Eco-Friendly Formulation and Characterization of Polianthes tuberosa Flower Extract Loaded ?-Glucan Nanosponges for Anti-Acne Cream Application

Polianthes tuberosa L. Botanical Profile.

Figure 1. Field photograph of Polianthes tuberosa plant

The perennial bulbous plant Polianthes tuberosa L. (commonly referred to as tuberose), is a member of the Asparagaceae family.Its aromatic flowers are widely used in perfumery and ornamental horticulture, and thrive prodigally in tropical and subtropical nations. The P. tuberosa has not just been utilized in traditional folk medicine due to its fragrant and aesthetically pleasing nature; the plant possesses antibacterial, anti-inflammatory and wound-healing properties as well. The quantities of bioactive chemicals may be different across various parts of the plant, such as flowers, stems, and tubers. The variation in phytochemical composition is another fact that supports the idea that different parts of plants can perform distinct biological roles[1,2].However, the problem is that these long-standing beliefs have not yet been scientifically proven, particularly, with regards to the bacteria associated with acne.

Alternative Anti-Acne Agents (Medicinal Plants).

Medicine has used herbal plants to treat the skin since time immemorial. Due to their anti-inflammatory effect, broad-spectrum antibacterial effect and low toxicity, plant-based medicines have recently gained much interest. Most of the secondary metabolites present in plants such as glycosides, tannins, phenolic compounds, alkaloids, saponins and flavonoids possess great antibacterial effects and may even kill the acne causing bacteria. These phytochemicals have many mechanisms of antibacterial activity, including cell membrane destruction, enzymes, protein production, and inflammatory pathway suppression. The other potential way that plant extracts can reduce the likelihood of the development of resistance is the synergistic effect of numerous bioactive compounds[1]. As such, medicinal plants are an ideal consideration to develop all-natural, eco-friendly, and cost-effective acnes treatment.

Acne Overview & Pathophysiology

Acne vulgaris is a persistent inflammatory condition of the pilosebaceous unit which can manifest in adolescents and young adults, but can continue well into adulthood. It is marked by the development of comedones, papules, pustules, nodules and in severe cases cysts. The condition is predominantly found in regions with high concentrations of sebaceous glands including the face, chest and back. The pathophysiology of acne is multifactorial and it includes hyperkeratinization of the follicles, hyper secretion of sebum, colonization by microbes and inflammation. The hyperkeratinization of the follicles results in the clogging of the hair follicles, and the excessive secretion of sebum provides a good environment to the bacteria to multiply. The microbial factors are central to the occurrence in the sebaceous follicles where the Cutibacterium acnes grows and secretes enzymes and pro-inflammatory mediators. This causes an immune reaction, which leads to the development of redness, swelling, and lesions and this is why inflammation is an important therapeutic goal in the management of acne[1,3].

Conventional Therapy weaknesses.

Traditional medicine to treat acne consists of topical and systemic antibiotics, retinoids, benzoyl peroxide and hormonal therapy. Though these treatment methods are popular and efficient, they are characterized by a number of limitations including skin irritation, skin dryness, erythema as well as peeling. The long-term effects of using antibiotics include the occurrence of antimicrobial resistance that decreases their use in the long-term. Also, systemic therapies have more severe adverse effects, which reduces compliance in patients. These disadvantages underscore the need to have safer and more efficient alternatives. Herbal medicines are getting attention in recent years because of their natural origin, improved safety profile, and ability to be used in the long-term, making them good candidates to manage acne.

Phytochemical Components of Polianthes tuberosa L.

Some of the physiologically active compounds of Polianthes tuberosa L. including glycosides, tannins, alkaloids, phenolics and saponins have been known to be anti-inflammatory, antioxidant and antibacterial respectively, destabilising microbial enzyme systems and membrane integrity and damaging bacteria cell membranes respectively. As these phytochemicals are synergistic and complementary, P. tuberosa extracts could be superior against bacteria[1,2]. It must also be knowledgeable of the phytochemical content of the different sections of the plants to be used in identifying the most effective antibacterial fractions.

Phytochemical analysis

The screening was conducted to qualitatively determine the major secondary metabolites, such as alkaloids, flavonoids, polyphenols, tannins, monoterpenes and sesquiterpenes, steroids, quinones and saponins, in tuber, stem and flower of Polianthes tuberosa L [1,2]. This was analyzed in order to determine variation in the phytochemical composition between the different plant parts.

