Asian Journal of Applied Chemistry Research

6(3): 41-52, 2020; Article no.AJACR.59950

ISSN: 2582-0273

Characterization of Bio-active Compounds Essential

for Blood Coagulation in the Crude Extracts of
Tradescantia zebrina, Tagetes minuta and
Codiaeum variegatum Leaves
Gakuo Grace1, Osano Aloys1 and Bakari Chaka1*
1
Department of Mathematics and Physical Sciences, Maasai Mara University, Kenya.

Authors’ contributions

This work was carried out in collaboration among all authors. Authors GG and OA designed the study,
performed the statistical analysis, wrote the protocol and wrote the first draft of the manuscript.
Authors OA and BC managed the analyses of the study. Authors GG and BC managed the literature
searches. All authors read and approved the final manuscript.

Article Information

DOI: 10.9734/AJACR/2020/v6i330164
Editor(s):
(1) Dr. Endang Tri Wahyuni, Gadjah Mada University, Indonesia.
Reviewers:
(1) Chérifi Fatah, University of Science and Technology Houari Boumediene, Algeria.
(2) Agnes Sri Harti, Kusuma Husada University of Surakarta, Indonesia.
(3) Amzad Hossain, University of Nizwa, Oman.
Complete Peer review History: http://www.sdiarticle4.com/review-history/59950

Received 15 June 2020

Original Research Article Accepted 20 August 2020
Published 28 August 2020

ABSTRACT

Aims: Many commercial drugs used for blood clotting are expensive and have associated side-
effects. The extracts of Tagetes minuta, Codieum variegatum and Tradescantia zebrine are used
for blood clotting. These extracts are highly efficient and have no known side-effects. This study
aimed at characterizing crude extracts of these plant species used to accelerate blood clotting.
Study Design: An independent measures experimental design was used in the study.
st st
Place and duration of study: The research was conducted between 21 September, 2018 and 21
May, 2019. The study was conducted in Maasai mara university, Kenya and Multimedia university
of Kenya.
Methodology: Extracts of these herbs were obtained and analyzed for absorption bands,
functional groups, bio-metal concentrations, physical-chemical parameters, phytochemicals and
antimicrobial activity. Test for blood clotting factors (calcium and vitamin K) was also conducted.
Results: All extracts had common functional group peaks at 2800-3500 cm-1 (carboxylic OH), 1680
_____________________________________________________________________________________________________

*Corresponding author: Email: [email protected];

 Grace et al.; AJACR, 6(3): 41-52, 2020; Article no.AJACR.59950

cm-1 (carbonyl), and 1035cm-1 (C-Ostretch). The extracts had an average pH of 6.590 ±0.702 and
conductivity of 0.580 ±0.079mS. The average solubility in distilled water was 16.670 ±1.534 g/100
ml water at 37°C. The extracts were found to be abundant in iron, copper and phytochemicals. All
extracts portrayed moderate inhibition to E. coli bacteria and C. albicans fungi but mild inhibition
towards S. aureus bacteria. The extracts had trace amounts of Vitamin K and moderate amounts
of calcium.

Keywords: Blood clotting; Tradescantia zebrina; Tagetes minuta; Codieum variegatum.

