INTRODUCTION:

Historically, medicinal plants have been a major source of cure for human diseases since time immemorial. According to the World Health Organization, 80 percent of the world’s population still relies mostly on traditional medicines for health treatment. The phytoconstituents, such as saponins, tannins, alkaloids, flavonoids, terpenoids, and sesquiterpenes, which work in synergy to produce the desired effect^1,2. Topical application of therapeutic agents provides an advantage of bypassing first pass metabolism, avoidance of the risks and inconveniences of intravenous therapy. Emulgel is a combination of emulsion and gel, which is a new approach for topical delivery of drugs. The presence of the gelling agent in water phase converts an emulsion into an emulgel³.

It has a double control release like emulsion and gel by penetrating the drug into the skin and providing a rapid onset of action. It has several complimentary properties for dermatological use such as being thixotropic, easily spreadable, emollient and pleasing appearance. Emulgels have good stability than other available topical preparation^4,5.

Thespesia populnea belonging to Malvaceae family is commonly known as Portia tree. The extensive literature survey revealed that T. populnea is a medicinal plant with diverse pharmacological spectrum. The various parts of the plant bark, leaves, flowers and fruits are reported to have shown in treatment of cutaneous infection such as scabies, psoriasis, eczema and having various therapeutic activities like antioxidant, antimicrobial, wound healing anti-implantation, diuretics, cytotoxic. The major phytoconstituents reported in T. populnea include Anthraquinone glycosides, cardiac glycosides, flavonoids, alkaloids and tannins. Air dried flowers of T. populnea contain Kaempferol, β-sitosterol, gossyptein, Rutin^6,7.

Hibiscus rosa-sinensis is a member of the Malvaceae family. The flower and leaf has a healing capacity⁸. Also exhibit analgesic, antioxidant, antibacterial, antifungal, anti-inflammatory, antidiabetic activities⁹. Leaves comprise β-sitosterols, stigmasterol, taraxeryl acetate and three cyclopropane compounds with derivatives¹⁰. There is no report on the formulation of polyherbal emulgel, combination of Thespesia populnea and Hibiscus rosa sinensis for antimicrobial activity. Hence, in the present study bioactive plant extracts emulgel is analysed for antimicrobial activity and its synergistic effect.

MATERIALS AND METHODS:

Plant collection and Authentication:

Thespesia populnea leaves were collected from local areas Belagavi whereas the flowers of Hibiscus rosa sinensis were collected from medicinal garden, Rani Chennamma College of Pharmacy. The Plant specimens were authenticated by Indian Council of Medical Research (ICMR) - National Institute of Traditional Medicine (NITM) Belagavi, Karnataka. The herbarium specimens of the same have been deposited in their herbaria with accession numbers RMRC-1709 and RMRC-1710 respectively.

Extraction method:^11,12

Thespesia populnea leaf extract by cold maceration: 100g of leaf powder is soaked in 500ml of 95% methanol for 72 hours. The extract was filtered using muslin cloth followed by whatman No.1 filter paper. The filtrate is concentrated by rotary evaporator.

Hibiscus rosa-sinensis flower extract by Soxhlet apparatus: 50gm of dried flowers powder is extracted successively with 500ml of methanol. The whole extract was obtained within the hours of 4-6 hours at 60°C. After filtration, the product-solvent was evaporated for dryness under reduced pressure using a Rotavapor.

Figure 1: Cold maceration and Soxhlet extraction methods

Preliminary phytochemical screening of plant extracts: ^{11, 13, 14}

The extracts were subjected to various preliminary phytochemical tests by using reported chemical test protocols as given below in table 1.

