‘‘Betel Leaf Ointment: Antimicrobial Activity”

A Dissertation submitted to the

Dr. Babasaheb Ambedkar Technological University, Lonere.

In partial fulfillment of the requirement for the award of degree of

Bachelor of Pharmacy
Submitted by,
PAWAR SAKSHI DADASO
Under the guidance of
Dr. Mangal S. Gaikwad
(M. Pharm, Phd)

AL- AMEEN EDUCATION AND MEDICAL FOUNDATION’S

DELIGHT COLLEGE OF PHARMACY
Sharad Campus, Pimple-Jagtap Road, Koregaon Bhima, Tal-Shirur, Dist-Pune

2024-2025

1
 AL- AMEEN EDUCATION AND MEDICAL FOUNDATION’S
DELIGHT COLLEGE OF PHARMACY
Sharad Campus, Pimple-Jagtap Road, Koregaon Bhima, Tal-Shirur, Dist-Pune

2024-2025

Certificate
This is to certify that investigations incorporated in this thesis titled, “Betel Leaf
Ointment: Antimicrobial Activity” submitted by Pawar Sakshi Dadaso for the partial
fulfillment of requirements of degree in Bachelor of Pharmacy in the Project work, University
of DBATU, Lonere. The research work was carried out in DELIGHT COLLEGE OF
PHARMACY, Koregoan Bhima Pune, under my guidance and supervision. The extent and
source of information / materials as has been obtained by candidate from other sources
has been duly acknowledged by her in the thesis. The research work of her is original and
Bonafide.

The project is now ready for examination. I hereby forward the same.

Date:
Place:

Guide
Dr. Mangal S. Gaikwad
(M. Pharm, Phd )
HOD
( Pharmacognosy and Phytocognosy)

2
 AL- AMEEN EDUCATION AND MEDICAL FOUNDATION’S
DELIGHT COLLEGE OF PHARMACY
Sharad Campus, Pimple-Jagtap Road, Koregaon Bhima, Tal-Shirur, Dist-Pune

2024-2025

Certificate
This is to certify that investigations incorporated in this thesis titled, “Betel Leaf Ointment:
Antimicrobial Activity” submitted by Pawar Sakhi Dadaso for the partial fulfillment of
requirements of degree in Bachelor of Pharmacy in the faculty of Pharmaceutical Sciences,
University of DBATU, Lonere, were carried out in DELIGHT COLLEGE OF
PHARMACY, Koregaon Bhima, under the guidance and supervision of Dr. Mangal S.
Gaikwad (M.Pharm,PHD), HOD (Pharmacognosy and phytochemistry), Delight College of
Pharmacy, Koregaon Bhima.

Date:
Place:

Dr. Sampat D. Navale

Principal
(M. Pharm, Ph.D.)

3
 AL- AMEEN EDUCATION AND MEDICAL FOUNDATION’S
DELIGHT COLLEGE OF PHARMACY
Sharad Campus, Pimple-Jagtap Road, Koregaon Bhima, Tal-Shirur, Dist-Pune

2024-2025

Statement by Candidate
I, the undersigned wish to state that the work presented in the current dissertation
titled, “Betel Leaf Ointment: Antimicrobial Activity” is my own contribution to
knowledge & research carried out under the guidance of Dr. Mangal S. Gaikwad and
HOD, Delight College of Pharmacy, Koregaon Bhima. The extent and source of
information / material as has been obtained from other sources has been duly
acknowledged in the thesis. The research work is original and has not been submitted in
part or full for any Diploma or Degree of this or any other University/Institute.

