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Abstract

Acne also known as acne vulgaris, is an inflammatory condition of the polysebaceous layer that is brought on by the buildup of keratin. Active oil glands under the skin cause acne. Androgen hormones function as stimulant for this activity. Cloves are herbal plants that contain essential oil chemicals. The chemical a composition of clove leaf oil is mainly composed of eugenol. Eugenol has pharmacological effects as antibacterial, analgesic and anti-inflammatory. Nanoemulsion is a nanocarrier that gets a lot of attention, because of its small particle size so that it can be thermodynamically stable and can increase drug penetration. Recent advances in nanoemulsion research have created new prospects in a number of industries, including pharmaceuticals, biotechnology, food, and cosmetics. This research was conducted by formulating clove leaf oil into nanoemulgel preparations, followed by evaluation of the preparations and testing of antibacterial activity against acne-causing bacteria. The result of the nanoemulgel formulation is pale yellow, smells of clove leaf oil, is transparent and homogeneous. Nanoemulgel viscosity 3434-3109 M.Pas, pH 6.15-6.41 and particle Size 75-340 nm. Physical studies of nanoemulgel been carried out for four cycles and there is no change in color, change in odor and phase separation. The results of the antibacterial activity of Staphylococcus epidermidis at NEG4 12.93 mm. The results of the antibacterial activity of Propionibacterium acnes at NEG4 18.58 mm. The results of the study concluded that clove leaf oil nanoemulgel with a was the best formula, because it is stable during storage and has the greatest antibacterial activity compared to emulgel.

Keywords

Clove leaf Oil, nanoemulgel, antibacterial and anti-acne.

Introduction

The skin is the first organ to be affected by external stimuli such as pain, touch, and other unpleasant sensations. As a result, the skin is more susceptible to disease. Acne is the most common skin disease worldwide. Acne also known as acne vulgaris, is an inflammatory condition of the polysebaceous layer that is brought on by the buildup of keratin. The etiology of this condition is influenced by the metabolites that bacteria like Propionibacterium acnes and Staphylococcus epidermis produce, which can react with sebum to exacerbate the inflammatory response. Active oil glands under the skin cause acne. Androgen hormones function as a stimulant for this activity. Acne has long been controlled with topical and systemic medications, including antibiotics and retinoids. In dermatology, antibiotic resistance, retinoid side effects, and drug allergies are on the rise. Alternative medicine based on medicinal plants has been investigated as a solution to this problem. Clove leaf is one of many herbal plants that are intended to treat acne. Clove plants contain essential oils in large enough quantities, both in flowers, bud and leaves. The main content of clove leaves is phenolic compounds, tannins, saponins, flavonoids, triterpenoids and alkaloids. The chemical composition of clove leaf oil is mainly composed of eugenol, which is 70 until 98% Eugenol compounds have pharmacological activities as analgesic, anti-inflammatory, antimicrobial, antifungal, antiseptic, and local anesthetics so that these compounds are widely used in the pharmaceutical industry. . The results of a previous study stated that clove leaf oil at a concentration of 20% had antibacterial activity against Propionibacterium acnes bacteria with an inhibition zone diameter of 18.58 mm. Nanoemulgel is a nanocarrier developed recently which has received a lot of attention due to its various benefits due to its small particle size that provides better absorption and increase drug bioavailability. Nanoemulgel is a thermodynamically stable system formed from aqueous phase, organic phase, surfactant and co-surfactant. It showed better nanoemulgel stability than conventional emulsions in flocculation, sedimentation, phase separation and creaming. Nanoemulgel drug delivery systems can be administered via different routes of administration, including oral, intranasal and topica. Emulsions and nanoemulsions differ mainly in particle size. The particle size in nanoemulgel is 75-340 nm. Nanoemulgel has a lower viscosity than hydrogel preparations, so it is able to produce proper dispersion and good dispersion. Advantages of nanoemulsion preparations when added to a gel base are that they are easy to apply and convenient to use so they are preferred by patients.

Different Layers Of Skin :-

The skin is composed of three main layers, the epidermis, dermis, and hypodermis, each with unique characteristics and functions.

       
            Figure No. 1. Layers of Skin.png
       

Figure No. 1. Layers of Skin

1.Epidermis: The epidermis is the outermost layer of the skin in humans and many other animals. It is composed of several layers of cells that provide a protective barrier between the internal tissues and the external environment. The main function of the epidermis is to prevent water loss and to protect against external damage, such as UV radiation, toxins, and microbes. The epidermis also contains cells that produce melanin, which is responsible for skin pigmentation, and cells that play a role in immune defence. The epidermis is the outermost layer of the skin and provides a protective barrier between the body and the external environment. It is composed of several layers of cells, including keratinocytes, melanocytes, and Langerhans cells, among others. The epidermis is responsible for the skin's colour, texture, and waterproofing, and contains sensory receptors that detect touch, pressure, and temperature.

2.Dermis: The dermis is the second layer of skin that lies beneath the epidermis. It is a thick layer of connective tissue that contains collagen fibres, elastin fibres, blood vessels, nerves, and various other structures. The dermis is responsible for providing strength, elasticity, and support to the skin. It also contains sensory receptors that are responsible for detecting touch, pressure, heat, and cold. The dermis plays a vital role in regulating body temperature and blood flow. Additionally, it contains cells that produce sweat and sebum, which are important for maintaining skin hydration and protection. The dermis is the middle layer of the skin and is composed of connective tissue, including collagen and elastin fibres. It contains blood vessels, nerves, and other structures that support the skin and provide it with nutrients and oxygen. The dermis is also responsible for regulating body temperature, detecting pain, and producing sweat and oil.

3.Hypodermis: The hypodermis, also known as subcutaneous tissue, is the deepest layer of the skin located beneath the dermis. It is primarily composed of adipose tissue, which serves as a layer of insulation and padding to protect the body's internal organs. In addition, the hypodermis plays a significant role in energy storage, as it serves as a reservoir for excess calories in the form of fat. The thickness and composition of the hypodermis can vary among individuals and can change over time due to factors such as age, gender, genetics, and lifestyle. The hypodermis is the deepest layer of the skin and is primarily composed of adipose tissue. It provides insulation and padding, helps to regulate body temperature, and serves as a reservoir for energy storage.

