Antioxidant Phenolic Compounds of Dracaena Cambodiana

The antioxidant activities of the petroleum ether, ethyl acetate, n-BuOH and water extract fractions from Dracaena cambodiana Pierre ex Gagnep were evaluated in this study. The ethyl acetate fraction contained the highest amount of total phenolics and total flavonoids, and showed the greatest DPPH˙, ABTS+ and Superoxide anion radical-scavenging capacities. The DPPH˙, ABTS+ and Superoxide anion radical-scavenging capacities of nine compounds isolated from the ethyl acetate fraction were also evaluated. The results indicated that these compounds contributed to the antioxidant activity of D. cambodiana. Therefore, D. cambodiana and these compounds might be used as natural antioxidants.


here has been
an increasing volume of evidence to suggest that many age-related human diseases such as heart disease, cancer, arthritis, immune system decline, brain dysfunction, and cataracts are the results of cellular damage by free radicals, and that antioxidants in our diet could play an important role in the prevention of such diseases [1,2].This has attracted much public interest in natural antioxidants and has led to an extensive search for effective antioxidants in nature [3][4][5][6][7].At the same time, numerous crude extracts and pure natural compounds from natural plants have been OPEN ACCESS reported to possess antioxidant and radical-scavenging capacities.For example, flavonoids, with a large distribution in nature, have strong antioxidant activity [8,9].

Dragon's blood is the red resin se reted by the stems of Dracaena, Daemonorops, Croton, and Pterocarpus [10].As a famous traditional medicine in the ancient Arab culture, dragon's blood has been used for the treatment of wounds, leucorrhea, fractures, diarrhea, and piles as well as intestinal and stomach ulcers for a long time [11].In China, dragon's blood is produced form Dracaena cochinchinensis (Lour.)and Dracaena cambodiana [12].Previous phytochemical studies on the plants of the genus Dracaena have led to the isolation of a number of phenolic compounds and a series of steroidal saponins [13], which are the same or similar to those compounds discovered in dragon's blood originated from plants of the genus.Nine compounds, namley 3,4-dihydroxyallylbenzene (1), p-hydroxybenzaldehyde (2), cambodianol (3), (2S)-3',7-dihydroxy-4'-methoxy-8-methylflavane ( 4), (2R)-4',7-dihydroxy-8-methylflavane ( 5), (±)-4',7-dihydroxy-3'-methoxyflavane ( 6), (2S)-4',7dihydroxyflavane (7), 7,4'-dihydroxy-homoisoflavane (8) and 4,4'-dihydroxy-2'-methoxychalcone (9) (Figure 1) were isolated from the ethyl acetate fraction of the D. cambodiana stem extract [14][15][16].Their structures are similar to those of compounds found in dragon's blood obtained from D. cochinchinensis.In continuation of our work, this report deals with the antioxidant properties of fractions and phenolic compounds derived from the ethanol extract of the stem of D. cambodiana for use as natural antioxidants.


Results and Discussion


Fractions

Table 1 shows the DPPH and ABTS radical-scavenging capacities of the fractions from D. cambodiana.The results illustrated that the DPPH and ABTS radical-scavenging capacities of various solvent fractions increased with increasing concentration, and all the regression equations were significant at p < 0.05, so all the fractions had some DPPH and ABTS free radical-scavenging capacities.The concentrations of the fractions (25-800 g/mL) or individual pure compounds (6.25-200 g/mL) were used in developing the respective standard curves.: y = 0.0050x + 0.0569 (R 2 = 0.9749 **); PET: y = 0.0003x + 0.0470 (R 2 = 0.9930 **); EAF: y = 0.0008x + 0.0718 (R 2 = 0.9992 **); BUF: y = 0.0005x + 0.0578 (R 2 = 0.9975 **); AF: y = 0.0004x + 0.0599 (R 2 = 0.9596 **).The symbol ** followed with the determination coefficient shows the significant level at 0.01, and the symbol * followed with the determination coefficient shows the significant level at 0.05, and no symbol followed with the determination coefficient shows no significance.ND represents not done."-" represents the data missed.

