Phytoformulation of Nyctanthes arbor-tristis Based Ointment for Enhanced Skin Regeneration in Wound Healing

Wound healing is a highly complex and dynamic biological process involving a coordinated sequence of hemostasis, inflammation, proliferation, and tissue remodeling, which aims to restore the structural and functional integrity of damaged skin. Delayed or impaired wound healing remains a major clinical challenge, particularly in chronic wounds associated with infections, inflammation, oxidative stress, and compromised collagen synthesis.

In recent years, phytoformulations derived from medicinal plants have gained significant attention as safe, cost-effective, and biologically active alternatives to synthetic wound care agents, owing to their rich reservoir of bioactive phytochemicals. Nyctanthes arbor-tristis, a well-known medicinal plant in traditional systems of medicine, possesses a wide range of therapeutic properties including anti-inflammatory, antioxidant, antimicrobial, and immunomodulatory activities.

The leaves and other parts of the plant are reported to contain flavonoids, tannins, saponins, iridoid glycosides, and phenolic compounds, which play a crucial role in accelerating wound contraction, enhancing epithelialization, and promoting collagen deposition. Incorporation of Nyctanthes arbor-tristis extract into a topical ointment base offers an effective phytoformulation strategy to ensure sustained release of active constituents, improved skin permeation, and localized therapeutic action.

Such bio-based ointment formulations not only aid in preventing microbial invasion at the wound site but also modulate inflammatory responses and stimulate tissue regeneration, thereby enhancing overall skin repair. Hence, the present study focuses on the development and evaluation of a Nyctanthes arbor-tristis-based ointment formulation as a promising natural therapeutic approach for enhanced skin regeneration and effective wound healing.

In addition, the antimicrobial activity of Nyctanthes arbor-tristis contributes significantly to wound management by reducing microbial load at the wound site, which is a critical factor in preventing infection-related complications. Its antioxidant properties help neutralize reactive oxygen species generated during inflammation, thereby preventing oxidative damage to newly formed tissues. The anti-inflammatory action further supports rapid wound closure by regulating cytokine release and minimizing prolonged inflammatory responses.

2.MATERIALS AND METHODOLOGY:

2.1. Collection and Preparation of Plant Material:

Fresh flowers of Nyctanthes arbor-tristis were collected early in the morning from a local area. The collected flowers were gently washed with distilled water to remove dust and other contaminants. The washed flowers were air-dried under shade for 1–2 hours to remove surface moisture before further processing.

2.2 Preparation of Flower Paste:

The semi-dried flowers were crushed using a sterile mortar and pestle or grinder to obtain a fine paste. This paste was used immediately for phytochemical extraction to prevent degradation of active constituents.

2.3         Extraction of Phytocompounds:

The prepared flower paste was mixed with coconut oil in a ratio of 1:5 (e.g., 20 g of flower paste with 100 mL of coconut oil). The mixture was transferred into a double boiler setup to ensure indirect heating.

2.4       Heating and Infusion Process:

The flower–oil mixture was heated gently at a temperature not exceeding 70–80 °C for 1–1.5 hours. The mixture was stirred intermittently to facilitate the release of bioactive phytocompounds into the oil medium.

2.5       Filtration of Infused Oil:

After heating, the mixture was allowed to cool slightly and then filtered using muslin cloth or Whatman No.1 filter paper. The solid residues were discarded, and the clear infused oil was collected for ointment preparation.

2.6       Preparation of Ointment Base:

Beeswax (10 g) was melted in a clean vessel using gentle heat. The filtered infused oil was slowly added to the melted beeswax with continuous stirring to ensure uniform mixing.

2.7 Formualtion of Herbal Ointment:

Stirring was continued until a homogeneous ointment consistency was obtained. The prepared ointment was 

allowed to cool, transferred into sterile containers, and stored at room temperature for further evaluation.

3. Results and Discussion:

3.1 phytochemical tests :

The presence of flavonoids and phenolic compounds contributes to antioxidant activity, which plays a crucial role in reducing oxidative stress during wound healing. Saponins enhance collagen synthesis and promote epithelialization. Alkaloids and glycosides may contribute to antimicrobial and anti-inflammatory activity, thereby accelerating wound repair.

