1. INTRODUCTION:

Wound healing remains a challenging clinical issue¹; with around 8.2 million individuals suffering from a variety of wounds worldwide^2-3. Common synthetic drugs cause unfavorable side effects (nitrofurazone, chlorhexidine, mupirocin, mafenide, silver nitrate, and povidone-iodine) including dermatitis from allergic touch, local discomfort, and systemic toxicity⁴. Innovative approaches like adverse pressure, biological, biophysical, and stem cell and bioengineered wound treatments have been developed, but their efficacy is deficient⁵.

Researchers are now exploring the potential of medicinal plants, since certain plant species phytoconstituents are recognized to be useful in treating wounds with minimal or no side effects. Using multiple medicinal plants in wound treatment has been reported to decrease wound closure time and increase wound quality. Herb-herb combinations (PHFs) have promising potential because of their numerous, multitargeted phytoconstituent groups, in the management and treatment of wounds⁶. Precise plant selection is necessary to prevent phytoconstituent counteraction and incompatibility, which can result in unfavorable effects⁷. Numerous risk factors, some of which may be controlled and prevented, and others of which cannot, cause wound healing to be considerably delayed. Modifiable risk factors include bacterial infection, diabetes, excessive alcohol use, prescription drugs, poor nutrition, stress, smoking, and trauma^8-9. Age, autoimmune conditions, and genetic abnormalities are examples of risk factors that cannot be changed. The most frequent risk factor is bacterial infection, causing inflammation and bacterial proliferation¹⁰. Because of these various risk factors as well as the intricate structure of the wound healing process, wound management remains a difficult issue that has to be given careful consideration¹¹. Polyherbal combinations have been demonstrated efficaciousness in treating wounds worldwide, but scientific confirmation of their medicinal advantages is lacking^12-13. The formulation of herbal medicines follows two principles: using one therapeutic plant in a base and using multiple plants known for wound healing, known as Polyherbal Formulations (PHFs). PHFs combine multiple medicinal plants or plant-derived ingredients for increased therapeutic potency. Various bases are employed in the process of preparation, such as oral PHFs made by mixing plant extracts with gum acacia, water, and tween-20¹⁴, or topical PHFs in cream, gel, or ointment. Numerous polyherbal preparations exist for various wound types¹⁵. The purpose of this study is to formulate and assess the polyherbal formulation using plant materials for effective and prominent wound healing activity in comparison to advertised product as better and invasive treatment approach.

2. MATERIAL AND METHOD:

2.1 Material:

2.1.1 Assortment and validation of plant material:

The medicinal plants Thunbergia erecta (flower) and Bryophyllum pinnatum (leaves) were collected in winter season form local areas of Bhopal. After cleaning, parts were dry under shade at room temperature till complete dryness. Dried plant parts were stored in air tight glass containers in dry and cool place to avoid contamination and deterioration. Authentication of medicinal plant Thunbergia erecta (Ref no AC/001/24) and bryophyllum pinnatum (Ref no AC/002/24) was performed by a plant taxonomist in order to confirm its identity and purity.

2.1.2 Animals:

The Institutional Animal Ethics Committee approved the use of animals for the present study (Ethical clearance number: PBRI/IAEC/15-12-2023/029). Healthy Wistar albino rats of both sexes weighing 200 to 220 g was used for the study. They were individually housed and were allowed free access to standard pellet diet and water ad libitum. Animals were periodically weighed before and after the experiment. The rats were anesthetized prior to and during infliction of the experimental wounds. The surgical interventions were carried out under sterile conditions using ketamine anesthesia (120 mg/kg). Animals were closely observed for any infection and those which showed signs of infection were separated and excluded from the study and were replaced.

2.2 Plant extraction using the Soxhlet extraction technique:

Thunbergia erecta and Bryophyllum pinnatum coarsely ground plant portions (300 gm) were then extracted progressively with various organic solvents, defatted with petroleum ether, and extracted again with methanol over a 36-hour period using a soxhlet apparatus. Each extract was dried completely under low pressure using a rotary evaporator to guarantee full extraction, and the dried residue was then kept for later use in an airtight container ¹⁶.

