Journal of Population Therapeutics

& Clinical Pharmacology

RESEARCH ARTICLE
DOI: 10.53555/jptcp.v31i2.3783

PRELIMINARY PHYTOCHEMICAL ANALYSIS AND IN VITRO

BIOLOGICAL ACTIVITIES OF OTOSTEGIA LIMBATA LEAVES
ETHANOLIC EXTRACT AGAINST ORAL PATHOGENS
Khursheed Ur Rahman1*, Ghulam Mujtaba Shah1, Muhammad Ajmal Shah2, Muhammad
Ikram3, Muhammad Fiaz1, Jan Alam1, Muhammad Sajid4
1*
Department of Botany, Hazara, University, Mansehra 21300, Pakistan.
2
Department of Pharmacy, Hazara University, Mansehra 21300, Pakistan.
3Department of Pharmacy, COMSATS University Islamabad, Abbottabad Campus 22060,

Pakistan
4
Department of Agriculture, Hazara University, Mansehra 21300, Pakistan.

*Corresponding author: Khursheed Ur Rahman

*Email: khursheed823@yahoo.com

Abstract
In the present investigation, we have accessed Phytochemicals, HPLC, Antibacterial, Antifungal and
Cytotoxic activity of Otostegia limbata. It diverted our attention to one of its potent species to be
unveiled in this research. The focus of this study was to examine systematically its biological
activities and seek out its chemical constituents. Qualitative phytochemical analysis removed
phenols, glycosides, flavonoids, alkaloids, quinone, carbohydrates, amino acid, terpenes and
coumarins are presence while tannins, saponins and sterols are absence. In Quantitative the O.
limbata leaves parts for isolation of active phtometabolites namely alkaloids, sterol, flavonoids,
tannins and phenols. The HPLC analysis showed that the samples contained eight identified
phenolic compounds, of which Gallic acid, Catechol, Hydroxybenzoic acid, Caffeic acid were most
abundant. The antibacterial activity of ethanolic extract displayed the highest inhibition region
against Streptococcus mitis that was 35 ± 0.1 (ZOI±SD) and the lowest inhibition region against S
.aureus that was 14 ± 0.3 (ZOI±SD) in 200mg/ml ± SD.The antifungal activity resulted that ethanol
shows the maximum inhibition zone against A.fumigatus that was 29 ± 0.1 (ZOI±SD) and the
minimum inhibition zone against Aspergillus flavus that was 19 ± 0.4 (ZOI±SD) in 200mg/ml ± SD.
The ethanolic extract of research plant were exposed to cytotoxic assay at concentrations is
50µg/ml, 100µg/ml and 150µg/ml and their results were calculated that indicates that at 50µg/ml is
60%, 100µg/ml is 70% and 150µg/ml is 80%. The current study suggested that, after the isolation of
individual components, O. limbata be investigated for assessing biological activity.

Key word: Phytochemicals, HPLC, Antibacterial, Antifungal and Cytotoxic activity, Otostegia
limbata.

Key findings: The selected medicinal plants Otostegia limbata which is used in the tooth pain we
are chick the effect of antimicrobial activity against oral pathogens, effect of cytotoxic activity
against Brime shrimps and also check the photochemical screening tests.

