Background
Methods
Plant material
Preparation of extract
Chemicals
Animals
Pharmacological investigations
Evaluation of anti-arthritic effect of Berberis orthobotrys on inhibition of protein denaturation using bovine serum albumin (BSA)
Evaluation of anti-arthritic effect of Berberis orthobotrys on inhibition of protein denaturation using egg albumin
Evaluation of anti-arthritic effect of Berberis orthobotrys on human red blood cell (HRBC) membrane stabilization
Evaluation of anti-arthritic activity of B. orthobotrys against turpentine oil induced joint edema in rats
Evaluation of anti-arthritic activity of B. orthobotrys against formaldehyde induced arthritis in rats
Evaluation of anti-arthritic activity of B. orthobotrys against complete Freund adjuvant (CFA) induced arthritis in rats
Anti-oxidant activity
Evaluation of reducing power of B. orthobotrys
Phytochemical investigations of n-butanol fraction
Total flavonoids content (TFC)
Fourier transformed infrared (FTIR) spectroscopic analysis
High performance liquid chromatographic (HPLC) analysis
Statistical analysis
Results
Effect of B.orthobotrys on inhibition of protein denaturation using BSA
Effect of B.orthobotrys on protein denaturation using fresh hen’s egg albumin
Effect of B.orthobotrys on membrane stabilization
Effect of B.orthobotrys against turpentine oil induced joint edema
Treatment groups | Increase in joint diameter (mm) | |||||
---|---|---|---|---|---|---|
1 h | 2 h | 3 h | 4 h | 5 h | 6 h | |
Arthritic control (3 ml/kg) | 9.44 ± 0.028 | 10.59 ± 0.022 | 12.34 ± 0.023 | 14.98 ± 0.027 | 16.38 ± 0.028 | 20.96 ± 0.028 |
Standard Aspirin (100 mg/kg) | 7.02 ± 0.026*** (25.63) | 6.83 ± 0.026*** (35.50) | 6.58 ± 0.035*** (46.67) | 6.37 ± 0.024*** (57.34) | 6.25 ± 0.028*** (61.84) | 6.02 ± 0.027*** (71.27) |
Aqueous-methanolic extract (50 mg/kg) | 7.40 ± 0.021*** (21.61) | 7.15 ± 0.021*** (32.48) | 6.95 ± 0.030*** (43.67) | 6.87 ± 0.026*** (54.13) | 6.71 ± 0.026*** (59.03) | 6.63 ± 0.024*** (68.36) |
Aqueous-methanolic extract (100 mg/kg) | 7.19 ± 0.021*** (23.83) | 6.87 ± 0.027*** (35.12) | 6.61 ± 0.022*** (46.43) | 6.54 ± 0.021*** (56.34) | 6.33 ± 0.026*** (61.35) | 6.10 ± 0.024*** (70.89) |
Aqueous-methanolic extract (150 mg/kg) | 6.84 ± 0.026*** (27.54) | 6.58 ± 0.025*** (37.86) | 6.31 ± 0.024*** (48.86) | 6.14 ± 0.022*** (59.01) | 5.82 ± 0.023*** (64.46) | 5.50 ± 0.025*** (73.75) |
n-butanol fraction (50 mg/kg) | 7.75 ± 0.018*** (17.90) | 7.68 ± 0.019*** (27.47) | 8.33 ± 0.024*** (32.49) | 8.29 ± 0.029*** (44.65) | 8.24 ± 0.029*** (49.69) | 8.19 ± 0.027*** (60.92) |
