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*Corresponding Author: J. Naga Sowjanya, Osmania University, Hyderabad, Telangana.
Citation: J. Naga Sowjanya, Raja rao, (2023), In Vitro And In Vivo Approaches to Evaluate Uncaria Tomentosa Bark Extract Loaded FDOFS on Osteoarthritis Models, Clinical Medical Reviews and Reports. 5(4); DOI:10.31579/2690-8794/156
Copyright: © 2022 Angel Hsu, this is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Received: 08 May 2023 | Accepted: 18 May 2023 | Published: 25 June 2023
Keywords: osteoarthritis; u tomentosa; assay; screening
Osteoarthritis is one the leading health concerns worldwide affecting two third million with no proper treatment ensured to restore the normal function and completely relieving the joint pain. Oral fast dissolving films have promising action and targeted delivery with high drug loading capacity. The present investigation involves the study the invitro and in vivo activity of developed Oral fast dissolving films of U. tomentosa bark extract with optimized F5 and F13 formulations. For invitro evaluation a three-dimensional OA model was prepared with first passage chondrocytes grown on trypsin EDTA media in 1: 3 ratios. The OA agarose model was prepared with C20A4 chondrocytes on agarose gel (25 ± 5oC) in phosphate buffer solution. Cultivation of chondrocytes was done with 1 mL of RPMI-1640 (10% FBS) which was added with 20% (IL-1β) solution on third day of incubation and media was replaced time to time. The incubated cell line with 20,000 cells/well in 96-well plates were treated with 5 µL of 0.5% MTT reagent on fifth day of incubation and absorbance was measured at 570 nm. The effects were studied for 7, 13, 27, 35 days for the study effects of FDOFs on the cell lines were (Control, IL-1β, F5, and F13 treated IL-1β injected types). The chondrocytes in agarose constructs cultured only in media (RPMI-FBS) without IL-1b, served as control. The GAG, HYP and DNA quantitation analyses along with DNA content assay were performed to study the arthritic effect of optimized FDOF’s i.e., F5. For in vivo studies Mon iodoacetate (MIA) induced arthritis models which is well established to understand weight bearing and response to tactile stimuli though the ongoing procedure is not known. The in vivo protocol was performed in seven-week-old male wistar rats with negative control of MIA and positive control as Celecoxib. The assessment of pain and thickness of the knee were estimated to be indicators of osteoarthritic potential. The study results revealed the F5 formulation has efficacy on the OA models which need a clinical investigation in humans.
Osteoarthritis (OA) is a major challenging concern affecting millions of people and is known to be a chronic progressive disease of joints and surrounding tissue [1]. In fact, there is no complete treatment available with medicine so far for this degenerative disease of joints. Yet there are many reported long term treatments that can relive the pain or reduce the incidence of pain which include hyaluronan (HA) injection (Intraarticular). The disease is characterized by loss of joint cartilage in slow manner leading to deformity, dysfunction [2] and pain which were not overcome by the conventional oral drug treatments. Thus, there is surge in alternative treatments to address this issue by modifying the oral drugs with slow-release polymers that can retain the drug for prolonged time and release [3]. Moreover, the Uncaria tomentosa (Cats Claw) extract is widely known for treating the arthritis and inflammatory bowel disease [4]. The spiroindoline alkaloids present in the plant include mitraphylline, pteropodine are responsible for the antiinflammatory, apoptotic and play a major role in rheumatoid arthritis. The oral fast dissolving films have fastest market approach over the conventional tablets and improved patient compliance, thus considered to be the better strategy to overcome problems of osteoarthritis especially for pain management [5]. The API can be soluble or insoluble which when designed using suitable film forming polymers, plasticisers and disintegrants enhance the oral bioavailability, thus this novel approach [6] can be expanded to design even the herbal novel drug delivery systems with improved patient compliance and site specific and can be conveniently used in geriatrics. Pain is the key driving factor and restriction that imparts the treatment strategy to orient towards [7] the replacement surgery. The pain is categorized as two types initially a dull aching and later a shorter throbbing pain which comes frequently occurring in three stages (early, mid, progressive). The treatment protocol primarily involves lifestyle changes, NSAIDS, steroidal injections and joint replacement therapy at chronic stage [8, 9]. A clear understanding of pain management to chronic is a better treatment option to be focussed on [10]. Thus, the evaluation protocols for osteoarthritis involve the determination of well-defined preclinical model of osteoarthritis such as invitro (pathogenic inflammatory mediators like a TNF-α, IL-6, and IL-1) and invivo protocols mediating the pain management strategy [11, 12].
The present investigation involved the development and evaluation of the novel fast oral dissolving films of Uncaria tomentosa bark extract using Box Behnken design [13, 26]. The evaluation and characterization [14] revealed the physicochemical nature and formulation parameters suitable for drug delivery. Further the in vitro and in vivo evaluation of efficacy of Uncaria tomentosa bark extract modified FDOFS was performed using various methods to prove that the product potential as a viable product for commercialisation and as alternative to existing product for the treatment of osteoarthritis.
Materials and Methods
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 evaluationAbout 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 (superDisintegrant) 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). minutes following first order kinetics with satisfactory mechanical properties (Table 1,2,3).
| 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 1: Composition of 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 2: Characteristic formulation features of Uncaria tomentosa FODFs
| . 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: | |||||
Table 3: 33 factorial design Uncaria tomentosa FDOFS.
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
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