In vitro and In-silico Study of the Plant Extract of Bixa orellana L. with the Evaluation of It's Antibacterial and Antioxidant Properties

Abstract

The disc diffusion assay was used in this study to determine the in vitro antibacterial activity of the
chloroform extract of Barleria lupulina leaves against a number of pathogenic bacterial strains.
Moderately significant antibacterial activity was found when comparing the zone of inhibition produced
by plant chloroform extracts to conventional antibiotic discs. MIC has been evaluated as well. Gallic acid
was used as the standard in the study of the total phenol content and was quantitatively estimated using
the Folin-Ciocalteu method. The assay for antioxidants has also been done using the peroxide method.
The physicochemical characteristics of the herbal extract were taken into consideration during
formulation. The molecular docking of the reported phytochemicals with the enzyme was examined
using Biovia Discovery Studio. The HDOCK server was used to determine the interaction's strength. The
model that had the significant confidence values after the docking process and optimum docking score
values showed efficient ligand receptor binding.

Full text

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In vitro and In-silico Study of the Plant Extract of Bixa orellana L. with
the Evaluation of It’s Antibacterial and Antioxidant Properties
Anindya Bagchi1*, Anusree Raha2, Prosenjit Mukherjee1, Monit Pal1 and Palash Chandra Biswas1
1Associate Professor, Department Pharmaceutical Chemistry, Netaji Subhas Chandra Bose Institute of
Pharmacy, Chakdaha (Affiliated to MAKAUT) West Bengal, India.
2Associate Professor, Department of Pharmaceutics, Netaji Subhas Chandra Bose Institute of Pharmacy,
Chakdaha (Affiliated to MAKAUT), West Bengal, India.
Received: 19 July 2023 Revised: 16 Dec 2023 Accepted: 22 Jan 2024
*Address for Correspondence
Anindya Bagchi
Associate Professor,
Department Pharmaceutical Chemistry,
Netaji Subhas Chandra Bose Institute of Pharmacy, Chakdaha
(Affiliated to MAKAUT)
West Bengal, India.
Email: tajuanindya@gmail.com
This is an Open Access Journal / article distributed under the terms of the Creative Commons Attribution License
(CC BY-NC-ND 3.0) which permits unrestricted use, distribution, and reproduction in any medium, provided the
original work is properly cited. All rights reserved.
The aim of this study was to determine the in vitro antibacterial activity of the chloroform extract of Bixa
orellana L. leaves against some pathogenic bacterial strains viz. S. aureus and P. aeruginosa by disc
diffusion assay method. The zone of inhibition produced by chloroform extracts of the plant comparing
with standard antibiotics discs showed moderately significant antibacterial activity. MIC was calculated.
Quantitative estimation of total phenol content also has been done using Folin- Ciocalteu method in
which Gallic acid was used as standard. Also antioxidant study has been done with peroxide method.
Herbal extract gel has been formulated with the evaluation of its physicochemical properties. The
molecular docking of the reported phyto chemicals with the enzyme was studied using biovia discovery
studio. The strength of the interaction was evaluated with the help of HDOCK server. Lowest docking
score gives an idea about ligand receptor binding process.
Keywords: Lipstick Plant, HDOCK, In-silico, Herbal extract, docking.
INTRODUCTION
Bixa orellana L. from the Bixaceae family, which accumulates several carotenoid derivatives, terpenoids and
flavonoids in the seeds and leaves [1]. The medicinal uses of annatto seeds include the treatment of diabetes, skin
infections, burns, fever, measles,gonorrhoea, diarrhoea and asthma [2]. In addition to their antimicrobial properties,
ABSTRACT
RESEARCH ARTICLE
RESEARCH ARTICLE

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the polyphenol content of annatto leaf extracts has shown other biological properties including antioxidant activity
[3, 4]. However very few reports have been seen involving antibacterial activity of chloroform extract from the leaves
of Bixa orellana L. The antioxidant study mainly has been done with DPPH method of assay. Also the in-silico study
of the isolated active constituents responsible for the antibacterial and antioxidant activity from the plant extract
have been reported in less numbers. Hence, considering the information mentioned above, this study was designed
to evaluate antioxidant study with peroxide method and antibacterial activity of chloroform extract of the plant
material followed by the in-silico study of the reported active constituents responsible for the activity.