Date:

Place:

Research scholar
Pawar Sakshi Dadaso

4
 ACKNOWLEDGEMENT

The secret of success is undaunted ardor, motivation, dedication, confidence on self and above
all the blessings of God. I bow in reverence to the Almighty for bestowing upon me all this kindness that
has helped me throughout the journey of my life. Success is an outcome of collaborated efforts aimed at
achieving different goals. I hereby take this opportunity to acknowledge all those who have helped me in
the completion of this dissertation work.
With a sincere note of gratitude, I wish to express my deepest thanks, heartfelt indebtedness and
regards to my respected teacher and guide, , Delight College of Pharmacy, Koregaon Bhima, for
granting me the permission to carry out my Project work at Delight College of Pharmacy, Koregaon
Bhima. His rich experience, scientific vision, passion for knowledge, insightful direction and invaluable
advices has always been a source of personal inspiration for me. With his illuminating guidance and
incessant suggestions, I have been successful in keeping my spirits high throughout the pursuance of my
project from an industry.
I would also like to express my heart-felt thanks to our respected Principal Dr. Sampat D.
Navale for his invaluable guidance sturdy support and making available the entire infrastructure and
facilities.
I take this opportunity to express my heart-felt gratitude to Founder President Hon. and
Hon’ble Secretary for providing the infrastructure and all the facilities required to carry out this
research work.
Words fail to express my feeling for my whole family, whose overwhelming support and blessings
has made this work possible. My parents have been my constant source of inspiration throughout my
life. I would like to dedicate this project with inexpressible gratitude to my parents, my family.

To the whose presence we all just feel, the almighty God, whose love and caring hands always
surround us, I thank you.

Research scholar

Pawar Sakshi Dadaso

5
 DECLARATION

I certify that,

a. The work contained in the project is original and has been done by myself under the general
supervision of my supervisor.
b. I have followed the guidelines provided by the Institute in writing the project.
c. I have conformed to the norms and guidelines given in the Ethical Code of Conduct of the
Institute.
d. Whenever I have used materials (data, theoretical analysis, and text) from other sources, I
have given due credit to them by citing them in the text of the thesis and giving their
details in the references.
e. Whenever I have quoted written materials from other sources, I have put them under
quotation marks and given due credit to the sources by citing them and giving required
details in the references.
f. The work has not been submitted to any other Institute for any degree or diploma.

6
INDEX

Sr. Page
Chapter
No. No.
1 Introduction 8
2 Aim & Objective 19
3 Plan of Work 21
4 Review Literature 23
5 Methodology 25
Evaluation
6 28
Parameter
7 Results 31
8 Conclusion 33
9 References 35
10 Errata 39

7
INTRODUCTION

8
1.INTRODUCTION:

Betel Plant
Betel (Piper betel) is a species of flowering plant in the pepper family Piperaceae,
native to Southeast Asia. It is an evergreen, dioecious vine, with glossy heart-shaped
leaves and white catkins. Betel plants are cultivated for their leaves which are most
commonly used as flavoring in chewing areca nut.

Etymology

The term betel was derived from the Tamil/Malayalam word vettila via Portuguese.

9
Cultivation

Betel leaf and Areca nut consumption in the world. The betel leaf is cultivated
mostly in South and Southeast Asia, from India to Papua New Guinea. It needs a
compatible tree or a long pole for support. Betel requires well-drained fertile soil.
Waterlogged, saline and alkali soils are unsuitable for its cultivation.

Proper shade and irrigation are essential for the successful cultivation of this crop.
Betel needs constantly moist soil, but there should not be excessive moisture.
Irrigation is frequent and light, and standing water should not remain for more than
half an hour. Dried leaves and wood ash are applied to the furrows at fortnightly
intervals and cow dung slurry is sprinkled. Application of different kinds of leaves
at monthly intervals is believed advantageous for the growth of the betel. In three to
six months, the vines reach 150 to 180 cm in height, and they will branch. Harvest
begins with the farmer plucking the leaf and its petiole with his right thumb. The
harvest lasts 15 days to one month.

Betel Leaves in Pune market, Maharashtra India

10
Scientific Classification

Kingdom: Plantae

Clade: Tracheophytes

Clade: Angiosperms

Clade: Magnoliids

Order: Piperales

Family: Piperaceae

Genus: Piper

Species: P.betle

Botanical Name: Piper betle L.

