INTRODUCTION:

The growing consciousness of the side effects of synthetic skincare products has prompted a worldwide trend toward the application of herbal formulations in dermatology and cosmetology. Herbal creams, being semi-solid emulsions of bioactive plant extracts, have become widely used alternatives because they are natural, biocompatible, have few side effects, and are multifunctional. These products provide a broad range of benefits, including antioxidant defence, antimicrobial effects, hydration, and repair of the skin barrier, and so they can be used for therapeutic as well as cosmetic applications.¹

Of the many medicinal plants studied for such use, Spathodea campanulata, or the African tulip tree, has particular potential. In the Bignoniaceae family, this flowering tree grows quickly and is widely cultivated throughout tropical areas. In folklore medicine, various parts of the plant—the leaves, bark, and particularly flowers—have been employed to heal wounds, skin infection, inflammation, and fever. Recent phytochemical research has shown that Spathodea campanulata flowers contain a high concentration of bioactive compounds like flavonoids, tannins, glycosides, phenolic acids, and saponins. These compounds have been reported to exhibit a wide range of pharmacological activities like antimicrobial, antioxidant, anti-inflammatory, and wound-healing activity.²

The formulation of a herbal cream from Spathodea campanulata flower extract is a new and environmentally friendly method of skincare. The bioactive compounds in the extract can neutralize free radicals, thereby preventing oxidative damage to skin cells. Moreover, the antimicrobial property of the extract could provide protection against skin pathogens, hence being of great use in the treatment of acne, minor cuts, and skin infections. In addition, herbal creams tend to promote skin hydration, increase elasticity, and facilitate regeneration of injured tissues without the irritation usually caused by artificial agents.³

This research seeks to systematically develop a stable and effective topical cream with Spathodea campanulata flower extract. The work entails a number of steps such as the extraction of phytoconstituents, formulation development, physicochemical analysis (e.g., pH, spreadability, viscosity, and stability), and biological tests such as antimicrobial and antioxidant assays, and possible in silico modeling to investigate molecular interactions. The general aim is to prove the therapeutic utility of Spathodea campanulata for external use and help expand the field of herbal skincare products.^{4, 5}

MATERIAL AND METHOD:

1. Materials

· Plant Material: Fresh flowers of Spathodea campanulata were collected from the campus of D.Y. Patil university, Ambi, Pune. The flowers were washed, shade-dried at room temperature for 5–7 days and coarsely powdered using a grinder.

· Solvents and Chemicals: 70% ethanol was used as the solvent for extraction. Liquid paraffin, Beeswax, Borax (sodium borate), Methylparaben (preservative), Rose oil (fragrance and therapeutic agent), Distilled water.⁶

2. Extraction of Spathodea campanulata Flowers

Powdered flowers (100g) were Soxhlet extracted with 70% ethanol as the solvent. The extraction was carried out for 6–8hours until the siphon tube drained clear. The extract was concentrated in a rotary evaporator and dried on a water bath at 45°C. The obtained extract was kept in a desiccator at 4°C until further application in cream formulation.⁷

3. Formulation of cream:

Table no.1: formulation of herbal cream

Sr No.	Ingredients	Quantity	Role
1.	African tulip flower extract	2ml	Drug
2.	Bees wax	5.45gm	Emulsifying agent
3.	Liquid paraffin	18.1ml	Lubricating agent
4.	Borax	0.36gm	Alkaline agent
5.	Methyl paraben	0.03gm	Preservative
6.	Distilled water	q.s.	Vehicle
7.	Rose oil	q.s.	Fragrance

Procedure:

a. Preparation of the Oil Phase:

· Weigh precisely 5.45g of beeswax and place it in a beaker.

· Place 18.1mL of liquid paraffin in the same beaker.

· Heat the blend on a water bath at 70–75°C until beeswax completely dissolves and a homogeneous oily phase is created.

· Incorporate a few drops of rose oil (q.s.) as a perfume when the wax has melted completely. Mix gently.

b. Preparation of the Aqueous Phase:

· In another beaker, dissolve 0.36g of borax and 0.03g of methyl paraben in a sufficient amount (q.s.) of distilled water, and warm to 70°C.

· Mix the aqueous solution with 2mL of African tulip flower extract. Mix well for dissolution or uniform dispersion.

c. Emulsification Process:

· Stir the oil phase continually while adding the hot aqueous phase slowly in small amounts to the oil phase.

· Keep the temperature at about 70°C while mixing to allow effective emulsification.

· Stir at constant pace with the help of a mechanical stirrer or hand blender until smooth cream starts to form.

d. Cooling and Finishing:

· After all of the aqueous phase has been added, stir the mixture while letting it slowly cool to room temperature.

· During cooling, the cream will thicken and become more stable.

