Evaluation of Anti-Diabetic Property of Extracts of Different Plant Parts of Salacia chinensis Linn

Plants from the Western Ghats Mountains (a global biodiversity hot-spot) in western India are increasingly gaining importance for their newfound disease curative properties. One such example is an extract of the plant, Salacia chinensis Linn., the compound Salacinol which (along with related compounds) is increasingly being used in the treatment of diabetes. Of late, demand for this extract has increased at a rapid pace, leading to widespread overharvesting of Salacia roots (the plant part predominantly used for extraction) and consequent population decline by over 50%. Such overexploitation in a global biodiversity hotspot threatens the ecological sustainability of this fragile ecosystem and global health care, as well as local livelihoods. One strategy to relieve the harvest pressure on wild population while attempting to cater to the ever increasing demand of raw material by the pharmaceutical industry is to utilize alternative plant parts and raise viable commercial cultivation. With a view to developing a sustainable harvest strategy, this paper presents an assessment of anti-diabetic activity of alternative plant parts (stems, seeds, leaves). We present the results of in-vitro evaluation of αglucosidase inhibition activity by S. chinensis extracts with respect to parameters like plant part, age of plant and effective solvent system. Promising αglucosidase enzyme inhibition results were obtained from crude extracts of stems and seeds. The highest inhibition levels demonstrated by aqueous extracts of roots and stems were 80.43 ± 1.14 % (IC50-22.17 μg/ml) and 81.2 ± 0.41 % (IC5022.23 μg/ml) respectively, whereas for successive aqueous extracts of seeds inhibition levels were 56.0 ± 1.30 % (IC50-79.04 μg/ml). By demonstrating that stems and seeds of S. chinensis can be used as an alternative to roots, our study has the potential to form the basis for a sustainable path forward for the harvesting of this plant for medicinal purposes.


Introduction
Salacia chinensis Linn.(Synonym-Salacia prinoides) (Figure 1), Family-Hippocrateaceae, is an evergreen climbing shrub or a small tree occurring in India, Sri Lanka, China, Malaysia, Java and Philippines [1][2][3].In Maharashtra (India), it occurs in pockets mainly around the Sahyadri -Konkan corridor area of the northern Western Ghats [1].Locally, it is referred to as 'Saptarangi', 'Saptachakri' or 'Ingali' [1,3].S. chinensis has gained importance as a rich repository of chemical constituents and is known to contribute to various medicinal properties.Phytochemical profiling reveals the presence of constituents such as salacinol [4], kotalanol, neokotalanol, neosalacinol [5], foliasalaciosides, foliachinenosides [6], mangiferin [7], proanthocyanidin [8], triterpenoids like lupanes, hopanes and freidelanes [9] and eudesmane-type sesquiterpenes [7].Extracts of S. chinensis have been shown to exhibit medicinal/curative properties with respect to several health conditions such as type 2 diabetes [4], tumors [10], mutagenicity [11], hepatitis, arthritis, cardiac disorders, mental disorders [12] and insulin resistance [13].In addition, they have been demonstrated to improve glucose tolerance [14] and regulate kidney carbohydrate metabolism [15].However, within traditional medicinal systems, the use of S. chinensis has been historically restricted to its use as an anti-diabetic agent.Salacinol, kotalanol, mangiferin, neosalacinol and neokotalanol have been shown to be major inhibitors of α-glucosidase enzyme [4,5], the enzyme that breaks down ingested carbohydrates and starches.These plant compounds therefore, act by reducing the absorption of glucose into the blood stream.The International Diabetes Federation (IDF) predicts the growth of diabetic patients from 366 million in 2011 to 552 million in 2030 [16].Current strategies for the management of type 2 diabetes have Biodiversity, Bioprospecting and Development limitations; for instance insulin therapy has been known to result in insulin resistance [17], anorexia nervosa, brain atrophy and fatty liver during chronic treatments [18].In contrast, the use of Salacia under prescribed dosage is considered completely safe [19].Commercial demand for plant material of Salacia (S. chinensis, S. oblonga, S. reticulata) currently exceeds 100 metric tons per year; 95% of this demand is supplied from the wild (uncultivated) populations of the Western Ghats.Currently, only the roots of the plant are utilized for commercial harvesting of medicinal products, effectively resulting in the destruction of the individual plant.So far, the potential of alternative plant parts as sources of the medicinal compounds remains unexplored.Our paper addresses this gap by evaluating the αglucosidase inhibitory activity of non-root parts of the plant, including stem, leaf and seed in addition to root.Variation in inhibitory activity by age of the plant and solvent system are also studied.

