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Department of Pharmaceutical Quality Assurance, MES’S College of Pharmacy, Sonai, Ahmednagar, Maharashtra, India.
The present study developed and optimized a betulin-loaded thermosensitive in situ gel to enhance the bioavailability of this poorly water-soluble pentacyclic triterpene. Preformulation studies confirmed betulin's identity (melting point: 258.4°C, ?max: 210 nm) and extremely poor aqueous solubility (0.048 mg/mL). Nine formulations were prepared using 3² factorial design with varying concentrations of Poloxamer 407 (18-22% w/v) and Butea monosperma gum (1.0-2.0% w/v). All formulations exhibited physiologically acceptable pH (6.1-6.4), gelation temperatures (27.7-35.8°C), and excellent drug content (93.8-97.8%). Response surface methodology yielded significant quadratic models (p<0.01) for gelation temperature and drug release. Optimized formulation F4 demonstrated ideal gelation temperature (32.8°C), superior mucoadhesive strength (3,580 dyne/cm²), and optimal sustained release (95.3% at 12h). Validation studies confirmed model accuracy with relative errors <2.5%. Stability studies showed excellent refrigerated stability over three months. This thermosensitive system offers a promising platform for enhancing betulin's therapeutic applications.
Parkinson's disease represents one of the most prevalent neurodegenerative disorders affecting millions of individuals worldwide, characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta region of the brain. This chronic and progressive movement disorder was first comprehensively described by James Parkinson in 1817 in his seminal monograph titled "An Essay on the Shaking Palsy," where he detailed six cases of patients exhibiting characteristic symptoms including tremor at rest, abnormal posture, and paralysis. The condition, initially termed "paralysis agitans," was later renamed Parkinson's disease by the French neurologist Jean-Martin Charcot in the late 19th century to honor James Parkinson's pioneering work in identifying and characterizing this distinct clinical entity. The historical recognition of Parkinson's disease marked a significant milestone in neurology, establishing it as a discrete nosological entity separate from other neurological conditions that presented with similar motor manifestations [1]. The historical trajectory of Parkinson's disease research demonstrates a gradual shift from purely clinical observation to sophisticated molecular and genetic investigations that have illuminated various aspects of disease pathogenesis. Modern neuroimaging techniques, including positron emission tomography and single-photon emission computed tomography, have enabled visualization of dopaminergic neuron loss in living patients, facilitating earlier diagnosis and monitoring of disease progression. Genetic studies have identified numerous genes associated with familial forms of Parkinson's disease, such as SNCA, LRRK2, PARK2, PINK1, and DJ-1, providing valuable insights into the molecular pathways involved in neurodegeneration. These genetic discoveries have revealed that Parkinson's disease results from complex interactions between genetic susceptibility factors and environmental influences, rather than being purely genetic or purely environmental in origin [3]. Parkinson's disease constitutes the second most common neurodegenerative disorder after Alzheimer's disease, affecting approximately 1 to 2 percent of individuals over 60 years of age globally, with prevalence increasing dramatically with advancing age. Epidemiological studies indicate that the global burden of Parkinson's disease has more than doubled over the past generation, with an estimated 6 to 10 million people currently living with the condition worldwide. This increase reflects not only improved diagnostic capabilities and awareness but also demographic shifts toward aging populations in many countries, as life expectancy continues to rise globally. The prevalence of Parkinson's disease shows significant geographical variation, with higher rates reported in industrialized nations compared to developing countries, though these differences may partly reflect variations in healthcare access, diagnostic practices, and epidemiological surveillance systems rather than true biological differences in disease susceptibility [4].