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1. INTRODUCTION
Pakistan has received a priceless gift from nature in the form of medicinal plants. Since ancient
civilizations, medicinal plants have occupied a permanent position for treating a variety of diseases
(Anand et al., 2019). Natural materials and their preparations make a significant contribution to
solving practical issues for people, animals, agricultural, veterinary, food goods, cosmetics, and
other industries (Drasar and Khripach, 2019). The family Lamiaceae includes one of the well-known
genera Otostegia, which is geographically widespread around the world. There are roughly 4000
species and 220 genera in this group of flowering plants. Otostegia limbata, also known as Rydingia
limbata (Benth.) Scheen & V. A. Albert and Ballota limbata is a significant medicinal plant of this
genus (Scheen and Albert, 2017). The common names "Spin aghzai," "Chiti booti," "Chitti jharri,"
"Spin azghay," and "Bui" are used to identify certain plant species.. The plant's distinctive features
include a cluster of pale yellow flowers, oblong leaves with a thick pointed tip, pointy bracts, and a
tiny petiole. Plant species contain a variety of chemical components, including the acids ballotenic
and ballodiolic, limbatolide A, B, C, and D (Sadaf et al., 2016). The Otostegia limbata is commonly
used as an ethnomedicine in Pakistan for a variety of ailments, including jaundice, cancer, scabies,
boils, goitre, ulcer, cuts, wounds, dental issues, and animal diseases (Rosselli et al., 2019).
Traditional healers use fresh leaf infusion to treat conditions including acidity, hypertension,
depression, ulcer, jaundice, gum disease, and ocular infection in Pakistan's Azad Jammu & Kashmir,
KPK, Punjab, and Himalayan regions (Rashid et al., 2015). To meet our everyday basic needs,
nature has bestowed upon us a wealth of valuable treasures. Plants are among the most crucial
sources. Plants have been used for therapeutic purposes for a very long time. These ancient
medicinal plants are essential to complementary medicine since they are used to cure a variety of
ailments. The Indian subcontinent is home to a rich trove of various plant species with a variety of
practicable medicinal characteristics. Herbal medicines also play a major role for gums and oral
problems. Herbal medications have unique recommendations and a long history of respectability.
Herbal medicine, which was once used to treat heart conditions like heart failure, plays a vital part
in the management and treatment of disorders like digitalis, which contains cardiac glycosides
(Arora and Arora, 2021). Even in this cutting-edge, technological age, doctors still recommend a
variety of medications with botanical origins. Up to 10% of local communities around the world
employ medicinal plants to treat various illnesses, yet only 1% of these plants have been identified
by scientists. The Alkaloids, tannins, and flavonoids, among other secondary metabolites, are widely
distributed in plants with antibacterial characteristics. Because medicinal plants are less poisonous
and have less negative effects, they are utilised to treat a variety of ailments (Morrison et al., 1980).
The development of caries and periodontal illnesses is significantly influenced by germs on the
tooth surface, according to the movie Dental Plaque (Gamboe et al., 2008). Mutans streptococci
have the capacity to produce extracellular polysaccharides from sucrose, mostly water-insoluble
glucan, using the glucosyl transferase enzyme, which allows them to colonise the tooth surface and
start the production of plaque (Bankova et al., 1992). This sucrose-dependent adherence and
accumulation of cariogenic streptococci is important to the establishment of a pathogenic plaque.
The microbial composition of the plaque surrounding the gingival margin and subgingival area may
change from being dominated by streptococcus to being more Actinomyces species and more
capnophilic and necessary anaerobic bacteria, including Porphyromonas gingivalis (Aga et al.,
1994). These microbes appear to play a role in periodontal disease and root caries, respectively.
Therefore, antimicrobial treatments for certain oral pathogens, especially those that might alter
plaque production, could be very effective in preventing dental caries and periodontal disorders. The
plant's ethanolic extract has numerous pharmacological properties, including anti-inflammatory,
anaesthetic, and cytostatic effects in addition to antibacterial activity. Streptococcus mutans is
another bacterium that it is antibacterial for (Koo et al., 2000). There is, however, little information
available regarding its antibacterial efficacy against other oral pathogens or its impact on dental
plaque formation in vitro. Over the past few decades, there has been a noticeable growth of bacteria
that are resistant to antibiotics. Antibiotic overuse and abuse are the primary causes of the rising
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prevalence of resistant microorganisms worldwide. It's interesting to note that traditional medicine,
including herbal medicine, has long been used in developing nations for healthcare and numerous
studies have confirmed its efficacy in controlling a variety of infectious diseases (WHO, 2002).
Plant extracts made from the leaves, stems, and roots serve as a valuable resource for the discovery
of powerful and innovative antibacterial and biofilm medications (Essawi and Srour, 2000).
Otostegia limbata is a spiny, 40–60 cm tall shrub with many branches (Fig. 1). It is known as "spin
azghay" locally in (Lower Dir) and thrives in dry environments. It is widely grown in Kashmir and
throughout Pakistan. O. limbata is useful for treating wounds and is effective against ophthalmia,
gum, and skin problems (Hedge et al., 1990). This study focuses on the crude methanolic extracts,
water, and hexane fractions of aerial portions (leaves) from J. regia and O. limbata's anti-
pseudomonal activity against P. aeruginosa planktonic and biofilm forms in vitro (Kale et al., 2011)
and (Abbasi et al., 2010).

