Journal of Molecular Structure 1250 (2022) 131678

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Journal of Molecular Structure

journal homepage: www.elsevier.com/locate/molstr

GC–MS characterization and evaluation of antimicrobial, anticancer

and wound healing efficiency of combined ethanolic extract of Tridax
procumbens and Acalypha indica
A. Sophia a, Md. Faiyazuddin b,c,∗, Prawez Alam d, Md. Talib Hussain e, Faiyaz Shakeel f,∗
a
Cauvery College for Women, Annamalai Nagar, Trichy, Tamil Nadu 620018, India
b
School of Pharmacy, Al-karim University, Katihar 854106, Bihar, India
c
Nano Drug Delivery®, Raleigh-Durham, NC 27705, USA
d
Department of Pharmacognosy, College of Pharmacy, Prince Sattam Bin Abdulaziz University, Al-Kharj 11942, Saudi Arabia
e
Department of Pharmacology & Toxicology, College of Pharmacy, University of Hail, Hail City, Saudi Arabia
f
Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia

a r t i c l e i n f o a b s t r a c t

Article history: Two weeding plants namely Tridax procumbens (TP) and Acalypha indica (AI) were studied for their pro-
Received 29 August 2021 found wound healing property, antimicrobial activity, and cytotoxicity. The topical formulation is based
Revised 26 September 2021
on the combined ethanolic crude extract of the weeds. The research aims to find the various phytochem-
Accepted 6 October 2021
icals present in the combinational extract and to evaluate the most abundant phytochemicals present in
Available online 9 October 2021
the combinational extract that provides the best pharmacological activity towards the profound wound
Keywords: healing by gas-chromatography mass-spectrometry (GC–MS) and to evaluate the extent of wound healing
Tridax procumbens property of the combinational extract by MTT assay for cell cytotoxicity. The GC–MS showed the presence
Acalypha indica of 50 compounds in the combined extract and the presence of a novel phenol, 2-(imidazo[1,2-a]pyridin-
Antimicrobial activity 2-yl)phenol which is an inflammatory cytokine release inhibitor reported for the first time in the paper.
GC-MS The agar well diffusion method showed an inhibition zone value of 8.5 ± 1.5 mm for a concentration
Inflammatory cytokine release
of 500 μg/ml of the combined extract against the E. coli bacteria establishing the antibacterial property
NF-κ B inhibitor
of the combinational extract. The MTT assay showed an IC50 = 216.45 μg/ml. Overall, the results of this
study showed potential antimicrobial and would healing effects of combined ethanolic extracts of TP and
AI.
© 2021 Elsevier B.V. All rights reserved.

1. Introduction cacies, wound healing activity, insecticidal, and anti-inflammatory

activity [5,6]. The extracts of the leaves of TP have been proved
Synergism of drugs is a biomimicry to healing, since plant ex- to decrease the time taken for blood clotting and thus helps in
tracts with a combination of phytochemicals has shown better haemostasis [6]. The variety of phytochemicals such as alkaloids,
healing properties with lesser or no side effects in combination [1]. carotenoids, flavonoids, hydroxycinnamates, lignans, benzoic acid
It has been seen that certain phytochemicals in combination had a derivatives, phytosterols, and tannins have been found in the leaves
better pharmacological effects than being separated as a single en- of TP [7,8].
tity used [2,3]. The plants chosen are two Indian weeds namely Tri- AI is ethnobotanically known to be used externally for skin ail-
dax procumbens (TP) (family: Asteraceae) and Acalypha indica (AI) ments and internally for medicinal purpose in different parts of
(family: Euphorbiaceae), which are ethnobotanically known to be Tamilnadu, India [9]. It also reportedly possesses diuretic, purga-
used for internal ulcers and external profound wounds [4]. The tive, anti-bacterial, antifungal, and anthelmintic properties [10]. AI
plant TP yields a juicy extract, when crushed and is tradition- is traditionally used for treating intestinal worms, gum problems,
ally known for its anticoagulant, antiviral effects, antioxidant effi- stomach aches, hernia, rheumatism, bronchitis, asthma, pneumo-
nia, scabies and skin diseases [11]. The plant AI is known by the
name kuppaimeni (kuppai–weed, meni-body) in Tamilnadu, India.
∗
Corresponding author at: Department of Pharmaceutics, College of Pharmacy,
The name means a weed found from anywhere for a good skin
King Saud University, Riyadh 11451, Saudi Arabia. health in Tamil etymology. It has been reported ethnobotanically
E-mail addresses: [email protected] (Md. Faiyazuddin), to be used [9]. The variety of phytochemicals such as alkaloids,
[email protected] (F. Shakeel).

