PROJECT WORK PRESENTATION

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 Formulation and Evaluation of Herbal Antimicrobial cream

Presented by: Research Guide:

Mr. Sarth Manohar Doiphode/ Dr. V.C Bhagat
Miss. Pranjal Manoj Sanas (M.Pharm Ph.D.)
Roll Number: 20/62
(Final Year B. Pharm)
(Semester- VIII/ 2019 Pattern)

Name of Domain: Industrial pharmacy

RAJGAD DNYANPEETH’S COLLEGE OF PHARMACY, BHOR (PUNE)

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2023-2024.
 CONTENTS
1. Introduction
2. Review of literature
3. Need of study
4. Aim & Objective
5. Material and methodology
6. Result and discussion
7. Conclusion
8. Summary
9. References
10. Future scope
11. Annexure

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 1. INTRODUCTION

• The skin is also known as cutaneous membrane which cover outer surface of body. Is the biggest organ of body in
weight accounting for 15% of body weight approximately (6-9 kg)

• The skin have three main functioning layers:

• Epidermis
• Dermis
• Hypodermis

Figure no 1-
Anatomy of skin

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• Epidermis:
It is the outermost layer of skin which acts as protective layer which has thickness of 0.1mm it mainly consist of
four types of cells Keratinocytes, Melanocytes, Merkel cells and langerhan cells

• Dermis:
Immediately below the epidermis the Dermis is located it is the largest portion of skin which has thickness of
1-4mm. It is made up of blood and lymph vessels, nerves, sweat and sebaceous glands and hair roots.

• Hypodermis:
It is the innermost layer of skin mainly composed of Adipose tissue, blood vessels and connective tissue It may
varies in thickness across the body.

• Functions of skin:

1. Temperature regulation
2. Acts as sensory organ
3. Synthesis of vitamin D
4. Protection of internal organs
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• The skin is the principal barrier against microbial invasion. It constantly interacts with the external environment
and is colonized with a diverse population of microbes.

• Bacteria, initially in low numbers, colonize different layers of the skin (ie, epidermis, dermis, Hypodermis). As
bacteria increase in number where it can disrupt the skin barrier, invasion by these colonizing bacteria ensures a
skin infection

• Skin serves as the first line of defense against microbial infection as a physical barrier; by secreting low pH,
sebaceous fluid and fatty acids to inhibit growth of pathogens, however some microbes can penetrate the skin
barrier. Infecting organisms may cause tissue damage and may incite an inflammatory response.

• The typical organisms that colonize the skin are usually Gram-positive species such as Staphylococcus
epidermidis, Corynebacterium species, S aureus and Streptococcus pyogenes. The later two species are
particularly significant because they contribute to a majority of skin infection.

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Tridax procumbens L.

figure no 2,- Tridax procumbens

• It is commonly known as coatbuttons or Tridax daisy is a species of flowering plant in the family Asteraceae. It is
best Known as widespread weed and pest plant. It is native to tropical Americas but has been introduced to
tropical and subtropical and mild temperature regions.

• Scientific classification
Kingdom: Plantae
Class: Asterales
Family: Asteraceae
Genus: Tridax
Species: T. procumbens
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Botanical name: Tridax procumbens
Chemical constituent:

• The plant is rich in minerals such as iron, copper, manganese, sodium and zinc and other trace minerals such as
magnesium, phosphorous, potassium, selenium and calcium. The Alcoholic extract contains phytochemicals
such as alkaloids, steroids, flavonoids, saponins and tannins.

Use in traditional medicine

• Traditionally Tridax procumbens has been in use in India for wound healing and as an antifungal agent
The juice extracted from the leaves is directly applied on skin.

• The plant is also used as treatment for boils, blisters, and cuts by local healers in parts of India.

