Pharmacogn Res.

2021; 13(3):121-128
A Multifaceted Journal in the field of Natural Products and Pharmacognosy Original Article
www.phcogres.com | www.phcog.net

Anticoagulant Activity of Crude and Phenolic Extracts of

Dalbergia ecastaphyllum (L.) Taub. Dried Leaves

Enrico Rogatto Rovetta1, Mara Angelina Galvão Magenta2, Thaís Moura Gascon3,
Fernando Luiz Affonso Fonseca3, Robson Miranda da Gama1, José Armando Jr1, Horácio Dorigan Moya1,*

ABSTRACT
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,
Enrico Rogatto Rovetta1, on blood coagulation. Materials and Methods: A statistical design of experiments was
Mara Angelina Galvão 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
Magenta2, Thaís Moura activated partially thromboplastin time (APTT) and the prothrombin time (PT) were analysed
Gascon3, Fernando Luiz in a Thrombolyzer®. Results: Treatment using ethanol 70%, a drug/solvent ratio of 5%, pH 9
Affonso Fonseca3, Robson and dynamic maceration for 6 hrs resulted in the best combination for the extraction of total
Miranda da Gama1, José phenols, while ethanol 70%, a drug/solvent ratio of 5% and dynamic maceration for 24 hrs
Armando Jr1, Horácio showed the best flavonoid extraction, with the pH being inconclusive. On average, all extracts
Dorigan Moya1,* 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
1
Centro Universitário FMABC,
study showed important results in the blood clotting process in vitro, increasing the mean
Pharmacy Laboratory, Santo André, time of APTT and PT, which suggests that Dalbergia ecastaphyllum (L.) Taub. has some potential
São Paulo, BRAZIL. as a possible drug in the anticoagulant class.
2
Universidade Santa Cecília, Santos, Key words: Anticoagulant activity, Dalbergia ecastaphyllum (L.) Taub., Flavonoids, Phenolic
São Paulo, BRAZIL. compounds, Plant extracts.
3
Centro Universitário FMABC,
Clinical Analysis Laboratory,
Santo André, São Paulo, BRAZIL.
4
Centro Universitário FMABC, INTRODUCTION
Analytical Chemistry Laboratory,
Santo André, São Paulo, BRAZIL. The blood tissue works in a perfect balance to control At least a quarter of current pharmaceutical
Correspondence
its own haemostasis by a complex coagulation process products were originally obtained from medical
that prevents leakage by blocking the passage through plants and most of these plants are responsible for
Dr. Horácio Dorigan Moya
the vessels. The defence mechanism for blood loss the origination of analgesic, anti-inflammatory and
Faculdade de Medicina da Fundação do through vascular injury is controlled by the haemo-
ABC - Av. Príncipe de Gales, 821 - CEP
antipyretic drugs.[5]
09060 - 650 Santo André, BRAZIL.
static system through vasoconstriction at the injury There are several mechanisms of action that are still
site, platelet aggregation and blood coagulation, which
Phone no: +55 11 4993-7292 unknown, therapeutic targets to be explored and
stabilizes and sustains the thrombus.[1]
Email id: [email protected] effects that can be of great use; the possibilities for
When the haemostatic system works properly, the the use of medical plants are therefore diverse.[6]
History
blood is kept inside the vessels by thrombus formation In Brazil, the flora biodiversity is abundant and
• Submission Date: 03-01-2021;
• Review completed: 23-02-2021; in the case of an endothelium injury and, after the consists of several bioactive substances that can
• Accepted Date: 15-05-2021 tissue is repaired, the thrombus is fragmented and
show important benefits in numerous pathologies,
the blood circulates normally.[2] However, when this
DOI : 10.5530/pres.13.3.3 coagulation disorders among them.[7]
process is unregulated, there may be complications
in the vascular system, such as ischemic diseases, Dalbergia is a large genus of the Fabaceae family; it
Article Available online
thrombosis or haemorrhage through intrinsic platelet consists of more than 200 species, which are wide-
http://www.phcogres.com
dysfunction, von Willebrand disease or by antiplatelet spread in tropical and subtropical regions. The parts
Copyright agent actions, for example.[3] Thus, it is of great interest most commonly used in the majority of research to
© 2021 Phcog.Net. This is an open- understand its pharmacological effect on traditional
to discover new antiplatelet and anticoagulant drugs,
access article distributed under the terms
of the Creative Commons Attribution 4.0 which could be more efficient in comparison to those medicine are the leaves, bark and root.[8] In Brazil,
International license. currently used, providing a prolonged effect and a coastal species is typical, occurring from the
showing less harmful effects on the patients, than drugs northeast to the southern region. Traditionally,
such as aspirin and clopidogrel, among others.[4] it has been used for uterine inflammation and

