This article has been collectively contributed by the Research & Development Team from the Healthcare Division of Sri Sri Tattva.
Dr. Ravi Kumar Reddy, Chief Scientific Officer
Dr. Hari Venkatesh K R, Head, R & D
Dr. Jothi Lakshmi, Asst. Manager, R & D
Mrs. Pushpa, Junior Manager, R & D
Abstract:
Low back pain (LBP) affects approximately 60–85% of adults during some point in their lives. Fortunately, for the large majority, symptoms are mild and transient, with 90% subsiding within 6 weeks. Chronic low back pain, defined as pain symptoms persisting beyond 3 months, affects an estimated 15–45% of the population. For the minority with intractable symptoms, the impact on quality of life and economic implications are considerable. Despite the high prevalence of low back pain within the general population, the diagnostic approach and therapeutic options are diverse and often inconsistent, resulting in rising costs and variability in management throughout the country. The terms lumbar osteoarthritis, disk degeneration, degenerative disk disease, and spondylosis are used in the literature to describe anatomical changes to the vertebral bodies and inter vertebral disk spaces that may be associated with clinical pain syndromes.
Many analgesics and Non – Steroidal Anti-inflammatory Drugs (NSAIDS) are available in the market used for the clinical treatment of inflammatory diseases. Most of them are known to produce untoward effects in the long run. Ayurveda offers a wide range of powerful herbs which deal effectively with pain and inflammation. Hence polyherbal formulations named Sandhimitra Vati , a proprietary product sold by Sri Sri Tattva was taken along with 2 other market brands (Market brand 1 and Market brand 2) for the current study to establish their analgesic and anti – inflammatory activity in rats and mice using four different preliminary models.
Acetic acid induced writhing in mice, Eddy’s hot plate induced thermal pain for analgesic, carrageenan induced rat paw oedema. Freund’s adjuvant study for anti-inflammatory study was selected for this preliminary screening. The results of the acetic acid induced model revealed that at a dose of 7.8 mg/kg p.o; the protective effect of Sandhimitra Vati was 78.68% (P<0.05) when compared to Market brand 1 and Market brand 2 having 42.10% (P<0.05) and 65.51% (P<0.05) respectively. The results of the hot plate model revealed that at the same dose, the percentage of protection of Sandhimitra Vati was found to be 91.50% (P<0.5) compared to Market brand 1 and Market brand 2 each having protection percentage of 75% (P<0.5) respectively. The results of the carrageenan induced oedema model revealed that at a dose of 270 mg/kg p.o; the percentage of protection of Sandhimitra Vati was 60% (P<0.01) compared to Market brand 1 and Market brand 2 having percentage of protection of 40% (P<0.05) and 46.66% (P<0.05) respectively. The result of Freund’s adjuvant study revealed that at a dose of 540 mg/ kg p.o; the percentage of protection of Sandhimitra Vati was 75.24% compared with Market brand 1 and Market brand 2 having percentage protection of 61.04% and 53.66%.
1. Materials and Methods
1.1. Collection of samples:
Sample of drugs “Sandhimitra Vati”, “Market brand 1” and “Market brand 2” were collected from sponsor Sriveda Sattva Private Limited, 21st KM, Kanakapura Road, Udayapura, Bangalore 560082.
1.2. Animals for experiments:
Adult wistar albino rats (150-200 g) and swiss albino mice (25-30 g) of either sex were obtained from Sri Raghavendra Enterprises, Bangalore, India. Maintained under standard in-house conditions, they were given a standard pellet diet and water ad libitum. All experiments were carried out in accordance with the guidelines laid down by the Institutional Animal Ethical Committee (IAEC/ABMRCP/2014-2015/11).