Health Benefits

Some useful phenol compounds of betel leaf are significantly remarkable

bioactive compounds such as hydroxychavicol (HC) and eugenol. However, HC
and eugenol have been acknowledged for specific attention from researchers and
scientists for their pharmacological and biological performance. Hydroxychavicol
and eugenol were found to be antimutagents. Moreover, HC also displayed an anti-
inflammatory activity. It can also be utilized as a therapeutic mediator for
cardiovascular ailments. Eugenol and amphipathic hydroxyphenyl propene have
been described as preventing the devel opment of various negative & positive gram
microorganisms and molds due to the presence of free hydroxy form in its chemical
structure. Additionally, the

11
healing use of Piper betle L. can cure urinary incontinence, pharyngitis, tussis, lung
disease, skin abscess, and lazy colon. The flow of mother’s milk can be increased by
applying the betel leaf coated with mustard oil on the breasts of a breastfeeding lady.

Health benefits of betel leaf

Betel leaves also displayed antibacterial, anti-nociceptive, anthelmintic,

antiallergic, anticancer, anti hypercholesterolemic, antidiabetic, anti-platelet effects.
It has been reported that PBL leaves act as a dental hygiene agent in Asian countries
mainly due to antimicrobial properties. As for nutritional aspects, leaves are the
source of carbohydrates (0.5–6.10%). It also contains a small quantity of calcium
(0.2–0.5%) and fats (0.4–1.0%) along with a good amount of vitamin B complexes
like riboflavin (1.9–30 μ g/100 g) and thiamine (10–70 μ g/100 g) and vitamin E
(3.20–3.69 mg/100 g).

12
Chemical compounds present in betel leaf and its uses

13
14
Flavor Profile

1. Strong Flavor Varieties: Certain varieties are known for their intense flavor,
often preferred for their bold taste in paan preparation. Examples include
Bangla and Magahi varieties.
2. Mild Flavor Varieties: Other varieties may offer a milder flavor profile,
suitable for consumers who prefer a gentler taste. Examples include Desi
and Sanchi varieties.
Microscopy

This microscopic study involves more detailed examination of a drug and it

can be used to identify the organized drugs. The piper betel leaf microscopic study
was focused towards Leaf constants or Diagnostic characters of leaf:

1. Vein-islet number: It is defined as the number of vein-islets per sq.mm of the

leaf surface mid way between the mid rib and margin.

2. Vein-termination: It is defined as the number of veinlet terminations per sq.mm

of the leaf surface mid way between mid rib and margin.

3. Stomatal number: It is average number of stomata per sq.mm of epidermis of

the leaf.

4. Stomatal index: It is the percentage which the number of stomata forms to the
total number of epidermal cells; stomata being counted as one cell. It is calculated
by using the following equation. S.I=S/E+S*100 Where S.I=stomata index S= no.
of stomata per unit area E=no. of epidermal cells in the same unit area.

5. Stomata: A stomata is minute epidermal opening present on Arial parts of the

plants, with following characteristics I. A central pore II. Two kidney shaped similar
cells containing known as guard cells and varying number of subsidiary cells
covering the guard cells.

6.Trichomes: Trichomes are the hairy outergrowth of the epidermal cells of the
leaf.

15
7.Transversion section of betel leaf: The transverse section was carried out
through the mid rib region. The arrangement of tissues in transverse and
longitudinal sections and type of cells and cell contents are revealed by suitable
histological study of a crude drug with the aid of microscope.

Powder microscopy: The dried piper betel leafs are make a fine powder by using
mortar pestle and pass it on the sieve. Then it is used for the tests.

Test for starch grains: Take a pinch of betel leaf powder and to this add N/50 iodine
solution and focus on the microscope. The hemicelluloses blue stain is present means
it shows that presence of starch grains.

Qualitative Chemical Test:

The extract of piper betel was subjected to following chemical tests for the
identification of the chemical groups present in them.