1. INTRODUCTION with vitamin K [17]. Anticoagulants such as

dabigitran, apixaban and rivaroxaban are simpler
Blood plays a key role in the metabolism of to use and less risky compared to warfarin [18].
humans. It is the main mode of transport
necessary for other metabolic processes to occur Some plants contain one or more blood clotting
[1]. Blood is lost when its conveyance vessels agents in varying concentrations. Some of the
(arteries, veins and capillaries) are cut. Bleeding blood clotting agents readily found in herbs
is the leading cause of death during accidents include calcium, vitamin K as well as other
[2]. During accidents, blood vessels are cut and crucial phytochemicals [19]. Tagetes minuta,
blood easily ooze out. The number of fatalities Tradescantia zebrina and Codiaeum variegatum
due to accidents (both minor and major) is still have successfully been used to treat diseases
high and require more attention. Several related with blood disorders. These extracts have
precaution measures have been enforced to traditionally been used for people of varying age
minimize these fatalities. Nevertheless, it is quite with little preparation required to formulate the
difficult to prevent bleeding after accidents have anti-clotting agents. Crude extracts of these
already occurred. It is therefore pertinent to plants were applied on the bleeding part to
minimize the bleeding process to ensure minimal hasten blood coagulation. There were also few, if
blood loss during accident. any cases of side effects or complications arising
from treatment using these extracts. A study was
Clot formation occurs in veins and arteries with hereby conducted to characterize, analyze and
the help of several factors [3,4]. During blood compare chemical compounds present in the
clotting, various factors and enzymes trigger extracts of these plants. Elucidation of the exact
conversion of blood in the affected site to species and amounts of phytochemicals in the
coagulate into a gel [5], preventing more three plants will go a long way in informing
bleeding. Blood clotting has three major steps; further pharmaceutic and pharmacognosy
vasoconstriction, platelets plug formation and clot studies. This will help to avoid potential drug
formation [6]. Clot formation occur when platelets toxicities while maximizing on extraction of blood
plugs have been formed [6]. Clot formation is clotting agents from the plants.
activated by a sequence of events called the
coagulation cascade which leads to formation of 2. MATERIALS AND METHODS
fibrin [7]. Several plant extracts have been found
to inhibit or prolong the blood clotting time [8]. 2.1 Design of Experiment
Drugs used for blood clotting include; antiplatelet An indipendent measures experimental design
agents such as aspirin, clopidogrel, dipyridamole was followed for this study. The plant specimen
and tidopine that work by inhibiting the were identified and assigned specimen numbers
production of thromboxane [9]. Antiplatelet are with the help of a botanist (from Maasai mara
not conducive for everyone especially for people university). Purposive sampling was used to
with liver or kidney diseases, bleeding disorders collect the plant samples whereby only the fresh
and peptic ulcers [10]. Other blood clotting leaves were targeted. The leaves were
agents are anticoagulants. Several coagulation pretreated to obtain crude extracts by maceration
factors are proteins [11]. These proteins are method. The extracts were then characterized
involved in several stages of blood coagulation for physical-chemical parameters, biometal
process and include thrombin, coagulation concentrations, absorption bands, functional
factors IX, IXa amongst others [12,13,14,15]. groups, phytochemicals, antimicrobial activities
They are synthesized in the liver with the help of and presence of blood clotting agents (calcium
vitamin K [16]. Anticoagulants such as warfarin and Vitamin K). Characterization and analysis
and heparin, slow clot formation by competing were done at Maasai mara university, Kenya

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 Grace et al.; AJACR, 6(3): 41-52, 2020; Article no.AJACR.59950

chemistry and biology labs. UV-VIS screening for Test for flavonoids: Onto the test samples, 2 g
presence of Vitamin K in the extracts was of vanillin powder was added and the mixture
conducted at Multimedia university, Kenya. agitated in an acidic medium.

2.2 Methods The procedure was confirmed by adding 3ml of

dilute ammonia solution to 2 ml of aqueous
2.2.1 Extraction of the sample extracts filtrate followed by 1 ml of concentrated sulfuric
acid. Formation of yellow deposits confirmed
The plants species Tagetes minuta, Tradescantia
presence of flavonoids.
zebrina and Codiaem variegatum were carefully
washed with distilled water and then air dried in a
Test for Tannins: About 0.1 g of the dry samples
cool shade away from direct sunlight for 3 days.
were boiled in 4ml distilled water in a boiling tube
The crude extracts of the samples were obtained
then filtered. A few drops of 0.1% ferric chloride
by squezzing the leaves.
solution were then added and observations of
2.2.2 Characterization of extracts change in color to brownish-green made.

