Anticoagulant Activity of Crude and Phenolic Extracts of Dalbergia ecastaphyllum (L.) Taub. Dried Leaves

Background: Ischemic diseases are one of the leading causes of death worldwide; therefore, the search for new compounds with safer and more effective anticoagulant and antiplatelet action becomes relevant. Objectives: To evaluate the impact of Dalbergia ecastaphyllum (L.) Taub. dry leaf extract, a species of the Fabaceae family that is commonly found in Brazil, on blood coagulation. Materials and Methods: A statistical design of experiments was carried out with selection of five factors and two levels, resulting in 32 different ethanolic raw extracts. The extracts were resuspended in saline and tested in a pool of human blood. The activated partially thromboplastin time (APTT) and the prothrombin time (PT) were analysed in a Thrombolyzer®. Results: Treatment using ethanol 70%, a drug/solvent ratio of 5%, pH 9 and dynamic maceration for 6 hrs resulted in the best combination for the extraction of total phenols, while ethanol 70%, a drug/solvent ratio of 5% and dynamic maceration for 24 hrs showed the best flavonoid extraction, with the pH being inconclusive. On average, all extracts were capable of increasing both APTT and PT, especially those in lower dilutions (1:5); however, the overall efficacy of the extracts is inconclusive. Conclusion: The species under study showed important results in the blood clotting process in vitro, increasing the mean time of APTT and PT, which suggests that Dalbergia ecastaphyllum (L.) Taub. has some potential as a possible drug in the anticoagulant class.


INTRODUCTION
The blood tissue works in a perfect balance to control its own haemostasis by a complex coagulation process that prevents leakage by blocking the passage through the vessels. The defence mechanism for blood loss through vascular injury is controlled by the haemostatic system through vasoconstriction at the injury site, platelet aggregation and blood coagulation, which stabilizes and sustains the thrombus. [1] When the haemostatic system works properly, the blood is kept inside the vessels by thrombus formation in the case of an endothelium injury and, after the tissue is repaired, the thrombus is fragmented and the blood circulates normally. [2] However, when this process is unregulated, there may be complications in the vascular system, such as ischemic diseases, thrombosis or haemorrhage through intrinsic platelet dysfunction, von Willebrand disease or by antiplatelet agent actions, for example. [3] Thus, it is of great interest to discover new antiplatelet and anticoagulant drugs, which could be more efficient in comparison to those currently used, providing a prolonged effect and showing less harmful effects on the patients, than drugs such as aspirin and clopidogrel, among others. [4] There are several mechanisms of action that are still unknown, therapeutic targets to be explored and effects that can be of great use; the possibilities for the use of medical plants are therefore diverse. [6] In Brazil, the flora biodiversity is abundant and consists of several bioactive substances that can show important benefits in numerous pathologies, coagulation disorders among them. [7] Dalbergia is a large genus of the Fabaceae family; it consists of more than 200 species, which are widespread in tropical and subtropical regions. The parts most commonly used in the majority of research to understand its pharmacological effect on traditional medicine are the leaves, bark and root. [8] In Brazil, a coastal species is typical, occurring from the northeast to the southern region. Traditionally, it has been used for uterine inflammation and anaemia. [9] In this paper, the optimization of plant material extraction and its impact on human blood coagulation by different fractions of total phenols and flavonoids is presented.

Plant material
The plant material consisted of Dalbergia ecastaphyllum (L.) Taub. leaves were collected at the Iporanga Allotment, which belongs to the Environmental Protection Area (APA) of Serra do Guararú, Guarujá-SP, Brazil in July 2016 (Sisgen code: AE5F32C). After collection, leaves were dried to a constant weight at 50°C (in a dry air chamber). Dry material was obtained by grinding the leaves to a powder using a knife mill (Mar-coni™ MA 048).

Phytochemical screening
Analytical tests were performed using dried leaf powder of Dalbergia ecastaphyllum (L.) Taub. to detect the presence of secondary metabolites (total phenols, alkaloids, tannins, flavonoids, saponins, anthraquinone and cardiotonic glycosides) using qualitative experimental methods. [10,11] Quantitative analysis An experimental statistical design was performed (DoE, Minitab®), selecting 5 factors by 2 levels (absolute ethanol or ethanol 70%; drug/ solvent ratio at 5% or 10%; pH 3 or pH 9; static or dynamic maceration for 24 hrs or 6 hrs), resulting in 32 different ethanolic raw extracts (ERE) ( Table 1 and 2). The ERE were stored in a freezer at -20°C for quantitative analysis and were evaporated to dryness in a rotary evaporator (Fisatom™ 802) to obtain the aqueous raw fraction (ARF) for the complete blood count (CBC). Non-purified extracts were resuspended in saline water to obtain a saline aqueous extract (SAE).