https://doi.org/10.1016/j.molstruc.2021.131678
0022-2860/© 2021 Elsevier B.V. All rights reserved.
A. Sophia, Md. Faiyazuddin, P. Alam et al. Journal of Molecular Structure 1250 (2022) 131678

catechols, flavonoids, phenolic compounds, saponins, and steroids has a low-moderate tropical climate with hot summers and cool
have been found in the roots and leaves of AI [12,13]. In order to winters. It has red soil, which is cultivable. The plants were col-
get the expected synergistic pharmacological effects, two extracts lected on a sunny day at temperature 32 °C and found as weed.
were combined. The plants were identified by LeafSnap, the first mobile app for
Profound wounds such as the diabetic foot ulcers are associ- identifying the plant species using automatic visual recognition
ated with peripheral neuropathy, peripheral vascular disease, and [29].
disruption of response to the infection [4]. Neuropathy lowers the
pain threshold so that it is often unaware of the existence of the 2.2. Materials, reagents, media and cultures
wound until the wound worsens leading to complications includ-
ing cellulitis, abscesses, osteomyelitis, gangrene, and septicaemia Dimethyl sulfoxide (DMSO), MTT and ethanol were obtained
[14]. The basic healing therapy is debridement, reducing the pres- from Sigma Aldrich (St. Louis, MO, USA). E. coli 443 strain was
sure on the area of the injury, manage the infection by diagnos- purchased from Microbial Type Culture Collection (MTCC) (Chandi-
ing the type of bacteria, providing adequate antibiotics and ulcer garh, India). Nutrient agar medium, nutrient broth and gentamicin
treatment using wound dressing clean and moist [15]. Thus, the antibiotic solution were obtained from HiMedia (Mumbai, India).
present study aims to provide a topical formulation that would Test samples, petri-plates, test tubes, beakers and conical flasks
combine anti-bacterial, anti-inflammatory, anticoagulant, antiviral were from Borosil (New Delhi, India). Dulbecco’s modified eagle
effect, and antioxidant efficacies to prevent affected individuals medium (MEM) and fetal bovine serum (FBS) were obtained from
from debridement and assisting in gradual and efficient healing Gibco (St. Louis MO, USA). The 96 well tissue culture plates and
[16]. wash beakers were purchased from Tarson (New Delhi, India). The
To the best of authors knowledge, the wound healing and an- water used in the study was distilled water which was collected
timicrobial effects of combined extracts of TP and AI have not been from the distillation unit.
studied in literature so far. Therefore, the objective of this work The medium was prepared by dissolving 2.8 g of the commer-
was to investigate the effective pharmacological activity (wound cially available nutrient agar medium in 100 ml of distilled water.
healing and antimicrobial effects) [17–20] of combinational ex- The dissolved medium was autoclaved at 15 lbs pressure at 121 °C
tracts which combines the pharmacology of skin health and wound for 15 min. The autoclaved medium was mixed well and poured
healing activity of the plant extracts. The work also focuses on onto 100 mm Petriplates (25–30 mL/plate) while still molten.
finding the various phytochemicals [21,22] in combined extract
using gas chromatography-mass spectrum (GC–MS) analysis to
2.3. Drying and extraction of samples
evaluate the phytochemicals which are responsible for profound
wound healing property [23]. The plant extracts contain the va-
The leaves of TP and the whole plant (except the roots) of AI
riety of known and unknown phytoconstituents [24,25]. For high-
were collected locally and dried for one day in sunlight (32 °C)
performance liquid chromatography mass spectrometry (HPLC/MS)
