STUDY ON THE ANTIOXIDANT ACTIVITIES OF THE AERIAL PARTS AND SOME COMPOUNDS ISOLATED FROM Archidendron clypearia ((Jack) I. Niels Part 1. THE ANTIOXIDANT ACTIVITIES OF EXTRACTS FROM Archidendron clyperia (Jack) I. Niels

The antioxidant activity of Archidendron clypearia was evaluated by different methods, including in vitro biological tests on liver cells of rats, total antioxidant activity and DPPH assay for free radical scavenging capacity. The curcumin standard, a typical antioxidant compound, was also compared. The extract and fractions of Achidendron clypearia had significant antioxidant properties with absorbance value, ED 50 and IC 50 value. Among five fractions, the ethyl acetate fraction brought the highest activity with the lowest ED 50 value of 0.63 µg/mL (about 6 times lower compare to the ED 50 of curcumin (4.43 µg/mL)) and IC 50 value of 1.81 (20 times lower compare to the IC 50 of curcumin).


Introduction
One of the most important properties of food or pharmaceuticals for disease prevention and cure is their antioxidant activity. The reactive oxygen species (ROS), which are chemically reactive molecules containing oxygen such as OH, HOO, O2, etc., are the highenergy and unstable molecules [1]. They tend to attach to macromolecules in the body such as lipid, DNA, protein, etc. to cause diseases like cancer, cardiovascular disease, diabetes, obesity and accelerate ageing [1]. Therefore, an additional antioxidant to maintain the stability of the free radicals in the body is necessary for health protection. The antioxidative compounds are usually able to scavenge the free radicals, slowing down the ageing process in the body, protecting liver function and preventing certain health complications [1]. Archidendron clypearia is a small tree or an evergreen shrub of the genus Archidendron, Mimosaceae. The Pako ethnic minority in Quang Tri province uses A. clypearia to treat some infections of upper respiratory tract, pharyngitis, laryngitis, tonsillitis, burns, scalds and other types of wounds, scabies, etc. [2]. So far, there have not been any comprehensive studies on the antioxidant activity of A. clypearia. To contribute to the research regarding A. clypearia as a traditional medicine, in this paper, we studied the antioxidant activity of fractions from A. clypearia collected in Quang Tri province, Vietnam.

Plant materials and laboratory animals
The plant materials were collected in Quang Tri province. The scientific name of the plant was determined as A. clypearia. The aerial parts of the plant were well washed, discarded the damaged parts, dried at 50 o C, and ground into powder.
Healthy 8-week old mice, weighing from 25 to 30 grams were kept in the laboratory room at 23 ± 2 o C, and standard 12 hours of day/dark light cycles and the humidity of 50-60 %. The animals were fed with standard food and tap water ad libitum [3].

Preparation of methanol extracts and fractions
A dried sample of A. clypearia (5.0 kg) was extracted with 15 L methanol (MeOH) three times at room temperature. The solutions were combined and evaporated under reduced pressure at 50 o C, resulting in 760 g of the crude methanol extract. The MeOH extract was distributed in water and then extracted with n-hexane, chloroform, ethyl acetate and nbutanol. The solvents were then distilled to get five fractions named H ( n -hexane, 100 g), C ( chloroform, 145 g), E ( ethyl acetate, 250 g), B (n-butanol, 105 g) and W (water, 145 g).

Tests for antioxidation activity in vitro on rat liver cells
Healthy BALB/c mice were used to separate the liver cells. The liver after being taken out using tweezers and surgical scissors was washed with PBS (phosphate buffer saline) containing 10 % of PSF antibiotics (Penicillin-Streptomycin-Fungizone) (Invitrogen). Liver cells were extracted in PBS. The extract was then centrifuged to removed the supernatant. The concentrated extract containing liver cells was dissolved in an NH4Cl solution to break down the erythrocytes and then centrifuged and redissolved in fetal bovine serum MEME containing 10 % FBS (fetal bovine serum) [4].
After being isolated, liver cells were distributed into a 96-well plate (1  10 4 cells per well) for overnight culture in an incubator with 5 % CO2 at 37 o C, followed by addition of a studied substance (extraction, fractions and curcumin) at different concentrations and then incubated for 2 hours. After that, 100 M H2O2 was added to each well and held for 2 hours. To determine the number of liver cells survived under the effect of H2O2 as well as the protective effects of the studied substance, MTT formazan (Thiazolyl blue formazan) 1 mg/mL (50 mL/well) was added to the wells and then incubated for 4 hours at 37 o C. The supernatant was then removed, and 100 L of DMSO was added to each well [5]. The optical density (OD) value of the formed formazan was measured at 492 nm wave length using Microplate Reader instrument. All experiments were replicated three times. The survival rate of liver cells was calculated as follows

Survival rate of liver cells (%) = OD(substance) − OD(H 2 O 2 ) OD(cells) − OD(H 2 O 2 ) . 100
where OD (substance) is the optical density value measured in the well containing substance; OD (H2O2) is the optical density value measured in the negative controlled well only cells killed by H2O2; OD (cells) is the optical density value measured in the well of healthy cells, not killed by H2O2.
ED50 values (Median effective dose -The dose required to achieve 50 % of the desired response in 50 % of the population) were determined using the Table Curve software.

