Formulation And Evaluation of An Herbal Topical Gel Incorporating Lagenaria Siceraria and Raphanus Sativus for Wound Healing Applications

 

 

 

Raphanus sativus commonly known as radish belongs to the family Brassicaceae. The seeds are rich in glucosinolates, flavonoids, sulfur compounds, and phenolics possessing antioxidant and antimicrobial properties.

Taxonomical Category	Classification
Kingdom	Plantae
Family	Brassicaceae
Genus	Raphanus
Species	Raphanus sativus

Although both Lagenaria siceraria and Raphanus sativus individually possess pharmacological activities relevant to wound healing, limited scientific evidence is available regarding their combined synergistic effect in a topical polyherbal gel formulation. Furthermore, systematic studies on formulation development and evaluation of these plants for wound healing applications remain scarce.

The present research focused on the formulation and evaluation of an herbal topical gel incorporating Lagenaria siceraria and Raphanus sativus for wound healing applications. The study highlights the therapeutic significance of medicinal plants possessing antioxidant, antimicrobial, and anti-inflammatory properties in promoting tissue repair and regeneration. The selected plants are rich in bioactive phytoconstituents such as flavonoids, phenolic compounds, glucosinolates, and terpenoids, which may contribute synergistically to enhanced wound healing activity.

Development of a polyherbal topical gel offers several advantages including ease of application, localized drug delivery, improved patient compliance, prolonged retention at the site of action, and reduced systemic side effects. The proposed formulation is expected to provide effective wound contraction, faster epithelialization, reduced microbial contamination, and enhanced collagen synthesis.

The findings of this study may provide scientific validation for the traditional medicinal use of Lagenaria siceraria and Raphanus sativus in wound management. Furthermore, the developed herbal formulation may serve as a safe, economical, and effective alternative to conventional wound healing therapies. However, further pharmacological, toxicological, and clinical investigations are necessary to establish its therapeutic efficacy and safety for future pharmaceutical applications.

2.1 Materials

2.1.1 Plant Materials

Fresh leaves of Lagenaria siceraria and seeds of Raphanus sativus were collected from the local market and authenticated by a qualified botanist. The collected plant materials were washed thoroughly with distilled water to remove dirt and foreign matter, shade dried at room temperature, and coarsely powdered using a mechanical grinder. The powdered materials were stored in airtight containers for further studies.

2.1.2 Chemicals and Reagents

All chemicals and reagents used in the study were of analytical grade.

Table 2.1: List of Chemicals and Reagents

S. No.	Chemical/Reagent	Purpose
1	Ethanol	Extraction solvent
2	Petroleum ether	Defatting agent
3	Methanol	Solvent
4	Distilled water	Preparation of solutions
5	Carbopol 934	Gelling agent
6	Triethanolamine	pH adjustment
7	Methyl paraben	Preservative
8	Propyl paraben	Preservative
9	DPPH reagent	Antioxidant assay
10	Nutrient agar	Antimicrobial study

2.2 Methods

2.2.1 Authentication of Plant Material

The collected plant materials were authenticated by a botanist based on macroscopic and microscopic characteristics. Voucher specimens were preserved for future reference.

2.2.2 Preparation of Plant Extracts

The shade dried powdered leaves of Lagenaria siceraria and seeds of Raphanus sativus were subjected to successive solvent extraction using petroleum ether followed by ethanol using Soxhlet apparatus.

The extraction process involved:

• Defatting with petroleum ether for removal of fatty impurities.
• Extraction with ethanol for isolation of phytoconstituents.
• Concentration of extracts using rotary evaporator.
• Drying and storage in desiccator until further use.

Procedure

Approximately 200 g of powdered plant material was packed in a Soxhlet extractor and extracted with petroleum ether for 6–8 hours. The marc obtained after defatting was dried and extracted with ethanol for 24 hours. The extracts were concentrated under reduced pressure and stored in airtight containers.

2.2.3 Percentage Yield of Extract

The percentage yield of extract was calculated using the following formula:

 

 

2.2.4 Phytochemical Screening

Preliminary phytochemical screening of ethanolic extracts was performed to identify the presence of various phytoconstituents such as alkaloids, flavonoids, tannins, glycosides, saponins, terpenoids, and phenolic compounds using standard qualitative methods.

 

 

 

 

Table 2.2: Phytochemical Tests

Phytoconstituent	Test Performed
Alkaloids	Mayer’s test
Flavonoids	Shinoda test
Tannins	Ferric chloride test
Saponins	Foam test
Glycosides	Keller-Killiani test
Phenolics	Lead acetate test
Terpenoids	Salkowski test

2.2.5 Evaluation of Antioxidant Activity

The antioxidant activity of plant extracts was evaluated by DPPH free radical scavenging assay.