n-butanol fraction (100 mg/kg) | 7.51 ± 0.023*** (20.44) | 7.48 ± 0.023*** (29.36) | 7.84 ± 0.021*** (36.46) | 7.76 ± 0.027*** (48.19) | 7.70 ± 0.025*** (52.99) | 7.65 ± 0.019*** (63.50) |
n-butanol fraction (150 mg/kg) | 7.32 ± 0.021*** (22.45) | 7.20 ± 0.021*** (32.01) | 7.39 ± 0.021*** (40.11) | 7.21 ± 0.021*** (51.86) | 7.04 ± 0.028*** (57.02) | 6.90 ± 0.023*** (67.08) |
Ethyl acetate fraction (50 mg/kg) | 7.96 ± 0.029*** (15.67) | 7.86 ± 0.028*** (25.77) | 8.44 ± 0.028*** (31.60) | 8.41 ± 0.031*** (43.85) | 8.05 ± 0.020*** (50.85) | 9.10 ± 0.023*** (56.58) |
Ethyl acetate fraction (100 mg/kg) | 7.74 ± 0.021*** (18.00) | 7.71 ± 0.026*** (27.19) | 7.96 ± 0.027*** (35.49) | 7.80 ± 0.028*** (47.93) | 7.75 ± 0.025*** (52.68) | 8.40 ± 0.021*** (59.92) |
Ethyl acetate fraction (150 mg/kg) | 7.43 ± 0.027*** (21.29) | 7.40 ± 0.026*** (30.12) | 7.73 ± 0.021*** (37.35) | 7.48 ± 0.017*** (50.06) | 7.22 ± 0.025*** (55.92) | 7.70 ± 0.030*** (63.26) |
Aqueous fraction (50 mg/kg) | 8.24 ± 0.023*** (12.71) | 8.11 ± 0.026*** (23.41) | 8.60 ± 0.026*** (30.30) | 8.92 ± 0.030*** (40.25) | 9.13 ± 0.027*** (44.26) | 9.58 ± 0.025*** (54.29) |
Aqueous fraction (100 mg/kg) | 7.98 ± 0.024*** (15.46) | 7.77 ± 0.026*** (26.62) | 8.10 ± 0.026*** (34.35) | 8.45 ± 0.027*** (43.59) | 7.78 ± 0.023*** (52.50) | 8.99 ± 0.025*** (57.10) |
Aqueous fraction (150 mg/kg) | 7.62 ± 0.021*** (19.27) | 7.54 ± 0.021*** (28.80) | 7.69 ± 0.028*** (37.68) | 7.95 ± 0.023*** (46.92) | 7.23 ± 0.033*** (55.86) | 8.30 ± 0.027*** (60.40) |
Effect of B.orthobotrys against formaldehyde induced arthritis
Treatment groups | Increase in paw diameter (mm) | ||||
---|---|---|---|---|---|
Day 2 | Day 4 | Day 6 | Day 8 | Day 10 | |
Arthritic control (3 ml/kg) | 8.49 ± 0.029 | 10.62 ± 0.023 | 12.56 ± 0.021 | 14.08 ± 0.020 | 16.92 ± 0.022 |
Standard Aspirin (100 mg/kg) | 4.61 ± 0.021*** (45.70) | 4.95 ± 0.023*** (53.38) | 4.07 ± 0.019*** (67.59) | 3.77 ± 0.028*** (73.22) | 3.63 ± 0.033*** (78.54) |
Aqueous-methanolic extract (50 mg/kg) | 4.76 ± 0.035*** (43.93) | 5.35 ± 0.026*** (49.62) | 4.41 ± 0.018*** (64.88) | 4.12 ± 0.021*** (70.73) | 4.09 ± 0.019*** (75.82) |
Aqueous-methanolic extract (100 mg/kg) | 4.51 ± 0.015*** (46.87) | 4.93 ± 0.023*** (53.57) | 4.10 ± 0.028*** (67.35) | 3.72 ± 0.029*** (73.57) | 3.60 ± 0.027*** (78.72) |
Aqueous-methanolic extract (150 mg/kg) | 4.28 ± 0.011*** (49.58) | 4.63 ± 0.016*** (56.40) | 3.63 ± 0.030*** (71.09) | 3.28 ± 0.023*** (76.70) | 3.03 ± 0.017*** (82.09) |
n-butanol fraction (50 mg/kg) | 4.96 ± 0.019*** (41.57) | 5.46 ± 0.020*** (48.58) | 4.85 ± 0.025*** (61.38) | 4.65 ± 0.024*** (66.97) | 4.54 ± 0.027*** (73.16) |