Acne

       
            acne.png
       

Acne is a skin condition that occurs when your hair follicles become plugged with oil and dead skin cells. It causes whiteheads, blackheads or pimples. Acne is most common among teenagers, though it affects people of all ages. Effective acne treatments are available, but acne can be persistent. The pimples and bumps heal slowly, and when one begins to go away, others seem to crop up. Depending on its severity, acne can cause emotional distress and scar the skin. The earlier you start treatment, the lower your risk of such problems. Acne signs vary depending on the severity of your condition:

  • Whiteheads (closed plugged pores)
  • Blackheads (open plugged pores)
  • Small red, tender bumps (papules)
  • Pimples (pustules), which are papules with pus at their tips
  • Large, solid, painful lumps under the skin (nodules)
  • Painful, pus-filled lumps under the skin (cystic lesions)

Acne usually appears on the face, forehead, chest, upper back and shoulders.

Causes

       
            Figure No. 2 Acne Causing to Skin.png
       

Figure No. 2 Acne Causing to Skin

How acne develops

Four main factors cause acne:

  • Excess oil (sebum) production
  • Hair follicles clogged by oil and dead skin cells
  • Bacteria
  • Inflammation

Acne typically appears on your face, forehead, chest, upper back and shoulders because these areas of skin have the most oil (sebaceous) glands. Hair follicles are connected to oil glands. The follicle wall may bulge and produce a whitehead. Or the plug may be open to the surface and darken, causing a blackhead. A blackhead may look like dirt stuck in pores. But actually the pore is congested with bacteria and oil, which turns brown when it's exposed to the air. Pimples are raised red spots with a white center that develop when blocked hair follicles become inflamed or infected with bacteria. Blockages and inflammation deep inside hair follicles produce cystlike lumps beneath the surface of your skin. Other pores in your skin, which are the openings of the sweat glands, aren't usually involved in acne.

Certain things may trigger or worsen acne:

  • Hormonal changes. Androgens are hormones that increase in boys and girls during puberty and cause the sebaceous glands to enlarge and make more sebum. Hormone changes during midlife, particularly in women, can lead to breakouts too.
  • Certain medications. Examples include drugs containing corticosteroids, testosterone or lithium.
  • Diet. Studies indicate that consuming certain foods — including carbohydrate-rich foods, such as bread, bagels and chips — may worsen acne. Further study is needed to examine whether people with acne would benefit from following specific dietary restrictions.
  • Stress. Stress doesn't cause acne, but if you have acne already, stress may make it worse.

Nanoemulgel

A type of structural fluid known as a nanoemulgel combines the characteristics of nanoemulsions and gels. Nanoemulgel are clear or translucent, thermodynamically stable dispersions of oil and water, with typical partical sizes ranging from 75 to 340 nm. Due to their large surface area and potential to improve drug solubility , bioavailability, and targeted distribution ,they are frequently used as drug delivery systems. Gels, on the other hand, are semisolid systems made up of a three-dimensional network of crosslinked polymer chains, and they can offer advantageous qualities like increased adhesion, extended residence time, and improved skin penetration. Nanoemulgel can offer a special set of benefits in drug delivery by combining these two systems, including increased stability, controlled release, and improved skin permeability. Nanoemulgel are a type of emulsion that has droplets in nanometer range. They are considered to have better stability and bioavailability than traditional emulsions due to their small droplet size. Nanoemulgel are commonly used in various industries, including the food, cosmetic, and pharmaceutical industries. Nanoemulgels are topical gels contain nanoemulsions. They are commonly used in dermatology for the treatment of skin conditions such as eczema, relief, pscoriasis and acne. They can also be used to deliver medications for pain relief, anti-inflammatory agents, and anti-infective agents. Nanogels are nanoparticles - based hydrogels that have been studied for their potential in drug delivery, tissue engineering and other biomedical applications. They are commonly used due to their biocompatibility , biodegradability and high drug loading capacity.

       
            Figure No. 3  Potent formulation component of nanoemulgel.png
       

Figure No. 3  Potent formulation component of nanoemulgel.

Advantages of nanoemulgel

Nanoemulgel preparations have various advantages over other topical as well as conventional preparation.                             

1. Incorporation of lipophilic drug

2 .Better loading capacity

3. Better stability

4. Controlled release

5. Better pharmacokinetic profile

 6. Better pharmacodynamics activity

7. Better patient compliance

8. Enhanced drug permeability through skin

  • Disadvantages of Nanoemulgel:

 1.Bubbles formed during emulgel formulation.

 2.For utilization in pharmaceutical application, surfactant used ought to be non-poisonous.

 3.Possibility of allergenic reactions.

 4.Skin irritation on contact dermatitis

4.Plan of Work

  1. Literature survey
  2. Procurement of drug and excipient.
  3. Preformulation studies.
  4. Phytochemical screening of drug extract.
  5. Formulation of Nanoemulgel.
  6. Evaluation Studies of Nanoemulgel
  7. Result and discussion.
  8. Summary and Conclusion.

5.Literature Survey

1. Yoan Dasawanti et al., was performed on the Nanoemulgel clove oil extract and conclude the aim of this study was the nanoemulgel of clove oil preparation has the particle size below 200nm.

2. W.E. Soliman et al., was performed on the enhancement of curcumin anti- inflammatory effect via formulation into myrrh oil- based Nanoemulgel.

3. B. Sgorbini et al., was performed on the determination of free and glucosidically-bound volatiles in plants. Two case studies: L menthol in peppermint and eugenol in clove (Syzygium aromaticum (L.) Merr. & L.M.Perry),” Phytochemistry .

4. Haro-Gonzalez et al., was performed on Clove essential oil (Syzygium aromaticum l.myrtaceae): Extraction, chemical composition, food applications, and essential bioactivity for human health.

5. M. S. Algahtani et al., was performed on Nanoemulgel for improved topical delivery of retinyl palmitate: Formulation design and stability evaluation.

6. Chellapa P, Mohamed AT, et al.,  was performed on Nanoemulsion and nanoemulgel as a topical formulation.

7. Aithal GC, Narayan R, Nayak UY. Et al., was performed on Nanoemulgel: A promising phase in drug delivery. Current Pharmaceutical Design.

8. Syamala U. et al., was performed on Development & optimization of allyl amine antifungal nanoemulgel.

9. Garg A, Gautam A, Ahmad J, Jain K, Komath S, Bano M et al., was performed on Topical nano-emulgel for skin disorders: Formulation approach and characterization. Recent Patents on Anti-Infective Drug Discovery.