· Place the prepared cream in a clean, sterilized, wide-mouth amber glass jar or container.

· Label and keep the cream in a dry, cool area.⁸

Evaluation of Cream:

1. pH Determination:

The pH of the African tulip flower extract-containing cream was determined by using pH indicator paper. A tiny amount of the cream (about 1 gram) was added to 5–10 mL of distilled water and shaken well to create an even suspension. A piece of universal pH paper was then dipped into the solution for a couple of seconds. Upon removal, the colour shift on the pH strip was compared with the standard colour chart immediately. The test aided in establishing whether the formulation lies within the optimal skin-compatible pH range of 4.5 to 6.5 so as to ascertain its appropriateness for topical use.⁹

2. Organoleptic Property: Organoleptic properties are the sensory attributes of a formulation that can be detected by the senses—sight, smell, touch.

· Colour: Colour is assessed visually under natural or standardized lighting conditions by observing the product's appearance in a clean, white background.

· Odour: Odour is evaluated by smelling the sample as it is or after soft rubbing on the skin (for creams), in order to identify the presence of any agreeable, neutral, or unpleasant odour.

· Texture: Texture and consistency (optional in organoleptic tests) are assessed by touch, to ensure the smoothness, grittiness, or greasiness of the formulation.

3. Viscosity Determination:

Viscosity was determined with a Brookfield rotational viscometer at 25±0.5°C. About 400 mL of the homogenated cream was put in a 600 mL beaker and allowed to come to temperature equilibrium for 15 minutes. A suitable spindle (LV 3 for medium bodied creams) was submerged to the calibration mark, and the machine was operated at different rpm; the torque reading was permitted to reach equilibrium for about 30 seconds before noting. Two further readings were also taken under the same conditions, and the mean was multiplied by the Brookfield spindle factor to get viscosity in cp.

4. Homogeneity:

The homogeneity of the African tulip flower extract-containing cream formulation was tested using both visual and touch assessment. Some of the cream was transferred and uniformly spread on a pristine glass slide and between fingers. The cream was inspected for the regular distribution of colour, consistency, and texture and the absence of lumps, phase separation, and grittiness. The product was deemed to be homogenous when it displayed an even, uniform, and smooth appearance with equal distribution of all ingredients, affirming stable formulation and adequate mixing.

5. Irritancy Study:

The irritancy test was performed to determine the safety and skin tolerance of the cream formulation with African tulip flower extract. A small amount of the cream was applied to a clean, shaved skin on the forearm or behind the ear of a healthy volunteer. The skin was then monitored for any appearance of redness, itching, inflammation, or irritation within 24 to 48 hours. The preparation was deemed non-irritant if no signs of skin irritation or annoyance were observed, which suggests that the cream is safe for external application.^{.10, 11}

6. Acid value:

Acid value of the cream formulation was found to determine the free fatty acid content, which can be a measure of product stability and shelf-life. Accurately about 1–2 grams of the cream was weighed and dissolved in a neutral alcohol-ether mixture (1:1). The solution was warmed gently if required for proper dissolution. This blend was titrated with 0.1 N sodium hydroxide (NaOH) solution with the aid of phenolphthalein as the indicator. The endpoint was marked by the occurrence of a stable pink color. The acid value was determined from the formula:

Acid Value = Volume of NAOH (mL)× Normality of NAOH× 56.1

Weight of sample (g)

7. Saponification value:

For determining the saponification value of the cream formulation, roughly 1–2 grams of sample was weighed precisely and transferred to a dry, clean flask. Next, 25 mL of 0.5 N alcoholic potassium hydroxide (KOH) solution was poured into the flask and refluxed for around 30 minutes so that the fats and oils are fully saponified. The flask was subsequently left to cool to the room temperature after refluxing. The mixture was titrated with 0.5 N hydrochloric acid with the use of phenolphthalein as an indicator, until the colour changed from pink to colourless. A blank titration with no sample was also conducted for comparison purposes. The amount of hydrochloric acid needed to neutralize the excess KOH was recorded and employed to determine the saponification value according to the official formula.¹²

(b – a) × 28.05

weight taken

b= volume of HCL for blank

a= volume of HCL for sample

w= weight of sample taken

8. Antimicrobial Testing:

In order to evaluate the African tulip flower extract or cream formulation's antimicrobial potential, the agar well diffusion method was utilized. Four typical bacterial strains—Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa) and Gram-positive bacteria (Staphylococcus aureus, Bacillus thuringiensis)—were used to conduct the research.

A. Preparation of Nutrient Agar Media Plates:

Nutrient agar is a general-purpose medium used for the cultivation of a wide variety of non-fastidious microorganisms. It provides essential nutrients for bacterial growth and is ideal for antimicrobial activity studies.

