2Doctor Thangs Products, Coimbatore, Tamilnadu, India
Keywords: CFA induced arthritis; Rheumatoid Factor; TNFα; herbal aerosol
Cardiospermum Halicacabumlinn (CH) is a climber belongs to the family Sapindaceae locally known as balloon vine and called as “modakathon” in Tamil. “Modaku” means crippling joint pain; “thon” means remedy. It is a deciduous, branched, herbaceous climber distributed throughout the plains of India. CH reported to contain arachidic acid, apigenin, apigenin-7-O-glucuronide, chysoeriol-7-O-glucuronide, luteolin, luteolin-7-O-glucuronide, saponin, quebrachitol, proanthocyanidin, beta sitosterol and stigmosterol [5]. Irulas in Thanjavur, Tamilnadu, India, provided evidence for the use of this plant as a traditional remedy that has been used for centuries to treat RA and it is still used by some locals to treat RA in Asian [6] communities, substantiated by many researchers [7-10]. Anti-inflammatory properties of CH have been researched [11], but no clinical trials have been reported so far. Experimental pharmacological studies of CH have shown antioxidant activity [12] analgesic and vasodepressant activities [13], antipyretic activity [14], Antimalarial activity [15], diuretic activity [16], anti-diarrheal activity [17], anti-diabetic activity [18] and anti-ulcer activity [19].
VitexnegundoLinn (VN) (Verbenaceae), grows gregariously in wastelands and is also planted as a hedge-plant. It was reported for the presence phytochemical constituents, flavonoids such as casticin, orientin, isoorientin, luteolin, lutecin‐7‐O‐glucoside, corymbosin; gardenins A and B [20]. VN has been documented for potent anti-inflammatory [21], anti-arthritic [22], anticonvulsant [23], hepatoprotective [24], laxative [25] and bronchial relaxant activities [26]. Though all parts of CH and VN are used, leaves are majorly used for treating arthritis in traditional medicine [27] and hence a dosage form was designed using the leaf extracts of the plants.
The existing topical formulations available have the disadvantages viz. difficulty in applying the formulation to the respective site, staining cloth due to greasy nature, more chance of contamination and more wastage due to inaccuracy of dose. The designed herbal aerosol spray formulation have the advantages viz. ease of administration, cause no contamination, no clean up procedure after using the product, protection of active constituents from oxygen and moisture, minimum pain and irritation experience during application, minimum side effects as it is a herbal medicine, stable as the constituents are enclosed in a pressurized container, rapid onset of action, accuracy of the administered dose, no first pass effect and GIT degradation and can be delivered directly on to the affected area. Further, exhaustive work on CH and VN has been done for its biological activity and mechanism of action through systemic application. But there is no report on preclinical studies of CH and VN for its anti-arthritic activity by topical application. Thus, keeping all this in view, a topical herbal aerosol spray was developed using MLECH and MLEVN and evaluated for anti-arthritic activity.
The overnight fasted animals were anaesthetized with ketamine (20 mg/kg, i.p) followed by withdrawal of blood samples from retro-orbital sinus and the collected blood samples were evaluated for hematological parameters. A portion of the blood samples were centrifuged at 10000 rpm for 10 min. The separated serum was analyzed for C-reactive protein (CRP) and Rheumatoid Factor (RF). Photographic and radiographic analysis was carried out using ALLANS fully computerized X-ray instrument (AXD-100i, Mumbai, India) for the confirmation of development of arthritis.