6.Drug and Excipient profile

    1. Drug Profile:

Clove:

Cloves are the aromatic flower buds of a tree in the family Myrtaceae, Syzygium aromaticum. They are native to the Maluku Islands, or Moluccas, in Indonesia, and are commonly used as a spice, flavoring, or fragrance in consumer products, such as toothpaste, soaps, or cosmetics.

       
            Figure No. 4 Clove Oil.png
       

Figure No. 4 Clove Oil

Scientific nameSyzygium aromaticum

Biological source: Clove consists of Dried flower bud of Eugenia Caryophyllus

FamilyMyrtaceae

GenusSyzygium

KingdomPlantae

OrderMyrtales

Sub kingdom : Tracheobionta

Super division : Spermatophyta

Division : Magnoliphyta

Class : Magnoliopsida

Subclass : Rosidae

Clove oil is an essential oil that’s derived from clove trees. The clove tree, known as Syzygium aromaticum, is native to Southeast Asia, although today you may find it growing in other locations, too. Clove oil, which ranges in color from colorless to light yellow and has a strong, spicy aroma, has been used for centuries in a variety of applications.

    1. Excepient Profile:

6.2.1. Tween 80:

       
            Tween 80.png
       

Chemical Name : Tween 80

Molecular Formula : C24H44O6

Molecular Weight : 428.60g/mol

Melting Point : -25ºC

Odour : Mild Alcoholic

pH : 5-7

Colour : Amber

It is a Surfactant reduces the interfacial tension between the mixtures of two immiscible liquids and changes the dispersion entropy, thus stabilizing thermodynamically unstable emulsion system. Selection of appropriate surfactant for nanoemulgel is based on the safety, stability, high drug loading capacity as well as good emulsification properties.

6.2.2. Propylene Glycol 340 :

       
            glycol.jpg
       

Chemical Name : Propylene Glycol 340

Chemical Formula : C3H8O2

Molecular Weight : 76g/mol

Melting Point : 59ºC

Boiling Point : 188.2ºC

Colour : Colourless

Odour : Odourless

It is a Cosurfactant may combine with surfactant and help in the emulsification process by disrupting the interfacial film. It may also help in solubilization of oil.

 6.2.3. Aquadest ad :   Water is a aqueous solvents act as the aqueous phase in emulsion preparation.

6.2.4. Carbapol 934 :

IUPAC Name : 2-Propenoic acid

Melting Point : >300ºC

Molecular Weight : 102.13g/mol

 Carbapol 934 are widely used gelling agent for nanoemulgel. They increased the thickness of the formulation and may interact with the surfactant to modify the viscosity of the formulation . It is added to the nanoemulsion preparation to change the physical state of nanoemulsion formulation from liquid to gel, thus solving the problem of low spreadability, low viscosity, and poor skin retention issue of nanoemulsion.

 6.2.5. Methyl paraben and propyl paraben :  It is used to protect the formulation from microbial attack and increase the shelf life of formulation, preservatives are added in the preparation.

6.2.6. Triethanolamine :

       
            fig-1.jpg
       

Chemical Name : Triethanolamine

Chemical Formula : N(CH2CH2OH)3

Odour : Ammoniacal

Melting Point : 21.60ºC

Boiling Point : 335.40ºC

Molecular Weight : 149.190g/mol It is used as a pH Balance of the formulation.

7.MATERIALS AND METHODS

7.1 Preformulation Studies of Drug:

        
            Figure No .5 Clove Bud.jpg
       
         

Figure No .5 Clove Bud

Organoleptic Properties of Drug:

The organoleptic properties of Clove like colour, odour, length of clove bud, pH of clove oil, Compostion of clove oil, Solubility of Clove extract was done.

7.2 Phytochemical Screening of Drug :


Phytochemical

Tests

Tests for Carbohydrates

(a) Molisch’s test: 1 ml of plant extract was added to 0.4 ml of Molisch’s reagent. Afterwards, 1 ml of conc. Sulphuric acid was added along the side of the test tube. A purple colour indicates the presence of carbohydrates (starch).

(b) Benedict’s test: 1 millilitre of plant extract and 1 millilitre of Benedict's reagent were heated for 5 minutes. The presence of carbohydrates (disaccharides) was shown by the formation of an orange precipitate.

Test for Proteins

(a) Xanthoprotic test: 0.25 ml of nitric acid was applied to 1 ml of plant extract. Appearance of white precipitate indicated the presence of proteins.

(b) Biuret test: 1ml of plant extract was taken in a test tube followed by 4% NaOH and 1% CuSO4. Violet pink colour development indicated the presence of proteins.

Test for Lipids

(a) Glycerol test: To 1 ml of 1% CuSO4.5H2O solution, 5 drops of the plant extract were added and mixed thoroughly. Then it received 5 drops of a 10% sodium hydroxide solution. A clear blue solution was obtained which indicates the presence of glycerol.

(b) Sudan III test: To 1ml of plant extract, few drops of Sudan III solution were added. Appearance of red colour indicated the presence of lipids.

Test for Alkaloids

(a) Wagner’s test: 1 ml extract was treated with Wagner’s reagent; formation of brown reddish precipitate indicates presence of alkaloids.

(b) Dragendorff’s test: 2 ml of Dragendorff’s reagent was added to 1 ml of plant extract. Formation of orange white precipitate indicated the presence of alkaloids.

Test for Saponin

Foam test: 1 ml of plant extract was taken in a test tube with small amount of water. Sodium bicarbonate was added to it and shaken vigorously for 5 min. Formation of foam indicated the presence of saponins.

Test for Flavonoid

To 0.5 ml of plant extract, 5 ml dilute ammonia was added followed by the addition of 1 ml concentrated sulphuric acid. A yellow coloration that disappeared on standing indicated the presence of flavonoids.

Test for Resin

In a dry test tube, to 0.5 ml of acetic acid and 2 drops of conc. Sulphuric acid were added. a purple colour that turned violet in about 10 minutes.

 

7.3 Materials and Equipments used in Formulation of Nanoemulgel:


Table No.1 Role of Ingredients

Sr. No.

Ingredients

Role of Ingredient

1.

Clove Leaf Oil

Drug

2.

Tween 80

Surfactant

3.

Propylene glycol

Co- surfactant

4.

Methyl paraben

Preservative

5.

