Plant secondary metabolites are sources for numerous natural products such as spices, vegetables, herbs, fruits. This is becoming more popular in the world of research because of their ability to detoxify free radicals from the biological system. Extractions of these secondary metabolites for biological or phytochemical investigations are faced with challenges such as poor yield of extracts and this depend on solvent of extraction. Phenolics compounds (flavonoids) isolation from plant source is a technique which include extracting solvent type and the method. These depend generally on the types of phenolics compound and the solvent [1]. Previous researches [2,3] revealed that extracts yield of flavonoid and phenolic content is largely dependent on the solvent polarity. The work of [4] reported highest content of phenolic compounds from rice bran and leaves of Moringa oleifera using aqueous methanol. Extracts from various plant materials such as rice bran, wheat bran, oat groats and hull, coffee beans, citrus peel and guava leaves extracted with 80% methanol was found to have high antioxidant activity [5].

Antioxidants are compounds with the ability to protect a biological system against potentials harmful effects of processes or reactions involving reactive oxygen and nitrogen species (ROS and RNS) [6]. Reactive oxygen species in biological system are related to free radicals, these are compounds that are generally not stable, energy molecules and very reactive. The sources of free radicals in a biological system are prooxidative enzyme systems, air pollutants, irradiation, smoking and glycoxidation [7,8]. From previous studies, it was reported that reactive oxygen species are the cause of numerous age related degenerative diseases such as cancer, asthma, arthritis, atherosclerosis, stroke, vasospasons, trauma, hyperoxia, heart attack, retinal damage, age pigment, dermatitis, cataractogenesis, hepatitis, periodontics [9,10]. An imbalance in the equilibrium between ROS and antioxidant leads to oxidative stress.

Flavonoids occur naturally as group of polyphenolic compounds mostly found in fruits, vegetables, flowers and in plants organs such as leaves, stem, root and rhizomes. Flavonoids possess antioxidative properties and mitigate against chronic diseases which are cause by ROS and lipid peroxidation during oxidative stress.

Curcuma longa and xanthorrhiza belong to family Zingiberaceae, order Zingiberales. They are widely distributed throughout the tropics especially Southeast Asia. They have aromatic leaves and rhizomes which are above the ground or subterranean [11]. They are both used as condiment, spices, traditional medicines, flavoring agents and as dyes sources [12]. They are also major ingredients in traditionally prepared tonics locally called “Jamu” which is commercially available in Malaysia [13]. There rhizomes are eaten raw or cooked as vegetable [14].

The aim of the study is to evaluate total antioxidant capacity, phenolic compounds and to determine the various effects of methanol concentrations on extraction yield on C. longa and C. xanthorrhiza rhizomes.

Chemicals and reagents

DPPH, 2, 4, 6-tri (2-pyridyl)-S-triazine (TPTZ), Trolox, Folin- Ciocalteu’s phenol reagent, and Naringenin were purchased from Sigma Co. St. Louis, Missouri, USA. Sodium carbonate, Methanol, Aluminum chloride, Iron (III) chloride hexahydrate, Gallic acid, Sodium nitrite and Acetic acid, Sodium hydroxide and Ascorbic acid were purchased from Merck, Darmstadt, Germany. All chemicals and reagents were of analytical grade.

Plant material

Curcuma longa and Curcuma xanthorrhiza rhizomes were planted at Tanam Pertanian Universiti, Universiti Putra Malaysia (University Agricultural Park) and the rhizomes were harvested after one month of growth. These rhizomes were identified and confirmed by the Herbal Garden Department, Universiti Putra Malaysia (UPM).

Sample preparation

Samples collected were washed and cuts into pieces and dried to a constant weight using the oven at 70°C. They were then pounded into powder form using pestle and mortar and stored in an air tight container.

Extraction

0.5g of each sample was weighed using analytical balance into a round bottom flask and 25 mL of different concentrations of methanol was added. The mixture refluxed at 60°C for 2hrs. The mixture was filtered using the Whatman No.1 filter paper and the filtrate was stored in 15 mL Amber bottle in -20°C refrigerator [15].

