Formulation and Evaluation of Herbal Hydrogel of Guava and Neem Leaf Extract for its Antimicrobial Property and its Wound Healing Activity

Chemicals/ Reagents and Equipment’s

List of Instruments and Equipment’s:

Preformulation Studies:         

Description of Neem (Azadirachta Indica A. Juss)

Description of Guava (Psidium guajava L.)

Experimental Methods:

Collection and authentication of Plant Material:

• Neem leaves and Guava leaves are collected from Local area of Chandagad, Dist-Kolhapur.
•  Neem and Guava leaves were authenticated from Shivraj College of Science Gadhinglaj, affiliated to Shivaji University, Kolhapur by HOD of Botany.

Fig no. 1: Collection and authentication of Plant Material

Drying and Extraction of Leaves

Extraction Procedure:

Extraction of Guava Leaf (Psidium guajava) Extract

Fresh guava leaves were collected and washed thoroughly with distilled water to remove adhering dirt and impurities. The leaves were then shade dried at room temperature for 7–10 days until a constant weight was obtained. The shade-dried leaves were pulverized using a mechanical grinder to obtain a coarse powder. Approximately 250 g of the coarsely powdered guava leaves was packed into a thimble and placed in a Soxhlet extraction apparatus. The extraction was carried out using ethanol as the solvent. Continuous hot percolation was performed for 6–8 hours until the siphon tube solvent became colorless, indicating complete extraction. The obtained ethanolic extract was filtered and concentrated by evaporation of the solvent at 25–30°C.The concentrated extract was further air-dried to remove residual solvent and then weighed. The dried extract was stored in an airtight container for further formulation studies. The percentage yield of the extract was calculated with respect to the initial dried plant material.

Fig no. 2: Psidium guajava leaves Extraction

Extraction of Neem Leaf (Azadirachta indica) Extract

Fresh neem leaves were collected, authenticated, and cleaned thoroughly with distilled water. The leaves were shade dried at ambient temperature for 7–10 days to prevent degradation of active constituents. The dried leaves were then ground into a coarse powder using a mechanical grinder. A weighed quantity of 250 g of neem leaf powder was placed into a thimble and subjected to extractionusingaSoxhletapparatuswithethanolastheextractionsolvent.Theextractionprocess was continued for 6–8 hours until complete extraction was achieved. The ethanolic extract obtained was filtered and concentrated by evaporating the solvent at 25–30°C. The concentrated extract was airdried to obtain as olidresidue. The dried neem leaf extract was weighed and preserved in an airtight container at room temperature for further experimental use. The percentage yield was calculated based on the weight of dried extract obtained.

Fig no. 3: Azadirachta indica Extraction

Solubility study of Extract

Solubility study is an important Preformulation parameter that helps in selecting appropriate solvent for extraction and formulation. The solubility behavior of herbal extract affects drug release, bioavailability, and overall stability of the formulation.

1. Guava leaf extract

Solubility is an important physicochemical property that influences the bioavailability of a drug. A small quantity of guava (Psidium guajava) leaf extract was placed in a test tube, followed by the addition of 5 ml of solvent (water, ethanol, chloroform, or phosphate buffer). The mixture was shaken vigorously and allowed to stand for a specified period. Solubility was assessed by visual observation and the results were recorded.

2. Neem Leaf Extract

Solubility is an important Physico chemical property that influences the bioavailability of a drug. A small quantity of neem (Azadirachta indica) leaf extract was placed in a test tube, followed by the addition of 5 ml of solvent (water, ethanol, chloroform, or phosphate buffer). The mixture was shaken vigorously and allowed to stand for a specified period. Solubility was assessed by visual observation and the results were recorded.

Phytochemical Screening

Phytochemical screening for alkaloids, carbohydrates, starch, glycosides, flavonoids, saponins, and proteins was carried out following the standard procedure. Organoleptic evaluation involves the assessment of crude drug or extracts using sensory organs. It provides preliminary identification and ensures purity and quality of the herbal material before formulation. These parameters help in detecting adulteration and maintaining consistency in raw material selection.

