Development, optimization, and invitro evaluation of novel fast dissolving oral films (FDOF's) of Uncaria tomentosa extract to treat osteoarthritis

Abstract

Fast dissolving oral films(FDOF's) are the popular dosage forms when placed in the oral cavity, disintegrate rapidly and dissolve to release the medication for improving oral absorption. Traditionally Uncaria tomentosa plant is used in the treatment of rheumatoid arthritis as Non-steroidal Anti-inflammatory agent along with analgesic properties. The plant extract is reported to be used in low dose as analgesic and anti-inflammatory agent in Rheumatoid Arthritis. No scientific data was reported on Uncaria plant formulation as FDOF's for treating osteoarthritis. This study aims to formulate fast oral dissolving film(FDOFs) Uncaria tomentosa bark extract and evaluate using Invitro studies for osteoarthritis. About 14 formulations were prepared using standard Box Behnken design protocol for optimization using the natural film formers, synthetic polymers, super disintegrants, plasticizers as independent variables with folding endurance and disintegration time as dependent variables by solvent casting method. Further the formulation characteristics including physical and mechanical behavior of films and drug release behavior was evaluated. The 3D countour plots and response curves suing the design expert software were investigated which proved that the optimized oral dissolving films of extract with Pullalan gum, HPMC (polymers), Propylene glycol, PEG 400 (Co-solvents) and Croscarmellose sodium (super Disintegrant) are stable and uniform with formulation characteristics. The drug release rates prove that F5 and F13 formulations showed 99.90% drug release within 30 min following first order kinetics with satisfactory mechanical properties. The study results suggest that the fast dissolving oral films of Uncaria tomentosa extract is novel, attractive and alternative to the available marketed products resulting in improved patient adherence in treatment of osteoarthritis.

1. Introduction

Fast dissolving oral films is new popular strategy of drug delivery with improved patient compliance [1]. The novel adapt of this drug delivery system has answered the problems of solubility, bioavailability, improved biological half life and therapeutic efficacy [2]. The oral disintegrating tablets are quite popular with fast disintegration and without interference but have limitation of disintegrating remnants until swallowed [3]. Thus the fast dissolving oral films gaining popularity and lot of drugs which suffer oral instability are being transformed [4]. They are unique in line commercial products with differentiation and meeting demands of pharmaceutical industry [5]. They work by dissolving or disintegrating in the mouth within seconds without the aid of water ensuring better patient compliance [6]. Dysphagia being the most common problem of oral administration in all age groups could be addressed easily with fast oral dissolving films(FODFs). The advantages include fast preparation, dosage accuracy, safe, efficacious which are convenient and portable without requiring the use of water or a spoon [6,7]. The polymers used are hydrophilic encapsulating hydrophilic or hydrophobic drug that enhances the feasibility to mask taste, prevent chocking, enhance stability and disintegration with local action [[8], [9], [10]].

The Uncaria tomentosa(Cats Claw, family- Rubiaceae), is enriched with phytoconstituents like indole alkaloids, glycosides, anthocyanidins, sterols, phenols and anthocyanidins [11,12]. The plant is pharmacologically active with antipyretic, antiviral, anti-inflammatory, immune booster and wound healing agent [13]. Thus the literature supports the use of Uncaria tomentosa as raw material dispensed in Public Hospitals of the Social Health Insurance as Complementary Medicine Service (CMS) in Peru. The root and bark extracts are widely used in conditions of allergies, inflammation, rheumatic infection and cancer [14].

2. Material and methods

2.1. Materials

The Uncaria tomentosa powder(Trimmed bark) was obtained from Kshipra Biotech Private Limited, certified distributor, Madhya Pradesh, India (batches: KB/021554). Methanol (HPLC grade) was used obtained from Sigma Aldrich Chemicals. Pullalan gum (Merck chemicals), Maltodextrin(sigma aldrich), HPMC(Sigma aldrich), PEG 400, PVA and Propylene glycol (Sigma Aldrich), Distilled water(lab scale). Further crosspovidone, Citric acid, Pregelatinized starch, cross carmellose sodium were available in the lab.

2.2. Extraction of Uncaria tomentosa powder

The trimmed bark powder [[15], [16], [17]]of coarse size #24 mesh was initially defatted twice using petroleum ether and then subjected to Soxhletion with methanol. The concentrated extract was dried, further stored in desiccators for further use. The Qualitative screening was performed as per standard protocol.

Standard Calibration Method using UV spectrophotometry: Accurately weighed amount of Uncaria tomentosa(10 mg) extract was dissolved in a few ml of water and then adjusted to10 ml with water and named as Stock I. Aliquots of 0.2, 0.4, 0.6, 0.8 and 1.0 ml were pipetted out into a series of 10 ml volumetric flasks and adjusted with water to make final concentration to range of 20–100 μg/ml. The samples [18,19] were analyzed by UV spectrophotometer (Shimadzu 3000). The process was carried out in triplicate and calibration curve was plotted for concentration vs absorbance.