Detection of alkaloids: A small quantity of the extract was treated with few drops
of dil.HCL and filtered. The filtrate was used following tests

Dragendraff’s test: The filtrate was treated with the Dragendraff’s reagent
obtained. Orange brown precipitate was obtained.

Detection of flavonoids:

Shinoda test: The extract was treated with 1gm of magnesium turnings and few
drops of conc. HCl and boiled for 5 min. the formation of orange colour shows
presence of flavonoids.

Detection of glycosides:

Test for cardiac glycosides:

Keller-Killani test: Extracts were dissolved in 3 ml of glacial acetic acid and add to
these 2 drops of ferric chloride. The contents are then transferred to a test tube
containing 2 ml of conc.H2S04. Re dish brown colour was formed at the junction of
two layers
16
Legal’s test: To the extracts add 1 ml of Sodium nitro prusside and NaoH. Colour changes
from pink to red were observed.

Test for Anthraquinone glycosides:

Borntrager test: about 1ml of extract was boiled with dil.H2SO4 and filtered. The
filter ate was extracted with chloroform. The chloroform layer was separated and
equal quantity of dil. Ammonia was added. Formation of pink or red colour in
organic layer indicates presence of glycosides.

Detection of carbohydrates:

Molisch’s test: The solution was treated with 2-3 drops of alcoholic alpha naphthol
and 2 ml of conc. H2SO4 was added along sides of the test tube appearance of violet
colour indicates the presence of carbohydrate.

Fehling’s test: About 2 ml of the solution was treated with 1ml of Fehling’s solution
and heat on the water bath. A reddish orange precipitate was produced to indicate
the presence of carbohydrate.

Detection of proteins and amino acids:

Biuret test: 1ml of solution was treated with equal volume of 5%NaoH and 1%
Cuso4 solution. A violet colour was produced.

Ninhydrin’s test: 1ml of solution was treated with ninhydrine reagent and heated,
purple colour was produced.

Detection of Phytosterols: A small amount of various extracts was dissolved in

5ml of chloroform separately. This solution was subjected to following tests.

Salkowski’s test: To 1ml of the solution, few drops of conc.H2SO4 added brown
colour was produced.

Libermann-Buchard’s test: The prepared chloroform solution was treated with 2

drops of conc.H2S04 acid, Followed by 3 drops of acetic anhydride. Emerald green
colour formed.

17
Detection of Saponins:

Foam test: The extract was diluted with 20 ml of distilled water and agitated in a
graduated cylinder for 15 minutes. Appearance of stable foam indicates the presence
of Saponins.

Detection of phenolic compounds

Test for phenolic compounds: To the extract, Dil. Ferric chloride was added, blue
colour was formed.

Test for tannins: To the extract 1% of gelatine and 10% of sodium chloride was
added then a white precipitate was formed. The presence of white precipitate in the
test solution, when treated with lead acetate solution indicates presence of tannins.
On treated with Bramer’s reagent, it produces a brown precipitate.

18
AIM & OBJECTIVE

19
2. AIM AND OBJECTIVE:

Aim:
To develop and evaluate the efficacy of a betel leaf ointment for wound healing
and skin care, leveraging the antimicrobial and anti-inflammatory properties of
Piper betle.

Objectives:
1. To formulate a stable and effective betel leaf ointment: Develop an ointment
with optimal concentration of betel leaf extract and suitable base.
2. To evaluate antimicrobial activity: Assess the ointment's antimicrobial
properties against various microorganisms.
3. To assess wound healing potential: Evaluate the ointment's efficacy in
promoting wound healing.
4. To ensure safety and stability: Conduct stability testing and assess potential skin
irritation or allergic reactions.
5. To explore potential applications: Investigate the ointment's potential use in
wound care, skin infections, and other dermatological conditions.

20
PLAN OF WORK

21
3.PLAN OF WORK :

Selection of topic

Literature survey

Selection of ingredient

Collection of ingredients

Formulation of ointment

Evaluation of ointment

22
LITERATURE
REVIEW

23
4.LITERATURE REVIEW:

Sr. Year of
Paper title and its author Name of journal
No publication
P. Tamilsankar, Dr. T. Gunasekar. Research Journal Of
Computer Aided Diseases Engineering And
1 2015
Technology.
Identification For Betel Leaf.