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Calotropis procera

figure no 3 - Calotropis procera

• Calotropis procera is considered as a medicinal plant, surrounded throughout India and in other tropical area.
Calotropis procera is native to Africa, Arabian Peninsula, Western Asia, the Indian Subcontinent, and Indo-
China

• Scientific classification
Kingdom: Plantae
Class: Asterids
Family: Apocynaceae
Genus: Calatropis
Species: C.procera
Botanical name: Calatropis procera 9
Chemical Constituent

• Several researchers have reported the presence of metabolites such as flavonoids, tannins, saponins, steroids, in
leaves of Calotropis procera. The plant also contains of several heavy metals such as manganese, lead,
chromium, iron, copper, nickel, cobalt, and cadmium

Use in traditional medicine

• Calotropis procera is a well known plant and has been traditionally used for skin disease and the leaf part is
used to treat jaundice. There are many reports on the geographical distribution, habitat, and morphological
characters of the plant. However, less work has been carried out on the leaves of this plant, which contain
potentially useful medicinal drugs.

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 2. REVIEW OF LITERATURE
1. Falguni K. Sheth et al.(2011): this article deals with classical and ethnic literature on the use of Calotropis
spp. on dermal fungal affections (Ringworm, Athlete’s foot) was tested. Author noticed the great prevalence of
skin disease in the contemporary society. It appears that the humid atmosphere of south Gujarat perhaps plays a
role in the precipitation of these diseases. It was also noticed that the fungal infections are very difficult to treat.
It was also learnt that though Calotropis spp. Are highly hailed in the classic and ethnic literature, for skin
ailments including fungal infections.

2. Vivek P. Navale et al. (2022): The article tells about Tridax procumbens It’s normally seen within the waste
lands and on roadsides. Ethnomedicinally this plant is also utilized in the treatment of varied style of diseases.
The plant shows various pharmacologic activities like antidiabetic, anticancer, medicine activity, anti- rheumy
activity, anti-inflammatory, immunomodulatory effects, anti-ulcer activity, inhibitor and hepatoprotective
activity. The preliminary phytochemical investigation showed the presence of flavonoids, saponins, alkaloids,
carotenoids and tannins.

3. Ranganathan K et al. (2023):The present study was to formulate and evaluate the herbal cream comprising
extracts of different medicinal plant experimental Using a Soxhlet apparatus, an aqueous extract of all the chosen
plants (Morinda Tinctoria, Tridax Procumbens) was created through a series of solvent extraction steps. A
topical herbal cream was created and tested for several qualities
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4. Deepika Chittoriya et al. (2020): Article deals withThe antifungal activity of medicinal weed plant Tridax
procumbens was studied against the fungal pathogen Helminthosporium sativum isolated from Luffa acutangula.
Leaf of the weed plant was screened with three different solvents including aqueous and two organic solvents of
ethanol and methanol.

5. Ashok Babu VL et al. (2022): This article tells about Fungal infections which have been a major concern across
the globe. The primary target of infection is skin. Most of the fungi causes skin diseases like candidiasis, eczema,
dermatitis, etc. Fungal infections are caused by fungus. The article intends to develop and evaluate the herbal
antifungal preparation which effective against various species of fungi.

6. S.Nagarju et al. (2020)- This article gives idea about tridax procumbens is widely used locally in many parts of
india. This is rich in alkaloids, steroids , carotenoids , flavanoids such as catechin, centaurein and bergenins and
minerals. The extract is used in diabetes , arthritis and applied to open wounds.

7. Vinod gubbiveeranna et al. (2016): The study tells us about Tridax procumbens which is a plant used majorly in
Indian traditional medicine. This is rich in alkaloids, steroids, carotenoids, flavonoids (such as catechins, centaurein
and bergenins), fatty acids, phytosterols, tannins and minerals. Concoctions of extracts from T. procumbens leaves,
stem, flower, and roots are used to treat patients suffering from diabetes, arthritis, inflammatory reactions and even
applied to open wounds.
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8.Lokesh prasad et al. (2017)- Plant derived substances and herbal medicines have recently attracted the
great interest towards their versatile application as medicinal plants are the richest source of bioactive
compounds used in traditional and modern medicine.

9.Dhotre Bhagyashree et al.(2020)- The main aim of present study herbal gel formulation containing extract
of plants for multi purpose property.