The DPPH

azyl) radi
al-scavenging assay is widely used to evaluate antioxidant capacity in a short time [17,18].The DPPH radical-scavenging capacities of the fractions of D. cambodiana stems are shown in Table 2.Among the fractions, the EC 50 values of the DPPH radical-scavenging capacities were found to be 5.007 mg/mL, 1.613 mg/mL, 2.441 mg/mL and 4.874 mg/mL for petroleum ether fraction (PEF), ethyl acetate fraction (EAF), n-butanol fraction (BUF), and aqueous fraction (AF), respectively.The order of DPPH radical-scavenging capacities was found to be EAF > BUF > AF > PEF.The EAF and BUF fractions showed the most potent activity, while the PEF was the least active scavenger, indicating that compounds with strongest radicalscavenging capacities were located in the medium polarity section.

Table 2 also illustrates the ABTS free radical scavenging capacity of the fractions of D. cambodiana stems.Among the fractions, the ABTS radical-scavenging capacity IC 50 values were found to be 1.510 mg/mL, 0.535 mg/mL, 0.884 mg/mL and 1.100 mg/mL for PEF, EAF, BUF and AF, respectively.The order of ABTS radical-scavenging capacities was found to be EAF > BUF > AF > PEF.Superoxide anion radical plays an important role in the formation of other reactive oxygen-species such as hydroxyl radical, hydrogen peroxide, or singlet oxygen in living systems [19], so evaluating the scavenging effects of D. cambodiana on superoxide radicals is one of the most important ways to clarify the mechanism of any antioxidant activity.Table 2 shows the superoxide anion radicalscavenging capacities of the fractions of D. cambodiana stems.All of the tested extracts exhibited superoxide radical-scavenging effects.Among the fractions, the order of the superoxide anion radicalscavenging capacities were found to be EAF > BUF > AF > PEF.

The overall antioxidant activities of plants are typic lly due to the presence of phenolic compounds.Generally, the antioxidant activities of phenolic compounds are realized by inactivating lipid free radicals and preventing decomposition of hydroperoxides into free radicals.The amounts of the total phenolics and the total flavonoids in the fractions of D. cambodiana stems are shown in Table 2. EAF showed the highest amount of total phenolics and total flavonoids, followed by BUF, AF, and PEF.Many studies have conclusively revealed that the antioxidant activity was related to the amount of total phenolics and total flavonoids [20,21].At the same time, we could find that this order was similar to that of their antioxidant and radical-scavenging capacities.Results in our study also demonstrated that the extent of antioxidant activity of D. cambodiana stems was in accordance with the amounts of phenolics and flavonoids present in this species.The results showed that the ethyl acetate fraction had the strongest DPPH, ABTS free radical, Superoxide radical-scavenging capacities and the highest amount of total phenolics and total flavonoids among the other fractions.We thus choose the ethyl acetate fraction to undergo further chromatography to isolate the sub-fractions containing compounds 1-9.


Sub-Fractions

Nine compounds were isolated from the ethyl a

ambodiana

The three test methods all demonstrated that compound 1 was the mo
t active free radical scavenger.This might relate to the double bond and the number of hydroxyls present in the aromatic ring.The results also support the notion that ortho-hydroxyl structures are crucial for the enhanced antioxidant activity because the ortho-quinone is easily formed [22,23].Compound 2 was the second most active free radical scavenger.Aside from hydroxyls, aldehydes might play an important role in the antioxidant activity.The antioxidant activities of compounds 4-9 had no evident differences.In addition, compound 1 showed higher inhibitory activity towards Staphylococcus aureus and methicillin-resistant Staphylococcus aureus, therefore, it has great potential for pharmaceutical applications.


Experimental


General

Optical density measurements were made with a S

n Province, Chin
in J

y 2007, dried immediately and crushed into pieces.
he specimen was identified by Associate Professor Dai Zheng-Fu of the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, where a voucher specimen (No. 20070701) of D. cambodiana was deposited.