1.3. Analgesic activity
1.3.1. Acetic acid induced writhing in mice
Swiss albino mice were randomly divided into five groups consisting of six animals per group. Group-I (Control) received 1% w/v of CMC (1 ml/kg, p.o.). Group II, III and IV received “Sandhimitra Vati”, “Market brand 1” and “Market brand 2” respectively. Group-V was treated with standard drug Diclofenac sodium (10 mg/kg, p.o.); after 30 minutes, each mouse received 1% v/v of acetic acid (0.1 ml/10 gm, i.p). The number of writhing as abdominal constriction, trunk twisting, and hind limb extension was cumulatively counted every 10 minutes over a period of 20 minutes immediately after the acetic acid injection. The analgesic activity was expressed as percentage of inhibition of writhing.
1.3.2. Eddy’s hot plate induced thermal pain
The mice were placed on a hot plate maintained at 55ºC ± 1ºC and the time between placement of the mouse on the platform and licking of the paws or jumping was recorded as hot plate latency. Mice with baseline latency higher than 10 second were eliminated from the study. Twenty four hours later Group I (Control) received 1% w/v of CMC (1 ml/kg, p.o.). Group II, III and IV received “Sandhimitra Vati”, “Market brand 1” and “Market brand 2” respectively. Group V was treated with the standard drug Diclofenac sodium (10 mg/kg, p.o.). Reaction time was measured after 30 minutes of drug administration.
1.4. Anti-inflammatory activity
1.4.1. Carrageenan induced rat paw oedema
The albino rats were randomly divided into five groups consisting of six animals per group. Group I (Control) received 1% w/v of CMC (1 ml/kg, p.o.). Group II, III and IV received “Sandhimitra Vati”, “Market brand 1” and “Market brand 2” respectively. Group V was treated with the standard drug Diclofenac sodium (10 mg/kg, p.o.). Subsequently, 30 minutes after the above treatment, 0.1 ml of 1% w/v of carrageenan was injected subcutaneously into the planter region of the right hind paw to induce oedema. The paw volume was measured at 0, 2, 3, 4, 6 and 24 h after carrageenan administration using plethysmometer and the percentage of inflammation was calculated.
2.4. Statistical analysis
Values were expressed in mean ±S.E.M. The data was analysed by one way ANOVA followed by Student ‘t’ test. P values lower than 0.05 were considered as statistically significant and those of p<0.01 and P<0.001 were highly significant.
2.RESULTS:
2.1. Acetic acid induced writhing in mice
The results revealed that Sandhimitra Vati significantly reduced writhing induced by 1% v/v of acetic acid at a dose of 7.8 mg/kg, p.o. (Table 1). The significant protective effect with Sandhimitra Vati was 78.68 % (P <0.05) reduction observed in the early phase compared with control. The significant protective effect with Market brand 1 and Market brand 2 were 42.10% (P <0.05) and 65.51 % (P < 0.05) reduction respectively. Diclofenac (10 mg/kg, p.o.) had 89.73 % (P < 0.01) inhibition acting as an analgesic.
Table 1: Effect of Sandhimitra Vati, Market Brand 1, Market Brand 2 and Diclofenac on writhing induced by acetic acid
| Group | Dose (mg/kg,p.o) |
No. of Writhing
|
|||
| Early phase
(0-10 minutes) |
Percentage protection (%) | Late phase
(10-20 minutes) |
Percentage protection
(%) |
||
| Control (CMC) | 5 ml/kg | 66.33 ± 4.91 | 0 | 106.5 ± 9.22 | 0 |
| Sandhimitra Vati | 7.8 | 13.5 ± 3.69** | 78.68 | 38.5 ± 8.00** | 63.84 |
| Market Brand 1 | 7.8 | 36.6 ± 6.93** | 42.10 | 53.5 ± 9.74** | 49.76 |
| Market Brand 2 | 7.8 | 21.83 ± .02** | 65.51 | 47.67 ± .64** | 55.23 |
| Diclofenac | 10 | 6.5 ± 4.20** | 89.73 | 35.33 ± .06** | 66.82 |
Data was presented as mean ± S.E.M. Statistical differences between control and treated groups were tested by one way ANOVA followed by Student’s t-test. The differences were considered significant at P < 0.05, P < 0.01.
