Evaluation of the antioxidant properties of Vitis vinifera juice extract in rifampicin dispersions

The purpose of this study was to evaluate the antioxidant properties of Vitis vinifera juice extract (grape) in rifampicin suspensions and to compare with that of ascorbic acid. Grape juice was extracted and used as antioxidant in both pure sample and commercial reconstituted samples of rifampicin. The rate of drug degradation was determined by spectroscopic method in a time dependent manner. Also, the antioxidant property of grape juice was compared with that of ascorbic acid. Phytochemical screening of the constituents in grape juice extract was carried out according to standard methods. The results revealed that rifampicin dispersions containing ascorbic acid (batches A1 to A5) showed significantly higher stability and had higher amount of drug remaining over time than the control (batch A6) containing no antioxidant (p < 0.05). However, the results revealed that grape fruit juice extract had higher antioxidant properties than the ascorbic acid (p < 0.05) in rifampicin dispersions. The results also indicated that increase in amount of both antioxidants significantly reduced the rate of drug degradation through oxidation, thereby causing a corresponding increase in the amount of rifampicin remaining over time. Grape juice extract contain alkaloids, carbohydrates, saponins, reducing sugars, steroids, tannins, proteins, flavonoids, resins, oils, terpenoids and acid compounds. Glycosides were however not found in the juice extract. Therefore, grape fruit juice extract could be used as a natural antioxidant in rifampicin suspensions.


INTRODUCTION
Natural antioxidants have gained considerable interest in recent years for their role in preventing the auto oxidation (El-Hawary et al., 2012).Vitis vinifera L. (grapes) (Vitaceae) is considered as a natural antioxidant source and contains many chemical constituents such as phenolic acids, flavonoids, anthocyanins, proanthocyanidins, sugars, sterols, amino acids, and minerals (Sokar, 1991;El-Hawary et al., 2012).Grapes are also used as demulcent, laxative, refrigerant, stomachic and diuretic.Moreover, it is useful in bilious dyspepsia, haemorrhage, dysuria, in chronic bronchitis, heart diseases and gout.
Grape juice is given to children to prevent constipation (El-Hawary et al., 2012;Bunea et al., 2012).Dried grapes or raisins are useful in thirst attendant on fevers, cough, catarrh, jaundice, and in sub-acute cases of enlarged liver and spleen.The pharmacokinetics of grapefruit revealed that fruit have a potent inhibitory effect on the intestinal cytochrome P450 system.For this reason, grapefruit has a high potential for interaction with drugs (Ulbricht and Seamon, 2010).The grape berries are important since they are consumed as fruits, wine, juice or raisins and are largely cultivated for the wine industry.Antioxidant activities of grapes are due to the presence of antioxidant components such as flavonoids, phenolic acids, anthocyanins and carotenoids (Guedes et al., 2001;Lutz et al., 2011;El-Hawary et al., 2012;Bunea et al., 2012).
Antioxidants are molecules capable of slowing or preventing the oxidation of other molecules (Hamid et al., 2010).Oxidation is a chemical reaction that transfers electrons from a substance to an oxidizing agent.Oxidation reactions can produce free radicals, which start chain reactions that damage cells (Hamid et al., 2010).Antioxidants terminate these chain reactions by removing free radical intermediates and inhibit other oxidation reactions by being oxidized themselves.As a result, antioxidants are often reducing agents such as thiols, ascorbic acid or polyphenols (Mendes-Pinto et al., 2004;Sies, 1997;Mulero et al., 2010;Hamid et al., 2010;Vinkovic et al., 2011).Antioxidants interact with and stabilize free radicals and may prevent some of the damage free radicals might otherwise cause.Free radical damage may lead to cancer.Examples of antioxidants include beta-carotene, lycopene, vitamins A, C, E and other substances (Sies, 1997;Hamid et al., 2010).
Rifampicin is the drug of choice in treatment of tuberculosis.Also, it is effective in treatment of various bacterial infections.Rifampicin is bactericidal agent against wide range of microorganism (Lubna et al., 2006;Dhanapal et al., 2012).It is slightly soluble in water, thus is suitable for suspension dosage form (Lubna et al., 2006).Rifampicin undergoes oxidation when exposed to light, air or moisture and hence requires incorporation of suitable antioxidant and should also be stored under nitrogen in an airtight container, protected from light, at a temperature not exceeding 25°C (BP, 2009).The aim of the work is to evaluate the antioxidant properties of grape fruit juice in rifampicin dispersions and to compare the antioxidant properties of grape juice with that of ascorbic acid in rifampicin suspensions.

