Anti-glycation and antioxidant properties of Abutilon indicum plant leaves

The study was done to investigate the in vitro anti-diabetic activity of Ethanol-water extract of Abutilon indicum plant leaves. The phytochemicals present in the extract was analyzed by using standard methods. In vitro antioxidant study was done using DPPH radical scavenging activity. The In vitro anti-diabetic studies were done by alpha amylase enzyme, alpha glucosidases enzyme inhibition studies and Glucose uptake in Yeast cells studies. The phytochemical analysis showed that the extract rich with proteins, polyphenols, flavonoids, alkaloids, tannins, polysaccharides. The in vitro antiglycation potential of extract was confirmed through alpha amylase enzyme, alpha glucosidases enzyme inhibition studies and Glucose uptake in Yeast cells studies. The results of the present study showed the significant antioxidant and antidiabetic activity. The potential pharmacological activity of Abutilon indicum might be the presence of phytochemicals.

A non-enzymatic reaction like Glycation takes place in amino groups of proteins and reducing sugars [7][8]. This process is associated with the pathogenesis of age and diabetes related complications. In living organisms, it represents post translational status of proteins, which can impair their functions. In the above glycation process, if the oxidative step is involved then it is called glycoxidation. During which, advanced glication end (AGE) products like free radicals, products of the autooxidation of the glycating sugar, and a heterogeneous group of substances are formed [9][10].
In India, Abutilon indicum plant commonly called as "Thuthi" in hindi, "Atibala" in Kannada, is a native plant of South Asia. Abutilon indicum (Linn.) is a perennial plant and it grows very well in even in dry and poor soils [11]. Natural sources or herbs or spices or medicines are effective in action without (sometimes considerably very less) side effects. From Vedic period, India is one of the most medico-culturally diverse countries in the world where the medicinal plant sector is a part of time-honored tradition had value even today. The usage of the plant higly cited in Ayurveda, Unani, Chinese medicine and Siddha practices. The various parts of the plant Abutilon such as roots, leaves, bark, seeds and even flowers are documented to possess various medicinal properties in ethnobotanical surveys conducted by ethnobotanists and in traditional systems of medicine such as Ayurveda [12]. The extracts of the whole plant Abutilon were scientifically evaluated for tonic and oja, vardhaka, augment and ojas, the subtle essence of all vital fluids, responsible for health, harmony and spiritual growth [13]. Since then large variety of compounds have been isolated from genus Abutilon and majorities of them are flavonoids, steroids, terpenoids and phenolics [14].

Material and methods
All required plant materials, chemicals and reagents used were of analytical grade were procured from authentic source, Merck Co, and S.d. fine chem., Mumbai, India.

Extraction of Abutilon indicum plant leaves (AIPL)
Abutilon indicum plant was identified by authorized Botanist. The plant leaves were collected and washed with double distilled water, crushed into paste form and stored in glass bottle. The 5gm of Abutilon indicum plant leaves paste mixed with 100 ml of 50:50 Ethanol and double distilled water and vortexed for 4 hours. The vortexed mixture was allowed to settle and filtered using glass wool. The supernatant volume reduced to required volume by using vacuum concentrator.

Phytochemical Analysis
The extraction of Abutilon indicum plant leaves (AIPL) obtained was analyzed for phytochemical components using following standard protocols. The proteins estimation was carried according to Bradford's method using BSA as standard and absorbance was read at 535nm. Total phenols were determined according to the method of Folin-Ciocalteu reaction using Gallic acid as a standard and absorbance was read at 750 nm 2 . Flavonoids estimation was done using Quercetin as a standard; absorbance was measured at 415 nm. Total Sugars estimation was done according to Dubois method and the absorbance was read at 520 nm. The alkaloid estimation and the tannin estimation were done as per the standard protocols [15][16][17][18][19][20][21]. The concentration was calculated accordingly using standard graph.

1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of AIPL
DPPH radical scavenging activity study was done in a dose dependent concentration of 0 to 10 μg each was mixed in 1 ml of freshly prepared 0.5 mM DPPH ethanolic solution and 2 ml of 0.1 M acetate buffer pH 5.5. The resulting solutions were then incubated at 37C for 30 min and measured spectrophotometrically at 517 nm. Ascorbic acid and αtocopherol was used as positive control under the same assay conditions. Negative control was without any inhibitor or AIPL. Lower absorbance at 517 nm represents higher DPPH scavenging activity. The % DPPH radical scavenging activity of extracts of AIPL was calculated from the decrease in absorbance at 517 nm in comparison with negative control [22].

