In vitro Evaluation of Terminalia arjuna on Calcium Phosphate and Calcium Oxalate Crystallization

Abstract

Urinary stones are one of the oldest and the most common afflictions in humans. This disease has tormented humans since the earliest records of civilization. Ten percent of men and 3 % of women have a stone during their adult lives. Calcium containing stones are the most common comprising about 75 % of all urinary calculi, which may be in the form of pure calcium oxalate (50 %) or calcium phosphate (5 %) or a mixture of both (45 %). A number of plants have been mentioned in the Indian ayurvedic system, which plays a vital role in the inhibition of kidney stones. In the present study, the inhibitory potency of crude extracts or fractions of successive solvent extractions of Terminalia arjuna bark was evaluated on various stages of formation of calcium phosphate and on the growth of calcium oxalate monohydrate crystals in vitro. Results obtained indicated that Terminalia arjuna bark has the potential to inhibit the formation of both calcium phosphate and calcium oxalate crystals in vitro. Butanol fraction of Terminalia arjuna extract was the most effective in inhibiting formation of calcium phosphate and calcium oxalate crystals in vitro.

Keywords: Calcium phosphate; TLC; Terminalia arjuna; calcium oxalate; saponin; urolithiasis.

Fig. 1

Effect of Terminalia arjuna extract on initial mineral phase formation of calcium phosphate (CaP) ions Percent inhibition of calcium (■) and phosphate (□) ions precipitation by aqueous extract of Terminalia arjuna in initial mineral phase formation. Volumes used of aqueous extract were 0.2, 0.4, 0.8, 1.0 and 1.5 ml. Maximum calcium ion inhibition of 48% was seen with 1.5 ml followed by 0.2, 1.0, 0.8 and 0.4 ml. However, for phosphate ions, maximum inhibition was found to be 68% with 0.2 ml followed by 1.5, 1.0, 0.4 and 0.8 ml

Fig. 2

Effect of Terminalia arjuna extract on growth of preformed mineral phase of calcium phosphate (CaP) ions Percentage inhibition of calcium (■) and phosphate (□) ions precipitation by aqueous extract of Terminalia arjuna in case of growth of preformed initial mineral phase formation. Here, maximum inhibition of calcium ion was at 1.5 ml (48%) followed by 1.0 (41%), 0.8 (37%), 0.4 (36%) and 0.2 ml (15%). Inhibition of phosphate ion was found to be maximum with 0.2 ml (50%) of extract followed by 1.540%), 0.8 (35%), 1.0 (29%) and 0.4 ml (24%).

Fig. 3

Effect of Terminalia arjuna extract on demineralization of calcium phosphate (CaP) ions Effect of aqueous extract of Terminalia arjuna on demineralization of calcium (■) and phosphate (□) ions in terms of percentage release of ions. In case of calcium maximum release was observed with 1.5 ml of the extract (197%). Release of calcium ions increased with the increase in the volume of the extract. Minimum calcium release was seen with 0.2 ml of aqueous extract which was 0%. In case of phosphate ions, again maximum release was observed with 1.5 ml of extract (380%) followed by 1.0 (243%), 0.8 (190%), 0.4 (38%) and least with 0.2 ml (2%).

Fig. 4

% inhibition of COM crystal growth by Terminalia arjuna extract Percentage inhibition of 20 % was found at 30 sec and 48% at 120 sec time interval when 15 μl of aqueous extract of Terminalia arjuna was used against growth of calcium oxalate monohydrate (COM) crystals.

Fig. 5

Inhibitory effect of Terminalia arjuna extract fractions on the initial mineral phase formation of calcium phosphate (CaP) ions Inhibitory activity of fractions obtained after performing successive solvent extraction against CaP initial mineral phase formation. Out of all the fractions obtained, hexane and dichloromethane fractions did not show any inhibition in calcium (■) and phosphate (□) ion precipitation. Ethyl acetate fraction showed 28.26% inhibition of calcium and 5 % inhibition of phosphorus in initial mineral phase formation. However, n-butanol fraction (0.1 ml) showed enhanced inhibition, 77% for calcium ions and 90% for phosphate ions

Fig. 6

Effect of n-butanol fraction on COM crystal growth. n-butanol fraction (10 μl of 10 times diluted fraction) showed inhibition of 29% and 69% at time intervals of 30 and 120 sec, respectively of calcium oxalate monohydrate (COM) crystal growth