Cite this article: Rovetta ER, Magenta MAG, Gascon TM, Fonseca FLA, Gama RM, Armando-Jr
J, Moya HD. Anticoagulant Activity of Crude and Phenolic Extracts of Dalbergia ecastaphyllum (L.)
Taub. Dried Leaves. Pharmacog Res. 2021;13(3):121-8.

Pharmacognosy Research, Vol 13, Issue 3, Jul-Sep, 2021 121

 Rovetta, et al.: Anticoagulant Activity of Dalbergia ecastaphyllum (L.) Taub.

anaemia.[9] In this paper, the optimization of plant material extraction Purified total phenol fraction
and its impact on human blood coagulation by different fractions of total The ARF originating from the best extraction condition (ethanol 70%;
phenols and flavonoids is presented. a drug/solvent ratio of 5%; pH 9 and dynamic maceration for 6 hrs)
was adjusted to pH 11 using NH4OH 50% solution and centrifuged at
MATERIALS AND METHODS 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
Plant material 10% solution, followed by filtration through a funnel with glass wool
The plant material consisted of Dalbergia ecastaphyllum (L.) Taub. and cotton.[15,16] After purification, the extract was resuspended in saline
leaves were collected at the Iporanga Allotment, which belongs to the water to obtain the purified total phenol SAE.
Environmental Protection Area (APA) of Serra do Guararú, Guarujá-SP,
Brazil in July 2016 (Sisgen code: AE5F32C). After collection, leaves were
Purified flavonoid fraction
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- The ARF originating from the best extraction condition (ethanol 70%;
coni™ MA 048). a drug/solvent ratio of 5%; pH 9 and dynamic maceration for 24 hrs)
was submitted to consecutive extractions with different polarity solvents.
Phytochemical screening The liquid-liquid partition was performed using 3 portions of petroleum
ether, followed by 3 portions of chloroform and, finally, 3 portions of
Analytical tests were performed using dried leaf powder of Dalbergia
ethyl acetate.[17] The purified fraction was corrected to pH 4 using HCl
ecastaphyllum (L.) Taub. to detect the presence of secondary metabolites
solution 10%, followed by filtration through a funnel with glass wool and
(total phenols, alkaloids, tannins, flavonoids, saponins, anthraquinone
cotton. After purification, the extract was resuspended in saline water to
and cardiotonic glycosides) using qualitative experimental methods.[10,11]
obtain the purified flavonoid SAE.
Quantitative analysis
An experimental statistical design was performed (DoE, Minitab®), Blood material
selecting 5 factors by 2 levels (absolute ethanol or ethanol 70%; drug/ Human blood was obtained from venous blood collected from 10
solvent ratio at 5% or 10%; pH 3 or pH 9; static or dynamic maceration healthy patients (Brazilian Ethics Committee Approval:
for 24 hrs or 6 hrs), resulting in 32 different ethanolic raw extracts (ERE) 08632919.9.0000.0082) from the Clinical Analysis Laboratory at Cen-
(Table 1 and 2). The ERE were stored in a freezer at -20°C for quantitative tro Universitário Saúde ABC and was kept in blood test tubes con-
analysis and were evaporated to dryness in a rotary evaporator (Fisatom™ taining sodium citrate or ethylenediamine tetra-acetic acid (EDTA) as
802) to obtain the aqueous raw fraction (ARF) for the complete blood anticoagulants.[18]
count (CBC). Non-purified extracts were resuspended in saline water to The human blood samples were divided into two groups and both
obtain a saline aqueous extract (SAE). coagulation and haemogram were mixed and analysed on purified and
non-purified SAE.
Total phenol quantification
The quantification of total phenol content of the dried leaf extract was Evaluation of coagulant activity
obtained by spectrophotometry analysis.[12,13] In order to better assess
The impact of purified and non-purified SAE plant extracts at four
which combination of factors had the greatest influence on the extraction
different dilutions (1:5, 1:10, 1:15 and 1:20) on prothrombin time (PT)
process of total phenols, the spectrophotometer was used to perform the
and activated partial thromboplastin time (APTT) was carried out using
standard curve in triplicate with a GEHAKA UV® device, model 330G, a haemostasis analyser (Start™ - Diagnostica Stago), a commercial kit
at a wavelength of 510 nm.[12,13] for PT (Thromboplastin Ds in lab haemostasis™) and a BioTécnica™ kit
for APTT. The evaluation of PT and APTT parameters are relevant to
Flavonoids quantification understand the impact of the plant extracts on coagulation.
The quantification of flavonoid content of the dried leaf extract was
For PT analysis, 100 μL of each of the four extract dilutions were added
obtained by spectrophotometry analysis.[14] The standard curve for
to test tubes containing 400 μL of plasma. The samples were incubated
flavonoids was obtained by quercetin and aluminium chloride
at 37°C for 60 sec in a Stago analyser™ to check the clot forming for
solutions. The absorbance was read in triplicate using a coagulation assays.
spectrophotometer GEHAKA UV® device, model 330G, at a wavelength
For APTT analysis, 100 μL of each of the four diluted extracts were
of 415 nm.[14]
added to test tubes containing 400 μL of plasma. The samples were incu-
bated for 180 seconds at 37°C, in a Stago analyser™. All PT and APTT
Table 1: Levels and factors of the statistical design of experiments for data using SAE were compared to blood and sodium citrate results and
32 trials (DoE, Minitab). were conducted in triplicate.
Factors Level (-1) Level (+1)
Solvent 70% ethanol 100% ethanol Blood count
Drug/solvent ratio 5:100 10:100 For the CBC, 100 μL of each of the four diluted SAEs were added to test
Time 6 hrs 24 hrs tubes containing 400 μL of blood. The samples were analysed in a CBC
analyser (Pentra™ 120) to obtain the haemoglobin (HGB) and platelet
pH Acid (pH = 3) Alkali (pH = 9)
(PLT) count; these results were compared to a blood control sample,
Maceration Static Dynamic
which was prepared with 100 μL of saline and 400 μL of blood.

