Hindawi

Scientifica
Volume 2023, Article ID 5967638, 6 pages
https://doi.org/10.1155/2023/5967638

Research Article
Quantitative and Qualitative Phytochemical Analysis of
Manilkara zapota (Sapodilla) Extract and Its Antibacterial
Activity on Some Gram-Positive and Gram-Negative Bacteria

Shahla Hashemi Shahraki ,1 Fereshteh Mohamadhasani Javar ,2 and Mehdi Rahimi 3

1
Biology Department, Faculty of Science, University of Sistan and Baluchestan, Zahedan, Iran
2
Department of Biology, Faculty of Science, Payame Noor University (PNU), Tehran, Iran
3
Department of Biotechnology, Institute of Science and High Technology and Environmental Sciences,
Graduate University of Advanced Technology, Kerman, Iran

Correspondence should be addressed to Shahla Hashemi Shahraki; shahlahashemi15@science.usb.ac.ir and Mehdi Rahimi;
mehdi83ra@yahoo.com

Received 5 September 2023; Revised 20 November 2023; Accepted 19 December 2023; Published 26 December 2023

Academic Editor: Vikram Dalal

Copyright © 2023 Shahla Hashemi Shahraki et al. Tis is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
A molecule’s antibacterial and antiviral action is exclusively linked to substances that selectively eradicate bacteria and viruses or
inhibit their growth without signifcantly damaging adjacent tissues. Te purpose of this research is to evaluate quantitative and
qualitative phytochemical analysis and the antibacterial efects of Manilkara zapota fruit extract on some Gram-positive
(Staphylococcus aureus, Enterococcus faecalis, Micrococcus luteus, Bacillus cereus, and Listeria monocytogenes) and Gram-
negative (Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae) bacteria in laboratory conditions. Qualitative
chemical screening was used to identify diferent classes of active chemical compounds, and quantitative analysis of the chemical
composition of the plant was used to measure the contents of favonoid, total phenol, anthocyanin, and antioxidant activity.
Antibacterial efects of Manilkara zapota ethanol extract were determined by disk difusion methods, minimum inhibitory
concentration (MIC), and minimum bactericidal concentration (MBC). Qualitative chemical screening revealed the presence of
favonoids, tannins, quinones, terpenoids, and glycosides while the presence of saponins was not observed. Te bacterial inhibition
zones against Listeria monocytogenes, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus, Micrococcus luteus,
Escherichia coli, Klebsiella pneumoniae, and Bacillus cereus are 15.44 ± 0.33, 12.23 ± 0.11, 8.85 ± 0.2, 14.22 ± 0.33, 15 ± 0.44,
9.33 ± 0.13, 10.33 ± 0.36 and 14.55 ± 0.45 mm, respectively. MIC and MBC of the extract in Gram-positive bacteria were 25 and 50,
and in Gram-negative bacteria were 50 and 100 mg/ml, respectively. Te fndings imply that Manilkara zapota extract includes
a good amount of plant compounds and can be a signifcant source for a variety of uses, including antibacterial.

1. Introduction compounds with newer and stronger formulations. Another

problem with the use of chemical drugs is the increase in
In recent years, there has been a great interest in antibacterial their side efects, which lead to diseases that can be more
herbal medicines, which can be attributed to the resistance of dangerous than the original disease [2]. Plants are consid-
pathogens and the increasing popularity of traditional ered sources of various chemical substances, efective sub-
medicine [1]. On the other hand, excessive and unbalanced stitutes for synthetic antibacterial agents with minimal side
use of chemical compounds has led to resistance to bacteria efects [3]. Plants are a source of potential and useful
and other microorganisms. Terefore, the efect of drugs has chemicals such as phenols, phenolic derivatives (quinones,
become weak and neutral and has led to an increase in the favones, favonoids, favonols, tannins, coumarins, etc.),
amount of drug consumption and a tendency to use terpenoids, essential oils, alkaloids, lectin, and polypeptides
2 Scientifca