MATERIALS AND METHODS
Rifampicin, hydrochloric acid (Sigma-Aldrich, Germany), ascorbic acid (BDH, Poole, England), distilled water from (UNN Water Resources Management Laboratories Ltd., UNN, Enugu State, Nigeria), and V. vinifera (grape) juice was processed in our laboratory.All the chemicals used were of analytical grade and was used as supplied without further purification.

Processing of grape juice
V. vinifera (grape) fruits were purchased from Ogige market Nsukka, in Enugu state, Nigeria in the month of February, 2008.The fruit was identified by a plant taxonomist, Mr. P.O.Ugwuozor of the herbarium section of Botany Department, University of Nigeria, Nsukka.The fruits were washed and peeled to remove the bark.The juice was squeezed out by hand, and filtered using a non adsorbent filter paper (Whatman no. 1).The extract was later pre-served with 30 % alcohol in a bottle and stored at room temperature (25°C) until used.

Phytochemical analysis
Phytochemical tests were carried out on the grape juice extract for the presence of alkaloids, saponin, steroids, terpenoids, glycosides, acidic compounds, carbohydrates, reducing sugars, tannins, proteins, fats and oil.The tests were carried out using standard procedures of analysis (Harborne, 1993;Sofowora, 1993;Trease and Evans, 2002).

Test for stability of rifampicin using ascorbic acid as the antioxidant
A 5 mg quantity of rifampicin was dispersed in 100 ml of distilled water, different concentrations of ascorbic acid was incorporated into the dispersions as presented in Table 1.The rifampicin dispersions were stored at room temperature (25°C) until used.The concentration of rifampicin remaining in the dispersion was determined in time dependent manner 1, 4, 8 and 12 days using a spectrophotometer (Jenway 6305, UK).The absorbance readings were recorded and the concentration of rifampicin in sample at any given time was calculated with reference to Beer's plot previously obtained for rifampicin at concentration range of 0.2 to 1 mg/% in 0.1 N HCl at a predetermined wavelength of 470 nm.The test was repeated using grape fruit extract as the antioxidant at concentrations listed in Table 1.Also, commercial reconstitutable samples of rifampicin was reconstituted with distilled water, ascorbic acid (batches C1 to C5) and grape fruit (batches D1 to D5), respectively was added to the dispersions and the amount of rifampicin remaining in each sample was also determined spectrophotometrically in time dependent manner with reference to Beer's plot.

Data and statistical analysis
Data were analyzed by one way analysis of variance (ANOVA) and differences in means were assessed using a two tailed student's ttest.P < 0.05 was considered statistically significant.

