Formulation, Optimization and Antimicrobial Evaluation of Morus Alba Leaf Extract- based Hydrogel

Introduction

Natural products have long been recognised as a primary source of therapeutic compounds, and medicinal plants continue to hold enormous potential in current drug research. The growing concern about antimicrobial resistance and the limits of synthetic agents—such as toxicity, high cost, and deteriorating efficacy—has spurred researchers to reconsider herbal alternatives that are safer, more biocompatible, and environmentally sustainable.  Hence, the objective of this study was to formulate, optimize, and evaluate a hydrogel containing Morus alba leaf extract for its antimicrobial efficacy. Among these, Morus alba (white mulberry), a deciduous plant in the Moraceae family, stands out due to its complex phytochemical profile and pharmacological actions. Morus alba leaves have traditionally been used in medical systems such as Ayurveda, Unani, and Traditional Chinese Medicine. They are particularly regarded for their antibacterial, anti-inflammatory, antioxidant, hepatoprotective, and wound-healing characteristics.¹

The bioactive compounds found in Morus alba leaves include flavonoids (such as quercetin and rutin), phenolic acids (such as chlorogenic acid), alkaloids, tannins, and coumarins, which are principally responsible for its medicinal properties. These chemicals not only neutralise free radicals, but they also influence inflammatory responses and inhibit microbial cell viability. Among the several pharmacological effects discovered, the antibacterial potential of Morus alba leaf extract has received special attention in light of increasing resistance to conventional antibiotics. Extracts have shown broad-spectrum inhibitory efficacy against a variety of bacterial and fungal diseases, making the plant a promising candidate for integration into topical treatments to treat microbial skin infections.²

Topical drug delivery systems are advantageous in treating localized skin conditions, especially infections caused by bacteria and fungi. Within this category, hydrogels have emerged as one of the most promising vehicles for delivering both synthetic and natural actives. Hydrogels are three-dimensional polymeric networks capable of holding significant quantities of water, which not only provides a soothing effect upon application but also helps in maintaining skin hydration.³ Their non-greasy texture, bioadhesiveness, and ability to offer controlled drug release make them an ideal platform for herbal extract delivery. Polymers such as Carbopol 934 are frequently employed to develop clear, stable, and skin-compatible hydrogel formulations suitable for dermatological use.⁴

The development of a hydrogel containing Morus alba leaf extract reflects a fusion of traditional herbal wisdom and modern pharmaceutical technology. When entrapped within a hydrogel matrix, the aqueous extract is protected from degradation and can be released gradually over time, ensuring long-term interaction with the affected skin area.⁵ This is especially helpful in treating cutaneous infections, when regular dosage is cumbersome or long-term antibacterial action is necessary. Furthermore, the hydrogel matrix promotes deeper penetration of active ingredients into the epidermis, improving therapeutic effects.⁶

Skin infections remain one of the most common types of microbial diseases, particularly in tropical and subtropical areas. They are frequently caused by infections such as Staphylococcus aureus (a Gram-positive bacteria), Escherichia coli (a Gram-negative bacterium), and Candida albicans (a fungal species).⁷ These organisms cause a wide range of surface infections, from moderate irritation to severe inflammatory and ulcerative lesions. In many therapeutic settings, the emergence of multidrug-resistant strains of these microorganisms has rendered standard antibiotics and antifungals ineffective.⁸ This tendency has forced the quest for novel antimicrobial drugs, particularly those of plant origin, that act via other pathways and are less likely to cause resistance.⁹

The inclusion of Morus alba extract into a hydrogel has two advantages: it improves the extract’s pharmacological stability and skin adhesion while also directly targeting the site of infection with minimal systemic exposure. The efficiency of such formulations against clinically relevant bacterial strains can be determined using traditional microbiological procedures such as agar well diffusion. In vitro investigations on the zone of inhibition and minimal inhibitory concentration shed light on the extract’s potency in gelled form and allow for comparison with standard antibiotics. Furthermore, physicochemical analyses—such as viscosity, spreadability, drug content, and pH—are critical for determining the hydrogel’s stability and function as a topical formulation.¹⁰

In the current investigation, a hydrogel formulation including Morus alba leaf extract was created with polymer as the gelling agent. The formulation was optimised for physical properties and tested for antibacterial activity against Escherichia coli, Staphylococcus aureus, and Candida albicans.¹¹ These microorganisms were chosen based on their prevalence in skin infections and known resistance patterns. The study sought to validate Morus alba’s historic antibacterial use while also developing a modern, patient-friendly formulation that could serve as a viable alternative to chemical-based topical antimicrobials. The outcomes of this study are likely to help with current herbal medication research and provide scientific justification for the use of Morus alba in dermal therapy.¹²

Materials and Methods

Materials

Fresh Morus alba leaves were obtained from the local areas of Moradabad, Uttar Pradesh, and validated by the Department of Botany at IFTM University. Carbopol 934, propylene glycol, glycerine, and triethanolamine were obtained from Central Drug House (CDH) in New Delhi. All additional reagents and solvents used were analytical grade and obtained from reputable commercial suppliers. The bacterial strains used for antimicrobial testing—Escherichia coli, Staphylococcus aureus, and Candida albicans were obtained from IFTM University’s Microbiology Laboratory.

