Phytochemical profile, in vitro antioxidant, and anti-protein denaturation activities of Curcuma longa L. rhizome and leaves

Curcuma longa L. is a famous spice cultivated in many countries with significant variations reported in its phytochemical contents and biological potential. For the first time, the present work is aimed to identify the major phytochemicals present in methanol:chloroform (MC) and petroleum ether (PE) extracts of Curcuma longa rhizome and leaves (by determining polyphenols and GC/MS analysis), and their in-vitro antioxidant and anti-protein denaturation potential. Results showed that the highest value (P < 0.05) of polyphenolic content was in MC extract of rhizome (51.46 ± 0.46mg GAE/g) followed by 31.20 ± 0.53mg GAE/g in MC leaves extract. The strong antiradical activity was evaluated in MC extract of rhizome with IC50 value of 92 ± 0.02 μg/mL. MC extracts of both the rhizome and leaves exerted a potent inhibitory effect against protein denaturation with IC50 values of 106.21 ± 0.53 and 108.06 ± 4.67 μg/mL (P > 0.5), respectively. GC/MS analysis showed that α-tumerone was the main component in the rhizome oil (32.44%), whereas in the leaf oil, palmitic acid was the prominent constituent (28.33%) and α-phellandrene recorded a comparable percentage (7.29). In conclusion, C. longa is a valuable source of natural antioxidants and anti-inflammatory constituents, as indicated by its high polyphenolic content and by its considerable in vitro antiradical and anti-protein denaturation potential.


Introduction
Curcuma longa L. (C. longa) is a famous spice of the ginger family (Zingiberaceae) with the tuberous rhizome widely used as food additives, cosmetic materials, and to cure many worldwide ailments [1]. Many previous and recent studies recorded numerous therapeutic benefits of C. longa rhizome when it is used as fresh or dry material, extracts, and pure components [2]. C. longa rhizome pharmacological activities are frequently imputed to its major active molecules especially those concentrated in its yellow-orange dye (curcumin) which has antioxidant [3], antimicrobial [4], anti-inflammatory [5,6], anticancer [7,8], and wound healing [9] properties.
Despite that the utilization of C. longa is limited to its rhizomes, some countries such as Malaysia and India also use its leaves as a spice. Also, it was reported that C. longa leaves have good contents of macronutrients (proteins, carbohydrates, and fiber) and considerable levels of minerals (Na, Ca, K, Mg, P, and Mn), therefore leaves are recommended as food additives in Brazil [10]. Furthermore, recent studies indicated that C. longa leaves contain active components which can prevent premature aging and oxidative stress [10][11][12], relieve skin inflammation [13], and act as antitumor agents [14]. With these significant medicinal benefits of C. longa leaves, the whole aerial part of C. longa including leaves is still neglected and considered as waste material and discarded post rhizome harvesting. There are limited reports on C. longa leaves compared to that carried out on rhizomes. Some previous reports studied the chemical composition of leaves and rhizomes of C. longa from different regions like Reunion Island in France [15], the lower Himalayan region [16], Plains of Northern India [17], southwest Nigeria [18], and south of Brazil [19]. Their results showed significant variations in the phytochemical contents of C. longa cultivated in different countries, and these differences were reflected in its quality and biological aspects. It is well-known that ecological conditions and geographical divergence are important factors directly affecting the plant growth, synthesis, and storage of plant products. It is worth noticing that there is no report on the chemical composition and biological activities of C. longa grown in Sudan. Therefore, the present work was designed to investigate phytochemicals, in vitro antioxidants against DPPH free radical activity, and in vitro anti-protein denaturation activity of C. longa rhizome and leaves cultivated in Sudan.

Plant materials and preparation of extracts
Fresh leaves and rhizomes of Curcuma longa were collected from seven months old C. longa cultivated in the botanical garden, Al Neelain University, Khartoum, Sudan. The samples were cleaned, dried at room temperature, and powdered for extraction. Ten grams of the dried powder of C. longa rhizome and leaves were soaked separately in 200 mL of petroleum ether (PE) and methanol:chloroform (MC) (1:1, v/v) for 72 h, after which the extracts were filtered and left for evaporation. All processes were conducted at room temperature.

Determination of total polyphenol content (TPC)
The TPC was determined by adopting the method described by Wolfe et al. [20]. TPC was expressed as Gallic acid equivalents (GAE, mg/g dry weight). The extract (1 mg/mL) was taken in a 10 mL glass tube and made up to a volume of 3 mL with ethanol of 0.5 mL, Folin-ciocalteau reagent (1:1 with water), and 4 mL of sodium carbonate (7.5%) added sequentially in each tube. The test solution was kept in dark for 30 min, cooled, and absorbance was measured at 765 nm. TPC was expressed as GAE mg/g dry weight using the following equation based on the calibration curve: Y = 0.0076x − 0.0785, R = 0.999.

