Machine Translated by Google

TYPE Review
PUBLISHED 19 December 2022 DOI
10.3389/fsufs.2022.1043823

Evaluation of the antimicrobial eects of Capsicum, Nigella

OPEN ACCESS
sativa, Musa paradisiaca L., and Citrus limetta: A
EDITED BY
Rabia Siddique,
review
Government College University,
Faisalabad, Pakistan

REVIEWED BY
Ayyaz Ahmad,
Muhammad Nawaz Sharif University of
Engineering & Technology, Pakistan Sonia Abid Bhatti1†, Muhammad Hammad Hussain2† Muhammad Zubair Mohsin2 ,

Muhammad Adil Farooq,

Waqas Qamar Zaman1*, Meijin Guo2 Muhammad
, Ali Mohsin2 ,
Khwaja Fareed University of
Engineering and Information
Waheed Iqbal3 , Shahida Anusha Siddiqui4,5 , Salam A. ,
Technology (KFUEIT), Pakistan
Akhtar Ali, Ibrahim6*, Saeed Ur-Rehman7 and Sameh A. Korma8,9
The University of Melbourne, Australia
Murtaza Ali,
Foshan University, China 1
Institute of Environmental Sciences and Engineering, School of Civil and Environmental Engineering,
*CORRESPONDENCE National University of Sciences and Technology (NUST), Islamabad, Pakistan, 2State Key Laboratory of Bioreactor
Waqas Qamar Zaman Engineering, East China University of Science and Technology, Shanghai, China, 3School of Food and Biological
[email protected] Engineering, Jiangsu University, Zhenjiang, China, 4Technical University of Munich Campus Straubing for Biotechnology
Salam A. Ibrahim
and Sustainability, Straubing, Germany, 5German Institute of Food Technologies (DIL e.V.), Quakenbrück, Germany,
[email protected] 6Food Microbiology and Biotechnology Laboratory, Food and Nutritional Sciences Program, North Carolina A&T
State University, Greensboro, NC, United States, 7School of Biochemistry and Biotechnology, University of the
†These authors have contributed equally Punjab, Lahore, Pakistan, 8Department of Food Science, Faculty of Agriculture, Zagazig University, Zagazig,
to this work Egypt, 9School of Food Science and Engineering, South China University of Technology, Guangzhou, China

SPECIALTY SECTION
This article was submitted to
Sustainable Food Processing, a
section of the journal
Frontiers in Sustainable Food Systems The extensive use of antibiotics and vaccines against microbial infections can result in
RECEIVED 14 September 2022 long-term negative eects on humans and the environment.
ACCEPTED 10 November 2022
However, there are a number of plants that have antimicrobial eects against various
PUBLISHED 19 December 2022
disease-causing microbes such as bacteria, viruses, and fungi without negative side
CITATION
Bhatti SA, Hussain MH, Mohsin MZ, Mohsin eects or harm to the environment. In this regard, four particular plants- Capsicum,
A, Zaman WQ, Guo M, Iqbal MW, Nigella sativa, Musa paradisiaca L., and Citrus limetta have been widely considered due
Siddiqui SA, Ibrahim SA, Ur-Rehman S
to their excellent antimicrobial eect and ample availability. In this review, we discuss
and Korma SA (2022)
Evaluation of the antimicrobial eects of their antimicrobial eects due to the presence of thymoquinone, p-cymene, pinene,
Capsicum, Nigella sativa, Musa paradisiaca alkaloids, limonene, camphene, and melanin. These antimicrobial compounds disrupt
L., and Citrus limetta: A review.
the cell membrane of microbes, inhibit cellular division, and form biofilm in bacterial
Front. Sustain. Food Syst. 6:1043823. doi: species, eventually reducing the number of microbes. Extraction of these compounds
10.3389/fsufs.2022.1043823
from the respective plants is carried out by dierent methods such as soxhlet, hydro-
COPYRIGHT
distillation, liquid-liquid extraction (LLE), pressurized liquid extraction (PLE), solid-phase
© 2022 Bhatti, Hussain, Mohsin, Mohsin,
Zaman, Guo, Iqbal, Siddiqui, Ibrahim, Ur-
extraction (SPE), supercritical fluid extraction (SFE), pulsed electric field (PEF),
Rehman and Korma. This is an open-access microwave-assisted extraction (MAE), enzyme-assisted extraction (EAE), ultrasound-
article distributed under the Creative
assisted extraction (UAE), and high-voltage electrical discharge. Suitable selection of
Commons Attribution License (CCBY).
the extraction technique highly depends upon the associated advantages and
The use, distribution or reproduction in other disadvantages.
forums is permitted, provided the original
author(s) and the copyright owner(s) are credited
and that the original publication in this In order to aid future study in this field, this review paper summarizes the advantages
journal is cited, in accordance with accepted
and disadvantages of each of these approaches. Additionally, the discussion covers
academic practice. No use, distribution or
reproduction is permitted which does not how antimicrobial agents destroy harmful bacteria. Thus,
comply with these terms.

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Bhatti et al. 10.3389/fsufs.2022.1043823

this review oers in-depth knowledge to researchers on the antibacterial properties of Capsicum,
Nigella sativa, Musa paradisiaca L. peels, and Citrus limetta.

KEYWORDS

antimicrobial agent, antimicrobial mechanism, solid waste management, extraction

techniques, Capsicum, Nigella sativa, Musa paradisiaca L., Citrus limetta

Introduction whether in underdeveloped, developing, or developed regions of the

world, these natural goods are widely accessible.
A global public health crisis has been caused by the rise in Additionally, these items are cost-effective in their use because they
resistant bacteria over the past few decades (Dhingra et al., 2020; Hu don’t need to be prepared using a time-consuming procedure in order
et al., 2020; Ibrahim et al., 2021). Although antibiotics are used to treat to fight infectious bacteria (Ncube et al., 2008).
illnesses by killing bacteria or limiting their growth, they can also have
several negative effects (Gyawali and Ibrahim, 2014). For example, Nigella sativa, Capsicum, peels of Musa paradisiaca L., and Citrus
because some disease-causing bacteria have acquired resistance to limetta contain effective antimicrobial agents against various infectious
particular antibiotics as a result of their widespread and sustained microbes such as the following: pathogenic bacteria (gram-negative
usage, these treatments are no longer as effective (Namita and Mukesh, and gram-positive), including S. aureus, Candida albicans, E. coli,
2012). In this regard, antimicrobial agents can eliminate resistant Gram-positive cocci, Microsporum canis, Trichophyton mentagrophytes,
pathogenic microbes without imposing any known side effects. Trichophyton interdigitale, four species of Trichophyton rubrum,
Staphylococcus epidermidis, Micrococcus luteus, Listeria
Antimicrobial agents disrupt the cellular structure of microbes, thus monocytogene, Bacillus cereus, Vibrio parahaemolyticus, Pseudo.
inhibiting various infectious diseases (Peterson, 2008) and are used to aeruginosa, Salmonella enteritidis, Sal. Typhimurium, and Shigella
treat humans with diseases that are primarily due to non-human flexneri (Hanafy and Hatem, 1991; Hosseinzadeh et al., 2007; Hannan
sources (Organization, 2019). According to WHO (world health et al., 2008; Halawani, 2009; Chaieb et al., 2011; Alshareef, 2019). The
organization), the overall consumption of antimicrobial agents is in microbes listed above are known to cause the following diseases:

between 4.4 and 64.4 Defined Daily Doses (DDD) per 1,000 persons per pneumonia, bloodstream infections, wound or surgical site infections,

day. As per WHO data, the national estimates of antimicrobial methicillin-resistant Staphylococcus aureus (MRSA), urinary tract
consumption in 65 countries and areas: six from the Region of America, infection, “traveler’s diarrhea,” immunologic infections, diarrhea
four from the African Region, three from the Eastern Mediterranean (sometimes bloody), fever, stomach cramps (Awaisheh and Ibrahim,
Region, 46 from the European Region: and six from the Western Pacific 2009; Awaisheh et al., 2013; Blinov et al., 2022) and various others as
Region are presented in Figure 1 (Organization, 2018). reported in earlier conducted studies (Edelson and Unanue, 2000; Otto,
2009; Doernberg et al., 2017; Liu and Ji, 2018). These diseases can be
prevented by using Nigella sativa, Capsicum, peels of Musa paradisiaca
According to WHO, residents of these low-income countries are L., and Citrus limetta as antimicrobial agents. We have chosen to focus
more likely to die of communicable and infectious diseases such as on these particular natural resources due to their global availability.
malaria, tuberculosis, diarrhea, HIV/AIDS, etc. (Philip, 2017). Therefore,
suitable preventive measures against these infectious and parasitic
diseases should be adopted. These measures may include self-
cleanliness, proper sanitation, safe food preparation and handling, and Worldwide, Capsicum grows on 1.93 million hectares of surface
the use of antimicrobial agents to immunize the body against infectious area. China is the world’s largest producer of Capsicum at ÿ16 million
and parasitic diseases (Mehta et al., 2014). Nature also plays its role in tons annually. Mexico is the second largest producer at ÿ2.3 million
fighting against disease-causing microbes (Ibrahim et al., 2011; Bor et tons, followed by Turkey and Indonesia with 2.2 and 1.8 million,
al., 2016). For example, many plants such as Nigella sativa, Capsicum, respectively (Penella and Calatayud, 2018). Nigella sativa is produced
Musa paradisiaca L., and Citrus limetta have attracted tremendous mainly by India on 9,000 hectares. It is also produced in Pakistan, Sri
public interest due to their efficient antimicrobial agents (Dutta et al., Lanka, Nepal, Bangladesh, Egypt, and Iraq (Huchchannanavar et al.,
2020b; El-Naggar et al., 2020; Ajijolakewu et al., 2021; Vanlalveni et al., 2019). The annual global production of Musa paradisiaca L. is ÿ41.265
2021; Vijayakumar et al., 2021). Furthermore, million tons per year with its primary producers being Brazil, China,
Ecuador, the Philippines, and India (Preedy and

