Introduction:
The increasing demand for natural and herbal-based skincare formulations has lead
researchers to explore plant-derived bioactive compounds with therapeutic benefits. Skin
disorders such as inflammation, oxidative stress, and microbial infections are common
concerns that necessitate the development of novel topical formulations with natural
bioactive ingredients. Traditional medicine has long utilized plant-based extracts for their
therapeutic benefits, including anti-inflammatory and antioxidant properties. In this context,
oats (Avena sativa) and betel leaf (Piper betle) have emerged as promising natural
ingredients for dermatological applications.[01]
Oats are well-known for their skin-soothing, moisturizing, and anti-inflammatory properties
due to the presence of bioactive compounds such as avenanthramides, phenolics, and
β-glucans. The active components in oats, particularly avenanthramides, are known for their
strong anti-inflammatory and antioxidant activities. Avenanthramides inhibit the release of
pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukin-6
(IL-6), thereby reducing redness, swelling, and irritation. Additionally, oats contain β-glucans,
which contribute to skin hydration and barrier repair, making them ideal for moisturizing
formulations. [02]
Betel leaf (Piper betle), a medicinal plant widely used in traditional medicine, is rich in
phenolic compounds, flavonoids, and essential oils. It possesses potent antioxidant,
antimicrobial, and anti-inflammatory properties, making it an ideal candidate for incorporation
into topical formulations aimed at protecting the skin from oxidative damage and
inflammatory conditions. the antioxidant potential of betel leaf extract, demonstrating its
ability to reduce lipid peroxidation in skin cells. efficacy of betel leaf extract as an antioxidant
in a moisturizing cream and reported enhanced stability and protective effects against
oxidative degradation. These findings support the use of betel leaf extract in novel skincare
formulations for improved skin health and longevity.[03]
The present research focuses on developing a novel oil-in-water (O/W) cream incorporating
oats and betel leaf extract to harness their synergistic anti-inflammatory and antioxidant
properties. This formulation aims to provide a natural and effective alternative for managing
skin inflammation and oxidative stress, potentially benefiting individuals with conditions such
as dermatitis, eczema, and premature aging.[04]
Topical Drug Delivery: [21]
In recent decades, various routes have been utilized for drug administration, including oral,
sublingual, rectal, parenteral, topical, and inhalation methods. Topical drug delivery involves
the direct application of a drug-containing formulation onto the skin to treat localized skin
conditions or the cutaneous manifestations of systemic diseases, such as psoriasis. The
primary goal is to confine the drug's action to the skin’s surface or within its layers,
minimizing systemic absorption. Semisolid formulations are the most commonly used in
topical delivery systems, but other forms like foams, sprays, medicated powders, solutions,
and adhesive patches are also employed in therapeutic applications.
Advantages:
● Bypasses first-pass metabolism, reducing drug degradation by the liver.
● Easy and convenient application, improving patient compliance.
● Minimizes risks associated with invasive drug delivery methods.
● Avoids challenges related to intravenous therapy, such as pH fluctuations, enzymatic
degradation, and variations in gastric emptying time.
● Ensures consistent drug efficacy with lower total daily doses through controlled
release.
● Reduces fluctuations in drug levels, maintaining steady plasma concentrations.
Limitations:
● Potential for skin irritation or dermatitis, depending on the drug or excipients used.
● Limited absorption for drugs with high molecular weight or poor lipid solubility.
● Slow drug absorption, leading to delayed onset of action.
● Suitable only for drugs requiring low plasma concentrations for therapeutic effects.
● Possible allergic reactions, particularly in sensitive individuals.
● Large drug molecules may struggle to penetrate the skin, reducing effectiveness.
PHYSIOLOGY OF HUMAN SKIN: [21]
Epidermis:
The epidermis is the outermost layer of the skin, made up of stratified keratinized squamous
epithelium. Its thickness varies across the body, with the thickest areas found on the palms
and soles. This layer lacks blood vessels and nerve endings, but its deeper sections receive
oxygen and nutrients from interstitial fluid, which originates from the dermis and is eventually
drained away as lymph.