Propyl paraben

Preservative

6.

Carbapol 934

Gelling agent

7.

Triethanolamine

PH balance

8.

Aquadest ad

Aqoues phase

 


Table No. 2 Equipment’s

Sr. No.

Equipment’s

Company Name

1.

Magnetic Stirrer

REMI 1MLH

2.

Homogeniser

REMI

3.

pH meter

KONVIO – NEER

4.

Sonicator

REMI


7.4 Procedure:  Formulation of Nanoemulgel

  1. The process of making nanoemulgel using spontaneous emulsification method consisting of an aqueous phase and organic phase.
  2. The aqueous phase consists of tween 80 (surfactant) and distilled water stirred constantly at a speed of 5000 rpm.
  3. The organic phase consisted of clove leaf oil and PG 340 (cosurfactant) was stirred at a constant speed of 5000 rpm.
  4. The organic phase was injected into the aqueous phase with constant stirring at 5000 rpm and sonicated for 30 minutes until a clear preparation was formed.
  5. In a separate container, carbopol 940 was dispersed in distilled water, then triethanolamine was added and homogenized at 500 rpm to form a transparent gel.
  6. The final step, nanoemulsion was added slowly into the gel base and homogenized at 2000 rpm for 10 hours .
  7. The nanoemulgel formula is shown in Table 2

Table No. 3 Nanoemulgel Formula

Ingredients

NEG1

NEG2

NEG3

NEG4

Clove leaf oil (in gm)

2

2

2

2

Tween 80  (in gm)

4.5

5.5

6.5

7.5

Propylene Glycol340 (in gm)

3.7

3.7

3.7

3.7

Methyl Paraben  (in gm)

0.05

0.05

0.05

0.05

Propyl Paraben (in gm)

0.05

0.05

0.05

0.05

Carbapol 934 (in gm)

0.9

0.8

0.6

0.5

Triethanolamine (in gm)

0.05

0.06

0.04

0.05

Aquadest ad (in gm)

38.75

37.85

37.06

36.15

Total

50

50

50

50


7.5  Evaluation of Nanoemulgel :

7.5.1 Organoleptic test  : Organoleptic tests were carried out on nanoemulgel which were visually observed for changes in color, smell and shape .

7.5.2 Homogeneity Test  : The homogeneity test was carried out on nanoemulgel by applying a certain number of samples to a glass object. The sample must show a homogeneous arrangement and there are no visible coarse grains .

 7.5.3 Viscosity measurement : Viscosity measurement aims to determine the viscosity of nanoemulgel and preparations. Viscosity measurement using NDJ-8S viscometer. Viscosity measurement was carried out by placing the preparation in a 50 ml beaker and using an appropriate spindle.

7.5.4 pH Measurement :  Determination of the pH of nanoemulgel and was carried out using a pH meter by calibrating it first with a buffer solution (pH 7.01) and (pH 4.01) until the instrument showed the pH value.

7.5.5 Particle size measurement :  Particle size measurement was carried out by E- SEM Testing which is Scanning electron microscopy . Particle size testing was carried out to determine the size of the globules formed in nanoemulgel and .

7.5.6 Invitro Drug Release Test : It involves drug release from carrier and ultimately absorbing into the skin Invitro drug release is quite pivotal to predict invivo drug release. The Invitro study of drug release was performed through diffusion .The drug release tends to increase with increase in aqueous content .

7.5.7 Physical stability study of nanoemulgel : The purpose of conducting a cycling test is to determine the stability. Cycling test is an accelerated test by storing the preparation at 4±20C for 24 hours, then transferred to 40±20C for 24 hours. This treatment is 1 cycle. The treatment was repeated for 4 cycles and the phase separation was observed. Physical conditions after the experiment were compared with before the experiment.

7.5.8 Spreadability Test : One of the most important characteristics of an nanoemulgel is its capacity to spread. It is a crucial aspect in therapy and serves as an assessment of how easy it is to use. Topical therapy's efficacy is determined by the patient's ability to apply the formulation in an even layer to deliver a dose . A lower dose would not produce the desired results, while a higher dose could cause unwanted side effects. The spread ability of a drug's formulation is crucial to its administration of the correct dose. When analyzing a formulation's spread ability, essential parameters to consider are the rate and duration of shear caused during smearing, as well as the temperature of the target location.

7.5.9 Antibacterial activity : Antibacterial activity testing was carried out on nanoemulgel  preparations. Using the agar diffusion method (Kirby-Bauer), this test was conducted. A sterile petri dish was first filled with 0.1 mL of inoculum, and then 15 mL of nutrient agar (NA) media was added while maintaining a temperature of 45– 50°C and homogenized until solid. Each petri dish was put of paper disk that had the test solution dripcoated on it. After 24 hours of incubation at 36–37°C, then the diameter of the clear zone was measured using a caliper which was expressed in millimeters. Tests are carried out triple.

8.RESULT AND DISCUSSION

8.1 Evaluation of preformulation studies of drug :

Organoleptic Properties of Clove oil :

Colour of clove oil  : Pale yellow

Odour of clove oil  :  Aromatic

Shape of clove bud : Round headed Nail like shape    

 Length of clove oil : 13-19mm

pH  of Clove oil  : 5-6

Solubility of clove bud oil : From the solubility test we observed the following solubility result as table no.4


Table No.4 Solubility Test

Solvent

Solubility

Water

Immiscible

Ethanol

Highly Miscible 

Methanol

Miscible

Chloroform

Miscible

8.2 Evaluation test of phytochemical screening of clove oil drug:


Phytochemical

Test

Observation

Test for Carbohydrates

(a) Molisch’s test: 1 ml of plant extract was added to 0.4 ml of Molisch’s reagent. Afterwards, 1 ml of conc. Sulphuric acid was added along the side of the test tube. A purple colour indicates the presence of carbohydrates (starch).

(b) Benedict’s test: 1 millilitre of plant extract and 1 millilitre of Benedict's reagent were heated for 5 minutes. The presence of carbohydrates (disaccharides) was shown by the formation of an orange precipitate.

 

Present

+ +

 

 

 

 

 

Present

+ +

Test For Proteins

(a) Xanthoprotic test: 0.25 ml of nitric acid was applied to 1 ml of plant extract. Appearance of white precipitate indicated the presence of proteins.