Gas chromatography/mass spectrometry (GC/MS) analysis
PE extracts of rhizome and leaves were analyzed by GC/MS (Model GC-MS-QP2010 Plus, Shimadzu, Japan). Separation was performed using Rtx-5MS capillary column (5% of diphenyl-95% of dimethylsilicone, 30 m × 0.25 mm × 0.25 m) and a temperature program of 50°C (1 min) ramped to 300°C (3 min) at 5°C/min. Identification of compounds was based on the comparison of the mass spectra with the GC/MS system data bank (NIST 08 library), published data, and retention indices. The relative amount of each compound was expressed as the percent peak area relative to the total peak area of the GC chromatogram.

Determination of antiradical activity
Anti-DPPH free radical scavenging activity was determined using the method described by Mensor et al. [21] with a modification. Extracts were prepared separately to get test solution of 1 mg/mL. Series of extract solutions of different concentrations (100-1,000 µg/mL) were prepared by diluting with methanol. Assays were performed in 96well microtiter plates. 140 mL of 0.6 × 10 −6 mol/L DPPH were added to each well containing 70 mL of sample. The mixture was shaken gently and left to stand for 30 min in dark at room temperature. The absorbance was measured spectrophotometrically at 517 nm using a microtiter plate reader (Synergy HT Biotek, logiciel GEN5). Blank was done in the same way using methanol and sample without DPPH and control was done in the same way but using DPPH and methanol without sample. The ability to scavenge DPPH radical was calculated by the following equation: where Abs sample is the absorbance of DPPH radical + sample; Abs blank is the absorbance of sample + methanol; and Abs control is the absorbance of DPPH radical + methanol. The IC 50 value was calculated from the linear regression plots of concentration of the test sample against the mean percentage of the antioxidant activity.

In vitro anti-protein denaturation activity
In vitro anti-inflammatory activity was assessed by determination of inhibition of albumin denaturation as described by Shallangwa et al. [22]. PE and MC extracts of C. longa rhizome and leaves were dissolved in 0.2 mL of dimethylsulphoxide (DMSO) and diluted with 2.6 mL of phosphate buffer (0.2 M, pH 7.4). The mixture was made up to 5 mL with 0.2 mL of egg albumin (from fresh hen's egg) and 2 mL of varying concentrations (100, 200, 400, 600, 800, and 1,000 μg/mL) of each extract. The reaction mixtures were incubated at 37°C for 15 min. Then, the denaturation was induced by keeping the mixture at 60°C in the water bath for 10 min. After cooling at room temperature, the turbidity was measured spectrophotometrically at 660 nm. Diclofenac sodium at the same concentrations as that of the extract was used as a reference drug. The inhibition percentage of albumin denaturation was calculated by the following equation and results were recorded as IC 50 values: where A sample = absorbance of sample or standard and A control = absorbance of negative control (DMSO).

Statistical analysis
All experiments (except GC/MS) were performed in triplicate and the obtained results were expressed as mean values ± standard deviation. One-way ANOVA was performed for determining significant differences between the four extracts and their antiradical activity.

TPC
The TPC values were expressed as mg GAE/g. Results are presented in

DPPH free radical scavenging activity
The anti-DPPH free radical scavenging activity of the MC and PE extracts of C. longa rhizome and leaves was determined and compared with standard Propyl Gallate. The inhibition percentage of the highest concentration used (1 mg/mL) against DPPH free radical is depicted in Figure 1 and IC 50 values are calculated ( Table 2). MC extract of rhizome revealed the highest antiradical activity with  DPPH inhibition percentage of 89 ± 0.01% and IC 50 value of 92 ± 0.02 µg/mL which were comparable to that obtained from the positive control (% DPPH inhibition of 90 ± 0.01 with IC 50 value of 77 ± 0.01 µg/mL), followed by MC leaves extract with inhibition percentage of 51.7 ± 0.00% and IC 50 value of 436 ± 0.30 µg/mL, whereas PE extracts of rhizome and leaves showed weak antioxidant activity with inhibition percentage of 10 ± 0.02 and 11 ± 0.03%, respectively.

In vitro protein denaturation inhibition
In vitro anti-inflammatory activity of rhizome and leaf extracts of C. longa and Diclofenac Sodium (reference drug) was evaluated using protein denaturation assay. The results are shown in Table 2. MC extracts of both the rhizome and leaves showed good inhibitory effect against protein denaturation with IC 50 values of 106.21 ± 0.53 and 108.06 ± 4.67 μg/mL, respectively, (P > 0.5) compared to that recorded by reference drug (IC 50 value of 53.18 ± 0.29 μg/mL). PE rhizome and leaves extracts revealed weak protein denaturation inhibitory effect with IC 50 values of 212.52 ± 2.22 and 246.42 ± 3.83 μg/mL, respectively.

GC/MS analysis of C. longa rhizome and leaves oils
PE extracts of both rhizome and leaves showed a lack in their polyphenol content by total phenol determination test. So it raised our attention to determine the main bioactive components responsible of their evaluated activities.

