Anti-Aging Activity and Non-Toxic Dose of Phytooxyresveratrol from Artocarpus lakoocha Roxb

Purpose: To determine the anti-aging activity and toxicity doses of phytooxyresveratrol extracted from Artocarpus lakoocha Roxb. Methods: Artocarpus lakoocha 100 g was extracted with 2 ml of 95 % ethanol to obtain phytooxyresveratrol (POV). Total phenolic content, as well as free radical scavenging and anti-glycation activities of POV were characterized in order to assess its anti-aging properties. The models of DNA nicking and bacterial reverse mutation (Ames) were applied to the extract in order to determine its effective and toxic doses, respectively. Results: Phytooxyresveratrol (POV) exhibited antiaging activity. It also showed high levels of phenolic content, radical scavenging activity and anti-glycation. A POV concentration of 25 µg/ml promoted strong anti-DNA nicking. Furthermore, it was non-toxic at concentrations ranging from 5 - 100 µg/ml. Conclusion: In vitro, phytooxyresveratrol (POV) extracted from Artocarpus lakoocha exerted effective anti-aging activity at a concentration of 25 µg/ml. Thus, the substance should be further studied for possible formulation into pharmaceutical products.


INTRODUCTION
Oxidative damage to nucleic acid, lipid membrane and protein are associated with progressive loss of cellular structure and function [1]. An imbalance of free radical species: reactive oxygen species and reactive nitrogen species are associated with aging and some chronic diseases such as neurodegenerative disorder and cancers [2,3]. Glycation is the reaction that occurs when the reducing portion of monosaccharides,such as glucose or fructose, binds to macromolecules of protein or lipid structure to form advanced glycation endproducts (AGEs) [4,5]. AGEs are related to the pathogenesis of skin aging and diabetic complications [4,6]. Phytocompounds play a crucial role in the process of anti-glycation and anti-oxidation, leading to the prevention of cellular aging [7].
Oxyresveratrol (OV) is a hydroxylated analog of resveratrol. It can be used to promote anticellular aging [7,8]. Artocarpus lakoocha is a plant used in traditional Thai medicine for anti-inflammatory therapy and as well as an anti-skin aging agent [8].
In this study, we investigated the effective anti-aging dose of phytooxyresveratrol (POV) isolated from Artocarpus lakoocha. To this end, total phenolic content, free radical scavenging and antiglycation were defined for anti-aging activity.

EXPERIMENTAL Experimental samples
Oxyresvenox (Sabinsa Co Ltd, Germany) was used as the oxyresveratrol standard. Phytooxyresveratrol was extracted from A. lakoocha which was obtained from Detox (Thailand) Co Ltd.

Extraction of POV
The heartwood of A. lakoocha was dried at 50 o C and then ground into powder, 100 g of which was mixed with 2 ml of 95 % ethanol in the dark. The mixture was left for 4 h and then centrifuged at 1500 rpm for 15 min. The dried extract was obtained by drying the supernatant in a rotary evaporator at 50 o C and 50 mmHg. Phytooxyresveratrol was isolated by a high performance liquid chromatography (HPLC) and its structure confirmed with the 1 H and 13 C-NMR spectroscopic methods.

Total phenolic content
Total phenolic content was assayed using Folin-Denis reagent [9]. The extract, POV (20 µl), was diluted with 780 µl of distilled water and then mixed with 50 µl of fresh Folin-Denis reagent. Thereafter, 150 µl of 7.5 % w/v of sodium carbonate was added and the mixture incubated at room temperature in the dark for 30 min. The spectrophotometric absorbance of the mixture was measured at 765 nm. Gallic acid equivalent (GAE) was used as a standard. Phenolic content of the sample was expressed as mg gallic acid/g sample equivalent based on the calibration standard curve of gallic acid standard.

1,1-diphenyl-2-picrylhydrazyl (DPPH)method
A working solution of DPPH was freshly prepared with 95 % ethanol with absorbance of 0.95 ± 0.01 at 540 nm, 180 µl of which was mixed with 20 µl of POV. The absorbance of the mixture was immediately measured spectrophotometrically (Shimadzu UV-2550, Japan) at a wavelength of 540 nm. Total antioxidant activity of the sample was expressed as mg Vit C/g sample equivalent based on the calibration curve of standard vitamin C [9].

Ferric reducing antioxidant power (FRAP) method
FRAP reagent was freshly set by mixing a solution of 2.5 ml of 10 mM 2,4,6-tripyridyl-striazine (TPTZ) solution, 2.5 ml of 20 mM ferric chloride hexahydrate (FeCl 3 .6H 2 O) and 25 ml of acetate buffer (pH 3.6). This method is based on the reduction of ferric complex (Fe 3+ -TPTZ) to the ferrous complex (Fe 2+ -TPTZ) at low pH [10]. The extract was mixed with FRAP reagent in 96 well plates of a multimode detector model of Beckman, DTX 880 and then incubated in dark conditions at room temperature for 15 min. Ferrous sulfate (Fe 2 SO 4 ) was used as reference standard at 595 nm. The FRAP activity was expressed as µmol Fe 2+ /g sample equivalent.