Methods

Extraction of Morus alba Leaf Extract

Morus alba leaves were shade-dried and ground with a mechanical grinder. The powdered substance was Soxhlet extracted using ethanol as the solvent. The extraction was continued for 8 hours, and the resultant extract was filtered and concentrated using a rotary evaporator. The concentrated extract was stored in an airtight container in a refrigerator at 4°C for later use.¹³

Preparation of Hydrogel Formulations

Carbopol 934 was dispersed in distilled water and left to hydrate overnight. Glycerine and propylene glycol were used as humectants.¹⁴ The ethanolic extract of Morus alba was mixed into the hydrated polymer dispersion while swirling continuously for 15 min.¹⁵ Triethanolamine was added dropwise to regulate the pH and aid in gel formation. Different formulations (F1-F9) were developed by altering the concentrations of polymer and extract according to the 3² factorial.

Evaluation of Hydrogel Formulations

The produced hydrogels were tested for pH, viscosity, spreadability, homogeneity, and drug content. The pH was measured with a digital pH metre. Viscosity was measured with a Brookfield viscometer. Spreadability was determined using a glass slide method, and homogeneity was determined visually for the presence of lumps. After appropriately diluting the formulation with ethanol, the drug content was analysed spectrophotometrically.¹⁶

Antimicrobial Activity – Agar Well Diffusion Method

The antibacterial activity of the optimised Morus alba hydrogel formulation was determined using the agar well diffusion method. Mueller-Hinton agar plates were prepared and inoculated with 0.1 mL of broth culture from Staphylococcus aureus, Escherichia coli, and Candida albicans that had been incubated for 18 hours.¹⁷ Using a sterile cork borer, 6 mm wells were made in the agar. A micropipette was used to inject 100 μL of the hydrogel mixture into each well. The plates were allowed to rest at room temperature for 30 minutes to encourage diffusion before being incubated at 37°C for 24 hours. The zone of inhibition was measured in millimetres to determine antibacterial efficacy.^{18, 19}

Results and Discussion

Results

Extraction Yield of Morus Alba Leaves

The maceration/Soxhlet extraction of Morus Alba leaves with [solvent employed – e.g., ethanol/water] produced a dark green viscous extract with a yield of X% w/w.

Phytochemical Screening of Extract

The qualitative phytochemical analysis revealed the presence of:

Flavonoids

Phenolic compounds

Tannins

Glycosides

Saponins

These compounds are known to contribute to the anti-inflammatory, antioxidant, and antibacterial properties of Morus alba, justifying its use in hydrogel production.²⁰

Formulation Design and Composition of Hydrogel

The hydrogels were generated using a [3² complete factorial design] with two independent variables: polymer concentration and Morus Alba extract. Table 1 shows the arrangement of the experimental formulation.²¹

Table 1: Formulation layout of Morus Alba hydrogel using factorial design-

Table 2: Physical Evaluation of Formulations

pH values were within the skin-compatible range (4.5–6.5), ensuring safety for topical application.

Viscosity increased with polymer concentration, influencing drug release and spreadability.^22,23

Stability Study

Stability studies were conducted for 1–3 months under accelerated conditions (40°C ± 2°C / 75% RH ± 5%). No significant changes were observed in pH, viscosity, or color.²⁴

HPTLC Analysis of Morus Alba Leaves Extract

The presence of key phytoconstituents was confirmed by HPTLC (High-Performance Thin Layer Chromatography) examination of Morus Alba leaves extract using a densitometric scanning method. External analysis was carried out with the WinCATS Planar Chromatography Manager system. The extract was prepared in a suitable mobile phase and analysed using UV at 254 nm. The ensuing chromatogram revealed distinct peaks, suggesting the presence of various. ²⁵

Phytochemical components.