The extracts were characteriseed for physico- Test for saponins: The sample was added to 3
chemicals (pH, conductivity, total solids, volatile ml distilled water and vigorously agitated until a
solids and solubility) using conventional stable, persistent froth formed. 3 drops of olive oil
methods. A pH meter (Hanna G114) and were then added and shaken vigorously.
conductivity meter (Jenway 6510) were used for Presence of emulsion indicated positive results.
pH and conductivity respectively. Four replicate
tests were conducted for each of the tests. Test for terpenoids (Salkowski’s test): About 3
ml of the samples were mixed with 1 ml of
For bio-metal analysis, the extracts were serially chloroform and 1ml of concentrated sulfuric acid.
diluted 200-folds using 20 ml aliquots distilled Formation of intense red-brown color indicated
water and filtering using Whatman #42 filter presence of terpenoids.
paper at each dilution stage. An Atomic
Test for alkaloids (Mayer’s test): About 3 ml of
Absorption Spectrometer (PG-990) was used.
ammonia solution was added onto the sample
The bio-metals were analyzed after formulation
followed by 10 ml of chloroform. The mixture was
of calibration curve using standard salts prepared
shaken well then filtered. The chloroform layer
for each of the bio-metal analyzed. Table 1
was then evaporated off and 3 ml of Mayer’s
summarizes the conditions used during the bio-
solution added to the remaining solution.
metal analysis.
Formation of a cream precipitate indicated
For functional group analysis, the extracts were positive test for alkaloids.
gradually concentrated by mild warming until all
the water was dried. The samples were then cast Test for steroids: The sample solution was
into pellets using potassium bromide pellet dissolved in 10 ml of chloroform followed by 3 ml
before analyzing for functional groups using IR of concentrated sulfuric acid. Formation of red
Spectrometer (Shimadzu 119). precipitates indicated presence of steroids.

2.2.2.1 Phytochemical screening of the extracts 2.2.3 Antimicrobial analysis of the extracts

Test for polyphenols: 3 ml of aqueous ferric Antimicrobial studies were conducted for both
chloride solution was added to 10 ml of the Gram-positive (S. aureus) and Gram-negative
sample solutions, shaken and observations bacteria (E. coli). C. albicans strain was used for
made. Formation of green coloration indicated antifungal analysis. All aseptic techniques were
presence of phenols. considered to minimize the contamination rates.

Table 1. AAS conditions used to analyze the bio-metals

Bio-metal Wavelength Bandwidth Lamp current Flame Sensitivity

Co 240.7 nm 0.4 nm 5.0 ma Air/Acetylene 0.05 mg/L
Cu 324.7 nm 0.4 nm 5.0 ma Air/Acetylene 0.03 mg/L
Fe 248.3 nm 0.2 nm 5.0 ma Air/Acetylene 0.05 mg/L
Zn 213.9 nm 0.4 nm 4.0 ma Air/Acetylene 0.01 mg/L

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 Grace et al.; AJACR, 6(3): 41-52, 2020; Article no.AJACR.59950

2.2.3.1 Media preparation burette and the solution titrated using DCPIP
solution. The end point was denoted by change
28.0 g of Muller-Hinton’s agar media was in colour from pink to green.
dissolved into 600 ml of sterile distilled water in a
media dispensing bottle. The mixture was 2.2.4.3 Screening for vitamin K by UV-VIS
gradually boiled to completely dissolve the spectroscopy (Shimadzu 1800).
media. Caution was taken not to break the media
bottle by loosening the bottle stopper 5 ml of the samples were added onto 2 ml of
occasionally to avoid pressure build up. The 0.2% solution 2,4-dinitrophenyl hydrazine (in
media was then sterilized by autoclaving along hydrochloric acid and absolute ethanol in ratio of
v
with petri-dishes and all apparatus to be used at 1:5 /v) and thoroughly agitated to mix. The
121°C and 15 psi pressure for 15 minutes. The mixture was then heated on water bath to
media was allowed to cool to 45°C before evaporate most of the solvent. 15ml of (ammonia
dispensing in sterile petri dishes. The media and alcohol in ratio of 1:1) was added to the
plates were allowed to cool, inverted and stored mixture, shaken gently and left to cool at room
in the refrigerator at 4°C for 24 hours. temperature. UV absorbance at 635nm
wavelength was recorded.
2.2.3.2 Antimicrobial tests
2.3 Data Analysis
Sterile media plates were sub-divided into six
equal parts using a marker pen and labelled Data obtained from analysing pH, temperature,
accordingly. The test microbes (bacteria and total solids, volatile solids, heavy metals was
fungi) were then spread aseptically on different subjected to statistical analysis. The degree of
media plates to prevent cross-contamination. freedom value was maintained at 8 with 95%
Sterile octo-discs impregnated with different confidence level being used for the statistics. The
extracts were then placed on the surface of the data was analysed using Ms Excel (2016) and
plates. The plates were then inverted and Originlab (version 6.5) statistical packages.
incubated at 37°C for 24 hours. After the
3. RESULTS AND DISCUSSION
incubation period, the inhibition zones were
noted and recorded in millimeters.
3.1 Physical-chemical Analysis of the
2.2.4 Analysis of blood clotting agents Plant Extracts