Principle

DPPH is a stable free radical showing deep violet color. Antioxidants present in plant extracts donate hydrogen atoms to DPPH radicals resulting in discoloration from violet to yellow. The decrease in absorbance indicates free radical scavenging activity.

Procedure

• DPPH solution was prepared in methanol.
• Different concentrations of plant extracts were prepared.
• 1 mL of extract solution was mixed with DPPH solution.
• The mixture was incubated in dark for 30 minutes.
• Absorbance was measured at 517 nm using UV spectrophotometer.

The percentage inhibition was calculated using the following formula:

 

 

Where:

• (A_0) = Absorbance of control
• (A_1) = Absorbance of sample

2.2.6 Evaluation of Antimicrobial Activity

The antimicrobial activity of extracts was evaluated by agar well diffusion method against selected bacterial strains.

Procedure

• Nutrient agar medium was prepared and sterilized.
• Bacterial cultures were inoculated on agar plates.
• Wells were prepared using sterile cork borer.
• Different concentrations of extracts were introduced into wells.
• Plates were incubated at 37°C for 24 hours.
• Zone of inhibition was measured in millimeters.

Table 2.3: Microorganisms Used

S. No.	Microorganism
1	Staphylococcus aureus
2	Escherichia coli
3	Pseudomonas aeruginosa

2.2.7 Formulation of Herbal Topical Gel

The herbal topical gel was prepared using Carbopol 934 as gelling agent.

Composition of Herbal Gel

Table 2.4: Composition of Herbal Gel Formulation

Ingredients	Quantity
Lagenaria siceraria extract	Required quantity
Raphanus sativus extract	Required quantity
Carbopol 934	1%
Propylene glycol	5%
Methyl paraben	0.2%
Propyl paraben	0.02%
Triethanolamine	q.s.
Distilled water	q.s. to 100 g

Procedure for Gel Preparation

1. Carbopol 934 was dispersed in distilled water with continuous stirring.
2. Preservatives were dissolved in propylene glycol.
3. Plant extracts were incorporated into the gel base.
4. Triethanolamine was added dropwise for pH adjustment and gel formation.
5. The prepared gel was stored in suitable containers for evaluation.

2.2.8 Evaluation of Herbal Gel

The formulated gel was evaluated for various physicochemical parameters.

(a) Physical Appearance

The gel was observed for color, homogeneity, consistency, and presence of grittiness.

(b) pH Determination

The pH of gel formulation was determined using a digital pH meter.

(c) Viscosity

Viscosity was measured using Brookfield viscometer at room temperature.

(d) Spreadability

Spreadability indicates ease of application of gel on skin surface.

The spreadability was calculated using the formula:

 

 

Where:

• (S) = Spreadability
• (M) = Weight tied to upper slide
• (L) = Length moved by glass slide
• (T) = Time taken

(e) Extrudability

Extrudability was determined by measuring the force required to extrude gel from collapsible tube.

(f) Homogeneity

Homogeneity was evaluated visually for uniform appearance and absence of lumps.

2.2.9 Evaluation of Wound Healing Activity

The wound healing activity of the formulated gel was evaluated using excision wound model in experimental animals.

Procedure

• Experimental animals were divided into different groups.
• Excision wounds were created under aseptic conditions.
• Gel formulations were applied topically once daily.
• Wound contraction was measured periodically.
• Percentage wound contraction was calculated.

The percentage wound contraction was determined using the formula:

 

2.2.10 Statistical Analysis

 

All experimental data were expressed as mean ± standard deviation (SD). Statistical analysis was performed using suitable statistical methods and significance level was considered at p < 0.05.

3.1 RESULTS

The present study was carried out to formulate and evaluate an herbal topical gel incorporating Lagenaria siceraria and Raphanus sativus extracts for wound healing applications. The extracts were evaluated for phytochemical constituents, antioxidant activity, antimicrobial activity, and wound healing potential. The prepared gel formulations were further evaluated for physicochemical parameters and stability.

3.2 Percentage Yield of Extracts

The ethanolic extracts obtained from Lagenaria siceraria leaves and Raphanus sativus seeds showed satisfactory percentage yields indicating efficient extraction of phytoconstituents.

