Evaluation of Antioxidant, Anti-Inflammatory and Antidiabetic Activity of Butter Fruit, Indian Jujube and Wood Apple

Free radicals are chemical species possessing an unpaired electron that can be considered as fragments of molecules and which are generally very reactive. They are produced continuously in cells as byproducts of metabolism [1, 2]. Reactive free radicals formed within cells can oxidize biomolecules and lead to cell death and tissue injury. The free radicals both the Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) are derived from both endogenous source (mitochondria, peroxisomes, endoplasmic reticulum, phagocytic cells etc.,) and exogenous sources (pollution, alcohol, tobacco, smoke, heavy metals, industrial solvents, paracetamol etc.,) [3]. Antioxidants are substances that scavenge the free radicals and stop the chain reaction. Phytochemical is a natural bioactive compound found in plants such as vegetables, fruits, medicinal plant, flowers, leaves and roots that work with nutrients and fibres to act as a defense system against diseases or more accurately, to protect against disease [4,5]. Some of the most important bioactive phytochemical constituents are alkaloids, essential oils, flavonoids, tannins, terpenoids, saponins, phenolic compounds and many more [6]. Many inflammatory diseases are free radical mediated and thereby the potential molecules in plant sources can inhibit the free radical initiated diseases. The oxidative stress results in various human diseases such as diabetes mellitus, neurodegenerative diseases, rheumatoid arthritis, cataracts, cardiovascular diseases, respiratory diseases as well as in aging process[7,8]. Therefore three different fruits were selected for evaluating the phytochemicals, proteins, carbohydrates, antioxidant, anti-inflammatory and antidiabetic activity.

MATERIALS AND METHODS

The fresh fruits were obtained locally and authenticated by the Department of Botany. Unless otherwise stated all chemicals used in these experiments were of analytical grade, obtained either from Sigma chemicals or SRL chemicals.

Preparation of fruit extracts

Three different fruits such as Butter fruit, Indian jujube and Wood apple were cleaned and washed with distilled water.  10g of pulp of each fruit was taken and cut into pieces and were ground to a paste in pestle and mortar using distilled water and is subjected to centrifugation at 2000 rpm for 20min. The same procedure was followed for the preparation of organic solvent extract using methanol The supernatant is filtered and evaporated to drynesss. . The yield was calculated and expressed in percentage. The extracts were dissolved at 1mg/ml concentration for performing various activities.

Determination of total phenolics by Folin-Ciocalteau assay

The concentration of total phenolics in the aqueous and methanolic fruit extracts was determined by the Folin-Ciocalteu assay. It involves reduction of the reagent by phenolic compounds, with concomitant formation of a blue complex, its intensity at 725 nm increases linearly with the concentration of phenolics in the reaction medium [9]. In this study Gallic acid was used as spectrophotometric standard. The phenolic contents of the extracts were determined from calibration curve and were expressed in mg of Gallic acid equivalents/ g sample.

Estimation of total flavonoids

Aluminium chloride colorimetric method [10] was used for flavonoids determination in aqueous and methanolic fruit extracts of Butter fruit, Indian jujube and Wood apple. The absorbance of the reaction mixture was measured at 420 nm with UV visible spectrophotometer. The content was determined from extrapolation of calibration curve of Quercetin. The concentration of flavonoid was expressed in terms of mg / g of sample.

Antioxidant activity by DPPH method

Determination of antioxidant activity by the DPPH method [11] was done for all the aqueous and methanolic fruit extracts. Diphenyl picryl hydrazyl (DPPH^.) was used as a stable radical for assessing antioxidant activity. Reduction of DPPH by an antioxidant result in a loss of absorption at 517 nm. Thus, the degree of discoloration of the solution indicates the scavenging efficiency of the added substances. Percentage of radical scavenging activity was calculated for all the extracts.

Determination of reducing power

The reducing power activity of all the aqueous and methanolic fruit extracts of Butter fruit, Indian jujube and Wood apple were evaluated according to the method of Oyaizu [12]. Compounds which have reductones converts potassium ferricyanide to ferrocyanide, which then react with ferric chloride to form ferric ferrous complex which can be measured at 700nm. Increase in absorbance of the reaction mixture indicated the reducing power of the samples.