2.2.4.1 Test of calcium contents All samples were found to be slightly acidic with
moderate conductivity values. A large portion of
5g of the crude extracts were mixed with 50ml of the total solids were found to be volatile. The pH
1M oxalic acid to precipitate out calcium ions of the extracts was lower than the normal blood
from the samples. The residue obtained was pH of 7.3-7.5 [20]. However, the margin between
then thoroughly filtered using more oxalic acid. the extracts pH and blood pH was considerable.
The residue was dried and heated gently in a Blood is an excellent buffer by itself and would
pre-weighed crucible (rinsed with ammonia quickly normalize the extracts pH when added
solution) until a white precipitate was obtained. onto a bleeding vein or artery. Kim et al., [21]
The mixture was weighed (crucible+residue) and reported application of garlic powder on bleeding
the mass of the residue deducted. The procedure surfaces as a blood coagulant, whereas garlic
was then repeated three more times and the has a pH of 6.0 [22]. All extracts had little
average value calculated. electrical conductivity in water solution. These
findings imply that the extracts had limited salts
2.2.4.2 Test of vitamin K available. Bowen and Remaley [23] showed that
abundance of salts lead to reactions between
About 0.1 g of sodium acetate was added onto samples and blood components. Some salts
10 ml of each of the extract solutions. The inhibit normal metabolic functions leading to
mixture was transferred to a separating funnel sickness or death. T. minuta extracts were found
which had been previously rinsed with acetone. to have the least conductivity values of
The mixture was shaken vigourosly while 0.307±0.002 mS. Szczurko et al., [24] reported
releasing the gas produced periodically for 1 low conductivity values in Ginkgo biloba water
hour. The upper part of the mixture was extracts used as blood coagulants. The
transferred into a conical flask. 50ml of composition of volatile solids in the total solids
dichlorophenolindophenol (DCPIP) was put in a was 44% (T. minuta), 46% (T. zebrina) and 47%