Table 1. Phytochemical Screening of Polianthes tuberosa Flower Extract Using Various Solvents

Note: + = detected, - = not detected

The research has shown that the flower extract of Polianthes tuberosa has a great potential in the treatment of acne because the extract can inhibit the acnes-causing bacteria and also has significant anti-inflammatory and antioxidant effects due to the presence of bioactive compounds including alkaloids, phenolics, flavonoids and quinones.

Green Synthesis Approach and Nanosponges.

The use of nanotechnology in drug delivery systems has become a new strategy in enhancing effectiveness of herbal formulations. Nanosponges are nanosized porous systems that improve the solubility of drugs, their stability, and the controlled release to increase the skin penetration and therapeutic effects. β-glucan-based nanosponges are particularly beneficial because of their biocompatibility, biodegradability, and their ability to deliver drugs to the skin. Moreover, the green synthesis of Polianthes tuberosa extract with 8-glucan nanosponges synthesized using a green approach is a new and efficient approach to acne treatment. Thus, the current research will design and describe the eco-friendly β-glucan nanosponges loaded with Polianthes tuberosa extract and assess their prospects of increased anti-acne effects[5,6].

This is to the best of our knowledge the first study to produce environmentally friendly 2 -glucan nanosponges loaded with Polianthes tuberosa flower extract and implant it in an anti-acne cream formulation. The research amalgamates herbal therapeutics, green synthesis, nanosponge technology, and topical drug delivery to offer a sustainable and effective method of treating acne.

Research Gap

Even though some herbal preparations have been examined in the treatment of acne, drawbacks like low skin penetration of active ingredients, instability of active ingredients and lack of sustained release systems are significant issues. Although a few studies have been done on the use of neem, aloe vera, tea tree oil, and herbal preparations based on turmeric, little has been done with the use of nanotechnology-based delivery system. Moreover, there is no report in the literature of the development of Polianthes tuberosa flower extract-loaded 8 -glucan nanosponges to be used as anti-acnes cream. Hence, the current research fills this research gap by creating a new eco-friendly nanosponge formulation to deliver drugs topically in an improved way.

2. Required Materials and Methods

Plant Material Collection

Figure 1: Fresh flowers of Polianthes tuberosa

Fresh Polianthes tuberosa (Family: Asparagaceae) flowers were picked in Kolhapur, Maharashtra, India and authenticated to by Mr. Yogesh Suresh Kolekar, Department of Pharmaceutical Quality Assurance, New College of Pharmacy ,Kolhapur based on standard taxonomical parameters in accordance with guidelines of the World Health Organization, confirming the identity and purity of the plant specimen. ensure botanical identification by washing with tap water and further with double distilled water to get rid of dust and other foreign contaminants. The shade-drying of the flowers was done individually at room temperature till a constant weight was reached. The dried flowers were then powdered in a mechanical grinder and kept in the clean, dry, airtight, glass containers until they were to be used again[11].

Plant Material Extraction.

Prior phytochemical screening was done by extracting the dried powder of the flower with various solvents such as n-hexane, ethyl acetate and methanol. The cold maceration technique was used to extract the samples and each powdered sample was dissolved in 1000 mL of the respective solvent and left to stand at room temperature over a period of 72 hours with frequent stirring to enable the phytoconstituents to diffuse into the solvent.

Figure 3. Extraction process of Polianthes tuberosa flower extract

The mixtures were filtered with the help of muslin cloth and then Whatman No.1 filter paper after the process of maceration and removal of plant residues. A steam bath at 50 o C was used to concentrate the filtrates to evaporate the solvents and get crude extracts. The extracts were concentrated and cooled and stored in airtight containers at 4 C to continue with phytochemical analysis.[13].

Percentage Yield = Weight of Powder Used / Weight of Extract Obtained?×100

Table 2. Percentage Yield of Different Extracts

Based on phytochemical screening results, the extract showing maximum bioactive constituents was selected for formulation of β-glucan nanosponges.

Materials for Nanosponge Formulation

The polymer used as nanosponge in the preparation of nanosponge was β-glucan and was purchased by a regular chemical supplier. Citric acid was utilized as a crosslinking agent[14]. Any chemicals and reagents utilized in the study were of analytical grade and did not undergo any additional purification[15].