and for four days at room temperature (28 °C). The dried leaves of
analysis, standards are required to identify the phytochemicals
TP and the whole plant (except the roots) of AI were crushed and
[24]. Hence, for HPLC/MS analysis, the large number of standards
subjected to cold extraction in ethanol by maceration for about
are required. However, in case of GC–MS analysis, the phytochem-
100 ml of the solvent. Different concentrations of the sample TP
icals are identified based on Wiley library and standards are not
in ethanol, AI in ethanol and TP+AI in ethanol were extracted
required [25,26]. Infrared devise is used to compare the spectra
for different concentrations, including 1 μg/ml, 5 μg/ml, 10 μg/ml,
with standard. Our combined extract had several unknown com-
15 μg/ml, 50 μg/ml, 100 μg/ml, 200 μg/ml, 250 μg/ml, 300 μg/ml,
pounds which were not possible to identify using infrared device.
400 μg/ml and 500 μg/ml. The maximum number of compounds
The purpose of the work was the chemical characterization and
were found to be soluble in alcoholic extract of individual plant
biological evaluation of the combined extract of TP + AI. Nuclear
[7,8,12,13]. Hence, ethanolic extract was used in this work. The
magnetic resonance (NMR) and Fourier transforms infrared (FTIR)
cold extraction in ethanol was done at TRI Biotech. Ltd., Tiruchi-
techniques are not sufficient to the detect the unknown phyto-
rappalli, India.
constituents present in the extract. NMR and FTIR techniques are
helpful in characterizing pure isolated compounds, which were not
isolated in this study. GC–MS is best technique for the identifica- 2.4. Gas chromatography-mass spectrum (GC–MS) analysis
tion of unknown compounds in plant extract. Therefore, GC–MS
analysis was performed to identify various phytochemicals in the The crude extracts of TP and AI in ethanol was combined and
combined ethanolic extract of TP + AI instead of HPLC/MS. The the GC–MS was performed. The greater the concentration in the
3–4,5 dimethylthiazol-2yl-2,5-diphenyl tetrazolium bromide (MTT) combined sample, the greater was the signal which was then pro-
assay was hence performed to find out the best concentration of cessed through the computer and the chromatogram was obtained
the crude ethanolic extracts for wound healing so that it can be [30]. The data obtained were compared with the compounds using
evolved into a topical formulation for profound wound healing. The Wiley library of chemical compounds [31]. The GC–MS profiling of
study also focuses on the antimicrobial activity of the both the the cold extract of TP+AI in ethanol was done at TRI Biotech. Ltd.,
combinational crude extract (TP+AI) and individual crude extract Tiruchirappalli, India.
especially on the ulcer creating bacteria Escherichia coli [27,28]. The
data obtained in this work will be helpful to the researchers work- 2.5. Antibacterial activity
ing in the area of phytochemistry.
The antimicrobial activity of extracts was evaluated using agar
2. Materials and methods well diffusion method. Petri plates containing 20 ml nutrient agar
medium were seeded with the 24 h culture of bacterial strains (E.
2.1. Plant materials coli 443). The wells were cut and different concentration of the
sample of TP, AI and TP+AI (500 μg/ml, 250 μg/ml, 100 μg/ml
The plants (TP and AI) were collected around the residential and 50 μg/ml of each) were added. The plates were then incu-
area in Neyveli Township, Tamilnadu, India located at 11.60°N and bated at 37 °C for 24 h [32]. The antibacterial activity was as-
79.48°E. It has an average elevation of 87 metres (285 ft). Neyveli sayed by measuring the diameter of the inhibition zone formed