Protein antiglycation
The antiglycation activity of POV extract was investigated as described in a previous study [8].

DNA nicking assay
DNA nicking was evaluated using supercoiled pUC 18 DNA model [11]. Supercoiled pUC 18 DNA was isolated from an E. coli strain, DH 5-alpha. Plasmid DNA 0.5 µg was added to Fenton reagent (consisting of a mixture of 30 mM H 2 O 2 , 50 µM ascorbic acid and 80 µM FeCl 3 ) and contained phytooxyresveratrol (either 5, 10, 25 or 50 µg/ml). The mixture was incubated for 30 min at 37 0 C and then stained with ethidium bromide on a 1 % agarose gel to obtain the intensity of DNA nicked.

Bacterial reverse mutation (Ames) test
This test was performed using plate incorporation method [12]. The toxicity of oxyresveratrol standard and phytooxyresveratrol were investigated for their capacity to induce reverse mutation by Salmonella typhimurium strains, TA98 and TA100. Doses ranging from 5, 25 -100 µg/ml of oxyresveratrol and phytooxyresveratrol were determined for their mutagenic toxicity. 4-Nitroquinoline-1-oxide (4NQO, 0.2 µg/ml) was used as positive mutagen control while 100 % DMSO was used as negative control.

Statistical analysis
All results were expressed as mean ± standard error (SE). The difference among groups was obtained by one-way ANOVA using SPSS program, version 13.0. Statistic significance was set at p < 0.05.

RESULTS
Total phenolic content and radical scavenging activity of phytooxyresveratrol and OxyResvenox (reference standard) are shown in Table 1. The phenolic contents of phytooxyresveratrol (isolate) and OxyResvenox (reference) were not statistically significant (p = 0.671). There was also no significant difference between the scavenging activities of phytooxyresveratrol isolate and OxyResvenox, based on DPPH and FRAP data (p = 0.227 and p = 0.373, respectively). Phytooxyresveratrol exhibited a high level of antiglycation with IC 50 value of 3.343 ± 0.414 µg/ml. Interestingly, phytooxyresveratrol had stronger antiglycation activity than aminoguanidine, a hydrazine-like compound that blocks the formation of advanced glycation end-products (AGEs) (p = 0.001), as shown in Fig 1, but is not statistically different from that of the standard OxyResvenox (p = 1.000). The standard gallic acid at 50 µg/ml (lane 7) served as positive control to prevent DNA damage in the mixed reaction. In this study it was found that the phytooxyresveratrol dose of 25 µg/ml (lane 5) had a beneficial anti-DNA damage activity but at a dose of 50 µg/ml (lane 6), the activity was lower. In the toxicity test, phytooxyresveratrol did not show any mutagenic activity against the test bacterial strains of T98 and T100 at concentrations 5, 25, 50 and 100 µg/ml of the isolate. Table 2 shows that the toxicity profiles of the test isolate and the standard, OxyResvenox, were not significantly different at concentrations of 5, 25 and 50 µg/ml. Treatment with 0.2 µg/ml 4NQO, a positive mutagen to T98(S9-) and T100(S9-), had high reverting colony per plate activity compared with control (DMSO), phytooxyresveratrol and reference (OxyResvenox). Thus, phytooxyresveratrol dose of 100 µg/ml, like OxyResvenox OxyResvenox (100 µg/ml) was less toxic than 100 % DMSO to T98(S9-) and T100(S9-) (p = 0.040 and p = 0.564, respectively).

DISCUSSION
Free radicals and glycation products have important roles in cellular aging development [6,13]. External radicals, especially ultra violet radiation-generating singlet oxygen, are linked to cellular aging [14].
In the present study, phytooxyresveratrol isolated from A. lakoocha showed both antioxidant and anti-glycation activities. This supports previous findings on the phytocompound [15,16]. As the results indicate, the high level of radical scanvenging and antiglycation activities of the phytooxyresveratrol isolate suggests that it is bioequivalent to the reference standard, OxyResvenox.
The similarity of the activities of both phytooxyresveratrol isolate and the oxyresveratrol standard indicate that the latter is a useful anti-aging agent. An imbalance of antioxidant molecules often leads to the formation of oxidant species [17,18]. The anti-aging activity can thus be attributed to the radical scanvenging and antiglycation properties of the compound.
Although the phytooxyresveratrol isolate demonstrated the capacity to prevent cellular aging and also exhibited good safety profile at the effective dose, there is a need to undertake studies to develop and clinically evaluate pharmaceutical formulations of phytooxyresveratrol derived from A. lakoocha in order to determine their suitability for anticellular aging applications.

CONCLUSION
Phytooxyresveratrol derived from A. lakoocha has the capacity to prevent cellular aging due to its antioxidant and anti-glycation activities.
At a dose of 25 µg/ml, the compound prevented DNA damage and was cellularly non-toxic. However, further studies are required to develop it into suitable pharmaceutical formulations.