Formula;

Actual yield

% Yield = -------------------------- x 100

Theoretical yield

2.3 An analysis of phytochemistry:

A comprehensive qualitative phytochemical analysis was used in the experiment to determine whether or not certain phytoconstituents were present. Medical reactions to tests were measured using the color intensity or the precipitate formation¹⁷.

2.4 Quantitative Phytochemical Estimation:

2.4.1 TPC:

The total amount of phenols in Thunbergia erecta alongwith Bryophyllum pinnatum extracts were measured performing the Folin-Ciocalteu Assay. Thunbergia erecta and Bryophyllum pinnatum 0.2mL of the stock solution's extracts were combined using 2.5 milliliters of Folin-Ciocalteu Reagent and two milliliters of sodium carbonate (7.5%). Distilled water was used to dilute this combination up to 7mL. The solutions were subsequently permitted to rest at room temperature for 2 hours prior to being spectrophotometrically analyzed at 760nm. Curves for calibration were created utilizing standard Gallic Acid Equivalent (GAE) mg/gm solutions. Gallic aid was produced in 20, 40, 60, 80, and 100g/mL concentrations. Reducing agents can affect the Folin-Ciocalteu reagent's sensitivity such as polyphenols. When they react, they turn blue. This blue color was determined using spectrophotometry¹⁸.

2.4.2 TFC:

The amount of flavonoids was measured performing the aluminium chloride technique. A solution of 0.5ml Thunbergia erecta and Bryophyllum pinnatum extracts was combined using two milliliters of distilled water. Next, 0.15 milliliters of sodium nitrite (5%) was introduced and thoroughly combined. After six minutes, add 0.15mL of 10% aluminum chloride and allowing it for duration of six minutes. And after that 2 milliliters, 4% of sodium hydroxide was introduced.The liquid was agitated, well combined. The absorption of the combination measured at 510nm with a UV spectrophotometer. Curves for calibration were created employing mg/gm of Rutin Equivalent (RE) standard solutions. Rutin was prepared at concentrations of 20, 40, 60, 80, and 100g/mL. The total flavonoid concentration was measured using a calibration curve in addition to the outcomes were expressed as milligrams of rutin equivalent per gram of weight of dried extract¹⁹.

2.5 Acute Toxicity Study:

Three animals of the same sex are used in each step of the three-step acute toxic class technique described in the guidelines. On average, 2-4 steps may be required to allow judgment on the acute toxicity of the test drug, depending on the animals' mortality and/or moribund stage. The material is given orally at one of the specified dosages to a cohort of experimental animals. Step-by-step protocol employing three animals of the same sex is used to test the drug. The next course of action, which entails treating three more animals at the same dose and three more animals at the next higher or lower dose level, will depend on whether compound-related mortality of the animals dosed at one step is present or absent. For every step, three animals are utilized. A starting dose of one of four preset dose levels—5, 50, 300, or 2000mg/kg body weight—must be chosen²⁰.

2.6 Formulation of topical gel:

Initially carbopol-934 was immersed in fifty mL warm water (A) for 2 hr and was homogeneously dispersed using magnetic stirrer at 600 rpm. In separate container carboxymethyl cellulose and methyl paraben was added into 50 ml warm water (B) and stirred continuously to make stiff gel. Both the mixtures A and B were combined with the continuous stirring. Then triethanolamine (Drop wise) was added to neutralize the pH and Formulations I, II, and III had an extract content of 1%, 2%, and 1%, respectively, of each extract were incorporated into dispersion to obtained gel. At this stage, permeation enhancer (Propylene glycol) was added. The final dispersion was agitated until smooth gel was formed without lumps^21-22.

Table 1: Composition of prepared herbal gel

Ingredient	Formulation I	Formulation II	Formulation III
Carbopol 940	1gm	1gm	1gm
Carboxymethyl cellulose	1gm	1gm	1gm
Propylene glycol	0.5ml	0.5ml	0.5ml
Methyl paraben	0.2ml	0.2ml	0.2ml
Thunbergia erecta	1gm	----	1gm
Bryophyllum pinnatum	----	1gm	1gm
Triethanolamine	q.s	q.s	q.s
Water	100ml	100ml	100ml

2.7 Characterization of extracts loaded Gel composition

2.7.1 Physical characteristics:

The prepared gel compositions were evaluated for visualization, Colour, Odour as well as homogeneity by visual observation²³.