Figure 1a. Otostegia limbata (Benth.) Boiss Figure 1b. Herbarium specimen

2. MATERIALS AND METHODS

2.1 Collection and authentication of plants
The plant sample was collected from district (Mansehra) during session June 2021 and Identified
with the help of flora and taxonomist expert by Prof. Dr Ghulam Mujtaba Shah, Chairman,
Department of Botany, Hazara University Mansehra KP, Pakistan. After identification the voucher
Number (15060) was assigned to the plant species and specimen were deposited in the Herbarium of
Hazara University (HUP) for permanent record (Fig. 2). The plant materials were washed with tap
water, separated and dried in shade for 15 days. These materials were used afterward for phyto-
chemical, and biological activities of in-vitro biological screening i-e antimicrobial activities against
oral pathogen and cytotoxic potential activity. The plant material was powdered with the help of
electrical grinder. The Whatman filter paper was used after the muslin cloth to filter the extracts.
Rotary evaporation will be used at 40°C to remove extra solvent from the filtrate. Until further
examination, the extract was kept in a container of amber colour.

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2.2 Extraction of plants material

Plant extracts were prepared using microwave extraction technology, according to a previously
reported procedure. The microwave's power setting was set at 9000 W. There are three basic stages
to this process. In the first step, 750 mL of ethanol and 100 g of each plant powder were added to
separate beakers in a 1000 mL container. The microwave was on for 2 minutes, then off for 30
seconds while the beakers were in it. Five times these procedures were carried out. The same
process will be used to complete two additional cycles. The muslin cloth will be used to filter the
extracts first, and then Whatman filter paper. Rotary evaporation at 40°C was used to remove extra
solvent from the filtrate. The extracts will be kept till further analysis in a container of amber colour
(Farrukh et al., 2022).

2.3 Phytochemical Analysis of Otostegia limbata

2.3.1 Qualitative analysis
Different protocols used to detect the presence or absence of different classes of phytochemicals
(Phlobatannins, Cardiac glycosides, Quinones, Steroids, Saponins, Coumarins, Tannins and
Terpenoids). Presence of these chemicals was detected by production of different colours (Chung et
al., 1998).

2.3.2 Quantitative analysis

The spectrometere was used in quantitative phytochemical analyses of total alkaloid contents, total
saponins, total flavonoids contents, total tannins contents and total phenolic contents by using
standered protocol methods (Haq et al., 2016).

2.4 High performance liquid chromatography (HPLC) analyses

In 20 ml of methanol (62.5%) and 5 ml of HCl were used to extract the ground plant material (6M).
The extract was sonicated for 15 minutes and then refluxed in a water bath for two hours following
nitrogen purging (Muhammad et al., 2020). Before injecting into HPLC, filter the extract twice via a
0.2 m Millex-HV membrane filter. The Shimadzu LC-20AT HPLC system includes a column oven,
an auto-sampler, and a diode array detector (SPD-M20A). Utilized was an analytical column with a
guard column (KJO-4282, Phenomenex): Purospher Star RP-18 endcapped 5 m 100 A° (250 x 4.60
mm, Merck, Germany). The composition gradient programme was used with just minor alterations
with the mobile phase consisting of (A) 0.1% acetic acid and (B) methanol (Qasim et al., 2017). The
flow rate was 0.8 mL per minute. By contrasting the retention times and UV-Vis spectra of
chromatographic peaks with those of genuine reference standards at 280 nm, phenolic chemicals
were identified (Jamshed et al., 2019).

2.5 Antimicrobial activity

Anti-microbiological action 100 mg/ml, 150 mg/ml, and 200 mg/ml of crude extract were the
chosen concentrations. Standard antibiotics were employed as the drug of choice for the positive
control for various bacterial and fungal infections, and DMSO was utilised for the negative control.
Drugs that were in powder form had been accurately weighed and dissolved in the proper dilutions
to the necessary 200 mg/mL concentration. The antibacterial assays were evaluated using the agar-
well diffusion method. Agar Mueller-Hinton was employed to prepare the media (Enjalric et al.,
1987).