Inhibition of Alpha Amylase Enzyme
The AIPL and standard drug Acarbose (100-500μg/ml) were added to 500 μl of 0.20 mM phosphate buffer (pH 6.9) containing α-amylase (0.5mg/ml) solution and were incubated at 25°C for 10 min. After this, 500 μl of a 1% starch solution in 0.02 M sodium phosphate buffer (pH 6.9) was added to each tube. The reaction mixtures were then incubated at 25°C for 10 min. The reaction was stopped with 1.0 ml of 3, 5 dinitrosalicylic acid colour reagent. The test tubes were then incubated in a boiling water bath for 5 min, cooled to room temperature. The reaction mixture was then diluted after adding 10 ml distilled water and absorbance was measured at 540 nm. Control represents 100% enzyme activity and were conducted in similar way by replacing extract with vehicle [23].

Inhibition of Alpha Glucosidase Enzyme
The inhibitory activity was determined in a dose dependent manner along with standard drug Acarbose (100 to 500ug) by incubating a solution of starch substrate (2% w/v maltose or sucrose) 1ml with 0.2 M Tris buffer (pH 8.0) and various concentration of AIPL for 5 min at 37°C. The reaction was initiated by adding 1ml of α-glucosidase enzyme (1U/ml) to it followed by incubation for 10 min at 37°C. Then, the reaction mixture was heated for 2 min in boiling water bath to stop the reaction. The amount of liberated glucose is measured by glucose oxidase peroxidase method [24][25][26].

Glucose Uptake in Yeast Cells
Yeast cells were prepared according to the method of Gupta et al., 2013 with minor modifications. Commercially procured baker's yeast was washed in distilled water by repeated centrifugation till the supernatant clear and a 10% (v/v) suspension was prepared in distilled water. Various concentrations of AIPL (10-50 μg) were added to 1mL of glucose solution (5, 10 and 25 mM) and incubated together for 10 min at 37°C. Reaction was started by adding 100 μl of yeast suspension, vortexed and further incubated at 37°C for 60 min. After 60 min, the tubes were centrifuged and glucose was estimated in the supernatant. Metformin was taken as standard drug. The percentage increase in glucose uptake by yeast cells was calculated. All the tests were performed in triplicate [27][28][29].

Statistical Analysis
All data are expressed as mean ± standard deviation of triplet (n=3). The significance of the experimental observation was checked by student's t-test and P < 0.05 was considered as statistically significant when compared to relevant controls.

Results and discussion
The ethanol and water extract of Abutilon indicum plant leaves found rich Polyphenols, polysaccharides and moderate amount of alkaloids, proteins and flavonoids. Vitamin -E (10μg/ml) 68 72

Evaluation of DPPH radical scavenging potential of AIPL
A dose dependent DPPH radical scavenging activity of AIPL was done where well known antioxidants Ascorbic acid and α-tocopherol are used as positive control. AIPL (25μg) showed 62%, Ascorbic acid (56%) and α-tocopherol (68%) at a highest dose of 10μg concentration ( Table 1). The above results showed AIPL is a potential DPPH scavenger in comparison with Ascorbic acid and α-tocopherol. The antioxidant property of AIPL could be due to the supply of hydrogen, which combined with radicals and thus forming a stable radical.

Evaluation of anti-glycation activity
The inhibition study for the production of AGEs was carried out for AIPL. The extract was able to inhibit the production AGEs by 60% at a maximum dosage of 25ug in comparison to pure Ascorbic acid (78%) and Vitamin -E (72%) at much lower dose ( Table 1). The ethanol -water crude extract of AIPL acted as a glycation inhibitor because of its free radical scavenging property. The effectiveness of AIPL in inhibiting DPPH radical formation and AGEs formation can speak about its uses for diabetic patients.
The effect of AIPL on the formation of fructosamine was studied by the reduction of NBT from the 1st day of incubation to the 3 rd day. AIPL inhibited fructosamine formation by 71% after 3 days of incubation. The possible explanation for the less formation of fructosamine in AIPL treated sample was that might have the ability to modify the amino or carbonyl groups in the Millard reaction that resulted in the inhibition of fructosamine formation.
The above two studies suggested that the inhibition of glycation exhibited by AIPL was not only due to its DPPH radical scavenging property but also due to the modification in the amino or carbonyl groups in the Millard reaction, which resulted in the inhibition of fructosamine formation.

Conclusion
The results of the present work indicated that the phytochemicals isolated from Abutilon indicum plant leaves extract (AIPL) possessed antioxidant, and anti-glycation properties. However, the in vivo antioxidant activity and the mechanism of action need to be further studied.