Discussion
Natural products especially those derived from plants represent the safest, effective, and alternative source for chemical drugs. With the high demand for plant products, scientists' interest tends to detect these products in each part of the plant rather than the main part used. In the current study, the chemical profile, in vitro antiradical scavenging activity, and anti-protein denaturation effect of PE and MC extracts of C. longa leaves and rhizomes were investigated.
The result of the chemical analysis showed that the highest TPC (51.46 ± 0.46 mg GAE/g) was determined in MC rhizome extract. This TPC is higher by 6.9-fold than what was reported in the ethanolic extract 80% of C. longa rhizome (7.45 mg GAE/g) [23], and by 75-fold than what was detected in the juice extract (0.68 mg GAE/g) of C. longa rhizome [24]. In contrast, Choi [25] results showed higher values of TPC, 228.7 ± 2.3 mg GAE/g in ethyl acetate and 140.7 ± 10.6 mg GAE/g in chloroform extracts of C. longa rhizome from Korea. Also, C. longa leaves in the present work had good levels (31.20 ± 0.53 mg GAE/g) of TPC in their MC extract, which was 2-fold more than that measured in C. longa leaves from Malaysia 15.33 mg GAE/g [11], and higher by 7.57-fold than that determined in fresh C. longa leaves from Brazil 4.12 ± 5.72 mg GAE/g [10]. These differences in TPC may be due to the difference in the environmental conditions and cultivation from one country to other. These differences were detected and reported earlier in TPC of strawberry and potato peel cultivated in different European regions [26]. GC/MS analysis of the oil of rhizome revealed that the oil was characterized with high percent (56.76%) of oxygenated sesquiterpene and α-tumerone represented 32.44% of the total oil. Previously α-tumerone was reported as the major compound of rhizome oil found in higher levels 44.1% [16], 35.9% [18], or in lower levels, 21.4% [15], 12.9% [17], and 8.52% [27] compared to that obtained in our study. GC/MS chromatogram of the leaves oil showed the absence of the main constituents detected in rhizome oil, instead,  The percent of each compound were expressed as percent peak area relative to the total peak area of the GC chromatogram.
leaves oil was rich in fatty acids (57.55%), especially unsaturated fatty acids, palmitic acid represented 28.33% of the total leaves oil content. Also, α-phellandrene, which was previously described as a prominent component of the oil of C. longa leaves was found in a comparable amount (7.28%) to that recorded earlier as 2.8% [15], 9.1% [17], and 41.99% [19] of C. longa leaves essential oil. Regarding antioxidant activity results, a strong DPPH scavenging activity (IC 50 = 92 ± 0.02 µg/mL) was evaluated in MC extract of the rhizome. This result was significantly higher than that reported recently through the evaluation of the antioxidant activity of four Curcuma species from Thailand and their isolated components [28]. They revealed that C. longa crude extract had an anti-DPPH activity with IC 50 = 134.9 ± 1.5 µg/mL. Moreover, curcumin, the main pigment in C. longa, was evaluated in their study which had the strongest antioxidant activity with IC 50 = 68.9 ± 0.6 µg/mL compared to the other constituents estimated. However, it was much lower in activity in comparison to the ethanolic extract activity of C. longa rhizome with IC 50 value of 27.2 ± 1.1 μg/mL [29].
MC extract of leaves displayed moderate antioxidant activity with a DPPH inhibition percentage of 51.7%. This result is comparable to that reported in aqueous extract of C. longa leaves from Korea with an inhibition percentage of 51.10% against DPPH activity [30].
The in vitro anti-inflammatory activity results showed that MC extracts of both rhizome and leaves recorded potent in vitro anti-inflammatory activity by suppressing albumin denaturation with IC 50 = 106.21 ± 0.53 and IC 50 = 108.06 ± 4.67 μg/mg (P > 0.5), respectively.
Protein denaturation is a process that happens when proteins lose their structure and biological function due to inflammation and could be induced in vitro by factors such as heat, stress, or some chemical compounds. Therefore, the denaturation of tissue proteins is recognized as a marker of inflammation [31]. It is worthwhile that searching for natural agents can prevent protein denaturation to develop a new anti-inflammatory drug. C. longa anti-inflammatory activity is well-documented. Many studies have been proven the potent anti-inflammatory properties of C. longa rhizome and its main constituent curcumin [28,[32][33][34]. Also, the antiinflammatory activity of C. longa leaves is recently reported, as the inhibitory effect of their essential oil on skin inflammation [13]. Many studies ascribed the pharmacological activities of C. longa to its polyphenol molecule contents, especially curcumin as a primary polyphenol [35]. In our study, high positive correlations were observed between anti-DPPH and TPC (r = 0.99), but also between TPC and anti-protein denaturation activity (r = 0.86). This indicated that C. longa phenolic molecules are the main contributor to antioxidant and anti-inflammatory activity in this plant.

Conclusion
Based on the findings obtained from this work, C. longa could be suggested as a promising source of natural  antioxidants and anti-inflammatory, as indicated by its strong antiradical scavenging activity of rhizome MC extract, potent anti-protein denaturation activity of MC rhizome and leaves extracts, and by its high content of polyphenols especially via MC extraction. Also GC/MS analysis showed considerable contents of the major components in the rhizome and leaf oils compared to that cultivated in other countries.