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FIGURE 1

National estimates of antimicrobial consumption in 65 countries and areas (Organization, 2018).

Watson, 2019). Brazil is the largest Citrus limetta producing country Biological properties of plant
globally with 15,912 tons followed by China with 5,450, the United
States with 9,237 and the European Union (EU) with 5,999 thousand The history of plants and/or plant components’ utilization to flavor
metric tons (Spreen, 2010). Annually, the global production of Musa and conserve food, and to prevent oxidation and treat health problems
paradisiaca L. is around 41.3 million tons and a large fraction of it is is dated back to thousands of years ago. Most interestingly, the
wasted as the peel. Notably, just in Brazil ÿ780 million tons of Musa knowledge of their biological properties has been transmitted over the
paradisiaca L. -waste is produced annually (Martínez-Ruano et al., centuries among human societies.
2018). According to Prabhakar and Singh (2019), ÿ57% of processed Considering this fact, it has been stated that the phytochemicals are
Citrus limetta contributes to peel waste Usage of the endocarp of Musa the valuable source of bioactive substances with significant
paradisiaca L. and Citrus limetta creates solid waste problems due to antimicrobial and antioxidant properties (Silva and Fernandes, 2010;
the production of their peels, whereas Nigella sativa and Capsicum do Radha and Laxmipriya, 2015).
not create such a problem. In this review, we address the handling of
the huge solid waste problem produced by Musa paradisiaca L. and
Citrus limetta by utilizing their peels as an antimicrobial agent against Anti-microbial agents present in plants
infectious diseases.
It has long been believed that plants have therapeutic properties.
At present globally, a gradual increase in the wide spread of Since prehistory, people have used hundreds of thousands of local
infectious diseases is quite evident. Therefore, it is need of time to plants in infusions and poultices to treat various ailments. The desire
adopt suitable preventive measures, such as regular use of antimicrobial to find novel antimicrobials has

agents. Therefore, it is proposed in this review paper that Capsicum, been prompted by the rise of microbial strains that are resistant to
Nigella sativa, Musa paradisiaca L., and Citrus limetta on account of antibiotics and the recurrence of newer, deadlier illnesses. To find
their abundant availability and antimicrobial constituents should be possible pharmacological leads, the whole natural resource base is
regarded as potential raw materials for the economical commercial being quickly evaluated (Ref: Antimicrobial Agents from Plants Reshma
production of various antimicrobial agents (Parthiban et al., 2011; Reghu, Pramod Sahadevan & Shiburaj Sugathan + Plant Products as
Somda et al., 2011; Chitranshi et al., 2020). These natural materials are Antimicrobial Agents Marjorie Murphy Cowanÿ ). Many plants and/or
non-toxic, produce only organic food waste, and are easily available plant components, including Nigella sativa, Capsicum, peels of Musa
and completely safe for producing antimicrobial agents. Furthermore, Paradisiaca L., and Citrus limetta are ascribed to the presence of anti-
the current study also discusses the antimicrobial compounds present microbial agents, which will be discussed herein.
in Capsicum, Nigella sativa, peels of Musa paradisiaca L., and Citrus
limetta along with the methods employed for their extraction. Moreover,
we also explain the overall antimicrobial mechanism of antimicrobial
agents present in Capsicum, Nigella sativa, Musa paradisiaca L., and Antimicrobial agent in Nigella sativa It
Citrus limetta. A schematic illustration of the content in this contains more than 100 valuable elements. According
to science N. sativa contains 20–85% of protein, 38.20%
of fat, 7–94% of fiber, and 31.94% of total carbohydrates
review is presented in Figure 2. that have antimicrobial effects. Due to the presence of thymoquinone

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FIGURE 2

Schematic illustration of antimicrobial eects of Capsicum, Nigella sativa, Musa paradisiaca L and Citrus limetta. (A) Thymoquinone. (B) Melanin.
(C) P-cymene. (D) a-Pine. (E) b-Pinene. (F) Alkaloids. (G) Lemon size. (H) Camphene.

thymohydroquinone in N. sativa it can fight against infectious a concentration of 0.5 g/100 g. Secondary metabolites such
disease-causing microbes (Bakal et al., 2017). The amount of as flavonoids, tannins, phlobatannins, alkaloids, glycosides,
the most significant bioactive component, thymoquinone, and terpenoids (Imam and Akter, 2011) have been found to be
found in the volatile oil obtained from the seeds of N. sativa present in banana peel. Ighodaro (2012) evaluated the
using various extraction techniques can range from 1.06 to antibacterial effectiveness of banana peel extract against
8.8 mg/g of oil, respectively (Yimer et al., 2019). human pathogenic microorganisms and exhibited good
inhibitory activity against S. aureus, Escherichia coli, and Proteus mirabilis.
Although banana peel is regarded as waste, peel extracts can
Antimicrobial agent in Capsicum Capsaicin, be utilized to create antibiotics (Saleem and Saeed, 2020).
also known as 8-methyl-N-vanillyl-6- nonenamide, is a
compound found in Capsicum plants (chili peppers), which
are used for food and medicine. Due to its antibacterial and
anti-virulence properties, as well as its multiple pharmacological
and physiological benefits (pain alleviation, cancer prevention, Antimicrobial agent in Citrus limetta peel
and advantageous cardiovascular, and gastrointestinal Peel from Citrus limetta can be utilized to make solid
benefits; Marini et al., 2015), capsaicin has recently attracted waste management more efficient as well as to create long-
a lot of attention. The capsaicinoids (2.5 mg/g; Othman et al., lasting antibacterial products. Citrus peel has been discovered
2011) found in capsicum, which include more than 20 alkaloids to have thousands more phenolic compounds than citrus pulp
(Bakht et al., 2020), are of major medical significance (Dutta et al., 2020a), and these phenolic compounds (444.55–
worldwide. Since capsicum has potent antibacterial effects, it 502.54 mg/L; Buyukkurt et al., 2019) have antimicrobial
is beneficial to utilize it in common food items. properties. In a 2009 study, Kekuda et al. investigated the
antifungal efficacy of the peels of three distinct citrus fruits,
C. limetta, C. sinensis, and C. limon, against Aspergillus
Antimicrobial agent in Musa paradisiaca L. species, and found that C. limetta was best at preventing the
Ethanol (0.5 g/100 g; Gorgus et al., 2016), which has a growth of the fungus that was tested (Shabnam et al., 2013).
microbe-killing effect, is present in Musa paradisiaca L. at

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Plants and/or plant components derived anti-tumor agent (Nickavar et al., 2014). P-cymene has anti-nociceptive,
antimicrobial compounds anticancer, antidiabetic, antiviral, antiparasitic, anti-inflammatory,
antifungal, antioxidant, and antibacterial effects (Balahbib et al., 2021).

