COMPARATIVE ANTIBACTERIAL AND ANTIFUNGAL STUDIES

WITH DIFFERENT PLANT EXTRACTS

Jayanti Mukherjee*, Peddi Varshitha, Pothulapalli Shruthi,
Nagulapally Sanjana, T. Rama Rao
ABSTRACT

In the present scenario, resistance of pathogenic microorganisms to the most of antimicrobial

(anti-bacterial and anti-fungal) agents is increasing. In this study we have compared the
antimicrobial effect of some medicinal plants like neem, turmeric and ginger. The methanol
extracts of these plants were prepared for determining their anti-bacterial and anti-fungal
activity. Then comparative analysis of antibacterial effects between those extracts were tested
in E. Coli and Bacillus subtilis culture. Also comparative ………… analysis of The
antibacterial activity was investigated using well diffusion method. ……………..All of the
extracts have shown antibacterial activity …………. The methanolic herbal extracts of
ginger, turmeric rhizomes and neem leaves exhibit promising antibacterial and
……….properties against E. Coli and Bacillus subtilis.

Key words: Antibacterial , antifungal, multidrug resistance, pathogenic microbes,

synergistic effect

INTRODUCTION

Since primitive times, man has gone in different ways to search for cures and relief from
various diseases by using several plants, plant products and plant-derived products. Medicinal
plants are essential to maintain our health. Antibiotics are extremely dangerous to host
organs, tissues and cells when used to treat illnesses. Herbs can be used to avoid the toxicity
that antimicrobial drugs create. Clove, neem, garlic, and turmeric are utilised in traditional
medicine. Comparative analysis of the antibacterial and ……..properties of ethanolic extracts
from a portion of these plants was the study's goal [1].

One of the greatest accomplishments of contemporary science is the development of

antibiotics to tackle with the microbial infections. The main concerns could be the
development of resistance of pathogenic bacteria to currently available antibiotics [2].
Infectious diseases are the reasons for the rising rates of mortality and morbidity in
developing countries. A significant risk is the establishment of multi-drug resistance bacteria
brought on by the incorrect use of broad-spectrum drugs. Plants with medicinal value that are
abundant in secondary metabolites that have therapeutic qualities are being investigated as
possible safer and more effective substitutes for produced pharmaceuticals. Different parts of
plant such as roots, leaves, stem, fruits contain bioactive compounds and are potent sources
of antimicrobial compounds [3]. Therefore, research into physiologically active compounds
derived from plant species commonly used in herbal medicine is becoming increasingly
relevant. These components might offer a potent new source of antibacterial action [4].
……….ANTIFUNGAL……….
There is a large range in the effectiveness of different herbal extracts against viruses, bacteria,
fungi and parasites. Unlike other antibiotics that are designed to target particular types of
bacteria, several herbs are effective against a broader variety of diseases. Rather of containing
a single active component, herbal extracts are made up of several different molecules. This
complexity poses a greater challenge for pathogens to develop resistance. Conversely,
pharmaceutical antibiotics frequently comprise isolated chemical constituents, which bacteria
can more readily adapt to and counteract [5]. Since herbal extracts often result in less side
effects than antibiotics, they are frequently seen as a safer alternative. It is crucial to
understand that many herbal medicines might potentially have adverse effects. Herbal
medicine is unique in that it treats the patient as a whole, treating all symptoms as well as the
underlying cause of illness. Herbal treatment aims primarily to improve general well-being
and restore equilibrium.
Herbal remedies are preferred over synthetic antibiotics since they are less likely to have side
effects. Our paper includes the work done to determine the antimicrobial activity of Turmeric
rhizomes, Ginger rhizomes and Neem leaves. ADD ANTIFUNGL FACTS

Ginger, scientifically known as Zingiber officinale. It belongs to family Zingiberaceae.

Ginger is a well-known plant used for flavouring and medicinal purposes for many years. It is
commonly used to treat health issues like pain, nausea, and vomiting. Research has shown
that ginger has antiemetic effects in various conditions. Over 100 compounds have been
found in ginger, with gingerols being the most important. Ginger has many biological
activities, including antioxidant and antimicrobial properties. Recently, ginger has also been
studied for its potential in treating cancer, chemotherapy-induced nausea, fatigue, and
improving quality of life [6,7].

