Antioxidative mechanism involved in the preventive efficacy of Bergenia ciliata rhizomes against experimental nephrolithiasis in rats

Figures & data

Figure 1. (A) HPLC chromatographic profiles of standard gallic acid sample. (B) HPLC chromatographic profiles of hydro-methanol extract of Bergenia ciliata rhizomes.

Table 1. Mean peak areas obtained for gallic acid from five calibration curves containing nine calibration standards (CS, 1–9).

Concentration (µg/mL)	Mean area ± SD
CS-1 (2.0)	37 920 ± 147
CS-2 (4.0)	75 901 ± 275
CS-3 (8.0)	151 562 ± 393
CS-4 (16.0)	303 222 ± 576
CS-5 (32.0)	607 045 ± 1417
CS-6 (64.0)	1 215 906 ± 3139
CS-7 (100)	1 896 291 ± 5499
CS-8 (150)	2 844 536 ± 9102
CS-9 (200)	3 796 582 ± 18 632

Table 2. Intra-day and inter-day precision and accuracy for gallic acid.

		Intra-day				Inter-day
QC level	Nominal conc. (µg/mL)	n	Mean conc. found (µg/mL)^a	% CV	% Accuracy	n	Mean conc. found (µg/mL)^b	% CV	% Accuracy
HQC	175	3	174.1	0.9	99.5	15	175.7	1.3	100.4
MQC	50.0	3	49.6	1.2	99.3	15	50.1	1.1	100.2
LQC	5.50	3	5.42	1.0	98.6	15	5.52	1.4	100.4

CV, coefficient of variation; n, total number of observations.

^aMean of three replicates at each concentration.

^bMean of three replicates for five precision and accuracy batches.

Table 3. Recovery results of gallic acid added to pre-analyzed samples (n = 3).

Original (µg/mg)	Amount added (µg/mg)	Amount found (µg/mg)	% Mean recovery (% CV)
85.0	68.0	153.2	100.1 (0.32)
85.0	85.0	168.9	99.4 (0.41)
85.0	102	185.5	99.2 (0.52)

Figure 2. Light microscopic images of calcium oxalate crystals in urine. (A) Control, (B) nephrolithic, (C) prophylactic treatment with cystone (150 mg/kg), (D) prophylactic treatment with cystone (300 mg/kg), (E) prophylactic treatment with extract of B. ciliata (150 mg/kg), and (F) prophylactic treatment with extract of B. ciliata (300 mg/kg).

Table 4. Prophylactic effect of oral administration of B. ciliata on biochemical parameters in urine.

I. Untreated control	II. Cystone control (CST)	III. B. ciliata (BCE)	IV. Ethylene glycol (EG)	V. EG + CST150	VI. EG + CST300	VII. EG + BCE150	VIII. EG + BCE300
Calcium (mg/dL) 14th day
6.67 ± 0.05	6.72 ± 0.07	6.75 ± 0.05	12.45 ± 0.49^a*	10.72 ± 0.17^b*	8.56 ± 0.16^b*	9.96 ± 0.12^b*	7.76 ± 0.08^b*
Calcium (mg/dL) 28th day
6.81 ± 0.03	6.78 ± 0.04	6.72 ± 0.02	15.32 ± 0.25^a*	12.33 ± 0.14^b*	9.19 ± 0.07^b*	10.83 ± 0.23^b*	7.96 ± 0.16^b*
Oxalate (mg/dL) 14th day
26.11 ± 0.70	26.37 ± 0.79	26.61 ± 0.65	46.14 ± 0.94^a*	42.20 ± 0.79^b†	34.06 ± 0.90^b*	37.88 ± 0.85^b*	30.04 ± 0.76^b*
Oxalate (mg/dL) 28th day
26.56 ± 0.41	26.42 ± 0.68	26.58 ± 0.44	91.43 ± 0.69^a*	72.22 ± 0.35^b*	45.03 ± 0.76^b*	53.38 ± 0.61^b*	31.60 ± 0.50^b*
Phosphate (mg/dL) 14th day
18.28 ± 0.02	18.32 ± 0.03	18.50 ± 0.02	73.30 ± 0.95^a*	60.11 ± 0.52^b*	38.30 ± 0.50^b*	45.30 ± 0.50^b*	21.20 ± 0.35^b*
Phosphate (mg/dL) 28th day
18.35 ± 0.04	18.42 ± 0.05	18.38 ± 2.35	137.63 ± 1.35^a*	105.11 ± 0.74^b*	65.50 ± 0.33^b*	80.40 ± 0.51^b*	26.80 ± 0.16^b*
Magnesium (mg/dL) 14th day
6.42 ± 0.02	6.34 ± 0.02	6.46 ± 0.02	3.72 ± 0.67^a*	4.42 ± 0.05	5.28 ± 0.03^b#	4.82 ± 0.07	5.94 ± 0.05^b*
Magnesium (mg/dL) 28th day
6.30 ± 0.02	6.37 ± 0.03	6.31 ± 0.04	1.43 ± 0.08^a*	3.02 ± 0.27^b#	4.90 ± 0.14^b*	3.85 ± 0.26^b*	6.10 ± 0.16^b*

Results are expressed as mean ± SEM; n = 5. No significant difference was noted between groups I and III.

