Formulation and Evaluation of Hydrogel Incorporated with Biophytum sensitivum Extract

Introduction

Chronic wound treatment remains a significant challenge due to high costs associated with wound care products, surgical interventions, and healthcare resources such as physician and nursing time. Despite the serious complications associated with diabetic wounds, such as foot amputation, increased morbidity, no specific effective therapy has been developed. Statistical analyses indicate that chronic skin wounds can have worse outcomes than several common types of cancer, including prostate, breast and colon cancers.¹

In wound management, incorporating antiseptics into hydrogels is a promising strategy to prevent wound colonization and contamination. At present, silver nanoparticles are the most commonly used antiseptics for this purpose; however, they are associated with toxicity.² To address this issue, new approaches are needed that incorporate antibiotics or antiseptics with antimicrobial activity into polymer-based hydrogels. In this context, the improved patient compliance and fewer side effects associated with herbal remedies have led to a shift from allopathic medicines to plant based alternatives. Recently, biofriendly plant-derived products have gained significant attention for their potential in preventing and treating various human diseases. Therefore, plants can be explored as effective alternatives to synthetic drugs. In India, the use of herbal medicines dates back to ancient times. However, there is a need to evaluate their use in wound management. Herbal medicines in wound management aid in disinfection, debridement and maintaining a moist environment suitable for natural healing.³

Hydrogels are widely recognized as a standard treatment option for necrotic and sloughy wounds. A key feature of hydrogels is that their high water content, which helps cool the wound surface, providing a soothing effect and promoting autolytic debridement of wound.

The present study focused on the design of a herbal formulation with reduced toxicity. An added advantage of this formulation is its antimicrobial potential, and its ability to retain water, hold the drug and absorb wound exudates. The incorporation of polymers like Carbopol and PEG (polyethylene glycol) in the hydrogel enhances its ability to cover and protect the wound from bacterial infections.

The plant used in the present study, Biophytum sensitivum Linn., is recognized for its diverse range of biological activities. In India, this plant is considered as a traditional herb and has been utilized for treating diabetes, ulcers, diarrhoea, oedema, burning sensations, asthma, and various skin diseases.⁴ Additionally, it is employed to treat snake bites. The phytochemical analysis of the plant indicates the presence of flavones including cupressuflavone and amentoflavone. In addition, it contains flavonoids such as luteolin 7-methyl ether and 3'-methoxyluteolin 7-O-glucoside, as well as two acids, 4-caffeoylquinic acid and 5-caffeoyl quinic acid. The plant has been reported to possess pharmacological activities-apoptotic,cell-mediated immune response, chemoprotective, immunomodulatory, hypocholesterolaemia, hypoglycaemic, anti-inflammatory, antitumor activities.⁵ Moreover, literature survey revealed a lack of scientific validation for this specific formulation and its stability.Therefore, an attempt was made to develop a gel using the alcoholic extract of the whole plant of Biophytum sensitivum Linn., along with conducting stability studies of the formulation.

Carbopol 934 is synthetic polymer (crossed linked polyacrylic acid polymer consisting of acrylic acid mono mers and allyl ethers of sucrose) which is hydrophilic in nature. It is also biocompatible, biodegradable and stable in nature, appropriate to serve as a vehicle in drug delivery systems. Its superior swelling and thickening properties have been leveraged by the pharmaceuti cal and cosmetic industries. Such polymers show great promise for being used as medicinal carriers, especially in controlled-release formulations.⁶ PEG-based biomaterials have several notable benefits, including excellent water solubility, biocompatibility, and lack of immunological response stimulation. These properties make PEG a highly versatile biomaterial, used in various forms such as bulk polymers, thin solid films, hydrogels, and nanoparticles.⁷ Therefore, we hypothesized that a herbal hydrogel could serve as an effective alternative to existing formulations for treating skin conditions.

Materials and Methods

Materials

Carbopol 934 -9003-01-4, PEG 400 -25322-68-3 and Triethanolamine -102-71-6 (Product code 13150) were purchased from Karnataka Fine Chem, Bengaluru. Methyl Paraben -99-76-3 (Product code 08120) and Methanol -67-56-1 were purchased from Yarrow Chem, Bengaluru.

Preparation of Biophytum sensitivum Linn. extract

Dried powder of whole plant material of B.sensitivum Linn. was extracted using Soxhlet apparatus for 48 hours at 450°C with 80% methanol as solvent. Then, the extract obtained was filtered, distilled and concentrated at 450°C. The concentrated extracts were stored at room temperature and used for further studies.⁸

Qualitative chromatographic analysis using thin layer chromatography (TLC)⁹

The principle of thin layer chromatography is based on adsorption phenomenon. In TLC, the mobile phase flows through the stationary phase and carries the dissolved solute along. The mobile phase used in the study was toluene, ethyl acetate, and formic acid in a 5:4:1 ratio. The standard Rf -value of quercetin is 0.46.

To determine the presence of flavonoids, quercetin was taken as the standard for estimation of major constituent in B.sensitivum Linn. For that, the mobile phase was prepared and allowed for saturation for 5-10 minutes. Then the sample and standard were applied on TLC plate and kept aside for drying. After drying, the TLC plate was immersed in the mobile phase and allowed for the formation of spots. The spots were detected and the Rf value was calculated.

