Antimicrobial, Anticoagulant and Anticancer Effects of Arum Palaestinum Flowers Extracts

Background: Wild plants are amply utilized in traditional medicine and folkloric food worldwide. Arum palaestinum Boiss. (AP) is one of the wild Palestinian plants which leaves have a long history in the Middle Eastern countries as food and medicine. Herby, the current study aimed to evaluate the antimicrobial, coagulation cascade activities, and anticancer effects of (AP) owers extract Methods: The aqueous extract of (AP) owers was screened on its antimicrobial activity using microdilution assay against eight pathogens. While, prothrombin time, activated partial thromboplastin time, and thrombin time tests were measured utilizing standard hematological methods. And Anti cancer effect was assessed by using Parameters of cell cycles and alph feta protein level that were investigated for (AP) owers fractionated with aqueous, DMSO, and methanol Results: The antimicrobial screening results revealed that the aqueous extract of (AP) has strong antibacterial effects against P. vulgaris and E. faecium compared with Ampicillin with MIC values of 6.25, 6.25 and 18 mg/ml, respectively. The aqueous extract of (AP) showed anticoagulant activity with signicant prolonged results in aPTT and TT tests at high concentrations (50 mg/ml and 25 mg/ml) and slightly prolonged results in the PT test at a high concentration (50 mg/ml). The anticancer results indicate a delay in cell cycle through decreased the cell proliferation rate following effects of the AP fractions. The delay in the S phase was in favor of the water fraction. Water and DMSO fractions while maintained the cells in the G2-M phase similar to the DOX, the ower extract in methanol accelerated the cells in the G2-M phase suggesting that (AF) ower extracts have anti-cancer properties. At the same time Aqueous extract decreased HCC aFP to 1.55-fold (P=0.0008). While DMSO and methanolic extract had no signicant effects on HCC aFP levels, compared to control untreated cells of 2519.16 ± 198.1 ng/ml. This data show that (AF) aqueous solution is potent inhibitor of alpha-fetoprotein secretion (P-value <0.05), which indicates its anti-carcinogenic effects Conclusion: These results showed that the aqueous extract of (AP) plant possesses bioactive components with antibacterial and anticoagulant properties, which may be exploited in the treatment of infectious diseases and blood coagulation disorders. Citrated samples from All also non-smoker individuals. One part of citrate was mixed with 9 parts blood to obtain a ratio equals 1:9. The citrated blood was centrifuged at 2500 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). 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 (1.5X10 8 cfu/ml); then it was diluted by taking 1000 μl of suspension and it was added to 2 ml of nutrient broth (0.5X10 8 cfu/ml). The Minimum Inhibitory Concentration (MIC) is the lowest concentration of an antimicrobial that inhibits the growth of a microorganism after hrs. serial tested coagulation assays aqueous of aPTT and TT in a manner with the highest effect at mg/ml. At the same time, a signicant in PT test plant extract These tests are used to evaluate the coagulation cascade, PT is used for evaluation of the extrinsic and common pathways of the coagulation cascade. APTT for the intrinsic common pathways, and TT for the brinogen to brin in the common pathway The results inhibition of clotting factors in the intrinsic and common pathways both aPTT and TT tests were signicantly. The increased in these tests plant Parameters of cell cycles were investigated for (AP) owers that were fractionated with aqueous, DMSO, and methanol. The incubation of these fractions of the (AP) owers with HEP3B cells was achieved for 48 hours. Doxorubicin (DOX), is an anticancer drug known to inhibit cell proliferations and DNA arrest were used as control.