2.7.2 Characterization of pH:

Formulated gel’s pH determined by utilizing Digital pH meter (EI)²⁴.

2.7.3 Estimation of Viscosity:

Using a Brookfield viscometer with spindle no. 7 at 100 rpm and 250C, the viscosity of the gel compositions was measured²⁵.

2.7.4 Spreadability:

A topical gel that is placed or rubbed on the skin's surface should have a sufficient spreading coefficient. A glass slide containing one gram of the formulation was used to assess this. A second, identically sized glass slide was positioned above it, and a 50mg mass was added to it such that the gel was sandwiched between the two slides and spread out at a specific distance. The amount of time it took the gel to move that far from its original position was recorded. Spreadability was calculated using the subsequent formula

S= M*L/T

Where, S-Spreadability, g.cm/s M-Weight put on the upper glass L-Length of glass slide T-Time for spreading gel in sec²⁶.

2.7.5 Skin irritation test:

Wistar rats of both sexes, weighing 150–200g on average, with their skin intact, were used. The rat was dehaired two to three days before to the experiment. The rat's skin was carefully shaved before the gel was administered. The animals received daily treatment for two to three days. During that time, any undesired skin changes—such as color changes or morphological changes—were monitored for twenty-four hours, and any erythema or edema on the treated skin were investigated^27-28.

2.8 Studies on wound healing:

A burn wound model with partial thickness was used in accordance with^29-30. Diethyl ether will be used to anesthetize the rats, and a sterile blade will be used to shave their back hair. 70% (v/v) ethanol was used to disinfect the shaved area. Then, 2 grams of hot, molten wax will be poured at 80 degrees Celsius to create a burn wound. The wax is going to stay on the skin until it solidifies³¹. Following the injury, the entire gel will be topically applied every day for 21 days, or until entire epithelization, whichever comes first. Following the guidelines of proper laboratory practice, the animals were housed in separate cages once they had fully recovered from anesthesia. The animals were split up into five groups at random, with six animals in each group. Once a day, the 500mg/rats of each gel therapy were administered topically^32-33.

G I: Group for control.

G II: Treated test group with Thunbergia erecta gel. (FI)

G III: Treated test group with Bryophyllum pinnatum gel. (FII)

G IV: Treated test group with (Thunbergia erecta and Bryophyllum pinnatum) (FIII)

G V: Gentamicin gel (Std. reference commercial preparation).

The monitoring of wound contraction will involve measuring the progressive changes and evaluating the wound exterior area. The wound surface area was then calculated after that trace was moved to a 1 mm2 graph sheet. The assessed surface area was subsequently used to compute the percentage of wound shrinkage using following equation, which took the wound's beginning size of 300 mm2 as 100%.

Percentage of wound contraction =

Initial wound area – Specific day wound area

------------------------------------------------------------- x100

Initial wound area

2.9 Antimicrobial Activity (Well Diffusion Assay):

2.9.1 Anti-bacterial Activity:

Assembling the Sample Dilutions:

After dilutions of the sample were made for concentrations of 100μg/ml, 150μg/ml, 200μg/ml, and 250μg/ml, to get the volume up to one milliliter, distilled water was employed.

Nutrient Agar Media Preparation:

One liter of purified water included 28grams of Nutrient Media dissolved in it. Prior to sterilization, the media's pH was measured. For fifteen minutes, the medium was autoclaved at 121 degrees Celsius and 15 pounds of pressure to disinfect it. Plates containing nutritional media were filled and exposed to laminar air flow until the agar solidified.