Dermis:
The dermis is a strong and elastic layer beneath the epidermis, composed mainly of
connective tissue. Its matrix contains collagen fibers, which help retain moisture and provide
tensile strength, and elastic fibers, which contribute to skin flexibility. Overstretching of these
fibers can result in permanent stretch marks, often seen in pregnancy and obesity. With
aging, collagen retention decreases, leading to the formation of wrinkles. The primary cells in
the dermis include fibroblasts, macrophages, and mast cells. The deepest layer of the
dermis contains areolar tissue and varying amounts of fat (adipose tissue).
Sebaceous Glands:
These glands consist of secretory epithelial cells that originate from the same tissue as hair
follicles. They produce sebum, an oily substance that lubricates the skin and hair.
Sebaceous glands are distributed throughout the body, except on the palms and soles. They
are most abundant on the scalp, face, armpits (axillae), and groin. In specific areas such as
the lips, eyelids, nipples, labia minora, and glans penis, some sebaceous glands function
independently of hair follicles, releasing sebum directly onto the skin’s surface.
Functions of the Skin: [21]
Protective Barrier:
The skin acts as a defense mechanism against harmful pathogens and external damage.
Langerhans cells, which are part of the adaptive immune system, contribute to immune
responses.
Sensory Perception:
It contains specialized nerve endings that detect temperature changes (heat and cold),
touch, pressure, vibration, and pain, allowing the body to respond to external stimuli.
Thermoregulation:
The skin plays a key role in heat regulation by controlling blood circulation. When blood
vessels dilate, heat is released, whereas constricted vessels help retain heat and minimize
heat loss through radiation, convection, and conduction.
Prevention of Fluid Loss:
The skin functions as a semi-impermeable barrier, minimizing water evaporation. A
compromised barrier, such as in burn injuries, can lead to excessive fluid loss.
Aesthetic and Communication Role:
The skin is an important visual feature, reflecting mood, health, and attractiveness, which
influences social interactions.
Storage and Synthesis:
It serves as a reservoir for lipids and water, while also facilitating the synthesis of vitamin D
through exposure to ultraviolet (UV) radiation.
Water Resistance:
The skin acts as a waterproof barrier, preventing the loss of essential nutrients and ensuring
the body's internal balance is maintained.
DISEASES OF SKIN: [18]
1. Vitiligo
Vitiligo is a skin disorder characterized by loss of pigment, leading to white patches on the
skin. It affects approximately 1% of the global population. The condition occurs due to the
destruction or malfunction of melanocytes, the cells responsible for producing melanin,
which gives skin its natural color.
2. Scabies
Scabies is a highly contagious skin condition caused by the human scabies mite. It can
affect individuals of all age groups, but it is more common in children and the elderly. The
mites, which are tiny parasites smaller than a pinhead, burrow into the skin, leading to
intense itching, red scaly patches, and scratch marks. If left untreated, the affected areas
may develop small pus-filled sores due to secondary infection.
3. Rosacea
Rosacea is a chronic skin condition that primarily affects the central face, often occurring in
middle-aged individuals. It is characterized by frequent facial flushing, followed by persistent
redness on the cheeks, nose, chin, and forehead. While the exact cause remains unclear,
researchers believe it is related to abnormal dilation of blood vessels in the skin.
4. Psoriasis
Psoriasis is a non-infectious, chronic skin disorder that affects about 2% of the population,
impacting both men and women equally. The condition is known for its unpredictable
flare-ups and periods of remission. It affects the multiple layers of the skin, causing red,
scaly patches, which may result in discomfort but do not leave scars.
5. Melanoma
Melanoma is a type of skin cancer that originates from pigment-producing cells
(melanocytes). It is not contagious, and if detected early, treatment outcomes are generally
positive. The term "melanoma" is derived from the Greek word "melas," meaning black,
referring to the dark pigment melanin, which determines skin color.