10.B.L. Aliero et al. (2012)- The result of this study validates the use of water extract of this species in
ethnomedicine and could provide a lead in isolation of anti bacterial agents from water extracts of calotropis
procera.

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 3. NEED OF STUDY
1. Worldwide there are number of infections are present like fungal, microbial, Viral (Corona) so we tried to
attempt to formulate the cream having maybe antimicrobial activity with lesser side effect

2. The combination these two extract shows the synergism effect means the combined effect of these two extracts
have greater effect than the individual effects of them.

3. There are number of methods use to prepare the extracts but we choose the hydroalcoholic extraction method.

4. The study on plant parts like Caltropis procera have lesser information and the least research has done on
individual so this study gives idea about it.

5. The reason behind the topic is to check the effect of antimicrobial study of Tridax procumbens and Calotropis
procera.

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 4. AIM & OBJECTIVES

The Aim of the study:

Formulation & Evaluation of herbal antimicrobial cream of Tridax procumbens and Calotropis procera.

The Objectives of the study:

1. Collection and authentication of plants material.

2. To prepare the extract of leaves of the plants by Soxhlet apparatus.
3. To perform phytochemical study and characterization of extracts.
4. To formulate and evaluate herbal antimicrobial cream of extracts of Tridax procumbens and Calotropis
procera .
5. To perform the anti microbial activity of cream.

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 5. MATERIAL AND METHODOLOGIES
1. Process of collection - Collection of plants of Tridax procumbens was from Bhor, Shirwal and Calotropis
procera was done from RDCOP Bhor Campus .
2. Drying Procedure – After the collection of the plants the suitable plant part ( leaves ) are cleaned
thouroughly cleaned, the Calotropis procera needs to hold for some time as leaves pass the sticky liquid
should be passed as it decrease the activity and after that they dried ,grind the properly for extraction.
3. Authentication- As the given plants dried in sheet of paper and send it for authentication to Botanical
survey of India Pune and received the certificate as given.
4. Method of Preparation of extract- The soxhlet extraction method was used to perform the extraction
process
a. Tridax procumbens- The extraction of plants is hydroalcoholic and done with ethanol and water with ratio
of 1:3 V/V and then obtained liquid extract is evaporated till the extract is obtained.
b. Calotropis procera- The extraction is done with ethanol and water with ratio 4:3 V/V for about 14 hrs and
then the liquid extract is taken on china dish and evaporate the water till the pure extract obtained after that
used for the cream formulation.
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5. Formulation of cream – The cream was prepared by using liquid paraffin, methyl paraben, bees wax, borax
and
water. The oil phase is made by melting bees wax on water bath after melting the liquid paraffin was added
and temperature was maintained at 700C. The aqueous phase was prepared by adding, methyl paraben and
borax in water and it was dissolved in water at 700C.Then the aqueous phase was added in oil phase while
constantly stirring and prepared extract was added simultaneously. After the cooling of cream the perfume
was added in cream and was packed in air tight container.

Formulation table – Role

INGREDIENTS QUANTITY
Anti-microbial activity
Tridax procumbens 3gm
Anti-microbial activity
Calatropis procera 2gm
Base
Bees wax 8gm
Preservative
Methyl paraben 0.2gm
Emollient
Liquid paraffin 25ml
Emulsifying agent
Borax 0.2gm
Vehicle
Water 10ml
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Fragrance
Figure 4 – Authentication certificate
1.Tridax procumbens
2. Calatropis procera

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Preparation of extract-

Figure 6- Prepared extracts

Figure 5- Extraction 19
 6. RESULT AND DISCUSSION
5. Phytochemical tests-

Test Methods Inference Result

1.Test for Tannins 2ml extract + 1% Yellow Tannins are

Lead acetate precipitate present.
solution

2. Test for 2ml extract + Black colour Phenols are

Phenolic few drops of Observed present.
compounds Ferric Chloride
solution
3. Test for 2ml extract + 1ml Yellow colour Alkaloids are
Alkaloids Hcl + Heat and precipitate present.
then Cool

4. Test for 1ml extract + Dark yellow Flavanoids are

Flavanoids Sodium solution present
hydroxide
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Figure no 7 Phytochemical study

