and in rocky areas (Feyissa, Asres, & Engidaworkm
2013). E. divinorum plant has medicinal values and
has been used traditionally by the Kalenjin community as an anti-venom and as a purgative drug (Kigen
et al. 2016). E. divinorum has also been used locally
as a source of natural dye. The roots and twigs of the
plant were used as toothbrushes, mouth disinfectant,
and to color the lips reddish brown (Maroyi 2011).
Phytochemicals in a plant extract are responsible for
the particular color produced by the natural dye. Phenols, flavonoids, and tannins are the most significant
classes of compounds in the dyeing process because
they account for the ability of a dye to fix to the fabric (color fastness) and the shade formed on the fabric
(Mongkholrattanasit et al. 2013). In addition, plants
rich in tannins have been used as bio-mordants (Amin
et al. 2020; Erdem ˙
Is ˛ mal et al. 2014; Prabhu & Teli
2014) and exhibit the potential to be used as alternatives to the toxic metallic mordants that are currently
in use ( ˙
Is ˛ mal & Yıldırım 2019). Qualitative phytochemical screening studies of E. divinorum has shown
that it contains flavonoids, alkaloids, phenols, tannins,
and terpenoids (Mwonjoria, Ngeranwa, Githinji, &
Wanyonyi 2018). However quantitative phytochemical
analysis of E. divinorum is still lacking.
The objective of this study was to determine the
suitable solvent for extraction of natural dye from
E. divinorum plant as well as the dyeing characteristics
on cotton fabric. Quantitative phytochemical analysis
of E. divinorum was also evaluated.
2 MATERIALS AND METHODS
2.1 Materials
The solvents (methanol, ethyl acetate, hexane and
dichloromethane) used for extraction were of analytical grade. Folin–Ciocalteu, tannic acid quercetin and
gallic acid were used as the analytical standards. All
chemicals used were purchased from Pyrex EastAfrica
Ltd. The cotton fabric (GSM 97.1) was obtained from
Rivatex East Efrica Ltd (REAL).
2.2 Methods
2.2.1 Collection and preparation of plant materials
The root bark of Euclea divinorum plant was collected
from Chemase escarpment in Nandi County. The collected plant materials were washed with tap water to
remove dust and other particles then air-dried in the
shade. The dry barks were ground to powder form in
an electric grinder and weighed using a weighing scale.
2.2.2 Dye extraction
Dye extraction was done using sequential maceration
of the ground samples with organic solvents (hexane, dichloromethane, ethyl acetate and methanol) and
direct aqueous extraction. The organic solvents were
used in order of their increasing polarity. Maceration
for each solvent was done for 24 hours followed by
filtration using Whatman No. 1 filter paper and the
solvents were evaporated using a rotary evaporator to
obtain a dry solid mass whose weight was determined
and percentage yield calculated. The aqueous extract
was filtered and directly used in the dyeing process.
2.2.3 Quantitative phytochemical analysis
Dichloromethane, ethyl acetate and methanolic
extracts were subjected to quantitative evaluation of
total tannins, phenols and flavonoids. All the spectrophotometric assays were measured by UV–Vis
spectrophotometer (Model No: DU’720PC, Beckman
Coulter). Measurements were done in triplicate and
averaged (Baliarsingh et al 2012). MS Excel software
was used to draw the standard curves and calculate the
correlation coefficient (R
2 ).
2.2.3.1 Determination of total tannins
Tannin content was estimated using the standard procedure described by Petchidurai (Petchidurai et al. 2019).
One milligram of the sample was dissolved in 1 mL of
distilled water. An aliquot of 1 mL of the sample was
mixed with 0.5 mL of 10% Folin–Ciocalteau’s reagent
and incubated for 3 minutes. 15% Na 2 CO 3 (1 mL) and
distilled water (8 mL) were added and incubated in the
dark for 30 minutes at room temperature. UV–Visible
spectrophotometric analysis of absorbance was done
at 725 nm in triplicate. A stock solution was prepared
using tannic acid standard (1 g) in a 1000 mL volumetric flask. The stock solution was used to prepare
tannic acid solutions in increasing concentrations (20,
40, 75, 100 and 125 mg/L). 0.5 mL of each concentration was measured using a micropipette and treated
like the sample, and then absorbance was measured. A
tannic acid calibration curve was plotted and the concentration of total tannins in the sample was expressed
as mg tannic acid equivalence/g (mg TAE/g) of the dry
sample extract.
2.2.3.2 Determination of total phenols
Total phenols was determined by the Folin–Ciocalteu
method using the standard procedure (Shi et al. 2019).
An aliquot of 0.5 mL of the sample was mixed with
10% Folin–Ciocalteu’s reagent (2.5 mL) and 15%
Na 2 CO 3 (2.5 mL), and then it was incubated in the dark
for 20 minutes at room temperature. UV–Vis analysis
of absorbance was done at 725 nm in triplicate. A stock
solution was prepared using gallic acid standard (1 g)
in a 1,000 mL volumetric flask. The stock solution was
used to prepare gallic acid solutions in increasing concentrations (20, 40, 75, 100 and 125 mg/L). 0.5mL of
each concentration was measured using a micropipette
and treated like the sample and then absorbance was
measured. The gallic acid calibration curve was plotted and the concentration of total phenols in the sample
was expressed as mg gallic acid equivalence/g (mg
GAE/g) of the dry sample extract.
2.2.3.3 Determination of total flavonoids
Total flavonoids was evaluated by aluminium chloride calorimetric method using a standard procedure
(Bahukhandi, Sekar, Barola, Bisht & Mehta 2019).