^aAs compared between untreated control (group I) and toxin treated (group IV).

^bAs compared between toxin treated (group V) and toxin + antidote treated (groups V–VIII).

Level of significance †p < 0.05, #p < 0.01, *p < 0.001.

Table 5. Prophylactic effect of oral administration of B. ciliata on biochemical parameters of serum.

I. Untreated control	II. Cystone control	III. B. ciliata (BCE)	IV. Ethylene glycol (EG)	V. EG + SD150	VI. EG + SD300	VII. EG + BCE150	VIII. EG + BCE300
Calcium (mg/dL) 14th day
10.40 ± 0.07	10.54 ± 0.05	10.87 ± 0.05	6.14 ± 0.25^a*	6.90 ± 0.12^b#	8.17 ± 0.05^b*	7.50 ± 0.20^b*	9.41 ± 0.06^b*
Calcium (mg/dL) 28th day
10.60 ± 0.05	10.75 ± 0.03	10.28 ± 0.07	3.40 ± 0.70^a*	5.02 ± 0.11^b#	7.10 ± 0.07^b*	5.90 ± 0.20^b*	9.20 ± 0.05^b*
Phosphate (mg/dL) 14th day
3.71 ± 0.02	3.50 ± 0.02	3.66 ± 0.01	6.46 ± 0.16^a*	6.01 ± 0.26	5.21 ± 0.15^b#	5.11 ± 0.25^b#	4.10 ± 0.16^b*
Phosphate (mg/dL) 28th day
3.77 ± 0.05	3.70 ± 0.08	3.14 ± 0.05	8.87 ± 0.52^a*	7.50 ± 0.42	6.62 ± 0.26^b#	6.15 ± 0.51^b*	4.21 ± 0.32^b*
Sodium (mmol/L) 14th day
149.40 ± 0.34	149.74 ± 0.47	148.80 ± 0.32	128.48 ± 0.75^a*	132.97 ± 0.62^b*	138.94 ± 0.16^b*	137.45 ± 0.71^b*	146.41 ± 0.26^b*
Sodium (mmol/L) 28th day
149.20 ± 0.25	149.80 ± 0.53	150.71 ± 0.70	117.86 ± 1.51^a*	123.84 ± 1.56^b*	132.79 ± 0.89^b*	131.30 ± 1.20^b*	143.23 ± 0.41^b*
Potassium (mmol/L) 14th day
5.54 ± 0.01	5.33 ± 0.02	5.69 ± 0.03	8.08 ± 0.16^a*	7.32 ± 0.06^b*	6.65 ± 0.05^b*	6.86 ± 0.15^b*	5.80 ± 0.06^b*
Potassium (mmol/L) 28th day
5.62 ± 0.02	5.75 ± 0.05	5.23 ± 0.03	10.48 ± 0.62^a*	8.94 ± 0.08^b*	7.24 ± 0.06^b*	8.02 ± 0.08^b*	5.87 ± 0.02^b*
Magnesium (mg/dL) 14th day
3.80 ± 0.05	3.25 ± 0.03	3.65 ± 0.02	2.04 ± 0.14^a*	2.48 ± 0.09^b†	3.02 ± 0.05^b*	2.91 ± 0.06^b*	3.65 ± 0.08^b*
Magnesium (mg/dL) 28th day
3.72 ± 0.02	3.89 ± 0.01	3.54 ± 0.05	1.05 ± 0.07^a*	1.76 ± 0.12^b*	2.69 ± 0.05^b*	2.24 ± 0.11^b*	3.62 ± 0.05^b*

Results are expressed as mean ± SEM; n = 5. No significant difference was noted between groups I and III.

^aAs compared between untreated control (group I) and toxin treated (group IV).

^bAs compared between toxin treated (group V) and toxin + antidote treated (groups V–VIII).

Level of significance †p < 0.05, #p < 0.01, *p < 0.001.

Table 6. Prophylactic effect of oral administration of B. ciliata on biochemical parameters in kidney of rats.