 

 

 

 

 

Table 3.1: Percentage Yield of Plant Extracts

Plant Material	Weight of Crude Drug (g)	Weight of Extract (g)	Percentage Yield (%)
Lagenaria siceraria	250	28.5	11.4
Raphanus sativus	250	24.2	9.68

The higher percentage yield of Lagenaria siceraria may be attributed to the presence of higher amounts of polar phytoconstituents such as flavonoids and phenolic compounds.

3.3 Preliminary Phytochemical Screening

Preliminary phytochemical investigation confirmed the presence of various bioactive constituents in both plant extracts.

Table 3.2: Phytochemical Screening of Ethanolic Extracts

Phytoconstituents	Lagenaria siceraria	Raphanus sativus
Alkaloids	+	+
Flavonoids	+++	++
Phenolic compounds	+++	+++
Tannins	++	++
Glycosides	+	+
Saponins	++	+
Terpenoids	++	++
Steroids	+	+

Key:
(+) Present
(++) Moderately Present
(+++) Highly Present

The phytochemical screening revealed that both extracts were rich in flavonoids and phenolic compounds which are known for antioxidant and wound healing activities.

3.4 Evaluation of Antioxidant Activity

The antioxidant activity of plant extracts was determined using DPPH free radical scavenging assay.

Table 3.3: DPPH Radical Scavenging Activity

Concentration (µg/mL)	Lagenaria siceraria (%)	Raphanus sativus (%)	Polyherbal Extract (%)
20	32.5	28.4	35.6
40	45.2	42.1	49.7
60	59.6	56.4	64.8
80	72.3	68.9	77.4
100	84.5	79.8	89.2

The polyherbal extract exhibited higher free radical scavenging activity compared to individual extracts, indicating synergistic antioxidant potential due to combined phytoconstituents.

3.5 Evaluation of Antimicrobial Activity

The antimicrobial activity of extracts was evaluated against selected pathogenic microorganisms using agar well diffusion method.

Table 3.4: Zone of Inhibition of Extracts (mm)

Extract	S. aureus	E. coli	P. aeruginosa
Lagenaria siceraria	16 ± 0.5	14 ± 0.4	13 ± 0.3
Raphanus sativus	15 ± 0.6	13 ± 0.5	12 ± 0.2
Polyherbal Extract	20 ± 0.4	18 ± 0.5	17 ± 0.4
Standard Drug	24 ± 0.3	23 ± 0.4	22 ± 0.5

The polyherbal extract showed enhanced antimicrobial activity against all tested microorganisms compared to individual extracts. The highest activity was observed against Staphylococcus aureus.

3.6 Evaluation of Herbal Gel Formulations

Three gel formulations (F1, F2, and F3) were prepared using different concentrations of Carbopol 934 and evaluated for physicochemical parameters.

3.6.1 Physical Appearance

All prepared formulations were smooth, homogeneous, and free from grittiness.

Table 3.5: Physical Characteristics of Gel Formulations

Formulation	Color	Consistency	Homogeneity	Grittiness
F1	Light green	Smooth	Good	Absent
F2	Green	Smooth	Excellent	Absent
F3	Dark green	Thick	Good	Absent

3.6.2 pH Determination

The pH of formulations was found within acceptable range for topical application.

Table 3.6: pH of Gel Formulations

Formulation	pH
F1	6.4
F2	6.8
F3	7.1

The pH values were compatible with skin pH and indicated suitability for topical administration without irritation.

3.6.3 Viscosity Study

Table 3.7: Viscosity of Gel Formulations

Formulation	Viscosity (cP)
F1	18,500
F2	24,300
F3	31,200

Viscosity increased with increase in Carbopol concentration. Formulation F2 exhibited optimum viscosity suitable for topical application.

3.6.4 Spreadability

 

Table 3.8: Spreadability of Gel Formulations

Formulation	Spreadability (g·cm/sec)
F1	13.2
F2	11.4
F3	8.6

Formulation F2 demonstrated good spreadability indicating easy application over skin surface.

3.6.5 Extrudability

Table 3.9: Extrudability of Gel Formulations

Formulation	Extrudability
F1	Excellent
F2	Good
F3	Moderate

The results showed that F2 possessed satisfactory extrudability and consistency.

3.7 Stability Study

The optimized formulation F2 was subjected to stability studies for one month at room temperature.

Table 3.10: Stability Study of Optimized Gel (F2)

Parameter	Initial	After 30 Days
Appearance	Smooth	Smooth
pH	6.8	6.7
Viscosity	24,300 cP	24,100 cP
Color	Green	No change

No significant changes were observed during stability studies indicating good stability of the formulation.

3.8 Evaluation of Wound Healing Activity

The wound healing activity of optimized formulation F2 was evaluated using excision wound model in Wistar albino rats.