Determination of Total Antioxidant Capacity

2,2-Diphenyl-1-picrylhydrazyl (DPPH) which is stable was used to determine the free radical scavenging capacity of the two samples using the method described by Norhaiza [16]. 0.1 mM solution of methanol DPPH was prepared and the absorbance was measured at 515 nm using (spectrophotometer UV-2602, Labomed, Inc. USA). 60 μL sample was added to 3 mL of Methanol and were incubated for 30 mins at room temperature in the dark. The change in absorbance was then measured. The experiment was done in three replicates. Percentage inhibition was calculated using the formula (%) inhibition = [(A₅₁₅ control – A₅₁₅ sample)/ A₅₁₅ control] × 100. The antioxidant capacity of the extracts using DPPH for free radical scavenging ability was expressed as mg Trolox equivalent per gram of dry weight of sample. Ascorbic acid (AR Grade) from Merck, Darmstadt, Germany was used as control.

Ferric reducing antioxidant potential (FRAP)

The FRAP reagent is made in ratio of 10:1:1 from the following reagent respectively 300 mM sodium acetate buffer at pH 3.6, 10 mM TPTZ solution, and 20 mM FeCl₃.6H₂O.The ability of the extracts to reduce the yellow TPTZ complex was conducted using [17]. 20 μL of extracts was added to 3mL of FRAP reagent that was prepared. The mixture was incubated in a water bath which has been maintained at 37°C for 30mins. The experiment was done in three replicates. A change in absorbance was measured at 593nm. The antioxidant capacity was calculated using the formula Antioxidant (%) = [(A593 sample – A₅₉₃ control) / A₅₉₃ sample] × 100 and FRAP reagent was used as control. Total antioxidant capacity was expressed as mg Trolox equivalent per gram of dry weight of sample. Ascorbic acid was used as positive control.

Total flavonoid assay

Aluminum chloride colorimetric method was used to deter the Total flavonoid content of the extracts. Total flavonoid of extracts was estimated using the method of Benzie IF [18]. 200 μL of extracts to 4 mL of distilled water, 0.3mL 5% NaNO₂ was then added and after 5mins 0.3mL of 10% AlCl₃ was added. After 6mins, 2 mL of 1 M NaOH was then added. 3.2 mL of distilled water was added to the mixture to make a total volume 10 mL. The mixture was mixed and the absorbance was taken at 510nm. Total flavonoid was expressed as mg Naringenin equivalents (Ng)/g samples.

Total phenolic acid assay

The Total phenolic content in the extracts was estimated using Folin – Ciocalteu method by Marinova et al. [19]. 100 μL of extracts was added to 9mL of distilled water. 1 mL of Folin – Ciocalteu’s phenol reagent was then added and the mixture was mixed well. After 5 mins, 10 mL of 7% of Na₂CO₃ was added. 4.9mL of distilled water was then added to make a total volume of 25 mL; the mixture was then incubated at room temperature for 90mins. Absorbance was taken at 750 nm and total phenolic acid content was expressed as mg gallic acid equivalents (mg GA/g dw) of dry samples.

Total polyphenol assay

The Total Polyphenol content in the extracts was estimated using Folin – Ciocalteu method by Singleton et al. [20]. The Folin – Ciocalteu was diluted 10 times. 100 μL of extracts was added to 2.5 mL of Folin – Ciocalteu’s phenol reagent and 2.5 mL of 7% of sodium carbonate was added after 5mins. The mixture was then incubated at room temperature for 1hr. Absorbance was taken at 725 nm and total polyphenol content was expressed as mg gallic acid equivalents per gram of dry samples (mg GA/g dw).

All experiments were replicated thrice and results are presented as mean ± standard deviation (SD) of three replicates and analyzed using One-way ANOVA, the difference between samples were determined by Duncan’s Multiple Range test (p<0.05) using SPSS Statistics version 21.