1.2 Materials And Methods:
1.2.1 Materials
High-purity active and analytical components such as Betulin were sourced from Sigma-Aldrich in the USA, while Poloxamer 407 (Pluronic F-127) was provided by BASF Corporation in Germany. Other essential chemical agents, specifically Benzalkonium chloride, Potassium bromide (KBr), and HPLC-grade Methanol, were also procured from Germany through Merck KGaA. Regional suppliers from India contributed several key ingredients, including Butea monosperma gum from a local source, Sodium chloride from S.D. Fine Chemicals, and both Potassium dihydrogen phosphate and Disodium hydrogen phosphate from Loba Chemie. Additionally, HiMedia Laboratories in India supplied the cellulose acetate membranes. To complete the material list, Ethanol was obtained from Changshu Yangyuan Chemical in China, and distilled water was prepared internally within the laboratory.
1.2.2 Organoleptic Evaluation of Betulin
The organoleptic properties of betulin were evaluated through visual and sensory examination to assess its physical characteristics. The color of betulin was determined by visual observation under natural daylight conditions, noting the appearance and any color variations. The odor was assessed by trained personnel through direct olfactory evaluation, where small quantities of betulin were placed in clean petri dishes and the characteristic smell was noted and described. The texture was evaluated by gentle handling of the betulin sample between fingers to determine its physical feel, consistency, and tactile properties, with observations recorded regarding its granular nature, smoothness, or any other relevant textural characteristics [72].
1.2.3 Experimental design
A 3² factorial design was employed to optimize the thermosensitive in-situ nasal gel formulation using two independent variables at three levels each: Poloxamer 407 concentration (X₁) at 18, 20, and 22% w/v, and Butea monosperma gum concentration (X₂) at 1, 1.5, and 2% w/v, with gelation temperature and cumulative drug release at 12 hours selected as dependent variables (Y₁ and Y₂ respectively) to evaluate the formulation performance. The design matrix consisted of 9 experimental runs generated using Design-Expert software, and the responses were analyzed using response surface methodology to understand the effect of independent variables on the dependent responses. The general regression equation for the responses was represented as
Y = b₀ + b₁X₁ + b₂X₂ + b₁₂X₁X₂ + b₁₁X₁² + b₂₂X₂²
where Y is the predicted response, b₀ is the intercept, b₁ and b₂ are linear coefficients, b₁₂ is the interaction coefficient, and b₁₁ and b₂₂ are quadratic coefficients for the independent variables X₁ and X₂ respectively [56].
Table 2.1: Experimental Design Variables for 3² Factorial Design
|
Independent variables |
Levels |
||
|
Low (-1) |
Medium (0) |
High (+1) |
|
|
Poloxamer 407 concentration (%w/v) |
18 |
20 |
22 |
|
Butea monosperma gum concentration (%w/v) |
1 |
1.5 |
2 |
|
Dependent Variables |
Goal |
||
|
Gelation temperature (°C) |
Target: 32-37°C |
||
|
Cumulative drug release at 12 hours (%) |
Maximize |
||
1.2.4 Formulation of Betulin Loaded Thermosensitive gel
The betulin-loaded thermosensitive in-situ nasal gels were prepared using the cold dissolution method by initially dispersing the required amount of Poloxamer 407 (18-22% w/v) in cold distilled water (4-5°C) with continuous stirring at 500 rpm for 2-3 hours until complete dissolution, followed by the addition of Butea monosperma gum (1-2% w/v) which was dissolved separately in small amount of water and mixed thoroughly to ensure uniform distribution. Betulin (0.1% w/v) was dissolved in minimum amount of ethanol and incorporated into the polymer solution along with other excipients including benzalkonium chloride (0.02% w/v) as preservative, sodium chloride (0.9% w/v) for isotonicity, and the pH was adjusted to 6.8 using phosphate buffer. The final formulation was stirred gently to avoid air entrapment, stored in refrigerator (2-8°C) overnight to ensure complete hydration of polymers, and different batches were prepared according to the experimental design matrix to evaluate the effect of polymer concentrations on gel properties [71].