2
A. Sophia, Md. Faiyazuddin, P. Alam et al. Journal of Molecular Structure 1250 (2022) 131678

Fig. 1. Tridax procumbens (TP) [A] and Acalypha indica (AI) [B] in their natural habitat conditions.

around the wells. Gentamicin antibiotic was used as a positive con- 3. Results and discussion
trol. The mean values were calculated. E. coli antibiotic susceptibil-
ity tests were performed at TRI Biotech. Ltd., Tiruchirappalli, India. 3.1. GC–MS analysis

In this study, the phytochemicals of cold extraction of TP+AI

2.6. MTT assay for cell cytotoxicity were analysed using GC–MS method for the first time. The list of
identified compounds along with other details is given in Table 1.
MTT assay is based on the ability of a mitochondrial dehydro- The representative GC–MS chromatogram of combined ethanolic
genase enzyme of viable cells to cleave the tetrazolium rings of the extract is presented in Fig. 2. GC–MS analysis showed the pres-
pale yellow MTT and form a dark blue coloured formazan crystals ence of 50 compounds in the combined extract in ethanol. Among
which is largely impermeable to cell membranes, thus resulting in them, almost 27 compounds are decene derivatives showing very
its accumulation within healthy cells. The solubilization of cells by long chain molecules (10 carbon atoms). The rest of the phy-
the addition of DMSO results in the liberation of crystals which tochemicals also showed long chain compounds with less than
are solubilized. The number of surviving cells is directly propor- ten carbon atoms and chemicals with at least one benzene ring.
tional to the level of formazan product created. The color can be The compounds of higher retention were identified. These com-
quantified using a multi-well plate reader [33]. MTT assay was also pounds were placed in descending order of highest to the lower
performed at TRI Biotech. Ltd., Tiruchirappalli, India. retention or A/H (area/height) values obtained from the GC–MS
Cytotoxicity evaluation was performed on human breast cancer curve and listed in Table 2. The compounds present in highest
cells (MCF-7). The MCF-7 cells (NCCS, Pune, India) were cultured amount were ergosta-5,7,22-trien-3-ol, (3.beta.,22E) (47.45%) fol-
in DMEM media, supplemented with 10% FBS, 100 ug/ml penicillin lowed by diethyl phthalate (10.67%), ergosta-5,8–dien-3-ol, (3.beta)
and 100 μg/ml streptomycin, and maintained under an atmosphere (5.60%), 2-(imidazo[1,2-a]pyridin-2-yl)phenol (2.84%), and tritria-
of 5% CO2 at 37 °C [34]. The cultured MCF-7 cells were harvested contane (2.75%).
by trypsinization, pooled in a 15 ml tube. Then, the cells were
plated at a density of 1 × 105 cells/well (200 μL) into 96-well tis- 3.2. Analysis of chemical structure by inhibition of nuclear factor
sue culture plate in DMEM medium containing 10% FBS and 1% kappa B pathway
antibiotic solution for 24–48 h at 37 °C. The wells were washed
with sterile phosphate buffer saline (PBS) and treated with vari- The compounds listed in Table 2 were studied for their anti-
ous concentrations of TP+AI plant extract samples in a serum free inflammatory and wound healing property than the rest of the
DMEM medium. Each sample was replicated three times and the phytochemicals since they were greater in amount when analysed
cells were incubated at 37 °C in a humidified 5% CO2 incubator through GC–MS. Several compounds were identified in combined
for 24 h. After the incubation period, MTT (20 μL of 5 mg/ml) extract, so A/H percentage was low. Generally, certain phytochem-
was added into each well and the cells incubated for another 2– icals exert their wound healing property by inhibition of nuclear
4 h until purple precipitates were clearly visible under an inverted factor kappa B (NF-κ B) pathway [36]. These phytochemicals were
microscope. Finally, the medium together with MTT (220 μl) were studied for their property of inhibition of NF-κ B pathway.
aspirated off the wells and washed with 1X PBS (200 μl). Further- A medicament having inhibitory activity against NF-κ B activa-
more, to dissolve formazan crystals, DMSO (100 μL) was added and tion, is reported to be comprised of a compound represented by
the plate was shaken for 5 min. The absorbance for each well was the following general formula (I) or a pharmacologically acceptable
measured at 570 nm using a micro plate reader (Thermo Fisher salt as an active ingredient:
Scientific, Waltham, MA, USA) and the percentage cell viability and
50% inhibitory concentration (IC50 ) value was calculated [35].