After the confirmation of arthritis development, Diclofenac sodium 10 mg/kg (p.o.) and the combined doses of MLECH and MLEVN (400 mg/kg each) (p.o.) were administered orally from 22 to 42 days to Group 3 (served as reference standard for oral route) and Group 4 respectively. Topical Diclofenac Diethylamine aerosol spray (8 mg/day) and topical herbal aerosol spray formulation (64 mg/day) were sprayed continuously for 3 sec from 22 to 42 days to the left knee joint region of Group 5 (served as reference standard for topical route) and Group 6 respectively. During the treatment period, the body weight of the animals and the rat paw volume of control and treatments were measured on 25th, 29th, 35th and 42nd day by using digital Vernier caliper (Mitetoyo digimatic caliper, Japan). At the end of 42nd day the pain test score of the animals were recorded visually 31. The overnight fasted animals were anaesthetized with ketamine (20 mg/kg, i.p) followed by withdrawal of blood samples from retroorbital sinus and the collected blood samples were evaluated for hematological parameters [32].A portion of the blood samples were centrifuged at 10000 rpm for 10 min. The separated serum was analyzed for urea, uric acid and serum biomarkers viz. CRP, RF (Omega diagnostics Limited, Scotland, UK), TNFα (ELISA Kit, Gen-Probe, France), IL-1ß and IL6 (ELISA Kit, R&D systems, USA). Biochemical investigations were carried out in a Autoanalyer (Photometer 5010 V5+, Robert Riely, Berlin) using Piramal healthcare limited reagent kit.
Photographic and radiographic analysis was carried out at the end of the arthritis experiment using ALLANS fully computerized X-ray instrument (AXD-100i, Mumbai, India) for the assessment of reduction in arthritis (Figures1A and 1B). The animals were sacrificed by cervical dislocation and the organs thymus, spleen and bone joints of ankle joint were isolated, weighed and [33] observed for histopathological changes under microscope and digitalimages were acquired [Figures1A and 1B].
S.No |
|
Parameters |
Value |
Units |
01 |
Flammability |
Flame Extension |
6 |
cm |
02 |
Physico chemical characteristics |
Vapour Pressure |
160 |
psig |
Density |
0.851 |
g |
||
pH |
4.20-4.33 |
-- |
||
Viscosity |
0.695 |
centipoise |
||
Compatibility of product concentrates with propellants |
No turbidity was observed |
|||
03 |
Performance |
Aerosol valve discharge rate |
0.284 |
g/3 sec |
Spray pattern |
6 |
cm |
||
Net content |
84 |
g |
||
Leakage test |
No leakage was observed |
|||
Particle size range |
13 -60 |
µm |
||
Total number of discharges per container |
98 puff |
3 sec for each puff |
||
Spray angle |
21 |
° |
||
04 |
Biological Evaluation |
Skin irritation test |
No edema or redness of skin observed |
S.N |
Parameters |
Topical herbal aerosol spray formulation containing 2% w/v of each MECH and MEVN |
|
||||||||||||||||
|
|
Storage condition |
Study Time period |
||||||||||||||||
Months |
Months |
Months |
|||||||||||||||||
0 |
1 |
2 |
3 |
6 |
0 |
1 |
2 |
3 |
6 |
0 |
1 |
2 |
3 |
6 |
Short term Six Months |
||||
1 |
Color |
No change in color |
No change in color |
No change in color |
|||||||||||||||
2 |
Odour |
No change in odour |