Preparation of Uncaria tomentosa extract FODFs:

Experimental Design: 3³factor design was used for optimization of fast dissolving film formulations. In this design, 3 factors were evaluated such as the type of polymer X₁,(Pullulan gum, Maltodextrin, HPMC 5 cps, and PVA) type of plasticiser X₂, (Propylene glycol and PEG 400) and type of superdisintegrants X₃, (Cross Povidone, Croscarmellose sodium and Pregelatinized starch) were selected as independent variables at one level over the dependent variables like folding endurance, disintegration time (sec), and in-vitro drug release (%) as shown in design layout Table A total 14 formulations [[20], [21], [22], [23]] were devised based on the above preparative protocol. The composition of various oral dissolving films is reported in design layout Table 1.

Table: 1.

Composition of FODFs with extract and polymers.

S. No	Formulation entry	Category	F1	F2	F3	F4	F5	F6	F7	F8	F9	F10	F11	F12	F13	F14
1	Cats claw extract	API	100	100	100	100	100	100	100	100	100	100	100	100	100	100
2	Pullulan gum	Natural thickening agent	100	–	–	–	100	–	–	–	100	–	–	–	50	–
3	Maltodextrin	Thickening agent	–	100	–	–	–	100	–	–	–	100	–	–	–	50
4	HPMC	Polymer	–	–	100	–	–	–	100	–	–	–	100	–	50	–
5	PVA	Polymer	–	–	–	100	–	–	–	100	–	–	–	100	–	50
6	Propylene glycol	Penetration enhancer	10	10	10	10	–	–	–	–	10	–	10	–	10	10
7	PEG 400	Penetration enhancer	–	–	–	–	10	10	10	10	–	10	–	10	–	–
8	Cross Povidone	Disintegrant	10	10	10	10	–	–	–	–	–	–	–	–	–	–
9	Croscarmellose sodium	Disintegrant	–	–	–	–	10	10	10	10	–	–	–	–	10	10
10	Pregelatinized starch	Disintegrant	–	–	–	–	–	–	–	–	10	10	10	10	–	–
11	Citric acid	Preservative	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5
12	Polysorbate 80	Surfactant, emulsifier	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5
13	Bronopol	Antimicrobial	3.5	3.5	3.5	3.5	3.5	3.5	3.5	3.5	3.5	3.5	3.5	3.5	3.5	3.5
14	Sucralose	Artificial sweetener	1.5	1.5	1.5	1.5	1.5	1.5	1.5	1.5	1.5	1.5	1.5	1.5	1.5	1.5
15	Distilled water		Q.S	Q.S	Q.S	Q.S	Q.S	Q.S	Q.S	Q.S	Q.S	Q.S	Q.S	Q.S	Q.S	Q.S

Quantities were mentioned in mg/film; solvents in ml.

Preparation of FDOF using Solvent casting method.

The FDOFs of Uncaria tomentosa extract were prepared by solvent casting method using Box Behnken design with formulation codes and their respective compositions are given in Table 1. The extract was mixed with measured amount of water (approx 7.5 ml per unit film) using over headed stirrer for 10 min, filtered through the muslin cloth [24]. To this filtered extract, successively measured amounts of polymer, plasticiser, superdisintegrants, preservative, saliva stimulating agent, sweetening agent, were added with constant stirring for 30 min resulting in thick viscous solution and was degassed using ultrasonicator(Lab india). The requisite quantity of solution was casted on a 30 × 45 cm² glass plate and was kept in hot air oven at about 80 °C for 15 min(Table 1). The film obtained was carefully removed from the glass plate and checked for any imperfections. Then the required size of film was cut to deliver the dose equivalent to 100 mg (2 × 3 cm²) per film. Further stored in airtight plastic containers to enable characterization and accelerated stability studies as per International Conference on Harmonisation (ICH Q1) guidelines [25].

2.3. Characterization of FDOF

2.3.1. Preliminary characteristics

• 1.Film forming capacity: It is the ability of a polymer to form films that can be separated from the surface on which they are casted. The films were characterized as very poor, poor, average, good, better, best depending upon their ability to form films.
• 2.Physical Appearance of films: The appearance of film was evaluated by visual observation. The parameters like surface texture, color, homogeneity and transparency of the films were evaluated.
• 3.Tackiness: Tackiness was evaluated gently by pressing the film between fingertips and results were noted in qualitative terms as tacky or non-tacky.

2.3.1.1. Key formulation characteristics

• 1.Thickness: All the formulations were evaluated for uniformity in thickness by using calibrated digital Vernier calipers. Ten films (pieces) from each formulation were taken randomly from different places of the plate. The thickness was measured and means value was calculated. The uniformity in thickness [26] is directly related to the accuracy of dose distribution in the film.
• 2.Surface pH study: The surface pH of fast dissolving strip was determined in order to investigate the possibility of side effects in vivo. As an acidic or alkaline pH may cause irritation to the oral mucosa, it was determined to keep the surface pH to neutral as close as possible. A combined pH electrode was used for this purpose. The film was allowed to swell by keeping it in contact with 1 ml of distilled water [27] for 3 min at room temperature. The pH was noted down by bringing the electrode in contact with the surface of the film, allowing it to equilibrate for 1 min and the pH was recorded. The experiments were performed in triplicate, and average values were reported [28].
• 3.Percentage Moisture Loss: Percentage moisture loss was calculated to check the integrity of films at the dry condition. The films were cut into 2 × 3 cm² and weighed accurately and kept in desiccators containing fused anhydrous calcium chloride. After 72 h the films were removed and weighed again. The decrease in the weight of the films [29] implicates the amount of moisture loss. The percentage loss in moisture(Equation (1)) was calculated by using the following formula:

$$\%\mathrm{M}\mathrm{o}\mathrm{i}\mathrm{s}\mathrm{t}\mathrm{u}\mathrm{r}\mathrm{e}\mathrm{l}\mathrm{o}\mathrm{s}\mathrm{s}=\frac{\left(\mathrm{I}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l}\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}-\mathrm{f}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{l}\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\right)\times 100}{\left(\mathrm{I}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l}\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\right)}$$ (1)

• 4. Percentage Moisture Absorption: The moisture uptake was determined by cutting films into 2 × 3 cm² patches. These films were put for one day in a desiccator containing a saturated solution of potassium sulphate (relative humidity 75%) at room temperature. The increase in the weight of the films was observed which was due to absorption of moisture. The moisture gain capacity(Equation (2)) by the films was calculated using the following formula:

$$\%\mathrm{M}\mathrm{o}\mathrm{i}\mathrm{s}\mathrm{t}\mathrm{u}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{p}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}=\frac{\left(\mathrm{I}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l}\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}-\mathrm{f}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{l}\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\right)\times 100}{\left(\mathrm{I}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l}\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\right)}$$ (2)

• 5.Morphological properties: The surface morphology of the optimized orally dissolving Uncaria tomentosa films were observed with scanning electron microscope (Hitachi S–3400 N type II model, Japan). Pictures were taken at an excitation voltage of 1.0 KV and a magnification of 1000x. All the formulations were stored at room temperature 25 ± 2 °C in air-tight containers [30].
• 6.Weight Variation: This test was carried out by taking 2 × 3 cm² of the film cut at three different places [31] from the casted film. The weight of each film was taken individually using electronic balance. An average of three readings was taken for weight variation study.
• 7.In-vitro disintegration of Films: In-vitro disintegration time of 2 × 3 cm² films was determined visually in a petri dish [32] containing 25 ml of phosphate buffer pH 6.8 at 37.0 ± 0.5 °C. The time when the film started to break or disintegrate was recorded, which is the disintegration time of the film [33]. The average disintegration time of six films from each formulation was noted.
• 8.Folding endurance: The folding endurance [34] which is related to the flexibility of a film was measured manually by firmly holding and folding the films repeatedly through the middle. The number of folds on the same crease, required to produce crack in the film was noted as the value of folding endurance.
• 9.Swelling Index: A pre-weighed drug loaded film was placed on a 2% agar plate. An increase in the weight of the film was noted until the constant weight [35] was obtained. The swelling index(Equation (3)) is calculated using the following formula.

$$\mathrm{S}\mathrm{w}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{d}\mathrm{e}\mathrm{x}=\frac{W_{\mathrm{t}}-W_{\mathrm{o}}}{\mathrm{W}\mathrm{o}}$$ (3)

where, W_t = weight of the film at time “t”; W_o = weight of film at t = 0s.

• 10. In vitro release studies: The release rate of the Uncaria tomentosa extract from the fast dissolving films F5, and F13 was determined by the help of USP Dissolution Test Apparatus-II. The release study was performed in 900 ml phosphate buffer pH 6.8, at 37 ± 5 °C with 50 rpm speed. An aliquot of the solution was collected and replaced with fresh medium at every 30 s to maintain the sink conditions. The aliquot was filtered through the Whatman's filter paper. The absorbance of the filtered solution was measured at 242 nm with constant withdrawing of samples from the vessel. The cumulative amount of drug release was calculated by using the calibration data. The process was carried out in triplicate [[29], [30], [31], [32], [33], [34]].
• 11.FT-IR interaction study: The plausible mechanisms between U. tomentosa extract and FDOFs, were predicted using the Fourier Transform Infrared (FTIR) spectrometer (Perkin Elmer, Frontier, USA) using the potassium bromide (KBr) pellet technique. The sample was scanned from the 4000–400 cm⁻¹ wave number [34].
• 12.Stability studies: For stability testing the FDOFs F5 and F13 were stored under controlled conditions of 40 °C ± 2 °C/75 ± 5% RH, over a period of 3 months according to the ICH guidelines. During storage the F5 and F13 were checked for their physical appearance, tackiness, surface pH, disintegration time, and dissolution profiles [[25], [26], [27], [28], [29], [30], [31], [32]].

13. Microbiological limit test: The most stable formulations F5 and F13 was subjected to microbiological examination as the herbal products are most susceptible for microbial contamination. This test was designed to determine total aerobic microbial, yeast and mould count for FDOFs of U. tomentosa microbiological limit test was performed for the development of safe and effective consumer product. The microbiological limit test was performed according to Indian pharmacopoeia specification [[24], [25], [26], [27]].