Thymoquinone, melanin, P-cymene, a-pinene, b-pinene, alkaloid,

limonene, and camphene are significantly present in Nigella sativa,
Capsicum, Musa Paradisiaca L., and Citrus limetta. Moreover, these
Pinene
plant-derived chemicals are known to act as antimicrobial agents with
numerous applications in the medical and food industries, which will Pinene is the natural compound found in citrus fruits. Pinene has
be discussed in the subsequent section. various potential benefits such as it has anti-inflammatory, antimicrobial,
antioxidant, and neuroprotective effects (Di Rauso Simeone et al.,
2020).
Pinene has anticoagulant, anti-inflammatory, anti-leishmania,
antimalarial, antimicrobial, antioxidant, antitumor, analgesic, and
Thymoquinone
antibiotic resistance modulation effects (Park et al., 2021).
Thymoquinone is a compound that has remarkable anti-sepsis
Pinene has the wound healing process by generating scars with
activity at a specific dose. Thymoquinone is the main component
effective tensile strength, accelerating wound closure, acting as an
present in Nigella sativa that is found to be effective against the Avian
adhesive of primary intention, and contributing to collagen deposition
influenza virus and coronavirus (Ulasli et al., 2014). Another study
(Salas-Oropeza et al., 2021).
found that Nigella sativa consumption makes it difficult for the
coronavirus to survive and replicate in the body (Ahmad et al., 2020).
Thymoquinone is reported as an inhibitor for the treatment of
coronavirus infections
Alkaloid
Alkaloid is a natural compound found in many plants.
by blocking the entry of virus into the cell (Xu et al., 2021).
The presence of alkaloids in plants protects them from the destruction
Studies reported that thymoquinone can serve as an anticancer, anti-
of various insects (Zhang et al., 2005). Indole alkaloids and isoquinoline
inflammatory, antioxidant and as an analgesic compound.
alkaloids are the primary classes of substances having antibacterial
Activator of transcription (JAK-STAT), janus kinase/signal transduction,
action among the numerous alkaloids, including pyridine alkaloids,
mitogen-activated protein kinase (MAPK), and nuclear factor kappa
indole alkaloids, steroidal alkaloids, and other alkaloids. Alkaloids
beta (NF-ÿÿ) signaling pathways can be inhibited by thymoquinone.
have antiviral, antibacterial, antifungal, cytotoxic, antioxidant,
Thymoquinone causes disruption in reactive oxygen species (ROS),
antifouling activities and anti-infective potential (Barghout et al., 2020;
pro-inflammatory cytokines, lipooxygenase (LOX) enzymes,
Diaz et al., 2020; Youssef et al., 2021).
cyclooxygenase (COX), myeloperoxidase, and elastase (Ali A. et al.,
2021).

Limonene
Melanin Limonene is a chemical found mostly in citrus fruits (Rodríguez et
Melanin is an efficient green agent that possesses anti-
al., 2011). Limonene has several health benefits and belongs to the
inflammatory, antioxidant, and antimicrobial activity with vast
group of terpenes. These chemicals have strong aromas which protect
application in the biomedical field (Silvestri et al., 2017; Avossa et al.,
plants from predators. Limonene also has anti-inflammatory and
2021). Antioxidants protect human bodies from free radicals, which
antioxidant effects (Miguel, 2010). Limonene can be used as a greener
may otherwise result in causing various diseases such as cancer and
and safer alternative to market-available antimicrobial agents (Ibáñez
coronary heart disease (Hamid et al., 2010).
et al., 2020).
Additionally, it is promising as an antibacterial agent against both
gram-positive and gram-negative pathogens (Liu et al., 2014).

Camphene
Camphene is a monoterpene found in many plants (Russo and
Marcu, 2017). Terpenes are those chemicals that have strong aromas
P-cymene P- which protect plants from predators (Miguel, 2010). Camphene can
cymene is a natural compound found in many plants. fight against various deadly pathogens, such as Enterococcus spp.
It is used as an important agent in many drugs. It is widely present in and S. aureus, methicillin-resistant Staphylococcus aureus (MRSA)
more than 200 types of foods, including grape fruit, orange juice, and have an antifungal effect against Trichophyton mentagrophytes
mandarin, raspberries, carrots, butter, oregano, nutmeg, and almost and several species of Paracoccidioides spp., Camphene have activity
every spice. P-cymene can serve as an analgesic, anti-inflammatory, against Mycobacterium tuberculosis and vancomycin resistance
antioxidant, and

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Enterococcus spp. Isolates (de Freitas et al., 2020). Figure 3 alkaloids. In Mayer’s test, the appearance of a yellow color
shows the chemical structures of thymoquinone, melanin, P- on using Mayer reagent indicates the presence of alkaloids.
cymene, a-pinene, b-pinene, alkaloid, limonene, and camphene. Whereas, in Wagner’s test a brown-reddish brown is
Table 1 shows the major antimicrobial compounds developed upon using Wagner’s reagent that indicates the
present in Nigella sativa and Capsicum along with the presence of alkaloids (Trease and Evans, 1989; Wallis, 1989;
adopted methodology and important findings reported in Pandey and Tripathi, 2014; Beena et al., 2016; Dhawan and
previous research studies. Whereas, Table 2 shows the major Gupta, 2017; Nortjie et al., 2022).
antimicrobial compounds present in Musa paradisiaca L.
Identification of flavonoids
and Citrus limetta along with the adopted methodology and
For the identification of flavonoids lead acetate and
important findings reported in previous research studies.
sulphuric acid test are usually performed. In Lead acetate
test; the extracts are mixed with a few drops of a lead acetate solution.
Extraction of antimicrobial The presence of flavonoids is indicated by a precipitate that
compounds is yellow in color. In the sulfuric acid test; after a couple of
drops of sulfuric acid are added to the extracts, an orange
The initial three basic processes in the extraction of color appears, indicating the presence of flavonoids (Trease
antimicrobial compounds reported in the literature are (1) and Evans, 1989; Wallis, 1989; Pandey and Tripathi, 2014;
pretreatment, (2) extraction, and (3) purification. Beena et al., 2016; Dhawan and Gupta, 2017; Nortjie et al., 2022).

Identification of steroids

Pretreatment Likewise, on adding a few drops of acetic anhydride to

the extracts causes samples to turn violet then blue and
The first step in the extraction of antimicrobial agents is lastly to green, which is a sign of the presence of steroids
the preparation of materials employing methods such as (Trease and Evans, 1989; Wallis, 1989; Pandey and Tripathi,
washing, grinding, dewatering, heating and microfiltration. 2014; Beena et al., 2016; Dhawan and Gupta, 2017; Nortjie et al., 2022).
Pre-treatment consists of three additional steps: physical
Identification of terpenoids
treatment, chemical treatment, and biological treatment.
The identification of terpenoids is usually done by
Salkowski’s test. This test is conducted by adding 3 mL of
Physical treatment concentrated sulfuric acid to an extract of 5 mg of the
Physical treatment involves pre-washing, drying, grinding targeted plant component. Eventually, the presence of
to get a homogeneous sample, heating, steaming, freeze- terpenoids is indicated by the appearance of reddish-brown
drying, and often boosting the kinetics of analytical extraction color (Trease and Evans, 1989; Wallis, 1989; Pandey and
as well as increasing the contact of the sample surface with Tripathi, 2014; Beena et al., 2016; Dhawan and Gupta, 2017; Nortjie et al., 202
the solvent system. When making the extract from plant
Identification of anthraquinones
samples, proper precautions must be taken to ensure that
For the identification of anthraquinones, Bontrager’s test
any potential active ingredients are not lost, altered, or
destroyed (Fabricant and Farnsworth, 2001; Cos et al., 2006; Sasidharan et al.,performed.
is usually 2011). The Bontrager’s test involves boiling 5
mg of the extract in a water bath for a short period of time
with 10% HCl. After filtering, it is allowed to cool followed by
Chemical treatment the addition of CHCl3 in an equal amount to the filtrate. The
Chemical treatment involves the use of salts, acids, bases, liquid is then heated before a few drops of 10% NH3 are added.
and solvents. The chemical extraction method relies on the kinds Anthraquinones are present when a pink color forms,
of functional groups each component in the mixture possesses. indicating their presence (Trease and Evans, 1989; Wallis,
Chemical processes can separate or purify materials when 1989; Pandey and Tripathi, 2014; Beena et al., 2016; Dhawan
the right reagents are used. There are several chemical tests and Gupta, 2017; Nortjie et al., 2022).
that can be used to identify the presence of alkaloids,
Identification of phenols
flavonoids, tannins, saponins, flavones, sterols, and terpenes,
For the identification of phenols, ferric chloride test and
which have been briefly discussed in the later part.
lead acetate test are usually performed. In the Ferric chloride
Identification of alkaloids test; 10 mL extract is mixed with a few drops of ferric
The extracts are individually filtered, dissolved in diluted chloride. The presence of phenol is indicated by a bluish-
hydrochloric acid, and analyzed for the presence of alkaloids. black color. In the Lead acetate test; 10 mg of extract is
Typically, Mayers and Wagner’s tests are used to identify added with a few drops of lead acetate solution. The presence of phenol is in

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FIGURE 3

Chemical structures of (A) thymoquinone, (B) melanin, (C) P-cymene, (D) a-pinene, (E) b-pinene, (F) alkaloid, (G) limonene, and (H) camphene.

by a yellow color (Trease and Evans, 1989; Wallis, 1989; Al-Owaisi et al., incubated overnight (Digrak et al., 1999; Parekh et al., 2005; Klancnik et
ÿ ´ ´

2014; Pandey and Tripathi, 2014; Beena et al., 2016; Dhawan and Gupta, al., 2010 ; Novakovic and Jankovi ). c, 2011

2017; Nortjie et al., 2022).

