, Salwa M. Raweh2
, Ali Gamal Al-Kaf3
, Hassan M. Ibrahim4
IN-VITRO ANTICOAGULANT EFFECT OF AERIAL PARTS EXTRACTS OF YEMENI FAGONIA SCHWEINFURTHII HADIDI
Hend Ahmed Alhaj1, Salwa M. Raweh2, Ali Gamal Al-Kaf3, Hassan M. Ibrahim4
1Modern and Global Pharma company, Sana’a, Yemen.
2Pharmacognosy Department, Faculty of Pharmacy, Sana’a University, Yemen.
3Medicinal Chemistry Department, Faculty of Pharmacy, Sana’a University, Yemen.
4Biology Department, Faculty of Science, Sana’a University, Yemen.
Background: Blood coagulation is a quick and effective process that results in the creation of clots, which demands to monitor. Many illness disorders include an abnormality in blood coagulation. This study examined the in vitro effects of methanol, ethyl acetate, and n-hexane extracts from aerial parts of Fagonia schweinfurthii Hadidi on healthy human volunteers' blood coagulation.
Methods: The Secondary metabolites were extracted from dried and crushed F. schweinfurthii aerial parts using n-hexane, ethyl acetate, and methanol, respectively. Additionally, the extracts were tested in vitro at different concentrations (10-100 µg/ml) on the blood coagulation profile, prothrombin time (PT), and activated partial thromboplastin time (a PTT) of apparently healthy human volunteers.
Results: Methanol, ethyl acetate, and n-hexane extracts of F. schweinfurthii aerial parts significantly (p˃0.05) prolonged PT and PTT in the blood of healthy human volunteers with Ethyl acetate and methanol extracts recorded the largest prolongation of PT and PTT correspondingly. The highest PT and PTT prolongation was achieved at 100µg/ml, and the least prolongation time was obtained at 10µg/ml.
Conclusion: These findings displayed that F. schweinfurthii aerial parts contain phytochemical constituents with anticoagulant characteristics and could be used to treat blood clotting disorders.
Keywords: Blood coagulation, F. schweinfurthii, Partial thromboplastin time, Prothrombin time.
INTRODUCTION
Medicinal plants are thought to be a rich source of ingredients for medication development and production. Furthermore, these plants play a significant role in the formation of human cultures all throughout the world. Additionally, some plants are thought to be important sources of nutrition, and as a result, some plants are indicated for their therapeutic potential1. Many secondary metabolites produced by plants have important biological activity. Anticoagulant action may be highlighted among these. Anticoagulants treat and prevent blood clots by interacting with the body's natural blood-clotting system. Anticoagulants treat patients with pulmonary embolism (PE), deep vein thrombosis (DVT), blood clots in the arteries or veins, atrial fibrillation, which causes strokes, and mechanical heart valves2.
F. schweinfurthii belongs to the family Zygophylla-ceae3. It is annual to biennial, up to 25 cm tall, spiny, erect, undershrub, with more and less granular leaves; thin, terete, triangular, glabrous branches; opposite, 1-3 foliate leaves. Petioles range from 3 to 30 mm in length, are deeply striated, and are very slender; stipules have two pairs of sharp, very short petioles. Geographically it is distributed in India, Pakistan, Iran, Eritrea, Ethiopia, Sudan, Somalia, and Kenya4,5. In Yemen, F. schweinfurthii is distributed in Tehama, Shara'b, Muthaikhira, Aden, Lahj, Abyan, Qatabah, Demt, al Hus'ein, Ashu'ib, Juban, Amran, Raydah, Huth, Haddah, E of Rada, Hadramout, Socotra6,7.
