Diabetes mellitus which was once considered a disease of the developed world has now become a worldwide diseases affecting both developed and developing countries. Non enzymatic glycation is the major cause of spontaneous damage to proteins leading to various complications due to formation of non-reversible Advanced Glycation End Products (AGEs) and oxidative stress. More than 400 traditional plants for diabetes mellitus have been recorded but very few have received scientific and medical evaluation for their efficacy. Medicinal plants having both antiglycation and antioxidant activity may be good therapeutic potential in the treatment of diabetic mellitus.

Determine the antiglycation activity of Cassia auriculata – flowers and Pillanthus embilica – Fruit which are being used in the treatment of diabetes mellitus in Ayurvedha medicine.

Collection of samples

Plant materials were collected from three different areas in Sri Lanka where given species is grown. Cassia auriculata flowers were collected from Anuradhapura, Katharagama and Mathugama and Phyllanthus embilica fruit from Anuradhapura, Bibile and Balangoda areas in Sri lanka. Commercially available dried sample of each plant material was collected from traditional herbal market in Colombo, Sri Lanka and identification of samples was carried out by a Botanist at Bandaranayke Memorial Ayurvedha Research Institute at Nawinna.

Preparation of plant materials

Fresh plant parts collected from different areas were air dried for 24 hours in room temperature and dried in a dehydrator at (Leader) at 50°C for 24 hrs and powdered using a domestic grinder to obtain fine particles and stored in air tight containers in a refrigerator.

Preparation of water extract

Water extracts of dried and milled powdered samples were prepared according to the traditional method practiced in Ayurveda to prepare ‘Kasaya’. As 60 g of the powdered samples were simmerly boiled in 960 ml to obtain the decoction of 240 ml.This water extract was filtered through a fine silk cloth. The filtrate was freeze dried and the powdered samples were kept at -4°C in a cold room in air tight containers.

Antiglycation activity was determined using the Bovian Serum Albumin assay [1] with slight modification. In all experiments the final reaction volume was 1.0 ml and carried out in 1.5 ml Eppendrof tubes. Bovian serum albumin 500 μl (1 mg/ml concentration) was incubated with glucose 400 μl (500 mM final concentration) and 100 μl sample, 100 μl phosphate buffer saline instead of the sample, as the sample control and 100 μl Arbutin as the reference standard. Negative control was carried out at the same time with Bovian serum albumin 500 μl (1mg/ml concentration), 400 μl phosphate buffer saline and 100 μl sample incubating under same conditions. The reaction was allowed to proceed at 60°C for 24 hours thereafter, stopped the reaction by adding 10 of 100% (W/V) trichloroacetic acid (TCA). The TCA added mixture was kept at 4°C for 10 minutes and centrifuged 4 minutes at 13000 rpm. The precipitate was redissolved with alkaline PBS (pH 10) and quantified for the relative amount of glycated BSA based on fluoresce intensity by Fluorescent Microplate Reader (Spectra Max Gemini EM). The excitation and emission wavelength used were at 370 nm and 440 nm respectively. Each sample was carried out in five concentrations and in triplicate. Percentage of inhibition was calculated using the formula given below and the sample concentration required for the 50% of inhibition was calculated using minitab 14.

% of inhibition=OD blank–(OD sample–OD sample negative)/OD blank×100

Antiglycation activity is inversely proportional to the sample concentration. Commercial sample showed the highest activity comparing to the other samples and it showed higher activity than the positive control Arbutin (Figure 1).

Figure 1: Antiglycation activity of Phyllanthusemblica fruit.

Commercial sample of Cassia auriculata flowers showed high antiglycation activity than the samples freshly collected and dried under laboratory conditions. Arbutin is the positive control (Figure 2).

Figure 2: Antiglycation activity of Cassia auriculata flower.

The presence of Gallic acid, Catechol, Ellagic acid, Phloroglucinol, Indol acetic acid, Vitamin C, β carotene, superoxide dismutase enzyme have been reported in the P. emblica fruit by Kalra [2], Singh et al. [3]. The phytochemical screening of aqueous and water-soluble fraction of ethanol extract of C. auriculata flowers revealed the presence of flavonoids, phenolic acids, sterods, triterpenoids, alkaloids, tannins and anthocyan by Hakkim et al. [4] in their study. High antioxidant activity of the water extracts of the P. emblica fruit and C auriculata flowers has been reported by the authors in a previous study should be due to the availability of these known antioxidant compounds. As oxidation plays an important role in the formation of Advanced Glycation End Products by converting Amadori products to reactive Intermediate carbonyl compounds, the antioxidant potential of the water extract of Emblica fruit would be involved in retarding the formation of AGEs in diabetes mellitus.

Hansen et al. [5] reported in their study as the P. emblica fruit containing high moisture content and sugar, during the drying process fermentation occurs and increases the free phenolic acids due to hydrolyzing of the bound phenolic compounds. This process may occur during the shade drying process than oven drying due to temperature fluctuation and slow drying.

The water extracts of commercial sample of Phyllanthus emblica and Cassia auriculata shade dried under natural environment showed high antiglycation activity than the samples dried under laboratory conditions. Effect on drying methods on total phenolic contents were studied, and they found air dried samples contains higher Total phenolic content than oven dried samples [6]. Thermal degradation of compounds may occur during hot air drying. Effect of the drying method could be affected for the variation of the antiglycation potential of the above samples. Maturity levels of the collected samples and the geographical variation also may contribute to the variation of glycation activity among the samples.

The commercial samples of Cassia auriculata flowers and Phyllanthus emblica fruits available in the traditional herbal market showed high antiglycation activity than the samples collected from other areas and dried under laboratory conditions. Among the two herbal plants Phyllanthus emblica fruits showed high activity.

In the previous studies of the authors, it is reported that all samples of C. auriculata flower and Pillanthus embilica – Fruit contained high phenolic contents and high antioxidant activities. The flower of Cassia auriculata and the fruit of Pillanthus embilica have a good therapeutic potential for diabetes mellitus.

Acknowledge the financial support given by the University Grant (Grant o.ASP/08/RE/2008/09) of University of Sri Jayewardenepura.