Table 3. Composition of β-Glucan Nanosponge Formulation [15]

Preparation of β-Glucan Nanosponges

Polianthes tuberosa flower extract of choice was integrated into β-glucan nanosponges, with the help of an environmentally friendly crosslinking technique to have a homogenous polymer solution. The flower extract that was chosen was added slowly to the polymer solution by stirring continuously[16].

Citric acid was incorporated as a crosslinking agent and the mixture was warmed at 80-90C in 3-4 hours to enable crosslinking and production of nanosponges. The centrifuged nanosponges were dried at 40 45 C, lysed in distilled water, and kept in airtight containers awaiting further analysis [17].

β-glucan powder
↓
Dissolution/dispersion in distilled water
↓
Continuous stirring
↓
Addition of crosslinking agent
↓
Crosslinking reaction
↓
Formation of nanosponge structure
↓
Centrifugation
↓
Washing/Purification
↓
Drying (freeze drying/oven drying)
↓
β-glucan nanosponges obtained

Figure 3: Preparation process of β-glucan nanosponges[16,17]

Microorganisms

The bacterial cultures were kept in standard laboratory conditions and subcultured before use to guarantee purity and viability and were cutibacterium acnes and Staphylococcus epidermidis. The choice of these microorganisms was to assess the antibacterial capability of the plant extracts against acnes-related bacteria.[18].

Calculation of 2.6max and Preparation of Calibration Curve.

UV-vis spectrophotometry was used to determine the maximum absorption wavelength ( λmax ) of Polianthes tuberosa flower extract. Stock solution of the extract was made by dissolving a known amount of extract in an appropriate solvent.[19].

The solution was placed in the wavelength of 200-800 nm in a UV-visible spectrophotometer to measure the λmax value. The highest absorbance of the extract was found at 278 nm.[20].

To prepare the calibration curve, the stock solution was diluted to various concentrations (2, 4, 6, 8, 10 and 12 g/mL) to prepare the various concentrations of extract. The absorbance of all concentrations was determined at 278 nm and a calibration was drawn between the concentration and absorbance.

The calibration curve was both linear (r 2 =0.998) and utilized to determine the drug content and entrapment efficiency.

The following formula was used to prepare Nanosponge Loaded Anti-Acne Cream.

Polianthes tuberosa flower extract-loaded β-glucan nanosponges were optimized and integrated into a topical cream base, which is used as an anti-acne. The oil phase components, including stearic acid, cetyl alcohol, and liquid paraffin, were melted at 70°C to 75°C[22]. The aqueous phase components, which included glycerin, methyl paraben, propyl paraben, triethanolamine and purified water were heated to the same temperature separately[23].

The oil phase was slowly mixed with the aqueous phase and stirred a uniform cream base was formed. The optimized nanosponge formulation was added to the cream base after it cooled down to about 40 C and was stirred at a constant rate to achieve a homogenous distribution of the optimized nanosponge formulation in the cream base [24].

The cream prepared was transferred into airtight containers and stored to be further evaluated.

Table 4. Composition of Nanosponge Loaded Anti-Acne Cream [24]

Objectives-

• To harvest and validate Polianthes tuberosa flowers.
• To extract flowers with other solvents.
• To conduct phytochemical screening of extracts.
• To make β-glucan nanosponges by green synthesis.
• To describe nanosponges in terms of particle size, zeta potential, morphology and entrapment efficiency.
• To prepare anti-acne cream nanosponge-loaded.
• To test the in-vitro drug release.
• To determine antimicrobial action against acnes causing bacteria.
• To carry out stability investigations of the final formulation.

Prepared Cream Evaluation.

The ready Polianthes tuberosa flower extract-impregnated nanosponge cream of 800 mg -glucan was assessed regarding different physicochemical parameters to make sure that the product is appropriate in terms of topical anti-acne use.

1. Appearance

The cream that was made was visually checked regarding color, smell, consistency, phase separation, and appearance. The formulation was monitored with regard to any grittiness, the presence of lumps or instability[25].

2. pH Determination

A digital pH meter was used to measure the pH of the cream formulation. A 1-gram amount of cream was placed in 100 mL of distilled water and left to stand after 2 hours. To be compatible with skin pH, the pH was measured at room temperature[26].

3. Spreadability

The slip and drag method was used to determine spreadability of the cream. Two glass slides were put in place with a fixed amount of cream on them and a known weight was placed on the slide that was on the top. The time that the upper slide took to travel a given distance was measured[27].