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Thin layer chromatography of extracts

• Tridax procumbens: Extract was loaded onto 4 × 7 cm pre-coated silica gel plates using a capillary tube, and ethyl acetate:
ethanol: water (8: 1.2: 0.8) was used as the mobile phase the Rf value was 0.6

Figure no 8- TLC study T.procumbens

• Calotropis procera: Extract was loaded onto 4 × 7 cm pre-coated silica gel plates using a capillary
tube, and chloroform: methanol (4:1) was used as the mobile phase system. The Rf value was 0.62

Figure no 9- TLC study C.procera

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Uv study of extracts :

1. Tridax procumbens: For Uv analysis stock I solution of extracts was prepared by dissolving 10mg of extract
in 10ml of ethanol and stock II was prepared by dissolving 1ml of stock I in 9ml of ethanol. The Uv analysis
of extracts were carried out using Uv visible spectrophotometer (Jasco V-530) The lambda max value was
found to be 294 nm which conforms the presence of phytochemicals.

Figure no 10 Uv study T. procumbens

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2. Calotropis procera: For Uv analysis stock I solution of extracts was prepared by dissolving 10mg of extract
in10ml of ethanol and stock II was prepared by dissolving 1ml of stock I in 9ml of ethanol. The Uv analysis of
extracts were carried out using Uv visible spectrophotometer (Jasco V-530) The lambda max value was found to

be 332 nm which conforms the presence of phytochemicals.

Figure no 11 Uv study C. procera

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7.Evaluation of cream –

1. pH- the pH meter was calibrated and measured the pH by adding cream dissolved in ethanol of 10ml and pH
rod dip in it.

2. Viscosity – The viscosity of cream formulations were determined using brook field viscometer with result is

viscosity of 120cp at 50rpm.

3. Homogeneity- The test was done by physical touch with hands.

4. Appearance- The appearance of the cream was found yellow green etc.

5. Washability- The washability of cream was determined by applying the cream over the skin and the extend

of easy washing with water with minimal force to remove the cream.

6. Irritancy test- The cream was applied on left hand dorsal side surface of 1sq cm and observed in equal
intervals upto 24hrs for irritancy and redness.
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7. Accelerated stability studies- It were performed on all the formulations by maintaining at room temperature

for 20 days with constant time interval. During the stability studies the parameters like homogeneity,
viscosity, physical changes.

8. Anti microbial study: The active components such as tannins, flavonoids (gallic acid, quercetin and
kaempferol), ethyl esters (9, 12-octadecadienoic acid ethyl ester, 5 α-cholestane, hexadecanoic acid ethyl
ester
and 9-octadecenoic acid ethyl ester), unsaturated fatty acids, phenols, saponins and sterols are responsible for
antimicrobial activity observed the cream showed moderate antimicrobial activity.

Figure no 12 Antimicrobial study Figure no 13 Antimicrobial study 26

Antimicrobial study

Sr. no Sample Concentration Zone of Zone of

inhibition inhibition(mm)
(mm) Candida
S.aureus albicans
1 Control
2 Standard 1mg/ml 42 4
Streptomycin
Fluconazole
3 A 10mg/ml 8 8

4 B 10mg/ml 10 2

5 C 10mg/ml 2 12

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Evaluation of cream-

Sr. No Evaluation parameter Observation

1 Colour Light green

2 Odor Perfumed odor

3 Homogeneity Homogeneous

4 Appearance Smooth

5 pH (6.5-7.72)

6 Skin irritation No irritation

7 Viscosity 120cp

8 Antimicrobial study Moderate activity

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 7. CONCLUSION

• The Herbal anti microbial cream with the best properties and having beneficial value was prepared by
simple mixing methods.

• The herbal extracts was prepared using Soxhlet apparatus

• The herbal extract shows presence of alkaloids, glycosides, flavonoids, tannins which may be
responsible for anti-microbial, which gives the betterment to herbal cream.

• TLC study and Uv spetra study of extracts conforms presence of various phytochemicals which also
may be responsible for antimicrobial activity

• The prepare Herbal cream, showed good Homogencity and consistency in study.