Test organisms
The Six bacterial strains (Streptococcus mutans (ATCC 25175), Streptococcus mitis (ATCC 23175),
Staphylococcus aureus (ATCC-6538), Pseudomonas aeruginosa (ATCC- 15442, Bacillus subtilis
(ATCC6633), and Escherichia coli (ATCC-25922), associated with dental infections were used for
antibacterial analyses. Similarly Aspergillus flavus (FCBP-0064), Aspergillus fumigatus (FCBP-
66), Aspergillus niger (FCBP-0198), Fusarium solani (ATCC 36031) and Candida albicans (ATCC
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26081) were used to detect antifungal activity. Cephradine 50µg was used as positive control for
antibacterial activity and same quantity of fluconazole used as positive control against fungal
strains. DMSO was used negative control against bacterial and fungal strains. The Department of
Microbiology at Hazara University in Mansehra, KPK, Pakistan and the Department of
Biotechnology at the University of Science and Technology in Bannu provided all the
microorganisms. Throughout the study, stock cultures of bacteria and fungi were kept in their proper
growth medium at 4 °C. Both antibacterial and antifungal studies were conducted using the agar
well diffusion method (Carron et al., 1987).

2.6 Cytotoxic activity

The cytotoxic activity was done by following standard protocol method (Meyer et al., 1982).

Required media
Brine shrimp eggs, sea salt, distilled water, a tray or container with partitions, plant extract, test
tubes, micro tips, and a magnifying glass.
Stock solution preparation
The 20 mg of Plant extract were dissolved in 2 ml of ethanol to create the stock solution.

Method
Following techniques allowed for the determination of the plant's potential for cytotoxicity. Brine
shrimp eggs were first placed in a plastic container or tray with a perforated partition and 3.8
grammes of sea salt was first dissolved in 1000 ml of distilled water. This media was then added,
and the container was placed at a temperature of 34–36 oC for one day to hatch the brine shrimp
eggs. As they emerged, the shrimp went to the opposite side of the container. Following the creation
of three concentrations 100 mg/ml, 500 mg/ml, and 1000 mg/ml stock solutions were added in
accordance with these concentrations and the test tubes were left for the remainder of the day to
allow the ethanol to evaporate. Next, 2 mg of sea salt was added to the test tubes to make the total
volume 5 mg, and ten newly hatched brine shrimp were then placed inside the test tubes using a
micro-pipette, and the tubes were then left The following day, using a microscope, the number of
alive and dead brine shrimp in each test tube was determined.

Statistical analyses
Data tabulated and analyzed by using statistic software statistic 8.1.

3. RESULTS
3.1 Qualitative phytochemical analysis of Otostegia limbate
Phytochemical analysis revealed that the crude extract of Otostegia limbate included many different
types of compounds such as phenolic and glycosides, as well as flavonoids and alkaloids, as well as
quinones, carbohydrates, amino acid, terpenoids and coumarins. However, the tannins, saponins and
sterols test results for the crude extract showed no change in colour (Table No.1)

Table No 1. Qualitative phytochemical analyses of ethanolic extract of Otostegia limbata

S.No Constituents Present (+) Absent (-)
1 Phenols +
2 Glycosides +
3 Tannins -
4 Flavonoids +
5 Alkaloids +
6 Saponins -
7 Quinones +
8 Sterols -
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9 Carbohydrates +
10 Amino acids +
11 TeT Terpenoids +
12 Coumarins +
Key = Negative sign (-) indicate absence, positive sign (+) indicate presence

3.2 Quantitative analysis of O. limbata

Quantitative phytochemical screening of the leaves parts of the O. limbate for isolation of active
phtometabolites namely alkaloids, sterol, flavonoids, tannins and phenols in (Table No.2).The
results revealed the bioactive constituents in leaves are alkaloids were in the range of (16.66 ± 1.33
mg/g) and sterol (14.68 ± 0.66 mg/g), flavonoids (11.5±0.33 mg/g), tannins is (14.30 ±0.10 mg/g)
and phenols is (56.73±0.25 mg/g).