2.7. Statistical analysis

All values are expressed as mean ± SD of at least three repli-

cates. The statistical analysis was performed using Graph Pad Prism Where ‘X’ represents a connecting group, ‘A’ represents hydro-
6.0 software (San Diego, CA, USA). gen atom or acetyl group, ‘E’ represents an aryl group or a het-

3
A. Sophia, Md. Faiyazuddin, P. Alam et al. Journal of Molecular Structure 1250 (2022) 131678

Table 1
The phytochemicals identified in the ethanolic extract of (TP+AI) by GC–MS technique.

Component name RT (Min) Area Area (%)

1-Decene 6.63 67,752 0.07

Benzaldehyde-2-hydroxy 7.88 600,302 0.58
1-Dodecene 11.34 335,515 0.32
Benzoic acid-2-hydroxymethyl ester 11.40 1,980,021 1.90
1-Pentadecene 15.91 641,946 0.62
Octadecane 16.09 65,040 0.06
Phenol-2,4-bis(1,1-dimethyl) 18.36 997,302 0.96
Diethyl phthalate 19.99 11,105,000 10.67
1-Hexadecene 20.06 1,031,039 0.99
Octadecene 20.21 71,961 0.07
1-Tetradecanol 21.68 144,990 0.14
Oxalic acid, cyclohexyl methyl tridecyl ester 22.01 471,737 0.45
Tetradecanoic acid 23.21 350,332 0.34
n-Hexadecanoic acid 26.64 1,294,002 1.24
1-Nonadecene 27.16 1,374,966 1.32
Nonadecane 27.27 77,599 0.07
Procaine 28.17 86,962 0.08
9,12-Octadecadienoic acid (Z,Z)- 29.31 464,932 0.45
Oleic acid 29.41 667,800 0.64
(E)−13-Docosenoic acid 29.49 161,080 0.15
Octadecanoic acid 29.77 450,278 0.43
1-Heptacosanol 30.25 1,049,528 1.01
Heptadecane 30.34 129,404 0.12
Eicosyl acetate 30.44 823,258 0.79
2-(imidazo[1,2-a]pyridin-2-yl)phenol 32.44 2,952,030 2.84
1,4,5,6-Tetrahydro-6-[phenylmethyl]−2H-1,2,4,5-tetrazine-3-thione 32.62 86,816 0.08
Naphtho(2,3-b)−1,4-diazabicyclo(2,2,2)octene 32.66 73,544 0.07
Heneicosane 33.16 157,439 0.15
Octacosyl acetate 33.26 455,290 0.44
Hexanoic acid, undecyl ester 34.37 350,233 0.34
Tetratriacontane 34.48 406,965 0.39
Hexadecanoic acid, 2–hydroxy-1-(hydroxymethyl)ethyl ester 34.61 1,409,433 1.35
Bis(2-ethylhexyl) phthalate 34.87 923,282 0.89
Tetracosane 35.76 213,382 0.21
1-(Piperidin-1-yl)hexadecan-1-one 36.08 425,177 0.41
Octadecanoic acid, ethenyl ester 36.18 901,613 0.87
Heptacosyl heptafluorobutyrate 36.45 323,594 0.31
Tetradecanoic acid, 2–hydroxy-1,3-propanediyl ester 36.56 592,406 0.57
Ergosta-5,7,9(11),22-tetraen-3-ol, (3.beta.,22E)- 36.74 1,818,047 1.75
Tritriacontane 36.99 2,857,819 2.75
Octadecanoic acid, 2,3-dihydroxypropyl ester 37.22 472,506 0.45
1,4-Benzenedicarboxylic acid, bis(2-ethylhexyl) ester 37.49 739,036 0.71
Ergosta-5,7,22-trien-3-ol, (3.beta.,22E)- 37.95 49,387,759 47.45
1-Hexacosanol 38.13 487,077 0.47
Hexatriacontane 38.18 675,050 0.65
5,6-Dihydroergosterol 38.26 1,547,133 1.49
Neoergosterol 38.49 1,103,727 1.06
1-(Piperidin-1-yl)octadecan-1-one 38.64 315,021 0.30
Ergosta-5,8–dien-3-ol, (3.beta.)- 39.42 5,830,093 5.60
γ -Ergostenol 39.89 1,689,676 1.62