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Table 2: Factorial planning matrix for the 32 trials and the quantification of total phenols and flavonoids from different treatments.
Extraction Solvent Drug/ Time pH Maceration Total phenols average Flavonoids average ±
solvent ratio ± SD (mg/g) SD (μg/g)
T1 70% ethanol 5 : 100 6 hrs Acid (pH=3) Static 135.22 ± 0.59 58.07 ± 0.17
T2 70% ethanol 5 : 100 6 hrs Acid (pH=3) Dynamic 159.38 ± 0.47 65.41 ± 0.17
T3 70% ethanol 5 : 100 6 hrs Alkali (pH=9) Static 119.38 ± 0.72 99.83 ± 0.17
T4 70% ethanol 5 : 100 6 hrs Alkali (pH=9) Dynamic 196.22 ± 0.76 155.24 ± 0.45
T5 70% ethanol 5 : 100 24 hrs Acid (pH=3) Static 123.22 ± 0.49 93.33 ± 0.17
T6 70% ethanol 5 : 100 24 hrs Acid (pH=3) Dynamic 107.88 ± 0.54 98.36 ± 0
T7 70% ethanol 5 : 100 24 hrs Alkali (pH=9) Static 152.22 ± 0.36 97.1 ± 0.17
T8 70% ethanol 5 : 100 24 hrs Alkali (pH=9) Dynamic 103.88 ± 0.62 117.25 ± 0.3
T9 70% ethanol 10 : 100 6 hrs Acid (pH=3) Static 174.88 ± 0.49 62.72 ± 0.15
T10 70% ethanol 10 : 100 6 hrs Acid (pH=3) Dynamic 135.28 ± 0.61 66.81 ± 0.09
T11 70% ethanol 10 : 100 6 hrs Alkali (pH=9) Static 98.53 ± 0.35 65.66 ± 0.15
T12 70% ethanol 10 : 100 6 hrs Alkali (pH=9) Dynamic 214.38 ± 1.06 134.7 ± 0.34
T13 70% ethanol 10 : 100 24 hrs Acid (pH=3) Static 132.22 ± 0.71 64.92 ± 0.15
T14 70% ethanol 10 : 100 24 hrs Acid (pH=3) Dynamic 172.55 ± 0.49 95.46 ± 0.23
T15 70% ethanol 10 : 100 24 hrs Alkali (pH=9) Static 111.44 ± 0.53 75.41 ± 0.09
T16 70% ethanol 10 : 100 24 hrs Alkali (pH=9) Dynamic 148.22 ± 0.72 118.96 ± 0.31
T17 100% ethanol 5 : 100 6 hrs Acid (pH=3) Static 91.22 ± 0.14 42.75 ± 0
T18 100% ethanol 5 : 100 6 hrs Acid (pH=3) Dynamic 85.55 ± 0.36 70.03 ± 0.17
T19 100% ethanol 5 : 100 6 hrs Alkali (pH=9) Static 39.22 ± 0.14 43.38 ± 0
T20 100% ethanol 5 : 100 6 hrs Alkali (pH=9) Dynamic 114.38 ± 0.27 80.94 ± 0.17
T21 100% ethanol 5 : 100 24 hrs Acid (pH=3) Static 67.22 ± 0.41 63.32 ± 0.17
T22 100% ethanol 5 : 100 24 hrs Acid (pH=3) Dynamic 139.05 ± 0.54 104.66 ± 0.3
T23 100% ethanol 5 : 100 24 hrs Alkali (pH=9) Static 34.05 ± 0.27 44.43 ± 0
T24 100% ethanol 5 : 100 24 hrs Alkali (pH=9) Dynamic 142.05 ± 0.36 86.61 ± 0.17
T25 100% ethanol 10 : 100 6 hrs Acid (pH=3) Static 63.11 ± 0.25 56.21 ± 0.09
T26 100% ethanol 10 : 100 6 hrs Acid (pH=3) Dynamic 83.03 ± 0.36 86.54 ± 0.09
T27 100% ethanol 10 : 100 6 hrs Alkali (pH=9) Static 13.61 ± 0 20.54 ± 0
T28 100% ethanol 10 : 100 6 hrs Alkali (pH=9) Dynamic 71.11 ± 0.36 52.75 ± 0.15
T29 100% ethanol 10 : 100 24 hrs Acid (pH=3) Static 38.11 ± 0.07 57.68 ± 0.15
T30 100% ethanol 10 : 100 24 hrs Acid (pH=3) Dynamic 74.36 ± 0.45 82.65 ± 0.15
T31 100% ethanol 10 : 100 24 hrs Alkali (pH=9) Static 45.69 ± 0.14 49.6 ± 0.09
T32 100% ethanol 10 : 100 24 hrs Alkali (pH=9) Dynamic 80.44 ± 0.25 80.24 ± 0.09

