Collection of the plant material
The flowers of (AP) plant were collected in April-May 2019 from the Tulkarem region in Palestine. The plant was characterized a pharmacognosist Dr. Nidal Jaradat in the herbal products laboratory at An-Najah National University. The plant was deposited in the Pharmacognosy Laboratory, Faculty of Medicine and Health Sciences at An-Najah National University (voucher specimen: Pharm-PCT-246). The study protocol complied with relevant international guidelines and legislation.
The collected flowers were washed with distilled water and later completely dried in shade at room temperature for 3 weeks. The dried parts were grounded into a fine powder using a mechanical blender and stored in tightly sealed special containers until use.
Preparation of (AP) sample
The dried (AP) flowers were chopped into small pieces and 100 g of (AP) flowers were boiled in 1 L of distilled water until the original volume is decreased to one fourth. After that, the decoction was filtered (Machrery-Nagel, MN 617 and Whatman no.1, USA) and the filtrate was placed into a freeze drier (Millrock Technology-BT85, China) apparatus until the aqueous extract turned into solid powder. The dried extract was then kept in a well-closed container for further use. In the next step, AP flower extracts were diluted with the water, DMSO and methanol solutions. To obtain the concentration of 0.1 mg/ml, 1 mg from each one of the preserved extracts was taken. Then it was diluted with 10 ml of the three solutions. Finally, 1 ml of the prepared dilution was obtained using a pipette. All preparations were packaged in suitable, locked containers and were sent to Hadassah Hospital Laboratories for further research.
The examined bacterial and fungal isolates were obtained from the American Type Culture Collection (ATCC). The selected species of microorganisms are frequently isolated in clinical settings in our region and some possess multidrug resistance. The isolates included three Gram-positive strains: Staphylococcus aureus (ATCC 25923), Methicillin Resistance Staphylococcus aureus (MRSA) a clinical strain, and Enterococcus faecium (ATCC 700221) and four Gram-negative strains: Klebsiella pneumoniae (ATCC 13883) Proteus vulgaris (ATCC 700221), Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853). Meanwhile, the fungal isolates included Candida albicans (ATCC 90028).
Blood samples collection and preparation
Citrated blood samples were collected from five healthy volunteers. All participants were not under any medications especially anticoagulant, they were also non-smoker individuals. One part of sodium citrate was mixed with 9 parts blood to obtain a ratio equals 1:9. The citrated blood was centrifuged at 2500 rpm for 15 minutes to obtain citrated-PPP (platelet-poor plasma). Prothrombin time (PT), activated partial thromboplastin time (aPTT), and Thrombin time (TT) tests were conducted on plasma within 2 h of blood collection. All results were obtained by a digital coagulation analyzer (coagulation analyzer Coa-DATA 4004, Germany). All measurements were conducted in duplicate and 1% DMSO was used as a negative control (15).
Hep 3B2.1-7 [Hep 3B, Hep-3B, Hep3B] was commercially purchased from ATCC (HB-8064) .Cytotoxicity evaluation was carried out by using the HEP3B cell line, which has the same genotype, phenotype and features of hepatocellular carcinoma (HCC). These cells were taken from a Japanese person liver who had HCC and hepatitis B. HEP3B are characterized by the secretion of α-fetoprotein (αFP) that is considered a tumor marker. αFP were assessed by a commercially available ELISA kit from (R&D Systems, Inc., USA). HEP3B condition was accomplished by using RPMI-1640 medium enhanced with 1% penicillin, 1% streptomycin, 1% l-glutamine and 10% fetal bovine serum; it was adapted to pH 7.2 by Dulbecco's Phosphate Buffered Saline (DPBS). The growing process of the cells was performed at 37°C in ESCO cell-culture incubator which had a humidified atmosphere of 95% air and 5% CO2 at 37° C.