Introduction
Herbal medicine is one of the traditional therapeutic methods that many people have used in their communities to treat and prevent harmful diseases (1). This kind of treatment is based on the use of different plants parts such as leaves, owers, seeds, bark, fruits, and roots in preparing of medicinal remedies to control or prophylaxis of disease and to nd a cure for it, which later developed into synthetic or semisynthetic drugs, based on the assumption that they are safer, more potent, selective and come with few side effects (2). Herbal medicine has played the most important role in drug discovery, mostly because it is offering potential lead compounds, which result from several processes on plants including isolation, puri cation, and molecular characterization (3).
Thus, one of the lead sources is plant secondary metabolites that are derived biosynthetically from plant primary metabolites. These secondary metabolites are bioactive chemicals that include alkaloids, glycosides, phenolic compounds, tannins, and terpenoids which continue to be good model molecules in drug discovery due to their potential medicinal applications for the treatment of numerous conditions and diseases from migraine up to cancers (4). From the foregoing discussion, we see the importance of herbal medicine in helping humanity in discovering and developing medicines for many diseases, especially the intractable ones (5). The molecular process by which the body forms clots to avoid bleeding is the coagulation cascade.
Platelets, endothelial cells, and leukocytes must be activated so that a suitable surface is provided for the adhesion of clotting proteins (6). Blood coagulation cascade reactions are propagated by complex enzymes containing a vitamin K-dependent serine protease and an accessory cofactor protein that are calcium-dependently assembled on the membrane surface (7). The traditional view of blood coagulation regulation consists of extrinsic and intrinsic pathways; extrinsic pathway depends on the initiation phase, include plasma factor VII/VIIa and transmembrane receptor tissue factor (TF), whereas intrinsic pathway requires ampli cation phase, that includes plasma FXI, FIX, and FVIII (8).
Antibiotics are usually used to treat various sorts of microbial infections and some microorganisms can develop more resistant strains by mutation or acquired resistance against some antibiotics. For example, most Gram (-ve) are inherently resistant to vancomycin. Bacteria are deemed resistant to an antibiotic if their development is not prevented by the maximum amount of the antibiotic that can be absorbed in the host dosage. However, microbial strains become resistant by the genetic alteration or/and the expression of proteins which usually occurred by the modi cation of target site, reduction of accumulation, or by the enzymatic inactivation (9). Arum palaestinum Boiss. (AP) (Araceae) is a perennial herbaceous plant which native to the Levant and other countries located in the Mediterranean Basin. It grows up to 0.82 ft and blooms in the spring, between March and April months The plant is well recognized by its dark purplish-black spadix enclosed by a reddish-brown spathe (10,11). In traditional Palestinian medicine (AP) aqueous extract has been used for the treatment of Bacterial infection, cough, cancer, constipation, intestinal worms, skin infections, blood circulation disorders, and renal stones.
To the best of the author's knowledge, no previous studies were conducted to evaluate the antimicrobial and antiplatelet activities of (AP) owers.

Collection of the plant material
The owers 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 owers were washed with distilled water and later completely dried in shade at room temperature for 3 weeks. The dried parts were grounded into a ne powder using a mechanical blender and stored in tightly sealed special containers until use.

Preparation of (AP) sample
The dried (AP) owers were chopped into small pieces and 100 g of (AP) owers were boiled in 1 L of distilled water until the original volume is decreased to one fourth. After that, the decoction was ltered (Machrery-Nagel, MN 617 and Whatman no.1, USA) and the ltrate 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 ower 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.

Microbial isolates
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 ve 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).
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 humidi ed atmosphere of 95% air and 5% CO2 at 37° C.

Antimicrobial test
The antimicrobial activity of (AP) owers 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 (1.5X10 8 cfu/ml); then it was diluted by taking 1000 μl of suspension and it was added to 2 ml of nutrient broth (0.5X10 8 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 nal 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).

Anticoagulant properties 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.

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 10 6 /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 Vconjugated to Fluorescein isothiocyanate (FITC) were done according to the manufacturer's instructions for viability assessments and apoptosis.. The apoptosis was then identi ed 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 quanti cation of DNA content. The HEP3B cells were xed 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 ow cytometry was used to evaluate them (Becton-Dickinson LSR II, Immuno-uorometry 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

Statistical assessment
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 signi cance was considered when the p-value was <0.05.

Results And Discussion
Antimicrobial activity The antimicrobial activity of (AP) crude aqueous extract was determined using micro-dilution assay against selected infectious pathogens belonging to the Gram-negative, Gram-positive and fungi strains. Fluconazole The results showed that the aqueous (AP) extract has antibacterial activity comparing with the positive antibacterial controls (Ampicillin and Cipro oxacin) and has not antifungal activity compared with the positive antifungal drug Fluconazole as reported in Table 1.
In fact, the aqueous (AP) extract possessed an antibacterial effect against all the tested bacterial strains while this extract revealed a strong antibacterial effect against P. vulgaris compared with Ampicillin with MIC values of 6.25 and 18 mg/ml, respectively. Also has the same antibacterial activity against E. faecium with MIC values of 6.25 mg/ml. However, the antibacterial MIC values of (AP) were lower than Cipro oxacin.  The in-vitro coagulation assays results showed that the aqueous extract of (AP) has prolonged aPTT and TT tests in a dosedependent manner with the highest effect at 50 mg/ml. At the same time, a signi cant increase in PT test was observed only at a high concentration of plant extract (50 mg/ml) as observed in Tables 2-4. These tests are used to evaluate the coagulation cascade, PT is used for evaluation of the extrinsic and common pathways of the coagulation cascade. APTT for the intrinsic and common pathways, and TT for the conversion of brinogen to brin in the common pathway (17,18). The results suggest that the inhibition of clotting factors in the intrinsic and common pathways because both aPTT and TT tests were prolonged signi cantly. The inhibition increased in these tests as the concentration of plant extract increased. PT was slightly prolonged, when a high concentration of plant extract was used, as a result of inhibition of the common pathway, since the main inhibition being in the intrinsic and common pathways.