(b) Biuret test: 1ml of plant extract was taken in a test tube followed by 4% NaOH and 1% CuSO4. Violet pink colour development indicated the presence of proteins.

 

Absent

_ _

 

Absent

_ _

Test for Lipids

(a) Glycerol test: To 1 ml of 1% CuSO4.5H2O solution, 5 drops of the plant extract were added and mixed thoroughly. Then it received 5 drops of a 10% sodium hydroxide solution. A clear blue solution was obtained which indicates the presence of glycerol.

(b) Sudan III test: To 1ml of plant extract, few drops of Sudan III solution were added. Appearance of red colour indicated the presence of lipids.

 

Absent

_ _

 

 

 

Present

+ +

Test for Alkaloid

a) Wagner’s test: 1 ml extract was treated with Wagner’s reagent, formation of brown reddish precipitate indicates presence of alkaloids.

(b)Dragendorff’s test: 2 ml of Dragendorff’s reagent was added to 1 ml of plant extract. Formation of orange white precipitate indicated the presence of alkaloids.

 

Present

+ +

 

Present

+ +

Test for saponin

Foam test: 1 ml of plant extract was taken in a test tube with small amount of water. Sodium bicarbonate was added to it and shaken vigorously for 5 min. Formation of foam indicated the presence of saponins.

Absent

_ _

Test for Flavonoid

To 0.5 ml of plant extract, 5 ml dilute ammonia was added followed by the addition of 1 ml concentrated sulphuric acid. A yellow coloration that disappeared on standing indicated the presence of flavonoids.

 

Present

+ +

Test for Resin

In a dry test tube, to 0.5 ml of acetic acid and 2 drops of conc. Sulphuric acid were added. a purple color that turned violet in about 10 minutes.

 

Absent

_ _


8.3 Evaluation parameter of Nanoemulgel:

8.3.1 Organoleptic test:

Organoleptic tests were observed visually on all Batches including color, odor and appereance. The organoleptic results are in Table.No.5


Table No . 5 Oganoleptic Test

Batch

Colour

Appearance

Odour

NEG1

Pale Yellow

Transparant

Pungent Odour

NEG2

Pale Yellow

Transparant

Pungent Odour

NEG3

Pale Yellow

Transparant

Pungent Odour

NEG4

Pale Yellow

Transparant

Pungent Odour


The results of organoleptic nanoemulgel  is yellow  in color, has a characteristic aroma of clove leaf oil and it is transparent.

8.3.2Homogeneity Test  :

The purpose of the homogeneity test was to determine the homogeneity aspect of nanoemulgel .The results of the homogeneity test are in Table No 6.


Table No.6  Homogeneity Test

Formula

Result

NEG1

Homogeneous

NEG2

Homogeneous

NEG3

Homogeneous

NEG4

Homogeneous


The results of the examination of the homogeneity of nanoemulgel  are that there are no coarse grains on the object glass, which means that all preparations are homogeneous.

8.3.3 Viscosity measurement:

Viscosity measurement aims to determine the viscosity of a preparation to flow. Viscosity measurement using a Brookfield NDJ-8S. The results of the viscosity measurements are shown in Table No.7


Table No. 7 Viscosity Measurement Test

Formula

Viscocity in cps

NEG1

3434

NEG2

3375

NEG3

3109

NEG4

3025


Viscosity value states the amount of resistance of a liquid to flow. The higher the viscosity value, the greater the resistance to flow. Carbopol is a type of gelling agent that provides excellent stability .

 8.3.4 pH Measurement  :

       
            Fig No. 6 pH Measurement.jpg
       

Fig No. 6 pH Measurement

The pH test aims to determine the safety of the preparation when used so as not to irritate the skin and also to determine the stability of the preparation. The results of the pH measurements are shown in Table No.8.


Table No. 8 pH Measurement Test

Formula

pH

NEG1

5.85

NEG2

5.68

NEG3

5.76

NEG4

5.92


The pH measurement results obtained at NEG1 , NEG2 , NEG3, NEG4 . The pH results obtained are in accordance with the pH of the skin, which is between 4.5 - 6.5 so it is safe to use and does not cause irritation to the skin.                                                  8.3.5 Particle size measurement :

       
            Fig No. 7  Fomulation for Particle size Measurement.jpg
       

Fig No. 7  Fomulation for Particle size Measurement

       
            Fig No. 8 E –SEM testing of NEG4 Formulation.png
       

Fig No. 8 E –SEM testing of NEG4 Formulation

Measurement of nanoemulgel  particles was carried out using a E- SEM test. when the preparation was completed. The results of the particle size measurements are shown in Table No.9.


Table No.9. Particle Size Measurement

Batch No.

Particle Size Range

NEG4

75.00 nm – 340.04 nm


Particle size is an important parameter in nanoemulgel preparations. The smaller the particle size will increase the surface contact area, the higher the surface contact, the faster the material enters and is absorbed into the skin so that it can produce the desired effect optimally.

8.3.6 Invitro Drug Release of Nanoemulgel :

Different nanoemulgel formulation were tested Invitro for drug release  are shown in below table No.10. and plotted.


Table No. 10 Drug Release of Nanoemulgel

Time in min

% Drug Release

NEG1

NEG2

NEG3

NEG4

0

0

0

0

0

120

46.37

43.56

45.66

48.98

240

68.39

62.87

64.77

69.79

360

80.24

76.23

79.98

82.77

480

96.78

93.58

94.67

97.03



       
            Fig No. 9 Graph of Drug release of Nanoemulgel.png
       

    Fig No. 9 Graph of Drug release of Nanoemulgel


Table No. 11 Stability test

 

Parameter of NEG4

Before Storage

After One Month

Colour

Pale Yellow

Pale Yellow

Odour

Pungent Odour

Pungent Odour

Appereance

Transparent

Transparent

Spreadability

28.17/9.27gm.cm/min

28.17/9.27gm.cm/min

Viscosity

3025

3025


Physical observations of nanoemulgel preparations have been carried out and there is no change in color, change in odor and phase separation. It is kept for stability in the freezing condition at 4ºC temp. It was concluded that the nanoemulgel and preparations were stable during storage.we have test the physical stability of optimized NEG4 batch. After one month of stability of formulation we observed that there is no change in colour, odour and separation phase.

8.3.8 Spreadability test:

Analyzing a formulation's spread ability, essential parameters to consider are the rate and duration of shear caused during smearing, as well as the temperature of the target location.From the observation of spreadability test of drug it shows that a lower dose would not produce the desired results, while a higher dose could cause unwanted side effects.