Broth and agar dilution Agar
dilution implies adding various quantities of the antimicrobial drug

Biological treatment to a nutrient-rich agar medium before applying a set number of cells to

Biological treatment uses bacteria and fungi to extract antimicrobial the agar plate’s surface. In broth dilution, Bacteria are introduced into a

agents. The antibacterial activity of an extract or a pure chemical can be liquid growth medium while being exposed to various concentrations of

assessed or screened using a number of biological laboratory techniques. an antimicrobial agent, which is frequently assessed in 96-well microtiter

Disk-diffusion and broth or agar dilution procedures are the most well- plate format. After 16–20 h of incubation, growth is evaluated and the MIC

known and fundamental techniques (Magaldi et al., 2004; Pfaller et al., value is recorded. This 3-day regimen is only applicable to aerobic

2004; Roden et al., 2005; Das et al., 2010; Bensah and Mensah, 2013; bacteria (Griffin et al., 2000; Wiegand et al., 2008; Wu C. et al., 2015;

Kokollari et al., 2015; Fierascu et al., 2019). Albano et al., 2020).

Disk-diusion method

The underlying idea behind this procedure is that an antibiotic

concentration gradient is created when an antibiotic-impregnated disc is Extraction techniques
placed on agar that has already been inoculated with the test bacterium.
The antibiotic is The second step is the extraction of required compounds from the
present in high concentrations near the disk’s edge and gradually material. The best extraction process must involve the thorough extraction
decreases as the space between them expands until the or3ganism is no of all the necessary metabolites or chemicals. If it’s going to be done
longer inhibited by the antibiotic, at which time it is free to proliferate. If repeatedly, it needs to be quick, easy, and repeatable. The choice of an
the antibiotic suppresses bacterial growth, a clear zone or ring forms appropriate extraction technique mostly depends on the work that has to
around the antibiotic disc be done

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TABLE 1 Nigella sativa and Capsicum as an antimicrobial agent.

Name of material Antimicrobial agent Adopted Findings References

methodology

Nigella sativa Thymoquinone and melanin Paper disc diffusion A clear antibacterial effect on the growth Bakathir and Abbas, 2011;

method of Staphylococcus was obtained Al-Sharif, 2019

Fixed oil and thymoquinone

-

Effective against many microbes Ali and Blunden, 2003

of N. Sativa including Escherichia coli, Shigella niger,

Staphylococcus Albus, Salmonella typhi,

and Vibrio cholera.

Most effective against Aspergillus species

Thymoquinone, p-cymene, Disc diffusion Thymoquinone is the main component Roy et al., 2006

p-tert-butylcatechol, and method that causes antimicrobial effects

pinene

Essential oil of N. sativa Plate diffusion Most effective against Bacillus subtilis El-Kamali et al., 1998

method

Crude alkaloid and water -

Gram-negative was most effective than Morsi, 2000

extract of the seed Gram-positive

Capsicum Low pH of fruit Disk diffusion Extracts of Capsicum fruits are suitable Careaga et al., 2003; Tano et

method and Well for antibacterial activities al., 2008; Koffi-Nevry et al.,

diffusion method 2012

Isopropanol extracts Growth inhibition Cinnamic and M-coumaric acids from Dorantes et al., 2000

test Chili extracts are effective against

bacterial species.

Capsaicin-induced alterations
-
Prevent microbial infections Tellez et al., 1993

in the pH

Presence of Capsaicin Inhibit CT Effective against V. cholera Omolo et al., 2014

production

Presence of Capsaicin and Disk diffusion Capsaicinoids are main component that Das et al., 2018

Capsaicinoids method causes antibacterial effect.

and on the target metabolites to be known. There are various sources, especially Nigella sativa, Capsicum, Musa paradisiaca
extraction processes like; Conventional techniques include peels, and Citrus limetta.
solvent extract, soxhlet extraction, vortexing, maceration,
centrifugation, hydro-distillation, cold pressing, and pressing Soxhlet extraction
and solid-liquid dynamic extraction. Non-conventional techniques
This extraction technique involves putting solid material
include; pressurized liquid extraction, microwave-assisted
inside the thimble present in the extractor. The solvent
method, ultrasound-assisted method, pulsed electric field,
condenses and fills the thimble as the vapor rises. This process
enzyme-assisted extraction, and supercritical fluid extraction.
of extraction is repeated again and again until the desired
Conventional techniques are more time-consuming and less
quantity is achieved. This technique was firstly used for the
environmental friendly while non-conventional are fast and
determination of fat in milk (Voon et al., 2012; Parashar et al.,
environment friendly techniques (Koubala et al., 2008; Azmir et
2014; Chen and Urban, 2015). Five types of soxhlet techniques
al., 2013a,b; Deng et al., 2015).
are mainly used for solvent extraction i.e., conventional soxhlet,
high-pressure soxhlet extraction, ultrasound-assisted soxhlet
extraction, automated soxhlet extraction, and microwave-
Extraction technologies for extracting assisted soxhlet extraction (Redfern et al., 2014; Wu C. et al.,
antimicrobial agents from Nigella sativa, Capsicum, 2015; Wu G. et al., 2015). Conventional soxhlet extraction is a
peels of Musa paradisiaca L., and Citrus limetta continuous process with slow processing time and an
environmental unfriendly technique (Azmir et al., 2013b;
This review presents an analysis of several extraction Lopresto et al., 2014). High-pressure soxhlet extraction is used
techniques to extract antimicrobial agents from different plant to fractionate polyethylene. The automated soxhlet technique saves

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TABLE 2 Musa paradisiaca L. and Citrus limetta as an antimicrobial agent.

Name of material Antimicrobial agent Adopted methodology Findings References

Musa paradisiaca L. Alcoholic extracts of Musa Agar diffusion method Effective against Staphylococcus and Pseudomonas Ahmad and Beg,

Paradisiaca banana fruit peel 2001; Karuppiah

and Mustaffa, 2013

Presence of Phenolic and Minimum Inhibition ß-sitosterol, 12-hydroxy strearic, malic and Fidrianny et al.,

Flavonoid compounds Concentration (MIC) and succinic acid from banana green peel shows 2014

paper disc diffusion method antibacterial activities

Presence of fatty acids Fermentation A suitable environmental condition should be Brooks, 2008

provided for high ethanol production

Methanol extract of M. Zone of inhibition test (ZIT), Methanol extracts of Banana peels are more Okorondu et al.,

paradisiaca peels minimum bacterial effective against bacteria than its ethanol extracts. 2010

concentration (MBC) and

minimum inhibitory

concentration (MIC)

Citrus limetta Aqueous and organic extracts of Solvent extraction procedure Methanolic extract of Citrus limetta have Shakya et al., 2019;

C. limetta peels antibacterial activity mostly against Escherichia Dutta et al., 2020a

coli, Pseudomonas aeruginosa, and Staphylococcus

aureus

Ethyl acetate and butanol Agar diffusion method Ethyl acetate stops the growth of bacteria and Khan et al., 2017

fractions of C. limetta peels butanol fractions stops the quorum sensing

extract control pigment production

Limonene (95.98%) Hydro-distillation, Disc Have strong antibacterial activity against Javed et al., 2013

Camphene (1.79%) and diffusion method food-borne pathogens

Terpenes

Peel extracts Well diffusion assay Pomegranate peel extract showed maximum Prakash et al., 2013

antibacterial activity among Orange, Banana,

Sweet lime, Apple, Papaya, and Mango

Acetone extracts Disc diffusion method Effective against Staphylococcus aureus Bacillus Al-Snafi, 2016

subtilis, and Bacillus cereus

time, sample consumption and is used for agricultural, food and adding an appropriate amount of water, and then heating it to a
industrial samples. Ultrasound-assisted soxhlet extraction is boil. Alternative methods include injecting direct steam straight
mainly used to extract fat from sunflowers, grapes and soya into the plant sample. By using indirect cooling with water, the
beans. However, this technique does not affect the quality of water and oil vapor combination is condensed.
extracted oil due to the adoption of mild conditions in the Oil and bioactive substances are automatically separated from
process (De Castro and Priego-Capote, 2010; Parashar et al., the water in a separator when the condensed mixture flows from
2014). the condenser. Its usage is constrained by restrictions on high-
The soxhlet technique was used to extract 6.77 ± 1.2, 3.66 ± temperature applications for heat-sensitive phenolic compounds,
1.43, and 4.81 ± 1.8% w/w of thymoquinone from Nigella sativa although it has several benefits such as the lack of organic
(Kausar et al., 2017). Capsaicin was extracted from Capsicum solvents in the process, the requirement for no dehydration of
by using this technique (Chuichulcherm et al., 2013). Citrus the plant materials, and faster extraction times (Vankar, 2004;
limetta which is a major phyto compound was extracted from Sa-Nguanpuag et al., 2011; Erkan et al., 2012; Chenni et al.,
Capsicum by using this technique (Baranitharan et al., 2020). 2016; Elyemni et al., 2019).
Through this technique, thymoquinone and capsaicin were
Hydro distillation extracted from Nigella sativa (El Khoury et al., 2019) and
Hydro distillation is a conventional method that is used for Capsicum, respectively (Jiang et al., 2013). Hydro distillation is
extracting bioactive compounds from different plant species. the most common method for obtaining thymoquinone from
This process involves packing plant materials in a still chamber, Nigella sativa (Liu et al., 2011; Oskouei et al., 2018).