Formulation of B.sensitivum Linn. extract incorpo rated hydrogel¹⁰

Measured quantities of polymers were dispersed in 50 mL of distilled water. For formulations F1, F2 and F3, Carbopol 934 was used, while PEG 400 was used for F4 and F5. The formulations were left undisturbed for four hours to allow the swelling of Carbopol 934 and PEG 400. Afterward, the mixing process was carried out using a magnetic stirrer. To that mixture, the required amount of methyl paraben was added by dissolving in 5 mL of distilled water. To adjust the formulation to the desired skin pH (6.8 - 7.0), 1.2 mL of triethanolamine was added dropwise with continuous stirring until a gel was formed. Further, 0.5 mg of B.sensitivum Linn. extract was incorporated into the gel, which was then stored for further study.

In this formulation, Carbopol 934 and PEG 400 were used as polymers, methyl paraben served as a preservative and triethanolamine as a gelling and pH adjustment agent.

Evaluation of B.sensitivum Linn. extract incorporated hydrogel

Appearance

The appearance, one of the physical properties of hydrogel formulations, was checked visually and observations were recorded.

Homogeneity

The homogeneity of the hydrogels was analysed through visual inspection of their physical properties, such as tone, lucidity and stage division and the observations were recorded.

Grittiness

The grittiness was assessed to observe if any visible particles were present in the prepared hydrogel formulation batches.

Washability

The formulated hydrogels were applied to the skin, and the extent of washing with water was assessed manually. The observations were recorded.

Determination of pH

The pH of the hydrogel formulations has been determined using digital pH meter. One gram of hydrogel was dissolved in 25 mL distilled water and the electrode was dipped into the hydrogel formulation. The measurements were noted.

Determination of viscosity¹¹

The viscosity of the prepared hydrogel was assessed using Brookfield digital viscometer. The viscosity was measured using spindle number 6 at 10 rpm, at 25℃.

Spreadability¹²

Spreadability is a term that describes the extent of area over which the gel spreads easily when applied. Spreadability was measured based on the time required for two slides, placed with the formulation in between and under a certain load, to slip apart. The shorter separation time indicates better spreadability.

Method: Two standard-sized glass slides (7×2) were selected. The formulated hydrogel, the spreadability of which is to be determined, was applied to one of the slides. The second slide was positioned over the first such that the formulation was sandwiched between them along a 7 cm length of the slide. About 100 g of hydrogel was taken on the upper slide, and was traced uniformly between the two slides to form a thin layer.

The lower slide was secured onto the apparatus board, while one end of the upper slide was attached to a string connected to a 20 g load via a simple pulley. The time taken for the upper slide to move 7 cm and separate from the lower slide in the direction of the weight was recorded.

Spreadability = m × l / t

Where, m= weight tied to the upper slide

l= length of glass slide

t= time taken in seconds

Extrudability¹³

Extrudability testing of the hydrogels was performed using gravimetric measurements. A specific quantity of hydrogel was filled into collapsible metal/plastic tubes, and the ends were crimped shut. The weight of each tube was recorded. The tubes were then positioned between two glass slides and secured using a clamp. Five grams of hydrogel was placed over the slides and the cap was removed. The material was extruded from the tubes in measured amounts, and the formulation's extrudability was tested. An extrudability of 90% or higher was considered excellent, above 80% was rated good, and above 70% was classified as fair.

Antimicrobial study of B.sensitivum Linn. extract incorporated hydrogel¹⁴

Standard Escherichia coli culture was procured from the Department of Microbiology, Acharya & B M Reddy College of Pharmacy, Bengaluru. A mixture of 0.3 g beef extract, 0.5 g peptone extract, and 1.5 g agar were dissolved in 100 mL of distilled water.

The flask was cotton plugged and was sterilized in the autoclave at 121℃ for 15 minutes. Using the spread plate method, the medium in the petri dish was inoculated with the required inoculum (1×10⁸ cells/mL). Then, 0.5 g of each hydrogel formulation was placed into the prepared bores, and 0.5 g of Azithromycin at a concentration of 0.05 mg/mL was used as the standard. The petri dishes were incubated for 24 hours at 37℃, after which the zones of inhibition were observed and recorded.

Stability study¹⁵

Determining the stability of the dosage form is essential. As per the ICH guidelines, stability studies were conducted on the most satisfactory formulation. The formulations were sealed and stored in wide-mouthed containers with aluminium packaging, then placed in a stability chamber maintained at 40±2℃ and 75±5% relative humidity for a period of two months. As the product is in the early stages of development, a two month stability study provides critical early stability data, supporting a risk-based approach to assess the formulations performance. A longer, six-month stability study will be necessary post approval to confirm the shelf-life of the product. This approach helps balance timely drug development with the assurance of final product quality.¹⁶

Results

Formulation of hydrogel

Table 1 presents five formulations of hydrogels containing Biophytum sensitivum Linn. extract, labelled F1 through F5, highlighting the variations in the constituents among these formulations.