2.2. Eddy’s hot plate induced thermal pain
The results of the hot plate test (Table 2) revealed that the reaction time for the mice had increased significantly from 2.0 to 3.83 seconds (Sandhimitra Vati) at a dose of 7.8 mg/kg, p.o. with protection found to be 91.50% (P < 0.5) at 30 minutes. Pre-treatment with Market brand 1 (7.8 mg/kg, p.o.) and Market brand 2 (7.8 mg/kg, p.o.) increased reaction time from 2.0 to 3.50 seconds and 2.0 to 3.50 seconds respectively with percentage protection found to be 75% (P < 0.5) at 30 minutes. Diclofenac (10 mg/kg, p.o.) significantly showed maximum protective effect of 100% (P < 0.01) at 30 minutes compared to control.
Table 2: Effect of Sandhimitra Vati , Market brand 1, Market brand 2 and Diclofenac on pain induced by hotplate
| Group | Dose (mg/kg,p.o) | Jump response
(At 30 minutes) |
Percentage protection | Jump response
(At 60 minutes) |
Percentage protection |
| Control (CMC) | 5 ml/kg | 2.0 ± 0.36 | 0 | 2. 0 ± 0.25 | 0 |
| Sandhimitra Vati | 7.8 | 3.83 ± 0.54* | 91.5 | 3.16 ± 0.40 | 58 |
| Market Brand 1 | 7.8 | 3.5 ± 0.34* | 75 | 3.33 ± 0.33 | 66.5 |
| Market Brand 2 | 7.8 | 3.5 ± .42* | 75 | 3.16 ± 0.74 | 58 |
| Diclofenac | 10 | 4.0 ± 0.25** | 100 | 3.83 ± 0.65 | 91.5 |
Data was presented as mean ± S.E.M. Statistical differences between control and treated groups were tested by one way ANOVA followed by Student’s t-test. The differences were considered significant at P < 0.05, P < 0.01.
2.3. Carrageenan induced rat paw oedema
The percentages of inhibition are reported in Table 3. For the control group, the injection of the carrageenan caused localised oedema, after 30 minutes. The swelling increased progressively after 3 h to a maximum volume of 0.018 ml and remained obvious 24 hours after injection. Rats pre-treated with Sandhimitra Vati (270 mg/kg, p.o.), significant decreased carrageenan-induced oedema at 6 hr (0.006 ± 0.001) ml was found to be 60 % (P <0.01). Whereas Market brand 1 (270 mg/kg, p.o.) and Market brand 2 (270 mg/kg, p.o.) achieved significant reduction in paw oedema at 6 hr were found to be 0.009 ± 0.002 ml (40 %, P < 0.05) and 0.008 ± 0.002 ml (46.66 %, P < 0.05) continued for 24 h (9.09 %, P < 0.05). Diclofenac (10 mg/kg, p.o.) attained their maximal effects at 6 hr was found to be 0.004 ± 0.001 ml (73.33 %, P < 0.001). Although both the Sandhimitra Vati and Diclofenac reduced the swellings, they still remain significantly visible after 24 hours.