6. Eczema (Atopic Eczema)
Atopic eczema is a chronic inflammatory skin condition often associated with genetic
predisposition. It commonly occurs in individuals with related conditions such as asthma, hay
fever, or seasonal allergies. Eczema affects the upper layer of the skin, leading to redness,
blistering, oozing, crusting, scaling, thickening, and occasional pigmentation changes.
CREAMS: [21]
Creams are topical formulations designed for application on the skin. They are defined as
viscous liquid or semi-solid emulsions, which can be classified as either oil-in-water (O/W) or
water-in-oil (W/O) emulsions, depending on their composition. The consistency of creams
varies based on the ratio of oil and water in the formulation.
These formulations serve multiple cosmetic and therapeutic purposes, including cleansing,
beautifying, improving appearance, and providing skin protection. Additionally, they are
widely used for the localized delivery of drugs to treat various skin conditions and disorders.
By targeting specific areas, creams enhance drug penetration into the skin or mucous
membranes for effective treatment.
As pharmaceutical products, creams are developed using techniques established in the
pharmaceutical industry. They can be classified into medicated and non-medicated
(cosmetic) creams. Based on composition, creams may be ayurvedic, herbal, or allopathic,
catering to different therapeutic needs. Typically, they contain one or more active drug
substances, which are either dissolved or dispersed in a suitable base. Traditionally, the term
"cream" is associated with semi-solid emulsions, such as oil-in-water (O/W) creams (e.g.,
vanishing cream) and water-in-oil (W/O) creams (e.g., cold cream).
TYPES OF SKIN CREAMS: [21]
1. Oil-in-Water (O/W) Creams:
● These creams consist of small oil droplets dispersed within a continuous water
phase. The oil is finely distributed throughout the aqueous medium, creating a light
and non-greasy formulation. O/W emulsions are commonly used in moisturizers and
pharmaceutical creams, as they are easily absorbed into the skin.
2. Water-in-Oil (W/O) Creams:
● These formulations contain water droplets dispersed within a continuous oily phase.
The oil serves as the dispersion medium, making these creams thicker and more
occlusive. W/O emulsions provide long-lasting hydration by forming a protective
barrier on the skin, making them ideal for cold creams and emollients.
Key Components in an Oil-in-Water (O/W) Cream [18]
Emollients
(e.g., Mineral oil, Jojoba oil, Shea butter)
Soften and smooth the skin by filling the gaps between skin cells. Improve skin hydration by
preventing excessive water loss. Provide a protective barrier against environmental damage.
Humectants
(e.g., Glycerin, Propylene glycol, Hyaluronic acid)
Attract and retain moisture from the environment into the skin. Prevent dryness and improve
skin flexibility. Enhance the overall hydration properties of the cream.
Surfactants (Emulsifiers)
(e.g., Polysorbate 80, Sodium lauryl sulfate, Cetomacrogol)
Help in mixing oil and water by reducing surface tension. Stabilize the emulsion and prevent
phase separation. Improve the spreadability of the cream on the skin.
Preservatives
(e.g., Methylparaben, Phenoxyethanol, Benzyl alcohol)
Inhibit the growth of bacteria, fungi, and [Link] the shelf life of the cream. Ensure
product safety for long-term use.
pH Adjusters
(e.g., Citric acid, Triethanolamine)
Maintain the optimal pH of the cream for skin compatibility. Prevent irritation caused by an
imbalanced pH. Enhance the stability and effectiveness of active ingredients.
Thickening Agents & Stabilizers
(e.g., Carbopol, Xanthan gum, Hydroxyethyl cellulose)
Provide viscosity and texture to the cream. Improve stability and prevent separation of
phases. Enhance the cream's feel and application on the skin.
Fragrances & Colorants
Improve sensory appeal by adding pleasant scents and colors. Make the product more
attractive to consumers. Should be skin-friendly and non-irritating.
Literature Review on Oats in Dermatological Applications:
(Sur et al., 2008).
