In vitro and ex vivo approach for anti-urolithiatic potential of bioactive fractions of gokhru with simultaneous HPLC analysis of six major metabolites and their exploration in rat plasma

Figures & data

Table 1. Validation data of developed method for catechin, diosgenin, gallic acid, quercetin, rutin and tannic acid.

S. No.	Metabolites	Linearity range (μg/mL)	Regression equation	Coefficient ofcorrelation	LOD (μg/mL)	LOQ (μg/mL)
1	Catechin	0.05–100	Y = 30539.0X + 3962.7	0.98	0.08	0.24
2	Diosgenin	0.1–100	Y = 2857.9X + 2213.6	0.98	0.17	0.51
3	Gallic acid	1–100	Y = 330.8X − 2222.8	0.98	1.05	3.2
4	Quercetin	0.1–100	Y = 2825.6X + 5561.1	0.99	0.38	1.15
5	Rutin	1–100	Y = 1744.6X + 806.1	0.99	0.91	2.78
6	Tannic acid	1–100	Y = 1816.4X − 953.8	0.99	0.21	0.63

Table 2. Precision and recovery data of developed method for catechin, diosgenin, gallic acid, quercetin, rutin and tannic acid.

Metabolite	Concentration(μg/mL)	Inter-day		Intra-day		Percentage of recovery
		Mean peak area ± SEM	%CV	Mean peak area ± SEM	%CV	Mean % of recovery ± SEM	%CV
		Catechin	1	175466.3 ± 211	0.2	175499.7 ± 0.19	0.19	98.33 ± 0.33	0.58
	40	985346.0 ± 235.2	0.04	985446.0 ± 253.5	0.04	98.64 ± 0.32	0.56
	80	2516146.0 ± 285.7	0.01	2516146.0 ± 230.1	0.01	97.32 ± 0.33	0.60
Diosgenin	1	14381.3 ± 100.6	1.20	14481.3 ± 80.2	0.95	98.16 ± 0.16	0.29
	40	139673.0 ± 216.3	0.26	139773.0 ± 88.1	0.10	98.56 ± 0.08	0.15
	80	249698.0 ± 60.9	0.04	249786.3 ± 51.1	0.03	98.31 ± 0.07	0.13
Gallic acid	10	2599.0 ± 42.5	2.83	2551.0 ± 27.1	1.83	98.50 ± 0.05	0.10
	40	9883.0 ± 23.8	0.41	9937.6 ± 33.4	0.58	98.23 ± 0.08	0.15
	80	23626.3 ± 113.7	0.83	23953.0 ± 41.1	0.29	98.28 ± 0.16	0.28
Quercetin	1	14267.3 ± 44.3	0.53	14300.6 ± 77.6	0.94	98.83 ± 0.16	0.29
	40	112741.3 ± 41.8	0.06	112708.0 ± 35.6	0.05	98.59 ± 0.05	0.08
	80	228678.3 ± 52.3	0.03	228745.0 ± 30.0	0.02	98.40 ± 0.11	0.20
Rutin	10	20524.6 ± 99.1	0.83	20534.6 ± 95.9	0.80	97.40 ± 0.66	1.10
	40	63989.0 ± 72.02	0.19	63965.6 ± 94.2	0.20	97.04 ± 0.48	0.80
	80	146348.0 ± 87.8	0.10	146348.0 ± 66.5	0.10	98.41 ± 0.05	0.09
Tannic acid	10	20603.3 ± 54.4	0.45	20480.3 ± 117.0	0.98	98.80 ± 0.21	0.36
	40	64877.3 ± 50.8	0.13	64947.3 ± 81.02	0.21	98.65 ± 0.15	0.26
	80	146273.7 ± 49.9	0.05	146307.0 ± 0.07	0.14	98.92 ± 0.17	0.31

Data are expressed as mean ± SEM, (n = 6).

Figure 1. Schematic representation of extraction and fractionation of aqueous extract (mother extract) of gokhru showing extractive values.