Table 3: Percentage protection of Sandhimitra Vati , Market Brand 1, Market Brand 2 and Diclofenac on carrageenan induced oedema
| Treatment | Dose
(mg/kg,p.o) |
Percentage protection (%) | |||||
| 0 h | 2 h | 3 h | 4 h | 6 h | 24 h | ||
| Control | – | – | – | – | – | – | – |
| Sandhimitra Vati | 270 | 0 | 21.42 | 50.00 | 56.25 | 60.00 | 9.09 |
| Market Brand 1 | 270 | 0 | 14.28 | 38.88 | 31.25 | 40.00 | 9.09 |
| Market Brand 2 | 270 | 0 | 14.28 | 38.88 | 43.76 | 46.66 | 9.09 |
| Diclofenac | 10 | 0 | 35.71 | 50.00 | 62.5 | 73.33 | 18.81 |
2.4. Freund’s adjuvant induced paw oedema changes
There is a decrease in percentage protection against rat paw volume in FCA injected arthritic control rats when compared to the standard and Sandhimitra Vati compounds treated rats. Diclofenac and Sandhimitra Vati treatment showed high protection for rat paw oedema volume when compared with the arthritic control group and values were depicted in Table 4. After 21 days it was found that the chronic inflammation induced by adjuvant showed an increase in paw oedema in arthritic control. Whereas, standard Diclofenac showed high protection against the paw oedema i.e.85.23 % after induction of Freund’s adjuvant; also Sandhimitra Vati showed 75.24 % protection against paw oedema compared to arthritic control.
Table 4: Effect of SAM samples on paw oedema in Freund’s adjuvant induced arthritis in albino rats
| Groups | % Protection against Paw edema | |||
| 1st Day | 7th Day | 14th Day | 21st Day | |
| Arthritis control
(1% CMC) |
0 | 0 | 78.98 | 31.33 |
| Diclofenac sodium
(15 mg/kg, p.o.) |
0 | 23.07 | 76.71 | 85.23 |
| Sandhimitra Vati (540 mg/kg, p.o.) | 0 | 12.5 | 70.91 | 75.24 |
| Market Brand 1
(270 mg/kg, p.o.) |
0 | 15.38 | 55.86 | 61.04 |
| Market Brand 2
(162 mg/kg, p.o.) |
0 | 65.38 | 45.48 | 53.66 |
All the values were expressed in Mean ± SEM (n=6) in term of percentage protection against paw oedema.
2.5 Haematology changes
The WBCs level was significantly (P < 0.5) decreased by Sandhimitra Vati compared with arthritic control. Migration of leucocytes into the inflamed area of arthritic rats was significantly (P ≤ 0.5) prevented by Sandhimitra Vati on day 21 (Table 5). RBCs level was significantly (P < 0.5) increased by SAM treated groups compared to arthritic control. Whereas haemoglobin levels had significantly (P < 0.01) increased in SAM treated group.
Table 5: Effect of SAM samples on blood parameters in Freund’s adjuvant induced arthritis in albino rats
| Blood parameters | |||
| WBCs
(Thousand/mm3) |
RBCs (millions/mm3) | Hemoglobin
(mg/dl) |
|
| Arthritis control
(1% CMC ) |
18.91 ± 1.40 | 4.69 ± 0.14 | 14.08 ± 0.20 |
| Diclofenac sodium
(15 mg/kg, p.o.) |
13.7 ± 1.27 | 6.36 ± 0.59 | 17.23 ± 0.59 |
| Sandhimitra Vati
(540 mg/kg, p.o.) |
10.7 ± 1.05* | 7.79 ± 1.07* | 16.68 ± 1.50 |
| Market Brand 1
(270 mg/kg, p.o.) |
14.26 ± 1.30 | 9.86 ± 1.31** | 20.3 ± 2.31 |
| Market Brand 2
(162 mg/kg, p.o.) |
15.6 ± 1.83 | 11.30 ± 0.65** | 23.08 ± 1.25** |
All the values were expressed in Mean ± SEM (n=6). The statistical analysis was carried out using one way ANOVA. Significant after analysis of variance (ANOVA) followed by Dunnett multiple comparison test. **P<0.5, **P<0.01 when compared to arthritic control group.