Table 2.2: Formulation Composition of Betulin-Loaded Thermosensitive In-Situ Nasal Gel Batches
|
Batch Code |
Betulin (%w/v) |
Poloxamer 407 (%w/v) |
Butea monosperma gum (%w/v) |
Benzalkonium chloride (%w/v) |
Sodium chloride (%w/v) |
Distilled water |
|
F1 |
0.1 |
18 |
1.0 |
0.02 |
0.9 |
q.s. to 100 mL |
|
F2 |
0.1 |
18 |
1.5 |
0.02 |
0.9 |
q.s. to 100 mL |
|
F3 |
0.1 |
18 |
2.0 |
0.02 |
0.9 |
q.s. to 100 mL |
|
F4 |
0.1 |
20 |
1.0 |
0.02 |
0.9 |
q.s. to 100 mL |
|
F5 |
0.1 |
20 |
1.5 |
0.02 |
0.9 |
q.s. to 100 mL |
|
F6 |
0.1 |
20 |
2.0 |
0.02 |
0.9 |
q.s. to 100 mL |
|
F7 |
0.1 |
22 |
1.0 |
0.02 |
0.9 |
q.s. to 100 mL |
|
F8 |
0.1 |
22 |
1.5 |
0.02 |
0.9 |
q.s. to 100 mL |
|
F9 |
0.1 |
22 |
2.0 |
0.02 |
0.9 |
q.s. to 100 mL |
1.3 Evaluation Of Formulations:
1.3.1 Physical Appearance and Organoleptic Properties
The physical appearance and organoleptic properties of the betulin-loaded thermosensitive gel formulations were evaluated by visual inspection of each batch for color, clarity, and homogeneity against a white and black background under natural daylight conditions to detect any phase separation, precipitation, or particulate matter. The odor of each formulation was assessed by trained personnel through direct olfactory evaluation to determine any characteristic smell or off-odors that might affect patient acceptability. The texture and feel of the gel were evaluated by gently handling the formulation to assess its consistency, smoothness, and tactile properties at room temperature, and the ease of administration through nasal route was subjectively assessed by observing the flow characteristics and spreadability of the formulation on a glass surface [74].
1.3.2 Gel Strength
The gel strength of betulin-loaded thermosensitive gel formulations was determined using a texture analyzer equipped with a cylindrical probe by first converting the formulation to gel state by heating to gelation temperature and allowing it to equilibrate in a 50 mL beaker at 37°C for 30 minutes. The gel strength was measured by penetrating the gel surface with a cylindrical probe of 1 cm diameter at a constant speed of 1 mm/sec to a depth of 4 mm, and the maximum force required for penetration was recorded in grams. The measurement was performed in triplicate for each formulation batch at different locations on the gel surface to ensure uniform assessment, and the average gel strength values were calculated and expressed as the force required for gel penetration, with higher values indicating stronger gel formation and better mechanical stability of the thermosensitive gel system [75].
Fig 3.1 Predicted Vs Actual plot showing Scattering of data around the system predicted line in gelation temperature
1.3.3 Mucoadhesive Strength
The mucoadhesive strength of betulin-loaded thermosensitive gel formulations was determined using a texture analyzer equipped with mucoadhesive attachment by mounting fresh sheep nasal mucosa on the lower probe and applying a known amount of gel formulation (0.5 mL) uniformly on the mucosal surface. The upper cylindrical probe was brought into contact with the gel-coated mucosa under a controlled preload force of 0.1 N for 60 seconds to establish proper contact and adhesion, followed by withdrawal of the upper probe at a constant speed of 1 mm/sec until complete detachment occurred. The maximum detachment force required to separate the gel from the mucosal surface was recorded as the mucoadhesive strength and expressed in Newtons (N), with measurements performed in triplicate using fresh mucosal tissue for each test to ensure accuracy and reproducibility. The mucoadhesive strength was evaluated at 37°C to simulate physiological nasal conditions, and higher values indicated better mucoadhesive properties essential for prolonged nasal residence time [76].