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(C. variagetum). Jiang et al., [25] showed that a Rzymski et al., [29] found out that in vitro addition
good composition of Ginkgo biloba extracts had of copper ions (CuSO4) in doses of 300-1000
volatile terpenes which might also be present in micrograms/ml elicited an anticoagulant effect,
the test samples above. Table 2 summarizes the though the thrombin time was not significantly
chemical parameters conducted for the three affected. Concentration of zinc in the plant
blood clotting plant extracts. extracts had a large disparity with T. minuta
having very low levels (15.10 mg/Kg wet sample)
Vishwas et al., [26] reported volatile solids whereas both C. variagetum (49.88 mg/Kg wet
concentrations of 6.3% in Homonoia riparia lour sample) and T. zebrina (59.20 mg/Kg wet
leaves extracts used to prolong blood clotting sample) had considerably higher values. Like
time. Solubility of blood coagulants is a crucial iron, addition of zinc ions to blood serum (in this
parameter in elucidating its effects and case using rats) was found to increase
effectiveness. Generally, blood is composed of recalcification, prothrombin and partial
several components, ferried by blood plasma thromboplastin times [32]. Doses ranging
which has a lot of water thus soluble in water. It between 0.3-1.0 mg/ml of zinc ions were used.
is practically impossible to determine the exact The thrombin clotting time was not altered even
solubility of blood in water due to the numerous by the highest concentration used (1.0mg/ml).
components and factors involved. Nevertheless, Going by these studies, T. zebrina would be best
the test extracts showed varying solubilities in placed as a potential blood coagulant. Cobalt
water at 37°C. C. variagatum had the largest ions do not significantly affect blood clotting
solubility value of 33.148±0.164 g/100 ml of process. However, Heemskerk et al., [33] found
water at 37°C. out that presence of cobalt ions in coagulants
alter blood clot retraction. C. variagetum extracts
3.2 Bio-metal Concentration of Blood were rich in cobalt (77.24mg/Kg of wet sample)
Clotting Plant Extracts thus expected to experience more blood clot
retraction processes.
The composition of metals in the estracts is vital
in predicting its efficacies and possibly toxicity 3.3 Functional Group Analysis of Plant
effects when applied to blood. Some metals are Extracts
inert in blood, others aid the body when
absorbed in the proper channel while others are All spectra of the test samples were found to
toxic even at minute concentrations. The have similar IR patterns. Presence of carboxylic
concentrations of four bio-metals crucial in blood groups and aromatic compounds as well as
coagulation in the test extracts are summarized conjugation as a result of numerous double bond
in Table 3. peaks in the fingerprint regions were observed.
From the spectra in Fig. 1, all samples had a
Iron was found to be the most abundant specie, -1
broad O-HRCOOH peak between 3600-2800 cm
especially in T. zebrina extracts. Greunz et al., highlighting carboxyllic acids, a sharp peak at
[27] reports that abundance of iron during blood -1
1650-1750 cm due to C=O. These postulates
coagulation process prolongs prothrombin, were confirmed by C-O-H stretch shallow peaks
thrombin and partial thromboplastin duration, in at around 1030-1050 cm-1. sp3 C-H peaks were
human plasma in vitro. This effect leads to -1
observed at around 2900 cm , followed by a
reduced blood loss as formation of blood clots large rift in the spectra. John et al., [34] reported
are prolonged. Ke et al., [28] found out that similar peaks in commercial blood coagulating
presence of iron in the coagulants provide drugs such as warfarin and heparin. Similar
optimal conditions for fibrinogen coagulability and peaks were also observed in garlic, turmeric and
fibrin monomer aggregation. However, Ke et al., Ginkgo biloba extracts, all used for blood
[28] did not cite the exact concentration of iron coagultion purposes [35]. The collated FT-IR
needed for these coagulopathies. Rzymski et al., spectra of the three plant extracts are
[29] found out that excessive addition of iron demonstrated in Fig. 1.
(FeSO4) in doses above 2-4 mg/ml significantly
-1
reduced platelet aggregation in rat blood serum. There were several peaks between 1600 cm
Copper is involved in synthesis of blood and 900 cm-1 indicating presence of aromatic
coagulant factor VIII [30]. T. minuta extracts were groups and double bonds. Further peaks at 600
found to have the highest amount of copper ions cm-1 and 500 cm-1 indicated presence of
(78.00 mg/Kg wet extract sample) for this crucial organometalloids and halides present in the plant
anti-hemophillic factor. Concentrations of copper extracts. Rodríguez-Torres et al., [36] found out
ions above 300 mg/kg of sample are toxic [31]. these peaks in the IR spectra of heparin drugs.

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 Grace et al.; AJACR, 6(3): 41-52, 2020; Article no.AJACR.59950

