Formulation and Evaluation of Rubia Cardifolia L. And Ocimum Sanctum L. Wound Healing Emulgel

Modern medicine has achieved remarkable progress; however, several diseases and disorders still lack effective therapies. Since ancient times, medicinal plants have played a crucial role in human health management. They provide a rich source of bioactive compounds such as phenolic acids, flavonoids, tannins, terpenoids, and alkaloids that possess antioxidant, antimicrobial, and anti-inflammatory properties, contributing to wound healing and tissue repair [1]. Traditional medicine systems like Ayurveda and Siddha have long used herbal formulations for treating wounds, skin ailments, and infections. The increasing resistance of pathogens to synthetic drugs and the side effects of modern therapeutics have renewed global interest in herbal-based medicines [2]. A wound is defined as a disruption of the normal structure and function of skin tissue caused by physical, chemical, thermal, microbial, or immunological injury [3]. Wounds are broadly classified as open or closed based on tissue damage, and as acute or chronic based on the healing process [4]. Acute wounds, such as surgical cuts, heal within a predictable period, whereas chronic wounds, like diabetic ulcers, fail to proceed through the normal phases of healing [5]. The wound healing process involves three overlapping and highly coordinated phases—inflammatory, proliferative (fibroblast), and remodeling. Initially, the inflammatory phase involves vasoconstriction and platelet aggregation to stop bleeding, followed by migration of neutrophils and macrophages to remove debris. The proliferative phase is characterized by fibroblast proliferation, collagen deposition, and angiogenesis. Finally, in the remodeling phase, collagen crosslinking increases the tensile strength of the tissue, leading to scar formation [6,7]. Various factors such as infection, age, diabetes, poor nutrition, and medications (like corticosteroids and NSAIDs) can impair wound healing [8]. Topical drug delivery systems are designed to deliver drugs directly to the affected skin site, offering localized action while minimizing systemic side effects. This route bypasses first-pass metabolism and provides a patient-friendly approach to treating skin infections, wounds, and inflammatory conditions [9,10]. Several dosage forms—ointments, creams, gels, and lotions—are used, but each has limitations. For instance, ointments are greasy, creams may lack stability, and gels alone are unsuitable for hydrophobic drugs [11]. Emulgels, an advanced hybrid system combining the properties of emulsions and gels, have emerged as a superior vehicle for topical delivery. They offer dual advantages—enhanced solubility of lipophilic drugs due to the emulsion phase and improved stability, spreadability, and aesthetic appeal due to the gel matrix [12]. Emulgels are thixotropic, non-greasy, easily spreadable, and washable, with improved bioavailability and controlled drug release [13]. This makes them particularly suitable for incorporating herbal extracts like Rubia cordifolia and Ocimum sanctum, which possess wound healing and antimicrobial activities. Ocimum sanctum (family: Lamiaceae), commonly known as Tulsi or Holy Basil, is an aromatic herb renowned for its therapeutic versatility. The plant contains bioactive constituents such as eugenol, ursolic acid, rosmarinic acid, orientin, and vicenin, which possess antioxidant, antimicrobial, and anti-inflammatory properties [14]. Studies have demonstrated that aqueous extracts of O. sanctum accelerate wound contraction, increase tensile strength, and enhance epithelialization in excision wound models, possibly by modulating cytokines such as TNF-α [15]. The herb is also known to improve local blood circulation and reduce microbial load, facilitating faster tissue regeneration. Rubia cordifolia (family: Rubiaceae), commonly known as Manjishtha or Indian Madder, is a perennial climbing herb traditionally used for treating skin diseases, inflammation, and wounds. The root contains anthraquinones (rubiadin, alizarin, purpurin), mollugin, and triterpenoids, which exhibit potent antioxidant, antibacterial, and collagen-promoting properties [16]. Research indicates that ethanolic root extracts of R. cordifolia significantly enhance wound closure, fibroblast proliferation, and tissue granulation [17]. Its phytochemicals stimulate fibroblast activity and angiogenesis, essential for granulation tissue formation, thereby accelerating wound repair. Conventional wound healing formulations, such as synthetic creams and antibiotics, often cause side effects including pigmentation and delayed healing. The need for biocompatible, non-toxic, and affordable alternatives has driven interest in herbal-based topical preparations [18]. Combining O. sanctum and R. cordifolia in a single formulation is scientifically justified, as their synergistic effects may enhance antimicrobial action, antioxidant defense, and tissue regeneration. However, both extracts are partially hydrophobic, which limits their solubility in simple gel formulations. Incorporating them into an emulgel system allows for uniform dispersion, improved drug release, and enhanced skin permeation [19]. Thus, the formulation of a herbal emulgel containing R. cordifolia and O. sanctum provides a novel and efficient approach for promoting wound healing while maintaining cosmetic acceptability. Emulgels possess multiple advantages, such as easy application, patient compliance, non-greasy texture, controlled drug release, and high stability. They enhance both hydrophilic and lipophilic drug penetration and provide sustained therapeutic action at the wound site [20]. Additionally, the formulation can be easily terminated by washing and avoids systemic side effects associated with oral therapy. The combination of O. sanctum and R. cordifolia in an emulgel base may therefore serve as a promising natural therapeutic option for managing wounds.The present research aims to formulate and evaluate an herbal wound-healing emulgel containing Rubia cordifolia L. and Ocimum sanctum L. extracts. The formulation is expected to exhibit enhanced wound contraction rate, antimicrobial activity, and stability while providing both medicinal and cosmetic benefits.