Table No.12 Spreadability test

Formula

Spreadability

NEG1

22.53/4.06gm.cm/min

NEG2

24.62/7.08gm.cm/min

NEG3

26.82/8.02gm.cm/min

NEG4

28.17/9.27gm.cm/min

 


8.3.9 Antibacterial activity:

       
            Fig No. 10.png
       

Staphylococus Epidermis 

  Fig No. 10 

       
            Fig No. 11.png
       

Propionibacterium acnes

   Fig No. 11


Table No. 12 Antibacterial activity

Batch No.

Inhibition Zone Diameter (mm)

Staphylococus Epidermis

Propionibacterium acnes

NEG4

12.93

18.58

 


Based on the table above, it can be seen that the higher the concentration of clove leaf oil used, the greater the inhibitory power produced. This proves that the eugenol contained in clove leaf oil can be used as an antibacterial, especially the bacteria that causes acne.

9.SUMMARY AND CONCLUSION

9.1 Summary :

The nanoemulgel formulations consisted of clove leaf oil, tween 80, PEG 400, aquadest, propyl paraben, methyl paraben, carbopol 940 and triethanolamine. Clove leaf oil is used as a carrier oil and an ingredient that has antibacterial, antiseptic and anti-inflammatory activities. Tween 80 (surfactant), PEG 400 (cosurfactant), carbopol 940 (gelling agent), triethanolamine (pH balance).

 The process of making nanoemulgel using spontaneous emulsification method consisting of an aqueous phase and organic phase.  The Preformulation studies and Phytochemcial screening of the clove drug were carried out. The evaluation of the parameters are shown as above table. We study the evaluation parameters of the formulation of nanaoemulgel. Organoleptic tests were observed visually on all formulas including color, odor and shape. The examination of the homogeneity of nanoemulgel  are that there are no coarse grains on the object glass, which means that all preparations are homogeneous. Viscosity value states the amount of resistance of a liquid to flow. The higher the viscosity value, the greater the resistance to flow. Carbopol is a type of gelling agent that provides excellent stability. The pH test aims to determine the safety of the preparation when used so as not to irritate the skin and also to determine the stability of the preparation. Particle size is an important parameter in nanoemulgel preparations. The smaller the particle size will increase the surface contact area, the higher the surface contact, the faster the material enters and is absorbed into the skin so that it can produce the desired effect optimally. Different nanoemulgel formulation were tested Invitro for drug release. Physical observations of nanoemulgel preparations have been carried out and there is no change in color, change in odor and phase separation. Analyzing a formulation's spread ability, essential parameters to consider are the rate and duration of shear caused during smearing, as well as the temperature of the target location. Antibacterial activity testing was carried out on nanoemulgel . By using the agar diffusion method (Kirby-Bauer). Nanoemulgels are topical gels contain nanoemulsions. They are commonly used in dermatology for the treatment of skin conditions such as eczema, relief, pscoriasis and acne. Nanogels are nanoparticles - based hydrogels that have been studied for their potential in drug delivery, tissue engineering and other biomedical applications. They are commonly used due to their biocompatibility , biodegradability and high drug loading capacity. Nanoemulgel system is more stable than other transdermal drug delivery system, because it decreases the   interfacial as well as the surface tension of the formulation, which make it superior from a conventional transdermal delivery system. Nanoemulgel has shown significant enhancement in the permeability of the drug through skin than other formulation since from nanoemulgel preparation, the drug can permeate the skin layer through both paracellular and transcellular route, whereas, in nanoemulsion, only transcellular permeation route is seen.

9.2CONCLUSION: 

From the results of the research that has been carried out, it is concluded that clove leaf oil can be formulated into nanoemulgel preparations and is stable during the cycling test and has a particle size below 340 nm. The Nanoemulgel formulation had the greatest antibacterial activity compared to the emulgel formulation. It shows the better spread ability while testing of the optimized batch of the formulation. So, it can be concluded that the formulation of Nanoemulgel has better characteristics than other Marketed Preparations for Anti- acne Treatment.