Equipment
The absorbance was measured by using a micro-plate reader (Versamax, Molecular devices).

Extraction
Harvested plant material was shade dried.Dried roots, stems, seeds and leaves were subjected to extraction.Extraction was carried out using 100 g of plant material in 400 ml of methanol (for methanolic extraction), and 100 g of plant material in 400 ml of water (for direct aqueous extractions).The mixtures were refluxed for 1 hour (1 hour time duration was determined as optimal by comparing extraction yields for 1, 2 and 3 hours durations and observing no increase in the extract beyond 1 hour).The procedure was repeated twice.For successive aqueous extraction, the spent raw materials of methanolic extraction were subjected individually to aqueous extraction with a 1 hour reflux.The extracts were then filtered, concentrated and dried under vacuum.

Enzyme inhibition assay
α -Glucosidase inhibition activity assay was performed as per Vogel and Vogel [20] with a few modifications.In brief, the pre-incubation mixture contained 80 mM phosphate buffer pH 7.0 and vehicle buffer or positive control or test sample.Following pre-incubation (37 º C, 30 minutes), substrate (sucrose 37 mM-0.316g dissolved in 25 ml of 80 mM phosphate buffer) was added to a final concentration of 23.125 mM, followed by re-incubation at 37 º C for 50 minutes.The reaction was arrested by transferring the reaction mixture to a boiling water bath for 2 minutes and then cooling to room temperature.250 µl of glucose reagent was added to 50 µl of reaction mixture and was incubated at 25 º C for 10 minutes.The absorbance was measured at 510 nm in a micro-plate reader (Versamax, Molecular devices) and enzyme inhibition percentage was measured using the formula, The enzyme inhibition activity demonstrated by different plant parts and by age of plant as well as solvent system is shown in (Table 1).Table 2 shows crude extract yield values for different plant parts (roots, stems, seeds and leaves) by age of the plant as well as cultivated status (i.e.whether wild or cultivated).Figure 2 and 3   Highest α-Glucosidase enzyme inhibition activity was obtained from plant roots, followed by stems, seeds and leaves (Table 1).Comparing enzyme activity across plant parts yielded promising findings.Successive aqueous extracts of stems and seeds (both 100 μg/ml) exhibited inhibition values of 73.44% and 56.00%, respectively, values that are comparable to those obtained from root extracts (80.99%).

Discussion
Enzyme inhibition activity of successive aqueous extracts of roots was significantly higher than the activity of direct aqueous extracts and methanolic extracts (p value<0.001).However, there was no significant difference between inhibition activities of successive aqueous extracts and direct aqueous extracts of stems (p value>0.05).Methanolic extracts of stems had significantly lower enzyme inhibition activity (p value<0.001)than the direct aqueous and successive aqueous extracts.
Even among plantation of young age (3 years), significant enzyme inhibitory activity was observed in root and stem extracts.As the age of plant increased, IC 50 value decreased, indicating higher enzyme inhibition (Figures 2 and 3).
Direct aqueous extraction resulted in a better yield of crude extracts in comparison of the other solvents used (Table 2).Our study has the potential for informing such practices by demonstrating the potential of stems and seeds.Future direction should focus on strategies to facilitate the use of these non-root plant parts as renewable source of raw material by developing sustainable agronomic practices like coppicing, pruning to increase harvestable biomass.Systematic cultivation will help farmers, cultivators to gain insights into how to harvest, how much to harvest and when to harvest.Industry can be benefited by assured supply of authentic raw material through contractual farming schemes of the government.

Figure 2 :
Figure 2: Effect of age on IC 50 (µg/ml) of successive aqueous extracts of roots.

Figure 3 :
Figure 3: Effect of age on IC 50 (µg/ml) of aqueous extracts of stems.

Table 1 :
represent the effect of age on IC 50 value of successive aqueous extracts of Salacia roots and aqueous extracts of Salacia stems respectively.Patwardhan A, Pimputkar M, Joshi R (2014) Evaluation of Anti-Diabetic Property of Extracts of Different Plant Parts of Salacia chinensis Linn.. J Biodivers Biopros Dev 1: 107.doi:10.4172/ijbbd.1000107α -Glucosidase inhibition activity of various plant parts across age for extracts from different solvent systems.