was swabbed on plates containing SDA. Filter paper discs loaded with 5 μL of extract (20 mg/ml in DMSO) as well as 2.5 μL of standard antifungal terbinafine (4 mg/ml) were placed on swabbed SDA plates. The plates were incubated at 28 °C for 24 h. After that the clear zones of growth inhibition around sample infused discs were measured using vernier caliper.
SRB cytotoxic assay against DU-145, HL-60 cancer cell lines
The cytotoxic potential of test samples to adherent cancer cell lines was determined by using SRB colorimetric assay [17]. All the cell lines were cultured in DMEM medium except for HL-60 that was cultured in RPMI-1640. Basic media were supplemented with 5% FBS and 1% antibody (AB) solution and maintained at 37 °C in 5% CO2 incubator to achieve the desired level of confluence. For adherent cells, after removing the old medium, cells were washed thrice with PBS and 1 mL of trypsin was added and put in the incubator for 5 min. Cells with trypsin were shaken along with 5 mL of fresh medium and transferred a falcon tube. For suspended cultures, cells were transferred to falcon tube and centrifuged for 5 min at 1500 rpm. Old medium was then replaced with 5 mL fresh medium. Cells were counted using Hemocytometer and seeding density was adjusted to 5 × 104 cells/mL.
During assay, 10 μL of extract was thoroughly mixed with 190 μL of culture in 96-well plate and incubated for 72 h at 33 °C. Then, 100 μL of 20% w/v TCA was added and incubated for 30 min at 4 °C to fix the cells. It was then washed three times with water, air dried and incubated with 100 μL of 0.4% w/v SRB in 1% acetic acid for 30 min at room temperature. Afterwards, wells were washed with 1% acetic acid, air dried and exposed to 200 μL of 10 mM Tris base for 5 min in the plate shaker. Absorbance of the microplate was measured at 492 nm using the plate reader. Taxol (400 μg/mL) was used as positive control while culture media having no visible growth was used as blank. 10% DMSO PBS used was negative control.
Cancer chemo preventive bioassays
Inhibition of TNF-α activated nuclear factor-kappa B (NF-ĸB) assay
The NF-κB assay was carried out in accordance to a previously published protocol [17]. In brief, the Hela cells nuclear extract was treated with each extract and TNF-α was used for evaluation for specific binding. After treatment with test samples, cells were treated with TNF-α (10 ng/mL) and the nuclear extract from the cells extracted using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents kit from Thermo Scientific (Pierce Biotechnology, Rockford, IL, USA). The specific binding ability of activated NF-κB p65 to the biotinylated-consensus sequence under the presence of test samples was assessed using the Pierce™ NF-κB p65 Transcription Factor Assay kit from Thermo Scientific (Pierce Biotechnology, Rockford, IL, USA). The binding activity was measured by detecting the chemiluminescent signal in a Fluostar Optima plate reader. The test samples exhibiting over 50% inhibition at 50 μg/mL were also tested at four concentration levels (0.05 μg/mL – 50 μg/mL), to obtain inhibitory concentration (IC50). Rocaglamide was used as a positive control and untreated cells as negative control.
Mitochondria transmembrane potential (MTP) assay
Variations on the mitochondria trans-membrane potential were noticed and measured by a fluorescence cell based assay as described previously [17]. In short, cells were cultured in black and clear bottom plates of 96-well plates at a density of 6 × 104 cells/mL and incubated for 24 h at 37 °C in a humified 5% CO2 incubator. Cells were then treated with the test samples and staurosporine (positive control) for 2 h. The cells were incubated with the lipophilic cationic dye (5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethyl benzymidazolyl carbocyanide) JC-1 for 30 min. After incubation, cells were washed with a buffer for removing unbound staining reagent. The clear bottom plates were then scanned with fluorescence imaging microscope. Black 96-well plates were analyzed by a plate reader (FLUO-star-Optima-fluorescence) with excitation and emission wavelengths of 485 nm and 530 nm respectively for JC-1 monomers while excitation and emission wavelengths of 560 nm and 595 nm respectively for J-aggregates. Measurements were performed in triplicate and are representative of at least two independent experiments.
Statistical analysis
The data is presented as mean ± standard deviation (SD). All the experiments were carried out in triplicate. SPSS Ver. 21 software was used for Post Hoc Multiple Comparison test in One Way ANOVA and IC50 was determined by using Table curve software 2D version 4. Origin 8.5 software was used for graphical presentation.