Extraction and Isolation of Antioxidant Compounds

The dried and crushed stems of D. cambodiana (13.3 kg) were exhaustively extracted three times with 95% ethanol (50 L) at room temperature for three weeks.The ethanol extract was then filtered through absorbent gauze, and the filtrate was concentrated under reduced pressure at 50 °C to remove ethanol.The extract was suspended in H 2 O (2 L) and partitioned with petroleum ether (1 L × 3), and the resulting supernatants were collected and filtered through absorbent gauze, followed by evaporation of the solvent at 50 °C under reduced pressure.The resulting liquid residue was labeled as the petroleum ether fraction.The defatted material remaining after petroleum ether extraction was partitioned successively with ethyl acetate (1 L × 3) and n-butanol (1 L × 3).The ethyl acetate and n-butanol extracts were separately combined and evaporated to dryness under reduced pressure, while the aqueous layer was lyophilised to dryness.These four fractions were designated as PEF (15.5 g), EAF (145.0 g), BUF (400.0 g), and AF (13.0 g), respectively.The EAF fraction (145.0) was subjected to column chromatography (CC) over silica gel eluted with a mixture of chloroform and methanol (100:1-0:100, v/v) of increasing polarity resulting in six fractions (Fr.1-Fr.6).Repeated CC on silica gel CC eluted with petroleum ether-acetone gradients (10:1-2:1, v/v) and Sephadex LH-20 (CHCl 3 -MeOH, 1:1, v/v), led to the isolation of compounds 1 (116.6 mg), 3 (120.9mg), 4 (26.3 mg), 5 (13.6 mg), 6 (10.2 mg) and 8 (8.3 mg) from Fr.2 (16.0 g).Fr.3 (24.0 g) yielded compounds 2 (

(50.3mg) and 9 (177.7 mg)
after CC with Sephadex LH-20 (CHCl 3 -MeOH, 1:1, v/v) and further purification with silica gel CC (CHCl 3 -MeOH, 30:1).The structures of these compounds were identified on the basis of physicochemical properties and spectroscopic data [14][15][16].


Assays


DPPH scavenging capacity

The DPPH radical-scavenging capacity was measured using the method of Zhang and Lu [24,25] with some modification.Two milliliter of an ethanol solution of DPPH (0.1 mM) was added to sample fractions (0.1 mL, 25-800 g/mL) or individual pure compounds (6.25-200 g/mL) in DMSO at differe perature, the absorbances of the resulting solutions were measured at 517 nm.Ascorbic acid was used as the positive control.The DPPH radical-scavenging capacity (%) was calculated using the following equation:
DPPH radical-scavenging capacity (%) = [1 − (A 1 − A 2 ) / A 0 ] × 100%
where A 0 was the absorbance of the control (without sample), A 1 was the absorbance in the presence of the sample, and A 2 was the absorbance without DPP

The EC 50 valuee, defined as the am
unt of antioxidant necessary to decrease the initial DPPH˙ concentration by 50%, were calculated from the results.Measurements were calibrated to a standard curve of prepared ascorbic acid (6.25-200 g/mL) y = 0.0074x − 0.0110, R 2 = 0.9958.


ABTS free radical scavenging assay

The ABTS free radical scavenging capacity assays were carried using a modified method as described by Fang and Re [26,27].Potassium persulfate was added to 7 mM of ABTS + and kept for 12-16 h at room temperature in dark.The ABTS + solution was diluted with PBS (potassium phosphate-buffered saline, pH 7.4) to an absorbance of 0.70 ± 0.02 at 734 nm before analysis.ABTS + solution (3.0 mL) was added to sample fractions (0.1 mL, 25-800 g/mL) or individual pure compounds (6.25-200 g/mL) in DMSO at different concentrations and mixed by hand for 20 s.Th was recorded at 734 nm on a Shimadzu UV-2550 spectrophotometer.Trolox ® was used as the positive control.The ABTS free radical-scavenging capacity (%) was calculated using the following equation:
ABTS free radical-scavenging capacity (%) = [1 − (A 1 − A 2 ) / A 0 ] × 100%
where A 0 was the absorbance of the control (without sample), A 1 was the absorbance in the presence of the sample, and A 2 was the absorbance without ABTS + .The

50 values, defined as the amount of an
ioxidant necessary to decrease the initial ABTS + concentration by 50%, were calculated from the results.Measurements were calibrated to a standard curve of prepared Trolox ® (6.25 − 200 g/mL) y = 0.0050x + 0.0569, R 2 = 0.9749.