REFERENCES

  1. Wasitaatmadja, Kelompok Studi Dermatologi Kosmetik Indonesia: Akne. Jakarta: Fakultas Kedokteran Universitas Indonesia, 2018.
  2. J. McLaughlin, S. Watterson, A. M. Layton, A. J. Bjourson, E. Barnard, and A. McDowell, “Propionibacterium acnes and acne vulgaris: New insights from the integration of population genetic, multi-omic, biochemical and host-microbe studies,” Microorganisms, vol. 7, no. 5, pp. 1–29, 2019, doi: 10.3390/microorganisms7050128.
  3. N. Baisaeng and S. Klayraung, “In Vitro Antibacterial Activity of Hydrogels Containing Tamarind Seed Husk Extracts against Propionibacterium acnes,” Pharm. Biomed. Mater. Technol., vol. 819, pp. 85–91, 2019..
  4. B. Sgorbini et al., “Determination of free and glucosidically-bound volatiles in plants. Two case studies: Lmenthol in peppermint (Mentha x piperita L.) and eugenol in clove (Syzygium aromaticum (L.) Merr. & L.M.Perry),” Phytochemistry, vol. 117, pp. 296–305, Sep. 2015.
  5. Haro-Gonzalez et al., “Clove essential oil (Syzygium aromaticum l. myrtaceae): Extraction, chemical composition, food applications, and essential bioactivity for human health,” Molecules, vol. 26, no. 21, 2021, doi: 10.3390/molecules26216387.
  6. Nanan Nurdjannah, “Diversifikasi Penggunaan Cengkeh,” Perspekt. Rev. Penelit. Tanam. Ind., vol. 3, no. 2, pp. 61–70, 2004.
  7. I. P. S. T. Lova, W. A. Wijaya, N. L. P. V Paramita, and A. A. R. Y. Putra, “Perbandingan Uji Aktivitas Antibakteri Minyak Atsiri Daun, Tangkai Bunga Dan Bunga Cengkeh Bali (Syzygium Aromaticum L.) Terhadap Bakteri Propionibacterium Acne Dengan Metode Difusi Disk,” J. Kim., vol. 12, no. 1, pp. 30–35, 2018.
  8. W. E. Soliman, T. M. Shehata, M. E. Mohamed, N. S. Younis, and H. S. Elsewedy, “Enhancement of curcumin anti-inflammatory effect via formulation into myrrh oil-based nanoemulgel,” Polymers (Basel)., vol. 13, no. 4, pp. 1–16, 2021.
  9. T. M. Shehata, H. M. Elnahas, and H. S. Elsewedy, “Development, Characterization and Optimization of the Anti-Inflammatory Influence of Meloxicam Loaded into a Eucalyptus Oil-Based Nanoemulgel,” Gels, vol. 8, no. 5, 2022.
  10. K. B. Sabjan, S. M. Munawar, D. Rajendiran, S. K. Vinoji, and K. Kasinathan, “Nanoemulsion as Oral Drug Delivery - A Review,” Curr. Drug Res. Rev., vol. 12, no. 1, pp. 4–15, 2019.
  11. B. Chatterjee, B. Gorain, K. Mohananaidu, P. Sengupta, U. K. Mandal, and H. Choudhury, “Targeted drug delivery to the brain via intranasal nanoemulsion: Available proof of concept and existing challenges,” Int. J. Pharm., vol. 565, no. May, pp. 258–268, 2019.
  12. A. M. Eid and M. Hawash, “Biological evaluation of Safrole oil and Safrole oil Nanoemulgel as antioxidant, antidiabetic, antibacterial, antifungal and anticancer,” BMC Complement. Med. Ther., vol. 21, no. 1, pp. 1–12, 2021.
  13. Y. Hisprastin and R. F. Nuwarda, “Review: Perbedaan Emulsi Dan Mikroemulsi Pada Minyak Nabati,” Farmaka, vol. 16, no. 1, pp. 133–140, 2018.
  14. P. Chellapa et al., “Nanoemulsion and Nanoemulgel as a Topical Formulation,” IOSR J. Pharm., vol. 5, no. 10, pp. 43–47, 2015.
  15. E. L. Syahfitri, R. J, and N. M, “Formulation and Antibacterial Activity Tests of Nanoemulsion Gel Black Cumin (Nigella Sativa L.) Ethanol Extract,” Asian J. Pharm. Res. Dev., vol. 8, no. 4, pp. 07–11, 2020.
  16. A. Wulansari, M. Jufri, and A. Budianti, “Studies on the formulation, physical stability, and in vitro antibacterial activity of tea tree oil (Melaleuca alternifolia) nanoemulsion gel,” Int. J. Appl. Pharm., vol. 9, pp. 135–139, 2017. 1782 International Journal Of Science, Technology & Management ISSN: 2722 - 4015 http://ijstm.inarah.co.id
  17. D. M. Hariyadi, N. Rosita, Isnaeni, S. Sudarma, and D. Rezania, “Virgin Coconut Oil Emulgel: Effect of VCO and Carbopol 940 Concentration on Characterization and Antibacterial Activity,” Res. J. Pharm. Technol., vol. 15, no. 5, pp. 2087–2092, 2022.
  18. A. G. Amalyuri, J. Reveny, and A. Dalimunthe, “Antibacterial Potential Of Ethanol Extract Of Tamarind Seed Bark (Tamarindus indica L.) And Formulation Of Anti-Acne Nanogel,” Int. J. Sci. Technol. Manag., vol. 3, no. 3, pp. 598–604, 2022.
  19. M. Jufri, R. Iswandana, D. A. Wardani, and S. F. Malik, “Formulation of Red Fruit Oil Nanoemulsion Using Sucrose Palmitate,” Int. J. Appl. Pharm., vol. 14, no. 5, pp. 175–180, 2022.
  20. V. D. Yadav, A. D. Shinde, and P. D. Jadhav, “Formulation and Evaluation of Liposomes Containing Fluconazole,” Int. J. Drug Deliv. Technol., vol. 12, no. 1, pp. 98–102, 2022, doi: 10.25258/ijddt.12.1.19.
  21. V. Verma and T. S. Easwari, “A Novel Approach of Leflunomide Nanoemulgel for Topical Drug Delivery System,” Int. J. Pharm. Investig., vol. 12, no. 2, pp. 199–204.
  22. R. Fitri, J. Reveny, U. Harahap, H. Dharmawan, and Nasri, “Anti-Acne Activity From Biocellulose Mask Formula Containing (Aloe Vera (L.) Burm.F) Essence Combined With Vitamin E,” Indones. J. Pharm. Clin. Res., vol. 4, no. 1, pp. 1–7, 2021.
  23. U. Harahap, A. Dalimunthe, T. Hertiani, M. Muhammad, Nasri, and D. Satria, “Antioxidant and antibacterial activities of ethanol extract of Vernonia amygdalina Delile. Leaves,” AIP Conf. Proc., vol. 2342, no. April, 2021, doi: 10.1063/5.0045447.
  24. M. S. Algahtani, M. Z. Ahmad, and J. Ahmad, “Nanoemulgel for improved topical delivery of retinyl palmitate: Formulation design and stability evaluation,” Nanomaterials, vol. 10, no. 5, 2020