Fig. 2. Representative GC–MS chromatogram of the crude ethanolic extract of (TP+AI).

4
A. Sophia, Md. Faiyazuddin, P. Alam et al. Journal of Molecular Structure 1250 (2022) 131678

Table 2
The compounds arranged in descending order of highest to the lower retention or A/H (area/height) values obtained
from the GC–MS curve.

S.N. A/H value Chemical name RT (Min) Area Area (%)

1. 20.49 Ergosta-5,7,22-trien-3-ol, (3.beta.,22E)- 37.95 49,387,759 47.45

2. 13.33 Ergosta-5,8–dien-3-ol, (3.beta.)- 39.42 5,830,093 5.60
3. 8.02 Tritriacontane 36.99 2,857,819 2.75
4. 7.98 Octadecanoic acid, ethenyl ester 36.18 901,613 0.87
5. 6.2 Tetradecanoic acid, 2–hydroxy-1,3-propanediyl ester 36.56 592,406 0.57
6. 6.05 Hexanoic acid, undecyl ester 34.37 350,233 0.34
7. 5.94 2-(imidazo[1,2-a]pyridin-2-yl)phenol 32.44 2,952,030 2.84
8. 5.93 Neoergosterol 38.49 1,103,727 1.06
9. 5.27 Heptacosyl heptafluorobutyrate 36.45 323,594 0.31
10. 5.15 γ -Ergostenol 39.89 1,689,676 1.62
11. 5.07 5,6-Dihydroergosterol 38.26 1,547,133 1.49
12. 4.97 Ergosta-5,7,9(11),22-tetraen-3-ol, (3.beta.,22E)- 36.74 1,818,047 1.75

Fig. 3. Chemical structure of 2-(imidazo[1,2-a]pyridin-2-yl)phenol obtained by GC–MS.

eroaryl group, and ring ‘Z’ represents an arene or a heteroarene wherein Rz represents hydrogen atom, a halogen atom, nitro group,
[36]. The combined extract in ethanol showed the presence of a cyano group, hydroxy group which may be substituted, an amino
novel phenol compound 2-(imidazo[1,2-a]pyridin-2-yl)phenol. The group which may be substituted, a hydrocarbon group which may
2-(imidazo[1,2-a]pyridin-2-yl)phenol is found to have a similar be substituted, a heterocyclic group which may be substituted, an
chemical structure as shown in Fig. 3 showing that it is a very good acyl group which may be substituted, an ureido group which may
inflammatory cytokine release inhibitor. be substituted, a thioureido group which may be substituted, or
It has also been reported that a medicament having inhibitory a diazenyl group which may be substituted [36]. Accordingly, the
action against NF-κ B activation comprises as an active ingredient a GC–MS showed the presence of various phytochemical compounds
substance selected from the group consisting of the compound and in the combined crude ethanolic extract of the plants with a sim-
a pharmacologically acceptable salt thereof, and a hydrate thereof ilar structure and listed in Table 3. The 19 of these molecules
and a solvate thereof, represented by the following formula were found to have similar general formula mentioned above as
a medicament having inhibitory activity against NF-κ B activation.