Table 2. Quantitative analysis of Otostegia limbata. All values are mean ± SEM of three
determinations. All values are expressed in mg/g.
S.No Extract Alkaloids Sterol Flavonoids Tannins Phenol
1 OLL 16.66 ± 1.33 14.68 ± 0.66 11.5±0.33 14.30 ±0.10 56.73±0.25
Key = OLL= Otostegia limbata Leaves, ND= Not detected

3.3 High performance liquid chromatography (HPLC) analyses

The HPLC analysis showed that the samples compound is contained eight identified phenolic
compounds, of which Gallic acid, Catechol, Hydroxybenzoic acid, Caffeic acid were most abundant.
The Gallic acid is 12.11 ± 0.25, Catechol is 3.01 ± 0.07 and Caffeic acid 7.57 ± 0.92. The highest
compound is Gallic acid is 12.11 ± 0.25 while the lowest compound is Catechol is 3.01 ± 0.07

Table 3. Phenolic composition (mg g-1 dry weight) of Otostegia limbata

S.No Compounds Retention time O. limbata
1 Pyrogallol 12.557 n.d
2 Gallic acid 15.192 12.11 ± 0.25
3 Catechol 18.145 3.01 ± 0.07
4 Hydroxybenzoic acid 19.136 n.d
5 Chlorogenic acid 23.59 n.d
6 Caffeic acid 24.756 7.57 ± 0.92
7 Coumaric acid 30.039 n.d
8 Ferulic acid 31.835 n.d

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Figure 2. HPLC chromatograms of standard compounds i.e. Pyrogallol (1), Gallic acid (2),
Catechol (3), Hydroxybenzoic acid (4), Chlorogenic acid (5), Caffeic acid (6), Coumaric acid (7),
and Ferulic acid (8) and Otostegia limbate extract.

3.4 Antibacterial activity of Otostegia limbata

Figure 2 displays the Otostegia limbata ethanolic extract's antibacterial activity. Streptococcus mitis
showed a maximum inhibition zone of 35 0.1, while S. aureus showed a minimum inhibition zone of
14 0.3 in 200 mg/ml standard deviation. Antibiotics had a maximum zone of inhibition of 45 0.4
against P. aerogenose and a minimum zone of inhibition of 28 0.5 against S. aureus in 100 mg/ml
SD. Table 3 of the results shows the results for the plant extract and antibiotic in terms of standard
and mean deviation values. When used as a negative control, DMSO does not inhibit bacterial strain
development (Table No. 4).

Table 4. Antibacterial activity of Otostegia limbata

Tests Antibiotics
Microorganism ZOI(mm) ± SD Leaves ZOI(mm) Means ± SD
Bacterial Cephradine 100mg/ml OLLEE OLLEE OLLEE
strains ZOI(mm) 100mg/ml ± SD 150mg/ml ± SD 200mg/ml ± SD
S. mutans 38 ± 0.1 18 ± 0.2 20 ± 0.6 22 ± 0.4
S .mitis 42 ± 0.3 14 ± 0.6 29 ± 0.2 35 ± 0.1
S .aureus 28 ± 0.5 8 ± 0.4 10 ± 0.5 14 ± 0.3
P.aerogenose 45 ± 0.4 25 ± 0.5 29 ± 0.3 32 ± 0.5
B.substilus 43 ± 0.6 19 ± 0.3 20 ± 0.1 22 ± 0.2
E .coli 40 ± 0.2 17 ± 0.1 24 ± 0.4 32 ± 0.6
Key= S.mutans= Streptococcus mutans, S.mitis= Streptococcus mitis, S .aureus = Staphylococcus
aureus, P.aerogenose= Pseudomonas aeruginosa, B.substilus= Bacillus subtilis,
E .coli = Escherichia coli and OLLEE= Otostegia limbata leaf ethanolic extracts,
SD =Standard deviations)

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Key= PSLEE (Otostegia limbata leaves ethanolic extract)

Figure 3. Graphical representation of antibacterial activity of Otostegia limbata

3.5 Antifungal activity of Otostegia limbata

The greatest inhibition zone against Aspergillus fumigatus was 29 0.1, and the minimum inhibition
zone against Aspergillus flavus was 19 0.4, according to an ethanol extract of the antifungal activity.
Antibiotics had a maximum zone of inhibition of 36.66 0.4 against C.albicans and a minimum zone
of inhibition of 29.66 0.2 against A. niger in (table 4) of the results shows the results for the plant
extract and antibiotic in terms of standard and mean deviation values. DMSO is utilised as a
negative control and exhibits no growth suppression or resistance to fungi. (Table 5).