Anticoagulant properties
These ndings suggest either the presence of different active ingredients in this plant, at which the inhibitions of intrinsic and common pathways were observed or by acting on a common factor that inhibits both pathways. This factor may be through the protein C pathway, as this pathway is used to inhibit factor Va (in the common pathway) and factor VIIIa (in the intrinsic pathway). It is suggested that this plant may contain active ingredients that activate protein C to become the activated form (APC), then APC inhibits factors Va and VIIa, that's why prolonged results in the intrinsic and common pathways were seen (19).
Also, it could be by the presence of an anti-thrombin substance (18).

Cytotoxicity
Arum palaestinum fractions inhibit DNA cell cycle of HEP3B cells Parameters of cell cycles were investigated for (AP) owers that were fractionated with aqueous, DMSO, and methanol. The incubation of these fractions of the (AP) owers with HEP3B cells was achieved for 48 hours. Doxorubicin (DOX), is an anticancer drug known to inhibit cell proliferations and DNA arrest were used as control.
In Figure 1 transferred from the inner face of the plasma membrane to the cell surface; thus, the presence of PS on the cell surface can be used to identify apoptotic cells. PS was stained using a uorescent compound of annexin-V, a protein known to have a high a nity for PS, as described in the materials and techniques section. After then, ow cytometry analysis was performed in order to detect the PS. Cells were also stained with propidium iodide (PI), a dye that can only enter the cell when the plasma membrane is damaged. Positive for PS but negative for PI, early apoptosis was assessed. Nonetheless, it was observed that cells in the late apoptotic and necrotic stages were positive for both PS and PI. Figure. 2 demonstrates that the untreated cells (HEP3B cells alone), which are considered the control cells, maintain a baseline apoptotic cell population of 39±5.3%. It also represents that cells receiving treatment with aqueous, DMSO and methanol fractions of the (AF) ower had a baseline apoptotic cell population of 57.3±2.5%, 20.6±3.9% and 18.3±1.5%, respectively. As can be observed, DMSO as well as the methanol fraction had diminished apoptosis compared to that of the untreated cells.
While the population of the late apoptotic/necrotic cells was recognized to increase in all the different fractions of the (AF) ower to 54.3±4.7%, 76.3±4.5%, 79.7±3.05%, for the aqueous, DMSO and methanol fractions respectively, compared to 41.7±1.5% in the untreated cells. All results showed statisticcally signi cant between all tested groups. Generally, the given data propose that the (AF) owers shift the cells to necrosis by prolongating the G2-M phase of the HEP3B cell cycle and con rm anticancer potentials of the (AF) fractions. (AF) ower extracts decrease alpha-fetoprotein (αFP) secretion from HEP3B cells αFP a tumor marker and amajor protein that is synthesized in the fetal liver has been speculated to be the fetal analog of serum albumin. Usually, it has very low levels in healthy adults, however high levels of it might hint different types of cancers such as liver cancer. Figure 3  The current work is regarded as a preliminary examination for the future creation of naturally occurring antiplatelet and antibacterial medicines, despite the fact that certain synergistic or antagonistic effects remain unknown. The anticancer mechanism of (AP) aqueous extract and isolated compounds, as well as a large-scale manufacture, must all be further documented by the signing of the written informed consent. We con rm that all methods were carried out in accordance with relevant guidelines and regulations. Furthermore, we con rm that all experimental protocols were approved by the IRB of An-Najah National University. Figure 1 The proportion of cells in the cell cycle phases after the processing of the different fractions of the (AP) ower extracts.