Table 1. Phytochemical screening of Thespesia populnea

Phytochemical constituent	Test	Inference
Carbohydrates	Molisch’s test	+++
Carbohydrates	Fehling’s test	+++
Phenolic compounds	Ferric chloride test	+++
Flavanoids	Shinoda test	+++
Alkaloids	Hager’s test	+++
Alkaloids	Wagner’s test	+++
glycosides	Legal’s test	++
Saponins	Foam test	++
Terpenoids	Biuret test	++
Terpenoids	Ninhydrin test	++

Highly present (+++), moderately present (++)

Table 2. Phytochemical screening of Hibiscus rosa sinensis

Phytochemical constituent	Test	Inference
Carbohydrates	Molisch test	+++
Alkaloids	Wagner’s test	+++
Saponins	Foam test	++
Tannins	Ferric chloride test	++
Phlobatannins	HCl test	+++

Highly present (+++), moderately present (++)

Preparation of emulgel:^15-18

The Emulgel was formulated in three different steps

Step 1 Preparation of primary emulsion: Take 5g of extract (4g +1g) + Coconut oil 5ml + Q. S of water in (4:2:1) ratio in a china dish and heat at 70⁰C with continuous stirring. In another china dish add span 80 and tween 80 in 1:3 ratio and incorporate glycerin and heat at 70⁰C with continuous stirring. Mix both phases at room temperature with continuous stirring.

Step 2 Preparation of gel base: For F1:2gm of carbopol 934 in 100ml of distilled water, make a gel by adding Triethanolamine drop by drop. For F2:2.5gm of carbopol 934 in 100ml of distilled water, make a gel by adding Triethanolamine drop by drop. For F3:3gm of Carbopol 934 in 100ml of distilled water, make gel by adding Triethanolamine drop by drop.

Step 3 Incorporation of emulsion into gel base: The prepared the emulsion was added to the gel in 1:1 weight ratio. For the consistency of the product triethanolamine was also added.

Table 3. Formulation of emulgel

Sl. No.	Ingredients	F1	F2	F3
1	Plant extract (Hibiscus rosa sinensis + Thespesia populnea)	4g+1g	4g+1g	4g+1g
2	Carbopol-934	2g	2.5g	3g
3	Coconut oil	2ml	2ml	2ml
4	Span 80	250mg	250mg	250mg
5	Tween 80	750mg	750mg	750mg
5	Sodium benzoate	0.5g	0.5g	0.5g
6	Triethanolamine	1ml	1ml	1ml
7	Methyl paraben	0 .05g	0.05g	0.05g
8	Glycerin	2%	2%	2%
9	Distilled water	Q. s	Q. s	Q. s

Figure 2. Prepared Emulgel formulation F1, F2 and F3 respectively

Evaluation parameters: ^19-22

Physical Examination: The organoleptic properties like colour, odour and state of prepared emulgel were evaluated.

pH determination: The emulgel was measured using digital pH meter. 1gm of gel was dissolved in 100 ml of distilled water and placed for 2 hours. The glass electrode is dipped into an emulgel.

Spreadability: Two sets of glass slides of standard dimensions were taken. The emulgel was placed over one of the slides. The other slide was placed on the top of the emulgel. The weight of 100gm was placed upon the upper slides so that the two slides were pressed uniformly to form a thin layer of emulgel. The time taken for the upper slide to slip off and separate away from the lower slide was noted. Spreadability was calculated using formula:

S = M. L / T Where,

M= weight tied to upper slide (1gm)

L = length of glass slide

T = Time taken to separate the slides

Tube Extrudability: The formulation was filled in aluminum collapsible tube with nozzle tube of 5mm opening and pressure was applied on tube by keeping weights. Tube Extrudability was then determined by measuring amount of emulgel extruded using the formiula:

Extrudability = Applied weight to extrude emulgel from tube (g) / Area (cm²)

Stability Studies: Stability studies were carried out according to International Conference on Harmonization (ICH) guidelines. Short term accelerated stability study was carried out for 3 months. The samples were stored at different temperature conditions i.e., refrigeration temperature (4-8°C), room temperature (25 ±2°C) and oven maintained at (45°C ± 2°C). Sample was withdrawn on monthly interval and analyzed for visual appearance, pH, spreadability and extrudability.