3.2 Formulation and Physicochemical content:

3.2.1Evaluation of Ointment

Appearance:

The formulated ointment was light green in color with characteristic odor and showed uniform consistency without phase separation.

pH:

The pH of the formulation was found to be 5.8 ± 0.2, which is compatible with skin pH.

Spreadability:

Spreadability was determined by slip and drag method and was found to be 6.5 ± 0.3 g·cm/sec indicating good spreadability.

Viscosity:

Viscosity measured using Brookfield viscometer was found to be 4500 ± 120 cps.

Washability:

The formulation was easily washable with water, indicating patient compliance.

Extrudability:

The ointment showed satisfactory extrudability from collapsible tube.

Stability Study:

The formulation remained stable without color change, phase separation or odor change at room temperature and 40°C for 30 days.

3.3 Antimicrobial Activity:

Agar well diffusion method was used against:

•           Staphylococcus aureus

•           Escherichia coli

•           Pseudomonas aeruginosa

Silver sulfadiazine was used as standard and base cream as negative control.

SCRATCH ASSAY CONTENT

The in vitro wound healing activity was evaluated using L929 fibroblast cell line.

The formulation showed:

•           24 h: 48% wound closure

•           48 h: 82% wound closure

This indicates enhanced fibroblast migration and tissue regeneration potential.

MTT ASSAY CONTENT

                        Cell viability was above 85% at all tested concentrations, confirming non-cytotoxic nature of the formulation.

3.1 Protein Quantification (Lowry Method results)

The keratin protein concentration in 1ml  of the extract was determined using the Lowry method. A standard curve was generated using Bovine Serum Albumin (BSA) standards, and the absorbance was measured at 750nm. The unknown sample (diluted 1:5) was compared against the linear regression equation derived from these standards.

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Table 1: OD values of BSA standard

Figure 1:  BSA standard curve (OD vs Concentration)

The sample OD value curve was compared using the BSA standard curve and 1ml of extract contained contained 1.197 mg/ml of keratin protein.

Figure 2 :  OD value of the sample compared with BSA standards

 3.2.FTIR results:

The FTIR spectra were obtained for the Nyctanthes arbor-tristis extract to identify the functional groups responsible for wound healing and skin regeneration. The presence of phenolic compounds and proteins was confirmed through the                                                      

Figure 3:  FTIR graph (spectrum of “Nyctanthes arbor-tristis”Extract)

The FTIR spectrum of the Nyctanthes arbor-tristis extract was analyzed to identify the functional groups responsible for its regenerative properties.

•           Amide A and Hydroxyl Groups: A broad peak observed at 3315.6 cm-1(Intensity: 45.320) corresponds to O-H stretching (phenolic compounds) and N-H stretching (Amide A). This indicates a high concentration of antioxidant flavonoids and peptide-based structures essential for reducing oxidative stress in wound sites.

•           Aliphatic Groups: The peak at 2918.2 cm-1(Intensity: 81.200) represents C-H stretching vibrations of alkanes, typically associated with the lipid components and long-chain fatty acids present in the plant wax that contribute to the ointment's emollient properties.

•           Amide I Band: A strong band at 1638.7 cm-1(Intensity: 52.150) is assigned to C=O stretching (Amide I). This region is critical for protein secondary structure. The peak at 1638.7 cm-1 suggests a stable protein framework within the extract, likely providing a scaffold-like effect for skin cell proliferation.

•           Glycosidic Linkages: The peak at 1042.3 cm-1(Intensity: 68.410) corresponds to C-O stretching, signifying the presence of iridoid glycosides (such as arbortristoside A and B). These compounds are documented to have significant anti-inflammatory and wound-healing activities.

Table 3: FTIR Peak Assignments for Nyctanthes arbor-tristis Extract

 Figure 4:  FTIR graph (microfilter syringe filtered extract)

N–H stretching and O–H stretching resulting in amide A presence at 3315.6 cm?¹ (Intensity: 45.320) and C=O stretching resulting in amide I esence at 1638.7 cm?¹ (Intensity: 52.150) was observed.

Table 4: Result comparison between both the forms of extract