Well Diffusion Assay:

Nutrient agar medium (NAM) was covered with a culture of E. coli strains of bacteria. Following the formation of the wells, volume make-up was carried out up to one millilitre in order to inoculate the samples (Formulation 1, Formulation 2, and Formulation 3) supplied in the various quantities. There was 100µl of the sample loaded. For the best results, At 37^OC, the plates were incubated for 48–72hours. Calibrated to 10 8 CFU/ml of microbes, the bacterial suspension was stored in the shaker. After that, a micropipette was used to extract 100µl of the inoculum (10 8 CFU/ml) from the broth, which was then transported to a fresh, hygienic, solidified Agar Media Plate. Using a sterile spreader, the inoculum was applied to the whole surface of the sterile agar to inoculate the agar plate. Four 6 mm wells were drilled using a sterile cork borer into the infected material. Different sample concentrations (100μg/ml, 150μg/ml, 200μg/ml, and 250μg/ml) were attached to each well. It was incubated for 18 to 24hours at 37^oC after being permitted to disperse for roughly 30 minutes at room temperature. Following incubation, plates were checked to see if a clear zone formed around the well, indicating that the chemicals under test had antimicrobial activity. A measurement was taken of the zone of inhibition (ZOI) in millimeters. The reversed Petri plate was positioned with a ruler on its back and used to measure zones to the closest millimeter. A few inches above a non-reflective black background was where the Petri plate was placed. The measurements included the well's diameter as well as the diameter of the zone of total inhibition as determined by unassisted vision^34-3⁵.

3. RESULTS:

3.1. Percentage Yield:

The percentage yield is essential in phytochemical extraction for determining efficiency of extraction for specific plants, sections, or solvents. Table 2 shows the amount of extracts produced by Thunbergia erecta and Bryophyllum pinnatum.

Table 2: Crude extract yield as a percentage of Thunbergia erecta and Bryophyllum pinnatum extract

Plant name	Solvent	Theoretical weight	Yield(gm)	% yield
Thunbergia erecta	Pet ether	300	1.56	0.52%
Thunbergia erecta	Methanol	350	5.58	1.86%
Bryophyllum pinnatum	Pet ether	300	1.30	0.43%
Bryophyllum pinnatum	Methanol	367	5.95	1.62%

3.2 Preliminary Phytochemical study:

Table 3: Phytochemical testing of Thunbergia erecta and Bryophyllum pinnatum

Experiment	Presence or absence of phytochemical test Thunbergia erecta		Phytochemical test present or absent *Bryophyllum pinnatum*
Experiment	Pet. Ether extract	Methanolic extract	Pet. Ether extract	Methanolic extract
Alkaloids
Dragendroff’s test	-	+	-	-
Mayer’s reagent test	-	+	-	-
Wagner’s reagent test	-	+	-	-
Hager’s reagent test	-	+	-	-
Glycoside
Borntrager test	-	+	-	+
Legal’s test	-	+	-	+
Killer-Killiani test	-	+	-	+
Carbohydrates
Molish’s test	-	-	-	-
Fehling’s test	-	-	-	-
Benedict’s test	-	-	-	-
Barfoed’s test	-	-	-	-
Proteins and Amino Acids
Biuret test	-	+	-	+
Flavonoids
Alkaline reagent test	-	+	-	+
Lead Acetate test	-	+	-	+
Tannin and Phenolic Compounds
Ferric Chloride test	-	+	-	+
Saponin
Foam test	-	+	-	+
Test for Triterpenoids and Steroids
Salkowski’s test	-	-	-	+
Libbermann-Burchard’s test	-	-	-	+

3.3 Quantitative Analysis:

Plant material contained flavonoids and phenolics, as demonstrated by preliminary phytochemical testing of crude extracts. Whole phenolic (TPC) and total flavonoid content (TFC) assays were carried out to determine their quantity.

3.3.1 Determination of Total Phenolic content (TPC)

Table 4: Gallic acid’s std. table

Concentration (µg/ml)	Absorbance
20	0.134
40	0.170
60	0.194
80	0.232
100	0.266

Figure 1: The graph depicts the standard curve for gallic acid.