MATERIALS AND METHODS

MATERIALS

The present study utilized Rubia cardifolia L. (Manjishtha) and Ocimum sanctum L. (Tulsi) extracts, procured from Amsar Pvt. Ltd., Indore, India. All chemicals and reagents used in the formulation and evaluation were of analytical reagent (AR) and high-performance liquid chromatography (HPLC) grade. The excipients used included Carbopol 940, Tween 80, Span 20, Propylene glycol, Liquid paraffin, Methyl paraben, and Propyl paraben, all sourced from Merck and Analab Fine Chemicals, Mumbai. Karanja oil was used as a natural emollient.

Table no. 2.1. List of excipients and Chemicals with source

All the glassware used were of borosilicate grade. Equipment used included an electronic balance (Shimadzu AX200), UV–Visible spectrophotometer (Shimadzu UV1700), pH meter (Equiptronics EQ-614), Brookfield Viscometer (LVDV), Franz diffusion cell (Orchid Scientific, India), FTIR (Shimadzu IRAffinity-1s), and stability chamber (Biomedica BMC-2122)

METHODS

2.2.1 Preformulation Studies

The preformulation studies included organoleptic evaluation, solubility analysis, UV and FTIR spectroscopy, and phytochemical characterization of both extracts to ensure compatibility and stability. UV spectral analysis was performed to determine the maximum absorbance wavelength (λmax) of the extracts, found to be 271 nm for Rubia cardifolia L. and 288 nm for Ocimum sanctum L. FTIR spectra were recorded to identify characteristic functional groups and to confirm the absence of chemical interaction between drug and excipients [30].

2.2.2 Preparation of Emulgel

The emulgel was prepared by the emulsification method followed by gel incorporation, as described by Mohamed and Modi and Patel [31]

Step 1: Preparation of Emulsion Phase

The oil phase consisted of liquid paraffin, Span 20, and the respective plant extracts, while the aqueous phase contained Tween 80, propylene glycol, and methyl paraben. Both phases were heated separately to approximately 70°C and then combined with continuous stirring using a mechanical stirrer (Remi RQ-121/D) until a uniform emulsion was obtained.

Step 2: Preparation of Gel Base

Carbopol 940 was dispersed in distilled water with continuous stirring and allowed to swell overnight. Triethanolamine was added dropwise to neutralize the pH and form a transparent gel base [32].