Reference

  1. Wasitaatmadja, Kelompok Studi Dermatologi Kosmetik Indonesia: Akne. Jakarta: Fakultas Kedokteran Universitas Indonesia, 2018.
  2. J. McLaughlin, S. Watterson, A. M. Layton, A. J. Bjourson, E. Barnard, and A. McDowell, “Propionibacterium acnes and acne vulgaris: New insights from the integration of population genetic, multi-omic, biochemical and host-microbe studies,” Microorganisms, vol. 7, no. 5, pp. 1–29, 2019, doi: 10.3390/microorganisms7050128.
  3. N. Baisaeng and S. Klayraung, “In Vitro Antibacterial Activity of Hydrogels Containing Tamarind Seed Husk Extracts against Propionibacterium acnes,” Pharm. Biomed. Mater. Technol., vol. 819, pp. 85–91, 2019..
  4. B. Sgorbini et al., “Determination of free and glucosidically-bound volatiles in plants. Two case studies: Lmenthol in peppermint (Mentha x piperita L.) and eugenol in clove (Syzygium aromaticum (L.) Merr. & L.M.Perry),” Phytochemistry, vol. 117, pp. 296–305, Sep. 2015.
  5. Haro-Gonzalez et al., “Clove essential oil (Syzygium aromaticum l. myrtaceae): Extraction, chemical composition, food applications, and essential bioactivity for human health,” Molecules, vol. 26, no. 21, 2021, doi: 10.3390/molecules26216387.
  6. Nanan Nurdjannah, “Diversifikasi Penggunaan Cengkeh,” Perspekt. Rev. Penelit. Tanam. Ind., vol. 3, no. 2, pp. 61–70, 2004.
  7. I. P. S. T. Lova, W. A. Wijaya, N. L. P. V Paramita, and A. A. R. Y. Putra, “Perbandingan Uji Aktivitas Antibakteri Minyak Atsiri Daun, Tangkai Bunga Dan Bunga Cengkeh Bali (Syzygium Aromaticum L.) Terhadap Bakteri Propionibacterium Acne Dengan Metode Difusi Disk,” J. Kim., vol. 12, no. 1, pp. 30–35, 2018.
  8. W. E. Soliman, T. M. Shehata, M. E. Mohamed, N. S. Younis, and H. S. Elsewedy, “Enhancement of curcumin anti-inflammatory effect via formulation into myrrh oil-based nanoemulgel,” Polymers (Basel)., vol. 13, no. 4, pp. 1–16, 2021.
  9. T. M. Shehata, H. M. Elnahas, and H. S. Elsewedy, “Development, Characterization and Optimization of the Anti-Inflammatory Influence of Meloxicam Loaded into a Eucalyptus Oil-Based Nanoemulgel,” Gels, vol. 8, no. 5, 2022.
  10. K. B. Sabjan, S. M. Munawar, D. Rajendiran, S. K. Vinoji, and K. Kasinathan, “Nanoemulsion as Oral Drug Delivery - A Review,” Curr. Drug Res. Rev., vol. 12, no. 1, pp. 4–15, 2019.
  11. B. Chatterjee, B. Gorain, K. Mohananaidu, P. Sengupta, U. K. Mandal, and H. Choudhury, “Targeted drug delivery to the brain via intranasal nanoemulsion: Available proof of concept and existing challenges,” Int. J. Pharm., vol. 565, no. May, pp. 258–268, 2019.
  12. A. M. Eid and M. Hawash, “Biological evaluation of Safrole oil and Safrole oil Nanoemulgel as antioxidant, antidiabetic, antibacterial, antifungal and anticancer,” BMC Complement. Med. Ther., vol. 21, no. 1, pp. 1–12, 2021.
  13. Y. Hisprastin and R. F. Nuwarda, “Review: Perbedaan Emulsi Dan Mikroemulsi Pada Minyak Nabati,” Farmaka, vol. 16, no. 1, pp. 133–140, 2018.
  14. P. Chellapa et al., “Nanoemulsion and Nanoemulgel as a Topical Formulation,” IOSR J. Pharm., vol. 5, no. 10, pp. 43–47, 2015.
  15. E. L. Syahfitri, R. J, and N. M, “Formulation and Antibacterial Activity Tests of Nanoemulsion Gel Black Cumin (Nigella Sativa L.) Ethanol Extract,” Asian J. Pharm. Res. Dev., vol. 8, no. 4, pp. 07–11, 2020.
  16. A. Wulansari, M. Jufri, and A. Budianti, “Studies on the formulation, physical stability, and in vitro antibacterial activity of tea tree oil (Melaleuca alternifolia) nanoemulsion gel,” Int. J. Appl. Pharm., vol. 9, pp. 135–139, 2017. 1782 International Journal Of Science, Technology & Management ISSN: 2722 - 4015 http://ijstm.inarah.co.id
  17. D. M. Hariyadi, N. Rosita, Isnaeni, S. Sudarma, and D. Rezania, “Virgin Coconut Oil Emulgel: Effect of VCO and Carbopol 940 Concentration on Characterization and Antibacterial Activity,” Res. J. Pharm. Technol., vol. 15, no. 5, pp. 2087–2092, 2022.
  18. A. G. Amalyuri, J. Reveny, and A. Dalimunthe, “Antibacterial Potential Of Ethanol Extract Of Tamarind Seed Bark (Tamarindus indica L.) And Formulation Of Anti-Acne Nanogel,” Int. J. Sci. Technol. Manag., vol. 3, no. 3, pp. 598–604, 2022.
  19. M. Jufri, R. Iswandana, D. A. Wardani, and S. F. Malik, “Formulation of Red Fruit Oil Nanoemulsion Using Sucrose Palmitate,” Int. J. Appl. Pharm., vol. 14, no. 5, pp. 175–180, 2022.
  20. V. D. Yadav, A. D. Shinde, and P. D. Jadhav, “Formulation and Evaluation of Liposomes Containing Fluconazole,” Int. J. Drug Deliv. Technol., vol. 12, no. 1, pp. 98–102, 2022, doi: 10.25258/ijddt.12.1.19.
  21. V. Verma and T. S. Easwari, “A Novel Approach of Leflunomide Nanoemulgel for Topical Drug Delivery System,” Int. J. Pharm. Investig., vol. 12, no. 2, pp. 199–204.
  22. R. Fitri, J. Reveny, U. Harahap, H. Dharmawan, and Nasri, “Anti-Acne Activity From Biocellulose Mask Formula Containing (Aloe Vera (L.) Burm.F) Essence Combined With Vitamin E,” Indones. J. Pharm. Clin. Res., vol. 4, no. 1, pp. 1–7, 2021.
  23. U. Harahap, A. Dalimunthe, T. Hertiani, M. Muhammad, Nasri, and D. Satria, “Antioxidant and antibacterial activities of ethanol extract of Vernonia amygdalina Delile. Leaves,” AIP Conf. Proc., vol. 2342, no. April, 2021, doi: 10.1063/5.0045447.
  24. M. S. Algahtani, M. Z. Ahmad, and J. Ahmad, “Nanoemulgel for improved topical delivery of retinyl palmitate: Formulation design and stability evaluation,” Nanomaterials, vol. 10, no. 5, 2020

Photo
Shweta Gaikwad
Corresponding author

Dattakala College of Pharmacy

Photo
Shivdas Gore
Co-author

Dattakala College of Pharmacy

Shweta Gaikwad*, Shivdas Gore, Emulgel and Nano Formulation & Evaluation of Herbal Nanoemulgel by using Clove Leaf Oil as Anti-Acne, Int. J. of Pharm. Sci., 2025, Vol 3, Issue 1, 605-621. https://doi.org/10.5281/zenodo.14625958

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