Total phenol quantification
The quantification of total phenol content of the dried leaf extract was obtained by spectrophotometry analysis. [12,13] In order to better assess which combination of factors had the greatest influence on the extraction process of total phenols, the spectrophotometer was used to perform the standard curve in triplicate with a GEHAKA UV® device, model 330G, at a wavelength of 510 nm. [12,13]

Flavonoids quantification
The quantification of flavonoid content of the dried leaf extract was obtained by spectrophotometry analysis. [14] The standard curve for flavonoids was obtained by quercetin and aluminium chloride solutions. The absorbance was read in triplicate using a spectrophotometer GEHAKA UV® device, model 330G, at a wavelength of 415 nm. [14] Purified total phenol fraction The ARF originating from the best extraction condition (ethanol 70%; a drug/solvent ratio of 5%; pH 9 and dynamic maceration for 6 hrs) was adjusted to pH 11 using NH 4 OH 50% solution and centrifuged at 4000 rpm for 15 min, followed by a liquid-liquid partition with 3 portions of chloroform. The purified fraction was corrected to pH 4 using HCl 10% solution, followed by filtration through a funnel with glass wool and cotton. [15,16] After purification, the extract was resuspended in saline water to obtain the purified total phenol SAE.

Purified flavonoid fraction
The ARF originating from the best extraction condition (ethanol 70%; a drug/solvent ratio of 5%; pH 9 and dynamic maceration for 24 hrs) was submitted to consecutive extractions with different polarity solvents. The liquid-liquid partition was performed using 3 portions of petroleum ether, followed by 3 portions of chloroform and, finally, 3 portions of ethyl acetate. [17] The purified fraction was corrected to pH 4 using HCl solution 10%, followed by filtration through a funnel with glass wool and cotton. After purification, the extract was resuspended in saline water to obtain the purified flavonoid SAE.

Blood material
Human blood was obtained from venous blood collected from 10 healthy patients (Brazilian Ethics Committee Approval: 08632919.9.0000.0082) from the Clinical Analysis Laboratory at Centro Universitário Saúde ABC and was kept in blood test tubes containing sodium citrate or ethylenediamine tetra-acetic acid (EDTA) as anticoagulants. [18] The human blood samples were divided into two groups and both coagulation and haemogram were mixed and analysed on purified and non-purified SAE.

Evaluation of coagulant activity
The impact of purified and non-purified SAE plant extracts at four different dilutions (1:5, 1:10, 1:15 and 1:20) on prothrombin time (PT) and activated partial thromboplastin time (APTT) was carried out using a haemostasis analyser (Start™ -Diagnostica Stago), a commercial kit for PT (Thromboplastin Ds in lab haemostasis™) and a BioTécnica™ kit for APTT. The evaluation of PT and APTT parameters are relevant to understand the impact of the plant extracts on coagulation.
For PT analysis, 100 μL of each of the four extract dilutions were added to test tubes containing 400 μL of plasma. The samples were incubated at 37°C for 60 sec in a Stago analyser™ to check the clot forming for coagulation assays.
For APTT analysis, 100 μL of each of the four diluted extracts were added to test tubes containing 400 μL of plasma. The samples were incubated for 180 seconds at 37°C, in a Stago analyser™. All PT and APTT data using SAE were compared to blood and sodium citrate results and were conducted in triplicate.

Blood count
For the CBC, 100 μL of each of the four diluted SAEs were added to test tubes containing 400 μL of blood. The samples were analysed in a CBC analyser (Pentra™ 120) to obtain the haemoglobin (HGB) and platelet (PLT) count; these results were compared to a blood control sample, which was prepared with 100 μL of saline and 400 μL of blood.

Statistical analysis
The statistical analysis was done by Student's t-test, performed by software Stata version 16.0. Variations in blood parameter values are expressed as mean ± standard deviation (SD) and a 95% (p<0.05) confidence level was considered as statistically significant for all parameters analysed.

Phytochemical screening
The results of phytochemical screening showed the presence of alkaloids, flavonoids, tannins and anthraquinone and coumarin glycosides (Table 3).

