Year : 2009 | Volume
: 1 | Issue : 5 | Page : 238--244
Determination of Radical Scavenging Activity of Hydroalcoholic and Aqueous Extracts from Bauhinia divaricata and Bougainvillea spectabilis Using the DPPH Assay
L Chaires-Martinez1, E Monroy-Reyes1, A Bautista-Bringas1, HA Jimenez-Avalos1, G Sepulveda-Jimenez2,
1 Food Research Center. Instituto Tecnológico Superior de Alamo Temapache (FRC-ITSAT). Km. 6.5 Tuxpan-Potrero del Llano. Alamo, Veracruz 92750
2 Centro de Desarrollo de Productos Bióticos del Instituto Politécnico Nacional. (CEPROBI-IPN). Yautepec, Morelos. México
Food Research Center. Instituto Tecnológico Superior de Alamo Temapache (FRC-ITSAT). Km. 6.5 Tuxpan-Potrero del Llano. Alamo, Veracruz 92750
Bauhinia divaricata and Bougainvillea spectabilis are medicinal plants widely distributed in Mexico and they are used because of its potential hypoglycemic action; however, no free radical scavenging activity (RSA) studies over these plants are known. Thus, aqueous and hydroalcoholic extracts from leaf and stem samples were evaluated for their RSA using 1,1-diphenylpicrylhydrazyl free radical (DPPH•). Total phenolics and flavonoids extracts were determined too. Statistical analyses were performed using the SPSS statistical program with the significance level set at P<0.05. Bauhinia divaricata stem aqueous extracts with total phenols content of 12.98 mg GAE/g DW had the highest amount between samples. The same behavior was shown in flavonoids determination. However, when RSA was estimated it was found that stem aqueous extracts from Bougainvillea spectabilis produced more DPPH absorbance reduction (95.66%), with an IC 50 (the concentration to inhibit the oxidation of DPPH by 50%) and AP (reciprocal of IC 50) values of 0.03 μg/mL and 33.33, respectively. These results were superior to common synthetic antioxidants used in the food industry like butylated hydroxyl toluene (BHT, IC 50=62 μg/mL) and can be useful for further applications of these plants or its constituents in pharmaceutical and alimentary preparations.
|How to cite this article:|
Chaires-Martinez L, Monroy-Reyes E, Bautista-Bringas A, Jimenez-Avalos H A, Sepulveda-Jimenez G. Determination of Radical Scavenging Activity of Hydroalcoholic and Aqueous Extracts from Bauhinia divaricata and Bougainvillea spectabilis Using the DPPH Assay.Phcog Res 2009;1:238-244
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Chaires-Martinez L, Monroy-Reyes E, Bautista-Bringas A, Jimenez-Avalos H A, Sepulveda-Jimenez G. Determination of Radical Scavenging Activity of Hydroalcoholic and Aqueous Extracts from Bauhinia divaricata and Bougainvillea spectabilis Using the DPPH Assay. Phcog Res [serial online] 2009 [cited 2021 Mar 7 ];1:238-244
Available from: http://www.phcogres.com/text.asp?2009/1/5/238/58045
Chemistry of natural products is a research field with endless potential, and is especially important in countries possessing great biodiversity  , as Mιxico. In recent years there is an intensive increase in researches objecting the evaluation of the antioxidant activity of extracts and other materials from natural sources ,,, since antioxidant compounds could be applied to treat and prevent cancer and cardiovascular diseases as well as to the aging process ,, . Currently available synthetic antioxidants like BHT, butylated hydroxyl anisole and tertiary butylated hydroquinones have been suspected to cause or prompt Chemistry of natural products is a research field with negative health effects. Hence, strong restrictions have endless potential, and is especially important in countries been placed on their application and there is a trend to possessing great biodiversity  , as Mιxico. In recent years substitute them with naturally occurring antioxidants  . there is an intensive increase in researches objecting the Several studies revealed that phenols, mainly the type of evaluation of the antioxidant activity of extracts and other flavonoids, from some medicinal plants, have antioxidant materials from natural sources ,,, since antioxidant properties and exert anticarcinogenic, antimutagenic, compounds could be applied to treat and prevent cancer antitumoral, antibacterial, antiviral and anti-inflammatory and cardiovascular diseases as well as to the aging process effects  due to their redox properties, acting as ,, . Currently available synthetic antioxidants like reducing agents, hydrogen donors, singlet oxygen quenchers and chelating metals ,,. Assays based on the scavenging of DPPH has been widely used to measure the antioxidant activity of different phenolic compounds and the results obtained are, in most cases, in agreement with those derived by lipid peroxidation assays in bulk oils ,, . DPPH is one of a few stable available organic nitrogen radicals and has a UV-vis absorption maximum at 515 nm. When a solution of DPPH is mixed with a substance that can donate a hydrogen atom, the reduced form of the radical is generated accompanied by loss of color , .
