All the chemicals, equipments and materials used for the experiment were of laboratory analytical grade with high purity. Equipments were sterilized prior to use. The statistical evaluation was done using ANNOVA[15].
Formulation of FDOF’s by Qbd design and evaluation
About 14 formulations were prepared by the Qbd Box Behnken design with the quadrate for optimization. Various natural film formers, synthetic polymers, super disintegrants, plasticizers were represented as independent variables with folding endurance and disintegration time as dependent variables using the solvent casting method. Further the formulation characteristics including physical and mechanical behaviour of films and drug release behaviour 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 minutes following first order kinetics with satisfactory mechanical properties(Table 1,2,3).
Table 1
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. |
Table 2
Characteristic formulation features 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) |
Table 3
33 factorial design Uncaria tomentosaFDOFs.
F. Code | Independent variable | Dependant variable |
X1 (Polymer) | X2 (Plasticizer) | 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) |
n-vivo studies: |
In-vitro Osteoarthritis evaluation
Culture and maintenance of C20A4 chondrocytes: Human Chondrocyte cell lines C20A4 were obtained from National Centre for Cell Sciences, Pune India. Chondrocytes were cultured in RPMI-1640 (10% fetal bovine serum (FBS), 10 U antibiotic/ml) (sigma Aldrich) medium at 37 oC under 5% CO2 in an incubator (5215, Shel Lab, USA). The growth medium was changed every third day and the chondrocytes were passage with trypsin-EDTA solution (0.05%) in a 1:3 ratio[16]. 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(Fig. 1, 2) was developed using C20A4chondrocytes of OA. Briefly, C20A4chondrocytes[17] were embedded in agarose (2% low-melting agarose-gelling temperature 25 ± 5oC) (Sigma, USA) prepared in phosphate buffer solution. Then, equal volumes of chondrocyte suspension in double strength RPMI-1640 (20% FBS) were 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 of interleukin-1β (IL-1β) (Sigma-Aldrich, USA) was added into the medium. The same amount of IL-1β was added during each medium change.
Cell viability assays
The cell viability[18–20] 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(Fig. 2) 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.
Analysis protocol
Establishment of the in-vitro [21–23]OA model and effectiveness of the release systems were evaluated after incubations for 7, 15, and 23 days. For determination of the effects of FDOFs, the cell lines were divided into four groups ’i.e Control, IL-1β, F5, and F13 treated IL-1β injected cell lines. The chondrocytes in agarose constructs cultured only in media (RPMI-FBS) without IL-1b, served as control. The media were collected and stored at -80 oC for GAG, hydroxyproline (HYP) and MMP-13 analyses. Agarose constructs with chondrocytes were digested with papain for GAG, HYP and DNA quantitation analyses. The DNA amounts of chondrocytes in agarose were used to normalize the results of GAG and collagen amounts.
DNA content assay
DNA amounts of papain digested samples were determined by Hoechst 33258 dye (Invitrogen, Germany) with Fluorometer (Modulus, USA). Calf thymus DNA was used as standard. The measurement was performed according to the protocol of the instrument and DNA concentrations were measured as (µg)[20].
Glycosaminoglycan (GAG) assay
Total sulphated glycosaminoglycan (sGAG) amounts both in papain digests of agarose-chondrocyte constructs and in media were determined by using 1,9 dimethylmethylene blue (DMMB) assay. Chondroitin sulphate[21] from bovine trachea (Sigma, USA) was used as standard. Total sGAG amounts of constructs were reported as sGAG/DNA (µg/µg) and sGAG concentrations of liquid media were reported as mg/ml.
Hydroxyproline (HYP) assay: The papain digests and collected media were used for hydroxyproline (HYP) assay to determine the collagen content. 4-hydroxyproline (Sigma) was used as standard. HYP: collagen converting factor is reported as 1:8 in literature. HYP content of constructs was reported as HYP/DNA (µg/µg) and HYP concentrations[22] of liquid media were reported as mg per mL.