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Liquid–liquid extraction not suitable for drugs and polar substances. It is also a costly technique
In liquid-liquid extraction separation of required material from (Mendiola et al., 2007; Abbas et al., 2008).
liquid solution is made through contact with another insoluble liquid. From mandarin, orange, lemon, grapefruit peel and sour orange
The required material is separated based on its solubility in different peel phenolic compounds were extracted by using supercritical fluid
liquids (Chen et al., 2017; El Blidi et al., 2019; Goyal and Singh, 2022). extraction (Omar et al., 2013; Trabelsi et al., 2016). Super critical fluid
extraction is used for obtaining thymoquinone from N. sativa (Liu et al.,
It is best suited for liquid samples. The technology is simple and 2011; Oskouei et al., 2018).
can be used at ambient temperature. However, the technology is time-
consuming, requires expensive chemicals, and takes a lot of labor
Pressurized liquid extraction
(Espinosa-Alonso et al., 2006).
Pressurized liquid extraction is mainly used for extracting phenolic
Liquid-liquid extraction is an energy e?cient technique (Blahušiak
compounds from plants. Liquid solvents are used in it with specific
et al., 2018) and can be used in biorefineries for lipid extraction from
temperatures and pressure. It is suitable for solid samples and polar
microalgae (Du et al., 2017) and for fractionation of pyrolytic bio-oils
compounds. However, pressurized liquid extraction is time-consuming
(Cesari et al., 2019).
technology that requires less solvent. Also due to the 10 g maximum
Earlier research study also reports lignin recovery via liquid extraction
sample weight limit, it is not appropriate for situations where very low-
(Stiefel et al., 2017). Moreover, the fractionation of citrus essential oils
level analyte detection is required (Jacobsen et al., 2004; Juan et al.,
(separating the terpenic hydrocarbons and oxygenated compounds) is
2010; Mustafa and Turner, 2011; Alañón et al., 2015; Alvarez-Rivera et
achieved through liquid-liquid extraction (Gonçalves et al., 2016).
al., 2020). It is mainly used to recover bioactive compounds from

grapes (Pereira et al., 2019). Extraction of phenolic compounds

(alkaloids) from Citrus limetta through pressurized liquid extraction
Solid-phase extraction The was carried out by Olabinjo et al. (2020).
solid-phase extraction technique is the simplest technique for
extracting and pre-concentrating trace levels of contaminants from
samples. The extraction efficiency of solid-phase extraction is
Pulsed electric field
enhanced by using nanomaterials as sorbents which provide more
pollutants capturing sites (Jacobsen et al., 2004; Silva et al., 2016; The pulsed electric field is an efficient non-thermal technique that

Azzouz et al., 2018). involves the application of high-voltage pulses.

The only difference between solid-phase extraction and liquid- These pulses cause rupturing of plant materials thus extraction of

liquid extraction is the medium used for separation; in solid-phase required components occurred from plant cells. Though it is a

extraction, one phase is a liquid phase and the other is a solid phase. sustainable technology, it takes a long time to process.

The solid surface consists of adsorbent material that absorbs specific This technology requires process parameters like energy inputs,

required material through the solution. The separation rate of solid- treatment temperature and field strength to be maintained (Liang et al.,

phase extraction technique is faster than liquid-liquid extraction 2002; Wu et al., 2005; Nguyen and Mittal, 2007; Puértolas et al., 2012;

technique. It is a simple technique. It can be employed for polar Garner, 2019). According to a study, Citrus limetta peels were subjected

compounds. It is not suitable for volatile samples (Abd-Talib et al., to pulsed electric fields in order to extract phenolic components

2014). It is an expensive technique than the liquid-liquid extraction (Luengo et al., 2013). In another study, phenolic compounds were

technique (Hernanz et al., 2008). Different methods were used to extract extracted from orange, grapefruit, and lemon peel through a pulse

thymoquinone from Nigella sativa including Solid-phase extraction electric field (El Kantar et al., 2018). Much more components were

(Kausar et al., 2017). extracted from the N. sativa’s oil when PEF was applied as a
pretreatment.
The major constituent was thymoquinone and cymene, whereas D-
Limonene and ÿ-Pinene were also present in small quantities (El-
Supercritical fluid extraction
Dakhakhny et al., 2000; Bakhshabadi et al., 2018).
Suppose the temperature and pressure of a specific liquid and gas
are above the critical point. In that case, the supercritical fluid extraction Enzyme-assisted extraction
technique should be used for extracting certain required materials from Specific enzymes are used in enzyme-assisted extraction to target
samples like essential oils. Certain parameters such as temperature, the cell walls of plant materials, destroying them and liberating
pressure, the flow rate of fluids, and the sample size significantly affect antimicrobial compounds. The effectiveness of the procedure as a
extraction yield (Santoyo et al., 2006; Ma et al., 2018; Yousefi et al., whole can be improved by combining this strategy with a number of
2019; Zizovic, 2020; Santos et al., 2021). other techniques (Nadar et al., 2018; Bilal et al., 2019; Habeebullah et
al., 2020). For instance, Naghshineh et al. (2013) found enzymatic
The supercritical fluid extraction technique saves time, it is extraction with high pressure treatment led to a higher level of pectin
sustainable and suitable for volatile samples. However, it is extraction form

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lime peel powder (Naghshineh et al., 2013). This technology has high technology has not yet been utilized (Barba et al., 2015; Li et al., 2019;
extraction rates and is sustainable. However, it has a high enzyme cost Rajha et al., 2019; Žuntar et al., 2019). Extraction of phenolic compounds
and is not appropriate for industrial use. (alkaloids) from grapefruit peel (El Kantar et al., 2019) and from the
Its operations must be handled with caution (Puri et al., 2012; Singh et mandarin peel (Buniowska et al., 2015), through high voltage electric
al., 2016). Camphene is the predominant essential oil of Aristolochia discharge was studied in respective studies.
manshurientsis Kom extracted from

enzyme-assisted extraction followed by hydrodistillation (Zhao et al.,

2018).
Comparative analysis of dierent extraction technologies for
Microwave-assisted extraction
extracting bioactive compounds Selecting an extraction
Organic compounds can be extracted from plant material with
technique
more ease and effectiveness using microwave technology than using
presents one of the biggest challenges in getting bioactive
traditional methods. It is a technology that saves time. It is a very
compounds from plants because many factors need to be taken into
efficient technology with a high yield. This technology is sustainable
account, including the solvent’s polarity, the chemical makeup of the
because it requires less heat (Huie, 2002; Kaufmann and Christen,
bioactive compound, yield, selectivity, temperature, solubility, extraction
2002; Wang and Weller, 2006; Chen et al., 2007; Cravotto et al., 2008;
time, and cost. Natural plant extracts can be obtained in a variety of
Sticher, 2008; Zhang et al., 2009). It involves a simple and economical
ways, but the elements of the process profoundly affect the final
process as compared to supercritical fluid extraction. It has a difficult
product’s quality. Among the extraction techniques, the supercritical
operation as compared to ultrasonic-assisted extraction (Hayat et al.,
extraction and/or the extraction utilizing pressurized fluids can be
2009). Extraction of Capsaicin through Capsicum was done through
highlighted. In these procedures, a highly compressible fluid is used
microwave-assisted extraction by Chuichulcherm et al.
as a solvent in low- or mid-temperature conditions. The utilization of
supercritical fluids is a possible technique for the extraction and
(2013). A study reported that phenolic compounds (alkaloids) were
fractionation of natural resources, particularly for food and
extracted from Citrus limetta through microwave-assisted extraction
pharmaceuticals.
(Arafat et al., 2020). In another study, phenolic compounds were
extracted from orange peel through microwave-assisted extraction
The key advantages of supercritical fluid extraction include high
(Nayak et al., 2015). Microwave-assisted extraction is used for
selectivity, ease of solvent removal, and use of mild temperatures
obtaining thymoquinone from N. sativa (Liu et al., 2011; Oskouei et al.,
(Correa et al., 2017). Using Capsicum frutescens L. (Chuichulcherm et
2018). Various alkaloids were extracted from plants using microwave-
al., 2013), investigated the extraction of capsaicinoids using microwave-
assisted extraction (Bergs et al., 2013; Ngo et al., 2017; Aqil et al., 2020).
assisted extraction (MAE) and ultrasound-assisted extraction (UAE), in
comparison to the conventional soxhlet method. They concluded that