Materials Required:

· Nutrient Agar Powder

· Distilled Water

· Conical flask

· Measuring cylinder

· Autoclave

· Petri plates (sterile)

· Bunsen burner or spirit lamp

· Laminar air flow cabinet (preferred for pouring)

Procedure:

1. Weighing the Medium:

· Weigh 28grams of Nutrient Agar powder (as per manufacturer's instructions) and transfer it into a 1-liter conical flask.

2. Dissolving:

· Add 1000mL of distilled water to the flask.

· Mix thoroughly and heat gently while stirring to dissolve the medium completely until a clear solution is obtained.

3. Sterilization:

· Cover the mouth of the flask with cotton plug or aluminium foil.

· Sterilize the solution by autoclaving at 121°C and 15 psi pressure for 15–20 minutes.

4. Pouring the Plates:

· After autoclaving, allow the medium to cool slightly (to about 45–50°C).

· Under aseptic conditions (preferably in a laminar airflow cabinet), pour approximately 20 mL of the sterile nutrient agar into each sterile Petri plate.

· Leave the plates slightly open until the agar solidifies, then close the lids.

5. Storage:

· Once solidified, plates can be used immediately or stored inverted (lid side down) in the refrigerator at 4°C for up to 1 week.

B. Preparation of Tryptic Soy Agar (TSA) Plates:

Tryptic Soy Agar (TSA) is a general-purpose, nutrient-rich medium that supports the growth of a wide variety of microorganisms, including both Gram-positive and Gram-negative bacteria.

Materials Required:

· Tryptic Soy Agar powder (commercially available)

· Distilled water

· Conical flask or media bottle

· Autoclave

· Sterile Petri dishes

· Measuring cylinder

· Bunsen burner or spirit lamp (for flame sterilization)

· Laminar airflow cabinet (for sterile pouring)

Procedure:

1. Weighing the TSA Powder:

· Accurately weigh 40 grams of TSA powder (as per manufacturer’s instructions) and transfer it to a clean 1-liter conical flask.

2. Dissolving the Medium:

· Add 1000 mL of distilled water to the flask.

· Heat gently with continuous stirring until the medium is completely dissolved and appears clear.

3. Sterilization:

· Cover the flask with a cotton plug or cap it with aluminium foil.

· Autoclave the solution at 121°C and 15 psi for 15–20 minutes to sterilize the medium.

4. Cooling and Pouring:

· Allow the autoclaved medium to cool to around 45–50°C (warm but not hot to the touch).

· Under aseptic conditions (preferably in a laminar airflow hood), pour about 20 mL of the medium into each sterile Petri dish.

5. Solidification and Storage:

· Let the plates sit with lids slightly ajar until the agar solidifies.

· Once solid, cover and store the plates in an inverted position (lid down) at 4°C until use.

C. Procedure for Making Wells in Agar Plates

1. Preparation of Inoculated Agar Plates:

· After preparing and solidifying the agar medium (e.g., Nutrient Agar or TSA) in sterile Petri dishes, allow it to cool and solidify at room temperature.

· Once solidified, swab the surface of the agar uniformly with the bacterial inoculum using a sterile cotton swab to create a lawn of the test microorganism.

2. Making the Wells:

· Using a sterile cork borer or gel punch (commonly 6 mm in diameter), gently press vertically into the agar to form a circular well.

· Remove the agar plug using a sterile needle or forceps, ensuring the bottom of the well is clean and flat.

· Repeat to make 3–5 wells per plate, spaced adequately to avoid overlapping of zones.

3. Labelling the Wells:

· Label each well on the underside of the Petri dish to indicate the test solution or concentration that will be added to it.

4. Adding Test Solutions:

· Add a measured volume i.e. 50, 100, 150, 200µL of the test sample, control, and standard into each respective well using a micropipette.

· Be careful not to overflow or touch the edges of the well.

5. Incubation:

· Allow the test solutions to diffuse into the agar for 15–30 minutes at room temperature before incubation.

· Incubate the plates in an inverted position at 37°C for 24hours.¹³

9. DPPH Assay:

The DPPH assay is one of the most common methods for the measurement of free radical scavenging activity of compounds or formulations. The reduction of the DPPH radical (purple colour) by antioxidants results in a colour change that is detectable, to yellow, which can be quantitated by a UV-Vis spectrophotometer.

1. Materials Required:

· DPPH (2,2-diphenyl-1-picrylhydrazyl) powder

· Methanol or ethanol (analytical grade)

· Sample formulation (cream or extract)

· Standard antioxidant (e.g., ascorbic acid or BHT)

· UV-Visible spectrophotometer

· Test tubes or microplates

· Pipettes and micropipettes

· Amber-coloured bottles (to protect DPPH from light)

2. Preparation of DPPH Solution:

· Dissolve 3.9mg of DPPH in 100mL of methanol to prepare a 0.1mM DPPH solution.