1.4 Results and Discussions:
1.4.1 Results of Thermosensitive Gel
All nine formulated thermosensitive gel batches (F1-F9) exhibited uniform physical appearance and organoleptic properties, demonstrating successful formulation development. Each batch displayed a clear gel with slight yellowish tinge attributed to betulin, maintaining transparency suitable for topical or ocular applications. The formulations were odorless, eliminating concerns regarding patient acceptability and compliance. All batches demonstrated smooth, homogeneous texture with viscous consistency, indicating proper polymer dispersion and absence of phase separation or aggregation. The uniformity across all formulations confirms the reproducibility of the manufacturing process and validates the compatibility of varying concentrations of Poloxamer 407 and Butea monosperma gum in the gel matrix.
|
Batch Code |
pH |
Gelation Temperature (°C) |
Gel Strength (g) |
Viscosity at 25°C (cP) |
Viscosity at 35°C (cP) |
|
F1 |
6.3 ± 0.12 |
35.8 ± 0.45 |
28.4 ± 1.52 |
165 ± 8.4 |
4,850 ± 185 |
|
F2 |
6.2 ± 0.15 |
31.5 ± 0.38 |
35.8 ± 1.68 |
245 ± 11.2 |
6,920 ± 238 |
|
F3 |
6.1 ± 0.15 |
33.2 ± 0.42 |
42.6 ± 1.85 |
325 ± 14.5 |
9,250 ± 285 |
|
F4 |
6.3 ± 0.13 |
32.8 ± 0.41 |
38.5 ± 1.72 |
285 ± 12.8 |
7,950 ± 258 |
|
F5 |
6.2 ± 0.11 |
29.2 ± 0.35 |
58.8 ± 2.15 |
485 ± 17.2 |
13,840 ± 385 |
|
F6 |
6.4 ± 0.14 |
30.8 ± 0.39 |
52.4 ± 1.98 |
420 ± 15.6 |
11,780 ± 342 |
|
F7 |
6.1 ± 0.16 |
30.5 ± 0.43 |
45.6 ± 1.88 |
365 ± 14.8 |
10,250 ± 308 |
|
F8 |
6.3 ± 0.12 |
27.7 ± 0.37 |
56.2 ± 2.05 |
465 ± 16.8 |
12,880 ± 368 |
|
F9 |
6.2 ± 0.13 |
29.5 ± 0.33 |
64.8 ± 2.28 |
545 ± 18.4 |
15,420 ± 412 |
Fig 4.1: Physicochemical Characterization of Formulated Thermosensitive Gel Batches
All values are expressed as mean ± SD
All formulations demonstrated excellent drug content uniformity ranging from 93.8% to 97.8%, well within the acceptable pharmacopeial limits of 90-110%, confirming uniform drug distribution and reproducible manufacturing process. The narrow standard deviations indicate consistent dose uniformity across batches, essential for therapeutic efficacy and regulatory compliance. Mucoadhesive strength progressively increased from 2,680 dyne/cm² (F1) to 6,450 dyne/cm² (F9) with increasing Butea monosperma gum concentration, attributed to enhanced hydrogen bonding between gum's hydroxyl groups and mucosal surface. Higher mucoadhesive strength ensures prolonged residence time at the application site, facilitating sustained drug release and improved bioavailability. F5, F8, and F9 exhibited superior mucoadhesion (>5,000 dyne/cm²), optimal for extended therapeutic action.
Fig 4.2: Contour plots showing effect of concentrations of poloxamer 407 and Butea Monosperma Gum on cumulative drug release at 12 hr Optimization
1.4.2 ANOVA for quadratic model for gelation temperature
The quadratic model demonstrated excellent fit for gelation temperature with high significance (p=0.0008, F=160.03) and superior adjusted R² (0.9900) and predicted R² (0.9546), indicating reliable predictive capability. Both independent variables significantly influenced gelation temperature: Poloxamer 407 (A) showed the most pronounced effect (p=0.0002, F=460.80), while Butea monosperma gum (B) exhibited moderate influence (p=0.0026). The significant quadratic term B² (p=0.0006) and interaction term AB (p=0.0462) confirmed non-linear relationships and synergistic effects. Contour and 3D response surface plots revealed that increasing Poloxamer 407 concentration decreased gelation temperature, while higher gum concentrations exhibited curvature effects. The minimal residuals and close predicted-versus-actual correlation validate the model's accuracy for optimizing formulation parameters.