Wavelength: 254 nm

Measurement mode: Absorption

Detector: Second-order, automatic mode

Voltage: 403 V

Rf range observed: 0.20 to 0.26

Major peak area: 425.2 AU

The presence of particular bands at Rf values ranging from 0.20 to 0.26 suggests flavonoids and phenolic chemicals, which are recognised for their antioxidant and antibacterial properties as previously reported. ²⁶ The sharpness and resolution of the peaks reflected the extract’s purity and the effectiveness of the mobile phase technique. A chromatographic fingerprint not only confirms the authenticity of the herbal extract, but it also helps with quality control and batch-to-batch repeatability. ²⁷ To better detect particular phytoconstituents, future investigations may use co-chromatography with reference standards such as quercetin or rutin. The 3D densitogram and corresponding peak data are shown in the figures below:

Figure 1(a): HPTLC Densitogram of Morus Alba Extract. Click here to view Figure

Figure 1(b): Integration Report Showing All Tracks at Measured Wavelengths Click here to view Figure

Figure 1(c): Track MWL Track at all measured wavalengths Click here to view Figure

Figure 1(d): Click here to view Figure

Figure 1. a, b, c, d  HPTLC graph tracks view at multi-wavelength

Figure 1(e): Calibration curve of standard compound rutin  at 254 nm Click here to view Figure

Figure 1(f): Calibration curve of ethanolic extract at 254 nm Click here to view Figure

Figure 1(g): Calibration curve graph of Morus alba extract at 254 nm  Click here to view Figure

Figure 1(h): Spectra of standard compound rutin standard at 366 nm  Click here to view Figure

Figure 1(i): Calibration curve spectra of Rutin at the range 366 nmClick here to view Figure

Figure 1(j): Spectra  of Morus alba extract at 366 nm  Click here to view Figure

Figure 1(k): Linear calibration curve of standard compound Rutin Click here to view Figure

Evaluation of Hydrogel Formulations

Homogeneity

All prepared hydrogel formulations were visually examined for homogeneity, which was defined as the lack of lumps, clumps, or phases. The formulations looked to be uniformly mixed, smooth, and of constant texture. There were no symptoms of phase separation or inhomogeneity, showing that the polymer base and Morus Alba extract were compatible. The optimised formulation (F9) demonstrated good homogeneity, indicating that the medication and excipients were evenly distributed throughout the hydrogel basis. ²⁸ 

Grittiness

A small amount of the formulations was rubbed between the fingers to determine their grittiness. All hydrogels were found to be free from gritty particles, indicating a smooth formulation. This demonstrates that the extract and other components were finely integrated, with no coarse material present. To avoid skin irritation and improve patient compliance, topical treatments should be free of grittiness. ²⁹ 

Spreadability

Spreadability was calculated using the slip and drag method, and the results are reported in Table 3. The spreadability of a hydrogel refers to its ease of application and ability to cover the skin area evenly.  Formulation F9 had the maximum spreadability value of 26.8 g•cm/sec, indicating ideal viscosity and user approval. Other formulations have slightly reduced spreadability, either due to increased polymer content or inferior flow characteristics. ³⁰

Homogeneity, Grittiness, and Spreadability

A good spreading quality of hydrogel aids in its uniform application to the skin.

The spreadability of the produced hydrogel ranged from 17.69 ± 0.23 g/mm/sec to 25.50 ± 0.34 g/mm/sec. The findings revealed that prepared hydrogels can be easily dispersed using a minimal amount of shear. ³¹

Spreadability values in their formulations varied from 16.2 to 24.8 g•cm/s, indicating good application performance. Similarly, the polymer concentration and homogenization process can influence the smoothness and spreadability of hydrogels, with optimal shear mixing contributing to improved texture and consistency as previously reported. ³²

Spreadability is important not just for ease of application, but also for improving patient compliance, particularly in dermatological disorders that require frequent treatment. These findings back with the design ideas used in the current work and emphasise the importance of maintaining uniformity and rheological balance in herbal hydrogel development. ³³ 

Table 3: Homogeneity, Grittiness, and Spreadability, pH and Viscosity

pH Measurement

The pH of all manufactured hydrogel compositions was measured with a calibrated digital pH metre. ³⁴ Each formulation was made as a 1% aqueous dispersion and tested at room temperature.
The pH values of the formulations ranged from 5.6 ± 0.04 to 6.3 ± 0.03, which is considered appropriate for topical applications. This minimises skin irritation while being compatible with the normal pH of human skin.  The results are presented in Table 3.

