Enhanced oxidative stress and the changes in antioxidant capacity, which are observed in both clinical and experimental DM, are thought to contribute to the etiology of chronic diabetic complications
. Indeed, an imbalance between formation of reactive oxygen species (ROS) and antioxidants in vivo has been suggested to play a major role in multiple chronic diseases including diabetes. ROS, which include free radicals such as superoxide anion radicals, hydroxyl radicals and non free-radical species such as H2O2 and singlet oxygen, are various forms of activated oxygen. These molecules are exacerbating factors in cellular injury, inflammation, cardiovascular diseases, diabetes and aging process
. There have been a growing number of investigations on the potential of antidiabetic medicinal plants as possible antioxidants to prevent or delay chronic diabetic complications. It is generally assumed that frequent consumption of plant derived phytochemicals from vegetables, fruit, tea and medicinal herbs may contribute to the shift in balance toward an adequate antioxidant status.
In the present study, six medicinal plants traditionally used against diabetes were evaluated for their antioxidant properties using standard bioassays such as DPPH, NO and SO radical scavenging potentials. Findings from the present investigation showed that AM, OT and FF were the most active plants for all the three assays as depicted by their low IC50 values. However, the degree of inhibition varies considerably and few of the extracts and fractions gave IC50 values higher than ascorbic acid, which is a known standard potent antioxidant. Additionally, a clear cut trend in activity for the fractions used was not observed. Nevertheless, the ethylacetate fraction for the three plants tested has been shown to be the most potent extracts. Several reports tend to show that numerous plant derived natural products are effective antioxidants, and many medicinal plants with a long history of use in folk medicine in different countries against a variety of diseases have turned out to be rich sources of antioxidants
[19, 20]. The advantage of natural antioxidants is their safety and that large oral doses are well tolerated
. Many antioxidant compounds, naturally occurring in plant sources have been identified as free radical or active oxygen scavengers. Recently, interest has considerably increased in finding naturally occurring antioxidant for use in foods or medicinal materials to replace synthetic antioxidants, which are being restricted due to their side effects such as carcinogenesis. Natural antioxidants can protect the human body from free radicals and retard the progress of many chronic diseases as well as lipid oxidative rancidity in foods. Hence, studies on natural antioxidants have gained increasingly greater importance
. In the investigations reported here, scavenging of the DPPH, SO anion and NO radicals which are commonly used procedures and validated against several other assays for antioxidant activity, including some with relevance for in vivo effects
[14, 22], were employed as radical scavenging assays. For inhibition of an enzymatic peroxidative process, inhibition of 15-lipoxygenase-mediated peroxidation of linoleic acid was used. It is to be noted that, that AM, FF and OT showed scavenging properties for all these in vitro assay methods. Lipoxygenase is the enzyme that peroxidizes polyunsaturated fatty acids such as linoleic acid or arachidonic acid to their respective hydro-peroxy derivatives. The six medicinal plants were tested for activity against this enzyme and it was found that AM, FF and OT were the most active fractions, showing good correlation with their antioxidant properties as noted against DDPH, SO and NO. The lipoxygenases have been implicated in a number of pathological states, and 15-lipoxygenase has been suggested to play a role in the development of atherosclerosis and diabetes, probably due to its ability to peroxidize low-density lipoprotein
[23, 24]. Investigators tend to suggest that an elevated level of lipid peroxides in the plasma of diabetic rats and lipid peroxidation is one of the characteristic features of chronic diabetes. Lipid peroxidation is a free radical induced process leading to oxidative deterioration of polyunsaturated fatty acids and this can be prevented by antioxidants. Additionally, inhibition of soybean 15-lipoxygenase is generally regarded as predictive for inhibition of the mammalian enzyme
[25, 26]. To this effect, it can be suggested that AM, FF and OT had profound antioxidant properties as supported by the assays carried out in this experiment. However, the phytochemical constituents present in the active extracts and fractions, which are responsible for the observed activity, need to be further investigated. Interestingly, we have recently found the presence of at least tannins, flavonoids and alkaloids in these plants which are known to possess potent antioxidant activity. Hence, the observed antioxidant activity might be due to the presence of any of these constituents or due to a synergistic effect.
A panoply of studies have focused on the factors involved in the pathogenesis of diabetic complications and most seeking effective therapies
[27, 28]. However, the exact cellular or molecular basis of these complications has not yet been fully elucidated
. Hyperglycemia is still considered the principal cause of diabetic complications. Its deleterious effects are attributable, among other things, to the formation of sugar-derived substances called advanced glycation end products (AGEs). AGEs are heterogeneous group of molecules formed from the non-enzymatic reaction of reducing sugars with free amino groups of proteins, lipids, and nucleic acids and their formation is markedly accelerated in diabetes because of the increased availability of glucose
. In this study, a simple screening method to measure the inhibitory effects of these extracts and fractions and aminoguanidine on formation of fluorescent AGEs in vitro is described. Our system used high concentrations of glucose to speed up the glycation reaction, thus allowing us to undertake the evaluation in an appropriate time-scale as glycation occurs very slowly under physiological conditions
In the present investigation, AM, OT and FF were found to inhibit the formation of AGE in vitro. It is suggested that the abilities to inhibit the formation of glycated end products is closely related to the abilities of the antioxidant properties of the plant extracts to scavenge radicals formed during the Maillard reaction which forms the basis of glycation. Interestingly, in the current work, extracts that were found to possess antioxidant properties against lipoxygenase activity were also found to possess antiglycation potential. Furthermore, a few of the extracts were observed to possess antiglycation activities similar to the standard drug aminoguanidine, also known as pimagedine which is a nucleophilic hydrazine compound
. Initially, it was thought that aminoguanidine prevented AGE formation by blocking carbonyl groups on Amadori products although it is now known to react with carbonyl groups from reducing sugars
. Aminoguanidine, guar gum and acarbose may act as an antioxidant, especially at high concentrations
. To this effect, it can be suggested that the antioxidant properties of the AM, FF and OT might to some extent justify the observed antiglycation properties similar to aminoguanidine mode of action.
The MTT assay is a test of metabolic competence based upon assessment of mitochondrial performance relying on the conversion of yellow MTT to the purple formazan derivative by mitochondrial succinate dehydrogenase in viable cells
. Increasing concentrations of the six extracts and fractions did not affect mitochondrial respiration as measured by the MTT cytotoxicity assay. Overall, the results of this assay measuring cell integrity showed that these medicinal plants which are widely consumed and used in traditional medicine is not toxic over this concentration range tested.
In this study the crude aqueous extracts were used as per the local tradition of the herbalists. The basis for performing extractions with different solvents was to corroborate and validate the inhibitory activity in the aqueous extractions performed in the traditional manner as well as to search for newer, more potent inhibitory compounds in the organic solvents. To this effect, it was important to assess the antioxidant activities of the crude water extracts in order to validate the medicinal uses of these plants. To this effect, if results from the present study could be extended and translated in vivo, then it can be safely suggested that ingestion of crude leaf decoctions of these medicinal plants might be beneficial in the long term management of diabetes. It is believed that these active phytochemicals from the plant extracts could restore the imbalance between formation of ROS and antioxidants observed in vivo which has been strongly implicated to play a major role in multiple chronic diseases including diabetes.