• The prepared formulation show moderate antimicrobial activity

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 8.SUMMARY

1. There are many formulations are available in the market but they contain synthetic ingredients and as per the
previous research articles these synthetic ingredients cause side effect on the skin including skin infections,
irritation, redness, viral infections, fungal infections and so more.

2. To solve this problem, we attempted the formulation of herbal anti-microbial cream. These anti-microbial
creams contain natural ingredients such as Tridax procumbens, Calotropis procera and other ingredients.

3. we prepared the hydroalcoholic extract of these plants by use of Soxhlet apparatus then formulated the Herbal
cream by adding aqueous phase to oily phase.

4. The prepared anti-microbial cream was then evaluated for various parameters such as physical appearance,
Consistency, pH.

5. Performed anti-microbial and anti fungal test with agar well method and it shows that moderate activity
against bacteria( S.aureus) and fungus(Candida albicans). This cream does not show irritancy and any other
problems

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 9. FUTURE SCOPE

• Creams typically have a balanced consistency that lies between lotions and ointments. This texture makes
creams versatile and effectively moisturizing without being too heavy or greasy.

• There is need of stability study and clinical study.

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 10. REFERENCES

1. Falguni K. Sheth and Minoo H. Parabia et al.: Ethnobotanical studies and validation of lead: a case study on evaluation of
Calotropis sp. on dermal fungal infections International journal of pharmacy June (2011)
2. Hina Batoola, Mumtaz Hussaina, Mansoor Hameeda, Rashid Ahmad et al: a review on calotropis procera its phytochemistry
and traditional uses Big data in agriculture (2020) 29-31
3. Atish K. Maldhure, Nilima A. Kalambe , Dnyansagar D. Bhokare et al: Antifungal activity of extract from the leaves of Tridax
procumbens Linn IJRBAT, Special Issue (2), Vol-5, July 2017
4. Deepika Chittoriya, Mamta Goyal and Shruti Ojha et al: Antifungal activity of leaf extracts of Tridax procumbens IJIRT Volume
7 Issue 2 July (2020)
5. Vivek P. Navale, Mayur K. Koljatkar , Prof. Gaurav Wadurkar et al Review on Tridax procumbens JETIR, Volume 9, Issue 12
December (2022)
6. Ashok Babu VL, Burhanuddin Madriwala, Jeevitha L, Merin Sara Mathew, Abhilash S, Anjani Singh and Shwetha K et al:
Development, characterization and in vitro antifungal evaluation of topical formulation of Tridax procumbens L. Leaf extract
Journal of Pharmacognosy and Phytochemistry (2022) 289-295
7. Lokesh Prasad MS, Kalaskar P Gurunath, SB Chandrasekar, Umashankar C, Pawar AT et al: Formulation and evaluation of
herbal formulations (Ointment, Cream, Gel) containing Tridax procumbens Journal of Scientific and Innovative Research
(2017) 97-100
8. Ranganathan K Jeevanandham S, Nandhini.A, Preethi.M, Rinshida P.A, Sakthivel.M, Mohammed Basith et al: Development
and evaluation of herbal cream JPTCP Vol. 30 No 19 (2023)
9. Shamim, Saad Ahmed and Lubna Fatima et al: Pharmacological actions and therapeutic uses of Aak (Calotropis procera): A
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Review The Pharma Innovation Journal 2019 40-47
10. Akhtar N, Shahiq uz zaman, Barkat AK , Haji M Khan S, Mahmood Ahmad 2011, Evaluation of various functional skin
parameters a topical cream of Calendula officinalis extract .

11. More BH et al.,2013 Evaluation of Sunscreen activity of Cream containing Leaves Extract Butaemonosperma for topical
application , International Journal Of Research in Cosmetic Science , page no 1-6.

12. Susan Budavari , ed (1989) Merek Index 11th ed page no -876Front Matter , Nomenclature Of Organic Chemistry :IUPAC
Recommendations and preffered names 2013.

13. Tortora GJ, Derrickson B: The integumentry system. United States of America , wiley publishers- 2013 page no- 157

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