[4]. Indeed, plants use a vast and largely unknown reservoir 2.2. Quantitative and Qualitative Phytochemical Analysis.
of substances to defend against microorganisms, insects, and Qualitative chemical screening was used to identify diferent
herbivores. According to the approach for the use of classes of active chemical compounds such as saponins,
medicines and herbal products, the study of the medicinal favonoids, tannins, quinones, terpenoids, and glycosides
properties of plants is of particular importance. [11]. To detect saponins, 1 ml of extract was added to 1 ml of
Evidence to support the therapeutic claims of plants distilled water and shaken vigorously. Te formation of foam
against various diseases can be discovered by identifying and confrms the presence of saponin. To detect favonoids, a few
evaluating plants and their plant compounds. Advanced drops of dilute sodium hydroxide (NaOH) solution were
methods like high-performance liquid chromatography added to 1 ml of the extract, whereby an intense yellow color
(HPLC) [5], gas chromatography (GC) [6], and thin layer appeared. If the solution becomes colorless after adding
chromatography (TLC) [7] are very useful for both quali- a few drops of dilute hydrochloric acid (HCl), this is a sign of
tative and quantitative detection of phytoconstituents. the presence of favonoids. For tannin, 1 ml of extract was
However, when these techniques are unavailable or in- mixed with 5% FeCl3. Te color of the black-green sediment
accessible, conventional phytochemical tests that are cost- confrms the presence of tannins. To reveal the presence of
efective, simple, and require few resources are often a good glycoside, 1 ml of the extract was added to sulfuric acid
alternative for initial phytochemical screening. Based on the (H2SO4) and glacial acetic acid (2 ml) containing a few drops
precipitation reaction, foamy appearance, and color change, of iron chloride. A purple ring may form below the brown
the qualitative analysis of the chemical compounds of the ring, indicating the presence of a glycoside. To test quinones,
plant is done [7]. 0.5 ml of concentrated hydrochloride was added to 1 ml of
Te Manilkara zapota plant belonging to the Sapotaceae extract. Te presence of quinones is indicated by the pro-
family has many medicinal efects. Diferent parts of the duction of a yellow precipitate. To identify terpenoids, 1 ml
Manilkara zapota tree are used in traditional medicine [8]. of extract was mixed with 5 ml of concentrated H2SO4 and
Te ability to purge the digestive system as well as diuretic 2 ml of chloroform in a mixture. Reddish-brown color in-
and tonic is the characteristic of the seeds [9]. Te tree’s bark dicates the presence of terpenoids. Quantitative analysis of
has antidiarrheal, astringent, and antibiotic efects. Te fruit the chemical composition of the Manilkara zapota fruit was
is also used for antidiarrhea and to treat pulmonary diseases. used to measure the content of favonoid, total phenol,
Te leaves are used to treat cough, cold, and diarrhea. Its anthocyanin, and antioxidant activity. Te basis of quanti-
seeds and leaves show signifcant antibacterial activity. Abdel tative photometric measurement of the absorption process is