Matrix metalloproteinase-13 (MMP-13) assay
MMP-13 concentrations in cell culture media were quantified by enzyme-linked immunosorbent assay (ELISA) (Cusa-bio) according to the protocol of the MMP-13 kit. MMP-13 concentrations were measured as ng/ mL[23].
In vivo Studies of formulations
Animals: Seven-week-old male Wistar rats (200–250 gr) were used for this study. The animals were provided with food and water ad libitum and were housed in a polycarbonate cage at 23 ± 3°C under a 12 h light/12 h dark photoperiod. Experiments were performed according to the guidelines for the use of laboratory animals approved by the Institutional animal ethical committee (IAEC), PGP Life Sciences, Hyderabad. (Approval No. PGP/OA/LS00129-042022/L3). During the study period, there were no abnormal symptoms or deaths from the administration of the test substance(Table 4) and there were no significant changes in body weight, so it was concluded that the test substance did not cause weight change or general symptoms[23, 24].
Table 4
Percent weight bearing on right hind paw
% Weight-bearing post-F5 administration in days |
| 7 | 13 | 20 | 27 | 35 |
Normal (N) | 51.33 ± 3.1 | 52.87 ± 3.7 | 49.12 ± 4.2 | 48.19 ± 3.8 | 51.57 ± 4.8 |
Negative Control (NC) | 31.58 ± 2.4 | 29.64 ± 1.9 | 31.69 ± 2.8 | 30.25 ± 2.9 | 26.25 ± 3.9 |
MIA with F5 at 150 mg/kg/day (M + F5-150) | 35.69 ± 2.9 | 32.69 ± 2.5 | 34.18 ± 2.1 | 36.58 ± 2.5 | 33.65 ± 4.2 |
MIA with F5 at 300 mg/kg/day (M + F5-300) | 41.85 ± 3.6 | 39.12 ± 2.8 | 40.68 ± 3.6 | 41.69 ± 3.4 | 40.23 ± 3.4 |
MIA with Celecoxib at 100 mg/kg/day (PC) | 43.95 ± 3.9 | 41.86 ± 2.3 | 44.25 ± 3.9 | 43.26 ± 3.3 | 41.93 ± 3.8 |
All the values are represented as Mean ± S. D (n = 3) |
Induction of OA with Monosodium Iodoacetate(MIA) injection and administration with F5: Rats were randomly assigned to one of the six groups as follows: Normal (N) (injection of saline, n = 6); Negative control (NC) (injection of 1.0 mg MIA, n = 6); M + F5-150 group, MIA (1.0 mg) + F5 (150 mg/kg/day, n = 6,); M + F5-30 group, MIA (1.0 mg) + F5 (300 mg/kg/day, n = 6); MIA (1.0 mg) + Celecoxib (100 mg/kg/day, n = 6, positive control). They acclimatized for ten days with the basal diet. On day 10, MIA (Sigma-Aldrich, MO, USA) was injected in a 1 mL syringe at a dose of 50 µL (60 mg/mL) in the right knee joint to induce OA. After injection of MIA, each experimental group was administered orally with either saline, 150 or 300 mg/kg F5, and 100 mg/kg Celecoxib (Kekule Pharma limited, Hyderabad) once daily for 5 weeks[25–27].
Percent weight bearing on right hind paw: Changes in weight distribution between the left and right hind paws were determined using in capacitance tester (Columbus Instruments, 950 N. Hague Ave OH, USA) on days 7, 13, 20, 27, and 35 after administration of F5. Rats were placed in an angled plexiglass chamber so that each hind limb was positioned on a separate force plate. The rats were allowed to acclimate to the apparatus and when stationary, readings were taken. The downward force (measured in grams) applied by each hind limb was assessed and averaged over a three-second period (each data point was the average of three readings). For weight-bearing measurements[26], the percent weight (in grams) borne on the right hind paw was determined using the following formula
Percent weight bearing= [right hind weight/ (left hind weight + right hind weight)] x100