Bioactivity comparison of extracts from various parts of common and tartary buckwheats: evaluation of the antioxidant- and angiotensin-converting enzyme inhibitory activities

Background Buckwheat flour and buckwheat sprouts possess antioxidant properties, and previous studies have reported on buckwheat flour displaying an inhibitory activity for angiotensin-I converting enzyme (ACE). Information is lacking on the bioactivity of other parts of the buckwheat, such as the seed hulls and plant stalks. This study investigates the ACE inhibitory activity and antioxidant activity of various parts of 2 types of buckwheat, namely, common buckwheat (Fagopyrum esculentum Moench) and tartary buckwheat (Fagopyrum tataricum Gaertn). Results The extract of common hulls extracted using 50% (v/v)-ethanol solvent presented a remarkable inhibitory activity. The value of IC50 is 30 μg ml-1. The extracts of both common and tartary hulls extracted using 50% (v/v)-ethanol solvent demonstrated an antioxidant activity that is superior to that of other extracts. Conclusion This study determined that the ethanolic extract of the hulls of common buckwheat presented more favorable antioxidant and ACE inhibitory abilities. However, the correlation of antioxidant activity and ACE inhibitory activity for all 18 types of extracts is low. The ACE inhibitory activity could have been caused by a synergistic effect of flavonoids or from other unidentified components in the extracts. The ethanolic extract of common hulls demonstrated remarkable ACE inhibitory activity and is worthy of further animal study.


Background
Food is not only a source of energy and nutrition for maintenance and growth of the body but is also a source of bioactive compounds that have beneficial effects on humans. For example, common buckwheat (Fagopyrum esculentum) and tartary buckwheat (Fagopyrum tataricum) are traditional foodstuffs available worldwide. They are also essential as functional food resources because of the high polyphenol and mineral contents in the seed. Numerous previous studies have discussed the antioxidant properties of buckwheat flour and buckwheat sprouts [1][2][3][4][5][6][7][8][9][10][11][12]. Campbell [13] reported that buckwheat comprises 3 classes of flavonoids: flavonols, anthocyanins, and C-glucosyl-flavones. ACEinhibitory activity of flavonoids isolated from other plants has been reported previously [14,15]. The only reports existent were of dipeptides from buckwheat flour displaying inhibitory activity for the angiotensin-I converting enzyme [16][17][18]. No information is available regarding the inhibitory activity of angiotensin-I converting enzymes in other parts of the buckwheat plant. From an agricultural perspective, identifying an alternative approach for using the entire buckwheat plant, such as the stalks, leaves, and hulls, is crucial. Therefore, the objective of this study was to evaluate systematically the ACE inhibitory activity and antioxidant activity of different parts and varieties of buckwheat, namely, common buckwheat (Fagopyrum esculentum Moench) and tartary buckwheat (Fagopyrum tataricum Gaertn).

Results
The IC 50 values of various extracts for ACE inhibition To ensure accurate function of the modified microplate fluormetric assay, we determined the IC 50 for Captopril, a known ACE inhibitor. Captopril is a common orally administered ACE inhibitor used to treat hypertension and congestive heart failure. The measured IC 50 was 1.9 ng ml -1 (8.7 nM), which was comparable to measurements shown in the literature (7 nM) [19]. Pihlanto-Leppala et al. [19] determined ACE activity by measuring hippuric acid liberated from the reaction of hippuryl-L-histidyl-L-leucine and rabbit ACE with HPLC. Table 1 shows the IC 50 of various extracts for ACE inhibition. The rutin equivalent activity was based on the IC 50 of rutin.   solutions. Three replicates (n = 3) were performed for each concentration.

Discussion
By comparing the extracts of the various extracting solvents, we discovered that extracts obtained from 50% v/v ethanol solvent exhibited the greatest ACE inhibitory activity. We evaluated the ACE activity of various extracts using a paired t test. The results showed that various ethanolic extracts presented significant differences of ACE inhibitory activities for all parts of the plant, except for the groats from common buckwheat, where the choice of solvent made no significant difference. Extracts from buckwheat plants (comprising stalks, stems, and leaves) showed poor ACE inhibitory activity for all solvents.
For the extracts with deionized water, groats presented optimal ACE inhibitory activity (lowest IC 50 values), as compared to the plants and hulls. The ACE inhibitory activity of water-extracts could be caused by water-soluble peptides. This was consistent with the findings reported by Aoyagi et al. [16]. The buckwheat flour contained water-soluble peptides, which inhibited ACE activity. The 50% ethanolic extract of common hulls produced the greatest inhibitory activity. The value of IC 50 (30 μg ml -1 ) was significantly lower than the IC 50 values of other extracts. Li et al. [17] reported that proteins of tartary buckwheat flour treated with pepsin, followed by chymotrypsin and trypsin hydrolysis, resulted in an ACE inhibitory activity with IC 50 at 140 μg ml -1 . Therefore, ethanolic-soluble ingredients demonstrate an inhibitory activity that is superior to that of water-soluble ingredients. Figure 3 shows the correlation plots of equivalent antioxidant activity and ACE inhibition. Comparing the rutin equivalent antioxidant activity and ACE inhibitory activity using the Pearson correlation showed that the correlation coefficient was 0.6, indicating that the correlation between these 2 bioactivities was low to medium. Quercetin and rutin, which are 2 major types of flavonoids in buckwheat [20], presented great antioxidant activity. We evaluated the inhibitory effect of commercially available quercetin and rutin. The IC 50 values were 39 μg ml -1 and 86 μg ml -1 for quercetin and rutin, Figure 2 The equivalent antioxidant activity of various extracts (n = 3). GC0 is groats of common buckwheat extracted with deionized water. GT0 is groats of tartary buckwheat extracted with deionized water. GC2 is groats of common buckwheat extracted with 20% ethanol. GT2 is groats of tartary buckwheat extracted with 20% ethanol. GC5 is groats of common buckwheat extracted with 50% ethanol. GT5 is groats of tartary buckwheat extracted with 50% ethanol. PC0 is plants of common buckwheat extracted with deionized water. PT0 is plants of tartary buckwheat extracted with deionized water. PC2 is plants of common buckwheat extracted with 20% ethanol. PT2 is plants of tartary buckwheat extracted with 20% ethanol. PC5 is plants of common buckwheat extracted with 50% ethanol. PT5 is plants of tartary buckwheat extracted with 50% ethanol. HC0 is hulls of common buckwheat extracted with deionized water. HT0 is hulls of tartary buckwheat extracted with deionized water. HC2 is hulls of common buckwheat extracted with 20% ethanol. HT2 is hulls of tartary buckwheat extracted with 20% ethanol. HC5 is hulls of common buckwheat extracted with 50% ethanol. HT5 is hulls of tartary buckwheat extracted with 50% ethanol. respectively. The antioxidant activity of crude extracts of common hulls per milligram was equivalent to 0.055 mg of quercetin only; thus, the high ACE inhibitory activity of the crude extracts could be caused by other unidentified components in the extracts or by a synergistic effect of flavonoids, as suggested by Lacaille-Dubois et. al. [14].