The antimicrobial activity of (AP) flowers aqueous extract was assessed using the well diffusion method. The bacterial suspension was prepared by picking some colony of overnight agar culture of the test organism and adding it to a test tube containing 5 ml of nutrient broth, then the turbidity was compared with that of McFarland nephelometer tube No. 0.5 (1.5X108 cfu/ml); then it was diluted by taking 1000 μl of suspension and it was added to 2 ml of nutrient broth (0.5X108 cfu/ml). The Minimum Inhibitory Concentration (MIC) is the lowest concentration of an antimicrobial that inhibits the growth of a microorganism after 18-24 hrs. The extract was subjected to serial broth dilution technique to determine their minimum inhibitory concentration for all tested microorganisms. The broth micro-dilution method was used to determine the antimicrobial activity of (AP). The plant extract was dissolved in 1 ml DMSO in a concentration of 100 % for aqueous extract. Then the final extract concentration was 100 µg/ml. After that, each well was inoculated with microbial inoculums which were prepared in the same medium after dilution of standardized microbial suspension adjusted 0.5 McFarland scale. After well mixing, the 96-well micro-titration plates are incubated under 37°C for 24 h. For all bacteria we tested here, was conducted four controls including; 1- +ve control which contains media + bacteria; 2- -ve control which contains media only; 3- Extract control (extract + media): to be sure there is no contamination and turbidity and the changes are not occurred due to extract itself (so extracts were serially diluted in this control); 4- DMSO: no extract used. The established tests were conducted in triplicates (16).
Prothrombin Time (PT) test
The in-vitro method was used for the test. 100 µl of pre-warmed plasma were incubated with 100µl plant extract for 5 minutes. All concentrations (50, 25, 5 and 1 mg/ml) from (AP) aqueous extract were tested. The clotting time was measured immediately after the addition of 200µl of pre-warmed thromboplastin reagent (Hemostat thromboplastin-SI. Human, Germany) (15).
Activated Partial Thromboplastin Time (aPTT) test
For this test, an in-vitro approach was used. For 5 minutes, 50 µl of pre-warmed plasma were incubated with 50 µl of plant extract. The concentrations of (AP) aqueous extract were evaluated at 50, 25, 5, and 1 mg/ml. The PTT reagent was then added and incubated for 3 minutes. The clotting time (aPTT, Human, Germany) was determined immediately after the addition of 50 µl CaCl2 reagent (15)
Thrombin Time (TT) test
The in-vitro technique was used for the test. 100µl of plant extract were incubated for 5 min with 100µl pre-warmed plasma. Different concentrations (50, 25, 5 and 1 mg/ml) from (AP) aqueous extract were tested. The clotting time was determined after 100 µl of pre-warmed thrombin reagent was added (Hemostat Thrombin Time. Human, Germany) (15).
Flow cytometry analysis
Following the culturing, the collected HEP3B cells were adjusted to 106/ml in staining buffer (in saline containing 1% bovine albumin). The labeling of fragmented DNA with propidium-iodide (PI) and the staining of phosphatidylserine with annexin V-conjugated to Fluorescein isothiocyanate (FITC) were done according to the manufacturer's instructions for viability assessments and apoptosis.. The apoptosis was then identified with annexin-V (+) but not propidium-iodide (-). Viable cells, on the other hand, were labeled with annexin-V (-) but propidium-iodide (-). In each experiment, unstained controls were used, such as IgG isotype controls and Fluorescence Minus One (FMO) controls. The use of propidium-iodide allowed for the investigation of the cell cycle by the quantification of DNA content. The HEP3B cells were fixed for at least 30 minutes in a cold 70% ethanol solution at 4°C. The cells were then rinsed twice in phosphate buffered saline (PBS). To dispose of the supernatant, it was calibrated to spin at 2000 rpm. The cells were treated with ribonuclease (50 l μl of 100 μg /ml RNase) to ensure that only DNA was stained. The cells were then stained with 5 μl of 50 μg Propidium iodide/100 ml and flow cytometry was used to evaluate them (Becton-Dickinson LSR II, Immuno-fluorometry systems, Mountain View, CA).(17)
Alpha Feto Protein (aFP) detection
aFP is produced whenever liver cells are regenerating. With chronic liver diseases, such as hepatitis and cirrhosis, aFP may be chronically elevated. Very high concentrations of aFP may be produced by certain tumors. This characteristic makes the aFP test useful as a tumor marker. Increased amounts of aFP are found in many people with the most common type of liver cancer called hepatocellular carcinoma and in a rare type of liver cancer that most commonly occurs in infants called hepatoblastoma. Secreted aFP concentrations in Hep3B cells culture medium were detected using Human alpha-Fetoprotein Quantikine ELISA Kit (R&D; DAFP00). Absorbance was measured at 450 nm using a Universal Microplate Reader
The obtained results of the studied (AP) aqueous extract were expressed as means ± standard deviation (SD). Averaged data were compared using a t-test. The statistical significance was considered when the p-value was <0.05.