Statistical analysis Table 3: Phytochemical screening (+) positive result; (-) negative result.
The statistical analysis was done by Student’s t-test, performed by software Secondary compounds Result
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 Alkaloids +
considered as statistically significant for all parameters analysed. Flavonoids +

RESULTS Tannins +
Phytochemical screening Anthraquinones glycosides +
The results of phytochemical screening showed the presence of alkaloids,
flavonoids, tannins and anthraquinone and coumarin glycosides (Table 3). Cardiotonic glycosides -

Quantitative analysis Coumarins glycosides +

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

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Table 4: Coagulation analysis results for control and diluted samples of raw and purified plant extracts.
Type of Group of HGB average ± SD PLT average ± SD
Sample PT average ± SD (s) APTT average ± SD (s)
extract metabolites (g/dL) (103/µL)
Control 15.4 ± 1.1 24.4 ± 2.7 13.0 ± 1.4 188.7 ± 10.0
1:5 17.4 ± 2.5 31.5 ± 3.4 11.4 ± 1.7 165.0 ± 10.1
Total Phenols 1:10 17.4 ± 0.4 27.6 ± 2.0 11.3 ± 2.5 153.7 ± 13.3
1:15 17.4 ± 1.7 25.1 ± 2.8 9.4 ± 2.7 130.3 ± 30.4
1:20 17.5 ± 1.1 26.3 ± 3.2 9.2 ± 1.2 138.0 ± 25.2
Raw
Control 15.6 ± 1.0 23.9 ± 2.2 13.0 ± 1.4 188.7 ± 10.0
1:5 16.7 ± 1.8 36.4 ± 6.2 10.9 ± 1.1 161.7 ± 11.1
Flavonoids 1:10 16.6 ± 1.3 28.5 ± 2.2 11.3 ± 1.2 152.3 ± 7.6
1:15 16.9 ± 1.5 27.2 ± 0.6 11.6 ± 1.7 145.3 ± 13.3
1:20 16.6 ± 1.4 26.7 ± 1.0 11.5 ± 2.1 150.0 ± 16.1
Control 14.6 ± 0.1 23.4 ± 1.3 11.2 ± 1.3 256.0 ± 58.9
1:5 16.3 ± 0.8 57.5 ± 25.7 9.2 ± 1.3 170.7 ± 50.9
Total Phenols 1:10 16.0 ± 0.6 28.5 ± 1.7 9.7 ± 0.5 192.0 ± 39.0
1:15 15.8 ± 0.6 29.2 ± 1.7 9.8 ± 0.5 145.7 ± 24.8
1:20 16.1 ± 0.5 27.8 ± 0.2 9.8 ± 1.6 194.0 ± 48.6
Purified
Control 14.6 ± 0.1 23.4 ± 1.3 12.8 ± 0.1 226.7 ± 33.5
1:5 17.2 ± 0.9 28.6 ± 1.4 11.1 ± 1.2 205.7 ± 50.9
Flavonoids 1:10 16.4 ± 0.3 25.8 ± 0.2 11.6 ± 0.6 183.0 ± 17.1
1:15 16.4 ± 0.4 25.6 ± 0.2 11.7 ± 0.4 176.0 ± 20.9
1:20 16.1 ± 0.4 25.3 ± 0.5 12.0 ± 0.3 184.7 ± 16.9