Quantitative analysis
The quantitative analysis of total phenols showed that the treatment using ethanol 70% as solvent, a drug/solvent ratio of 5% and dynamic

Secondary compounds Result
Alkaloids +

Flavonoids +
Tannins +    maceration for 6 hrs resulted in the best combination for the extraction of this group of metabolites. The pH factor showed a relatively equal impact from pH 3 and pH 9. Ethanol 70%, a drug/solvent ratio of 5%, pH 9 and dynamic maceration for 24 hrs was shown to be the best multifactorial condition for flavonoid extraction (Table 4, Figure 1 and 2).

Coagulation analysis
The non-purified SAE that was rich in total phenols showed major variations in APTT at the lowest extract dilution (1:5), while presenting a non-linear behaviour in the other dilutions. Some deviations were observed in PT for all dilutions; however, the average measurements were constant. The non-purified SAE that was rich in flavonoids showed a greater APTT variation at the lower dilution (1:5); however, observing the variations in PT parameters, a non-significant increase was exhibited for all dilutions (Figure 3, Table 4). Most parameter variations did not show statistical relevance, with the exception of the most concentrated dilution for APTT and the total phenol 1:10 extract for PT (Table 5).
In non-purified SAE that was rich in total phenols, the blood count parameters exhibited variations in all dilutions, presenting less HGB reduction at the lowest dilution (1:5), while the other extracts showed greater HGB reduction as the dilutions increased. The PLT count showed similar results, with a smaller decrease in PLT count at the lowest dilutions (1:5 and 1:10) and a greater reduction in PLT count at the highest dilutions (1:15 and 1 :20). Variations in the non-purified flavonoid-rich SAE dilutions were observed and there was higher HGB dosage at the highest dilutions, especially in the treatment using the 1:15 dilution. However, the PLT count showed the opposite results, where a decreasing PLT count was observed at the lower dilutions (1:5 and 1:10) and a greater reduction was noted at higher dilutions ( Figure 4, Table 4). Most HGB variations did not show statistical relevance, with the exception of the total phenol 1:20 extract; however, PLT variations were statistically relevant for both extracts in all dilutions ( Table 5). The purified SAE that was rich in total phenols showed a significant increase in APTT at 1:5 dilution, which was not observed in the other dilutions. When analysing the variations in PT, all dilution results were similar and showed some increase over the control sample. The purified SAE that was rich in flavonoids showed a greater variation for both APTT and PT at the lowest extract dilution (1:5), while a decreasing in time was observed for both parameters in the intermediate dilutions ( Figure 5, Table 4). Almost all parameter variations showed statistical relevance, with the exception of the total phenol 1:5 extract and the flavonoids 1:20 for APTT ( Table 5). The blood count parameters showed some decrease in HGB in all dilutions; however, the 1:15 sample showed the best result with lower reduction. When analysing the changes in the PLT count, there was no direct correlation to the extract dilution, since the 1:10 and 1:20 dilutions presented the best results with a smaller variation in the PLT number. The blood count parameters showed some reduction in HGB dosage for the highest diluted extracts, whereas the 1:5 dilution demonstrated the greatest reduction. When observing the PLT count, the scenario is the opposite, with a smaller decrease in PLT numbers at the lowest dilution (1:5) and a greater reduction at the higher dilutions ( Figure 6, Table 4). Most PLT variations did not show statistical relevance, with the exception of the total phenol 1:15 extract. However, HGB variations were statistical relevant for almost all flavonoid extracts, with the exception of the 1:5 dilution (Table 5).

DISCUSSION
The secondary compounds produced by plants have demonstrated important pharmacological activities in several areas of application; thus, the medicinal interest in raw plant materials that are rich in these substances is growing. [19] Several studies on species of the Dalbergia genus have shown the presence of secondary compounds and subsequently evaluated their biological actions.

Phytochemical Screening
The Fabaceae family has been studied by many authors and the genus Dalbergia is one of the most investigated among them, since this genus has relevant production of secondary metabolites, such as total phenols and flavonoids. Ismail et al. [8] and Yemitan and Adeyemi [5] studied the pharmacological effects of Dalbergia saxatilis Hook. F. roots and phytochemical screening reported the presence of secondary compounds, such as alkaloids, flavonoids, tannins, triterpenoids, cardiotonic and saponin glycosides. Chandra, Sachan and Pal [20] detected the presence of several carbohydrates and total phenolic compounds, including flavonoids, in Dalbergia sissoo Roxb. leaves when investigating the effects on diarrhoea episodes. Hasan et al. [21] evaluated several pharmacological aspects of Dalbergia candenatensis (Dennst.) Prain leaf extracts and the phytochemical screening conducted on this species identified the presence of total phenolic compounds including tannins and flavonoids, but obtained negative results for the presence of saponin glycosides. When comparing the results of the present study with those observed in previous studies involving other Dalbergia species, the agreement of some secondary compounds among these species is notorious, since     most were also present, suggesting that this genus has congruent characteristics regarding the production of phenolic compounds.