· Store the solution in a dark amber bottle to prevent degradation by light.

3. Sample Preparation:

· For extracts: Prepare different concentrations in methanol.

· For cream formulations: Weigh a small amount (e.g., 0.5–1g), mix with 10mL methanol, and vortex or stir thoroughly. Filter or centrifuge the mixture to obtain a clear supernatant for testing.

4. Assay Procedure:

a) Test Setup:

· In clean test tubes or cuvettes, add 1mL of DPPH solution to 1mL of the sample solution.

· Prepare a blank with 1mL sample + 1mL methanol (no DPPH).

· Prepare a control with 1mL DPPH + 1mL methanol (no sample).

· Prepare standards using ascorbic acid or another known antioxidant.

b) Incubation:

· Shake gently and incubate the mixture at room temperature in the dark for 30 minutes.

c) Measurement:

· Measure the absorbance at 517nm using a UV-Vis spectrophotometer.

5. Calculation:

The formula to calculate the DPPH free radical scavenging activity (%) is:

DPPH Scavenging Activity (%)

= (A_control −A_sample) × 100^14,15

A_control

RESULTS:

1. pH determination:

The pH of the formulated Spathodea campanulata cream was measured using a digital pH meter at 25±1°C. A 1% w/w dispersion of the cream in distilled water was prepared and evaluated in triplicate. The observed pH values were as follows:

Table no.2: Results of pH

Trial	pH value
1	4.3
2	4.2
3	4.1

Mean pH ± SD: pH = 4.2 ± 0.1

2. Organoleptic Characteristics:

The formulated Spathodea campanulata cream was evaluated for appearance, color, odour, and texture through visual and sensory inspection. The observations are summarized below:

Table no.3: Results of organoleptic characteristics

Parameters	Observations
Colour	Brownish
Odour	Pleasant
Texture	Smooth

3. Homogeneity:

The cream was found to be smooth, uniform, and free from any lumps, gritty particles, or phase separation, indicating excellent homogeneity.

4. Viscosity:

The viscosity of the formulated cream was measured using a Brookfield viscometer at 25±0.5°C, using spindle LV-3 at different rotational speeds. The results are as follows:

Table no.4: Results of viscosity

RPM	Viscosity
10 RPM	2391± 0.577cp
12 RPM	1924.5± 0.578cp
20 RPM	1500± 2.08cp
60 RPM	1027.2± 0.87cp
100 RPM	534± 0.577cp

5. Irritancy study:

The formulated Spathodea campanulata cream was tested for skin irritation by applying a small amount (about 0.5g) to the forearm skin of healthy human volunteers. The test area was observed after 24hours for any signs of redness, itching, swelling, or rashes.

Table no.5: Irritancy study results

Parameters	Observation
Redness	Absent
Itching	Absent
Swelling	Absent
Rashes	Absent

6. Acid value:

The acid value of the formulated cream was determined by titration method and calculated using the formula:

Acid Value = Volume of NaOH (mL)× Normality of NAOH× 56.1

Weight of sample (g)

Weight of sample=10gm

Normality of NaOH= 0.1N

Table no.6: Acid value results

Trials	Volume of NaOH	Acid value
1	1.7	0.9537
2	1.9	1.0659
3	2.1	1.1781

Average Acid value for the cream formulation is = 1.0659 ± 0.1121

7. Saponification value:

The saponification value of the formulated Spathodea campanulata cream was determined by titration method using alcoholic potassium hydroxide solution, and calculated by the formula:

(b – a) × 28.05

weight taken

b – Blank value

a – Titre value

Table no.7: Saponification value

Trial	Saponification value (mg KOH/g)
1	145.6
2	146.5
3	148.8

Average saponification value for cream is = 146.97± 1.65

8. Anti-microbial Assay:

The antimicrobial activity of the formulated Spathodea campanulata cream was evaluated using the agar well diffusion method against selected bacterial strains. The zone of inhibition (in mm) was measured after 24 hours of incubation at 37°C for bacteria.

Fig no:1-Bacillus T.

Fig no:2- E-coli

Fig no:3 Streptococcus aureus

Microorganisms	Zone of inhibition ZOI (in mm)
Microorganisms	50µg/ml	75µg/ml	100µg/ml	200µg/ml
Bacillus thuringiensis	10	11	12	15
Escherichia coli	No zone of inhibition	5	6	10
Pseudomonas aeruginosa	No zone of inhibition
Streptococcus aureus	No zone of inhibition

Sr no	Control	Sample
-	-	250 µg/ml	500 µg/ml	750 µg/ml	1000 µg/ml
% Antioxidant activity	-	7.704654896	13.964687	25.89620118	30.12306046

Formulation and Evaluation of Herbal cream using Spathodea campanulata