No change in odour |
No change in odour |
|||||||||||||||
3 |
pH |
4.22 |
4.22 |
4.22 |
4.22 |
4.21 |
4.22 |
4.20 |
4.20 |
4.19 |
4.18 |
4.22 |
4.16 |
4.10 |
4.08 |
4.02 |
|||
4 |
Density (g/cm3) |
0.851 |
0.851 |
0.851 |
0.851 |
0.851 |
0.851 |
0.851 |
0.851 |
0.851 |
0.851 |
0.851 |
0.848 |
0.836 |
0.830 |
0.822 |
|||
5 |
Viscosity (centipoise) |
0.695 |
0.695 |
0.695 |
0.695 |
0.695 |
0.695 |
0.694 |
0.692 |
0.690 |
0.688 |
0.695 |
0.674 |
0.638 |
0.614 |
0.596 |
|||
6 |
Net content (g) |
84 |
84 |
84 |
84 |
84 |
84 |
84 |
84 |
84 |
84 |
84 |
84 |
84 |
84 |
84 |
|||
7 |
Leakage test |
No leakage observed |
No leakage observed |
No leakage observed |
|||||||||||||||
8 |
Microbial load (Bacteria & Fungi) |
No microbial growth was observed at 24, 48 and 72 h |
No microbial growth was observed at 24, 48 and 72 h |
No microbial growth was observed at 24, 48 and 72 h |
|||||||||||||||
9 |
Sterility test |
No microbial growth was observed at 24, 48 and 72 h |
No microbial growth was observed at 24, 48 and 72 h |
No microbial growth was observed at 24, 48 and 72 h |
Groups |
Initial body weight (g) |
Body wt after 22 days of CFA induction |
Body wt after treatment |
Body wt after treatment |
Body wt after treatment |
Body wt after treatment |
Weight gain (g) |
Normal Control (1% w/v SLS) |
146.26 ± 1.47 |
174.16 ± 1.53 |
171.50 ± 0.84 |
175.66 ± c 0.88 |
182.00 ± 1.03 |
194.16 ± 0.98 |
47.91 ± 1.78 |
Arthritic control |
143.45 ± 1.22 |
158.30 ± 1.25 |
160.50 ± 1.25 |
161.66 ± 1.08 |
163.16 ± 0.94 |
164.33 ± 1.17 |
20.88 ± 2.15 |
Diclofenac sodium 10 mg/kg (p.o.) |
148.53 ± 0.68 |
160.66 ± 0.84b |
170.00 ± 0.96b |
173.33 ± 1.62b |
180.5 ± 0.56b |
186.33 ± 0.61b |
38.30 ± 1.08b |
MLECH 400 mg/kg & MLEVN 400 mg/kg (p.o.) |
146.26 ± 1.47 |
155.00 ± 1.06b |
169.50 ± 0.42b |
172.83 ± 0.30b |
174.5 ± 1.11b |
184.33 ± 0.91b |
37.4 ± 1.54b |
Diclofenac topical aerosol spray (8 mg/day) |
144.43 ± 1.81 |
154.50 ± 1.28b |
174.33 ± 1.66b |
177.83 ± 1.49b |
188.33 ± 0.88b |
193.50 ± 0.61b |
51.6 ± 1.14b |
Topical herbal aerosol spray formulation (64 mg/day) |
143.16 ± 1.01 |
154.60 ± 1.17b |
171.00 ± 0.73b |
176.80 ± 1.42b |
184.16 ± 1.60b |
187.00 ± 1.13b |
42.56 ± 2.01b |
Groups |
Dose |
Rat paw volume (mm) |
|||||
Before treatment |
After treatment |
||||||
Initial |
After 21 days |
25th day |
29thday |
35th day |
42nd day |
||
Normal control |
1% w/v SLS |
4.91±0.19 |
5.26± 0.23 |
4.90 ± 0.31 |
5.25±0.17 |
5.39 ± 0.13 |
5.52 ± 0.16 |
Arthritic control |
1% w/v SLS |
4.98± 0.30 |
10.48± 0.26 |
10.75 ± 0.10 |
10.26 ± 0.08 |
10.41 ± 0.07 |
10.42 ± 0.10 |
Diclofenac sodium (p.o.) |
10 mg/kg |
5.33 ± 0.16 |
10.48± 0.22 |
9.36 ± 0.07b |
8.93 ± 0.09b |
7.71 ± 0.26 |
6.91 ± 0.32b |
MLECH + MLEVN |
400 + 400 mg/kg |
4.94± 0.17 |
10.62± 0.33 |
9.66 ± 0.07b |
8.04 ± 0.21b |
7.59 ± 0.18b |
7.30 ± 0.16b |
Diclofenac topical aerosol spray |
8 mg/day |
5.05± 0.14 |
10.41± 0.21 |
9.72 ± 0.06b |
8.84 ± 0.15b |
7.71 ± 0.15b |
7.55 ± 0.17b |
Topical herbal aerosol spray formulation |
64 mg/day |
4.87± 0.18 |
10.39± 0.25 |
9.98 ± 0.15a |
7.91 ± 0.34b |
7.87 ± 0.16b |
6.91 ± 0.24b |
ap < 0.05, treatment groups Vs arthritic control
bp < 0.01, treatment groups Vs arthritic control