Fig. 1. FT-IR spectra of the blood coagulant plant extracts

Essien et al., [37] observed presence of organo- formation [41]. Only T. zebrina sample had
halide peaks in the roots of Fagara steroids and terpenoids. Xavier et al., [42]
xanthoxyloides used as blood coagulants. observed presence of steroids in the extracts of
Homonoia riparia lour leaves used in blood
3.4 Screening of Phytochemicals in the coagulation. T. zebrina and C. variagetum
Plant Extracts extracts tested positive for saponins. Several
plant extracts known to increase blood coagultion
Several phytochemical compounds have been such as Ginkoga biloba, green tea, garlic and
observed to aid in blood clotting process. turmeric have all been found to have varying
Vishwas et al., [26] showed that presence of concentrations of saponins [43,44]. It is however
alkaloids, glycosides, phenols and flavanoids in not clear how saponins affect blood coagulation.
Homonoia riparia lour leaves extracts increased
3.5 Antimicrobial Activity of the Plant
blood clotting time. The study further found out
that extraction using ethanol gave the highest Extracts
effectiveness. The extracts of T. zebrina, T. The test extracts were found to exhibit moderate
minuta and C. variagetum were all found to be antifungal activity against C. albicans. The
abundant in phytochemicals. These findings are performance against E. coli was moderate while
summarized in Table 4. the extracts showed mild suppression of S.
aureus bacteria. The diameters for the inhibition
Archana et al., [38] indicaated that tannins have zones of the plant extracts against the mibrobes
the ability to reduce blood clotting at controlled are illustrated in Table 5.
doses. All test extracts were found to test
positive for tannins. Green tea, proven to have a All the plant extracts had close inhibition against
lot of tannins and other polyphenols have over the microbes. Effective inhibition against E. coli
time been used to aid in blood clotting processes and C. albicans by these extracts as observed in
[39]. This study also showed all three test Table 5 implies that the extracts are suitable in
samples to contain phenolic compounds. Further preventing diseases as a result of these
research by Li et al., [40] has shown that microbes. During bleeding, human blood and its
interaction between polyphenols (including components are exposed to many external
tannins and flavonoids) prolongs the interaction pathogens, such as microbes responsible for
between thrombin and these compounds. These numerous diseases. The extracts of these three
effect leads to increased thrombin times for herbs can thus be applied as a remedy to these
efficient blood coagulants. However, some microbes while still aiding in blood clotting, in
flavonoids were reported to inhibit thrombin vitro.

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Both calcium and vitamin K are directly involved proportional to blood clotting efficacy based on
in formation of the coagulation cascade that the crucial role this factor plays in the process.
leads to fibrin formation [45,46]. Calcium, Most blood coagulants contain vitamin K in
phospholipids and clotting factors Xa and IXa varying amounts. Warfarin drug used in blood
are involved in activation of prothrombin to coagulation contains loads of vitamin K1 [52].
thrombin [47,48,49]. Thrombin is then used in Venugopala et al., [53] reported high abundance
activation of fibrinogen to fibrin [50]. Fibrin is the in vitamin K in coumarin extracts (used in blood
final mesh that prevents bleeding [50]. Calcium coagulation). Venugopala et al., [54] reported
is a catalyst in this process and its deficiency that vitamin K conversion cycle was interfered by
in the blood slowls down blood coagulation coumarin extracts leading to hepatic production
process [51]. The levels of vitamin K in the three of partially carboxylated and decarboxylated
extracts were screened by UV-VIS as shown in proteins with procoagulant activity. The vitamin K
Fig. 2. antagonists also inhibit conversion cycles of
other coagulant factors such as anticoagulant
3.6 Calcium and Vitamin K Analysis proteins C and S [55]. The exact concentrations
of vitamin K and calcium in the plant extracts are
Alongside, fibrinogen (found in blood plasma), illustrated in Fig. 3. From the figure, calcium
calcium and vitamin K as well as other levels were higher than vitamin K levels in all the
compounds are key in blood coagulation extracts. Both T. minuta and C. variagetum had
process. These factors are required at various more calcium and vitamin K levels compared to
points of the process, without which the process T. zebrina.
is altered. Both calcium and vitamin K are directly
involved in formation of the coagulation cascade The order of vitamin K abundance in the extracts
that leads to fibrin formation [45,46]. Calcium, was T. zebrina (0.34mg/L), T. minuta (0.27mg/L)
phospholipids and clotting factors Xa and IXa are and C. variagetum (0.19mg/L). There is little
involved in activation of prothrombin to thrombin information of the exact amount of vitamin K
[47,48,49]. Thrombin is then used in activation of needed for blood clotting. However, Kaku et al.,
fibrinogen to fibrin [50]. Fibrin is the final mesh [56] found out that for optimal blood clotting,
that prevents bleeding [50]. Calcium is a catalyst about 8.4mg/dL of calcium ions are required.
in this process and its deficiency in the blood Using these standards, all the extracts qualify as
slowls down blood coagulation process [51]. The suitable blood clotting agents. It has been noted
levels of vitamin K in the three extracts were that in the presence of calcium ions and reduced
screened by UV-VIS as shown in Fig. 2. vitamin K (as vitamin KH2), carboxylation
reactions cause a conformation change in
T. zebrina sample was found to have a higher coagulation proteins leading to binding of
absorbance citing more abundance in the cofactors on phospholipid surfaces [55]. A fibrin
extracts. Abundance of vitamin K is directly clot is thereafter formed.