n-butanol fraction (100 mg/kg) | 4.68 ± 0.023*** (44.87) | 5.11 ± 0.021*** (51.88) | 4.41 ± 0.021*** (64.88) | 4.13 ± 0.021*** (70.66) | 4.02 ± 0.021*** (76.24) |
n-butanol fraction (150 mg/kg) | 4.38 ± 0.026*** (48.40) | 4.28 ± 0.020*** (59.69) | 3.68 ± 0.023*** (70.70) | 3.55 ± 0.021*** (74.78) | 3.30 ± 0.023*** (80.49) |
Ethyl acetate fraction (50 mg/kg) | 5.40 ± 0.033*** (36.39) | 5.70 ± 0.022*** (46.32) | 5.32 ± 0.030*** (57.64) | 5.00 ± 0.021*** (64.48) | 4.91 ± 0.022*** (70.98) |
Ethyl acetate fraction (100 mg/kg) | 5.25 ± 0.021*** (38.16) | 5.50 ± 0.021*** (48.21) | 4.75 ± 0.017*** (62.18) | 4.54 ± 0.022*** (67.75) | 4.48 ± 0.022*** (73.52) |
Ethyl acetate fraction (150 mg/kg) | 4.75 ± 0.020*** (44.05) | 5.05 ± 0.017*** (52.44) | 4.37 ± 0.024*** (65.20) | 4.00 ± 0.021*** (71.59) | 3.85 ± 0.022*** (77.24) |
Aqueous fraction (50 mg/kg) | 5.88 ± 0.022*** (30.74) | 6.13 ± 0.021*** (42.27) | 5.71 ± 0.024*** (54.53) | 5.37 ± 0.022*** (61.86) | 5.25 ± 0.024*** (68.97) |
Aqueous fraction (100 mg/kg) | 5.43 ± 0.024*** (36.04) | 5.96 ± 0.030*** (43.87) | 5.18 ± 0.027*** (58.75) | 4.85 ± 0.028*** (65.55) | 4.75 ± 0.027*** (71.92) |
Aqueous fraction (150 mg/kg) | 4.98 ± 0.023*** (41.34) | 5.58 ± 0.027*** (47.45) | 4.47 ± 0.024*** (64.41) | 4.25 ± 0.021*** (69.81) | 4.20 ± 0.022*** (75.17) |
Effect of B.orthobotrys against CFA induced arthritis
Treatments | Increase in paw diameter (mm) | |||
---|---|---|---|---|
Day 1 | Day 5 | Day 10 | Day 15 | |
Arthritic Control (3 ml/kg) | 7.21 ± 0.035 | 16.22 ± 0.029 | 20.50 ± 0.024 | 23.72 ± 0.023 |
Normal Control | 4.19 ± 0.160*** | 4.24 ± 0.166*** | 4.24 ± 0.097*** | 4.27 ± 0.079*** |
Standard Aspirin (100 mg/kg) | 5.90 ± 0.034*** (18.16) | 7.29 ± 0.030*** (55.05) | 6.34 ± 0.029*** (69.07) | 5.83 ± 0.029*** (75.42) |
Aqueous-methanolic extract (150 mg/kg) | 5.13 ± 0.029*** (28.84) | 6.63 ± 0.029*** (59.12) | 5.69 ± 0.027*** (72.24) | 4.51 ± 0.033*** (80.98) |
n-butanol fraction (150 mg/kg) | 5.37 ± 0.030*** (25.52) | 6.97 ± 0.033*** (57.02) | 5.75 ± 0.029*** (71.95) | 5.16 ± 0.032*** (78.24) |
Ethyl acetate fraction (150 mg/kg) | 5.51 ± 0.030*** (23.57) | 7.68 ± 0.044*** (52.65) | 6.47 ± 0.032*** (68.43) | 5.55 ± 0.039*** (76.60) |
Aqueous fraction (150 mg/kg) | 5.82 ± 0.038*** (19.27) | 8.22 ± 0.034*** (49.32) | 6.95 ± 0.045*** (66.09) | 6.02 ± 0.042*** (74.62) |
Treatments | Body weight gain (g) | |||
---|---|---|---|---|
Day 1 | Day 5 | Day 10 | Day 15 | |
Arthritic Control (3 mL/kg) | 262 ± 3.647 | 223 ± 3.906 | 218 ± 3.555 | 208 ± 4.534 |
Normal Control | 269 ± 6.038ns
| 270 ± 5.544*** | 270 ± 5.955*** | 274 ± 6.112*** |
Standard Aspirin (100 mg/kg) | 243 ± 3.040** | 246 ± 3.597*** | 243 ± 2.926*** | 245 ± 3.776*** |