Anti-microbial activity assay: ^{12, 23-28}

The in vitro antimicrobial activity was performed by minimum inhibition concentration method (MIC). The antimicrobial activity of methanolic extract of Thespesia populnea and hibiscus rosa sinensis were done to check the synergistic effect.

RESULT AND DISCUSSION:

Table 4. Results of prepared formulations

Sl. No.	Parameter	F1	F2	F3
1	Colour	Green colour	Green colour	Green colour
2	Odour	Characteristic	Characteristic	Characteristic
3	Appearance	Smooth	Smooth	Smooth
4	pH	6.7	6.8	6.9
5	Spreadability (cm/sec)	11.33	8.5	13.6
6	Extrudability (gcm/sec)	66.66	71.42	76.92
7	Stability test	No separation occurs so its formed to be stable

From the table 4, the prepared formulation emulgel F1, F2 and F3 were found to be smooth in texture with good spreadability and extrudability. All the formulations were stable maintaining the integrity of the product.

Table 5. Results of antimicrobial activity of methanolic plant extracts by MIC method

Sl. No	Samples (T+H) µg/ml	Fungal Strain (Candida albicans)	Bacterial Strain (E. coli)
1	100	S	S
2	50	S	S
3	25	S	S
4	12.5	S	R
5	6.25	S	R

S= Sensitive, R= Resistant

Standard values

Candida albicans-Fluconazole 16µg/ml

E-coli-Ciprofloxacin 2µg/m

Table 6: Results on synergistic activity

Strains	*Thespesia populnea* (T)	*Hibiscus rosa sinensis* (H)	Combination (T+H)
E coli	250µg/ml	25µg/ml	25µg/ml
Candida albicans	25 µg/ml	25µg/ml	6.25µg/ml

Figure 3. Graph of synergistic antimicrobial activity of two plant extracts

Figure 4. MIC dilution test of plant extracts against E. coli and C. albicans

According to table 6, the plant extract combination (T + H) showing sensitive synergistic effect at the range of 25µg/ml as compared to individual effect. The combination (T+H) showing better antibacterial activity at 25ug/ml compared to individual plant extract, specific to that of Thespesia populnea at 250ug/ml, whereas the plant extract combination (T+H) showing better antifungal activity at 6.25µg/ml compared to individual plant extract i.e., 25µg/ml as depicted graphically in figure 3.

CONCLUSION:

The present work was aimed to formulate and evaluate polyherbal emulgel prepared by using methanolic extract of Thespesia populnea and Hibiscus Rosa-sinensis. Combination of herbal drugs shows better antimicrobial effect. Based on the results and discussion, it was concluded that the prepared formulations were found to be smooth in texture with no evidence of phase separation during the stability study. From the result, F3 showed desired spreadability and extrudibility compared to F1 and F2. As both individual plant extract possibly cannot increase the extent of efficiency of medicinal and cosmetic property compared to polyherbal plant extract. Considering that, we used combination of Thespesia populnea and Hibiscus rosa sinensis plant extract for the preparation of emulgel. Hence it can be concluded that the prepared polyherbal emulgel was stable and can be safely used topically in order to protect skin against microbial activity.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank Rani Chennamma college of Pharmacy, Belagavi for carrying out this project work. We would also like to thank ICMR-NITM, Belagavi and Maratha mandal’s central research laboratory (CRL), Belagavi for providing the required facilities.

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Received on 13.03.2023 Revised on 09.04.2024

Accepted on 22.11.2024 Published on 27.03.2025

Available online from March 27, 2025

Research J. Pharmacy and Technology. 2025;18(3):1058-1062.

DOI: 10.52711/0974-360X.2025.00152

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Figure 1: Cold maceration and Soxhlet extraction methods

Preliminary phytochemical screening of plant extracts: 11, 13, 14

The extracts were subjected to various preliminary phytochemical tests by using reported chemical test protocols as given below in table 1.

Preliminary phytochemical screening of plant extracts: ^{11, 13, 14}