Table 3. Percentage yield of extract and metabolite content in different extract of gokhru.

Extract/Fractions	Yield (%w/w)	Percentage in dried extract (%w/w)
		Catechin	Diosgenin	Gallic acid	Rutin	Tannic acid	Quercetin
		Mother extract	14.5 ± 0.25	3.52 ± 0.54	4.34 ± 0.55	0.97 ± 0.25	3.42 ± 0.24	5.85 ± 0.15	0.99 ± 0.24
Hexane fraction	2.1 ± 0.31^a	0.004 ± 0.014^a	0.12 ± 0.23^a	–^a	2.8 ± 0.32^c	0.3 ± 0.45^a	–^a
Toluene fraction	2.6 ± 0.78^a	0.72 ± 0.26^a	1.29 ± 0.47^a	0.18 ± 0.14^b	6.1 ± 0.51^a	3.46 ± 0.32^a	–^a
DCM fraction	1.5 ± 0.17^a	2.02 ± 0.94^a	1.23 ± 0.74^a	0.57 ± 0.47^ns	10.34 ± 0.44^a	7.18 ± 017^a	0.17 ± 0.32^b
n-Butanol fraction	1.3 ± 1.23^a	2.19 ± 0.47^a	12.75 ± 0.18^a	0.77 ± 0.33^ns	6.12 ± 0.36^a	15.81 ± 0.47^a	1.95 ± 0.41^a
Water	6.1 ± 0.66^a	5.34 ± 0.65^a	1.35 ± 0.61^a	2.03 ± 0.47^a	5.76 ± 0.27^a	11.17 ± 0.19^a	0.78 ± 0.19^ns

Data are expressed as mean ± SEM, (n = 6).

^a p < 0.001.

^b p < 0.01.

ns ^cp > 0.05 as compared to mother extract.

Figure 2. Effect of different concentrations of extract and fractions of gokhru on calcium oxalate crystallization (A) in synthetic urine and (B) in plasma.

Figure 3. SCIM image sequences of calcium oxalate precipitate showing no increase in crystal size in (A) mother extract (C) n-butanol fraction treated and (B) without drug treated reaction solution, (D) calcium oxalate growth curves exhibit the step-like crystals size with respect to incubation time

Table 4. Robustness of the developed method for catechin, diosgenin, gallic acid, quercetin, rutin and tannic acid.

Parameters		Amount metabolite spiked to plasma (μg/mL)	Mean % of recovery ± SEM
			Catechin	Diosgenin	Gallic acid	Quercetin	Rutin	Tannic acid
			Change in initial mobile phase composition (v/v) Initially, acetonitrile: 0.5% formic acid in water (100:0)	(5:95)	40	98.1 ± 0.31	96.5 ± 0.52	97.2 ± 0.31	95.5 ± 0.42	97.5 ± 0.25	96.8 ± 0.24
(10:90)	40	99.3 ± 0.42		98.8 ± 0.34	99.2 ± 0.44	98.2 ± 0.44	98.4 ± 0.43	98.5 ± 0.21
(15:85)	40	97.8 ± 0.39		97.2 ± 0.28	96.9 ± 0.28	96.5 ± 0.38	97.5 ± 0.62	96.8 ± 0.35
Change in column temperature (°C)	25	40	97.2 ± 0.24	97.8 ± 0.33	96.5 ± 0.26	96.8 ± 0.43	97.5 ± 0.41	96.5 ± 0.36
	30	40	98.8 ± 0.36	99.2 ± 0.66	98.7 ± 0.45	98.9 ± 0.65	98.5 ± 0.22	99.5 ± 0.45
	35	40	97.7 ± 0.62	96.9 ± 0.53	97.5 ± 0.28	97.5 ± 0.35	96.7 ± 0.35	97.8 ± 0.31
Change in flow rate (mL/min)	0.9	40	97.1 ± 0.53	96.4 ± 0.49	98.2 ± 0.45	97.5 ± 0.41	97.8 ± 0.17	97.5 ± 0.24
	1.0	40	98.4 ± 0.23	98.8 ± 0.51	99.2 ± 0.41	99.7 ± 0.15	98.4 ± 0.15	98.9 ± 0.17
	1.1	40	96.2 ± 0.41	97.2 ± 0.51	97.9 ± 0.44	98.2 ± 0.21	97.5 ± 0.24	96.8 ± 0.18

Data are expressed as mean ± SEM, (n = 6).