Preliminary Phytochemical Screening Test for Neem leaf Extract

Table no. 6: Preliminary Phytochemical Screening Test for Neem leaf Extract

Preliminary Phytochemical Screening for Guava leaf extract :( Psidium guajava)

Table no. 7: Preliminary Phytochemical Screening for Guava leaf extract (Psidium guajava)

Fourier Transforms Infrared Analysis:

Fourier Transform Infrared (FTIR) spectroscopy is a technique used to identify the functional groups present in a compound by analyzing how it absorbs infrared radiation at various wavelengths. Each functional group exhibits absorption at a specific characteristic wave number.

Method/ Procedure:

• The Guava and Neem leaves extract was dried and finely powdered
• Approximately 2-3 mg of sample was mixed with potassium bromide (KBr)
• The mixture was compressed into a thin transparent pellet using a hydraulic
• Press the pellet was placed in the FTIR sample holder
• The spectrum was recorded in the range
• The obtained spectra were analyzed for characteristic functional group.

Guava Leaf Extract:

The potential structural modifications induced by the guava leaf extract sample were examined using infrared (IR) spectroscopy. The extract was analyzed within the spectral range of 400 to 4000cm⁻¹. The solid form of the guava leaf extract was carefully placed on the sample holder for analysis, and the spectra of the polymer were recorded under similar experimental conditions for comparison.

Neem Leaf Extract:

The potential structural modifications induced by the neem leaf extract sample were investigated using infrared (IR) spectroscopy. The analysis was carried out over arangeof400to 4000cm⁻¹. The solid form of the neem leaf extract was placed on the sample holder, and the corresponding spectra of the polymer were obtained under the same conditions to ensure consistency in comparison.

Formulation of hydrogel of guava and neem leaf extract:

An accurately weighed quantityofCarbopol934 was dispersed in distilled water and allowed to hydrate overnight. The combined neem (Azadirachta indica) and guava (Psidium guajava) leaf extracts were then incorporated into the hydrated gel base along with aloe vera gel and methyl paraben, using continuous stirring on a magnetic stirrer to ensure uniform dispersion. Triethanolamine was added drop wise to adjust the pH of the formulation to a range of 6.0–7.0, while maintaining constant stirring until a smooth and homogeneous polyherbal hydrogel was formed.

Formulation Table:

Table no. 8: Formulation Table

Evaluation Parameters:

Physical appearance: The physical appearance of the herbal hydrogel was assessed through visual inspection.

pH: A standard digital pH meter can be used to measure the pH of a herbal hydrogel at room temperature by taking an appropriate amount of the formulation, diluting it with a suitable solvent, and placing it in an appropriate beaker.

Fig no. 4: Dilutions For pH determination

Fig no. 5: Digital pH meter

Viscosity:

To determine the viscosity of the formulated herbal gel in order to evaluate its flow behavior and consistency for proper application.

Principle:

Viscosity is the measure of a fluid’s resistance to flow. It is determined using a viscometer, where the resistance offered by the gel to the rotating spindle is measured. Proper viscosity ensures easy application, stability, and effective drug release from the gel.

Material and Instruments:

Prepared herbal gel formulation

Brookfield Viscometer Spindle (appropriate type, e.g., spindle no.6 or7)

Beaker Thermometer

Procedure:

 Transfer the gel into a clean, dry beaker. Set up the rook field Viscometer and select an appropriate spindle. Immerse the spindle into the gel without trapping air bubbles. Set the desired speed (rpm) and allow the instrument to run. Note the dial reading once it stabilizes. Record the viscosity value (in centipoises, cP). Repeat the measurement for accuracy and take the average value.

Formula:

Viscosity (cP) =Dial Reading ×Factor

Spredability:

The herbal hydrogel was placed between two Petri dishes. One gram of herbal hydrogel was placed in a Petri dish, and another Petri dish was placed on top. A100 g weight was then placed on the upper Petri dish for 60 seconds. After 60 seconds, the diameter of the circles formed by the spread herbal hydrogel was measured in triplicate. The average of these readings was used to calculate the value using the following formula.