3. Content uniformity assay

For content uniformity test, the films of size 2 × 3 cm² were cut from different positions of casted film. Each film of 6 cm² was placed in a volumetric flask containing 60 ml of distilled water and was sonicated for 30 min using an ultrasonicator. The volume was made up to 100 ml and the absorbance of the solution was measured by UV spectrophotometer at 242 nm after appropriate dilutions [27,28].

4. In-vitro osteoarthritis evaluation

Culture and maintenance of C20A4 chondrocytes: Human Chondrocyte cell lines C20A4 were obtained from the National Centre for Cell Sciences(NCCS), Pune India. The Chondrocytes were cultured in RPMI-1640 (10% fetal bovine serum (FBS), 10 U antibiotic/ml) (sigma Aldrich) medium at 37 °C under 5% CO₂ in an incubator (5215, Shel Lab, USA). The growth medium was changed every third day and the chondrocytes were passaged with trypsin-EDTA solution (0.05%) in a 1:3 ratio. First passage chondrocytes were used for cell culture studies.

Establishment of three-dimensional in-vitro osteoarthritis model: For in-vitro experiments, a three dimensional (3D) OA agarose model was developed using C20A4chondrocytes of OA. Briefly, C20A4 chondrocytes were embedded in agarose (2% low-melting agarose-gelling temperature 25± 5 °C) (Sigma, USA) prepared in phosphate buffer solution. Then, equal volumes of chondrocyte suspension in double strength RPMI-1640 (20% FBS) was mixed with agarose to obtain a final chondrocyte concentration of 10⁶ cells per mL in each well of a 24-well plate. About 1 mL of RPMI-1640 (10% FBS) was then added into each well. Medium was refreshed every 3 days. On the third day of in-vitro cultivation, 20 ng/mL interleukin-1β (IL-1β) (Sigma-Aldrich, USA) was added into the medium. The same amount of IL-1β was added during each medium change [[36], [37], [38], [39], [40], [41]].

Cell viability assays: The cell viability of C20A4 chondrocytes, as well as IL-1β, induced OA model of C20A4 was assessed in contact with optimized formulations F5, and F13 at different concentrations ranging from 0 to 500 μg/mL (0, 10, 50, 100, 200, 350, and 500 μg/mL. The C20A4, and C20A4/IL-1β cells were plated at a density of 20,000 cells/well in 96-well plates. C20A4/IL-1β cell were used after inducing OA for a period of 5 days of IL-1β treatment. After 24 h of culture incubation, F5 and F13 were suspended in PBS and incubated or 24 h at 37 °C. Approximately 5 μL of 0.5% 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution was added to each well, and the plates were left for 3 h. All wells were incubated for 45 min with 1 μL of DMSO, and the absorbance was measured at 570 nm (8 points per well) on a Sunrise Tecan microplate reader.

4.1. Statistical analysis

ANOVA (one-way analysis of the variance) and student t-test were used to perform the statistical analysis. When p < 0.05, then there assumed to be a statistical differences.

5. Results and discussion

Extractive yield, Physical nature and Qualitative screening: The extract was obtained as pale yellow powder with 1.68% yield. Further the qualitative screening revealed the methanolic extract is rich with alkaloids, flavanoids, terpenoids, saponins, tannins, steroids, glycosides and phenols which are responsible for pharmacological activity (see Fig. 1).

Fig. 1.

(a) Uncaria tomentosa bark powder; (b) Methanolic extract; (c) Freez dried concentrate of methanolic extract of U. tomentosa after defatting.

5.1. Formulation and optimization of the uncaria tomentosa extract FDOFS

Visual characterization, thickness, pH, percent moisture loss and gain: The visual characterization of the films appeared to be uniformly thick, good, non tacky(Fig. 2, Table 2). The films were found to glass plate, clear, transparent, soft without air bubbles, easily removable from glass plate and possess uniform texture along all edges. Further the thickness was found to be within the limits ensuring uniform dose distribution. The pH was found to be neutral ensuring no irritation as shown in Table 2. The moisture loss capacity was found to be in the range of 5.3 ± 0.47 to 26.1 ± 1.89 percentages which was found to increase with increase in polymer concentration. The lower moisture aids in stability and become a completely dried and brittle film. The moisture gain percent was found to be in the range of 6.2 ± 0.54 to 17.2 ± 0.99. The low moisture gain ensures the less microbial contamination and bulkiness.

Fig. 2.

FDOF's of Uncaria tomentosa extract A) Blank FDOF,s B) Optimized formulation F5 C) Optimized formulation F13.

Table 2.

Physical formulation characteristics of Uncaria tomentosa FODFs.