MATERIALS AND METHODS
Materials
Methanol (Merck, India), Ethanol (Lobachem, India), Chloroform (Lobachem, India) were used during the extraction
process. Gallic Acid (Lobachem, India) and Folin- Ciocalteu Reagent (Lobachem, India) had been used for the
phenolic content estimation of the extract and Hydrogen Peroxide (Merck, India) was used for the antioxidant study
by peroxide method. Hydroxy Propyl Methyl Cellulose (Lobachem, India), Propylene Glycol (Lobachem, India),
Propyl Paraben (Lobachem, India), Methyl Paraben (Lobachem, India) were used for the formulation of herbal
extract. The plant sample i.e. leaves were collected and air dried under shade at room temperature, ground with
electric grinder intofine powder and stored in air tight container for further use. Powdered sample was mixed with
methanol: water of 4:1 ratio (solvents) for extraction in 1:1 ratio. After that the material was filtered by using
Whatman No.1 filter paper and the filtrate was mixed with (2-3) drops of 2M HCl and then mixed with the equal
volume of chloroform as same as of filtrate. After the formation of bottom organic layer, it was taken and separated
followed by evaporation of the solvent for obtaining the dried residue. The resulting chloroform extract solution was
used for further antibacterial and antioxidant activity [5].
Phyto chemical screening of Extract
Following phyto chemical tests has been carried with the extract:
Terpenoids Test
Salkowski test
Dry extract was mixed with chloroform and few drops of conc. H2SO4was added, if red-brown colour formed at the
interface can confirm the presence of triter penoid.
Alkaloid’s Test
Dragendorff test
Few drops of Dragandroff’s reagent was added to the 2 ml of the filtrate containing the extract, the formation of a
reddish brown precipitate can signified positive result.
Wagner’s test: Small amount of extract was mixed with few drops Wagner’s reagent where formation of reddish
brown precipitate can confirm positive result.
Hager’s test
Small amount of extract was mixed with few drops of Hager’s reagent and the formation of yellow colour precipitate
may indicate signified positive result.
Mayer’s test
Few ml of extract was mixed with few drops of Mayer’s reagent where the formation of a creamy precipitate may
confirm positive result.
Glycosides Test
If the extract gives positive result to Fehling (Fehling test A and Fehling test B) solutionthen it can confirm the
presence of glycoside part.
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Flavonoids Test
Ammonium test
Small amount of extract was mixed with dilute ammonia solution (1 ml, 1% v/v) and it was allowed for layer
separation. If yellow colour is seen in the ammonia layer it may signified positive result.
Alkaline test: If small amount of extract was treated with a few drops of 20% (w/v) sodium hydroxide solution, the
generated dark yellow material, which becomes colourless by the addition of dilute hydrochloric acid may signified
positive result.
Steroids Test
Salkowski test
Small amount of extract was mixed with 2 ml of chloroform and 2 ml of concentrated sulphuric acid, if the generated
chloroform layer was red coloured and the acid layer was yellow-green fluorescence in appearance it can signified
positive result.
Phenols Test
Ferric chloride test: Small amount of extract was treated with 3-4 drops of 10% (w/v) ferric chloride solution and the
formation of black green colour may signified the presence of phenolic compound [6].
Quantitative analysis of total Phenolic content
Quantitative analysis of total phenolic in extracts was determined with the Folin- Ciocalteu reagent. Standard used
for the analysis was Gallic acid. Concentration of (10-50) mg/ml of gallic acid was prepared in methanol.
Concentration of 1mg/ml of plant extract was prepared in methanol and from that 0.5ml of sample was introduced
into test tubes and mixed with 2 ml of Folin- Ciocalteu reagent and 2ml of 10% of sodium carbonate solution. The
tube was covered and allowed to stand for 30 min at room temperature before the absorbance was measured at 760
nm spectrometrically. The Folin- Ciocalteu reagent is sensitive to reducing compounds including polyphenols,
thereby producing a blue colour upon reaction. This blue colour is measured spectrophotometrically. Accordingly,
total phenolic content was determined [7].
Antioxidant activity of the plant extract
A solution of H2O2 (40 mM) was prepared in phosphate buffer (100 mM, pH 7.4). Extract (1mg/ml) was added to a
H2O2 solution (0.6 mL) and absorbance was measured. Ascorbic acid was used as standard/positive control. Samples
without hydrogen peroxide were used as a negative control. Absorbance was determined spectro photometrically at
230 nm. The abilities to scavenge the hydrogen peroxide were calculated using the equation: % scavenged (H2O2) =
(Ao − A1)/Ao × 100 Where Ao is the absorbance of the control and A1the absorbance of the sample[8].
