Studies on oxalic acid and oxalate oxidase enzyme in costus pictus D.Don

Abstract

Costus pictus D.Don is a native of Mexico and referred as Mexican cane or spiral ginger. It is commonly known as insulin plant in Kerala. The hypoglycemic property of Costus pictus was well established. The aerial part of Costus pictus is used in Mexican folk medicine to treat renal diseases. The decoction of fresh leaves and stems of Costus pictus is widely used by the Cuban population as a traditional medicinal remedy for urinary problems like stones, pain and sepsis.

The sour taste of Costus pictus is due to the presence of oxalic acid in the leaves. Excess oxalate intake through oxalate rich foods leads to nephrolithiasis, a condition in which oxalate crystallises to form stones in the kidney, bladder and urethra. Based on the above fact, a basic study on Costus pictus is required for safer use of this plant in medical preparations. In this context the present study entitled “Studies on oxalic acid and oxalate oxidase enzyme in Costus pictus D.Don” was carried out at Centre for Plant Biotechnology and Molecular Biology. The objective of the study was to estimate oxalic acid and oxalate oxidase enzyme activity of Costus pictus D.Don leaf sample at different stages of maturity and to study the effect of drying and extraction with various solvents on oxalic acid content and oxalate oxidase enzyme activity.

Fully opened leaves from top 1st to 3rd, 4th to 6th and 7th to 9th were collected for analysis of oxalic acid and oxalate oxidase enzyme. The samples were designated as stage 1, stage 2 and stage 3 respectively. Oxalate was isolated by two different methods, Baker and Burrows method. Oxalate content was determined by iron ferron method. Oxalate oxidase enzyme was assayed by 4-aminoantipyrine method and protein content was determined by Bradford method.

The study revealed that oxalate and oxalate oxidase activity was maximum in second leaf stage followed by first leaf stage and third leaf stage. Drying causes substantial loss of oxalate content and complete loss of oxalate oxidase activity. With various solvents water recovered more oxalate followed by methanol and ethanol while oxalate oxidase activity was maximum in ethanol followed by methanol and water. The storability of oxalate oxidase was poor in ethanol followed by methanol and water.

The crude oxalate oxidase enzyme showed maximum activity at pH 5.2 while the partially purified enzyme showed maximum activity at pH 5.8. The crude and partial purified enzyme showed maximum activity at 45oC. The activity of the partially purified enzyme showed a hyperbolic relationship with oxalic acid concentration only up to 0.8 mM above which the enzyme showed decrease in activity due to substrate inhibition. The activity of the crude enzyme was steadily decreased with increase in oxalic acid concentration due to excess availability of oxalate in the crude extract which inhibits the oxalate oxidase activity. The Km and Vmax of partially purified oxalate oxidase was 0.065 mM and 427.5 units g-1 5 min-1 at 40oC respectively. Oxalate oxidase in Costus pictus requires Cu2+/Mn2+ for its activity.

The ethanol or methanol extract of second leaf stage of Costus pictus can be used for isolating active principles. The oxalate oxidase from Costus pictus can be used as a cheap source of oxalate oxidase enzyme which is used in oxalate determination in biological fluids. More over the sensitivity of oxalate determination employing oxalate oxidase from Costus pictus will be more as oxalate oxidase in Costus pictus has Km 20 times lesser than the commercially available barley oxalate oxidase enzyme.