Figure 1: Average effects chart for total phenols. Figure 2: Average effects chart for flavonoids.

maceration for 6 hrs resulted in the best combination for the extraction for all dilutions (Figure 3, Table 4). Most parameter variations did not
of this group of metabolites. The pH factor showed a relatively equal show statistical relevance, with the exception of the most concentrated
impact from pH 3 and pH 9. Ethanol 70%, a drug/solvent ratio of 5%, dilution for APTT and the total phenol 1:10 extract for PT (Table 5).
pH 9 and dynamic maceration for 24 hrs was shown to be the best In non-purified SAE that was rich in total phenols, the blood count
multifactorial condition for flavonoid extraction (Table 4, Figure 1 and 2). parameters exhibited variations in all dilutions, presenting less HGB
reduction at the lowest dilution (1:5), while the other extracts showed
Coagulation analysis greater HGB reduction as the dilutions increased. The PLT count showed
The non-purified SAE that was rich in total phenols showed major similar results, with a smaller decrease in PLT count at the lowest dilutions
variations in APTT at the lowest extract dilution (1:5), while presenting (1:5 and 1:10) and a greater reduction in PLT count at the highest dilutions
a non-linear behaviour in the other dilutions. Some deviations were (1:15 and 1:20). Variations in the non-purified flavonoid-rich SAE
observed in PT for all dilutions; however, the average measurements dilutions were observed and there was higher HGB dosage at the high-
were constant. The non-purified SAE that was rich in flavonoids showed est dilutions, especially in the treatment using the 1:15 dilution. How-
a greater APTT variation at the lower dilution (1:5); however, observing ever, the PLT count showed the opposite results, where a decreasing PLT
the variations in PT parameters, a non-significant increase was exhibited count was observed at the lower dilutions (1:5 and 1:10) and a greater

124 Pharmacognosy Research, Vol 13, Issue 3, Jul-Sep, 2021

 Rovetta, et al.: Anticoagulant Activity of Dalbergia ecastaphyllum (L.) Taub.

Figure 3: Mean alteration results of the parameters PT and APTT for Figure 4: Average changes of the HGB and PLT parameters for
non-purified SAE rich in phenols and flavonoids. non-purified SAE rich in phenols and flavonoids.