Ultrasound-assisted extraction the UAE approach was the best technique for extracting capsaicinoids
from C. frutescens. Therefore, even though the amounts of capsaicinoids
Ultrasound-assisted extraction is a technique that involves
in MAE and UAE were 5.28 and 4.01 mg/g of C. frutescens, respectively,
throwing sound energy to plants and agitating it thus extracting the
UAE needed the least amount of energy and was deemed the best
required materials through a plant cell. In this technology, low energy
approach. The low-quality oil produced by traditional solvent extraction
and chemicals are required. High yield can be obtained from it, and it
methods necessitates intensive purifying procedures, which cause
is a time-saving technology. Proper optimization in ultrasound
thermal deterioration and the loss of important components. There has
frequency, the nominal power of the device, input power, propagation
been a shift toward more environmentally friendly extraction techniques,
of cycle, and system geometry is required for maximum yield (Azmir et
such supercritical fluid extraction, as a result of the use of organic
al., 2013a; Barba et al., 2015; Sneha et al., 2022). Studies report the
solvents and the need to recover the solvent (SFE).
extraction of Capsaicin from Capsicum, phenolic compounds (alkaloids)
from orange peel and citrus fruit peel through ultrasound-assisted
extraction (Chuichulcherm et al., 2013; Omar et al., 2013; Boukroufa
et al., 2015; Nishad et al., 2019). Different methods were used to extract SFE is evolving into a successful and widely utilized method for

thymoquinone from Nigella sativa including Ultrasound-assisted extracting valuable natural compounds from challenging materials
(Ghahramanloo et al., 2017). Particularly, maceration, microwave-
extraction (Kausar et al., 2017).
assisted extraction (MAE) and ultrasound-assisted extraction (UAE)
have been considered as popular extraction techniques; Zhang and his
High-voltage electrical discharge A high- coworkers concluded that MAE was the most effective with a yield of
voltage electrical discharge is a non-thermal method that relies on 7.04 ± 0.14 mg/g chelerythrine and 17.10 ± 0.4 mg/g sanguinarine with
ambient temperature and uses pulsed rapid discharge voltages. It is a 5 min of extraction time (Zhang et al., 2005). High hydrostatic pressure
technique that saves time and uses less energy. However, at the (HHP), ultra-sonics and pulsed electric fields (PEF) were used for
industrial and pilot level, this extracting

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bioactive compounds from grapes and the finding of this study was; this technique can filter out a variety of undesirable particles of various
the total phenolic content of samples was 50% higher than in the sizes and is comparatively inexpensive (McGaw et al., 2002; Jin et al.,
control samples (Corrales et al., 2008). Typically, phenolics have 2007; Mushore and Matuvhunye, 2013; Sahle and Okbatinsae, 2017).
antibacterial and antioxidant properties. Due to their unique molecular
structure, can offer superior platforms for making antimicrobial
products (Siddiqui et al., 2022).
High pressure (HP), pulsed electric fields (PEF), and low pasteurization Ultrafiltration

(LPT) were used for extracting, pulsed electric field and low Ultrafiltration (UF) is a type of membrane filtration, that forces a
pasteurization; the former was the most effective treatment for the liquid against a semi-permeable membrane by hydrostatic pressure.
extraction of bioactive components from orange juice (Plaza et al., Water and solutes <0.005 micro meter pass through the barrier, whereas
2011). Conventional soxhlet extraction, MAE and ultrasound assisted suspended solids and solutes >0.1 micro meter are trapped. Compared
extraction (UAE) were used for extracting bioactive compounds from to traditional methods, ultrafiltration technology can accomplish more
agiorgitico red grape pomace. This study concluded that UAE was the work in 50% less space (Pampanin et al., 2012; Wang et al., 2013; Tang
most effective one (Drosou et al., 2015). There have been several et al., 2018; Kellogg et al., 2019; Martinenghi et al., 2020).
research investigations on the extraction of bioactive substances using
traditional and non-conventional (green) methods, including soxhlet
extraction, microwave, supercritical fluid extraction, ultrasound
extraction, etc. Each methodology has pros and cons. Paper chromatography
In this method, separations are performed using a piece of paper
For instance, the greatest yield (26.52%) and total phenolic content that serves as both a support and a medium for separation. The filter
(15,263.32 mg Eq gallic/100 g DW) from citrus reticulata blanco cv. paper is placed in the chromatographic chamber with the solvent, and
sainampueng peel were obtained via the extraction of bioactive the sample is put close to the bottom of the filter paper. By means of
components under the ideal UAE conditions. UAE therefore capillary action, the solvent flows while also transferring soluble
demonstrated greater extraction efficiency in terms of yield under the molecules.
same extraction circumstances, which was 1.77 times higher than MAE Paper chromatography has the benefit that separations can be done
(Saini et al., 2019). easily on sheets of filter paper, which serves as both a substrate and a
medium for separation. The high degree of reproducibility of Rf
(retention factor) values can also be obtained on paper through this
Purification technique (Thumar et al., 2010; Galanakis, 2012; Ingle et al., 2017;
Muhamad et al., 2017; Tang et al., 2018; Martinenghi et al., 2020).
Purification of antimicrobial agents is generally the final step.
There are various purification methods that can be used to purify
antimicrobial agents. The choice of a specific purification method in
any study depends on the degree of purification required for specific
antimicrobial agents. In the case of polar compounds, purification is Thin-layer chromatography Using this
done mainly by filtration, ultrafiltration, paper chromatography, thin- method, samples are separated based on their interactions with a
layer chromatography, gas chromatography and high-performance thin layer of adsorbent that is adhered to the plate of low molecular
liquid chromatography. Whereas, non-polar compound purification is weight substances. Compounds are separated using various
mainly performed by; liquid-liquid extraction, reversed phase adsorbents. The particular benefit of TLC over paper chromatography
purification chromatography and through Hydrophobic interaction is its adaptability, speed, and sensitivity (Thumar et al., 2010; Galanakis,
chromatography (Thumar et al., 2010; Galanakis, 2012; Ingle et al., 2012; Tang et al., 2018; Martinenghi et al., 2020).
2017; Tang et al., 2018; Martinenghi et al., 2020).

Gas chromatography
Utilizing this method, volatile substances are separated.
Filtration Through its distribution in the gas phase, the chemical species’ rate of
Filtration is a technique that involves pouring a combination of kinetics is identified. While the liquid phase is still, the gas phase is
solid and liquid onto a membrane or a filter paper, which permits the moving. The chemical species’ rate of migration is estimated based on
passage of liquid (the filtrate) and results in the collection of the solid, how widely distributed it is in the gas phase.
this process is used to separate solids from liquids. During filtration, For instance, a species that disperses 100% of itself into the gas phase
the size of the materials that can be removed relies on the filter’s pore will migrate at the same rate as the gas that is flowing, whereas a
size. With just one filter, species that disperses 100% of itself into the stationary

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phase will not move at all. In gas chromatography, a sample is Antibiotic resistance mechanisms in disease causing
vaporized and then injected into the chromatographic column’s head.
bacteria
The flow of the inert, gaseous mobile phase carries the sample through
the column. A liquid stationary phase that is adsorbed onto the surface
Microbial infections are on the rise globally, and the human
of an inert solid is present in the column itself (Thumar et al., 2010;
population is at risk due to microbes because these microbes are the
Galanakis, 2012; Ingle et al., 2017; Tang et al., 2018; Sreedharan et al.,
major cause of worldwide mortalities, as shown in Table 3. Basically,
2019; Martinenghi et al., 2020).
disease causing bacteria have resistance against various antibiotics
due to myriad mechanisms. These mechanisms are: (1) Bacteria can
have resistance through transformation, transduction, and conjugation
phenomenon.
High-performance liquid chromatography (2) Through the processes of phosphorylation, adenylation, or
HPLC is an analytical technique used for the separation and
acetylation bacteria deactivate the antibiotics. (3) Bacteria prevent the
measurement of organic and inorganic solutes in any samples,
interaction of drugs and antibiotics. (4) They can cause efflux of the
particularly biological, pharmaceutical, food, environmental, industrial, antibiotic from the cell (Munita and Arias, 2016; Reygaert, 2018;
etc. This method divides compounds by how they interact with the
Abushaheen et al., 2020). Recently, resistance to antibiotics has been
solvent of the mobile phase and the solid particles of a densely packed
the biggest challenge which threatens human communities. Meanwhile,
column. To help in identifying the chemicals, the Diode Array Detector the occurrence of the evolution of resistance endangers the
examines the analytes’ absorption spectra (Thumar et al., 2010;
effectiveness of existing antibiotic drugs (Baym et al., 2016; Marston et
Galanakis, 2012; Ingle et al., 2017; Tang et al., 2018; Martinenghi et al.,
al., 2016).
2020).
Therefore, several strategies have been proposed to overcome the
antimicrobial resistance. One of the recommended methods