Groups |
Pain test |
Mobility score |
Stance score |
|
Extension |
Flexion |
|||
Arthritic control |
9.66 ± 0.21 |
8.50 ± 0.34 |
1.16 ± 0.16 |
1.33 ± 0.21 |
Diclofenac 10 mg/kg p.o. |
4.33 ± 0.21b |
3.83 ± 0.16 b |
2.83 ± 0.16 b |
3.16 ± 0.16b |
MLECH 400 + MLEVN 400 p.o. |
6.16 ± 0.16b |
4.16± 0.16 b |
2.16 ± 0.16a |
2.50 ± 0.22b |
Diclofenac topical aerosol spray (8 mg/day) |
4.50 ± 0.22b |
3.50 ± 0.34 b |
3.00 ± 0.36b |
2.83 ± 0.16b |
Topical herbal aerosol spray formulation (64 mg/day) |
5.50 ± 0.22b |
4.33 ± 0.21 b |
2.33 ± 0.33a |
2.10 ± 0.16b |
aP< 0.05 treated groups vs. arthritic control;
bP< 0.01 treated groups vs. arthritic control
Groups |
Hb (mg %) |
WBC (x103/mm3) |
RBC (x106/mm3) |
ESR (mm/h) |
Before treatment |
After treatment |
||||||
Before treatment |
After treatment |
Before treatment |
After treatment |
Before treatment |
After treatment |
Before treatment |
After treatment |
Urea (mg/dl) |
Uric acid (mg /dl) |
Urea (mg/dl) |
Uric acid |
|
Normal control |
15.56± 0.27 |
16.13± 0.15 |
6.61± 0.27 |
6.78± 0.06 |
9.71± 0.22 |
10.26±0.15 |
4.83±1.47 |
5.00±0.63 |
16.16±0.91 |
2.66± 0.09 |
16.60± 0.91 |
2.86±0.07 |
Arthritic control |
10.9±0.17a |
10.26± 0.10 |
11.78±0.32a |
10.2± 0.2 |
7.66±0.30a |
5.67± 0.20 |
36.16± 1.19a |
40.33± 1.43 |
44.35±1.50a |
6.96± 0.16a |
44.8± 3.62 |
7.15±0.13 |
Diclofenac sodium (10 mg/kg) |
11.01± 0.03a |
15.91±0.21b |
11.95±0.35a |
7.53± 0.17b |
7.23± 0.19a |
9.01± 0.23b |
33.5± 1.17a |
13.66± 1.11b |
43.75±1.49a |
7.20± 0.11a |
25.93±1.25b |
3.43±0.16b |
MLECH 400 mg/kg & |
10.73± 0.16a |
15.48±0.26b |
12.68±0.47a |
8.76±0.06b |
7.71± 0.22a |
9.20± 0.12b |
34.33± 1.28a |
28.66± 1.08b |
43.88±1.41a |
7.13± 0.16a |
32.56±1.79b |
4.46±0.10b |
Diclofenac topical aerosol spray 8 mg/day |
11.43± 0.20 a |
15.43± 0.19b |
11.65±0.29 a |
7.81± 0.07b |
7.32± 0.18a |
9.23± 0.19 b |
35.33± 1.64 a |
24.83± 0.79 b |
43.75 ±0.65a |
7.35 ±0.13b |
27.25±0.60b |
3.36±0.10b |
Topical herbal aerosol spray formulation 64 mg/day |
11.41± 0.21a |
15.11± 0.66b |
11.86±0.37a |
8.26± 0.04b |
7.48± 0.07a |
9.81± 0.25b |
36.33± 0.61a |
22.66± 0.66b |
44.25±1.31a |
6.90± 0.12a |
31.16±0.84b |
4.41±0.09b |
ap< 0.01 arthritic control Vs normal control
bp< 0.01 treated groups Vs arthritic control
Groups |
TNFα (pg/ml) |
Interleukin levels (pico g/ml of serum) |
|
IL-1ß |
IL-6 |
||
Normal control |
1.36 ± 0.23 |
1.38 ± 0.24 |
19.58 ± 1.14 |
Arthritic control |
44.90 ± 2.36a |
67.70 ± 1.09a |
728.61 ± 31.99a |
Diclofenac sodium 10 mg/kg (p.o.) |
2.36 ± 0.45b |
23.34 ± 1.34b |
323.33 ± 16.55b |
MLECH 400 mg/kg + MLEVN 400 mg/kg (p.o.) |
29.04 ± 0.63b |
45.11 ± 1.08b |
512.72 ± 24.87b |
Diclofenac topical aerosol spray (8 mg/day) |
6.28 ± 0.71b |
20.99±0.60 b |
313.95±14.66 b |
Topical herbal aerosol spray formulation (64 mg/day) |
19.79 ± 0.81b |
36.55 ± 1.25b |
428.12 ± 10.17b |
ap< 0.01 arthritic control Vs normal control
bp< 0.01, treatment groups Vs arthritic control
Quality control test performed for topical herbal aerosol spray formulation complied with USP standards and the stability study performed for 6 months for the formulated topical herbal aerosol spray was found to be stable and complied with ICH guidelines.