F. code	Film forming capacity	Appearance of films	Tackiness	thickness (mm)	Surface pH	% moisture loss	% moisture gain
F1	Good	Transparent	Non-tacky	0.06 ± 0.013	6.71 ± 0.35	15.6 ± 1.31	12.9 ± 157
F2	Very good	Transparent	Non-tacky	0.07 ± 0.021	6.52 ± 0.23	9.3 ± 1.12	10.7 ± 0.83
F3	Very good	Transparent	Non-tacky	0.08 ± 0.025	6.35 ± 0.18	13.9 ± 2.44	17.2 ± 0.99
F4	Good	Transparent	Non-tacky	0.09 ± 0.019	7.01 ± 0.21	11.2 ± 2.86	11.5 ± 0.83
F5	Very good	Transparent	Non-tacky	0.06 ± 0.023	7.03 ± 0.31	6.5 ± 0.75	6.2 ± 0.54
F6	Very good	Transparent	Slightly tacky	0.08 ± 0.016	6.84 ± 0.17	19.3 ± 1.56	12.5 ± 1.13
F7	Good	Transparent	Non-tacky	0.09 ± 0.019	7.69 ± 0.42	14.9 ± 1.22	11.9 ± 1.05
F8	Good	Transparent	Non-tacky	0.08 ± 0.021	6.29 ± 0.25	9.7 ± 0.54	7.3 ± 0.85
F9	Good	Transparent	Non-tacky	0.09 ± 0.025	7.62 ± 0.29	13.9 ± 1.29	15.2 ± 1.36
F10	Good	Transparent	Non-tacky	0.08 ± 0.016	7.42 ± 0.24	17.5 ± 158	13.7 ± 1.59
F11	Very good	Transparent	Slightly tacky	0.07 ± 0.023	7.48 ± 0.39	18.4 ± 1.66	12.4 ± 1.09
F12	Good	Transparent	Non-tacky	0.09 ± 0.026	6.85 ± 0.32	15.4 ± 1.27	13.8 ± 0.95
F13	Very good	Transparent	Non-tacky	0.07 ± 0.018	7.19 ± 0.39	5.3 ± 0.47	6.9 ± 0.58
F14	Average	Transparent	Slightly tacky	0.09 ± 0.024	7.75 ± 0.21	26.1 ± 1.89	17.2 ± 1.32

The data is represented as Mean ± S.D (n = 3).

5.1.1Films Physical form

.

5.2. Optimization of FDOFs of Uncaria tomentosa based on 3³ factorial designs

The model was fit to the data with a three-level, three-factor Box-Behnken experimental design requires 14 runs. The independent variables (14 runs) and the responses observed are tabulated in Table 3. The ranges of Y1 and Y2 for all batches were 165.5 ± 12.4–240.5 ± 18..3% and 34.1 ± 2.31–75.3 ± 4.51 sec-respectively. The responses observed for the 14 formulations were fitted randomly to first-order, second-order and quadratic models using Design Expert software(Design Expert 8.05 b). Further the comparative values of R2, standard deviation (SD), observed responses for Box–Behnken design for Y1 as folding endurance, in %; and Y2 as disintegration time within 30 min are tabulated in Table 3. The Best Fit model was found to be quadratic and regression equation of each response increased as per the factor. It is evident that all three independent variables, namely the concentration of polymer (X1), the amount of plasticiser (X2) (Fig. 4) and superdisintegrants (X3), have interactive effects on the two responses(Y1 and Y2).

Table 3.

3³ Factorial design Uncaria tomentosa FDOFs. With dependent and independent variables.

F. Code	Independent variable			Dependant variable
	X1 (Polymer)	X2 (Plasticiser)	X3 (Superdisintigrant)	Y1 (Folding endurance)	Y2 (Disintegration time in sec)
F1	Pullulan gum	Propylene glycol	Cross Povidone	170.5 ± 11.6	41.2 ± 1.26
F2	Maltodextrin	Propylene glycol	Cross Povidone	205.6 ± 13.6	52.3 ± 2.31
F3	HPMC	Propylene glycol	Cross Povidone	190.4 ± 15.5	48.1 ± 2.54
F4	PVA	Propylene glycol	Cross Povidone	165.5 ± 12.4	56.5 ± 2.95
F5	Pullulan gum	PEG 400	Croscarmellose sodium	215.2 ± 13.9	35.9 ± 0.58
F6	Maltodextrin	PEG 400	Croscarmellose sodium	210.3 ± 11.2	59.7 ± 3.65
F7	HPMC	PEG 400	Croscarmellose sodium	220.5 ± 13.4	47.8 ± 2.28
F8	PVA	PEG 400	Croscarmellose sodium	200.7 ± 15.1	63.8 ± 3.84
F9	Pullulan gum	Propylene glycol	Pregelatinized starch	185.6 ± 14.3	66.1 ± 3.24
F10	Maltodextrin	PEG 400	Pregelatinized starch	210.1 ± 14.7	54.1 ± 1.92
F11	HPMC	Propylene glycol	Pregelatinized starch	240.3 ± 11.4	48.3 ± 3.25
F12	PVA	PEG 400	Pregelatinized starch	210.7 ± 15.8	58.1 ± 2.82
F13	Pullulan gum + HPMC (50:50)	Propylene glycol	Croscarmellose sodium	240.5 ± 18.3	34.1 ± 2.31
F14	Maltodextrin + PVA (50:50)	Propylene glycol	Croscarmellose sodium	185.3 ± 14.7	75.3 ± 4.51

The data is represented as Mean ± S.D (n = 3).