Spreadability was calculated using the formula:

S=M×LT

Where:

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

4. Viscosity

Taking the viscosity of the cream was done at room temperature on Brookfield viscometer with the proper speed of the spindle. The values of viscosity were measured in centipoise (cP).[28]

5. Washability

To find out the ease of removal, a small amount of cream was spread on the skin and rinsed with tap water. The formulation was noted because of its washability properties[29].

6. Homogeneity

Homogeneity of the prepared cream was checked by both visual inspection and rubbing a small portion between fingers to ascertain the existence of coarse particles or phase separation [30].

7. Drug Content

A solution of a known amount of the cream was prepared in an appropriate solvent and filtered. To measure drug content, UV-visible spectrophotometry was used to measure the absorbance at the corresponding wavelength.

Drug Content (%) = Theoretical amount of drug /Actual amount of drug present x100.

A standard calibration curve was used to calculate the drug content [31].

8. Skin Irritation Study

The study on skin irritation was conducted on animal skin/human volunteers (depending on the study model you are doing) that was healthy after seeking ethical approval where necessary.

A dab of cream was put on the skin area and monitored over 24-48 hours to see whether there was any irritation including:

Redness, Swelling , Itching , Inflammation

Any formulation that did not result in irritation was regarded as safe [32].

Characterization

1. Particle size and Polydispersity Index (PDI)

Dynamic Light Scattering (DLS) was used to measure the average particle size and polydispersivity index (PDI) of the nanosponges prepared. The dilution of samples with distilled water and the particle size analyzer was performed at room temperature [33].

2. Zeta Potential

A zeta potential analyzer was used to measure the zeta potential of the nanosponge formulation to determine the surface charge of the particles as well as their stability [34].

3. Surface Morphology

The surface morphology of prepared nanosponges was studied with the help of Scanning Electron Microscopy (SEM). Dried samples were placed on aluminum stubs, coated with gold and examined with respect to shape, porosity and surface morphology [35].

4. FTIR Analysis

The FTIR spectroscopy has been used to determine the functional groups and also to gauge the compatibility between Polianthes tuberosa flower extract and 2 glucan Spectra were recorded in the range of 4000–400 cm?¹.[36]0

5. Entrapment Efficiency

Based on the results of the UV- visible spectrophotometry, the trapment efficiency was calculated by measuring the concentration of free drug in the supernatant following the centrifugation procedure [37].

EE (%) = (Total Drug − Free Drug / Total Drug) × 100

6. Drug Release Study in vitro.

In-vitro drug release experiment was conducted in a Franz diffusion cell in presence of phosphate buffer (pH 5.56.8) kept at 37 +0.5 C. Samples were taken at specific times (0, 1, 2, 4, 6, 8, 12 and 24 hours) and measured by UV-visible spectrophotometry.

Determination of Antibacterial Activity

1. Preparation of Bacterial Inoculum.

Polianthes tuberosa flower extract-loaded β-glucan nanosponges were tested against Cutibacterium acnes and Staphylococcus epidermidis using the antibacterial activity. Nutrient agar media was used to culture the bacterial strains and incubated in the presence of 37o C (within a range of 1o C) in sterile conditions.

The bacterial suspensions were made using 0.90 (w/v) sodium chloride solution and brought to the same level of transparency as the McFarland turbidity standard by using UV-visible spectrophotometry at 580 nm. The final concentration of bacteria was kept at about 1.5x10 8 CFU/mL.[39].

2. Paper Disc Diffusion Process.

The developed nanosponge formulation was tested against its antibacterial properties using the paper disc diffusion technique. Agar plates prepared in sterile were inoculated with 0.1 mL of a standardized bacterial suspension using a sterile spreader to evenly spread the bacteria.

Sterile paper discs (6 mm diameter) were wetted with:

• Polianthes tuberosa nano-formulation as a flower extract loaded in the nanosponge.
• Crude flower extract
• Clindamycin hydrochloride (positive control)
• 5% Tween 80 (negative control) [39]

The discs were put on the agar surface with the utmost care and incubated at 37 C at a time of 24 hours. The diameter of the zones of inhibition was measured after incubation with the help of a digital caliper. Each experiment was conducted thrice[40].