Equivalent antioxidant activity
However, the presence of strong in vitro activities does not necessarily imply that hull extracts can provide powerful anti-hypertensive and antioxidant drugs. These preliminary bioactive results of ethanolic extracts of common hulls warrant additional animal studies.
A microplate fluometric assay was measured using a Flex Station 3-microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Preparation of the extracts
According to the regulations of the Department of Health, Taiwan, solvents extracted for functional foods or medicinal herbs may contain up to 50% (v/v) ethanol. Thus, we extracted various buckwheat plant samples using 3 different solutions: deionized water; 20% (v/v) ethanol; and 50% (v/v) ethanol, at 60°C for 2 h.
The grains were dehulled manually and divided into groats and hulls. The buckwheat plants (comprising leaves, stems, and stalks), groats, and hulls were ground separately. Thirty-gram aliquots of each ground sample were extracted using 300 ml of the various extracting solvents, under constant magnetic stirring in a 60°C water bath for 2 h. After extraction, the slurry was filtered, and subsequently centrifuged at 5000 rpm for 20 min. The supernatant was collected and frozen at −20°C and concentrated to dryness under reduced pressure. The dried crude extracts were stored at −20°C. Fresh solutions were prepared before each assay. The crude extracts were reconstituted using the respective extracting solvents, and then diluted to various concentrations using an ACE assay buffer.

Angiotensin-I-converting enzyme inhibitory assay
This study used a microplate fluometric assay modified according to the method proposed by Schwager et al. [21] to provide rapid screening and to consume fewer reagents. The fluorometric assay involved using the hydrolysis of hippuryl-L-histidyl-L-leucine (HHL) by ACE to form hippuric acid and histidyl-leucine (HL). Adding aqueous NaOH stopped the reaction. The product HL was reacted with OPA [22], and fluorescence was detected at an excitation of 365 nm and an emission of 460 nm.
A lyophilized angiotensin-converting enzyme derived from porcine kidneys was reconstituted with an assay buffer to a concentration of 12.5 mU ml -1 . The assay buffer (pH 8.3) comprised potassium di-hydrogen phosphate (0. 1 M), di-sodium hydrogen phosphate (0.1 M), and zinc chloride (10.0 mM). A brief description of the assay is as follows: the ACE solution (20 μl) was incubated using an assay buffer (50 μl) containing HHL (5.0 mM) and various concentrations of buckwheat extracts (10 μl). The reaction was conducted at 37°C for 30 min, and then stopped by adding aqueous NaOH (50 μl, 1.0 M). An OPA reagent (50 μl) was reacted with the HL produced from enzymatic hydrolysis for 10 min, and fluorescence intensity was subsequently measured at 365 nm excitation and 460 nm emission. To correct for intrinsic fluorescence of ACE and the extracts, a blank was prepared by adding NaOH immediately after adding ACE, followed by adding HHL (50 μl) and the extract solution (10 μl).
The relative ACE activity was calculated as follows: Flourescence intensity of inhibitory experiments À blank Flourescence intensity of 100% activity assay À blank The relative ACE activity% versus the logarithmic concentrations was plotted. The concentration of 50% inhibition of ACE activity (IC 50 ) was determined for varied extracts by using linear regression analysis of logarithmic plots. We conducted at least 3 replicates for each extract.

Ferric-reducing antioxidant power assay
The antioxidant activities of the extracts were determined according to the procedures recommended by Benzie and Strain [23]. In brief, 40 μl of flavonoidcontaining solutions were mixed in a 96-well plate with 300 μl of a reagent solution containing 0.8 mM of tripyridyltriazine and 1.7 mM of FeCl 3 in 300 mM sodium acetate (pH 3.6). The samples were incubated for 15 min at 37°C, and the absorbance at 593 nm was recorded on a Flex Station 3-microplate reader.

Statistical analysis
Statistical differences between the groups were evaluated using the t test for paired data. The p-values were considered significant at p < 0.05. We used Microsoft Excel W 2007 software for statistical analysis.

Conclusion
In conclusion, the findings showed that the ethanolic extract of hulls of common buckwheat presented powerful anti-hypertensive and antioxidant properties. It is worthy of further animal study and further investigation on components of the extract.