Bougainvillea is a genus of flowering plants native to South America and are popular ornamental plants in all over the world  . Narayanan  , Senapati  and Menakshi  found that the alcoholic extract of B. spectabilis has significant hypoglycemic effect in alloxan-induced diabetic albino mice and that it is free from any acute toxicity. In another work, Edwin  reported the free radical scavenging activity of ethanol extracts from B. glabra. It is claimed that D-pinitol (3-O-methylchiroinositol) extracted from B. spectabilis exert insulin-like effects , .
The Bauhinia genus comprises about 500 species of shrubs, small trees, and lianas in the tropics. It can be found in the rainforests and tropical regions of Africa, Asia and Latin America. Many plants of the genus are used in traditional medicine for their interesting biological activities such as analgesic, antidiabetic, antiinflamatory, antimicrobial, astringent and diuretic effects , . There are several works dealing with the antioxidant capacity of the Bauhinia genus, mainly over B. candicans  , B. forticata  , B. variegata  and B. purpurea  . In this project, Bauhinia divaricata L. was studied.
In summary, Bougainvillea spectabilis and Bauhnia divaricata are species whose infusion extracts have been used in hypoglycemic treatments in Mιxico, but today no free radical scavenging activity reports on these natural sources are known. Thus, in this work it was carried out the determination of the phenols and flavonoids content and the evaluation of the antioxidant activity of aqueous and methanolic extracts from leaves and stem samples of these plants, in order to contribute to the knowledge for further applications in food and pharmaceutical industries.
Materials and Methods
1,1-diphenyl-2-picrylhydrazyl, gallic acid, cathequin, 2,6 di-tert-butyl-4-methylphenol (BHT), butylated hydroxyanysole (BHA), sodium carbonate, sodium nitrite, aluminum chloride, sodium hydroxide and methanol were purchased from Sigma Co. (St. Louis, MO, USA) and Folin Ciocalteu reagent from Fluka Chemical Co. (Buchs, Switzerland).
Leaf and stem of Bauhinia divaricata L. and Bougainvillea spectabilis were collected manually throughout 2008 in Alamo-Veracruz, Mιxico and they were ground and milled into flour. Voucher specimens are deposited at the Herbarium "IZTA" of FES-Iztacala, Universidad Nacional Autonoma de Mιxico under the number 42167.
Portions of 200 mg of the leaf and stem samples were separately homogenized with 20 mL of 95% methanol for 50 min at room temperature. For the aqueous extraction, 200 mg of the samples were separately homogenized with 20 mL of deionized water and then boiled at 98 °C for 3 h. After this procedure, all samples were filtered under vacuum using Whatman No. 1 paper and filtrates were frozen and lyophilized and were finally stored at -18 °C until their use , .
Total phenols determination
Total phenols were determined by Folin-Ciocalteu colorimetric method , . To 0.5 mL of 50% hydroalcoholic solution of each extract was mixed 0.5 mL of the Folin-Ciocalteau reagent and 0.5 mL of 10% Na 2 CO 3 , and the absorbance was measured at 760 nm after 1 h incubation at room temperature, with an Aquarius Cecil CE7200 spectrophotometer. The results were given in mg GAE/100 g DW of gallic acid equivalent. The standard curve was prepared using 0, 50, 100, 150, 200, and 250 mg/L solutions of gallic acid in methanol:water (50:50, v/v).