Devyani Chandrakant Kirve. An

2 Overview Of Betel Leaf : Green 2024 Journal Of Pharmacognosy
Gold Of India.
And Phytochemisty.

Arani Datta, Shreya Ghoshdastidar

and Mukesh Singh. International Journal of
3
Antimicrobial Property of Piper 2011 Pharma Sciences and
betel Leaf against Clinical Isolates Research (IJPSR)
of Bacteria

Devjani Chakraborty, Barkha Shah. International Journal of

4 Antimicrobial, Antioxidative and 2011 Pharmacy and
Antihemolytic activity of Piper
Pharmaceutical Sciences.
betel.

24
METHODOLOGY

25
5.METHODOLOGY :
MATERIALS :
The herbal plant material was collected from the local market. The betel leaf
were collected from local area market. While the chemicals and reagents used from
Delight College of Pharmacy Koregaon Bhima, Pune. Maharashtra, India-412216.

INGREDIENTS QUANTITY ROLE

Betel Leaf Extract 4g Active Ingredient

Polyethylene 1g Binder
Glycol
Methyl Paraben 0.09g Preservatives
Bees Wax g Emolient
Coconut Oil 2ml Moisturizer
Rose Oil 2ml Fragrance
Petroleum Jelly 1.5g Lubricant
TABLE: Formulation Of Betel Leaf Ointment.

METHODS:

STEP 1: Betel Leaf Extraction

Method: Soxhlet Extraction

1. Take 50g of dried powder of betel leaf.
2. Place in soxhlet thimble or wrap in filter paper.
3. Add 200 ml of ethanol for extraction.
4. Assemble the soxhlet apparatus.
5. Start heating gently; the solvent will evaporate ,condense, and extractthe
compound cyclically.
6. Run the cycle for 6-8 hours , until the solvent in the siphon becomes colourless.
7. Store the filtered extract in a sterile container for formulation.
26
STEP 2: Ointment Preparation
1. Melt bees wax and coconut oil in a beaker.
2.Add polyethylene glycol to the melted mixture and stir until dissolved.
3.Add betel leaf extract to the mixture and stir well.
4. Add methyl paraben to mixture and stir until dissolved.
5.Add rose water and allow the mixture to cool and solidify at room temperature
6. Fill the container into clean, dry containers.

Fig: ointment preparation Fig: Extraction Of Betel Leaf

27
EVALUATION
PARAMETER

28
6.EVALUATION PARAMETER:

(A) PHYSICAL PARAMETERS:

1. APPEARANCE & COLOUR:
Result: The ointment was found to be smooth,uniform & light greenish to
brown in colour, confirming proper mixing of ingredients.

2. ODOUR & TEXTURE :

Result : The ointment exhibited a mild herbal fragrance and had a non-sticky,
smooth texture.

3. PH MEASUREMENT:

TEST NAME : PH Test

Procedure: 1g of ointment was dissolved in 10ml of distilled water, and the PH

was measured by a PH meter.

Result: The PH of ointment was found to be 4.5 ,which is suitable for therapeutic
application.

4.SPREADABILITY:

TEST NAME: SPREADABILITY Test

Procedure: A specific amount of betel leaf ointment is placed between two glass
plates, a weight is applied, and the diameter of the spread ointment is measured to
calculate spreadability.

Result: The betel leaf ointment showed satisfactory spreadability with a measured
diameter of 6 cm, indicating ease of application and uniform coverage.

29
5. IRRITANCY TEST:

Procedure: The betel leaf ointment underwent a patch test on human skin.

Result: The betel leaf ointment showed no significant irritation or adverse

reactions, indicating good skin tolerance.