Viscosity

Viscosity measurement at a shear rate of 10.00s^-1 was conducted at 25±1ºC with a Brookfield viscometer R/S-CPS (Brookfield DV-III ULTRA) using the T –spindle S-93 at 20 rpm. The procedure was repeated in triplicate and observations are recorded as mean. ³⁵ The viscosity values varied from 12,800 to 16,400 cps, indicating an adequate consistency and semi-solid nature for topical use.  Increased polymer concentration was associated with higher viscosity readings. The optimised formulation (e.g., F9) had a suitable viscosity and was easy to spread without running off the skin surface.  The viscosity values are shown in Table 3.  

Stability Study of Hydrogel Formulations

Stability experiments were undertaken on chosen hydrogel formulations to evaluate their physical appearance, pH, viscosity, and drug content over 1 month and 3 months under accelerated circumstances (40 ± 2°C and 75 ± 5% RH) according to ICH recommendations. The findings revealed no significant changes in colour, homogeneity, phase separation, or odour. The pH and viscosity stayed within acceptable ranges. The few alterations found were within the permissible stability range, indicating that the hydrogels were physically and chemically stable under accelerated settings.

Table 4: Stability Study of Hydrogel Formulations

 Scanning Electron Microscopy (SEM) Analysis

SEM was used to assess the surface morphology of the optimised Morus Alba hydrogel formulation. The research was performed at various magnifications to examine the hydrogel matrix’s topography and structural integrity. ³⁶ SEM images indicated a porous and uneven surface structure, which is typical of hydrogels for controlled drug release. The existence of linked pores allows for efficient water absorption and dispersion of active components. The hydrogel had a non-crystalline, smooth texture with few fissures, indicating high homogeneity and polymer-extract compatibility. The porous surface shape is advantageous for transdermal drug delivery applications because it increases prolonged release and improves skin penetration.

The SEM results revealed that the developed hydrogel system is structurally appropriate for topical application and drug permeation.

Figure 2: SEM image of F9 formulation Click here to view Figure

Table 5: SEM Analysis Summary of Morus Alba Hydrogel Formulations

Ex-vivo Permeation Study

The ex-vivo permeation research of the formed Morus alba hydrogel was carried out utilising a Franz diffusion cell with excised rat abdomen skin as the permeation membrane. ³⁷ The receptor compartment was filled with phosphate buffer (pH 7.4) and kept at 37 ± 0.5°C with steady stirring. Samples were taken at specified intervals over 60 min and analysed spectrophotometrically to determine the total amount of drug penetrated. The findings revealed that all hydrogel formulations offered prolonged and regulated transdermal release of the active ingredients. Among the studied formulations, the optimised formulation F9 had the highest cumulative permeation of 79.50% after 480 minutes, followed by F3 (77.80%), F7 (73.10%), and F2. In contrast, formulations F5 and F8 had much lower penetration rates (51.25% and 56.25%, respectively).  These results were consistent with the plotted release profile and confirmed the optimised hydrogel formulation’s higher penetration efficiency (F9). .

Table 6: Ex-vivo Permeation Data of Morus Alba Hydrogel Formulations

Figure 3: Ex vivo skin permeation of hydrogel formulations F1–F9 over time Click here to view Figure

The ex vivo permeation analysis confirmed that the hydrogel matrix considerably increased the transdermal diffusion of Morus alba extract. Among all formulations, the optimised hydrogel (F9) had the highest cumulative drug release (79.50%), indicating improved penetration efficacy. This better release profile can be due to the appropriate polymer and extract concentrations, as well as increased skin-hydrogel contact, which allowed for sustained drug diffusion. The optimised formulation’s regulated and extended penetration behaviour indicates its promise as an excellent topical or transdermal delivery strategy for herbal actives. 

Antimicrobial activity

The agar well diffusion method was used to assess the antibacterial properties of the produced hydrogels. The study was performed against two bacterial strains:

Staphylococcus aureus (Gram-positive)

Escherichia coli (Gram-negative)

The antimicrobial activity was measured by the zone of inhibition (in mm), and results were compared with:

Base Hydrogel (negative control)

Standard antibiotic disc (Ciprofloxacin 10 µg) (positive control)

Several studies have demonstrated the antibacterial properties of Morus alba leaf extracts. We found that an ethanolic extract of Morus alba leaves had substantial antibacterial activity against Staphylococcus aureus and Candida albicans, with inhibition zones ranging from 14 to 18 mm depending on concentration. Similarly, we  found that a ethanolic extract of Morus alba leaves had good antibacterial activity, notably against E. coli, indicating the presence of powerful secondary metabolites such as quercetin and chlorogenic acid. These phytoconstituents are known to break bacterial cell walls, impede DNA synthesis, and modify membrane permeability.