Aqueous-methanolic extract (150 mg/kg) | 265 ± 3.479ns
| 269 ± 4.176*** | 277 ± 4.179*** | 285 ± 4.320*** |
n-butanol fraction (150 mg/kg) | 259 ± 4.841ns
| 260 ± 5.109*** | 261 ± 4.831*** | 263 ± 4.802*** |
Ethyl acetate fraction (150 mg/kg) | 250 ± 2.922ns
| 251 ± 2.839*** | 252 ± 2.379*** | 253 ± 2.498*** |
Aqueous fraction (150 mg/kg) | 242 ± 2.205** | 242 ± 2.227** | 243 ± 2.577*** | 246 ± 2.782*** |
Hematological parameter | Arthritic control | Normal control | Standard (Aspirin) | Aqueous- methanolic extract | n-butanol fraction | Ethyl acetate fraction | Aqueous fraction |
---|---|---|---|---|---|---|---|
Hb (g/dl) | 9.36 ± 0.145 | 14.26 ± 0.240***
| 12.60 ± 0.058***
| 13.40 ± 0.115***
| 13.36 ± 0.176***
| 12.63 ± 0.133***
| 11.70 ± 0.115***
|
RBCs (× 106 / μl) | 4.97 ± 0.079 | 7.42 ± 0.266***
| 6.72 ± 0.133***
| 6.80 ± 0.038***
| 6.53 ± 0.104***
| 6.30 ± 0.038***
| 5.78 ± 0.059**
|
WBCs (× 103 / μl) | 9.43 ± 0.203 | 5.23 ± 0.120***
| 7.70 ± 0.208***
| 6.20 ± 0.265***
| 6.80 ± 0.346***
| 7.50 ± 0.208***
| 8.50 ± 0.058*
|
Platelets (× 103 / μl) | 1225.00 ± 105.39 | 311.00 ± 3.215***
| 732.33 ± 4.631***
| 394.00 ± 3.512***
| 492.33 ± 3.528***
| 591.66 ± 2.728***
| 692.00 ± 3.786***
|
ESR (mm / 1st h) | 20.33 ± 0.882 | 3.00 ± 0.577***
| 12.33 ± 1.202***
| 9.00 ± 0.577***
| 11.00 ± 0.577***
| 12.33 ± 0.882***
| 15.00 ± 0.577**
|
RF (IU / ml) | 48.33 ± 2.028 | 14.00 ± 0.00***
| 25.33 ± 2.333***
| 20.00 ± 1.155***
| 25.66 ± 0.882***
| 32.66 ± 1.453***
| 35.33 ± 1.764***
|
Reducing power of B.orthobotrys
Total flavonoid contents in n-butanol fraction of B.orthobotrys
FTIR analysis of B.orthobotrys n-butanol fraction
Peak | Bond | Functional group |
---|---|---|
3354.21 cm−1 - 3334.92 cm−1
| OH Stretching vibrations | Phenols |
2956.87 cm−1 – 2872.01 cm−1
| CH Stretching vibrations alkanes | Alkanes |
1722.43 cm−1
| C = 0 carboxylic acid stretching vibrations | Carboxylic acids |
1459.76 cm−1
| CH3 bending vibrations | Methyl |
1384.89 cm−1 – 1367.53 cm−1
| C-O Stretching vibrations | Carbonyls |
1037.7 cm−1
| C-F stretching vibrations | Carbon fluorine |
Phenolic profile of B.orthobotrys n-butanol fraction
Peak No. | RT (min) | Area Count | Amount (ppm) | Compounds |
---|---|---|---|---|
4 | 2.887 | 21.493 | 1.13 | Quercetin |
6 | 4.927 | 335.728 | 12.08 | Gallic acid |
12 | 12.767 | 305.143 | 14.03 | Caffeic acid |
13 | 17.153 | 297.613 | 3.86 | p-coumaric acid |
14 | 20.760 | 340.918 | 3.56 | M-coumaric acid |
15 | 21.987 | 152.704 | 10.94 | Ferulic acid |
17 | 25.280 | 197.301 | 4.53 | Trans-4-hydroxy-3-methoxy cinnamic acid |
18 | 26.713 | 278.642 | 3.61 | Sinapic acid |