Step 3: Incorporation of Emulsion into Gel Base

The prepared emulsion was slowly incorporated into the gel base under mechanical stirring to obtain a smooth, homogeneous emulgel. The final formulation was stored in air-tight containers at room temperature

2.3 Evaluation of Emulgel

The prepared formulations were subjected to various physicochemical and biological evaluations to determine their performance, stability, and efficacy [33].

2.3.1 Physical Appearance and Homogeneity

The prepared emulgel was inspected visually for color, consistency, and phase separation. Homogeneity was assessed by simple visual observation.

2.3.2 pH Determination

The pH of the emulgel was measured using a calibrated pH meter (Equiptronics EQ-614). Measurements were performed in triplicate, and the average was reported to ensure skin compatibility.

2.3.3 Viscosity

Viscosity was determined using a Brookfield Viscometer (LVDV) at room temperature using spindle number 64 at 10 rpm. The readings ensured appropriate rheological behavior for topical application [34].

2.3.4 Spreadability

The spreadability of the emulgel was evaluated using a glass slide method, where the time required for two slides to separate under a specific weight was recorded. Spreadability was calculated using the formula:

S=M×LTS = \frac {M \times L} {T}S=TM×L?

where M = weight tied to upper slide, L = length moved, and T = time in seconds.

2.3.5 Drug Content Determination

The drug content was analyzed spectrophotometrically by dissolving 1 g of emulgel in phosphate buffer (pH 7.4), followed by suitable dilution and absorbance measurement at respective λmax values [35].

2.3.6 In Vitro Drug Release

The drug release profile was determined using a Franz diffusion cell fitted with an egg membrane. The receptor compartment was filled with phosphate buffer (pH 7.4) maintained at 37 ± 2°C and stirred magnetically. Samples were withdrawn at predetermined intervals, filtered, and analyzed by UV spectrophotometry.

2.3.7 Antimicrobial Activity

The antibacterial activity of the formulated emulgel was determined using the well diffusion method on E. coli using Muller Hinton agar medium. The diameter of the inhibition zone was measured in millimeters and compared with a standard antibiotic (Amikacin) as a positive control. Methanol served as the negative control. The average inhibition zone was found to be 10.43 mm, indicating promising antimicrobial potential [36]

2.3.8 Stability Studies

Stability testing was carried out at 37°C, 45°C, and 60°C for three months in a stability chamber. The formulations were observed for color change, phase separation, viscosity variation, and drug content degradation at intervals of 15 days [37].

2.4 Statistical Analysis

All experiments were performed in triplicate, and data were analyzed using Design of Experiment (DOE) and ANOVA for optimization of formulation parameters. Results were expressed as mean ± standard deviation [38].

RESULT AND DISCUSSION

3.1. Pharmacognostic study of extract

Table No. 3.1: Pharmacognostic study of Ocimum Sanctum L. and Rubia Cardifolia L. extract

 3.2. Phytochemical tests –

3.2.1. Phytochemical tests of Ocimum Sanctum extract:

Table No.3.2: Phytochemical test of Ocimum Sanctum L.

(+) =present of constituent, (-) = constituent not present  

The results of preliminary phytochemicals tests are shown in above table. The phytochemicals screening of the extract of Ocimum Sanctum L. Extract indicates the presence of glycosides, Saponins, and terpenoids.

 3.2.2. Phytochemicals test of Rubia Cardifolia extract:

Table No.3.3: Phytochemicals test of Rubia cardifolia L.

(+) = present of constituent, (-) = constituent not present  

The results of preliminary phytochemicals tests are shown in above table. The phytochemicals screening of the extract of indicates the presence of glycosides, Saponins, and carbohydrates. 

Fig no.3.1 phytochemicals test

UV Spectra of Ocimum Sanctum extract – Standard calibration curve of Ocimum Sanctum extract was Drawn by plotting absorbance V/S concentration. Maximum of Ocimum Sanctum was found to be 282nm. The absorbance value is given in table.

Table no. 3.4 UV Absorbance of Ocimum Sanctum L.