 

 

 

 

 

 

Table 3.11: Percentage Wound Contraction

Day	Control	Standard	Herbal Gel F2
4	18.2%	35.4%	32.1%
8	39.5%	62.7%	58.4%
12	58.3%	85.6%	81.2%
14	71.4%	96.8%	93.5%

The herbal gel formulation showed significant wound contraction compared to control group and demonstrated activity comparable to standard drug treatment.

3.9 DISCUSSION

The present study successfully formulated and evaluated a polyherbal topical gel containing Lagenaria siceraria and Raphanus sativus extracts for wound healing applications. Preliminary phytochemical screening confirmed the presence of important bioactive constituents such as flavonoids, phenolics, tannins, terpenoids, and saponins which are known to possess antioxidant, antimicrobial, and anti-inflammatory activities.

The DPPH assay demonstrated significant free radical scavenging activity of the polyherbal extract, indicating strong antioxidant potential. Oxidative stress is one of the major causes of delayed wound healing; therefore, antioxidant activity plays an important role in tissue repair and regeneration.

The antimicrobial study revealed effective inhibition against both Gram-positive and Gram-negative bacteria. The enhanced antimicrobial activity of the polyherbal extract may help prevent microbial infection at wound site and accelerate healing.

Among all formulations, F2 containing 1.5% Carbopol 934 showed optimum physicochemical properties including acceptable pH, viscosity, spreadability, and extrudability. Stability studies confirmed that the formulation remained stable during the study period without significant changes.

The excision wound model demonstrated significant wound contraction and faster healing in animals treated with the herbal gel formulation. The observed wound healing activity may be attributed to synergistic action of phytoconstituents present in Lagenaria siceraria and Raphanus sativus which promote collagen synthesis, epithelialization, antioxidant defense, and microbial inhibition.

Overall, the results suggest that the formulated polyherbal gel possesses promising wound healing potential and may serve as a safe and economical alternative to conventional wound care therapies.

ACKNOWLEDGEMENT

I express my sincere gratitude and deep sense of respect to my esteemed guide, Dr Deepesh Lall, Associate Professor, LCIT School of Pharmacy, Bilaspur, Chhattisgarh, for his valuable guidance, constant encouragement, scholarly suggestions, and continuous support throughout the completion of this research work. His inspiring supervision and motivation greatly contributed to the successful accomplishment of this study. I would also like to express my heartfelt thanks to the Principal, faculty members, and staff of LCIT School of Pharmacy for providing the necessary facilities, laboratory support, and academic environment required for carrying out this research work successfully.

I am grateful to Chhattisgarh Swami Vivekanand Technical University for providing the opportunity to undertake this research work as a part of the Master of Pharmacy programme.

I would like to acknowledge my friends and classmates for their encouragement, cooperation, and valuable support during the course of this work. Finally, I express my deepest gratitude to my parents and family members for their unconditional love, blessings, patience, and moral support, without which this work would not have been possible.

Funding Statement

The present research work was carried out without receiving any specific grant or financial assistance from any government agency, commercial organization, or funding body. The study was conducted using the facilities available at LCIT School of Pharmacy as a part of academic research work for the award of the degree of Master of Pharmacy.

Conflict of Interest

The author declares that there is no conflict of interest regarding the publication of this research work. The research was conducted purely for academic and scientific purposes without any commercial or financial relationships that could influence the outcomes of the study.