Avenanthramides: Polyphenols with anti-inflammatory and antioxidant properties that help
reduce redness, itching, and irritation. It demonstrated that avenanthramides inhibit
pro-inflammatory cytokines, reducing skin irritation and redness. It found that
avenanthramides help reduce oxidative damage and improve skin elasticity.
(Draelos, 2012).
Beta-glucan: A polysaccharide that enhances skin hydration, barrier function, and wound
healing. It highlighted that oat polyphenols neutralize free radicals, preventing UV-induced
damage and collagen breakdown. Oat lipids and proteins contribute to skin repair and barrier
reinforcement, preventing irritant penetration and water loss.
(Reynertson et al., 2015)
Saponins: Natural cleansing agents that provide a gentle, non-irritating alternative to
synthetic detergents. It confirmed that oat extracts significantly improve skin barrier function,
reducing transepidermal water loss (TEWL) in inflamed skin. Oat-based formulations have
been shown to enhance skin hydration and smoothness, reducing fine lines and wrinkles.
Literature Review on Betel Leaf Extract in Dermatological Applications:
(Deld et al., 2019).
Betel leaf is rich in phenolic compounds, flavonoids, and tannins, which contribute to its
potent antimicrobial activity against bacteria, fungi, and viruses. Studies have demonstrated
its effectiveness against Staphylococcus aureus and Pseudomonas aeruginosa, two
common skin pathogens associated with acne, wound infections, and dermatitis.
(Peter et al., 2020).
The anti-inflammatory effects of betel leaf extract have been linked to the inhibition of
pro-inflammatory cytokines, reducing redness, swelling, and irritation in various
dermatological conditions. Betel leaf also promotes collagen synthesis and fibroblast
proliferation, which accelerates wound healing and skin regeneration.
(Ruers et al., 2021).
Betel leaf contains eugenol, hydroxychavicol, and other bioactive compounds that exhibit
strong antioxidant activity, neutralizing free radicals and protecting the skin from oxidative
stress and premature aging. Its ability to reduce UV-induced damage makes it a potential
ingredient in natural sunscreens and anti-aging formulations.
AIM
The aim of this thesis is to formulate and evaluate a novel nourishing cream incorporating
oats and betel leaf extract, focusing on its stability, antimicrobial, and anti-inflammatory
properties. The study aims to develop a safe, effective, and herbal-based skincare
formulation, assess its physicochemical characteristics, and validate its therapeutic potential
through appropriate scientific and analytical methods.
Objectives:
Formulation Development: To develop a stable nourishing cream incorporating oats and
betel leaf extract.
Physicochemical Evaluation: To assess the stability, texture, pH, viscosity, and
spreadability of the formulated cream.
Antimicrobial Activity: To evaluate the antimicrobial efficacy of the cream against common
skin pathogens.
Anti-inflammatory Properties: To analyze the anti-inflammatory potential of the formulation
through appropriate biological assays.
Shelf-life and Storage Stability: To determine the stability and shelf-life of the formulation
under different storage conditions.
Natural Formulation: Develop a herbal-based nourishing cream using oats and betel leaf
extract.
Alternative to Synthetics: Provide a natural, non-toxic alternative to commercial skincare
products.
Safe and Non-Toxic: Ensure the cream is free from harmful chemicals, relying only on
natural ingredients.
Rationale:
Need for Natural Skincare Products:
The increasing consumer preference for herbal-based skincare products highlights the
demand for safe, effective, and chemical-free formulations. Synthetic ingredients in
conventional creams may cause skin irritation, allergies, or long-term adverse effects.