Total flavonoids determination
Total flavonoids were determined by the Aluminum chloride colorimetric method  , where 250 mL of each sample was mixed with 1.25 mL of deionized water and 0.075 mL of 5% sodium nitrite. After 6 min, 0.15 mL of 10% aluminum chloride was added and after another 6 min the product was mixed with 0.5 mL of 1M sodium hydroxide and 2.5 mL of deionized water. Total flavonoids were measured at 510 nm  . The results were given in mg CE/100 g DW of cathequin equivalent. The standard curve was prepared using 5, 10, 20, 40, 60, 80 and 100 mg/L solutions of cathequin in methanol:water (50:50, v/v).
Estimation of RSA using the DPPH assay
The %RSA of phenols was determined using the DPPH assay , . The decrease of the absorption at 516 nm of the DPPH solution after addition of the antioxidant was measured in a cuvette; concentration of the extracts was varied in the reaction mixtures adding 0, 200, 400, 600, 800 or 1000 μL of each of them to a 3.9 mL hydroalcoholic solution of DPPH (25 ppm), completing a final volume of 4.9 mL with methanol. The absorption was monitored at the start and continuously every 30 sec until a constant value (plateau) was reached. Then, DPPH was calculated through a calibration straight line obtained in a range of concentrations of this substance. The results are expressed as %RSA= [Abs 516 nm (t = 0) -Abs 516 nm(t = t')] Χ 100/Abs 516 nm(t = 0). Measurements were performed in triplicate. Absorbance values were corrected for radical decay using blank solutions. The quality of the phenol antioxidants in the extracts was measured by determining the IC 50 (the concentration to inhibit the oxidation by 50%) of the pooled phenol extracts for each sample. IC 50 was determined graphically from the sigmoidal-shaped curve of antioxidant concentration (μg/mL) vs. %RSA. For comparison purposes the results were expressed as AP, defined as the reciprocal of IC 50 (AP=1/IC 50 ). The higher number of AP the better quality antioxidants  .
Statistical analyses of results
To verify the statistical significance of the studied parameters, means and standard deviation of three measurements were determined. Where it was appropriate, differences between groups were tested by 2-way ANOVA using SPSS v11.0 software (SPSS Inc. Chicago, IL). Significant differences (P values 0.05) were assessed using Tukey`s test  .
Total phenols determination
Comparison of total phenols content [Table 1] between Bauhinia divaricata leaf and stem extracts show slightly significant difference in each solvent used though not different enough to be statistically significant (P). However, significant differences were found between solvents used, being total phenols content in aqueous extraction higher than that with hydroalcoholic extraction procedure. The same behavior was found in Bougainvillea spectabilis samples although there are significant differences between samples and solvents used. Bahuinia divaricata stem aqueous extracts with total phenol contents of 12.98 ± 0.29 mg GAE/g DW had the highest amount among the plants analyzed in this study.
Total flavonoids determination
Studying the main group of phenolic compounds, the total flavonoid content was analyzed. Results are exhibited in [Table 2] and they indicate that total flavonoids contents in leaf and stem extracts from Bauhinia divaricata shown no statistical significant difference for the aqueous extraction procedure, whereas for hydroalcoholic extracts there is more flavonoids in leaf than in stem. Aqueous extraction method allows obtaining higher total flavonoids content in comparison with hydroalcoholic extraction procedure. In case of flavonoid concentration in Bougainvillea spectabilis it was found that leaf aqueous extracts presented the highest values (5.33 ± 0.08 mg CE/g DW); but, Bauhinia divaricata leaf aqueous extracts had the highest among all the samples.