(B) ANTIMICROBIAL TEST:

Procedure:
The agar well diffusion method is used, where Betel leaf ointment is placed in a
well on an agar plate inoculated with microorganisms,( Staphylococcus aureus)
and the zone of inhibition is measured after incubation.

Result: The Betel leaf ointment showed significant antimicrobial activity against
tested microorganisms, with a zone of inhibition against Staphylococcus aureus.

Fig: Microbial Test For Betel Leaf Ointment

30
RESULT

31
7.RESULT:
Sr.no Physical Parameter Observations
1. Appearance Greenish colour
2. PH 4.5
3. Homogeneity Homogenous
4. Irritancy test Not irritant
5. Consistency Smooth semisolid texture
6.. Washability Easily removed by water
7. Antimicrobial Test activity Significant
antimicrobial

Fig: Formulation Of Betel Leaf Ointment

32
CONCLUSION

33
8.CONCLUSION:

The betel leaf ointment has emerged as a potential natural remedy with promising
therapeutic applications, showcasing a unique blend of traditional knowledge and
modern pharmaceutical potential. Its significant antimicrobial activity,
demonstrated through rigorous testing, highlights its capacity to combat a range of
pathogens, making it an attractive option for wound healing and skin infections.
Additionally, the ointment's good spreadability and suitable consistency ensure
ease of application and uniform coverage, enhancing its practicality for topical use.
Furthermore, the absence of notable irritancy in patch tests supports its safety
profile, alleviating concerns about adverse reactions. With its natural ingredients
and potential benefits, the betel leaf ointment warrants further investigation to fully
explore its efficacy and safety in humans. As research continues to uncover the
properties and potential applications of this ointment, it may offer a valuable
alternative to conventional treatments, aligning with the growing interest in natural
and holistic healthcare solutions. Ultimately, the betel leaf ointment represents a
promising avenue for innovation in natural therapeutics, with potential
implications for skincare, wound management, and beyond.

34
REFERENCES

35
9.REFERENCES:
1. P. Tamilsankar, Dr. T. Gunasekar. Computer Aided Diseases
Identification For Betel Leaf. International Research Journal Of
Engineering And Technology. 2015;02(09): 2577-2581.

2. Rakesh Kumar Gupta, Proshanta Guha, Prem Prakash Srivastav.

Phytochemical And Biological Studies Of Betel Leaf (Piper betel L.):
Review On Paradigm And Its Potential Benefits In Human Healths. Acta
Ecologica Sinica. 2022.

3. Joshu Jakson , Gerome M. Romero, Diana Hawkins, Richard G.

Cornwall And Georgi L. Lukov. Identifying The Structural Components
Responsible For The Antiproferative Properties Of
Hydroxychavicol. MDPI
.2023;(3):552-560.

4. Devyani Chandrakant Kirve. An Overview Of Betel Leaf : Green Gold

Of India. Journal Of Pharmacognosy And Phytochemisty.
2024;13(2):240-248.

5. Indrajeet Kumar, Rahul Verma. Betel Plant Leaf Age And Disease
Detection Using K-mean Clustering. International Journal Of Innovative
Research In Science, Engineering And Technology. 2020;9(5).

6. Md Atikul Islama,b, In Min Hwangc, Naeem Khand, Ok Yeon Songa, Ji

Young Jeonga, Ji Hyeon Sona, Nargis Jamilae, and Kyong Su Kim.
Authentication of Leaves and Petioles of Piper betle L. Varieties via
Elemental Composition and Multivariate Chemometric Analysis . ISSN.
2020.

7. Kambham Venkateswarlu1, N.Devanna, N.B.L.Prasad. Microscopical

36
 and Preliminary Phytochemical Screening of piper betel. Pharma Tutor
Magazine. 2014;2(4):112-118.

8. K. Y. Pin, A. Luqman Chuah, A. Abdull Rashin, M. A. Rasadah, C. L.

Law And T. S. Y.Choong. Solid-Liquid Extraction Of Betel Leaves.
Journal Of Food Process Engineering. 2011;549-565.

37
ERRATA

38
10. ERRATA:
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