Fig.1: Zingiber officinale

Turmeric, also known as Curcuma longa, is a plant that grows naturally in the forests of
South Asia and Southeast Asia, particularly in southern India. It belongs to Zingiberaceae
family. Different parts of the turmeric plant are utilized for various purposes. The rhizomes
are especially valuable due to their unique biological properties, colour, and scent. The leaves
of turmeric serve as protective covers for food while cooking. Turmeric is composed of
volatile and non-volatile compounds. Volatile compounds are extracted from raw materials
like essential oils, while non-volatile compounds, such as turmeric powder, are commonly
used as spices. This research focuses on extracting essential oils from the rhizome to
highlight the volatile substances present. Turmerone, Turmerone, Tr-Curcumin, and Curlone
are key components in turmeric oils, contributing to their antioxidant and antimicrobial

properties. Turmeric offers various health benefits, including anti-inflammatory, antitumor,

and antiprotozoal properties, making it a popular ingredient in the cosmetics and
pharmaceutical industries [7,8].

Fig.2: Curcuma longa

Azadirachta indica, also known as Neem, is a plant from the Meliaceae family. It is used in
traditional medicine for its therapeutic properties. Neem has antibacterial, antifungal, and
antiviral activities. It also has various pharmacological benefits such as being an antioxidant,
antimalarial, anti-inflammatory, and anti-diabetic. Neem leaves can be used to treat eczema,
acne, diabetes, and other conditions. Our study aims to explore the ANTIBACTERIAL AND
ANTIFUNGAL ACTIVITIES of neem leaves on harmful bacteria [8,9].
 Fig.3: Azadirachta indica
Materials and methodology

Strains used
Escherichia coli- The bacterium Escherichia coli, also known as E. coli, is facultatively
anaerobic, rod-shaped and gram-negative. Theodor Escherich originally reported this
bacterium in 1885. As a typical part of the gut flora, the majority of E. coli strains
inadvertently invade the digestive tracts of humans and animals. But certain strains of E. coli
have developed into dangerous strains by gaining virulence factors from bacteriophages,
transposons, plasmids, and/or pathogenicity islands [9]. A large number of E. coli genomes
have been sequenced; these genomes show variations in size and genetic diversity between
commensal and pathogenic bacteria, suggesting a wide range of variations within the same
bacterial species. They are made up of commensals, or non-pathogenic microorganisms [11].

Bacillus subtilis- Numerous Bacillus strains can be found in the environment, including the
human gut, soil, air, and fermented foods. The spore form of Bacillus probiotics can
withstand harsh environmental conditions, allowing for long-term life in settings that would
normally be fatal for vegetative bacteria. Probiotic Bacillus spores have been shown to
germinate, develop, and resporulate throughout the gastrointestinal tract. Numerous strains of
Bacillus, such as B. clausii, B. pumilus, B. polyfermenticus and B. subtilis, have been the
subject of recent investigations. Because Bacillus stains can transfer genes linked to antibiotic
resistance, enterotoxins, and biogenic amines (BA), employing them as probiotics has
significant drawback [12]. Bacillus species have the ability to release compounds that
promote plant development and health in addition to forming endospores that are incredibly
resilient to adverse environmental conditions. Therefore, the effective use of beneficial
microorganisms offers a blueprint for improving stress tolerance and climate change
adaptation [13].
Colonization of roots by Bacillus subtilis is beneficial to both the bacterium and the host
plant. By means of root exudates, plants release about 30% of the fixed carbon they make.
After bacteria colonize the roots, plants receive a supply of nutrients in return. The substances
and activities produced by the bacteria promote plant growth and protect the hosts from
stress. As a means of long-term colonization, Bacillus subtilis creates a thin biofilm on the
roots [14].

YEAST

Nutrient agar medium (Semi solid media) USED FOR ANTIBACTERIAL

ACTIVITIES
Beef extract -2gm
Sodium chloride- 1gm
Peptone- 2gm
Agar -3gm
Water -200ml
Chemicals used
Methanol

Collection of parts of medicinal plants

Ginger, neem and turmeric were gathered from different places OF TS. Ginger was collected
from the local market, while turmeric was collected in the college store. NEEM????? The
neem leaves, ginger, and turmeric rhizomes were then dried in room temperature. [2,14]
BOTANICAL AUTHENTIFICATION REPORTS WAS DONE FOR 3 PLANTS AT ……
(FIG NOS 5……..)
Extraction techniques

The active ingredients of the plants were systematically extracted by cold maceration
technique. Neem leaves, ginger and turmeric rhizomes were air-dried for a period of 3-4 days.
Neem leaves were grounded into a fine powder and dissolved in 100ml of solvent (methanol)
for 2 days. Ginger and turmeric rhizomes were peeled, cut into small pieces, dried and soaked
in 100ml of methanol for 2 days respectively. The mixture was stirred daily and after 48
hours, the ingredients were filtered using muslin cloth. [2,14]

Methanolic Extraction
After 48 hours of cold maceration, the solution was filtered using muslin cloth and the filtrate
was concentrated on the water bath to evaporate the methanol. [2,14] THEN THESE
EXTRACTS WERE USED FOR DETERMINING ANTIBACTERIAL AND ANTIFUNAL
ACTIVITIES.