Monem et al. reported that Manilkara zapota leaves have according to the Beer–Lambert law. UV-visible spectroscopy
antihyperglycemic and hypocholesterolemic activities. Tey can be used to estimate the favonoid content. About 0.025 g
also showed that aqueous and alcoholic leaf extracts have of powder is ground with ethyl alcohol : acetic acid (99 :1, v : v).
antioxidant activity [10]. After centrifugation and placing in a water bath at 80°C, the
Since medicinal plants are natural sources of chemicals, absorbance of the sample was read in a UV-visible spec-
in this research, the antibacterial efects of Manilkara zapota trophotometer at wavelengths of 270, 300, and 330 nm [12].
extract on some bacteria (Listeria monocytogenes, Entero- To determine the anthocyanin content, 0.001 g of fruit
coccus faecalis, Pseudomonas aeruginosa, Staphylococcus powder was ground with 10 ml of acidic methanol and
aureus, Micrococcus luteus, Escherichia coli, Klebsiella placed in the dark for 24 hour. After centrifugation, the
pneumoniae, and Bacillus cereus) in laboratory conditions absorbance was read at a wavelength of 550 nm [13]. An-
were investigated. tioxidant potential was evaluated by the DPPH (2,2-
diphenyl-1-picrylhydrazyl) method by Ishaque et al.
2. Materials and Methods Te absorbance was measured at 517 nm to evaluate the
amount of residual DPPH [14]. Total phenol content was
2.1. Experimental Research. Tis study complied with rele- estimated by the Folin-reagent method. Te absorbance was
vant institutional, national, and international guidelines and measured at a wavelength of 725 nm by using a UV-vis
legislation of Iran. In this research, Manilkara zapota fruit spectrophotometer [15].
was collected from Chabahar Gardens in Zahedan City. To
prepare the extract, the fruits were frst dried in the shade
and then powdered. Te resulting powder was extracted by 2.3. Antibacterial Activity. Bacterial strains of Listeria
the maceration method. During this process, 100 g of the monocytogenes, Enterococcus faecalis, Pseudomonas aerugi-
powder was mixed with an organic solvent of 80% ethanol nosa, Staphylococcus aureus, Micrococcus luteus, Escherichia
and placed in a shaker at a temperature of 25°C for 24 hour. coli, Klebsiella pneumoniae, and Bacillus cereus were ob-
To separate the large parts, the content was passed through tained from the Kerman University of Medical Sciences. Te
the Whatman No. 1 flter paper. After these steps, the reference culture of the mentioned microorganisms was kept
concentrated solution was placed in a 40°C incubator to in the refrigerator, and the culture was renewed every month
obtain a dry powder from the extract. Te obtained powder in nutrient agar. To validate the antibacterial efect of
was stored in a dark glass container in the refrigerator until Manilkara zapota plant extract, the methods of difusion in
use. Te obtained powder was used for quantitative and agar with the help of disk (disc difusion), minimum in-
qualitative phytochemical analysis and antibacterial hibitory concentration (MIC), and minimum bactericidal
activity. concentration (MBC) were used. At frst, 200 mg of powder
Scientifca 3