Comparative thin layer chromatography (TLC) of methanolic extract

The TLC analysis of the methanolic extract showed an Rf value of 0.46, which matches the Rf value of standard quercetin (Figure 1). Analysis was performed using a mixture of toluene, ethyl acetate, and formic acid in a 5:4:1 ratio, confirming the presence of flavonoids.

Evaluation of hydrogel formulations

Appearance

The physical appearance of the hydrogel formulations was visually evaluated, revealing a greenish coloured gel with a characteristic odour and texture.

Grittiness and Homogeneity

The homogeneity and grittiness of the prepared formulations were assessed by analysing their physical, chemical, and mechanical properties using a homogenizer. The gels appeared clear and transparent before the addition of the extract. After incorporating the extract, the uniform distribution of the extract within the hydrogel was confirmed by consistent colour and appearance. No visible coagulation of extract particles was observed in the formulations.

Washability and Extrudability

Among the batches of obtained formulations of hydrogels, Carbopol incorporated formulations F2 and F3 exhibited excellent washability and extrudability.

Determination of pH

The various formulations of B.sensitivum Linn. hydrogel showed the following pH values: F1 - 7.08±0.06, F2 - 6.58±0.07, F3 - 6.50±0.07, F4 - 7.11±0.02, and F5 - 7.03±0.06.

All the five formulations of topical hydrogel demonstrated varying pH values; however, all the pH values were within the range of skin pH 6.5-7.0.

Determination of viscosity

The viscosity values (Cps) of the formulations were as follows: F1 - 9344 Cps, F2 - 9689 Cps, F3 - 9523 Cps, F4 - 8819 Cps, and F5 - 8850 Cps.

Spreadability

The spreadability values (g.cm/sec) of the formulations were as follows: F-1 - 14.0 g.cm/sec, F-2 - 7.9 g.cm/sec, F-3 - 7.8 g.cm/sec, F-4 - 16.3 g.cm/sec, and F-5 - 13.9 g.cm/sec.

Antimicrobial studies

The antibacterial activity against gram-negative organism (E. coli) was evaluated for all the five formulations and compared with a standard solution of Azithromycin. The diameters of the zones of inhibition were measured. Among the formulations, F2 demonstrated a zone of inhibition same as that of Azithromycin, as shown in Figure 2. A comparative analysis of the zones of inhibition for all formulations is presented in Figure 3.

Stability Studies

Stability studies were conducted on the optimized hydrogel fromulation containing B.sensitivum Linn. No physical changes were observed during the study period, as shown in Table 2. The formulation was evaluated for spreadability, viscosity, and pH over a duration of two months.

Discussion

To utilise the therapeutic properties of B.sensitivum Linn., for biomedical applications-particularly in dermatology and wound healing-the study focused on incorporating its extracts into hydrogel formulations. A thorough evaluation was conducted on the hydrogels that were thus prepared, considering multiple factors including appearance, homogeneity, texture, spreadability, pH, viscosity, and extrudability. These analyses are important as they establish the hydrogel formulations' physical properties and applicability.

Based on the results, the optimised hydrogel formulation demonstrated strong antibacterial activity, on par with Azithromycin standard solution. This shows that when added to hydrogel, B.sensitivum Linn. extract could be a strong antibacterial agent. Further stability experiments showed that, under accelerated conditions simulating storage at elevated temperature and humidity for two months, the prepared hydrogel remained stable without appreciable deterioration.

It was found that the hydrogel formulation's potential for topical applications-particularly in wound healing was improved with the addition of Carbopol polymer. Carbopol is well-known for its ability to enhance the adherence and viscosity of gels, properties that are crucial for increasing therapeutic efficacy on the skin surface and prolonging the formulation’s contact time.

Conclusion

The plant Biophytum sensitivum Linn., selected for this study, has demonstrated significant potential in the treatment of skin conditions. According to literature reviews, this plant has a long history of use in managing various illnesses, including wound healing. An attempt was made in this study to formulate and evaluate a hydrogel incorporating B.sensitivum Linn. extract. using various polymers like Carbopol 934 and PEG 400. Preliminary phytochemical tests and TLC were performed to confirm the major constituents such as flavonoids, phenols, etc. All the formulations were evaluated for appearance, homogeneity, texture, spreadability, pH, viscosity and extrudability. Antimicrobial studies were conducted to evaluate the efficiency of the prepared formulations (F1 to F5). Among them, F2 was found to be the optimised formulation based on its antimicrobial activity compared to the standard Azithromycin solution, as well as its performance in various hydrogel evaluation parameters. Stability studies showed that B.sensitivum Linn. extract incorporated hydrogel (F2) did not show any significant changes in terms of degradation, when stored at 40±2℃, 75±5% RH for two months. Therefore, the F2 formulation containing Carbopol polymer showed promise as a suitable candidate for topical application, especially for wound healing.

Conflict of Interest

The authors declare that they have no known competing f inancial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors thanks to Management, Acharya & BM Reddy College of Pharmacy, Bengaluru and Rajiv Gandhi University of Health Sciences, Bengaluru, India for providing required facilities to carry out this research work.