3.3. Antibacterial activity

The results for antibacterial evaluation of TP, AI and (TP+AI)

are listed in Table 4. The images for zone of inhibition of E. coli
using TP, AI and (TP+AI) extracts are displayed in Fig. 4. The an-
tibacterial test by agar diffusion method showed that the zone
of E. coli inhibition was greater in the combined extract than in
the crude ethanolic plant extracts taken separately [32]. For ex-
ample, for the 500 μg/ml extract which showed the greatest zone

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A. Sophia, Md. Faiyazuddin, P. Alam et al. Journal of Molecular Structure 1250 (2022) 131678

Table 3
The list of phytochemicals with similar structures having inhibitory activity against NF-κ B activation identified by GC–
MS technique.

S.N. Component name RT (Min) Area Area (%)

1 Benzaldehyde-2-hydroxy 7.88 600,302 0.58

2 Benzoic acid-2-hydroxymethyl ester 11.40 1,980,021 1.90
3 Phenol-2,4-bis(1,1-dimethyl) 18.36 997,302 0.96
4 Diethyl phthalate 19.99 11,105,000 10.67
5 Oxalic acid, cyclohexyl methyl tridecyl ester 22.01 471,737 0.45
6 Procaine 28.17 86,962 0.08
7 2-(imidazo[1,2-a]pyridin-2-yl)phenol 32.44 2,952,030 2.84
8 1,4,5,6-Tetrahydro-6-[phenylmethyl]−2H-1,2,4,5-tetrazine-3-thione 32.62 86,816 0.08
9 Naphtho(2,3-b)−1,4-diazabicyclo(2,2,2)octene 32.66 73,544 0.07
10 Bis(2-ethylhexyl) phthalate 34.87 923,282 0.89
11 1-(Piperidin-1-yl)hexadecan-1-one 36.08 425,177 0.41
12 Ergosta-5,7,9(11),22-tetraen-3-ol, (3.beta.,22E)- 36.74 1,818,047 1.75
13 1,4-Benzenedicarboxylic acid, bis(2-ethylhexyl) ester 37.49 739,036 0.71
14 Ergosta-5,7,22-trien-3-ol, (3.beta.,22E)- 37.95 49,387,759 47.45
15 5,6-Dihydroergosterol 38.26 1,547,133 1.49
16 Neoergosterol 38.49 1,103,727 1.06
17 1-(Piperidin-1-yl)octadecan-1-one 38.64 315,021 0.30
18 Ergosta-5,8–dien-3-ol, (3.beta.)- 39.42 5,830,093 5.60
19 γ -Ergostenol 39.89 1,689,676 1.62

Table 4
The zone of inhibition obtained by sample TP, AI, and TP+AI against E. coli.

Zone of inhibition (mm)

Test Test
Mean ± SD
organism sample
500 μg/ml 250 μg/ml 100 μg/ml 50 μg/ml AB

E. coli TP 6.5 ± 0.2 5.5 ± 0.5 4.5 ± 0.5 2.5 ± 1.5 11 ± 1.0
AI 7.5 ± 0.5 6 ± 0.8 5.5 ± 0.5 3.5 ± 0.6 11 ± 1.0
TP+AI 8.5 ± 1.5∗ 7.5 ± 0.5∗ 5.5 ± 1.5 4.5 ± 0.5∗ 10 ± 1.0

SD: standard deviation,.

∗
significant- p< 0.05.