Antioxidant activity by TBA method

Thiobarbituric acid (TBA) reacts with malondialdehyde (MDA) to form a diadduct, a pink chromogen, which can be detected spectrophotometrically at 532 nm as per Halliwell and Gutteridge [13]. The egg yolk was used for the study of invitro lipid peroxidation. The percentage of antilipid peroxidation activity  per gram of sample is calculated for the aqueous and methanolic fruit extracts.

Estimation of total sugars

Total soluble sugars was estimated for all fruit extracts according to the procedure of phenol-sulphuric acid method [14]. To suitable extract, 1ml of phenol and 5ml of conc. H₂SO₄ was added and incubated for 30 mins. The absorbance was measured at 470nm in spectrophotometer. Total soluble sugars were expressed in terms mg of glucose/g of sample. Glucose (0-25micro g) was used as reference standard.

Estimation of total protein content

The total protein content was estimated using biuret method [15]. The peptide bond in the polypeptide chain reacts with copper sulphate in an alkaline medium to give a purple colour which can be measured at 540nm. The calibration curve was plotted using standard gelatin solution. The total protein content was expressed in terms of mg/ml of sample.

ANTI-INFLAMMATORY ACTIVITY

Protein denaturation assay

In this assay either egg albumin or bovine serum albumin (BSA) are used as protein. Denaturation of protein is induced by keeping the reaction mixture at 70°C in a water bath for 10 minutes [16]. The reaction mixture consists of fruit pulp extract, 0.2 ml of 1% egg albumin or  bovine serum albumin, and volume is made up to 5ml by using phosphate buffered saline pH 6.4. Distilled water instead of extracts with above mixture is used as a negative control. Afterward, the mixtures is incubated at 37 °C for 15 min and  then heated at 70°C for 5 min. After cooling the absorbance is measured at 660 nm. Acetyl salicylic acid  or diclofenac sodium or ibuprofen or indomethacin is taken as a positive control. The  percent inhibition for protein denaturation is calculated using following equations:

% Inhibition of denaturation = (C-T/C) X100.

Where T is the absorbance of test sample and C is the absorbance of negative control (without the test sample or reference drug).

Protease inhibitory activity

In this assay different enzymes and different protein can be used, enzymes are  trypsin and casein are used as protein [17]. The reaction mixture (2 ml) contain 0.06 mg trypsin, 1 ml 20 mM Tris HCl buffer (pH 7.4) and 100ug concentration of sample. The mixture is incubated at 37°C for 5 min and then 1 ml of 0.8% (w/v) casein is added. The mixture is incubated for an additional 20 min at 37⁰c. 2 ml of 70% perchloric acid  is added to terminate the reaction. Cloudy suspension is centrifuged at 3000 rpm for 10 minutes or 2500 rpm for 5 minutes. 1ml of sample from each tube is taken and  2.5 ml of Na₂Co₃ of 0.4M and 0.5 ml of  1:2 FC is added and absorbance of the supernatant is read at 660 nm against buffer as blank. The percentage inhibition of protease activity is calculated using the following equation.

Percentage inhibition = (Abs control –Abs sample) / Abs control X100

Membrane stabilization method

a) Heat induced hemolysis

The stability of RBC membrane against heat is tested according to heat induced hemolysis assay method [18]. The reaction mixture  consist of  test sample of 100μg and the volume is make up to 1ml using saline and 1ml of 10% erythrocyte suspension, instead of test sample only vehicle is added to the control test tube.  Acetyl salicylic acid (aspirin) is used as a standard drug. This reaction mixture is mixed gently by inversion.  The reaction mixture is incubated in water bath at  56 ⁰ C for 30 minutes . At the end of the incubation the tubes are cooled and centrifuged at 2000 rpm for 5 min and the absorbance of the supernatant is measured at 560 nm. The percentage inhibition of hemolysis is calculated as follows:

Percentage inhibition of haemolysis =C-T/C×100

b) Hypotonic solution induced haemolysis

This experiment of membrane stabilization was carried out on RBC cells according to hypotonic solution induced hemolysis assay mathod [19]. A number of different agents can be used as hypotonic solutions, including hypo saline (50mM NaCl in 10mM sodium phosphate buffer saline-pH7.4) and distilled water. Reaction mixture contain erythrocyte suspension, fruit pulp extract, saline and hypotonic solutions. Control is prepared by without plant extract. Acetyl salicylic acid or indomethacin or diclofenac can be used as reference standard drug. This mixture is incubated at 37⁰ C for 30 minutes and centrifuged at 3000rpm for 5 minutes. Finally the haemoglobin content of the supernatant solution is estimated by spectrophotometrically at 560 nm. The percentage of Red blood cell membrane stabilization or protection is calculated by the following equation:

% protection = [ C-T/C ]X 100

 ANTI-DIABETIC ACTIVITY

α-glucosidase inhibition assay

In this experiment 100ug concentration of fruit pulp extract was taken in a test followed by addition of 20µl of  buffered α-glucosidase  and incubated at 37^o C for 10minutes, then 0.1M  125µl  of phosphate buffer pH6.8 was added and incubated for 20 min at room temperature then  5Mm of 20µl  pNPG  was added and incubated at room temperature, for 30 min, then 0.1M of 50µl  Na₂CO₃ was added to stop the reaction [20]. Then the absorbance of the p-nitrophenol was  measured at 405 nm and the percentage inhibition was calculated as follows:

% Inhibition= Ac−At\Ac x 100

RESULTS AND DISCUSSION

In recent days emphasis has been on the use of natural, edible sources for maintenance of the health. Fruits, vegetables and other parts of plants are considered as one of the antioxidants rich sources for combating free radical mediated diseases. Fruits are rich in vitamins, minerals, fibres, sugars, phytochemicals, water and small amounts of protein and fats. The antioxidants present in fruits are known to combat the oxidative stress induced diseases amyotrophic lateral sclerosis, Huntington’s disease, Parkinson’s disease, Alzheimer’s disease, cardiovascular diseases, cancer, diabetes and osteoarthritis. Several antioxidant mechanisms exist in the cell, which keeps the oxidative balance in the cell in equilibrium. These mechanisms have evolved in due course of evolution to protect the cell against oxidative imbalance. Inflammation is a vital response of human immune system. Nevertheless, the state of chronic inflammation can have several secondary consequences in biological response associated with enhanced risk of chronic diseases and disorders. Oxidative stress also leads to inflammation and diabetes [21]. In the present study aqueous and methanolic extracts of three different fruit samples were investigated for their antioxidant, anti-inflammatory and antidiabetic activity. The drugs available in market for treatment of inflammation and diabetes shows many side effects [22, 23]. Therefore, the fruits with many bioactive compounds are safer for prevention of many oxidative stresses induced inflammatory diseases.

Extraction of Butter fruit, Indian jujube and Wood apple fruits were carried out using methanol and distilled water. The yeid was maximum in Indian jujube with 24.7% and minimum in aqueous extract of Wood apple. The percentage of yield, protein, carbohydrate and phytochemicals, antioxidant, anti-inflammatory and antidiabetic activity is depicted in Table.1 Total Phenolics, flavonoids, sugars was estimated for all the six fruit extracts at 100 μg concentration as shown in Figure 1 & 2 and Table 1. Aqueous extract of Wood apple  with  2.1 mg gallic acid equivalent/g and 0.09mg/g of sample showed maximum amount of phenolics and flavonoids followed by other extracts. The phenols contain hydroxyls that are responsible for the radical scavenging effect mainly due to redox properties. These results give a reason for the activity of these fruits as antioxidant and how these fruit extracts enable to scavenge the free radicals. Despite their wide distribution, the health effects of dietary polyphenols have come to the attention of nutritionists only in recent years. Researchers and food manufacturers have become more interested in polyphenols due to their potent antioxidant properties, their abundance in the diet, and their credible effects in the prevention of various oxidative stress associated diseases. Flavonoids are important secondary plant metabolites, widely present in fruits, vegetables and herbs known for their antioxidant and health protective properties. Free radical scavenging potential, reducing power capacity and anti-lipid peroxidation activity was carried out for all the six fruit extracts at 100 µg concentrations and the results are shown in Figure 3, 4 & 5.  The aqueous extract of wood apple showed maximum free radical scavenging activity of 83% followed by other extracts.  The activity of extracts is attributed to their hydrogen donating ability. Increasing the number of hydroxyl or catechol groups increases radical scavenging activity. In presence of other H-donating groups (sulfhydryl, amide) in molecule also accelerates this activity. The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity. The good reducing activity was observed in aqueous wood apple extract with absorbance of 0.40 at 700nm. The reducing power activity is due to the presence of reductones (Phenolics) which have been shown to exert antioxidant action by breaking the free radical chain by donating a hydrogen atom. Reductones are also reported to react with certain precursors of peroxide, thus preventing peroxide formation. Lipids and proteins are more susceptible to oxidative damage. The mechanism of free radical mediated damage during ulcer involves lipid peroxidation, which destroys cell membranes with the release of intracellular components, such as lysosomal enzymes, leading to further tissue damage. These free radicals also promote inflammation and diabetes (28). At 100 µg concentration, the aqueous extract of wood apple showed highest inhibition of lipid peroxide generation with 52.6%.