Hence, there is a need for natural alternatives with proven moisturizing, antimicrobial, and
anti-inflammatory properties.[05]
Potential of Oats and Betel Leaf Extract in Dermatological Applications:
Oats (Avena sativa) Recognized for its hydrating, soothing, and anti-inflammatory effects,
making it suitable for sensitive and dry skin [Link] Leaf (Piper betle) Known for its
antimicrobial, antioxidant, and anti-inflammatory properties, which can help in preventing and
treating skin infections and inflammation.[05]
Contribution to Pharmaceutical and Cosmetic Industries:
The study aligns with the growing demand for herbal-based cosmeceuticals, offering a new
formulation with potential commercial applications. The findings will contribute to the
development of innovative skincare solutions that are both effective and safe for long-term
use.[06]
Research Gap:
Despite the successful formulation of a stable nourishing cream with oats and betel leaf
extract, certain research gaps remain. The formulation lacks extensive clinical trials to
validate its long-term safety and efficacy. Further optimization is needed for large-scale
production while maintaining stability and effectiveness. Additionally, consumer acceptance
studies are required to evaluate sensory attributes such as texture, absorption, and overall
user experience.[07]
Materials & methodology:
Ingredient Compney
Oats milk powder Saipro Company
Aloe vera gel Kudos aloe vera gel
Borax Astron Company
Olive oil Figaro olive oil
Bees wax Chemdyes Corporation
Jojoba oil JKA pharmacy
Betel leaf oil Avi natural
Rose water Blue Heaven
Instruments Compney
Centrifuge Plasto Crafts Industry Pvt. Ltd.
Ultrasonic Bath ANM Industries
U.V Spectrophotometer Shimadzu
Weighing Balance Uni Bloc
Ph meter Welltronix
Frans diffusion cell Orchid scientific
Autoclave
Incubator
Name of ingredient 2.5% (F1) 5% (F2) 7.5% (F3)
Aqueous phase
Oats milk power 0.5 gm 1 gm 1.5 gm
Aloe vera 8 gm 8 gm 8 gm
Borax 1.2 gm 1.2 gm 1.2 gm
Water q.s. q.s. q.s.
Multani Matti q.s. q.s. q.s.
Oil phase
Olive oil 3 ml 3 ml 3 ml
Bees wax 1.5 ml 1.5 ml 1.5 ml
Jojoba oil 0.3 ml 0.3 ml 0.3 ml
Betel leaf oil 0.6 ml 0.6 ml 0.6 ml
Rose water q.s. q.s. q.s.
Total weight 20 gm 20 gm 20 gm
Method of Preparation:
Step 1: Preparation of Oil Phase
Weigh and mix the oil-soluble ingredients (olive oil, bees wax, jojoba oil). Heat the mixture to
70-75°C to ensure complete solubilization of ingredients.
Step 2: Preparation of Aqueous Phase
Weigh and dissolve water-soluble ingredients (oats milk powder, alovera gel, borex), such as
hydrophilic emulsifiers, preservatives, humectants, and any water-soluble actives, in purified
water. Heat this phase to 70-75°C to match the temperature of the oil phase.
Step 3: Emulsification Process
Slowly add the oil phase into the aqueous phase with continuous stirring. Maintain the
temperature around 70°C during mixing to ensure uniform dispersion of oil droplets in water.
Step 4: Cooling and Stability Enhancement
After emulsification, gradually cool the mixture to room temperature while continuing to
stirring. Add heat-sensitive ingredients such as fragrances, colorants, and antioxidants (betel
leaf extract, rose water). Continue stirring until the emulsion reaches room temperature and
achieves the desired consistency.
EVALUATION PARAMETERS OF CREAMS:
1. Physical Appearance:
The cream’s physical characteristics, including color, texture, and smoothness, are observed
and graded accordingly.
2. pH Determination:
The pH of the cream formulation is measured using a standard digital pH meter at room
temperature. A 1 gm of the cream is diluted with 100 ml of water and placed in a suitable
beaker for accurate measurement.
3. Spreadability:
To assess spreadability, a specific amount of the cream is placed between two glass slides,
and a 100 g weight is applied for specific time. The ease with which the cream spreads
under this pressure is then evaluated.
S= m*l/t
Where, m = weight applied to upper slide.
l = length moved on the glass slide.
t = time taken.
4. Saponification Value:
A 2 g sample of the cream is refluxed with 25 mL of 0.5 N alcoholic KOH for 30 minutes.