3.3.1.1 TPC of extract

Table 5: TPC of extracts

	TPC (mg/gm equivalent to gallic acid)
Extracts	*Thunbergia erecta*	*Bryophyllum pinnatum*
Absorbance (mean±SD)	0.179±0.07	0.196±0.09
TPC	78	95

3.3.2 Total Flavonoids content (TFC) estimation

Table 6: Rutin’s std.table

Concentration (µg/ml)	Absorbance
20	0.156
40	0.172
60	0.190
80	0.202
100	0.240

Figure 2: A graph showing the Rutin standard curve

3.3.2.1 TFC of extort:

Table 7: Total Flavonoid Content in extracts

TPC (mg/gm equivalent to rutin)
Extracts	*Thunbergia erecta*	*Bryophyllum pinnatum*
Absorbance (mean±SD)	0.156±0.010	0.170±0.009
TFC	24	38

3.4 Evaluation parameter of herbal gel formulation:

3.4.1 Organoleptic possessions:

Table 8: Organoleptic properties

Parameters	Results
Appearance	Semisolid gel
Colour	A little yellowish grey gel
Homogeneity	Absence of aggregates

An evaluation of the gel, including colour, appearance and homogeneity, was conducted. Gel was found to possess a slightly yellowish grey color to it upon testing. Gel showed the same colour, as well as Appearance like I.P. criteria for these traits and the outcomes were stated in Table No.8.

3.4.2 Measurement of Ph:

Table 9: pH

Formulations	Results
Formulation1	6.2
Formulation 2	5.8
Formulation 3	6.5

Every developed formulation had a pH between 5.8 and 6.5. The developed gel formulation's pH was deemed suitable in order to minimize the possibility of skin irritation upon application. The outcomes were displayed in Table No.9.

3.4.3 Determination of Viscosity

Table 10: Viscosity determination

Formulation	Results (cps)
Formulation1	1453±0.71
Formulation 2	1431±0.25
Formulation 3	1486±0.86

A crucial fluid quality that characterizes a liquid's reluctance to flow and is associated with internal friction in the fluid is called viscosity. This rheological characteristic aids in figuring out the gel's consistency and drug dispersion rate. A Brookfield viscometer with spindle number 7 was used to determine the viscosity of the generated gel; the results are shown in Table No. 10.

3.4.4 Spreadability:

Table 11: Spreadability test

Formulation	Results (gm.cm/sec)
Formulation1	22.44
Formulation 2	21.30
Formulation 3	23.19

When a gel is administered to the skin or a contaminated region, its spreadability refers to the area to which it spreads easily. Many gel formulations spreadabilities were investigated. The compositions produced excellent spreadability, as seen in Table no.11.

3.4.5 Acute skin irritation study:

Table 12: study on skin irritation

Formulation	Results
Formulation1	Not irritant observed
Formulation 2	Not irritant observed
Formulation 3	Not irritant observed

The results of the skin irritation test show that produced gels did not cause any dermatological reactions or cause irritation, redness, or edema when applied.

3.5 Wound contraction studies:

An additional metric for evaluating wound healing is wound contraction. The table displayed a significant reduction in wound size.

Table 13: Percentage wound closure in diverse treatment groups

Region of wound through diverse days of surveillance (%)
	4^th day	8^th day	12^th day	16^th day	21^st day
Control	8.31± 0.7160	8.48± 0.8152	8.44± 0.7819	8.39± 0.8852	8.42± 0.9825
F I	9.22± 0.4042	23.25±0.4350	34.19±0.7087	69.89±0.5417	70.94± 0.5522
F II	8.12± 0.7821	17.18±0.5232	49.01±0.5512	71.90±0.5510	73.29± 0.5810
F III	10.87±0.8595	26.39±0.9520	55.17±0.6677	88.09±0.3358	89.31± 0.3828
Reference Standard (Gentamicin gel)	11.89±0.7487	30.03±0.7275	59.00±0.6425	88.99±0.6432	91.55± 0.2525

Graph 1: Evaluation of wound healing activity

3.6 In-vitro antimicrobial activity:

(a) F 1 (b) F 2

Figure 3: (c) F 3

Table14: Antimicrobial activity

Conc. (µg/ml)	Inhibitory zone (in mm)
Conc. (µg/ml)	Formulation 1	Formulation 2	Formulation 3
25	10mm	8 mm	12mm
50	13 mm	11 mm	14 mm
75	15 mm	17 mm	16 mm
100	20 mm	19 mm	25 mm

The in vitro antibacterial actions of extorts of F1, F2 along F3 samples have been investigated. The well diffusion assay was utilized to measure the antimicrobial action against E. coli at concentrations of 25, 50, 75, and 100μg/ml. Formulation 1 extract revealed best inhibitory zones of 20mm diameters at a concentration of 100μg/ml in opposition to E. coli. In the same way, Formulation 2 extract exhibited greatest inhibitory zones of 19 mm in diameters at 100μg/ml concentrations alongside E. coli and Formulation 3 displayed the best zones of inhibition against E. coli, measuring 25mm in diameter at a dose of 100μg/ml.