Fig. 4.

Contour plot graphs of the effect of factors X1 (Polymer) & X2 (Plasticiser) on Disintegration time (a); factor X1 (Polymer) & X3 (Superdisintegrant) on Disintegration time (b); and factor X2(Plasticiser) & X3 (Superdisintegrant) on Disintegration time (c).

Optimization and validation The formulations were optimized and the best optimized formulations were selected based on the standards i.e., maximum cumulative release with minimum disintegration time with optimum concentration of polymer, plasticiser and superdisintegrants using design expert software. The optimum super Disintegrant values of Pullulan gum concentration (50.0%, w/v), PEG 400, propylene glycol (50.0%, w/v) and CMC concentration (2.5%, w/v) were clear indicated by contour plots and optimal response was observed(Fig. 3, Fig. 4). These effect the disintegration time to be 35.9 ± 0.58 s and 34.1 ± 2.31 s respectively for F5 and F13 formulations which is shortest amongst the 14 formulations. The optimized formulations correlate with the formulation characteristics in accordance with the theoretical predictions as shown in Table 4.

Fig. 3.

Contour plot graphs of the effect of factors X1 (Polymer) & X2 (Plasticiser) on Folding endurance (a); factor X1 (Polymer) & X3 (Superdisintegrant) on Folding endurance (b); and factor X2(Plasticiser) & X3 (Superdisintegrant) on Folding endurance (c).

Table 4.

Statistical analysis of weight variation, Swelling index and extract content uniformity.

Name	Weight variation	Swelling Index	Uniformity of extract content
F1	71.2 ± 3.42	39.6 ± 1.89	87.01 ± 5.48
F2	75.3 ± 4.26	26.3 ± 1.04	79.35 ± 5.41
F3	68.2 ± 5.04	32.6 ± 1.23	93.58 ± 6.35
F4	58.3 ± 4.13	28.1 ± 1.18	94.55 ± 6.18
F5	39.2 ± 2.07	11.3 ± 0.54	99.39 ± 5.78
F6	52.8 ± 2.98	18.6 ± 0.48	91.08 ± 5.12
F7	63.9 ± 3.84	17.6 ± 0.63	85.34 ± 4.69
F8	49.3 ± 2.74	26.7 ± 1.08	71.39 ± 4.75
F9	54.3 ± 2.63	35.6 ± 1.33	75.04 ± 4.58
F10	43.1 ± 2.17	28.3 ± 1.42	63.28 ± 5.12
F11	58.2 ± 3.16	24.6 ± 1.92	69.32 ± 4.08
F12	56.3 ± 3.44	20.4 ± 1.08	85.54 ± 5.89
F13	37.2 ± 2.87	13.7 ± 0.87	99.46 ± 5.36
F14	74.1 ± 4.69	32.6 ± 1.63	90.72 ± 5.17

The data is represented as Mean ± S.D (n = 3).

3D response surface graphs(Fig. 5, Fig. 6) were obtained from the design expert software. All the variables such as polymer concentration, plasticiser and superdisintegrants were reasonably optimal in accordance with the disintegration time and folding endurance. Further effect was minimal at optimized concentrations of the variables pertaining to better performance of the selected variables as shown in Fig. 5, Fig. 6. Thus the optimization of the variable resulted in best fit model for the formulation.

Fig. 5.

3D response surface graphs of the effect of factors X1 (Polymer) & X2 (Plasticiser) on Folding endurance (a); factor X1 (Polymer) & X3 (Superdisintegrant) on Folding endurance (b); and factor X2(Plasticiser) & X3 (Superdisintegrant) on Folding endurance (c).

Fig. 6.

3D response surface graphs of the effect of factors X1 (Polymer) & X2 (Plasticiser) on Disintegration time (a); factor X1 (Polymer) & X3 (Superdisintegrant) on Disintegration time (b); and factor X2(Plasticiser) & X3 (Superdisintegrant) on Disintegration time (c).

UV spectrophotometric analysis- Standard calibration plot.

The UV spectrometric analysis was performed on the Uncaria tomentosa extract to determine λ_max which was obtained by subjecting 100 μg/ml of the sample extract over the range of 350–500 nm which gave λ_max at 242 nm in phosphate buffer (pH 6.8) as shown in Fig. 3B. Further the standard calibration curve gave a linear relationship which was obtained from Beer–Lambert's plot of Uncaria tomentosa, shown in Fig. 7 [A, B]. Further the UV spectrometric analysis analyzed few active components like phenols(199.3 nm), Terpenoids at (221–242.1 nm), Glycosides like (260.5 nm) and alkaloids(291.3 nm) as shown in Fig. 3.

Fig. 7.

A) UV spectrophotometry analysis and B) Calibration curve of a methanolic extract of Uncaria tomentosa.

Weight variation, Disintegration time, Folding endurance and Swelling index.