After cooling, 1 mL of phenolphthalein is added, and the mixture is immediately titrated with
0.5 N HCl. The titration reading is recorded as ‘a’. A blank titration is performed under
identical conditions without the test substance, and the reading is recorded as ‘b’.
Saponification value= (B-S)N×56.1/W
Where,
B = Volume of HCl (mL) for blank titration
S = Volume of HCl (mL) for sample titration
N = Normality of HCl
W = Weight of oil/fat used (g)
56.1 = Molecular weight of KOH
5. Acid Value:
A 10 g sample of the cream is dissolved in 50 mL of a 1:1 mixture of alcohol and ether, taken
in a pre-weighed flask. The flask is connected to a reflux condenser and gently heated until
the sample dissolves completely. 1 mL of phenolphthalein is then added, and the solution is
titrated with 0.1 N NaOH until a faint pink color appears, which persists for 30 seconds upon
shaking.
Acid value = n*5.61/w
Where,
n = the no. of ml of 0.1 N KOH solution.
w = the weight of substance in gram.
6. Viscosity:
The viscosity of the formulated cream is measured using a Brookfield Viscometer to assess
its consistency and flow properties.
7. Homogeneity:
The uniformity of the formulation is evaluated through visual inspection and tactile
assessment to ensure a smooth and even texture.
8. Ease of Removal:
The ease with which the cream can be removed is tested by washing the applied area with
tap water, observing how effectively it rinses off.
9. Dye Test:
A sudan red dye is incorporated into the cream, and a drop of the dyed cream is placed on a
microscope slide, covered with a cover slip, and examined under a microscope. If the
dispersed globules appear red while the background remains colorless, it confirms an
oil-in-water (O/W) emulsion. If the background is red and the globules remain colorless, it
indicates a water-in-oil (W/O) emulsion.
10. After-Feel Assessment:
The emolliency, slipperiness, and residue left on the skin after applying a fixed amount of
cream are evaluated to determine the product’s sensory characteristics.
11. Type of Smear:
The nature of the film or smear formed on the skin after applying the cream is observed to
assess its spreadability and texture.
12. Irritancy Study:
A 1 cm² area on the dorsal surface of the left hand is marked, and the cream is applied to
this specific area. The site is observed at regular intervals for up to 24 hours to check for
signs of irritation, erythema (redness), or edema (swelling), and the results are documented.
13. Accelerated Stability Study: For accelerated stability testing of cream, the samples are
first placed in a freezer at a temperature of -20°C to simulate cold storage conditions.
Afterward, the samples are removed and allowed to equilibrate to room temperature (25°C),
mimicking typical storage and handling conditions. Finally, the cream is subjected to heating
in a 40°C–50°C water bath to simulate high-temperature exposure, which can accelerate
potential degradation. Throughout the process, the samples are monitored for any changes
in texture, appearance, separation to assess the cream's stability under varying temperature
conditions.[18]
14. Franz Diffusion Cell [17]
I) Preparation of the Membrane:
● Select an appropriate dialysis membrane or biological skin (e.g., cellulose
membrane). Hydrate the membrane by soaking it in the receptor medium for 30
minutes to 1 hour before use. Trim the membrane to fit securely between the donor
and receptor compartments of the Franz diffusion cell.
II) Assembly of the Franz Diffusion Cell:
● Fill the receptor compartment with an appropriate dissolution medium (e.g.,
phosphate buffer pH 7.4). Place the hydrated membrane between the donor and
receptor compartments, ensuring no air bubbles. Secure the compartments tightly to
prevent leakage.
III) Addition of the Cream Formulation:
● Accurately weigh and apply a fixed amount of the cream sample (e.g., 1 g) onto the
surface of the membrane in the donor compartment. Cover the donor compartment
with parafilm or an occlusive cover to prevent evaporation.
IV) Experimental Conditions:
● Maintain the receptor compartment at 37 ± 0.5°C using a thermostatic water bath to
mimic physiological conditions. Stir the receptor solution continuously at 300-600 rpm
using a magnetic stirrer to ensure uniform drug distribution.
