Phytochemical analysis and biological activities of ethanolic extract of Curcuma

Curcuma longa is an important dietary plant which possess several pharmacological activities, including antioxidant, antimicrobial, anti-inflamatory, anticancer and anti clotting etc. The aim of the present study was to determine the phenolic profile of Curcuma longa and in vitro antioxidant and antidiabetic activities. In HPLC chromatogram of Curcuma longa rhizome extract 15 phenolic compounds were identified namely Digalloyl-hexoside, Caffeic acid hexoside, Curdione, Coumaric, Caffeic acid, Sinapic acid, Qurecetin-3-D-galactoside, Casuarinin, Bisdemethoxycurcumin, Curcuminol, Demethoxycurcumin, and Isorhamnetin, Valoneic acid bilactone, Curcumin, Curcumin- O -glucuronide respectively. The ethanolic extract displayed an IC 50 value of 37.1±0.3 µg/ml against alpha glucosidase. The IC 50 value of DPPH radical scavenging activity was 27.2 ± 1.1 μg/mL. It is concluded that ethanolic extract of Curcuma long is rich source of curcumin and contain several important phenolics. The in vitro antioxidant and alpha glucosidase inhibitory effect of the plant justifies its popular use in traditional medicine.


Introduction
Diabetes mellitus (DM) is the disorder with servere micro and macro complication that results in significant deaths. It is main causes of death in the world. There are limited effective therapies to cure diabetes. The use of insulin and other antidiabetic agents cause unpleasant side effects, thus there is need to find safe natural products to treat diabetes. Long term complications such as organs failure are the result of chronic hyperglycemia of diabetes (Olatunde et al., 2014). DM also represented by lipidaemia and oxidative stress (Ghazanfar et al., 2014). Although many traditional medicinal plants are effective in decreasing blood sugar, most of these are not practically utilized in severe diabetes (Ranilla et al., 2010). Many plants have shown biological activities and utilized as standardized extracts (Dar et al., 2019;Silva et al., 2019;Pontes et al., 2019).
Turmeric (Curcuma longa) is a common plant which belongs to family Zingberaceae (Thomas-Eapen, 2009). The rhizome of the plant are dried, ground and boiled to get yellow powder which is used as food color in curry powder in Asian Countries (Goel et al., 2008). Turmeric powder is a food preservative due to its antioxidant action and adds to the flavor and fragrance of food (El Demerdash et al., 2012;Aggarwal et al., 2007). Turmeric contains curcumin, demethoxycurcumin, bisdemethoxycurcumin and rich in volatile oil (Shehzad et al., 2013). Curcuma longa possess several pharmacological activities, including antioxidant, antimicrobial, anti-inflamatory, anticancer, anti clotting etc. (Akbik et al., 2014;Aggarwal and Harikumar, 2009;Widowati et al., 2018). The phenolic composition of Cucuma longa has not been studied in detail. The present study was therefore, aimed to evaluate the phenolic composition of ethanol extract of Curcuma longa. The antioxidant and antidiabetic activities of Curcuma longa were also determined.

Plant extract
Rhizome of the plant was collected locally, identified by a botanist and a voucher specimen was submitted at the Herbarium of the University of Poonch, Department of Botany (Ref. No. BOT/2018/35).
Finely grounded rhizome (25g) was soaked for 3 days in ethanol (500 ml) with constant stirring and filtered with the help of filter paper. The extraction procedure was repeated three times and the filtrates were pooled. The filtrate was evaporated by rotary evaporator (45 °C) producing 3.5g (14% w/w) extract.

HPLC analysis of phenolic compounds
The dried ethnaol extract of Curcuma longa was dissolved in ethanol (1 mg/mL), filtered and subjected for analysis by Shimadzu HPLC system as reported by Zeb (2015). The best separation was achieved in 40 min using gradient elution of methanol, deionized water and acetic acid on a Zorbax plus C18 column (4.6 × 100 mm, 3.5 μm) at 25 °C. The chromatography peaks were confirmed by comparing its retention time with those of reference standards and by DAD spectra (200-500 nm).

Determination of alpha glucosidase inhibitory activity of extract
Alpha glucosidase inhibitory activity was determined by the method described by Sancheti et al., (2011) .

DPPH radical scavenging activity of extract
Scavenging of the DPPH radical (ethanolic solution of 0.25 mM) was assayed in vitro (Hatano et al., 1988). The results were expressed as percent inhibition calculated from the control.

Statistical analysis
The results were expressed as mean ±standard deviation. The data was analyzed by ONE WAY ANNOVA followed by Duncan multiple range test (DMRT) where necessary. Satista 7.1 was used as software package.
increased with increasing concentration of extract which indicates that extract possess in vitro antidiabetic activity (Figure 2). The IC 50 for inhibition of alpha glucosidase was 37.1±0.3 µg/ml for ethanolic extract obtained from rhizome.   Blood glucose is elevated when carbohydrate rich diet is consumed as the complex carbohydrate is rapidly absorbed in human intestine due to action α-glucosidase enzyme which breaks disaccharides into absorbable monosaccharides.
The inhibitors of α-glucosidase inhibits the digestion of disaccharides and enable overall smooth glucose profile (Casirola and Ferraris, 2006). The natural products have great diversity in their structure and are potential inhibitor of alpha glucosidase. The phenolic rich ethanolic extract of Curcuma longa has higher potential to inhibit alpha glucosidase and thus can be effectively utilized in diabetes.
The antioxidant activity of the extract was tested by widely used DPPH method. The ethanolic solution of DPPH free radical is reduced on treatment with antioxidants present in the extract. In the DPPH assay, Curcuma longa showed excellent scavenging against the radical (Figure 3). The activity was the highest at maximum concentration, with an IC 50 value of 27.2 ± 1.1 μg/mL (r 2 = 0.96). The pure gallic acid showed an IC 50 value of 3.1± 0.7 μg/mL (r 2 = 0.99). Antioxidants act at different stages (prevention, interception, and repair) and by different mechanisms as reducing agents by donating hydrogen, by quenching of singlet oxygen, and by acting as chelators and trapping free radicals (Devasagayam et al., 2004). The high DPPH radical scavenging activity of Curcuma longa suggests use against diseases arising from free radical attack.

Conclusions
The results showed that Curcuma longa rhizome can be considered as potential source of substances with anti-oxidant and anti-diabetic activities. The findings suggest research continuity with the fractions of the crude extract in oxidative stress and other degenerative diseases. These results clearly demonstrated that Curcuma longa has the potential to be selected as an alternative medicinal and food plant that can be utilized in health food products, functional tea and pharmaceutical products.