Table 5. Antifungal activity of ethanolic extracts of Otostegia limbata

Tests Antibiotics
Microorganism ZOI(mm) ± SD Leaves ZOI(mm) Means ± SD
Fungal strains Fluconazole OLLEE OLLEE OLLEE
100mg/ml ZOI(mm) 100mg/ml ± SD 150mg/ml ± SD 200mg/ml ± SD
F.flavus 35.33 ± 0.5 10 ± 0.2 12 ± 0.6 19 ± 0.4
A.fumigatus 32.33 ± 0.3 12 ± 0.6 15 ± 0.2 29 ± 0.1
C.albicans 36.66 ± 0.4 13 ± 0.4 15 ± 0.5 19 ± 0.3
A.niger 29.66 ± 0.2 16 ± 0.5 20 ± 0.3 21 ± 0.5
F.solani 30.33 ± 0.1 12 ± 0.3 15 ± 0.1 19 ± 0.2
Key= F.flavus = Aspergillus flavus, A.fumigatus = Aspergillus fumigatus, C.albicans = Candida
albicans, A.niger = Aspergillus niger, F.solani s= Fusarium solani and OLLEE= Otostegia
limbata Leaf ethanolic extracts,
S.D =Standard deviations)

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Figure 4. Graphical representation of antifungal activity of Otostegia limbata

3.6 Cytotoxic Brine shrimps assay

The cytotoxic activity of O. limbate extracts at various doses (50, 100 and 150µg/ml) was
conducted. It was shown that O. limbate extracts had a cytotoxic impact on brine shrimps when
evaluated for 72 hours under controlled conditions. Results after 24 hours show that brine shrimp
mortality is inversely related to extract concentrations. The O. limbate reported a maximum lethality
of 80 % at 150 µg/ml, as seen in the (table no. 6). The highest mortality was reported in 80 % at 150
µg/ml (Fig 5). The bioactive components in both plants make them more cytotoxic than plant
extracts.

Table 6. Cytotoxic activity of Otostegia limbata

Concentrations Total no Living Dead Shrimps Death %
µg/ml Shrimps Shrimps ± SD ± SD
OLLEE 10 4 ± 0.2 6 ± 0.3 60
50 µg/ml
OLLEE 10 3 ± 0.3 7 ± 0.1 70
100 µg/ml
OLLEE 10 2 ± 0.1 8 ± 0.2 80
150 µg/ml

Key=OLLEE= Otostegia limbata Leaf ethanolic extracts, SD =Standard deviations)

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Figure 5. Graphical representation of cytotoxic activity of Otostegia limbata