V) Sample Collection:
● Withdraw aliquots (e.g., 1 mL) from the receptor compartment at predetermined time
intervals (e.g., 0, 15, 30, 60, 120, 240, 360 minutes). Immediately replace the
withdrawn volume with an equal amount of fresh receptor medium to maintain sink
conditions. Filter the collected samples (if necessary) before analysis.
VI) Drug Release Analysis:
● Analyze the collected samples using a UV-Visible spectrophotometer or HPLC at a
pre-determined wavelength (λmax) specific to the drug. Measure the cumulative drug
release at each time point. Plot a drug release profile (cumulative drug release vs.
time) to determine the release kinetics.
VII) Cumulative Drug Release (%) = (Ct×Vr)+(∑Cp×Vs)/D ×100
● Ct = Drug concentration at time
Vr = Volume of receptor medium
Cp = Previously collected sample
Vs = Volume of each sample withdrawn
D = Initial drug content in the formulation
15. Total Phenol Content (TPC) using Folin-Ciocalteu Assay: [19]
Preparation of Gallic Acid Standard:
I) Stock Solution Preparation:
● Weigh 10 mg of gallic acid and dissolve in 10 mL of methanol or distilled water to
prepare a 1000 µg/mL (1 mg/mL) stock solution.
II) Standard Dilutions:
● Pipette appropriate volumes of the stock solution into separate volumetric flasks (10
mL each) and dilute with methanol/water to obtain concentrations of 10, 20, 40, 60,
80, and 100 µg/mL.
III) Standard Curve Preparation:
● Take 1 mL of each standard solution and treat it with 1 mL of Folin-Ciocalteu reagent.
Allow it to stand for 5 minutes, then add 2 mL of 7.5% sodium carbonate solution.
Incubate at room temperature for 30 minutes in the dark. Measure absorbance at 765
nm using a UV-Vis spectrophotometer.
IV) Plot Calibration Curve:
● Use the obtained absorbance values to plot a standard curve of absorbance vs.
concentration (µg/mL). Express total phenolic content as mg Gallic Acid Equivalent
(GAE) per gram of sample.
sample preparation:
I) Sample Preparation:
● Weigh 1 g of cream and dissolve it in 10 mL of methanol or ethanol using sonication
for 10-15 minutes. Filter the extract and collect the supernatant.
II) Reagent Preparation:
● Prepare Folin-Ciocalteu reagent (1:10 dilution with water) and 7.5% sodium
carbonate solution.
III) Reaction Setup:
● Take 1 mL of sample extract in a test tube. Add 1 mL of Folin-Ciocalteu reagent and
allow it to stand for 5 minutes. Add 2 mL of sodium carbonate solution and mix well.
Incubate the mixture at room temperature for 30 minutes in the dark.
IV) Measurement:
● Measure the absorbance at 765 nm using a UV-Vis spectrophotometer.
V) Quantification:
● Calculate the total phenolic content using a standard calibration curve of gallic acid
and express results as mg gallic acid equivalent (GAE) per gram of cream.
16. Antioxidants activity by DPPH method: [19]
Preparation of Ascorbic Acid Standard:
I) Stock Solution Preparation:
● Weigh 10 mg of ascorbic acid and dissolve in 10 mL of methanol or distilled water to
prepare a 1000 µg/mL stock solution.
II) Standard Dilutions:
● Dilute stock solution in methanol/water to obtain final concentrations of 10, 20, 40,
60, 80, and 100 µg/mL.
III) Reaction Setup:
● Mix 1 mL of each ascorbic acid standard solution with 2 mL of DPPH solution (0.1
mM in methanol). Incubate in the dark for 30 minutes at room temperature. Measure
absorbance at 517 nm using a UV-Vis spectrophotometer.
IV) Plot Calibration Curve:
● Use absorbance values to create a standard curve of absorbance vs. concentration.
Calculate percentage inhibition using the formula:
% Inhibition= (A0-A1/A0) × 100
A0= Absorbance of control (DPPH solution without sample)
A1= Absorbance of sample
Sample preparation:
I)Sample Preparation:
● Extract 1 g of cream with 10 mL of methanol, sonicate, and filter the solution.