About 14 formulations were subjected to weight variation test which are depicted below in Table 4. The weight variation was found to be between 37.2 ± 2.87–75.3 ± 4.26 mg. The folding endurance is the ability of the film to rupture under high pressure which was performed for all the formulations as shown in Table 3, Fig. 8 which was found to be 165.5 ± 12.4–240.5 ± 18.3% with PEG 400 and propylene glycol as plasticizers. Further the disntegration time was studied using the phosphate buffer which was found to be 34.1 ± 2.31–75.3 ± 4.51 s. The swelling index was found to be 11.3 ± 0.54–39.6 ± 1.89 with F5 and F13 as optimized formulations with best formulation characteristrtics.

Fig. 8.

Statistical evaluation of A)Weight variation; B) Disintegration time; C) Folding endurance; D) Swelling index of FDOFs (F1 to F14).

Effect of the film-forming agents or the polymer: The Pullulan gum, maltodextrin, HPMC and PVA were used as film forming agent. Amongst the three the Pullulan gum was found to be best film forming polymer with improved film forming capability where the drug was uniformly dispersed and easy peeling was achieved through the petridish. Thus F5 and F13 were optimized. With water insoluble polymers, dispersion of the drug uniformly throughout the film was not achieved.

Effect of plasticiser: The plasticizers maintain the flexibility of the films and PEG 400, Propylene glycol were used as plasticizers for FDOF,s which gave slight bitter taste hence sucralose was used as sweetener to mask the taste.

In-vitro Drug release:

The in vitro drug release was studied and it was found that the films with PEG400 and propylene glycol with pullulan gum and HPMC was observed to be faster in drug release(Fig. 9). The release from the malt dextrin and PVA was compared to be slower than the pullulan gum. The increase in the polymer concentration leads to decreased time in wetting and dissolve in the polymer matrix.

Fig. 9.

Cumulative Percent release of U. tomentosa extract from FDOFs F1–F14; (a) F1 to F7; (b) F8 to F14.

5.2.1. Release kinetics

The release kinetics(Table 5) of the optimized formulations like F5 and F13 were studied, following first order kinetics indicating the water solubility of the drug from the porous matrices based on R² value 0.98 and 0.982 respectively. The Higuchi value is high for both the formulations indicating the non-fickian diffusion. The Korsemeyer peppas model with R² value < 0.89 (for F5 and F13) clearly indicate the immediate release nature of the drug serving the fast dissolving nature(Table 6, Fig. 10, Fig. 11). Further the Hixson model with R² value 0.98 for both the formulations indicating the dissolution of the drug in planar model. Thus the drug release kinetics proves the non-fickian immediate release nature of the drug.

Table 5.

Release kinetics of F5 optimized formulation.

Time (hr)	Cumulative % drug released	% Drug remaining	Square root time	log Cumu. % drug remaining	log time	log Cumu % drug released	% Drug released	Cube Root of % drug Remaining(Wt)	Wo-Wt
0.00	0.00	100	0.000	2.000	0.000	0.000	100	4.642	0.000
0.05	35.1	64.9	0.224	1.812	−1.301	1.545	35.1	4.019	0.623
0.12	58.9	41.1	0.342	1.614	−0.933	1.770	23.8	3.451	1.191
0.20	80.4	19.6	0.447	1.292	−0.699	1.905	21.5	2.696	1.946
0.28	94.6	5.4	0.532	0.732	−0.548	1.976	14.2	1.754	2.888
0.37	98.6	1.4	0.606	0.146	−0.436	1.994	4	1.119	3.523
0.45	99.6	0.4	0.671	−0.398	−0.347	1.998	1	0.737	3.905
0.50	99.8	0.2	0.707	−0.699	−0.301	1.999	0.2	0.585	4.057

The data is represented as Mean ± S.D (n = 3).

Table 6.

Release kinetics of F13 optimized formulation.

Time (hr)	Cumulative % drug released	% Drug remaining	Square root time	Log Cumu % drug remaining	Log time	Log Cumu % drug released	% Drug released	Cube Root of % drug Remaining(Wt)	Wo-Wt
0	0	100	0.000	2.000	0.000	0.000	100	4.642	0.000
0.05	42.7	57.3	0.224	1.758	−1.301	1.630	42.7	3.855	0.787
0.12	69.8	30.2	0.342	1.480	−0.933	1.844	27.1	3.114	1.528
0.20	86.3	13.7	0.447	1.137	−0.699	1.936	16.5	2.393	2.249
0.28	95.4	4.6	0.532	0.663	−0.548	1.980	9.1	1.663	2.979
0.37	99.2	0.8	0.606	−0.097	−0.436	1.997	3.8	0.928	3.714
0.45	99.7	0.3	0.671	−0.523	−0.347	1.999	0.5	0.669	3.973
0.50	99.9	0.1	0.707	−1.000	−0.301	2.000	0.2	0.464	4.178

The data is represented as Mean ± S.D (n = 3).

Fig. 10.

Release kinetic profile of F 5 optimized formulation.

Fig. 11.

Release kinetic profile of Optimized formulation F13.