Estimation of RSA Using the DPPH Assay
These results shown the DPPH scavenging kinetics measured at 515 nm for 20 min [Figure 1] and [Figure 2]. Stem aqueous extracts from Bougainvillea spectabilis produced more DPPH absorbance reduction (95.66 %) than hydroalcoholic extracts and this value was higher than Bauhinia divaricata results in samples analyzed [Table 3]. Bauhinia divaricata aqueous extracts exhibited a %RSA values in the range of 53-75% in stem and leafs versus 74-84% in hydroalcoholic extracts. The IC 50 and AP values showed that stemaqueous extracts from Bougainvillea spectabilis has the better quality of antioxidants, because at lower concentrations produce high radical scavenging activity (0.03 μg/mL and 33.33, respectively). IC50 values of the standard compounds were 28.83 for gallic acid, 3.3 for cathequin, 62 for BHT and 363 μg/mL for BHA.
Taking into account the use of Bauhinia divaricata and Bougainvillea spectabilis to design and formulate disease-preventing products in a future, it was observed that the phenol content range observed in the composition of the plant extracts may be completely acceptable due to several points: they were obtained from two sources (leaf and stem) and they were extracted by different methods (aqueous and hydroalcoholic procedures), rending different content of dry weight. In comparison with other vegetables resources, the phenol content values determined in this work for Bauhinia divaricata ranged between 11.99 to 12.98 mg/g, and for Bougainvillea spectabilis ranged 9.64 to 11.1, being values lower than the ones reported for other medicinal plants [Table 4], like Camellia sinensis (288.5 mg/g), Sesbania grandiflora (54.5 mg/g) and Mellilotus officinalis (289.5 mg/g) , . However, when %RSA is evaluated, Bougainvillea spectabilis has the highest activity (95.66%). It is seen that although Bougainvillea spectabilis had lowest contents of phenolics the %RSA is the highest between several medicinal plants. These results were complemented with the IC 50 evaluation, where lower values are indicative of a higher free radical scavenging activity of a sample. Aqueous and hydroalcoholic stem extracts from Bougainvillea spectabilis shown strong activities, with IC 50 values of 0.03 and 0.061 μg/mL. Their activities were 110 and 54-fold more potent than cathequin and 2066, and 1016-fold more potent than the known synthetic antioxidant BHT (62 μg/mL) reported here and elsewhere  . In the case of Bauhinia divaricata stem hydroalcoholic extracts showed IC 50 values of 0.035 μg/mL. In other reports, B. variegata , B. purpurea, B. candicans, B. monandra, B. forticata and B. angulosa exhibited IC 50 values ranged between 60 to 2000 μg/ mL and the authors indicate that these values represent a good potential as free radical scavengers ,,. Therefore, results from Bauhinia divaricata and Bougainvillea spectabilis shown that they could represent good options for antioxidants sources.
The mechanism of reduction of the DPPH molecule  is based on a scavenging activity. In this system, the structure (both planar and spatial) of the antioxidant compound, present in the extract, is important for its capacity of donating hydrogen ions. Compounds able to donate hydrogen are derived from the shikimate pathway, as for example, flavonoids  , which were determined in Bougainvillea spectabilis and Bauhinia divaricata. These molecules are not produced by plants whose extracts display a very high IC 50 in the DPPH test, opposite to the results found in this work. Plants with high IC 50 are, in fact, very rich in compounds of the acetate pathway, like terpenoids and fatty acids, which are unable of scavenging the DPPH free radical, but are able to avoid oxidative damage of cell membranes  .
The properties of the solvents with different polarities used in this work significantly affect the yield of total phenolics and flavonoids and the antioxidant activity, and correspond with the reviewed results of other medicinal plants. The results shown that water is the best solvent to extract total polyphenols compounds from leaves and stem of Bauhinia divaricata L. and Bougainvillea spectabilis, and that they have a very potent antioxidant activity, compared with the pure catechins and gallic acid used as positive controls. These results can be useful for further applications of Bauhinia divaricata and Bougainvillea spectabilis or its constituents in pharmaceutical preparations after performing clinical in vivo researches. With this kind of investigations it would be easier the establishment of natural extracts supposed to functionalize formulations to treat and prevent the human damages occurring due to free radicals and also to replace synthetic antioxidants in industry.
We thank to CEPROBI-IPN for allowing Bautista-Bringas to carry out part of the technical work at its labs and to Biol. Ma. Edith Lopez Villafranco for her technical help in taxonomical identification of the plant material.
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