to achieve this goal is the usage of natural compounds that are derived
from plants. These plant extracts seem to be ideally suited to overcome
Reverse-phase chromatography In reverse-
the emergence of antibiotic resistance in multidrug resistance bacteria
phase chromatography (RPC), molecules are separated from one
(Ncube et al., 2008; Rossiter et al., 2017).
another through hydrophobic interactions between the ligands attached
to the stationary phase and the solute molecules in the mobile phase
of the liquid chromatography. Better solubility for polar analytes is
provided by reverse-phase chromatography, which also uses non-toxic
Anti-microbial properties of Capsicum, Nigella
solvents, provides a means for removing impurities and mobile phase
sativa, peels of Musa paradisiaca L., and Citrus
additives, and provides rapid sample recovery with minimal solvent
limetta
evaporation (Nair and Kanfer, 2006; Thumar et al., 2010; Galanakis,
2012; Tang et al., 2018; Martinenghi et al., 2020).
Plants have a plethora of constituents that impose antimicrobial
effects (Gyawali et al., 2011; Mickymaray et al., 2016; Dewapriya et al.,
2018; Casciaro et al., 2019; Ali M. Y. et al., 2021). Two major groups of
antibiotics extracted from plants are; (1) phytoanticipins and (2)

Hydrophobic interaction chromatography phytoalexins. Phytoanticipins inhibit microbial actions, whereas

Phytoalexins are generally antioxidants (Sukalingam et al., 2017, 2018).
Based on their hydrophobicity, molecules are separated using
Moreover, various secondary plant metabolites are known to cause a
hydrophobic interaction chromatography (HIC). Due to the use of less
significant antimicrobial effect which will be discussed in later section.
denaturing conditions and matrices, HIC is a good separation approach
for removing proteins while preserving biological activity. This
technique can improve the mass spectrometry’s sensitivity of detection.
When compared to reversed-phase liquid chromatography, the flow
rate in this approach can be larger because the mobile phase’s viscosity
Antimicrobial mechanism of bioactive
is low. Low viscosity mobile phases enable the use of columns that are compounds
twice as long as those required for reverse phase HPLC. The sample
separates very quickly due to the eluent’s viscosity, which is also These secondary metabolites are grouped into three types; (1)
correlated with a lower separation impedance. Compared to reverse phenolic compounds, (2) terpenes, and (3) alkaloids (Crozier et al.,
phase HPLC, basic solutes can be loaded more readily (Thumar et al., 2006; Mickymaray, 2019; Roaa, 2020). These antimicrobial extracts
2010; Galanakis, 2012; Sagar et al., 2012; Tang et al., 2018; Martinenghi cause cell wall disruption and lysis, cell wall construction, inhibit
et al., 2020). biofilm formation, inhibit microbial DNA replication, inhibit energy

synthesis, inhibit bacterial

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TABLE 3 Infectious diseases caused by microbes.

Infectious disease Microbe that causes Death rate References

the disease

Methicillin-Resistant Staphylococcus Aureus (MRSA), Gram-positive bacteria 76% Doernberg et al., 2017; Sizar and Unakal, 2020

Vancomycin-Resistant Enterococci (VRE), and

Clostridium difficile

Pneumonia, bloodstream infections, wound or surgical Gram-negative bacteria 14% Centers for Disease Control and Prevention, 2011;

site infections, and meningitis Sizar and Unakal, 2020

Candida can cause infections if it grows out of control Candida albicans 19–24%. Akpan and Morgan, 2002; Magill et al., 2014

or if it enters deep into the body (for example, the

bloodstream or internal organs like the kidney, heart,

or brain)

Cholecystitis, Bacteremia, Cholangitis, Urinary tract E. coli 4.2% Makvana and Krilov, 2015; Khalil et al., 2018

infection (UTI), traveler’s diarrhea, and other clinical

infections such as neonatal meningitis and pneumonia.

Dermatophytosis Trichophyton interdigitale 4–8% Kardjeva et al., 2006; Mügge et al., 2006; Zhang

et al., 2019

Nosocomial infections Staphylococcus epidermidis 20% Otto, 2009; Turnidge et al., 2009

Food-borne bacterial illness cause to unborn babies, Listeria monocytogene 15–20% Edelson and Unanue, 2000

newborns and people with weakened immune systems

Diarrheal and toxico-infections Bacillus cereus 619 death from 1998 to 2015 Jessberger et al., 2020; McDowell et al., 2020

Superficial and ear infections in humans Vibrio alginolyticus 35–50% Reilly et al., 2011

Human infectious diseases including keratitis, burn Pseudo. aeruginosa 18–61% Kang et al., 2003; Cao et al., 2017

infections, urinary tract infections (UTIs), sepsis, as

well as acute and chronic infections of human airways.

Gastroenteritis and focal infections Salmonella typhimurium 3 million each year Coburn et al., 2007; Jung et al., 2020

Diarrhea (sometimes bloody), fever, and stomach Shigella flexneri 13.2% Khalil et al., 2018; Duncan-Lowey et al., 2020

cramps.

toxins to the host, and induce reactive oxygen species microbes ( Tsuchiya and Iinuma, 2000 ; Sanver et al., 2016 ).
production. Despite all these effects, these antimicrobials also Terpenes contain isoprene that disrupts microbial membranes
prevent antimicrobial resistance and synergetics to antibiotics, (Guimarães et al., 2019; Moghrovyan et al., 2019).
which helps kill pathogenic organisms (Tariq et al., 2019; Zheng
et al., 2020; Vaou et al., 2022), as shown in Figure 4.
Induction of reactive oxygen species
production
Promote cell wall disruption and lysis Reactive oxygen species are formed by the partial reduction
Cell disruption mechanism takes place via two approaches of molecular oxygen that helps in antimicrobial activity and
that are mechanical and non-mechanical approach (Figure 5). provides a defense mechanism against various microbes. The
The detail of the antimicrobial mechanism is given in separate method of catechins involves augmentation of the production
subsequent paragraphs. Firstly, compounds that functionalized of oxidative stress that disrupts cell walls (Fathima and Rao,
this disruption are delineated as follows. 2016; Van Acker and Coenye, 2017; Zou et al., 2017). Sources
Phenolic compounds belong to the family of aromatics of reactive oxygen species, antioxidant defenses, and subsequent
containing a hydroxyl functional group, which in reaction with biological effects depending on the level of reactive oxygen
the microorganisms disrupts their cell wall (Ganesan and Xu, species production are shown in Figure 6.
2017a, 2018). These aromatics are moved to the microbe’s cell
surface, and thus they disrupt their cell walls. Flavonoids are
phenolic compounds that form a complex relationship with Inhibition of biofilm formation
the bacterial cell wall and thus disrupt its structure (Ganesan Biofilms developed by bacteria are 100–1,000 times more
and Xu, 2017b,c). Rutin, naringenin, sophoraflavanone, and resistant to the antimicrobial drugs (Kahaliw et al., 2017; Mishra
tiliroside are the flavonoids that disrupt S. aureus and S. mutans et al., 2020). In some studies, it was indicated that flavonoids

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FIGURE 4
Antimicrobial mechanism of bioactive compounds.

FIGURE 5
Antimicrobial mechanism by promoting cell wall disruption and lysis (Moo-Young, 2019).

aggregate the multicellular composites; thus, inhibiting lipids, respiration and the transport mechanism. For all
bacteria growth. Flavonoids such as; (1) galangin, (2) these functions, membrane integrity plays a major role in
isovitexin, (3) 3- O-octanoyl-epicatechin, and (4) 5, 7, and 40- bacterial species, so the membrane disruption leads to
trihydroxyflavanol cause the aggregation of S. aureus and bacterial death (Lazar and Walker, 2002; Klis et al., 2006; Reygaert, 2014).
S. mutans; therefore inhibiting their growths (Awolola et al., 2014; Rabin et al., 2015).

Inhibition of bacterial toxins against the host

Inhibition of cell wall construction Catechins and some other flavonoids cause disruption
The bacterial cell wall is the main target of any of bacteria cell walls thus resulting in an inability to
antimicrobial agent because the cell wall is the main source of biosynthesis
discharge of
toxins (Lee et al., 2011; Chang et al., 2019).

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FIGURE 6
Endogenous and exogenous sources of ROS, antioxidant defenses, and biological eects that depends on ROS production.