Table 4.3: Model fit summary for Gelation Time
|
Source |
Sequential p-value |
Lack of Fit p-value |
Adjusted R² |
Predicted R² |
|
|
Linear |
0.0317 |
0.5780 |
0.3124 |
||
|
2FI |
0.6574 |
0.5152 |
-0.3165 |
||
|
Quadratic |
0.0014 |
0.9900 |
0.9546 |
Suggested |
|
|
Cubic |
0.0791 |
0.9998 |
0.9957 |
Aliased |
Regression equation obtained for gelation time is as follows:
Gelation Time = 29.1778 + -2.13333 * A + -0.933333 * B + 0.4 * AB + 0.433333 * A^2 + 2.63333 * B^2
Table 4.4: ANOVA summary for quadratic model for gelation time
|
Source |
Sum of Squares |
df |
Mean Square |
F-value |
p-value |
|
|
Model |
47.42 |
5 |
9.48 |
160.03 |
0.0008 |
significant |
|
A-Poloxamer 407 |
27.31 |
1 |
27.31 |
460.80 |
0.0002 |
|
|
B-Butea monosperma gum |
5.23 |
1 |
5.23 |
88.20 |
0.0026 |
|
|
AB |
0.6400 |
1 |
0.6400 |
10.80 |
0.0462 |
|
|
A² |
0.3756 |
1 |
0.3756 |
6.34 |
0.0864 |
|
|
B² |
13.87 |
1 |
13.87 |
234.04 |
0.0006 |
|
|
Residual |
0.1778 |
3 |
0.0593 |
|||
|
Cor Total |
47.60 |
8 |
Fig 4.5: Contour plot showing effect of concentrations of poloxamer 407 and Butea monosperma gum on gelation temperature
CONCLUSION:
The present study successfully developed and characterized a betulin-loaded thermosensitive in situ gel formulation utilizing Poloxamer 407 and Butea monosperma gum as primary polymeric components to address the critical challenge of betulin's poor aqueous solubility and limited bioavailability. Comprehensive preformulation studies confirmed betulin's identity through organoleptic evaluation, melting point determination (258.4°C), and UV spectrophotometry revealing absorption maximum at 210 nm. The calibration curve demonstrated excellent linearity with correlation coefficient of 0.9997, establishing a reliable analytical method for quantification. Solubility studies revealed betulin's practically insoluble nature in aqueous media (0.048-0.052 mg/mL), providing strong rationale for developing an advanced delivery system. Differential scanning calorimetry and FTIR spectroscopy confirmed complete drug-excipient compatibility, with the physical mixture retaining all characteristic peaks without significant shifts or new peak formation, indicating absence of chemical interactions. Nine formulations (F1-F9) were developed using 3² factorial design with varying concentrations of Poloxamer 407 (18-22% w/v) and Butea monosperma gum (1.0-2.0% w/v). All formulations exhibited uniform physical appearance with clear, slightly yellowish transparent gel, smooth texture, and physiologically acceptable pH (6.1-6.4). Physicochemical characterization revealed gelation temperatures ranging from 27.7 to 35.8°C, with formulations F5-F9 demonstrating optimal values near physiological temperature. Gel strength increased progressively from 28.4g to 64.8g with higher polymer concentrations, while viscosity exhibited characteristic thermosensitive behavior with dramatic increases from 25°C to 35°C.
REFERENCES
Nikhil Birgale*, J. G. Wagh, Development and Optimization of Betulin loaded thermosensitive In-situ Nasal gel to treat Parkinson's disease, Int. J. of Pharm. Sci., 2026, Vol 4, Issue 5, 7768-7781. https://doi.org/10.5281/zenodo.20438551
10.5281/zenodo.20438551