4. DISCUSSION
The qualitative and quantitative phytochemicals analysis of ethanolic extract was used for detection
of phytochemicals. Most of the phytochemicals (alkaloids, terpenes, coumarins, saponins, cardiac
glycosides, phlobatannins, flavonoids, quinone, steroids and tannins) were qualitatively and
quantitatively detected. Ethanol observed as a best solvent used for the extraction of different
phytochemicals. Our results are agreed with the findings of they compare different solvent for
phytochemical extraction and found that ethanol is the best solvent for extraction of different
phytochemical (Lezoul et al., 2020). Presence of these biologically active compounds shows the
medicinal value of Otostegia limbata as these phytochemicals have different medicinal properties.
Otostegia limbata's phytochemical analysis found that it contains phenols, glycosides, flavonoids,
alkaloids, quinones, carbohydrates, amino acids, terpenoids, and coumarins but not tannins,
saponins, or sterols, which are thought to be the phytochemicals that give plants their antimicrobial
properties (Anthony et al., 2010). Numerous biological processes, including antibacterial,
antioxidant, and inflammatory ones, have been connected to flavonoids. They are also known to be
able to suppress cell growth and regulate enzymatic activity. They are well recognised to act as a
plant's defence mechanism against encroaching diseases (Oikeh et al., 2020). Tannins bind to
proline-rich proteins to create complexes that prevent the creation of proteins in cells. It is
recognised that the combined effects of tannins, flavonoids, alkaloids, and saponins can stop
pathogen growth. Alkaloids are renowned for their anaesthetic, anti-inflammatory, and
cardioprotective effects (Javed et al., 2020). Tannins are significant phenolic substances that are
well known for their antibacterial properties. The capacity of tannins to precipitate proteins, block
the availability of the substrate to the bacterial cells, directly attack the microbial cells, and restrict
the uptake of iron by microorganisms is what gives tannins their specific ability to combat
dangerous bacterial diseases (Nwankwo et al., 2014). Coumarin's ability to suppress
anticholinesterase is what makes them beneficial for treating Alzheimer's disease. Phlobatanins used
to cure treating swelling, new wounds, and lymphatic diseases (Kiani et al., 2019). These chemicals
are found in P. stewartii, according to phytochemical tests, which suggests that this plant is used to
treat a variety of illnesses. The Otostegia limbata ethanolic extract show the significant activity
against all selected oral bacteria strains Table 3, (Streptococcus mutans, Streptococcus mitis,
Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, and Escherichia coli), this
might be due to the antibacterial compound present in our plant are well extracted by ethanol and
thus inhibiting the growth of selected bacteria. Our findings are similar to that codded (Philip and
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Mahalakshmi, 2019). Using the disc diffusion method, the antibacterial activity of three medicinal
plant extracts, including Azadirachta indica, Melia azedarach, and Spilanthes acmella, was
examined against Streptococcus mutans and Staphylococcus aureus.
In order to combat the bacteria that cause denture plaque, plant extracts may be a safe substitute for
dangerous medications and chemicals. Similar outcomes were categorized as well (Saquib et al.,
2019). They discovered that the two plants' ethanolic extracts had a growth-inhibiting impact on all
four strains of periodontal pathobionts. Tannerella forsythia was the target of C. zeylanicum's
highest antibacterial activity, whereas Aggregatibacter actinomycetemcomitans was the target of C.
zeylanicum's lowest antibacterial activity among all the groups under study (MIC = 12.5 3.25
mg/mL, MBC = 75 8.23 mg/mL, respectively). Candida albicans is significantly inhibited by
ethanol extract, although Aspergillus flavus is the fungus that is most resistant to our plant. This
action demonstrates the antifungal constituents' presence in O. limbata and their absorption by the
fungi strains. If A. flavus activity is lower than usual, it may be because this strain is resistant to the
component found in the chosen plant. Our findings concur with the findings (Mohammed et al.,
2019). They discovered that S. marianum extract works well against Candida species at 400-800
g/mL doses. As a result, it was discovered that the plant's fruit sections might be a natural source of
antifungal and antibacterial agents. The ethanolic extract of O. limbata showed zone of inhibition
against Aspergillus flavus, Aspergillus fumigatus, Aspergillus Niger, Fusarium solani and Candida
albicans.

5. CONCLUSIONS
By using qualitative phytochemical screening, it was discovered that Otostegia limbata possesses
active secondary metabolites such as alkaloids, terpenes, coumarins, saponins, cardiac glycosides,
phlobatannins, flavonoids, quinone, steroids, and tannins. According to the results of the
antimicrobial assay, Otostegia limbata plant ethanol extract is a useful tool for testing new
antimicrobial medications for the treatment of oral microorganism-related disorders. From the data
taken together, it can be inferred that HPLC is a flexible, repeatable chromatographic method for the
quantification of medicinal products. Regarding the quantitative and qualitative estimation of active
compounds, it has a wide range of applications in various sectors. At 150 g/ml of concentration, the
plant likewise exhibits the highest potential for cytotoxic activity. The high performance liquid
chromatography potential of Otostegia limbata yields impressive results.

Acknowledgments
The authors are grateful to the Faculty of Health and Biological Science Hazara University
Mansehra, Pakistan for providing the facilities for phytochemical analysis, Antimicrobial activity,
cytotoxic activity analysis and funding support for this research.

Conflicts of interest
There is no conflict of interest.

Data availability statement

All required data is provided in the manuscript; no external dataset is required or utilized.

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