Stability of rifampicin dispersions
The results of the percentage amount of rifampicin remaining in the dispersion are shown in Figures 1 to 4. The results show that batch A1 containing 10 mg of ascorbic acid had 52.5 and 18.8% rifampicin remaining at 1 and 12 days, respectively while batch A5 containing 30 mg of ascorbic acid had 52.5 and 32.9% of rifampicin.However, batch A6 containing no antioxidant had 32.5 and 12.9% at 1 and 12 days, respectively as shown in Figure 1.Also, (Figure 2) batch B1 containing 2.8 ml of grape fruit had 68.8 and 3.3% rifampicin remaining at 1 and 12 days, respectively.Batch B4 containing 7 ml of grape juice had 77.9 and 54.6% rifampicin remaining at 1 and 12 days, respectively while 95.8 and 69.2% rifampicin were remaining at 1 and 12 days, respectively  for batch B5 formulated with 8.4 ml of grape juice.However, the negative control containing no grape juice (batch B6) exhibited 32.5 and 12.9% rifampicin remaining at 1 and 12 days, respectively.Also batches C1 to C5 (Figure 3) formulated with commercial rifampicin powder for reconstitution and containing 10 to 30 mg of ascorbic acid showed significant reduction in the amount of rifampicin remaining with time just like the rifampicin pure samples as above, however batches D1 to D6 (Figure 4) also formulated with commercial rifampicin powder for reconstitution, and containing grape fruit juice had 100% amount of drug remaining at 12 days for batches C4 and C5 as shown in Figure 4.

Phytochemical constituents of grape juice
The results of phytochemical constituents of V. vinifera (grape) juice are shown in Table 2.The results revealed the presence of alkaloids, saponins, tannins, flavonoids, resins, acidic compounds, steroids and terpenoids in very high quantity.Reducing sugars were found in moderate quantity, while carbohydrates, proteins, fat and oil were found in low quantity.Glycosides were however not found.

Stability of rifampicin dispersion
The results of antioxidant effect of ascorbic acid in rifampicin dispersions studied over time show that ascorbic acid exhibited good antioxidant properties.However, rifampicin dispersions containing ascorbic acid (batches A1 to A5) showed higher stability (p < 0.05) and amount of drug remaining over time than the control (batch A6) containing no antioxidant.Also, the results of the antioxidant properties of grape fruit juice revealed that it had significantly higher antioxidant properties than the ascorbic acid (p < 0.05) in rifampicin dispersions as shown in Figures 2 and 4. The results also indicated that increase in amount of both antioxidants significantly reduced the rate of drug degradation through oxidation, thereby causing a corresponding increase in the amount of rifampicin remaining over time.
The antioxidant activities of grapes are due to the presence of antioxidant components such as flavonoids, phenolic acids, anthocyanins and carotenoids (El-Hawary et al., 2012;Bunea et al., 2012).These constituents  terminate chain reactions by removing free radical intermediates and inhibit other oxidation reactions by being oxidized themselves (Sies, 1997;Hamid et al., 2010).Antioxidants interact with and stabilize free radicals and may prevent some of the damage free radicals might otherwise cause.This natural antioxidant have several advantages over synthetic antioxidants, grape fruit juice is an edible fruit juice therefore, it is physiologically inert hence, may have better biocompatibility over synthetic antioxidants, it is relatively cheap and readily available compared to most antioxidants used in drug formulation.These have led to sudden interest in grape juice as antioxidant in drug formulation.

Phytochemical constituents of grape juice
The results of the phytochemical screening of the grape juice indicate the presence of very important phytochemicals at different concentrations.The juice extract contain alkaloids, carbohydrates, saponins, reducing sugars, steroids, tannins, proteins, flavonoids, resins, oils, terpenoids, acid compounds, but glycosides were however, not found in the juice extract.Phytochemicals are non-nutritive plant chemicals that have protective or disease preventive properties.Plants produce these chemicals substances to protect themselves, and they are also believed to protect humans against certain diseases (Edeoga et al., 2005).

Conclusion
Grape fruit juice extract was successfully used as an antioxidant in rifampicin dispersions.The results revealed that grape juice had significantly higher antioxidant properties than ascorbic acid in rifampicin dispersions.Natural antioxidants such as grape juice extract may have advantages over the synthetic and semi synthetic ones due to the fact that they are edible fruits, relatively inexpensive, better biocompatibility among others.More research into this field of study is however required in order to scale up all its aspect.

Table 1 .
Contents of rifampicin preparations.