2.4
Evaluation of the total antioxidant activity using the phospho-molybdenum method The total antioxidant activity of studied samples was assessed using the phospho-molybdenum method, which determines the electron transfer capability of the antioxidant compounds in the test sample. The total antioxidant activity of studied samples was determined according to the method described by Lopi et al. [6] with certain modifications. In brief, a 0.3 mL aliquot of the sample was mixed with 3 mL of the reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate), and then the mixture was incubated at 95 o C for 90 min. The mixture was then cooled down to 25 o C and the absorbance was measured at the wave length of 695 nm against a blank that contained 3 mL of the reagent solution without the sample. The total antioxidant activity was expressed as the absorbance of the sample. The higher absorbance value indicates the higher antioxidant activity. Gallic acid and curcumin were used for comparison.

Evaluation of DPPH radical scavenging activity
The DPPH radical scavenging activity determines the hydrogen transfer capability of the antioxidant compounds in the test samples. The DPPH free radical scavenging activity of each sample was determined using the Jasco V-630 Spectrophotometer according to the method described by Lopi et al. [6] and Leong et al. [7] with certain modifications. The samples were dissolved in 1.5 mL methanol at various concentrations (25, 50, 75 and 100 µg/ mL) and mixed with 1.5 mL of 100 µM DPPH (100 µM DPPH dissolved in methanol before using). The reaction mixture was shaken for 1 minute and incubated at room temperature for 30 minutes. The absorbance was then measured at a wave length of 517 nm. Three mililitres of methanol was used as a blank. The DPPH radical scavenging activity (%) of the sample was calculated using the following formula where SADPPH (%) is the inhibition of DPPH activity; Ac is the optical density of the blank; As is the optical density of the sample.
All experiments were repeated three times to avoid errors. Radical scavenging activity was evaluated using the IC50 value.

In vitro antioxidation of methanol extraction and fractions
The antioxidant activity of A. clypearia was determined based on the ED50 values. In this study, curcumin was used as a control. The results are shown in Table 1. The results revealed that A. clypearia had good activity with ED50 value of 2.18 µg/mL and was comparable to curcumin -an antioxidant proven on a scientific basis and was medicinally used.
Thereafter, five fractions: H (n -hexane), C (chloroform), E (ethyl acetate), B (n -butanol) and W (water) of A. clypearia were examined regarding the antioxidation in vitro on rat liver cells, and the results are given in Table 2. All five extracts showed good results in terms of antioxidant activity, in which the ethyl acetate fraction demonstrated the best activity with the lowest ED50 value of 0.63 µg/ mL. This value is six times lower than that of curcumin (4.43 µg/mL). In the other words, ethyl acetate fraction exhibited better antioxidant activity than curcumin.
The ED50 values of n-hexane and n -butanol fractions were nearly equal (9.10 µg/mL and 9.30 µg/mL, respectively). The chloroform fraction had the ED50 value of 14.91 µg/mL, while the water fraction demonstrated a low antioxidant activity in this test.
It was hypothesized in the previous studies that the antioxidant performance of a substance or a mixture of substances may follow the mechanism of hydrogen transfer or electron transfer or both [1]. However, one model only evaluates one side of the antioxidant capacity. Therefore, we continued to investigate the antioxidant activity of A. clypearia regarding both hydrogen and electron transfer.

Total antioxidant capacity in terms of electron transfer
The total antioxidant capacity was determined by assessing the electron-donating capacity of the sample using the phospho-molybdenum method. In principle, this method based on the reduction of Mo(VI) to Mo(V) by the antioxidant compounds and the formation of a green Mo(V) complex at a low pH with a maximal absorbance at 695 nm. A high absorbance value indicates that the sample exhibits high antioxidant activity [5]. A comparison of the antioxidant activity of five A. Clypearia fractions and curcumin is presented in Fig. 1.

Fig. 1. Total antioxidant capacity of extracted fractions from A. clypearia
The result showed that the antioxidant capacity based on the electron transfer mechanism of all methanol extraction and fractions from A. clypearia was lower than that of curcumin.

Antioxidant capacity in terms of hydrogen transfer
The antioxidant capacity of A. clypearia fractions was determined using the DPPH radical scavenging method as described in part 2.5. The scavenging activity of the fractions is presented in Table 3. The results in Table 3 showed the antioxidant capacity of all five fractions of A. clypearia, in which, four fractions had higher activity compared with curcumin, including chloroform, ethyl acetate, n -butanol and water fractions with the IC50 values of 12.98, 1.81, 6.39, 13.40 µg/mL, respectively (the IC50 of curcumin was 38.50 µg/mL). Especially, the ethyl acetate fraction demonstrated the greatest activity with the lowest IC50 value of 1.81 µg/ mL, which was 20 times lower than the IC50 of curcumin.
In general, based on the three in vitro models, the five fractions extracted from A. clypearia demonstrated the comparable antioxidant activity with, and in many cases, better than curcumin. This was the basis for our future studies.

Conclusion
The methanol extract of A. clypearia (Jack.) I. Niels. showed a significant antioxidant activity with the ED 50 of 2.18 µg/mL in in vitro tests on isolated rat liver cells while the value of curcumin is 1.87 µg/mL. From the five fractions studied, that of ethyl acetate extract showed the highest antioxidant activity with the lowest ED50 and IC50 (about 6 times and 20 times, respectively lower than that of curcumin). These results play an important role in further research regarding the discovery of new therapeutic compounds from nature. As far as we know, this is the first report on the antioxidant activity of A. clypearia species from Vietnam.