REFERENCES

1. Adewumi SS, Akinpelu BA, Akinpelu DA, Aiyegoro OA, Alayande KA, Agunbiade MO. Studies on wound healing potentials of the leaf extract of Terminalia avicennioides on Wistar rats. S Afr J Bot. 2020;133:285-97. doi:10.1016/j.sajb.2020.07.039.
2. Ahmad F, Hasan I, Chishti DK, Ahmad H. Antibacterial activity of Raphanus sativus Linn. seed extract. Glob J Med Res. 2012;12(11):25-34.
3. Alawdi SH, Shehab M, Al-Mekhlafi AG. Formulation of herbal gel preparations and evaluation of their wound curing activities. Saudi J Biomed Res. 2019;4(8):279-84. doi:10.21276/sjbr.2019.4.8.1.
4. Attar UA, Ghane SG. In vitro antioxidant and RP-HPLC analysis of phenolics from wild bottle gourd. S Afr J Bot. 2019;125:360-70. doi:10.1016/j.sajb.2019.08.017.
5. Beevi SS, Narasu ML, Gowda BB. Polyphenolic profile and antioxidant activity of Raphanus sativus L. Plant Foods Hum Nutr. 2010;65(1):8-17. doi:10.1007/s11130-009-0148-6.
6. Boateng J, Catanzano O. Advanced therapeutic dressings for effective wound healing. J Pharm Sci. 2015;104(11):3653-80. doi:10.1002/jps.24610.
7. Bodeker G, Ryan TJ, Ong CK. Traditional approaches to wound healing. Clin Dermatol. 1999;17(1):93-8. doi:10.1016/S0738-081X(98)00056-X.
8. Chouhan D, Dey N, Bhardwaj N, Mandal BB. Emerging approaches for wound healing and skin regeneration. Biomaterials. 2019;216:119267. doi:10.1016/j.biomaterials.2019.119267.
9. Dan MM, Sarmah P, Rao VD, Dattatreya A. Wound healing: Concepts and updates in herbal medicine. Int J Med Res Health Sci. 2018;7(1):170-81.
10. Demilew W, Adinew GM, Asrade S. Evaluation of wound curing activity of Acanthus polystachyus. Evid Based Complement Alternat Med. 2018;2018:2047896. doi:10.1155/2018/2047896.
11. Dorle AK. Beneficial effects of Lagenaria siceraria fruit epicarp in animal models. Nat Prod Res. 2008;22(4):345-52.
12. Enoch S, Leaper DJ. Basic science of wound healing. Surgery. 2008;26(2):31-7. doi:10.1016/j.mpsur.2007.11.005.
13. Gautam MK, Purohit V, Agarwal M, Singh A, Goel RK. In vivo wound healing potential of Aegle marmelos. ScientificWorldJournal. 2014;2014:740107. doi:10.1155/2014/740107.
14. Ghule BV, Ghante MH, Yeole PG. Analgesic and anti-inflammatory activities of Lagenaria siceraria. Pharmacogn Mag. 2006;2(8):232-8.
15. Gonzalez ACDO, Costa TF, Andrade ZDA, Medrado ARAP. Wound healing: A literature review. An Bras Dermatol. 2016;91(5):614-20. doi:10.1590/abd1806-4841.20164741.
16. Guo S, DiPietro LA. Factors affecting wound healing. J Dent Res. 2010;89(3):219-29. doi:10.1177/0022034509359125.
17. Harborne JB. Phytochemical Methods. London: Chapman and Hall; 1984.
18. Herman A, Herman AP. Herbal products in postsurgical wound healing. Planta Med. 2020;86(11):732-48. doi:10.1055/a-1162-9988.
19. Heggers JP, Pelley RP, Robson MC. Beneficial effects of Aloe in wound healing. Phytother Res. 1993;7(S1):S48-S52.
20. Jamuna S, Paulsamy S, Karthika K. In vitro antioxidant activity of Hypochaeris radicata. J Appl Pharm Sci. 2012;2(7):149-54.
21. Kumar D, Sharma C, Singh B, Singh D. Pharmacognostical and pharmacological profile of Lagenaria siceraria. Br J Pharm Res. 2015;7(5):340-52.
22. Malik M, Sharma V, Gupta R. Role of medicinal plants in wound healing. J Ethnopharmacol. 2018;219:1-12.
23. Marinova G, Batchvarov V. Evaluation of DPPH radical scavenging methods. Bulgarian J Agric Sci. 2011;17(1):11-24.
24. Medina A, Scott PG, Ghahary A, Tredget EE. Pathophysiology of chronic non-healing wounds. J Burn Care Rehabil. 2005;26(4):306-19.
25. Muniandy K, Gothai S, Tan WS, Arulselvan P. In vitro wound healing potential of Alternanthera sessilis. Evid Based Complement Alternat Med. 2018;2018:3142073.
26. Nagoba B, Davane M. Study protocols in herbal wound healing research. J Ayurveda Integr Med. 2019;10(4):316-8.
27. Olsson M, Järbrink K, Divakar U, Bajpai R, Upton Z, Schmidtchen A, et al. Economic burden of chronic wounds. Wound Repair Regen. 2019;27(1):114-25.
28. Rasik AM, Shukla A. Antioxidant status in delayed wound healing. Int J Exp Pathol. 2000;81(4):257-63.
29. Schreml S, Szeimies RM, Prantl L, Landthaler M, Babilas P. Wound healing in the 21st century. J Am Acad Dermatol. 2010;63(5):866-81.
30. Wilkinson HN, Hardman MJ. Cellular mechanisms of wound healing. Open Biol. 2020;10(9):200223. doi:10.1098/rsob.200223.