Fig no. 6: Spredability determination

Spredability (S) = M.L/T

Where:

S=Spredability

M=Weight tied to the upper slide (g)

L=Length moved by the glass slide (cm) T = Time taken (sec)

Anti-microbial activity:

Agar well diffusion method:

The antimicrobial activity of the prepared neem and guava leaf extract hydrogel was evaluated using the agar well diffusion method. Nutrient agar medium was prepared by dissolving 3.8 g of agar powder in 100 ml of distilled water, followed by heating until complete dissolution. The medium was sterilized by autoclaving at 121°C for 15 minutes and then cooled to approximately 50°C in a water bath. The стерile medium was poured into Petri plates under aseptic conditions and allowed to solidify. The plates were incubated at 37°C for 24 hours to confirm sterility. Sterile wells of6 mm diameter and approximately 5 mm depth were punched into the solidified agar using a sterile cork borer. Test microorganisms, including Escherichia coli, Staphylococcus aureus, Candida albicans, and Aspergillus Niger, were separately inoculated on to the agar surface using the spread plate technique. The neem–guava hydrogel was prepared in different concentrations (20, 40, 65, and 80 mg/ml equivalent of extract content). A standard reference (such as Aqua Clob-GM creamat 20 mg/ ml) was used for comparison. A fixed volume of each hydrogel formulation was carefully introduced into the respective wells. The inoculated plates were incubated at 37°C for 24 hours for bacterial strains and at appropriate conditions for fungal strains. After incubation, the antimicrobial activity was assessed by measuring the diameter of the zones of inhibition (in mm) around each well. The results were recorded, and the effectiveness of the hydrogel formulation was determined by comparing the inhibition zones with the standard.

Stability Studies

The prepared neem–guava herbal hydrogel was packed in 10 g containers and subjected to accelerated stability studies. The samples were stored at 40° C ± 2 ° C and 75 ± 5 % relative humidity for a period of 45 days. Samples were withdrawn at 15-day intervals and evaluated for various parameters, including physical appearance, color, odor, pH, Spredability, consistency, and any signs of phase separation or microbial growth.

RESULT AND DISCUSSION:

Preformulation Studies:

Plant Authentication

• Neem leaves and Guava leaves are collected from Local area of Chandagad, Dist.-Kolhapur
• Neem and Guava leaves was authenticated from Shivraj College of Science Gadhinglaj, affiliated to Shivaji University, Kolhapur By HOD of Botany

1. Organoleptic Character: 

Guava leaf extract

Table no. 9: Guava leaf Extract

Table no. 10: Neem leaf Extract

1. Solubility:

Guava leaf extract:

Table no. 11: Guava leaf extract

Neem leaf extract:

Table no. 12: Neem leaf extract

2. Phytochemical Screening:

Phytochemical screening for alkaloids, carbohydrates, starch, glycosides, flavonoids, saponins, and proteins was carried out following the standard procedure. Organoleptic evaluation involves the assessment of crude drug or extracts using sensory organs. It provides preliminary identification and ensures purity and quality of the herbal material before formulation. These parameters help in detecting adulteration and maintaining consistency in raw material selection.

Phytochemical Screening Test for Guava leaf Extract

Table no. 13: Phytochemical Screening Test for Guava leaf Extract

Fig no. 7: Phytochemical Screening of Guava leaf Extract

Fig no. 8: Phytochemical Screening of Guava leaf Extract

Phytochemical Screening Test for Neem leaf Extract

Table no. 14: Phytochemical Screening Test for Neem leaf Extract

Fig. no. 9: Phytochemical Screening of Neem Leaf Extract

1. Compatibility Study 

FTIR of Plant extract

1. 1. FTIR of Plant Extract (Neem Leaf extract)

FTIR analysis of the Neem ethanolic extract wascarriedoutinthespectralrangeof4000–600 cm⁻¹ to identify the functional groups present in the extract. The spectrum showed the following major characteristic peaks:

Table no. 15: Peaks and Functional groups present in plant extract

FTIR Interpretation of Neem Leaf Extract

TheFTIRspectrumofneemleafextractrevealedthepresenceofseveralimportantfunctional groups responsible for its pharmacological activity. A broad absorption peak observed in the range of 3320–3400cm⁻¹ corresponds to O–H stretching vibration, indicating the presence of phenolic compounds and alcohols. This confirms the presence of flavonoids and tannins, those are well known for their antioxidant and anti-inflammatory activities. The peaks observed at 2920–2850 cm⁻¹ indicate C–H stretching of aliphatic compounds, suggesting the presence of terpenoids and long-chain hydrocarbons, which contribute to antimicrobial and healing properties. A peak around 1730–1700 cm⁻¹ corresponds to C=O stretching, indicating the presence of Aldehydes, ketones, or ester compounds, which may play a role in biological and pharmacological activity. The peaks between 1600–1500 cm⁻¹ correspond to aromatic C=C stretching, suggesting the presence of aromatic rings, commonly found in flavonoids and other polyphenolic compounds. The peaks at 1240–1210 cm⁻¹ and 1100–1030 cm⁻¹ correspond to C–O stretching vibrations, confirming the presence of alcohols, ethers, and glycosidic linkages.

Fig. no. 10: FTIR of Neem leaf extract

1. 1. FTIR of Plant Extract (Guava Leaf extract)

FTIR analysis of the Guava leaves extract was carried out in the spectral range of 4000–600 cm⁻¹ to identify the functional groups present in the extract. The spectrum showed the following major characteristic peaks:

Table no. 16: Peaks and Functional groups present in plant extract

FTIR Interpretation of Guava Leaf Extract

The FTIR spectrum of guava leaf extract reveals the presence of various bioactive functional groups responsible for its medicinal properties. Abroad peak observed at~3335cm⁻¹corresponds to O–H stretching vibrations, indicating the presence of phenolic compounds and alcohols. This confirms the presence of flavonoids, tannins, and polyphenols, which contribute to strong antioxidant and antimicrobial activities. The peak at ~2980cm⁻¹ is attributed to C–H stretching of aliphatic compounds, suggesting the presence of terpenoids and other hydrocarbon chains, which are known for their biological and therapeutic effects. The band at ~1639 cm⁻¹ corresponds to C=O stretching or aromatic C=C vibrations, indicating the presence of amide groups, conjugated carbonyl compounds, or aromatic structures, commonly associated with proteins and polyphenolic compounds.

The peak around~1382cm ^-1 represents C–H bending vibrations, which may be linked to Alkanes and phenolic compounds. The absorption peak at ~1170cm⁻¹ indicates C–O stretching, confirming the presence of alcohols, esters, or ethers. Strong peaks intheregionof~1039–1004cm⁻¹correspondtoC–O stretching vibrations, suggesting the presence of glycosides and carbohydrates, which are important for various biological activities. The peak at ~872cm⁻¹ is due to aromatic C–H out-of-plane bending, further confirming the presence of aromatic compounds.

Fig no. 11: FTIR Of Guava Leaf Extract

Formulation of Herbal Hydrogel:

Fig. no. 12: Formulation of Herbal Hydrogel

Evaluation Test:

1. Physical appearance: A] Color

Table no. 17: Color study of developed formulation

2. Odor:

Table no. 18: Odor study of developed formulation

3. Appearance:

Table no. 19: Appearance study of developed formulation

Consistency:

Table no. 20: Consistency study of developed formulation

1. Homogeneity:

Table no. 21: Homogeneity study of developed formulation

2. Grittiness:

Table no. 22: Grittiness study of developed formulation

1. Spredability:

Table no. 23: Spredability study of developed formulation

Fig. no. 13: Graphical representation of Spredability study of

Developed formulation

1. Viscosity:

Table no. 24: Viscosity study of developed formulation

Fig. No. 14: Graphical representation of Viscosity study of developed formulation

2. Measurement of pH:

Table no. 25: pH study of developed formulation

Fig. No.15: Graphical representation of pH study of developed formulation

2. Washability:

Table no.26: Washability study of developed formulation

Antimicrobial Activity of combined extract of Psidium guajava and Azadirachta indica

To evaluate the antimicrobial activity of plant extract against Escherichia coli and Staphylococcus aureus. The tests ample diffuse into agar medium inoculated with microorganisms. If the extract has antimicrobial activity, it inhibits microbial growth and forms a zone of inhibition around the well.

Table no. 27: Antibacterial testing of combine extract

S: Sensitive      R: Resistant

The entire tests were performed according to standard methods.

Fig no 16: Antimicrobial activity of liquid extract against Staphylococcus aureus and Escherichia coli.

Fig no.17: Antimicrobial activity of Gel extract against Staphylococcus aureus and Escherichia coli