Compounds such as catechins, pinocembrin, kaempferol, Antimicrobial mechanism of

kaempferol-3-O-rutinoside, gallocatechin gallate, quercetin antimicrobial agents
glycoside, genistein, and proanthocyanidins neutralize
bacterial toxic factors initiating from N. gonorrhoeae, V. Antimicrobial mechanisms of antimicrobial agents present
cholerae, E. coli, B. anthracis, V. vulnificus, S. aureus, and C. in Nigella sativa, Capsicum, Musa paradisiaca L., and Citrus
botulinum (Shahnazari et al., 2011; Ahmed et al., 2016). limetta are discussed below;

Antimicrobial mechanism of thymoquinone

Inhibition of energy synthesis The
Thymoquinone plays a major role in fighting against
production of energy or adenosine triphosphate (ATP) is
microbes. Basically, thymoquinone causes resistance in the
important for the development of bacteria because it is the
biofilm formation in bacterial species because biofilm
main source of a living system. The reaction of flavonoids
formation is the important activity of microbes in their virulence strategy.
such as isobavachalcone and 6-prenylapigenin with S. aureus
Thymoquinone also causes hindrance in the oxidative activity
results in disruption of the bacterial cell wall (Freudenberg
of microbes thus reducing the number of microbes (Harzallah
and Mager, 1971; Kuete et al., 2011; Xie et al., 2015). et al., 2011; Forouzanfar et al., 2014; Khan, 2018; Fan et al., 2021).
Microbes cause many infectious diseases like AIDS,
dengue, MERS, MRSA, and VRE, etc. Thymoquinone prevents
the murine cytomegalovirus (CMV) replication in the liver and
Inhibition of microbial DNA replication spleen of the infected mice. Medication with thymoquinone
Antimicrobial agents inhibit DNA replication thus stopping increases the number of CD4+ T cells which plays a major
the further division of microbes. Moreover, bioactive role in building the immune system. Thymoquinone increases
compounds present in plants such as kaempferol, quercetin, levels of serum interferon-ÿ (IFN-ÿ) and macrophages.
apigenin, genistein, nobiletin, myricetin, chrysin, tangeritin, Thymoquinone also fights against various fungal pathogens
and 3, 6, 7, 30, 40-pentahydroxyflavone are DNA disrupters like Candida albicans and Aspergillus fumigatus (Khan, 2018;
(Gotoh et al., 2008; van Eijk et al., 2017; Vijayakumar et al., 2018). Wang et al., 2021; Qureshi et al., 2022).

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Antimicrobial mechanism of melanin in it. Limonene is also one of the main causes of the

Melanin constitutes a heterogenous group of phenolic polymers, dissipation of proton motive force in microbes. Limonene being an
which plays a major role in fighting against microbes. antimicrobial agent has a broad application in the food sector. Studies
Melanin is an important antimicrobial agent which stops the proliferation reported that limonene can kill gram-positive and gram-negative bacterial
of pathogens around the infected area. Melanin can fight against food species. Limonene is also found to be effective against fungal pathogens
pathogens; thus, contributes a major role in the food and health sector. (Cai et al., 2019; Han et al., 2019, 2020; Gupta et al., 2021).
Melanin also helped in antimicrobial drug discovery. Melanin extracted
from hair and further dopped in metal ions demonstrated the potential of
antibacterial activity (Mackintosh, 2001; Alviano et al., 2004; Nosanchuk
and Casadevall, 2006; Eliato et al., 2021).

Antimicrobial mechanism of camphene

Camphene is a lipophilic antimicrobial compound that by penetrating
in cell membranes of bacterial species becomes the reason of cell death.
Antimicrobial mechanism of P-cymene Camphene inhibits microorganism by disruption in many cell activities,
P-cymene is an important antimicrobial agent which is found in more and the breakdown of the membrane thus cellular components and ions
than 100 plants and contributes to the food and medicine sector. P- start leaking from the bacterial body. Also, camphene is the major source
cymene is used in the preparation of fungicides and pesticides. P-cymene of depletion of ATP in bacterial species. Studies reported that camphene
affects the bacterial membrane, pH and ATP thus contributing to the file has antibacterial, antiviral, and antifungal activities (Zhou et al., 2016; Er
of antimicrobes. Studies reported that p-cymene inhibits the growth of E. et al., 2018; de Freitas et al., 2020; Hachlafi et al., 2021).
coli, L. monocytogenes, S. enterica, S. aureus, Vibrio parahaemolyticus,
and Streptococcus mutans (Kordali et al., 2008; Marchese et al., 2017;
Miladi et al., 2017; Balahbib et al., 2021).

Conclusion
Antimicrobial mechanism of pinene Pinene belongs to Microbial diseases are a major threat to the human population. The
the group of terpenes and has antimicrobial properties. Pinene worldwide death rates due to microbes are: Pseudomonas aeruginosa
inhibits microorganisms by interfering in their metabolic process and 61%, E. coli 4.2%, Staphylococcus epidermidis 20%, Candida albicans
modulation of gene (Tyc et al., 2017; do Amaral et al., 2020). Pinene has 24%, Shigella flexneri 13.2%, Sal. Typhimurium 3 million each year, and
its antimicrobial activity (Dhar et al., 2014; Zhang et al., 2021) against C. Vibrio alginolyticus 50%. However, these death rates can be reduced by
neoformans, C. neoformans phospholipase and esterase, C. Albicans, R. using antimicrobial agents on a regular basis. This review evaluated the
oryzae, and MRSA (Silva et al., 2012; Kovac et al., 2015 ). antimicrobial effects of four plant resources- Capsicum, Nigella sativa,
Musa paradisiaca L., and Citrus limetta. These natural resources contain
ÿ

thymoquinone, p-cymene, pinene, alkaloid, limonene, camphene, and

melanin, which serve as antimicrobial agents by disrupting the cell
membrane of microbes, inhibiting the cellular division and inhibiting the
Antimicrobial mechanism of alkaloid Alkaloids disrupt formation of biofilm in bacterial species, thus reducing the number of
the cell membrane of microbes, inhibit cellular division, inhibit efflux microbes. Soxhlet, hydro-distillation, solid-phase extraction (SPE), liquid-
pump, and inhibit biofilm formation thus act as a lead compound in the liquid extraction (LLE), pressurized liquid extraction (PLE), supercritical
development of antimicrobial agents. Studies reported that alkaloids can fluid extraction (SFE), pulsed electric field (PEF), microwave-assisted
kill Pseudomonas aeruginosa, Streptococcus mutans, faecalis and extraction (MAE), enzyme-assisted extraction (EAE), ultrasound-assisted
pyogenes, Lactobacillus acidophilus, Candida albicans, and extraction (UAE), and high-voltage electrical discharge are the various
Staphylococcus aureus (Orhan et al., 2010; Özçelik et al., 2011; Mittal techniques used for extracting critical antimicrobial agents from
and Jaitak, 2019; Othman et al., 2019; Zielinska et al., 2019 ; Huang et al., Capsicum, Nigella sativa, peels of Musa paradisiaca L., and Citrus
2022). limetta. A brief description of all these techniques was given along with
´

their advantages and disadvantages in order to ease the selection of

appropriate technology for the extraction of naturally available
antimicrobial compounds.

Antimicrobial mechanism of limonene

Limonene belongs to the family of monoterpenes. Limonene by
accumulating in the membrane of microbes causes disruption

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Author contributions Acknowledgments

SB, WZ, MH, and AM equally contributed to the concept We would like to acknowledge the support of the
of the study, its framework, the coordination and activities Agricultural Research Station at North Carolina Agricultural
of subgroups, and writing of the manuscript. MM, MG, MI, and Technical State University (Greensboro, NC, United States).
SS, SI, SU-R, and SK equally contributed to the activities of
subgroups and the review of the manuscript. All authors
contributed to the article and approved the submitted version.
Conflict of interest

The authors declare that the research was conducted in the

Funding absence of any commercial or financial relationships that
could be construed as a potential conflict of interest.
This work was funded in part by the USDA/NIFA through
the Agricultural Research Program at North Carolina
Agricultural and Technical State University (Evans-Allen Publisher’s note
Program, project number NC.X-291-5-15-170-1), by an 1890
Capacity Building Program Grant (No. 2020-38821- 31113/ All claims expressed in this article are solely those of
project accession No. 021765), and (Project Nos. the authors and do not necessarily represent those of their
NC.X337-5-21-170-1 and NC.X341-5-21-170-1) from the affiliated organizations, or those of the publisher, the editors
National Institute of Food and Agriculture (NIFA). SI would and the reviewers. Any product that may be evaluated in this
like to acknowledge the support of the Agricultural Research article, or claim that may be made by its manufacturer, is not
Station at North Carolina Agricultural and Technical State guaranteed or endorsed by the publisher.
University (Greensboro, NC, United States).

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