Figure 4. HPLC chromatogram of standard marker compounds at (A) 254 nm (tannic acid, rutin and quercetin) and at (B) 278 nm for gallic acid, catechin and diosgenin.

Table 5. System suitability of developed HPLC method for catechin, diosgenin, gallic acid, quercetin, rutin and tannic acid.

System suitability parameters	Proposed method
	Catechin	Diosgenin	Gallic Acid	Quercetin	Rutin	Tannic acid
	Retention time	9.1	19.6	5.1	21.4	13.6	11.0
Capacity factor	0.38	0.67	0.87	0.14	0.65	0.18
Theoretical plate	4338	2458	2987	8794	3547	4254
HETP (mm)	0.021	0.034	0.047	0.019	0.039	0.024
Peak asymmetry @ 10% height	1.15	1.56	1.32	2.15	1.89	1.12
Tailing factor	1.12	1.20	1.21	0.87	1.87	0.98

Figure 5. HPLC chromatogram of extract at (A) 254 and (B) 278 nm indicating the presence of marker metabolites (tannic acid, rutin and quercetin, gallic acid, catechin and diosgenin) in mother extract of gokhru.

Table 6. Liquid–liquid extraction of catechin, diosgenin, gallic acid, quercetin, rutin and tannic acid from serum.

Solvent used	Percentage recovery
	Catechin	Diosgenin	Gallic acid	Quercetin	Rutin	Tannic acid
	Solvent selection
Ethyl acetate	87.3 ± 0.12^a	91.2 ± 0.16^b	25.2 ± 0.16^a	85.3 ± 0.33^a	84.3 ± 0.31^b	41.2 ± 0.15^a
Methyl-tert-butyl ether	61.2 ± 0.21^a	82.3 ± 0.14^a	52.3 ± 0.25^a	48.2 ± 0.16^a	87.6 ± 0.33^b	58.3 ± 0.16^a
Ethyl acetate: isopropyl alcohol (1:1, v/v)	32.9 ± 0.14^a	61.7 ± 0.41^a	71.5 ± 0.31^b	47.3 ± 0.11^a	41.1 ± 0.41^a	34.2 ± 0.33^a
Acetonitril:methyl-tert-butyl-ether:ethyl acetate (1:1:1, v/v/v)	92.2 ± 0.34^a	95.4 ± 0.21^b	93.7 ± 0.25^a	94.6 ± 0.26^a	93.4 ± 0.42^a	96.5 ± 0.16^a
Methanol	85.6 ± 0.24^b	78.5 ± 0.32^a	72.9 ± 0.23^a	84.3 ± 0.24*	73.5 ± 0.33^a	68.2 ± 0.12^a
Preconditioning of solvent
Basification	37.3 ± 0.33^a	70.5 ± 0.33^a	48.2 ± 0.16^a	87.3 ± 0.32^b	82.7 ± 0.18^a	87.6 ± 0.25^b
Acidification	91.2 ± 0.17^a	38.4 ± 0.24^a	84.2 ± 0.13^b	21.5 ± 0.21^a	41.6 ± 0.17^a	41.2 ± 0.13^a
Basification and acidification	98.4 ± 0.32^a	96.2 ± 0.31^a	95.5 ± 0.17^a	96.5 ± 0.19^a	95.5 ± 0.25^a	95.6 ± 0.27^a

Data are expressed as mean ± SEM, (n = 6).

^a p < 0.001.

^b p < 0.0.

Figure 6. Optimized extraction method for extraction of metabolite marker from serum.