I. Untreated control	II. Cystone control (CST)	III. D. biflorus (DBE)	IV. Ethylene glycol (EG)	V. EG + CST150	VI. EG + CST300	VII. EG + BCE150	VIII. EG + BCE300
Calcium (mg/gm tissue weight)
1.08 ± 0.02	1.06 ± 0.01	1.12 ± 0.02	3.95 ± 0.20^a*	2.95 ± 0.02^b*	2.05 ± 0.06^b*	2.42 ± 0.06^b*	1.25 ± 0.07^b*
Oxalate (mg/gm tissue weight)
1.94 ± 0.02	1.88 ± 0.02	1.90 ± 0.01	5.65 ± 0.15^a*	4.40 ± 0.11^b*	3.28 ± 0.07^b*	3.66 ± 0.17^b*	2.12 ± 0.05^b*
Phosphate (mg/gm tissue weight)
2.27 ± 0.01	2.32 ± 0.01	2.36 ± 0.02	5.03 ± 0.24^a*	4.26 ± 0.05^b*	2.46 ± 0.02^b*	3.60 ± 0.07^b*	2.48 ± 0.02^b*
Lipid peroxidation (nmoles MDA/mg protein/60 min)
56.20 ± 0.66	56.60 ± 0.22	56.41 ± 0.43	179.60 ± 1.56^a*	140.40 ± 1.26^b*	95.40 ± 1.12^b*	95.80 ± 1.59^b*	58.60 ± 1.11^b*
Total protein (mg/gm tissue weight)
242.78 ± 0.24	242.24 ± 0.26	242.62 ± 0.21	341.62 ± 1.33^a*	307.70 ± 0.79^b*	271.74 ± 0.46^b*	291.70 ± 0.60^b*	250.88 ± 0.36^b*
Reduced glutathione (µg/100 mg tissue weight)
21.06 ± 0.27	21.14 ± 0.20	21.30 ± 0.21	7.70 ± 0.62^a*	12.12 ± 0.45^b*	15.08 ± 0.39^b*	15.34 ± 0.49^b*	20.77 ± 0.33^b*
Total ascorbic acid (mg/gm tissue weight)
115.20 ± 0.30	115.56 ± 0.44	115.61 ± 0.41	20.04 ± 1.16^a*	54.32 ± 0.66^b*	86.14 ± 0.26^b*	67.12 ± 0.77^b*	110.23 ± 0.23^b*
Catalase (µmoles H2O2 consumed/mg protein/min)
17.84 ± 0.10	17.53 ± 0.07	17.95 ± 0.08	6.60 ± 0.35^a*	9.28 ± 0.21^b*	13.77 ± 0.11^b*	12.06 ± 0.33^b*	17.50 ± 0.27^b*
Superoxide dismutase (units/mg protein)
3.04 ± 0.02	3.07 ± 0.03	3.42 ± 0.01	0.86 ± 0.02^a*	1.37 ± 0.02^b*	1.98 ± 0.02^b*	1.85 ± 0.03^b*	2.91 ± 0.02^b*
Glutathione peroxidase (nmoles NADPH consumed/mg protein/min)
18.22 ± 0.10	18.44 ± 0.15	18.24 ± 0.14	5.54 ± 0.44^a*	9.12 ± 0.25^b*	12.40 ± 0.15^b*	11.42 ± 0.25^b*	17.50 ± 0.16^b*
Glutathione reductase (nmoles NADPH consumed/mg protein/min)
45.20 ± 0.50	45.36 ± 0.51	45.42 ± 0.35	17.60 ± 0.76^a*	27.92 ± 0.80^b*	34.74 ± 0.70^b*	32.30 ± 0.65^b*	43.94 ± 0.53^b*

Results are expressed as mean ± SEM; n = 5. No significant difference was noted between groups I and III.

^aAs compared between untreated control (group I) and toxin treated (group IV).

^bAs compared between toxin treated (group V) and toxin + antidote treated (groups V–VIII).

Level of significance *p < 0.001.

Figure 3. Light microscopic architecture and calcification in the kidney section with von Kossa stained: ×100 of (A) control, (B) nephrolithic, (C) prophylactic treatment with cystone (300 mg/kg), and (D) prophylactic treatment with extract of B. ciliata (300 mg/kg).

Figure 4. Light microscopic architecture and calcification in the kidney section with Pizzalato stained: ×100 of (A) control, (B) nephrolithic, (C) prophylactic treatment with cystone (300 mg/kg), and (D) prophylactic treatment with extract of B. ciliata (300 mg/kg).