The total antioxidant capacity of methanol extracts of Curcuma longa and Curcuma xanthorrhiza consume in Southeast Asia were evaluated using DPPH and FRAP assay, the phenolic contents ( Phenolic acids and polyphenol) and the effects of solvent concentration on flavonoid extracts are shown below.

Research in natural products have continue to grow due to the alternatives it provide to synthetic products which are either too expensive or have negative side effects. Zingiberaceae (Curcuma longa and Curcuma xanthorrhiza) has been used in folkloric medicine in the management and treatment of swelling, headache, toothache, stomach ache, rheumatism and arthritis [21].

The extraction of bioactive compounds from plant is dependent on types and concentration of solvent. For the extraction of total phenolics and flavonoids from ginger parts, methanol is more efficient compare to other solvent [22]. Antioxidant activity of young ginger extracts is significantly affected by extracting solvent [23].

For this research, we evaluated total antioxidant capacity and phenolics content in C. longa and C. xanthorrhiza using DPPH and FRAP method. In Figure 1 the free radical scavenging potential of C. longa and C. xanthorrhiza was assayed and the activity was compared to Ascorbic acid which is a standard free radical scavenger. The activity of C. longa to scavenge Trolox was found to be (270.1 mg /TE/g dw) while C. xanthorrhiza was found to have (240.4 mg /TE/g dw) and Ascorbic acid was (283.7 mg /TE/g dw).

Figure 1: DPPH radical scavenging activites of C. Longa and C. xanthrorrhiza consumed in Southeast Asia expressed as Trolox equivalent in methanol extracts. Values are means ± SD (n=3). Bar represents standard error.

Figure 2 shows the activity of C. longa and C. xanthorrhiza to reduce FRAP. C. longa was found to have a higher activity of FRAP with (231.7 mg/TE/g dw) while C. xanthorrhiza activity was (100.4 mg /TE/g dw) compared to ascorbic acid activity (284.2 mg /TE/g dw).

Figure 2: Ferrous ion chelating activity of C. Longa and C. xanthorrhiza consumed in Southeast Asia expressed as Trolox equivalent methanol extracts. Values are means ± SD (n=3). Bar represents standard error.

Figure 3 shows the total phenolic acid content of both C. longa and C. xanthorrhiza expressed as Gallic acid equivalent with (42.7 mg / GA/g dw) and (22.0 mg /GA/g dw) respectively. Figure 4 shows total polyphenol content of both C. longa and C. xanthorrhiza also expressed as Gallic acid equivalent with (39.4 mg/GA/g dw) and (37.3 mg /GA/g dw) respectively.

Figure 3: Total phenolic acids determination for C. Longa and C. Xanthorrhiza consumed in Southeast Asia expressed as gallic acid equivalent in methanol extracts. Values are means ± SD (n=3). Bar represents standard error.

Figure 4: Total polyphenols determination for C. Longa and C. xanthorrhiza consumed in Southeast Asia expressed as gallic acid equivalent in methanol extracts. Values are means ± SD (n=3). Bar represents standard error.

Total flavonoid content of C. xanthorrhiza and C. longa rhizomes extracted with different methanol concentrations is shown in figure 5 and 6 respectively. Total flavonoid content was expressed as Naringenin equivalent. It was observed that flavonoid activity in both samples were solvent concentration dependent with the highest activity at 90% (v/v) concentration.

Figure 5: Total flavonoid content of C. xanthorrhiza rhizome extracted with different methanol concentrations expressed as Naringenin equivalent. Values are means ± SD (n=3). Bar represents standard error.

Figure 6: Total flavonoid content of C. Longa rhizome extracted with different methanol concentrations expressed as Naringenin equivalent. Values are means ± SD (n=3. Bar represents standard error.

C. longa and C. xanthorrhiza rhizomes extract revealed strong antioxidant capacity with strong free radical scavenging activity with reference to DPPH and FRAP. Also it can be concluded that flavonoids content is directly proportional to solvent concentration. C. longa and C. xanthorrhiza could be a good source of natural antioxidants.