5.2.2. Stability

A stability indicating study was performed on the optimized formulations for three months at 40 °C ± 2 °C/75 ± 5% RH as per ICH Q1A (R2) where the films were kept under observation for the parameters like physical changes, drug content and percent drug release. The Uncaria tomentosa extract FDOF's were stable in both physical and chemical states and also shown a no significant change in the drug content, represented in Table 7 and Fig. 12.

Table 7.

Stability indicating studies of optimized formulations F5 and F13.

Formulation	Physical appearance	Tackiness	Disintegration time (s)	Surface pH
F5	Transparent	Non-tacky	36.2 ± 0.54	7.09 ± 0.23
F13	Transparent	Non-tacky	35.2 ± 3.52	7.15 ± 0.34

Fig. 12.

Dissolution profiles of F5 and F13 at storage conditions of 40 °C ± 2 °C/75 ± 5% RH.

The data is represented as Mean ± S.D (n = 3)

5.2.3. FT-IR interactional study

The FTIR studies could reveal the important components of the methanolic extract i.e., phenols, terpenoids, saponins, flavonoids, tannins, steroids, glycosides and alkaloids. Further the Optimized formulations F5 and F13 were analyzed which showed no prominent interactions indicating the stability of the formulations as shown in Fig. 13.

Fig. 13.

FTIR spectra of (a) U. tomentosa extract and (b) Formulation F5 (c) F13.

5.2.3.1. Microbial limit tests

The microbial limit tests have been performed for the optimized formulations F5 and F13 which tested the inhibition capacity of the films against the microbes and the respective zone of inhibition was measured. The results suggest the microbial growth is within the limits as shown in Table 8.

Table 8.

Microbial limit test for F5 and F13.

Formulation code	Test	Limits	Result
F5	Total microbial plate count	Not more than 105 cfu/g of sample	203 cfu/g
	Total yeast and mould	Not more than 105 cfu/g of sample	125 cfu/g
F13	Total microbial plate count	Not more than 105 cfu/g of sample	197 cfu/g
	Total yeast and mould	Not more than 105 cfu/g of sample	113 cfu/g

Content Uniformity: The content uniformity was evaluated using UV spectrophotometric analysis whose results could suggest that the drug loading capacity is high in F5 and F!3 formulations. Thus they can be concluded as optimized formulations as shown in Fig. 14.

Fig. 14.

Content Uniformity o f U. Tomentosa FDOFs.

6. In-vitro osteoarthritis evaluation

The in-vitro osteoarthritic model was evaluated using five different methods with study on human chondrocyte cell line C20A4 culture. The study results suggest that the regeneration capacity of the FDOF as depicted in Fig. 15.

Fig. 15.

Culture of Human chondrocyte cell lines C20A4 in RPMI media without IL-1β treatment (a); With IL-1β treatment (b).

Cell viability assays.

The potency of the optimized formulations F5 and F13 showing increased cell viability in chrondrocytes within the cellular environment was observed. The range of percentage cell viability increased gradually from 10 to 500 μg/ml concentration reciprocating the cell viability from 37.48 to 93.86% for F5 and 32.71–82.34% for F13 optimized formulations respectively, thereby indicating the in-vitro osteoarthritis potential of the F5 an F13 formulations. Depicted in Table 9, Fig. 15, Fig. 16[A-C]. This proves that the formulations have increased activity against IL-1 β.

Table 9.

Percent cell viability of IL-1β of F5 and F13 formulations.

F5 in μg/ml	% Viability		F13in μg/ml	% Viability
	C20A4	C20A4/IL-1β		C20A4	C20A4/IL-1β
0	100	37.48	0	100	32.71
10	99.98	41.26	10	99.99	35.72
50	99.92	52.99	50	99.91	39.87
100	99.84	64.28	100	99.83	47.08
200	99.73	79.58	200	99.79	58.06
350	99.62	86.11	350	99.65	66.29
500	99.54	93.86	500	99.52	82.34
ED50		43.55	ED50		61.24

Data is represented as Mean ± S. D.

Fig. 16.

Results of cell Viability assays for optimized formulations F5 and F13.

7. Conclusion

The popularity of the fast dissolving oral films increased dissolution in short time is attractive proposition for a condition like osteoarthritis. Hence the traditional known herbal remedy i.e Uncaria tomentosa extract was chosen as API to be formulated as oral fast dissolving film. The results suggest a positive approach of the fast-dissolving films of Uncaria tomentosa as alternative to conventional tablets for clinical use in the treatment of osteoarthritis with short onset of action and minimized side effects. The solvent casting method is simple procedure with minimum ingredients resulting in cost effectiveness. The drug release also proves the advantage of short onset of action within 30 min to reach the plasma levels. The invitro study on osteoarthritis model proves the potential of FDOFs of Uncaria tomentosa extract as drug. A definite invitro-invivo correlation has to further established for dosage and pharmacological effect for treating osteoarthritis to reach the competitive market.

Declarations

Author contribution statement

Naga Sowjanya Juluru: Designed and performed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.Raja Rao: Contributed reagents, materials, analysis tools or data.

Funding statement

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability statement

Data will be made available on request.

Declaration of interest's statement

The authors declare no conflict of interest.