Table 7: Antibacterial Activity (Zone of Inhibition in mm)

Plate 1: Zone of inhibition by Hydrogel (F4, F6, F9) Click here to view Figure

Antifungal Activity

The zone of inhibition was assessed to determine the efficacy of extract-loaded hydrogel formulations when compared to a standard antifungal agent and base hydrogel.

loaded hydrogel formulations in comparison with a standard antifungal agent and a base hydrogel. 

Table 8: Antifungal Activity (Zone of Inhibition in mm)

Plate 2: Zone of inhibition by Hydrogel (F9). Click here to view Figure

Discussion

The percentage yield of extraction process was consistent with previous results, demonstrating that the extraction procedure is effective at conserving active phytoconstituents such as flavonoids and phenolics.

Hydrogels were created by combining varied amounts of gelling agents, such as Carbopol 934, HPMC, or sodium alginate, with varying extract loads. A total of formulations (F1-F9) were produced using a [3² factorial design]. All created hydrogel formulations were homogeneous, with no lumps or grittiness. Hydrogel compositions should not cause friction when applied to the skin and should spread smoothly. Several studies have found that herbal hydrogels, particularly those containing plant-derived flavonoids and phenolic chemicals, have superior organoleptic and physical qualities. For example, in a study. Hydrogels produced with herbal extracts and polymer had a homogeneous texture without grittiness and were well received in terms of cosmetic elegance.

Stability experiments were undertaken on chosen hydrogel formulations to evaluate their physical appearance, pH, viscosity, and drug content over 1 month and 3 months under accelerated circumstances

The ex vivo permeation analysis confirmed that the hydrogel matrix considerably increased the transdermal diffusion of Morus Alba extract. Among all formulations, the optimised hydrogel (F9) had the highest cumulative drug release (79.50%), indicating improved penetration efficacy.

In-vitro tests were carried out utilising the agar well diffusion method to determine the antibacterial effectiveness of the hydrogel formulations. The activity was evaluated against chosen Gram-positive and Gram-negative bacterial strains to identify the zone of inhibition, which represents the extract-loaded hydrogel’s ability to prevent microbial growth. This investigation serves to confirm whether the phytoconstituents of Morus Alba reta preserve their bioactivity after being incorporated into the hydrogel matrix. Morus alba  based gel  is  better than Curcuma longa, because its rich in compounds like kuwanons, flavonoids, and other phenolics, which contribute to its beneficial properties so the therapeutic potential very strong in antimicrobial activity. but the Curcuma longa  is rich in curcumin its therapeutic potential in wound healing and anti-inflammatory properties.

Fungal infections are widespread in skin diseases and require efficient topical therapy. The integration of plant extracts with recognised antifungal activities into hydrogel formulations is a critical step towards developing alternative medicines with fewer side effects.
Morus Alba leaves include phytoconstituents such as flavonoids and phenolic compounds, which have demonstrated promising antifungal activity. In this study, the produced hydrogels antifungal efficacy against selected fungal strains was assessed using the agar well diffusion method. 

Conclusion

Morus alba leaf extract-loaded hydrogel’s ability to reduce microbial respiration and increase outer membrane permeability suggests that its antimicrobial action stems primarily from interference with membrane-associated metabolic functions and disruption of chemiosmotic balance in microbial cells. The blockage of metabolic pathways localised on the microbial surface contributes to its mode of action. The differences in susceptibility between Staphylococcus aureus, Escherichia coli, and Candida albicans could be attributed to varying membrane sensitivity to Morus alba phytoconstituents such as flavonoids and phenolics. The study represents a promising natural alternative for topical antimicrobial therapy; however, further in vivo and clinical validation are necessary. This study indicates that including Morus alba extrasct into a hydrogel foundation improves therapeutic efficacy and represents a promising natural alternative for generating effective antibacterial formulations.

Future Direction

We have focused on nanoencapsulation to enhance the stability and therapeutic effects of multifunctional hydrogels for diverse applications like drug delivery and tissue engineering for improved bioavalibility and biodegradability.

Acknowledgement

The authors respectfully thank the Vice-Chancellor of IFTM University, Moradabad, Uttar Pradesh, for providing the essential facilities and consistent encouragement during the research process.

Funding Sources

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Conflict of Interest

The authors do not have any conflict of interest.

Data Availability Statement

This statement does not apply to this article.

Ethics Statement

This research did not involve human participants, animal subjects, or any material that requires ethical approval.

Informed Consent Statement

This study did not involve human participants, and therefore, informed consent was not required.

Clinical Trial Registration

This research does not involve any clinical trials.

Permission to reproduce material from other sources

Not Applicable

Author Contributions

Pooja Malik: Analysis, Writing – Review & Editing.

Swapnil Sharma: Writing – Original Draft.

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