4. DISCUSSION:

The phytochemical screening of Thunbergia erecta and Bryophyllum pinnatum indicated the existence of active metabolites like flavonoids, saponins, glycosides, phenols, proteins and amino acid. The study conducted a quantitative phytochemical assay, calculating total phenolic and flavonoid substance. The TPC was computed using gallic acid as a measure, and TFC was estimated using rutin as a measure. In the investigation on acute toxicity; no toxicity symptoms were observed up to 2000 mg/kg body weight, so a 1/10th and 1/5th dose of 500 mg/kg was fixed for the study. The gel was prepared according to the existing literature, which were taken as reference^36-37. The produced gel formulation's pH ranged from 5.8-6.5, deemed acceptable to prevent skin irritation. The gel's viscosity was gauged utilizing a viscometer from Brookfield, spindle no: 7 and resulted as Formulations 1- 1453, 2-1431, and 3-1486 cps. The spreadability of the gels was also studied, with all formulations producing good results. Skin irritation tests showed no irritation, redness, or edema, and no dermatological reactions. The in vitro antibacterial activities of F1, F2, and F3 were tested against E. coli using a well diffusion assay, with Formulation 3 showing the best inhibitory zones of 25 mm in diameters at 100μg/ml. Further, the study conducted wound healing studies on a 21-day wound model, evaluating the wound curing activity by expurgation model. Different groups were tested, including control, Thunbergia erecta gel (Formulation I), Bryophyllum pinnatum gel (Formulation II), polyherbal gel (FIII), and Gentamicin gel (Standard Marketed Preparation as a Reference). Results showed that higher quantities of herbal extracts resulted in greater wound contraction. The polyherbal formulation displayed increased activity as a result of the plant components' synergistic impact. The topical gels demonstrated versatility in contraction of the healing wound.

5. CONCLUSION:

In a pharmacological assessment, the polyherbal gel—a blend of these two extorts—displayed synergistic wound-healing action in contrast to the individual effects of all herbal medications. The merged outcomes of Bryophyllum pinnatum and Thunbergia erecta on wound curing were synergistic, and the synthesized herbal gel demonstrated considerable wound healing activity. According to the current study, polyherbal gel has a greater capacity for wound healing than separate gels, such as gel containing Bryophyllum pinnatum and Thunbergia erecta. In polyherbal gel, the proportion of wound contracture is higher. The current study serves as a foundation, and further research will be necessary to fully realize the potential of Bryophyllum pinnatum gel and Thunbergia erect for numerous activities related to wound healing.

6. FUTURE PROSPECTS:

The future prospects for the formulation and evaluation of a polyherbal gel containing Thunbergia erecta and Bryophyllum pinnatum extracts for wound healing is promising. This innovative gel leverages the synergistic effects of both plants bioactive compounds, offering a natural and potentially more effective alternative to synthetic wound healing products. Future work could explore optimizing the formulation for enhanced stability and efficacy, conducting extensive clinical trials to confirm its therapeutic benefits, and evaluating its safety profile. Additionally, expanding research into the molecular mechanisms underlying the wound healing properties of these extracts could lead to the development of novel therapeutic agents and broader applications in dermatology and regenerative medicine. This research could also pave the way for sustainable, plant-based wound care solutions with fewer side effects, aligning with the growing demand for natural healthcare products.

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Received on 08.02.2024 Revised on 17.05.2024

Accepted on 25.07.2024 Published on 24.12.2024

Available online from December 27, 2024

Research J. Pharmacy and Technology. 2024;17(12):5816-5824.

DOI: 10.52711/0974-360X.2024.00884

Group	4^th Day	8^th Day	12^th Day	16^th Day	21^st Day
Control
Formulation I Thunbergia erecta gel
Formulation II Brvophyllum pinnatum gel
Formulation III Polyherbal gel
Reference Standard(Gentamicin gel)