Superoxide radical-scavenging capacity

The superoxide anion radical-scavenging capacities of the sample were tested by the method of Sakanaka et al. [28].Briefly, phosphate buffer solution (1.0 mL, 65.0 mM, pH 7.8), xanthine solution (0.1 mL, 7.5 mM), ydroxylammonium chloride solution (0.1 mL, 10.0 mM), and sample fractions (0.1 mL, 0.1 mg/mL) or individual pure compounds (0.1 mg/mL), redistilled water (0.4 mL) and protein xanthine oxidase solution (0.3 mL, 20.0 μg/mL) were mixed in turns.The mixture was incubated for 20 min at 25 °C, followed by the addition of anhydrous p-aminobenzenesulfonic acid (0.5 mL, 19.0 mM) and α-naphthylamine solution (0.5 mL, 1.0%).The reaction mixture was kept at room temperature for 20 min, and the absorbency (A 1 ) was tested at 530 nm.The sample was substituted by redistilled water and repeated the procedures mentioned above, the absorbency (A 0 ) of the blank was tested.The standard curve for the superox

e anion radical-scavenging capacity was develop
d using α-tocopherol standard solution (0-1000 μM) y = 0.00004x + 0.00090, R 2 = 0.9592.The superoxide radical-scavenging capacity was shown with α-tocopherol equivalent antioxidant capacity (mol α-tocopherol/g DW).


Determination of the amount of total phenolics

The amount of total phenolic contents was determined according to the Folin-Ciocalteu method [29] with some modifications.Briefly, sample fractions (1.0 mL) were was mixed with distilled water (9.0 mL) in a 25 mL volumetric flask.Then Folin-Ciocalteu's phenol reagent (1.0 mL) was added to the mixture which was then shaken.The mixture was kept for 5 min, followed by the addition of 7% Na 2 CO 3 solution (10 mL).The mixed solution was then diluted to 25 mL with distilled water and mixed thoroughly.After 90 min of reaction at room temperature, the absorbance versus a blank was measured at 750 nm.The standard curve for total phenolics was developed using galli

acid standard solution (0-100 mg/L) y = 99.5670x
0.0425, R 2 = 0.9952.The total phenolics in the extract and the fractions were expressed as g gallic acid equivalents (GAE)/g extract.All samples were tested in three times, and the results were averaged.


Determination of the amount of total flavonoids

The amount of total flavonoids was determined using the method described by Jia et al. [30].Briefly, sample fractions or standard solution of rutin (1 mL) was mixed with distilled H 2 O (4 mL) in a 10 mL volumetric flask, followed by the addition of 5% NaNO 2 solution (0.3 mL).After 5 min, 10% AlCl 3 solution (0.3 mL) was added.At 6 min, 1 M NaOH solution (2 mL) was added to the mixture.Immediately, distilled H 2 O (2.4 mL) was added to the reaction flask and the contents mixed well.The absorbance versus a blank was measured at 510 nm.Measurements were calibrated to a standard curve of prepa

d rutin standard solu
ion (0-0.5 mg/L) y = 1.0503x + 0.0176, R 2 = 0.9941.Total flavonoids of the extracts and the fractions were expressed on an extract weight basis as mg/g rutin equivalents (RE).All samples were analyzed in three replications.


Statistical Analysis

Results were expressed as mean ± standard deviation (S.D.) of replicate solvent extractions and triplicate of assays and analyzed by SAS.One-way analysis of var

nce (ANOVA)
ith Tukey's test was carried out to test significant differences between levels of treatment.Significant levels were defined using the values p < 0.05 and p < 0.01.Pearson correlations between variables were established using SAS.


Conclusions

All the tested extracts of D. cambodiana showed antioxidant and radical-scavenging capacities, and the order of antioxidant and radical-scavenging capacities among the extracts assayed through all the three methods was found to be EAF > BUF > AF > PEF.This order was similar to the total phenolics and total flavonoids contents of the fractions.However, the antioxidant and radical-scavenging capacities of some compounds isolated from the stems of D. cambodiana were discordant in the three methods.Therefore, we should use at least two methods to evaluate whether a sample has antioxidant activity.The EAF and compound 1 had the lowest IC 50 values for DPPH, ABTS radical-scavenging capacities and showed the highest superoxide anion radical-scavenging capacities.The DPPH˙, ABTS + and Superoxide anion radical-scavenging capacities of most compounds are higher than the fractions.The results not only confirmed that these compounds were the main constituents contributing to the antioxidant activities of the ethyl acetate fraction, but also indicated that the extracts and compounds from D. cambodiana might be used as natural antioxidants and alternatives to synthetic antioxidants and seem to be applicable in both