Guaiacum officinale L. (Thai name: Keaw Jao Jom) is a plant belonging to the family Zygophyllaceae. It is a tree with grayish bark. The leaves are opposite with round apex and terete petiole. The flowers are light-blue. For phytochemical constituents, it is reported that bark,1 flowers2 and leaves3 of Guaiacum officinale L. mainly contain saponin such as guaianin and guaiacin. Saponin is found to have pharmacological activities including immunostimulant, anti-carcinogenic properties, anti-rheumatoid and hypocholesterolaemic effect.4,5
Recently, treatments of many diseases by alternative medicine or natural products are interesting. There are reports on biological activities of Guaiacum officinale L. such as anti-HIV activity of methanolic leaf, twig and seed extracts.5 The anti-oxidant activity of wood resin has been carried out.6 However, there is no report on the anti-oxidant activity of ethyl acetate, ethanol and water extracts of this plant. Therefore, the objectives of this study are to investigate total phenolic contents and free radical scavenging activity of the extracts from Guaiacum officinale L. The results from this study may be helpful for value addition and development of this plant as cosmetic, drugs and supplementary food.
MATERIALS AND METHODS
Gallic acid (Sigma-Aldrich), ascorbic acid (Sigma-Aldrich), sodium carbonate (Na2CO3), 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Sigma-Aldrich), Folin-Ciocalteu (Sigma-Aldrich), ethanol AR grade (Merck) and ethy acetate AR grade (Merck)
Plant preparation and extraction
Bark, twig and leaf of Guaiacum officinale L. were collected from campus of Suan Sunandha Rajabhat University, Bangkok, Thailand. Voucher specimens were deposited and identified at herbarium of National Park, Wildlife and Plant Conservation Department, Ministry of Natural Resources and Environment, Bangkok, Thailand. Plant parts were dried in hot air oven at 60 °C until absolutely dry. They were then ground as fine powder. 600 ml of different solvents (ethyl acetate, water and ethanol) were added to 30 g plant powder (bark, twig and leaf). The plant powders were then extracted by Soxhlet extraction apparatus at 60 °C for 4 hours. The extracts were evaporated in water bath at 60 °C and frozen dry in order to obtain crude extracts. The crude extracts were kept in dark container at room temperature until uses.
Determination of total phenolic contents
Total phenolic contents were determined by Folin-Ciocalteu Colorimetry method (adapted from Amin et al., 2006).7 Gallic acid is used as standard (concentration of 12.5-100 mg/ml). The crude extracts were dissolved in methanol. Folin-Ciocalteu reagent was added to 0.5 ml of different extracts (1 mg/ml) and left for 5 minutes. 2 ml Na2CO3 was then added to the solution. The solutions were mixed and adjusted volume as 5 ml by distilled water and left at room temperature for 2 hours. The solutions were then measured the absorbance at 760 nm wavelength. Total phenolic contents in the extracts were determined by comparison of standard curves between mg of Gallic acid equivalent/g extract.
DPPH free radical scavenging activity study
Free radical scavenging activity of the extracts was investigated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity assay which is followed the study by Likhitwitayawuid et al. (2006).8 100 μm DPPH was prepared in methanol. The extracts were prepared in methanol at the concentration of 1 mg/ml. 180 μl DPPH solution and 20 μl extract solution were pipetted into microplate. The solutions were then incubated in dark room at room temperature for 20 minutes. These solutions were measured for absorbance by microplate reader (SPECTRO star Nano, BMG LabTech) at 517 nm wavelength. The absorbance of samples (As) was obtained. Methanol was used as blank and vitamin C was used as standard. Percentage inhibition of free radical was calculated by the following equation; % radical scavenging = [(Ac – As)/Ac] × 100. The graph illustrating the relationship between concentrations of extracts and % radical scavenging is plotted and then IC50 was calculated.
The dry weight and percentage of yield
The dry weight and percentage of yield of plant parts (bark, twig and leaf) extracted by different solvents (ethyl acetate, ethanol and water) is shown in Table 1. The percentages of yield of ethyl acetate, ethanol and water bark extracts are 0.83, 1.23 and 4.67 %, respectively. The percentages of yield of ethyl acetate, ethanol and water twig extracts are 1.97, 15.03 and 8.99 %, respectively. The percentages of yield of ethyl acetate, ethanol and water leaf extracts are 1.44, 14.50 and 7.07 %, respectively.
Total phenolic contents
Total phenolic contents were calculated from standard curve of gallic acid (y=4.1867x+0.0215, R2=0.996). It is found that ethyl acetate twig extracts have the highest total phenolic content (20.3±0.0031 μg GAE/1 μg extracts), followed by ethanol and water twig extracts (12.9±0.0006 and 10.3±0.0010 μg GAE/1 μg extracts, respectively).
DPPH free radical scavenging activity
The results found that ethanolic twig extracts have the highest anti-oxidant activity with IC50 of 0.45±0.0188 mg/ml, followed by ethyl acetate twig extracts and ethanolic bark extracts (IC50 of 1.16±0.0054 and 1.96±0.0243 mg/ml, respectively). However, the standard vitamin C has anti-oxidant activity with IC50 of 0.0034±0.0002 mg/ml (Table 2.)
According to the results, it is found that twig extracts from Guaiacum officinale L. have the highest total phenolic content and anti-oxidant activity in comparison to those of bark and leaf extracts. Interestingly, ethyl acetate twig extracts have the highest total phenolic content followed by those of ethanol and water twig extracts. DPPH free radical scavenging activity study revealed that ethanolic twig extracts have the highest anti-oxidant activity with the lowest IC50 value in comparison to those of other extracts. When compared to those of vitamin C, the plant extracts have higher IC50 value. This indicates that Guaiacum officinale L. extracts have lower anti-oxidant capacity than those of vitamin C.
Lowe et al. (2014) reported anti-HIV activity of methanol and ethyl acetate extracts of leaf, twig and seed of Guaiacum officinale L. It was found that ethyl acetate extracts has higher HIV inhibitory activity than those of methanol extracts and leaf extracts have the highest anti-HIV activity.5 This report correspond with the present study in which ethyl acetate leaf extracts exhibit the higher anti-oxidant capacity than those of ethanol and water leaf extracts of this plant.
There is a study on anti-rheumatoid and anti-oxidant activity of resin from wood of Guaiacum officinale L. which was found that the extracts have high anti-rheumatoid and anti-oxidant activity in an animal model.6 This findings is similar results to the anti-oxidant activity of this plant in the in vitro study.
The results from this study revealed that twig extracts have higher total phenolic content and anti-oxidant activity in comparison to those of bark and leaf extracts. However, there is a few reports on twig extracts of this plant. The studies mainly focused on the biological activity and phytochemical constituents of bark, wood (resin) and flower extracts. Therefore, there should be further study on phytochemical constituents of twig extracts of Guaiacum officinale.
The results from this study revealed that Guaiacum officinale L.extracts exhibit anti-oxidant capacity. Therefore, there should be the investigations on other biological and pharmacological activities such as anti-microbial activity and anti-inflammatory activity. In additions, phenolic compound and flavonoids are beneficial to human health and disease prevention including cardio-vascular disease and cancers .9,10 The findings from this study provide information for selection of this plant for health promotion and further development as supplementary food.