Antimicrobial assessment
The antibacterial activity was carried out using disc diffusion method. Bacterial strains vizS. aureus and P. aeruginosa
were purchased from Microbial Type Culture Collection and Gene Bank (MTCC) Chandigarh. Bacteria was cultured
in sterile nutrient broth medium which had been autoclaved at 121°C under a pressure of 15 atmospheres for 15min.
and left to grow for 48 h at 37°C in an incubator. The bacterial cultures obtained were diluted with autoclaved
Nutrient. This culture served as the inoculums for the antimicrobial experiments. For the evaluation of antibacterial
property nutrient agar plates were prepared by mixing (28 g) in 1000 ml distilled water boiled to dissolve the
medium completely. Nutrient agar solution was sterilized by autoclaving at 1210C for15 min at 15 lb pressure. After
cooling (450C), agar solution (25 ml)were poured into sterilized Petri dishes and left to solidify. Agar plates were
inoculated with an overnight bacterial culture, using spread plate method after appropriate serial dilutions. The
plant extract was aseptically put into the wells (100 μl approx.) made in agar plates making lawns of different test
cultures viz.S. aureus and P. aeruginosa. The Nutrient agar plates were then incubated at 370C for 24 h. The diameter
of inhibitory zone surrounding disc and antimicrobial activity of the plant extract solution (2mg/ml and 3mg/ml) was
then measured after 24 hours. Two cross sectional points and the average was taken as the inhibition zone and the
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68447
size ofthe zone diameter was measured in millimetres. The plates were then photographed individually. The results
were compared with the standard drug, Streptomycin. [9-11].
In-silico study
Molecular docking method has been used to identify the phytoc hemical from the plant extract that act as a ligand
and form a strong covalent bond with the microbial protein to successfully inhibit the microbe. The discovery studio
module of the biovia software is using for identify molecular interaction and perform molecular docking. In this
process, first the pdb files for the phyto chemicals (Kaempferol) found in the Bixa orellana plant were downloaded
from the website drug bank .The protein3-oxyacyl-[acyl carrier protein] reductase (FabG) for unknown ligand data
base code(4BNW)was collected fromRCSB protein data bank. Molecular docking was done using the HDock
Server.The enzyme molecule was treated as the receptor molecule and the phyto chemical was treated as the ligand.
The high positive value of those indicators presented a good interaction between the ligand and the receptor. Thus,
the interaction with high values might indicate the major phyto chemical responsible for curing the disease [12].
Kaempferol interact with3-oxyacyl-[acyl carrier protein] reductase (FabG) therefore inhibiting the biosynthesis of
fatty acids by Pseudomonas aeruginosa and responsible for antibacterial activity.
Method of formulation of herbal extract gel
The required amount of gelling agent was accurately measured and dispersed in a small amount of water with
continuous stirring to produce a uniform dispersion. Then the drug was dissolved in a suitable solvent here using
propylene glycol and added to the above dispersion. Other substances such as methyl paraben and propyl paraben
were also added with continuous stirring. The final weight of the gel formulation was adjusted to 10 g with distilled
water. The gel was stored in container with wide mouth.
Evaluation of gel formulation
Physical Characterizations
The composition of gel using different gelling agents tested for colour, odour, homogeneity, in which the gels were
placed in containers.
Measurement of Surface pH
The pH formation of gel was measured using a digital pH meter. 1 g of gel was dissolved in 25 ml of distilled water
in a beaker. The electrode was then immersed in the beaker solution and allowed to simmer for 1 minute and further
reading was observed.
Spread ability
Indicates the level of area where the gel spreads easily when applied to the affected skin. The therapeutic potential of
the gel also depends on its spreading value. It is periodically displayed in seconds taken by two slides to move from
the gel placed between the slides under the direction of a specific load (20 g). The formula for calculating gel spread
ability is: S = M * (L / T)
Where,
M = Weight tied to the top slide (20 g)
L = Length of the glass slide slipped
T = Time taken to split the slides.
Tube Extrudability
In this experiment was taken a closed folding tube containing the composition of the ciprofloxacin gel. The gel was
pressed tight at the end and a clamp was placed at the end of the tube to prevent any loosening. A weight of 500 g
was placed on tube and removed from the cap. The gel was pulled out. The formula for calculating tube
extrudability is: E = (M / A)
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Where,
E = Tube extrudability
M = Weight applied on tube (500 g)
A = Extrude gel area [13].