People living in desert regions have traditionally utilized the plant to treat various conditions, including ear infections, skin eruptions, heal sores, skin illnesses, fever, and pain relief8. They have traditionally been used to treat hemorrhoids, inflammation, ulcers, leprosy, open wounds, and fever in internal and external formulations. When powder from the entire parts of F. schweinfurthii is applied to boils and skin eruptions, it induces healing; when the whole plant is boiled in water, its bath is beneficial for allergies and other skin problems; and the decoction is administered orally as a blood purifier9. Fagonia sp. has been reported to have anticancer, antibacterial, antiviral, analgesic, anti-inflammatory, antipyretic, coolant, antioxidant, and thrombolytic activities10. Phytochemical screening revealed that F. schweinfurthii extracts contain alkaloids, cardiac glycosides, flavonoids, carbohydrates, tannins, saponins, steroids, and amino acids5. The anticoagulant activity of Fagonia arabica extracts (water, ethanol, methanol, isopropyl alcohol (IPA), butanol, ethyl acetate, acetone, chloroform, petroleum ether, and hexane) was studied in vitro and revealed that the aqueous extract and its fifth fraction had the greatest anticoagulant effect (31 minutes and 27 minutes, respectively)15. Furthermore, Ismail et al.,14 demonstrated that Fagonia cretica had marked anticoagulant activity with a coagulation time of 86.9 seconds. The effects of F. schweinfurthii on blood coagulation have received little to no scientific attention. As a result, this study aims to investigate the possibility and potential of F. schweinfurthii as a medicinal plant for the treatment of blood coagulation disorders.
MATERIALS AND METHODS
Drugs and chemicals
The reagents used were all of the analytical quality. Prothrombin Time (PT) and activated partial thromboplastin time (PTT) reagent kits were obtained from SEIMENS.
Plant Material:
F. schweinfurthii aerial parts were collected in September 2019 from local areas in Sana'a, Yemen. Dr. Hassan Ibrahim identified the plant in the Biology Department, Sciences College, Sana'a University.
Preparation of plant extract:
One kilogram of fresh aerial parts was properly rinsed with tap water, dried at room temperature, and then using an electric blender, reduced to a fine powder. The powder was stored at cool place and protected from light prior to future uses. In 3.76L conical flasks, powdered aerial parts weighing 752g were sequentially soaked in hexane, ethyl acetate, and methanol. The samples were immersed at room temperature for three to seven days with continuous agitation. These procedures were repeated three times to obtain higher extraction yields. The extracts were then filtered, concentrated in a rotary evaporator at 40°C, and dried in an oven at 37°C. Then the dried extracts were weighed and kept at 4°C for later investigation.
In vitro anticoagulant activity assay:
Using the traditional coagulant tests PT and PTT, the anticoagulant activity of F. schweinfurthii aerial parts extracts was examined. Nine parts of healthy human blood were drawn into a one-part sample which would be measured. Heparin and extracts of F. schweinfurthii aerial parts were made in different concentrations (100, 75, 50, 25, 10, and 10g/ml), and all of the samples were dissolved in a 0.9% (w/w) solution of NaCl. Plasma alone was the negative control, whereas heparin was a positive control. For the PT assay, citrated normal plasma was combined with a sample solution, which was then incubated for 3 minutes at 37°C. After pre-incubating the PT test reagent for 3 minutes at 37oC, 0.20 ml was added to the mixture, and the clotting time was noted. Then, PTT assay reagent 0.10 ml, pre-incubated for 3 min at 37oC, was added to the mixture and incubated for 5 min at 37oC. After that, 0.10 ml CaCl2 (0.025 mol/L) pre-incubated for 3 min at 37oC was added, and clotting time was recorded12. The experiment was carried out in triplicate.
Statistical analysis
The data was analyzed using SPSS version 26. The mean ±SD from three distinct trials was used to express all the results performed in triplicate. One Way -ANOVA test was used to detect statistical differences between the means in all studies, Followed by Dunnett’s multiple comparaison test. The statistically significant difference was denoted by p<0.05, p<0.01, and p<0.001.
RESULTS
The anticoagulant activity of methanol, n-hexane, and ethyl acetate extracts of F. schweinfurthii aerial parts was determined in vitro using the traditional coagulation assays prothrombin time (PT) and activated partial thromboplastin time (a PTT) assays, with the use of normal citrated human plasma as a negative control and heparin as a reference. Many different concentrations (10, 25, 50, 75, and 100 µg/ml) of F. schweinfurthii aerial parts methanol, ethyl acetate, and n-hexane extract were prepared, and the results are given in Table 1 and Table 2 and Figure 1 and Figure 2).