In our earlier acute oral toxicity study, carried out separately for MLECH and MLEVN was found to be safe upto a dose of 2000 mg/kg and hence 1/20th, 1/10th and 1/5th dose of 2000 mg/kg viz.100, 200, 400 mg/kg were used for anti-arthritic activity study. It was also observed from our earlier study, that the combination (100 + 100, 200 +200, 400 + 400 mg/kg) of MLECH and MLEVN showed significant inhibition of inflammation in a dose dependent manner than the individually administered
CFA-induced arthritis is the most widely used chronic test model which is associated with an immune-mediated inflammatory reaction and rat is unique in developing polyarthritis after CFA treatment and hence they were used for the study [38]. In first phase, irritant nature of the adjuvant induces inflammation and hence measurement of 3 to 5 day effect of treatment represents the efficacy on inflammation of primary lesions such as edema and soft-tissue thickening. The second phase, reduction in body weight due to immunogenic responses [39].
In the present study, administration of Diclofenac sodium, MLECH and MLEVN (400 +400 mg/kg, p.o.), topical Diclofenac topical aerosol spray and topical herbal aerosol spray formulation in rats significantly reduced the progression of RA when compared with arthritic rats. Significant alterations in flexion pain test score, mobility score and stance score was observed for all the treated group of rats when compared with arthritic control rats. This alteration of arthritic test scores support the anti-arthritic activity of the MLECH & MLEVN and topical herbal aerosol spray formulation.
Information related to the pathology of arthritis was obtained during the study from body weight changes and hematological parameters. Restoration of body weight of rats was observed after treatment of MLECH and MLEVN and topical herbal aerosol spray formulation. Decrease in body weight during inflammation in arthritic control group was reported [40] due to deficient absorption of nutrients through intestine. It was reported that treatment of anti-inflammatory drugs normalizes the process of absorption and hence the restoration of body weight in MLECH, MLEVN and Diclofenac sodium treated groups may involve improvement of intestinal absorption of nutrients and reduction in the distress caused by the severity of arthritis. It has been reported that a moderate rise in the WBC count occurs in arthritic conditions due to an IL-1B mediated rise in the respective colony stimulating factors. The present study reveals that MLECH, MLEVN and Diclofenac sodium treatments tend to normalize the WBC count. Characteristic hematological alterations such as increased Hb count and decreased ESR by MLECH and MLEVN support the anti-arthritic effect. It is proposed that the reduction in the Hb count during arthritis results from reduced erythropoietin levels, a decreased response of the bone marrow erythropoietin and premature destruction of red blood cells and increase in ESR is due to formation of endogenous protein, globulin and fibrinogen [41]. Increase in serum levels of CRP, RF37 TNF-α, IL1β and IL6 are characteristic feature of RA [42] and hence, in vitro determination of serum biomarkers such as CRP, RF, TNF-α, IL1β and IL6 were performed for the arthritic control and all the treated groups. Significant reduction of serum biomarker levels in treated rats when compared with arthritic control rats support the anti-arthritic activity.
Radiological observations of the rats provided the evidence for the inhibition of arthritis after treatment of Diclofenac sodium p.o, MLECH and MLEVN (400 +400 mg/kg, p.o.), topical Diclofenac topical aerosol spray and topical herbal aerosol spray formulation.
Synovial Inflammation and hyperplasia is considered to be the important pathological condition of rheumatoid arthritis [43]. Histological examination of joint showed significant reduction of inflammation in treated group of rats when compared with arthritic control rats.
Significant reduction in thymus and spleen weight of rats after treatment with MLECH & MLEVN and topical herbal aerosol spray formulation support the anti-arthritic activity.
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