was dissolved in 1 ml of ethanol (80%) to prepare the extract Table 1: Qualitative phytochemical composition of Manilkara
solution. In the solution of the ethanolic extract of the tested zapota.
plant, several blank disks with a diameter of 6 mm were Phytochemicals +/−
inserted and remained in the solution for one hour. By sterile Saponins −
tweezers and under aseptic conditions, the discs were re- Flavonoids +
moved from the extract and transferred to a sterile plate to Tannins +
dry completely [16]. Discs soaked in consumable solvent Quinones +
were considered as test controls. To perform the disc dif- Terpenoids +
fusion test, one loop of the reference culture of each bac- Glycosides +
terium was transferred to the medium of nutrient broth and + indicates presence; − indicates absence.
incubated for 24 hour. Ten, the resulting antibacterial
suspension was diluted to reach a turbidity equal to half
Table 2: Quantitative analysis of total phenol, anthocyanin, fa-
McFarland. In the next step, with the help of a sterile swap,
vonoid contents, and antioxidant activity in extracts of Manilkara
a sample was taken from this suspension and cultured in zapota
Mueller–Hinton agar medium. Finally, with the help of
sterile tweezers, discs were placed on the medium at a dis- Parameter Amount
tance of 15 mm from the edge of the plate and the plate was Total phenol contents (mg/g GA) 0.54 ± 0.02
incubated for 24 hour at 37°C. Te solvent was considered as Anthocyanin contents (mmol/g DW) 40 ± 0.98
negative control and chloramphenicol antibiotic as the Flavonoid contents 270 (%) 5 ± 0.03
positive control. Te diameter of the inhibition zone of the Flavonoid contents 300 (%) 94 ± 0.5
Flavonoid contents 330 (%) 34 ± 0.44
extract was measured by using a millimeter ruler [17]. Te
Antioxidant activity (%) 59 ± 0.13
minimum inhibitory concentration (MIC) and minimum
bactericidal concentration (MBC) were determined by the
broth dilution method. At frst, diferent dilutions of the Te results of the efect of Manilkara zapota extract on
studied fruit extracts were prepared. Te mentioned di- some bacteria (Listeria monocytogenes, Enterococcus faecalis,
lutions included 10 dilutions of 0.5 to 200 mg/ml, which Pseudomonas aeruginosa, Staphylococcus aureus, Micrococcus
were prepared by the sequential dilution method and from luteus, Escherichia coli, Klebsiella pneumoniae, and Bacillus
the stock solution of the extracts. To determine MIC, 1 ml of cereus) are shown in Table 3. Te results show that Manilkara
24 hour bacterial suspension grown in nutrient broth and zapota extract prevented the growth of all tested bacteria. Tis
brought to half McFarland turbidity was added to each inhibitory efect on inhibiting the growth of Staphylococcus
dilution. Te tubes were incubated for 24 hours at a tem- aureus, Micrococcus luteus, Listeria monocytogenes, and Ba-
perature of 37°C. After this period, the lowest concentration cillus cereus bacteria was the highest. Te least inhibitory efect
in which bacterial growth was inhibited was selected as MIC. of Manilkara zapota extract was on Klebsiella pneumoniae,
To calculate the minimum bactericidal concentration (MBC) Pseudomonas aeruginosa, and Escherichia coli.
of the extract from the MIC test treatment tubes, 100 μl was Te results of the minimum inhibitory concentration
placed on the Mueller–Hinton agar medium without the (MIC) and minimum bactericidal concentration (MBC) of
extract and spread on the surface of the plate with the help of Manilkara zapota extract are given in Table 4. Tese results
a sterile glass spreader. Te plate with the lowest concen- show that the concentration of 25 mg/ml of the extract has
tration of infected extract in which bacteria did not grow an inhibitory efect on the bacteria Staphylococcus aureus,
after a 24 hour incubation period at 37°C was determined as Enterococcus faecalis, Micrococcus luteus, Listeria mono-
MBC [18, 19]. Te data obtained from this study were cytogenes, and Bacillus cereus, while the concentration of
statistically analyzed using SPSS software and one-way 50 mg/ml of the extract has an inhibitory efect on the
ANOVA statistical test. bacteria Pseudomonas aeruginosa, Klebsiella pneumonia, and
Escherichia coli.
3. Results Te results of minimum bactericidal concentration
(MBC) of Manilkara zapota fruit extract on Staphylococcus
Preliminary phytochemical screening of various bioactive aureus, Enterococcus faecalis, Micrococcus luteus, Klebsiella
compounds (tannins, saponins, favonoids, quinones, ter- pneumoniae, Bacillus cereus, Listeria monocytogenes, and
penoids, and glycosides) was performed in the Manilkara Pseudomonas aeruginosa were 50, 50, 50, 100, 50, 50, and
zapota extract. Among them, the presence of tannins, fa- 100 mg/ml respectively. Tese results indicate that among
vonoids, quinones, terpenoids, and glycosides was observed, the tested bacteria, there was the least sensitivity in the case
while the presence of saponins was not observed (Table 1). of Klebsiella pneumoniae, Pseudomonas aeruginosa, and
Table 2 shows the amount of total phenol, anthocyanin, Escherichia coli.
favonoid content, and antioxidant activity of Manilkara
zapota fruit extract. Te data show that the amounts of total 4. Discussion
phenol, anthocyanin, favonoid 270, 300, 330 content, and
antioxidant activity of Manilkara zapota extract were In this study, it was found that the extract of Manilkara
0/54 ± 0.02, 40 ± 0.98, 5 ± 0.03, 94 ± 0.5, 34 ± 0.44, and zapota fruit showed a good antibacterial efect. Various
59 ± 0.13, respectively. chemical compounds present in medicinal plants can lead to
4 Scientifca