Table 2. Physical-chemical parameter of the test extracts

Parameters Samples (n = 8)
T. zebrina T. minuta C. variegatum
pH 6.519±0.036 6.573±0.016 5.871±0.000
E. conductivity (mS) 0.587±0.001 0.307±0.002 0.860±0.001
Total solids (g/L) 5.192±2.567 5.268±0.996 5.251±0.957
Volatile solids (g/L) 2.416±0.027 2.358±0.028 2.472±0.020
Solubility (g/100ml water 16.667±0.175 7.870±0.076 33.148±0.164
at 37°C)

Table 3. Concentration of biometals in blood coagulating plant extracts

Biometal Sample concentration (mg/Kg wet sample)

T. zebrina T. minuta C. variegatum
Iron 180.00 68.40 56.40
Copper 54.00 78.00 46.00
Zinc 59.20 15.10 49.88
Cobalt 43.86 45.38 77.24

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Table 4. Phytochemicals present in the test plant extracts

Phytochemicals Samples
T. minuta T. zebrina C. variegatum
Tannins + + +
Alkaloids + + -
Steroids - + -
Phenols + + +
Saponins + - +
Flavonoids + - -
Terpenoids - + -

Table 5. Inhibition zone diameters of plant extracts against test microbes

Samples Inhibition zone diameters (mm)

E. coli S. aureus C. albicans
T. zebrina 11.50 6.00 12.00
T. minuta 10.50 7.50 13.00
C. variagetum 12.00 6.00 10.50

0.65
0.6
0.55
Absorbance (a.u)

0.5
0.45
0.4
0.35
0.3
0.25
0.2
620 625 630 635 640 645 650
Wavelength (nm)

T. zebrina T. minuta C. variagetum

Fig. 2. UV-VIS spectra of vitamin K in plant extracts

C. variagetum 0.19
3.86

T. minuta 0.27
5

T. zebrina 0.34
2.46

0 1 2 3 4 5 6
Concentration (mg/L)

Vitamin K Calcium

Fig. 3. Analysis of calcium and vitamin K in plant extracts

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4. CONCLUSIONS DOI: 10.1002/jbt.21885

5. Galvatilde A, Henriques S, Pestka D,
T. minuta, T. zebrina and C. variagetum crude Lukasik K, Skarzynski D, Mateus L. Equine
extracts were found to have varying amounts of luteal function regulation may depend on
bio-active compounds necessary for blood the interaction between cytokines and
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all the plant extracts were biometals (especially vitro study 1. Biology of Reproduction.
iron, copper and calcium), phytochemicals and 2012;86:6.
vitamin K. The three extracts also exhibited 6. Alli N. Bleeding disorders (part 1). South
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albicans fungi and E. coli bacteria. DOI: 10.7196/SAMJ.2017.v108i1.13019
7. Adams R, Bird R. Review article:
DATA AVAILABILITY STATEMENT Coagulation cascade and therapeutics
update: Relevance to nephrology. Part 1:
Overview of coagulation, thrombophilias
All data used in this study is found within the
and history of anticoagulants. Nephrology.
article and the supplementary sheetes provided.
2009;14(5):462-470.
8. Abebe W. Review of herbal medications
ACKNOWLEDGEMENTS with the potential to cause bleeding: dental
implications, and risk prediction and
The authors are grateful to the assistance of Mr. prevention avenues. EPMA J. 2019;10(1):
Patrick Lumumba, Mr. Rutto Kenneth, Mrs. 51-64.
Mesoppirr Linda and Mr. Kirui Simon for their DOI: 10.1007/s13167-018-0158-2
assistance during lab analysis. 9. Eikelboom JW, Hirsh J, Spencer FA,
Baglin TP, Weitz JI. Antiplatelet drugs:
COMPETING INTERESTS Antithrombotic therapy and prevention of
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