RESULTS AND DISCUSSIONS
Phyto chemical Screening
The result showed that the chloroform extract has primarily flavonoids and phenolic part which is generally
important for antibacterial and antioxidant activity. Ocimum tenuiflorum was used as positive control to find out the
validity of the reagent used for phyto chemical screening.
Quantitative estimation of phenolic content
The absorbance value of the plant material is 0.911Now by plotting the value on the equation the conc. was found
out to be is 63.26 ug/ml.
Antioxidant assessment
Percent Scavenged; % (H2O2) = [(A0− A1)/A0× 100]
Ao= Absorbance of the control = 0.515
A1= Absorbance of the plant sample = 0.235
So according to equation
0.515- 0.235 = 0.28/0.515=0.5436×100 = 54.36%.
From the above result it has been seen that the plant extract has showed significant effect in the dose 2mg/ml.
In-silico Study
These are docking score of 10 modules involving docking of ligand and receptor. Module 1 shows the least value so
it could be judge as the best fit.
CONCLUSION
According to the results obtained in this investigation, it can be concluded that the chloroform extract of the selected
plant entity is having the moderate antibacterial activity in two different pathogenic species. Also the plant extract
shows the antioxidant activity have been measured spectro photometrically which may cause scavenging of free
radicals forms inside the biological systems. Total phenolic content have been measured which may influence the
both antimicrobial and antioxidant activity. Insilco study has been done for a specific pathogenic bacteria enzyme
whose activity can be blocked by a reported phyto constituents leading to the antibacterial activity. Herbal extract
formulation has been prepared with its evaluation of physicochemical properties. In future the herbal formulation
can be tested for the antibacterial activity and further Insilco study can be done with different phyto constituents
responsible for the different activities if the protein/enzyme structure of the pathogenic entity is known, responsible
for the different types of diseases.
ACKNOWLEDGEMET
The authors are highly thankful to the respected principal sir Dr. Arnab Samanta for providing the necessary facility
for the completion of the research work.
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Table 1: Composition of Gel Formulation
INGREDIENTS
FORMULATION (g)
Plant extract (dry)
1
Hydroxy propyl methyl cellulose
1
Propylene glycol
2 ml
Methyl paraben
0.1
Propyl paraben
0.2
Distilled water
upto
10
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Table 2: Phyto chemical Screening
Plant Name Terpenoids Alkaloids Glycoside Flavonoids Steroids
Phenolic
content
Putranjiva roxburghii - D H M W - + - +
+ - - -
Ocimum tenuiflorum
+
+
-
-
+
+
+
+
+
D= Dragandroff’s reagent M= Mayer’s reagent
H= Hager’s reagent W= Wagner’s reagent
(+) signify positive result
(-) signify negative result
Table 3: Uv-Spectroscopic analysis of Gallic acid
Conc. of Gallic acid(ug/ml)
Observed Absorbance
10
0.087
20
0.286
30
0.47
1
40
0.532
50
0.747
Table 4: Assessment of Antibacterial activity
Name of the Drug/Extract Name of the bacteria Zone of the inhibition
2mg/ml 3mg/ml
Control S. aureus 3.2 mm -
P. aeruginosa 2.9 mm -
Plant Extract S. aureus 5.2 mm 2.6 mm
P. aeruginosa 2.4 mm -
Table 5: In silico assessment
Expected Confidence score- 0.5-0.7.
Table 6: Physical Characterizations of Gel formulation
Physical characterizations
FORMULATION
COLOUR
ODOUR
HOMOGENECITY
F1
Yellowish white
Pleasant
Homogenous
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Table 7: Surface pH of Gel formulation
Measurement of surface pH
FORMULATION
SURFACE pH
F1
6.0
Table 8: Spreadibility of Gel formulation
Spreadability
FORMULATION
SPREADIBILITY (g.cm/sec)
F1
12.8
Table 9: Tube Extrudability of Gel formulation
Tube extrudability
FORMULATION
TUBE EXTRUDABILITY (g/cm
2
)
F1
74
F1 =Plant extract
Fig 1: Standard curve of Gallic acid
Fig 2:Standard drug on S
. aureus
Fig 3
: Standard drug on
P. aeruginosa
Fig 4:Plant Extract on
S. aureus
Fig 5: Plant Extract o
n
P. aeruginosa
Fig 6:
Protein structure of 3
-
oxyacyl
-
[acyl carrier protein]
reductase (FabG) for unknown liganddata base code
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(4BNW)
Fig 7:Binding site of ligand in enzyme structure
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