reduction was noted at higher dilutions (Figure 4, Table 4). Most HGB DISCUSSION
variations did not show statistical relevance, with the exception of the
The secondary compounds produced by plants have demonstrated
total phenol 1:20 extract; however, PLT variations were statistically
important pharmacological activities in several areas of application; thus,
relevant for both extracts in all dilutions (Table 5).
the medicinal interest in raw plant materials that are rich in these
The purified SAE that was rich in total phenols showed a significant substances is growing.[19] Several studies on species of the Dalbergia
increase in APTT at 1:5 dilution, which was not observed in the other genus have shown the presence of secondary compounds and subse-
dilutions. When analysing the variations in PT, all dilution results were quently evaluated their biological actions.
similar and showed some increase over the control sample. The puri-
fied SAE that was rich in flavonoids showed a greater variation for both Phytochemical Screening
APTT and PT at the lowest extract dilution (1:5), while a decreasing in The Fabaceae family has been studied by many authors and the genus
time was observed for both parameters in the intermediate dilutions Dalbergia is one of the most investigated among them, since this genus
(Figure 5, Table 4). Almost all parameter variations showed statistical has relevant production of secondary metabolites, such as total phenols
relevance, with the exception of the total phenol 1:5 extract and the and flavonoids.
flavonoids 1:20 for APTT (Table 5). Ismail et al.[8] and Yemitan and Adeyemi[5] studied the pharmacological
The blood count parameters showed some decrease in HGB in all effects of Dalbergia saxatilis Hook. F. roots and phytochemical screening
dilutions; however, the 1:15 sample showed the best result with lower reported the presence of secondary compounds, such as alkaloids, flavo-
reduction. When analysing the changes in the PLT count, there was no noids, tannins, triterpenoids, cardiotonic and saponin glycosides.
direct correlation to the extract dilution, since the 1:10 and 1:20 dilu- Chandra, Sachan and Pal[20] detected the presence of several carbohy-
tions presented the best results with a smaller variation in the PLT drates and total phenolic compounds, including flavonoids, in Dalbergia
number. The blood count parameters showed some reduction in HGB sissoo Roxb. leaves when investigating the effects on diarrhoea episodes.
dosage for the highest diluted extracts, whereas the 1:5 dilution dem- Hasan et al.[21] evaluated several pharmacological aspects of Dalbergia
onstrated the greatest reduction. When observing the PLT count, the candenatensis (Dennst.) Prain leaf extracts and the phytochemical
scenario is the opposite, with a smaller decrease in PLT numbers at screening conducted on this species identified the presence of total
the lowest dilution (1:5) and a greater reduction at the higher dilutions phenolic compounds including tannins and flavonoids, but obtained
(Figure 6, Table 4). Most PLT variations did not show statistical relevance, negative results for the presence of saponin glycosides.
with the exception of the total phenol 1:15 extract. However, HGB varia- When comparing the results of the present study with those observed
tions were statistical relevant for almost all flavonoid extracts, with the in previous studies involving other Dalbergia species, the agreement of
exception of the 1:5 dilution (Table 5). some secondary compounds among these species is notorious, since

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Table 5: Statistical analysis results of blood parameter for samples of raw and purified plant extracts.

Group of PT APTT HGB PLT

Type of extract Sample
metabolites (p value) (p value) (p value) (p value)
1:5 0.269 0.046 0.272 0.045
1:10 0.046 0.165 0.359 0.022
Total Phenols
1:15 0.172 0.768 0.112 0.034
1:20 0.082 0.477 0.023 0.032
Raw
1:5 0.415 0.030 0.103 0.035
1:10 0.364 0.065 0.185 0.007
Flavonoids
1:15 0.272 0.069 0.324 0.011
1:20 0.391 0.119 0.339 0.024
1:5 0.021 0.083 0.134 0.130
1:10 0.012 0.015 0.115 0.192
Total Phenols
1:15 0.019 0.009 0.139 0.040
1:20 0.007 0.005 0.285 0.232
Purified
1:5 0.007 0.009 0.085 0.583
1:10 0.0003 0.039 0.023 0.114
Flavonoids
1:15 0.001 0.048 0.009 0.090
1:20 0.002 0.079 0.006 0.124

Figure 5: Mean alteration results of the parameters PT and APTT for Figure 6: Average changes in the parameters HGB and PLT for purified
purified SAE rich in total phenols and flavonoids. SAE rich in total phenols and flavonoids.

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 Rovetta, et al.: Anticoagulant Activity of Dalbergia ecastaphyllum (L.) Taub.