Table 3: Size of inhibition zone diameter of Manilkara zapota extracts against some bacteria.
Size of inhibition
Bacteria Control (−) Control (+)
zone diameter (mm)
Escherichia coli 9.33 ± 0.13 5 ± 0.53 33 ± 0.55
Staphylococcus aureus 14.22 ± 0.33 − 36 ± 0.71
Enterococcus faecalis 12.23 ± 0.11 6 ± 0.03 36 ± 0.88
Micrococcus luteus 15 ± 0.44 6 ± 0.66 21 ± 0.32
Klebsiella pneumoniae 10.33 ± 0.36 − 23 ± 0.44
Bacillus cereus 14.55 ± 0.45 5 ± 0.45 26 ± 0.55
Listeria monocytogenes 15.44 ± 0.33 − 26 ± 0.33
Pseudomonas aeruginosa 8.85 ± 0.2 5 ± 0.23 26 ± 0.23
− indicates the absence of antimicrobial activity in the extract.

Table 4: Minimum inhibitory concentration and minimum bactericidal concentration of Manilkara zapota extracts on bacteria.
Minimum inhibitory concentration Minimum bactericidal concentration
Bacteria
(MIC) (mg/ml) (MBC) (mg/ml)
Escherichia coli 50 100
Staphylococcus aureus 25 50
Enterococcus faecalis 25 50
Micrococcus luteus 25 50
Klebsiella pneumoniae 50 100
Bacillus cereus 25 50
Listeria monocytogenes 25 50
Pseudomonas aeruginosa 50 100

the antibacterial activity of plant extracts. Phytochemical essential oils are hydrophobic, it can be concluded that these
analysis of Manilkara zapota extract showed the presence of substances cannot penetrate and access the active points
tannins, favonoids, quinones, terpenoids, and glycoside inside Gram-negative bacteria. For this reason, Gram-
compounds. Te antibacterial activity of Manilkara zapota negative bacteria usually show more resistance to plant
fruit extract may be due to the presence of the above plant compounds compared to Gram-positive bacteria. Bangar
chemicals. Tannins and terpenoids are plant metabolites et al. reported that chemical compounds in Manilkara
known for their antimicrobial activity [20]. zapota, such as anthocyanins, alkaloids, favonoids, poly-
Similar to our results, Tiago et al. reported a complete phenolic compounds, tannins, and triterpenoids, play an
inhibition of the growth of Staphylococcus aureus, Pseudo- important role in antibacterial activity [26]. Tannins are
monas aeruginosa, and Escherichia coli caused by the water-soluble chemical compounds that are widely found in
aqueous extract of Manilkara zapota leaves [21]. Bhargavi the plant kingdom and exhibit strong antibacterial prop-
et al. reported that methanolic and aqueous extracts of erties. Various mechanisms that exist in tannins to inhibit
Manilkara zapota roots showed antibacterial activity against antibacterial growth include deprivation of iron through
Staphylococcus aureus and Escherichia coli [22]. iron chelation, disruption of metabolic activities of bacteria
Acetone and methanol extracts of Manilkara zapota through inhibition of oxidative phosphorylation, and dep-
seeds were active against Gram-positive and Gram-negative rivation of essential compounds for bacterial growth.
organisms. Te MIC values of potent extracts against sen- Polyphenol compounds also have strong antibacterial ac-
sitive organisms were between 53 and 380 μg/ml [23]. In this tivity [27]. Inhibition of RNA and DNA, cytoplasmic
study, concentrations of about 12.5 mg/ml of Manilkara membrane depolarization, and inhibition of macromolecular
zapota fruit extract prevent the growth of Gram-positive synthesis are some of the underlying antibacterial mecha-
bacteria, while a higher concentration is needed to afect nisms of polyphenol compounds. Terefore, it seems that the
Gram-negative bacteria. Terefore, Gram-positive bacteria compounds in Manilkara zapota fruit cause the antibacterial
are more sensitive than Gram-negative bacteria. Tis dif- property of Manilkara zapota. In general, according to the
ference in the response of Gram-negative and Gram-positive above, the authors suggest that Manilkara zapota fruit extract
bacteria can be related to the diference in the structure of can be used in the pharmaceutical industry as well as in the
the outer membrane [24]. Gram-positive bacteria do not food industry as a biological preservative. More research is
have an outer membrane compared to Gram-negative needed to detail their mechanism of action.
bacteria. Te corresponding outer membrane acts as a bar-
rier with limited permeability that prevents the penetration 5. Conclusions
of some antibiotic components and drugs into the cell [25].
Te presence of polysaccharides in the outer membrane Manilkara zapota fruits were once widely consumed, but
prevents the passage of large and hydrophobic molecules. today, not much attention is paid to them. Terefore, it is
Since most of the efective compounds in the extracts and urgently necessary to raise public awareness of this plant’s
Scientifca 5

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