A healthy human's normal value of PT and PTT was 11.8 and 30.20 seconds, respectively. Heparin, as positive control showed considerable anticoagulant activity (PT>60 s, PTT>170 s). The results showed significant PT prolongation (p˂0.01, p˂0.0001) of the methanol, ethyl acetate, and n-hexane extract at (25, 50, 75, 100µg/ml) concentrations. In contrast, at a concentration of 10µg/ml, the methanol, ethyl acetate, and n-hexane extract did not significantly (p˃0.05) prolong PT compared with the plasma. In the PTT assay, there was an insignificant prolon-gation (p˃0.05) of PTT at a concentration of 10µg/ml of methanol and ethyl acetate extracts.
However, n-hexane extract did not significantly prolong PTT at 10 µg/ml and 25µg/ml concentrations (p˃0.05). On the other hand, methanol and ethyl acetate extracts produced significant prolongation (p˂0.01, p˂0.0001) of PTT at 25, 50, 75, 100µg/ml concentrations. While, n-hexane extract showed a significant prolongation (p˂0.01, p˂0.0001) of PTT only at 50, 75, 100µg/ml.
The highest PT and PTT prolongation times were noted at 100µg/mL compared with other concentrations under testing. The methanol extract exhibited the highest PTT Prolongation activity compared with other tested extracts. At the same time, ethyl acetate recorded the largest PT prolongation activity.
DISCUSSION
Anticoagulant and procoagulant medications are commonly used in healthy and pathological states, including cardiovascular disease, diabetes mellitus, and bleeding disorders. Although many of these medications have been developed over time, the majority are frequently associated with unfavorable side effects. New procoagulants and anticoagulants are still needed with fewer side effects13. The current study considered the in vitro anticoagulant effects of F. schweinfurthii aerial parts methanol, n-hexane, and ethyl acetate extract using classical PT and PTT assays. The current study examined the in vitro anticoagulant effects of F. schweinfurthii aerial parts methanol, n-hexane, and ethyl acetate extract using classical PT and PTT assays. The results indicated a considerable prolongation of the PT and PTT by n-hexane, ethyl acetate, and methanol extract depending on the concentration with an optimal prolongation of PT and PTT at 100 µg/mL and the minimal prolongation time was obtained at 10 µg/m. These results agreed with Ismail et al.,14 and Chourasia et al.,15 that Fagonia cretica leaves extract and aqueous extract of Fagonia arabica and its fractions exhibited a concentration-dependent anticoagulant activity.
A comparison of the effect of the three extracts on the PT and PTT showed that the ethyl acetate and methanol extract exhibited the highest anticoagulant action. These results agreed with Duric et al.,16 that methanol extract showed the highest anticoagulant activity. The largest anticoagulant action was shown with ethyl acetate (medium-polar) and methanol (polar) extracts compared to the non-polar n-hexane extract, suggesting that the primary anticoagulant elements of F. schweinfurthii aerial parts are polar. Anticoa-gulant activity F. schweinfurthii aerial parts extracts may be attributed to saponin, flavonoids, tannin, triterpenoids13,15, and coumarins17,18. Coagulation parameters PT and PTT are utilized to identify the clotting mechanism. Prothrombin time (PT) is a useful test for determining how well the components of the extrinsic coagulation pathway are functioning. The PTT is also used to evaluate the activity of components involved in intrinsic and common pathways. In clinical examination, prolonged PTT and PT values imply an abnormality in the activity of particular clotting factors; for instance, an exceptionally prolonged PTT but a normal PT value suggests the need to assay contact pathway factors VIII, IX, and XI.
The common pathway's factors V, X, and prothrombin (factor II) are implicated if both the PT and PTT are affected19. Therefore, the extended PT and PTT induced by therapy with F. schweinfurthii propose that factors V, X, and prothrombin of the common coagulation pathway activity have been inhibited.
CONCLUSION
According to the study, F. schweinfurthii has anticoagulant effects that can be used to treat blood coagulation disorders. Additional in vivo study is required to investigate the mechanism by which the anticoagulant components of the plant impact their activity.
ACKNOWLEDGMENTS
The authors would like to thank Dr. Safwan Alaghbari, the manager of the Research and Development Centre of Modern and Global Company, for supporting this study.
AUTHOR’S CONTRIBUTIONS
All authors agreed to be held accountable for every aspect of the study, helped with data analysis, composed, revised, reviewed the paper, and gave their final approval before publication.
CONFLICT OF INTEREST
No conflict of interest associated with this work.
REFERENCES