Biochemical changes:
After 21 days of Freund’s adjuvant induced, the total protein levels of serum showed significant (P < 0.01) changes in the entire group compared with arthritic control where as albuminutes level significant (P < 0.01) increased in Sandhimitra Vati compared to control and all treated groups (Table 6). The LDH levels shows significant (P < 0.01) increase in entire group compared to arthritic control. The creatinine level is significantly (P < 0.01) enhanced in SAM-001-D2 group compared to arthritis rats. Whereas, urea level is significantly (P< 0.01) increased in entire group except Market brand 1 compared to arthritis rats (Table 6). As a result of inflammation induced by adjuvant, the levels of SGPT and SGOT were increased in all arthritis rats as compared to treated rats. After Sandhimitra Vati compounds treated the levels of these enzymes were significantly (P< 0.01) decreased in the entire group as compared to control rats. SAM-001-D2 treatment prevented biochemical changes to a greater extent than the arthritic control. The MDA level shows non-significant changes in all the groups compared to arthritic control (Table 6).
Table 6: Effect of SAM samples on biochemical parameters in Freund’s adjuvant induced arthritis in albino rats
| Groups | Total protein (g/dl) | Albuminutes (g/dl) | LDH
(U/L) |
Creatinine (mg/dl) | Urea
(mg/dl) |
SGOT
(U/L) |
SGPT
(U/L) |
MDA
(n.mol/ml) |
| Arthritis control
(1% CMC ) |
6.43 ±
0.21 |
2.01 ±
0.27 |
459.9 ±
5.28 |
0.75 ±
0.034 |
42.16 ±
1.62 |
189.5 ±
5.05 |
107.33 ±
1.66 |
5.88 ±
0.48 |
| Diclofenac sodium
(15 mg/kg, p.o.) |
7.71 ±
0.17** |
2.8 ±
0.42ns |
415.66 ±
4.54** |
0.61 ±
0.04 ns |
31.33 ±
1.99** |
119.66 ±
2.66** |
86.5 ±
2.14** |
1.16 ±
0.13 ns |
| SAM-001-D2 Sandhimitra Vati
(540 mg/kg, p.o.) |
7.96 ±
0.21** |
3.63 ±
0.43** |
336.25 ±
4.07** |
0.51 ±
0.06** |
27.33 ±
1.11** |
132.66 ±
6.32** |
54.0 ±
2.30** |
1.56 ±
0.58 ns |
| Market Brand 1
(270 mg/kg, p.o.) |
7.61 ±
0.08** |
2.66 ±
0.42 ns |
343.76 ±
2.60** |
0.63 ±
0.03 ns |
39.5 ±
1.05 ns |
168.33 ±
2.77** |
81.5 ±
2.56** |
2.73 ±
0.53 ns |
| Market Brand 2
(162 mg/kg, p.o.) |
7.68 ±
0.08** |
2.75 ±
0.07 ns |
385.65 ±
6.23** |
0.6 ±
0.06 ns |
29.83 ±
1.37** |
150.0 ±
2.85** |
74.5 ±
2.23** |
4.76 ±
.0.24 ns |
All the values were expressed in Mean ± SEM (n=6). The statistical analysis was carried out using one way ANOVA. Significant after analysis of variance (ANOVA) followed by Dunnett multiple comparison test. ns– Non significant, **P<0.01 when compared to arthritic control group
Conclusion:
Sandhimitra Vati exhibited a significant increase in analgesic activity by acetic acid induced and hot plate induced thermal pain compared with Market brand 1 and Market brand 2. In addition, Sandhimitra Vati achieved maximum percentage protection against carrageenan induced paw oedema compared with Market brand 1 and Market brand 2. According to our findings, Sandhimitra Vati showed significant reduction in rat paw oedema volume and it could also normalize the haematological and biochemical abnormalities in both developing and developed phases of FCA induced arthritis compared to Market brand 1 and Market brand 2. This can be a very effective remedy and further studies are needed in this direction to evaluate the efficacy of Sandhimitra Vati. This will clinically establish the efficacy with regard to lower back pain due to lumbar osteoarthritis, disk degeneration, degenerative disk disease, and spondylosis.