Fig 3.2 Standard calibration curve of Ocimum sanctum in Water

IR spectrum of Ocimum sanctum:

Fig.3.3 IR graph of Osmium sanctum L.

Table no. 8.5 IR Interpretation of Osmium Sanctum L.

UV Spectra of Rubia Cardifolia:

Standard calibration curve of Rubia Cardifolia extract was Drawn by plotting absorbance V/S concentration. Maximum of Rubia Cardifolia was found to be 271nm. The absorbance value is given in table.3.4.

Absorbance-

Table 3.6 UV absorbance Rubia Cardifolia L.

Fig 3.5 Standard calibration curve of Rubia Cardifolia in Water

IR spectra of drugs Rubia Cardifolia –

Fig.no 3.6 IR graph of Rubia Cardifolia

Interpretation of IR graph -                            

Table 3.7 IR identification of Rubia Cardifolia L.

3.3. Evaluation of Emulgel-

3.3.1. Appearance of Emulgel:

Colour, Homogeneity, phase separations and texture were found to be acceptable limit. The phase separation does not occur in any Formulation and have a good Consistency and excellent homogeneity, which is indicates the good and very stable Formulations.                  

TableNo.3.8: color, homogeneity and phase separations of Emulgel.

3.3.2. Viscosity: The viscosity of Emulgel formulation is done as per standard procedure. It indicates increases the concentration of Carbopol 940 results in increase in the viscosity. The viscosity of Emulgel was found as shown in table no 8.9

Table No. 3.9: Viscosity of Emulgel

Values as shown, Mean ± SD. (N=3)

Fig.no.3.7 3D Graph of viscosity and rheological property

Fig.no 3.10 viscosity parameter as per dependent factors

• The model value F 172.12 implies that model is significant, there is only 0.01% chance of F value due to noise can occur value is also less than 0.005 which shows model is significant. 
• Viscosity = +1987.00+202.75A-105.00B-49.25C+87.75AB-52.25AC+32.75BC-35.38A² 

3.3.3. pH: The pH evaluations of topical dosage form are very important as it may cause irritation to skin if varied from the normal the pH of the skin condition. The pH of all batch formulation is done as per standard procedure. It indicates more polymers like Carbopol give consistency and having acidic nature. The pH range of all the formulation was found to be within acceptable range. The pH of all batches was found as shown in table 8.11.

Table No. 3.11 pH of Emulgel

Values as shown, Mean ± SD. (N=3)

3.3.4. Spreadability: The Spreadability of Emulgel batches was done as per standard procedure.                   

The Spreadability was how in table 8.12.

Table No.3.12: Spreadability of Emulgel

Values as shown, Mean ± SD. (N=3)

3.3.5. Drug diffusion study- In vitro drug diffusion is studied using standard IP procedure of Drug diffusion cell up to 6 hr time.  

TableNo.3.13: Drug diffusion of Emulgel at 360 min.

Fig.3.9 Drug release counter plot graph

Fig.no.3. 10 Drug release 3D Graph

Fit Statistics as per ANNOVA-

TableNo.3.14: ANNOVA statistics for drug release

The Predicted R² of 0.9527 is in reasonable agreement with the Adjusted R² of 0.9932; i.e. the difference is less than 0.2 and Adel. Precision measures the signal to noise ratio. A ratio greater than 4 is desirable. ratio of 50.842 indicates an adequate signal. This model can be used to navigate the design space. 

? Drug release Equation = +93.00-1.07A-1.08B-4.10C-0.8550AB+1.76AC+2.00BC-

6.81A²+0.4212B²-7.11C² 

3.3.6. Antimicrobial study- The well diffusion method was used to evaluate the antibacterial activity to determine the antimicrobial action and efficacy of formulation comparing marketed antibiotic activity drug.  

Fig.no. 3.11 antimicrobial study by zone inhibition method

3.3.6.1. Antimicrobial study parameters-

Table no.3.15 parameters of antimicrobial study

3.3.6.1. Zone of inhibition calculation –

Table no.3.16 calculation of zone inhibition