Quantitative Analysis
The quantitative multifactorial analysis using DOE helped investigate the effects of different variables at the same time. The tool consists of a series of tests in which deliberate changes are made to the input variables, supporting the identification of conditions that affect plant material extraction and then determining the settings for factors that optimize results. The use of DOE corroborated the decision-making process, based on highly complex analyses and statistical studies. After DOE analysis, the extractive condition had a definitive result for all five factors, showing that ethanol 70%, a drug/solvent ratio of 5%, pH 9 and dynamic maceration for 24 hrs is statistically the best extraction of flavonoids present in Dalbergia ecastaphyllum (L.) Taub. in the case of total phenols, DOE analysis had a definitive result for four factors, showing that ethanol 70%, a drug/solvent ratio of 5% and dynamic maceration for 6 hrs is statistically the best extraction. Despite results tending to show pH 3 as a better extractive condition, the difference for pH 9 was almost inexistent; therefore, the most effective pH for the extraction of total phenols is still inconclusive.

Coagulation Analysis
There were several coagulation analyses conducted in the Fabaceae family species. The study conducted by Pereira and Brazón [22] evaluated the impact of Brownea grandiceps Jacq. aqueous flower extracts on human blood coagulation by measuring PT and APTT, observing that low concentrations of the extract decreased the PT, while higher concentrations contributed to increasing it, suggesting that the extract acts as a procoagulant and as anticoagulant depending on compounds concentration, although there was no evidence of which compounds could be involved in such an action.
Cordier et al. [23] also evaluated the coagulation and haemolysis processes in several Fabaceae species, such as Dichrostachys cinerea (L.) Wright and Arn. roots, Cassia petersiana Belle., Mundulea sericea Willd. A. Chev. roots, Medicago sativa L. leaves and Dalbergia melanoxylon Guill and Perr. bark. The authors observed a significant increase both in PT and APTT, with Cassia petersiana Belle. and Dalbergia melanoxylon Guill. and Perr. The results on blood parameters showed that Dalbergia melanoxylon Guill and Perr. significantly increased haemolysis in the studied concentrations, when using aqueous and methanolic extracts.
Hasan et al. [21] studied Dalbergia candenatensis (Dennst.) Prain leaf extract to evaluate the impact on human blood by comparing the PT to warfarin, a classic synthetic drug used as an anticoagulant. The results showed an increasing in proportional PT to the extract concentration used in the assay; thus, it was suggested that this event is dose-dependent. The same authors conducted a haemolysis test and evaluated different concentrations of extracts to inhibit the haemolytic process, identifying that higher-dose extracts resulted in greater haemolysis inhibition. Wadekar et al. [24] studied the effects of aqueous and alcoholic extracts of Dalbergia sissoo Roxb. bark on human plasma by measuring the PT and found that clot dissolution increased as the sample concentration increased, showing a moderate anticoagulant activity. The same authors suggested a possible mechanism for this anticoagulant effect and pointed out that phenolic compounds -especially tannins and flavonoids -are involved with the anticoagulant property, although did not specify how it happens. The results of previously described studies show that the Dalbergia genus has compounds that certainly act on the coagulation process and PLT inhibition in human blood in vitro, which corroborates the results found in the present study, since both non-purified and purified extracts analysed at different dilutions with ethanol 70%, a drug/solvent ratio of 5%, pH 9 and dynamic maceration for 6 and 24 hrs caused an increase in PT and APTT in human blood in vitro. Therefore, Dalbergia ecastaphyllum (L.) Taub. leaf extract probably has some level of intervention on the coagulation process, especially in the APTT levels for the lower dilution samples, while no major variations were observed in PT for most samples. Red cells and PLT count showed a reduction in all samples; however, the overall safety of the leaf extract is inconclusive. Wadekar et al. described the mechanism of action as being related with the phenolic compounds, which could be the same mechanism observed in this study. [24] CONCLUSION From the present study, it can be suggested that the purified Dalbergia ecastaphyllum (L.) Taub. dried leaf SAEs mainly induced an anticoagulant activity. The flavonoids and total phenols present in these extracts might be responsible for the development of an important role on the coagulation cascade and platelet maintenance; nonetheless, more investigation is required to identify which molecule interferes in that process. In addition, it can be inferred that Dalbergia ecastaphyllum (L.) Taub. leaves have a strong potential as a future drug for coagulation disorder treatments.