3. Minimum Effective Concentration (MEC).

The most active formulation in terms of antibacterial activity was again tested on minimum effective concentration (MEC). The disc diffusion procedure was performed on various concentrations of 1-10mg/mL on Cutibacterium acnes and Staphylococcus epidermidis.

The minimal concentration that created a visible zone of inhibition of the tested microorganisms was taken as the MEC[41].

4. Cell Morphological Observation

In an attempt to analyze the influence of the nanosponge formulation on bacterial cell morphology, overnight fixation of treated bacterial samples with 2% glutaraldehyde was used.

The samples were further processed with:

• 2% tannic acid
• Cacodylate buffer
• 1% osmium tetroxide

The samples were dried with the help of graded ethanol (50, 70, 80, 95, and 100) and freeze-drying and gold coating.

Scanning Electron Microscopy (SEM) was used to observe the morphological changes in bacterial cells.

5. Bioautography Assay

The analysis of the Polianthes tuberosa flower extract was conducted using bioautography to determine the antibacterial compounds.

A thin-layer chromatography (TLC) was performed on Silica Gel GF254 plates, and the solvent system was ethyl acetate:ethanol (7:3).

Following the process of solvent evaporation, the chromatogram was transferred to inoculated nutrient agar and allowed to stay 30 minutes to enable diffusion of active compounds. The agar plates were incubated in presence of 37 C temperature in 24 hours and the zones of inhibition were noted. The respective Rf values of active compounds were documented.[43].

Stability Studies

Stability tests were performed based on ICH guidelines at:

• 25°C ± 2°C / 60% ± 5% RH
• 40°C ± 2°C / 75% ± 5% RH

during a time of 3 months. [45]

Statistical Analysis

Each experiment was repeated (n=3) and results reported as mean and standard deviation. One-way ANOVA was used to carry out statistical analysis and a p-value below 0.05 was regarded as significant[46].

3. Workflow of formulation

Fresh Flower Collection
↓
Washing and Shade Drying
↓
Powder Preparation
↓
Solvent Extraction
↓
Phytochemical Screening
↓
Selection of Optimized Extract
↓
Preparation of β-Glucan Nanosponges
(Crosslinking using Citric Acid)
↓
Characterization of Nanosponges
• Particle Size
• Polydispersity Index
• Zeta Potential
• SEM Analysis
• FTIR Analysis
• Entrapment Efficiency
↓
Formulation of Nanosponge-Loaded Anti-Acne Cream
↓
Evaluation of Cream
• Appearance
• pH
• Spreadability
• Viscosity
• Washability
• Homogeneity
• Drug Content
• Skin Irritation Study
↓
In-vitro Drug Release Study
↓
Antimicrobial Activity Study

↓
Stability Study
↓
Final Optimized Anti-Acne Cream Formulation

Figure 5. Workflow of formulation development of Polianthes tuberosa extract-loaded β-glucan nanosponge anti-acne cream

4. Results and Discussion

Figure 6. Visual appearance of optimized Polianthes tuberosa extract-loaded nanosponge gel for topical application.

Table 5. Evaluation Parameters of Prepared Cream

Table 6. Characterization Results of β-Glucan Nanosponges

Figure 7. SEM image showing porous spherical β-glucan nanosponges

Figure 8. TEM images showing morphology and porous structure of optimized nanosponges

Figure 9. FTIR spectra of extract, polymer, and optimized formulation

Table 7. Comparative Cumulative Drug Release Profile.

Interpretation: Nanosponge formulation shows sustained release compared to crude extract.

Figure 10: Cumulative percentage drug release of nanosponge formulation compared with crude extract.

Table 8. Standard Calibration Curve Data of Polianthes tuberosa Flower Extract

Regression equation: y = 0.0572x + 0.011 , R² = 1.000

Figure 11: Calibration curve of Polianthes tuberosa flower extract at 278 nm.

Figure 12. Representative agar well diffusion plates showing antimicrobial activity of different samples against acne-causing bacteria. (A) Crude extract, (B) Blank formulation/control, (C) Standard drug, and (D) Nanosponge formulation showing zones of inhibition against Cutibacterium acnes and Staphylococcus aureus.

Table 9. Zone of Inhibition Against Acne-Causing Bacteria

Figure 13. Bar graph showing zone of inhibition against acne-causing bacteria

Table 10. Stability Study Results

Figure 14. Stability analysis of formulation under ICH conditions