The anti-inflammatory activity was studied by performing various assays. The Protein denaturation results in loss of biological activity of protein molecules. Protein denaturation has been correlated with the formation of inflammatory disorders like rheumatoid arthritis, diabetes and cancer. Therefore, ability of substance to prevent the protein denaturation may also help to prevent the inflammatory disorders. In this assay either egg albumin or bovine serum albumin (BSA) are used as protein. Denaturation of protein is induced by keeping the reaction mixture at 70°C in a water bath for 10 minutes. In the present study, aqueous extract of wood apple showed maximum inhibition of protein denaturation with 57% followed by methanolic extract of Indian jujube with 48% as depicted in Figure 6. It is demonstrated that proteases implicate the tissue damage during the inflammatory reactions. Proteases abundantly exist in lysosomal granules of neutrophils. Therefore, protease inhibitors provide the significant anti-inflammatory activity. The aqueous extract of Wood apple had significant protease inhibitory activity of 52%. During inflammation, lysis of lysosomal membrane may occur which release their enzyme components that produce a variety of disorders. Therefore, membrane stabilization of lysosomes is important to control the inflammatory response. This will lead to prevention of leakage of its constituents. The membrane stabilizing activity of the extracts can be determined through heat induced haemolysis and hypotonic solution induced haemolysis using human erythrocytes. Since human red blood cell membranes are similar to lysosomal membrane, the inhibition of hypotonicity and heat induced lysis of red blood cell membrane will be taken as a measure of the mechanism of anti-inflammatory activity. Hypotonic solution causes the excessive accumulation of fluid within the red blood cells which resulting in the rupturing of its membrane. In both experiments aqueous extract of wood apple exhibited highest membrane stabilization activity with 69 and 75% respectively. Alpha-glucosidase inhibitors are oral anti-diabetic drugs used for diabetes mellitus type 2 that work by preventing the digestion of carbohydrates (such as starch and table sugar). Carbohydrates are normally converted into simple sugars (monosaccharides) by alpha-glucosidase enzymes present on cells lining the intestine, enabling monosaccharides to be absorbed through the intestine. Hence, alpha-glucosidase inhibitors reduce the impact of dietary carbohydrates on blood sugar. Alpha-glucosidase inhibitors inhibit the absorption of carbohydrates from the small intestine. They competitively  inhibit enzymes  that convert complex  non-absorbable carbohydrates into simple absorbable carbohydrates. These enzymes include sucrase, maltase, glucoamylase and iso-maltase [24]. The enzyme inhibition was found to be maximum in aqueous extract of wood apple fruit.

Table 1: Biochemical, phytochemical, antioxidant, anti-inflammatory and antidiabetic activity of aqueous and methanol fruit extracts.

Figure 1: Phenolic content in different fruit extracts at 100µg concentration

Figure 2: Flavonoid content in different fruit extracts at 100µg concentration

Figure 3: Percentage of Radical scavenging activity in different fruit extracts at 100µg concentration by DPPH method

Figure 4: Reducing power activity in different  fruit extracts at 100µg concentration

Figure 5: Percentage of antilipid peroxidation in different fruit extracts at 100µg concentration

Figure 6: Percentage inhibition of protein denaturation in different fruit extracts at 100µg concentration

Figure 7: Percentage inhibition of  protease activity in different fruit extracts at 100µg concentration

Figure 8: Percentage inhibition of  heat induced hemolysis activity in different fruit extracts at 100µg concentration

Figure 9: Percentage inhibition of  hypotonicity induced hemolysis activity in different fruit extracts at 100µg concentration

Figure 10: Percentage inhibition of  α- glucosidase activity in different fruit extracts at 100µg concentration