The sample extract (1 mL) was mixed with methanol
137
2013). E. divinorum plant has medicinal values and
has been used traditionally by the Kalenjin community as an anti-venom and as a purgative drug (Kigen
et al. 2016). E. divinorum has also been used locally
as a source of natural dye. The roots and twigs of the
plant were used as toothbrushes, mouth disinfectant,
and to color the lips reddish brown (Maroyi 2011).
Phytochemicals in a plant extract are responsible for
the particular color produced by the natural dye. Phenols, flavonoids, and tannins are the most significant
classes of compounds in the dyeing process because
they account for the ability of a dye to fix to the fabric (color fastness) and the shade formed on the fabric
(Mongkholrattanasit et al. 2013). In addition, plants
rich in tannins have been used as bio-mordants (Amin
et al. 2020; Erdem ˙
Is ˛ mal et al. 2014; Prabhu & Teli
2014) and exhibit the potential to be used as alternatives to the toxic metallic mordants that are currently
in use ( ˙
Is ˛ mal & Yıldırım 2019). Qualitative phytochemical screening studies of E. divinorum has shown
that it contains flavonoids, alkaloids, phenols, tannins,
and terpenoids (Mwonjoria, Ngeranwa, Githinji, &
Wanyonyi 2018). However quantitative phytochemical
analysis of E. divinorum is still lacking.
The objective of this study was to determine the
suitable solvent for extraction of natural dye from
E. divinorum plant as well as the dyeing characteristics
on cotton fabric. Quantitative phytochemical analysis
of E. divinorum was also evaluated.
2 MATERIALS AND METHODS
2.1 Materials
The solvents (methanol, ethyl acetate, hexane and
dichloromethane) used for extraction were of analytical grade. Folin–Ciocalteu, tannic acid quercetin and
gallic acid were used as the analytical standards. All
chemicals used were purchased from Pyrex EastAfrica
Ltd. The cotton fabric (GSM 97.1) was obtained from
Rivatex East Efrica Ltd (REAL).
2.2 Methods
2.2.1 Collection and preparation of plant materials
The root bark of Euclea divinorum plant was collected
from Chemase escarpment in Nandi County. The collected plant materials were washed with tap water to
remove dust and other particles then air-dried in the
shade. The dry barks were ground to powder form in
an electric grinder and weighed using a weighing scale.
2.2.2 Dye extraction
Dye extraction was done using sequential maceration
of the ground samples with organic solvents (hexane, dichloromethane, ethyl acetate and methanol) and
direct aqueous extraction. The organic solvents were
used in order of their increasing polarity. Maceration
for each solvent was done for 24 hours followed by
filtration using Whatman No. 1 filter paper and the
solvents were evaporated using a rotary evaporator to
obtain a dry solid mass whose weight was determined
and percentage yield calculated. The aqueous extract
was filtered and directly used in the dyeing process.
2.2.3 Quantitative phytochemical analysis
Dichloromethane, ethyl acetate and methanolic
extracts were subjected to quantitative evaluation of
total tannins, phenols and flavonoids. All the spectrophotometric assays were measured by UV–Vis
spectrophotometer (Model No: DU’720PC, Beckman
Coulter). Measurements were done in triplicate and
averaged (Baliarsingh et al 2012). MS Excel software
was used to draw the standard curves and calculate the
correlation coefficient (R
2 ).
2.2.3.1 Determination of total tannins
Tannin content was estimated using the standard procedure described by Petchidurai (Petchidurai et al. 2019).
One milligram of the sample was dissolved in 1 mL of
distilled water. An aliquot of 1 mL of the sample was
mixed with 0.5 mL of 10% Folin–Ciocalteau’s reagent
and incubated for 3 minutes. 15% Na 2 CO 3 (1 mL) and
distilled water (8 mL) were added and incubated in the
dark for 30 minutes at room temperature. UV–Visible
spectrophotometric analysis of absorbance was done
at 725 nm in triplicate. A stock solution was prepared
using tannic acid standard (1 g) in a 1000 mL volumetric flask. The stock solution was used to prepare
tannic acid solutions in increasing concentrations (20,
40, 75, 100 and 125 mg/L). 0.5 mL of each concentration was measured using a micropipette and treated
like the sample, and then absorbance was measured. A
tannic acid calibration curve was plotted and the concentration of total tannins in the sample was expressed
as mg tannic acid equivalence/g (mg TAE/g) of the dry
sample extract.
2.2.3.2 Determination of total phenols
Total phenols was determined by the Folin–Ciocalteu
method using the standard procedure (Shi et al. 2019).
An aliquot of 0.5 mL of the sample was mixed with
10% Folin–Ciocalteu’s reagent (2.5 mL) and 15%
Na 2 CO 3 (2.5 mL), and then it was incubated in the dark
for 20 minutes at room temperature. UV–Vis analysis
of absorbance was done at 725 nm in triplicate. A stock
solution was prepared using gallic acid standard (1 g)
in a 1,000 mL volumetric flask. The stock solution was
used to prepare gallic acid solutions in increasing concentrations (20, 40, 75, 100 and 125 mg/L). 0.5mL of
each concentration was measured using a micropipette
and treated like the sample and then absorbance was
measured. The gallic acid calibration curve was plotted and the concentration of total phenols in the sample
was expressed as mg gallic acid equivalence/g (mg
GAE/g) of the dry sample extract.
2.2.3.3 Determination of total flavonoids
Total flavonoids was evaluated by aluminium chloride calorimetric method using a standard procedure
(Bahukhandi, Sekar, Barola, Bisht & Mehta 2019).
The sample extract (1 mL) was mixed with methanol
137