Fig. 4. Effect of TP (A), AI (B) and TP+AI (C) against E. coli.

of inhibition than the other concentrations; TP showed an inhi- 3.4. MTT assay for cell cytotoxicity
bition diameter 6.5 ± 0.2 mm, AI showed an inhibition diameter
7.5 ± 0.5 mm and the combined extract TP+AI showed an inhibi- Cell viability (%) of (TP + AI) combined extract against MCF-7
tion diameter larger than the extracts taken separately showing a breast cancer cells was determined using MTT assay at 1, 5, 10, 15,
value of 8.5 ± 1.5 mm. All studied concentrations showed signif- 50, 10 0, 20 0, 30 0, 40 0 and 500 μg/ml concentration. The results
icant antibacterial effects for combined extract compared to their of cell viability (%) are shown in Fig. 5. The maximum cytotoxicity
individual counterparts (p<0.05). However, the antibacterial effects for combined extract was found to be 72.60 ± 2.89% at the con-
of all plant extracts were found to be inferior to standard antibi- centration of 500 μg/ml (Table 5). However, the control did show
otic (positive control). Overall, these results showed that combined any cytotoxic effects. In general, the cytotoxic effects of combined
extract had synergistic antibacterial effects and can be utilized in extract were found to be enhanced with increase in the concentra-
the treatment of bacterial infections. tion of the extract. The IC50 value for combined extract was also

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A. Sophia, Md. Faiyazuddin, P. Alam et al. Journal of Molecular Structure 1250 (2022) 131678

Fig. 5. Concentration-dependent cell viability curve for (TP+AI) plant extract on MCF-T breast cancer cells (mean ± SD, n = 3.0).

Table 5 ethanolic crude extract would be efficient in profound wound heal-

Cytotoxicity (% inhibition) values for different con-
ing and also in inhibiting the proliferation of cancer cells. The an-
centrations of (TP+AI) combined plant extract
compared with control (mean ± SD, n = 3.0). tibacterial test showed that AI is more effective against the E. coli
bacteria than TP, yet the combined extract TP+AI showed an im-
Concentration Cytotoxicity (%) ± SD
proved inhibition against E. coli. All these factors combine to pro-
(μg/ml)
TP + AI extract Control duce a significant wound healing property due to a synergic phar-
1 1.58 ± 2.35 macological action which is a biomimicry to healing. Hence, this
5 7.24 ± 1.74 paper would suggest on the further study of the phenolic com-
10 14.92 ± 1.97 pound’s occurrence in individual plant extracts namely T. procum-
15 23.13 ± 2.54 bens and A. indica and its efficacy in profound wound healing such
50 30.81 ± 4.14 0.00 ± 0.00
100 35.34 ± 2.83
as diabetic foot as a topical formulation. The isolation and purifi-
200 46.42 ± 1.74 cation of compounds from the combined extract is going on and
300 59.25 ± 3.76 these data will be used for future publications.
400 68.50 ± 1.74
500 72.60 ± 2.89
Credit author statement

Faiyaz Shakeel: Conceptualization, Supervision, A. Sophia:

determined by the interpolation of concentration-dependent cell
Methodology, Investigation, Writing original draft, Md. Faiyazud-
viability curve (Fig. 5). The IC50 value for combined extract was
din: software, Supervision, Project administration, Prawez Alam:
determined as 216.45 μg/ml against the MCF-7 breast cancer cells.
software, Supervision, Project administration,. All authors have
These results suggested the potential of combined (TP+AI) extract
read, edited and approved the final manuscript.
in the treatment of breast cancer.

Declaration of Competing Interest

4. Conclusion

Authors declare no conflict of interest associated with this arti-

The present study focuses on the synergic combination of phy-
cle.
tochemicals where the plant extracts as crude source is found
to have an efficient healing effects. GC–MS analysis showed the
presence of a novel phytochemical 2-(imidazo[1,2-a]pyridin-2- Acknowledgments
yl)phenol that may have occurred due to synergism of the extracts.
The MTT Assay was performed on human breast cancer cells to test Authors are thankful to TRI Biotech. Ltd., Tiruchirappalli, India,
the extent of inhibition against the cancer cells, obtaining a IC50 which was founded with a noble vision to help and develop rural
value of tested sample: 216.45 μg/ml implies that the combined students with scientific advancement in the field of life sciences.

7
A. Sophia, Md. Faiyazuddin, P. Alam et al. Journal of Molecular Structure 1250 (2022) 131678

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