most were also present, suggesting that this genus has congruent charac- analysed at different dilutions with ethanol 70%, a drug/solvent ratio of
teristics regarding the production of phenolic compounds. 5%, pH 9 and dynamic maceration for 6 and 24 hrs caused an increase in
PT and APTT in human blood in vitro.
Quantitative Analysis Therefore, Dalbergia ecastaphyllum (L.) Taub. leaf extract probably has
The quantitative multifactorial analysis using DOE helped investigate the some level of intervention on the coagulation process, especially in the
effects of different variables at the same time. The tool consists of a series APTT levels for the lower dilution samples, while no major variations
of tests in which deliberate changes are made to the input variables, were observed in PT for most samples. Red cells and PLT count showed
supporting the identification of conditions that affect plant material a reduction in all samples; however, the overall safety of the leaf extract is
extraction and then determining the settings for factors that optimize inconclusive. Wadekar et al. described the mechanism of action as being
results. The use of DOE corroborated the decision-making process, related with the phenolic compounds, which could be the same mecha-
based on highly complex analyses and statistical studies. nism observed in this study.[24]
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 CONCLUSION
9 and dynamic maceration for 24 hrs is statistically the best extraction From the present study, it can be suggested that the purified Dalbergia
of flavonoids present in Dalbergia ecastaphyllum (L.) Taub. in the case of ecastaphyllum (L.) Taub. dried leaf SAEs mainly induced an anti-­
total phenols, DOE analysis had a definitive result for four factors, showing coagulant activity. The flavonoids and total phenols present in these
that ethanol 70%, a drug/solvent ratio of 5% and dynamic maceration for
extracts might be responsible for the development of an important role
6 hrs is statistically the best extraction. Despite results tending to show
on the coagulation cascade and platelet maintenance; nonetheless, more
pH 3 as a better extractive condition, the difference for pH 9 was almost
investigation is required to identify which molecule interferes in that
inexistent; therefore, the most effective pH for the extraction of total
process. In addition, it can be inferred that Dalbergia ecastaphyllum (L.)
phenols is still inconclusive.
Taub. leaves have a strong potential as a future drug for coagulation
disorder treatments.
Coagulation Analysis
There were several coagulation analyses conducted in the Fabaceae ACKNOWLEDGEMENT
family species. The study conducted by Pereira and Brazón[22] evaluated
the impact of Brownea grandiceps Jacq. aqueous flower extracts on The authors are grateful to the Pharmacy, Clinical Analysis and
human blood coagulation by measuring PT and APTT, observing that Analytical Chemistry laboratories of Centro Universitário FMABC, for
low concentrations of the extract decreased the PT, while higher concen- providing facilities for carrying out the study.
trations contributed to increasing it, suggesting that the extract acts as a
procoagulant and as anticoagulant depending on compounds concen- Financial support and sponsorship
tration, although there was no evidence of which compounds could be Nil.
involved in such an action.
Cordier et al.[23] also evaluated the coagulation and haemolysis processes CONFLICT OF INTEREST
in several Fabaceae species, such as Dichrostachys cinerea (L.) Wright and The authors declare no conflict of interest.
Arn. roots, Cassia petersiana Belle., Mundulea sericea Willd. A. Chev.
roots, Medicago sativa L. leaves and Dalbergia melanoxylon Guill and ABBREVIATIONS
Perr. bark. The authors observed a significant increase both in PT and
APTT: Activated Partially Thromboplastin Time; ARF: Aqueous Raw
APTT, with Cassia petersiana Belle. and Dalbergia melanoxylon Guill.
Fraction; CBC: Complete Blood Count; DOE: DoE, Minitab®; EDTA:
and Perr. The results on blood parameters showed that Dalbergia mela-
Ethylenediamine Tetra-Acetic Acid; ERE: Ethanolic Raw Extracts; HGB:
noxylon Guill and Perr. significantly increased haemolysis in the studied
Haemoglobin; PLT: Platelet; PT: Prothrombin Time; SAE: Saline Aque-
concentrations, when using aqueous and methanolic extracts.
ous Extract.
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GRAPHICAL ABSTRACT SUMMARY

The present study consisted of Dalbergia ecastaphyllum (L.)

Taub. dried and powdered leaves as plant material, submitted
to several extract conditions (agitation, extractor liquid
concentrations, its pH, plant drug proportions and extraction
time) which resulted in 32 treatments. Afterwards were
analyzed by spectrophotometry to stablish the most efficient
extraction condition for total phenols and flavonoid fractions.
From the most efficient extract, dilutions were carried out and
analysis were conducted to evaluate their impact on human
blood coagulation parameters (PT, APTT, hemoglobin and
platelet counting).

Cite this article: Rovetta ER, Magenta MAG, Gascon TM, Fonseca FLA, Gama RM, Armando-Jr J, Moya HD. Anticoagulant Activity
of Crude and Phenolic Extracts of Dalbergia ecastaphyllum (L.) Taub. Dried Leaves. Pharmacog Res. 2021;13(3):121-8.

128 Pharmacognosy Research, Vol 13, Issue 3, Jul-Sep, 2021