II) Reagent Preparation:
● Prepare 0.1 mM DPPH solution in methanol.
III) Reaction Setup:
● Mix 1 mL of sample extract with 2 mL of DPPH solution. Incubate the mixture in the
dark for 30 minutes at room temperature. Measurement: Measure the absorbance at
517 nm using a UV-Vis spectro- photometer.
IV) Calculation of % Inhibition:
● % Inhibition= (A0-A1/A0) × 100
A0= Absorbance of control (DPPH solution without sample)
A1= Absorbance of sample
17. Antimicrobial assay [20]
The antimicrobial activity of the formulated creams was assessed using the disc diffusion
method. Mueller-Hinton Agar (MHA) and Sabouraud Dextrose Agar (SDA) were prepared,
sterilized, and poured into sterile Petri dishes, where they were left to solidify. The test then
inoculated onto the respective agar plates using pore method.
After incubation, the zones of inhibition (IZDs) were measured in millimeters to evaluate
antimicrobial activity. A clear inhibition zone around the disc indicated susceptibility of the
microorganisms to the cream formulation, whereas the absence of such a zone suggested
resistance.
Result:
Physicochemical properties:
Physicochemical properties of cream have a significant impact on its efficacy and application
on skin. Physicochemical properties of herbal cream which was prepared are listed in table
parameter F1 F2 F3
colour Faint yellow Faint yellow Faint yellow
Odour pleasant pleasant pleasant
Physical state Semi solid Semi solid Semi solid
homogeneity homogeneous homogeneous homogeneous
After feel Emollient Emollient Emollient
Irritancy test Non-irritancy Non-irritancy Non-irritancy
Greasiness Non-greasy Non-greasy Non-greasy
consistency smooth smooth smooth
Washability Easily washable Easily washable Easily washable
Dye test O/W O/W O/W
pH of the Cream:
The pH of the cream was found to be in range of 5 – 7.5 which is good for skin pH. All the
formulations were shown pH nearer to skin required.
Formulation pH
F1 7.2
F2 7.1
F3 7.1
Spreadability
F1
F2
F3
Acid value and Saponification value:
The results of acid and saponification value of all formulation of cream are presented below
table and showed satisfactorily values.
Parameter F1 F2 F3
Acid value 35.3 12.9 14.24
Saponification 78.54 95.37 106.5
value(mg KOH/g)
Microbial Assay:
Accelerated Stability Study:
Parameter F1 F2 F3
PH Unchanged Unchanged Unchanged
Phase separation Not seen Not seen Not seen
homogeneity homogeneous homogeneous homogeneous
Drug release:
Time (min) Absorbancee (280nm)
10 0.159
20 0.226
30 0.235
45 0.268
60 0.323
90 0.346
120 0.348
Total phenol content:
Galic acid (standard)
Concentration ug/ml Absorbance (765nm)
10 0.148
50 0.185
100 0.312
500 0.409
Cream
Concentration ug/ml Absorbance (765nm)
1000 (F2) 0.275
1000 (F3) 0.214
Anti oxidant activity:
Ascorbic acid (standard)
Concentration ug/ml Absorbance (517nm)
DPPH 0.931
5 1.056
10 0.71
50 1.051
100 0.693
1000 0.4
1500 0.408
2000 0.06
Concentration (ug/ml) Absorbance
Oats
Dpph 1.011
50 1.018
100 1.02
1000 1.025
Betel leaf oil
Dpph 0.999
10 0.785
50 0.523
100 0.329
500 0.274
Concentration ug/ml Absorbancee
Cream (F2)
Dpph 0.952
10 0.952
100 0.884
500 0.849
Cream (F3)
Dpph 0.952
10 0.969
100 0.879
500 1.008
1000 0.99
Streptomycin Zoi (mm)
5 7.2
10 7.8
15 7.9
20 8.2
25 8.5