CULTURE MEDIA PREPARATION

Procedure:
Inoculum preparation
Stock cultures of bacteria were created in the lab. The stock culture of gram-negative bacteria
Escherichia coli and gram-positive Bacillus subtilis were prepared. These cultures were
stored on nutrient agar media and kept at 4°C. An active culture was made in nutrient broth
for the experiment by transferring cells from the stock cultures into a test tube. The same
process was repeated for each sample of bacterial and fungal cultures. Agar disc diffusion
was carried out for the assay [15].
Method for Disc Diffusion:
The antibacterial properties of the provided samples were evaluated using a technique called
the disc diffusion method. First, a nutrient agar media was prepared by mixing it with
distilled water at a specific concentration. The media and petri plates were sterilized before
pouring the media into the petri plates and allowing it to solidify. After solidification, bores
were made in
Herbal extracts Zone of inhibition in E. Coli Zone of inhibition in Bacillus
subtilis

Ginger 12 1.15cm
Turmeric 1.1cm 1.15cm
Neem 1.2cm 1.2cm
the petri plates and the herbal extract was poured into them to check for antimicrobial
activity. The petri plates were then left undisturbed for 15-20 minutes before being incubated
in an incubator set at 37°C for 24 hours. The zone of inhibition was observed and the
diameter was measured to determine the growth of bacteria and the activity of that specific
concentration of
plant extract on the disc [16].
Table.1: Comparision of Zone of inhibition of different herbal extract
 12.1 E.coli Bacilus subtilis

Zone of Inhibition (mm)

11.9
11.7
11.5
11.3
11.1
10.9
10.7
10.5
turmeric Ginger Neem
E.coli 11 12 12
Bacilus subtilis 11.5 11.5 12

Figure. 4: Zone of inhibition of herbal extracts

Figure. 5: Zone of inhibition of neem, turmeric, ginger

Tests Ginger Turmeric Neem
a) Test for Reducing Sugars
 Benedict’s Test + + +
 Fehling’s Test + + +

b) Test for Monosaccharides

 Barfoed’s Test - - -

c) Test for Amino acids

 Ninhydrin test - - -

d) Test for proteins

 Biuret test - - -
 e) Test for Alkaloids
 Dragendorff’s test + + -

f) Test for Tannins

 Ferric chloride test - + +

Table.2: Preliminary Phytochemical Screening

 + means the phytoconstituents are present

 - means the phytochemicals are absent

ADD ANTIFUNGAL RESULTS IN TABULAR FORM

Results and Discussion
Plant extracts contain a large number of bioactive compounds. The phytochemical screening
is important and thus the screening of compounds exhibiting the antimicrobial activity is
necessary. The zones of inhibition are visualized on the plates (Table 5). This method is
considered to be convenient for obtaining the reliable information on the antimicrobial
activities.
The results of antimicrobial activity of methanolic extract of ginger, turmeric and neem is
given the above table. It shows that ginger and neem are equally effective against E. Coli and
neem is more effective against Bacillus subtilis (Fig 4). This indicates that these herbal
extracts have potential antimicrobial activity. Combination of these extracts might show
enhanced activity.

CONCLUSION
From the above study, it can be concluded that all the plant extracts showed promising
antimicrobial activity against harmful pathogenic strains. The methanolic herbal extracts of
ginger, turmeric rhizomes and neem leaves exhibit promising antibacterial properties against
E. Coli and Bacillus subtilis. Extracts exhibiting variations in the antimicrobial activity is due
to differences in the composition of bioactive compounds extracted depending upon the
polarity of the solvent.

Variations in the antibacterial activity of the extracts are attributed to the presence of a wide
range of active constituents. It is plausible that the combination of these extracts may lead to
enhanced activity compared to individual extracts. Therefore, these extracts can be utilized as
additives in food and as preservatives to regulate the growth of microorganisms, thereby
safeguarding the well-being of both humans and animals. Further research is going on in this
area to isolate and characterize the bioactive compounds of neem, turmeric and ginger which
may provide a scope of developing more effective drugs for combating infectious diseases.

The use of herbal preparations for food preservation, the treatment of infectious disorders
brought on by pathogenic bacteria, and the prevention of microbial deterioration of food
products has been suggested by research on herbal extracts. Potential uses for the isolated and
characterised active chemicals with biological activities of these herbal plants include the
development of a novel medication.

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