Plant Foods Hum Nutr DOI 10.1007/s11130-013-0384-7
ORIGINAL PAPER
Effect of Flavonoids and Saponins Extracted from Black Bean (Phaseolus vulgaris L.) Seed Coats as Cholesterol Micelle Disruptors Rocio A. Chávez-Santoscoy & Janet A. Gutiérrez-Uribe & Sergio O. Serna-Saldívar
# Springer Science+Business Media New York 2013
Abstract Strategies for reducing plasma cholesterol have been focused on reducing the absorption or synthesis of cholesterol. The aim of this study was to correlate the content of flavonoids and saponins in black bean (Phaseolus vulgaris L.) seed coats extracts with a potential effect in lowering cholesterol absorption by the inhibition of cholesterol micellar solubility. Extracts with different flavonoids and saponins concentration were obtained from a Box-Behnken design used to optimize extraction temperature, stirring time and solvent composition. Major flavonoids and saponins were quantified by HPLC-PDAELSD and confirmed through mass spectrometry. Contrary to the flavonoid content, saponins were correlated to the inhibitory effect of cholesterol micelle solubility as an approach to a potential reduction of cholesterol absorption. Extracts with the highest saponin content strongly inhibited cholesterol micellization with values of 55.4±1.9 %, while stigmasterol used as control, only reached 12±2.3 % at the same concentration (5 mg/ml). The optimal extracting conditions for saponins were 25 °C, during 3 h in 85 % aqueous-methanol. Correlations of inhibitory effect of cholesterol micellar solubility with the content of each identified saponin suggested that the reduction of cholesterol micellization depends on the C-22 substitution of saponins. Keywords Optimization . Seed coats . Flavonoids . Saponins . Cholesterol absorption
R. A. Chávez-Santoscoy : J. A. Gutiérrez-Uribe (*) : S. O. Serna-Saldívar Centro de Biotecnología FEMSA. Escuela de Biotecnología y Alimentos. Tecnológico de Monterrey-Campus Monterrey, Av. Eugenio Garza Sada 2501 Sur, C.P. 64849 Monterrey, N.L., México e-mail:
[email protected]
Abbreviations TFC TSC
Total flavonoid content Total saponin content
Introduction Hyperlipidemia is a group of metabolic disorders characterized by elevated levels of cholesterol and triglycerides in plasma. The prevalence of hyperlipidemia has increased worldwide due to an increased consumption of diets rich in saturated and trans fatty acids [1]. Strategies for the reduction of plasma cholesterol have been focused to the inhibition of endogenous cholesterol synthesis or cholesterol absorption [2]. In general, cholesterol absorption inhibitors may act as bile acid sequestrants, cholesterol solubility disruptors, and Acyl-CoA cholesterol acyltransferase (ACAT) inhibitors [3]. It has been reported that amaranth protein concentrate has bile acid binding capacities [4]. The most studied phytochemicals which inhibit cholesterol absorption are phytosterols, because of their similar structure to cholesterol [5–7]. However, more research is needed in terms of other natural compounds which exert similar effects. Common beans (Phaseolus vulgaris L.) are the second most important legume crop in the world. Black bean seed coat has been studied because of its high levels of polyphenols [8, 9], anthocyanins [10] and other important antioxidant compounds [11–13]. Flavonoids are the most abundant polyphenols of black bean seed coat. Furthermore, quercetin, kaempferol and catechin have been associated with the decrease of total cholesterol (TC) levels, the inhibition of LDL lipid peroxidation and the increase of HDL [14]. Saponins are secondary plant metabolites that exist in a wide variety of edible legumes [15–17]. They are amphiphilic molecules
Plant Foods Hum Nutr
containing a triterpenoid aglycone and carbohydrate moieties linked through ether and ester linkages at one or more glycosylation sites. Saponins have been detected in significant amounts in black bean tissues [16]. Saponins are divided in groups according to their structure: group A saponins have glycosyl groups attached to the C-3 and C-22 positions of the aglycone whereas saponins of group B are glycosylated only in the C-3 position while C-22 contains an hydroxyl group [16]. Group B saponins also exist in the plant as conjugates of 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP) at C-22. Saponins have been shown to increase fecal steroids in man and in experimental animals, and in some instances to lower plasma cholesterol [17], probably as a result of bile acid binding [18]. The aim of this study was to correlate the content of flavonoids and saponins in black bean (Phaseolus vulgaris L.) seed coats extracts with a potential effect in lowering cholesterol absorption by the inhibition of cholesterol micellar solubility. Extracts with different flavonoids and saponins concentration were obtained from a Box-Behnken design used to optimize extraction temperature, stirring time and solvent composition.
Materials and Methods Seed-Coat Sample Phaseolus vulgaris L. var. San Luis was obtained from Sinaloa, Mexico during the month of March of 2011. The seeds were stored at 4 °C and relative humidity of 85 %. Black beans were wiped with a flannel and then soaked in a plastic bag with distilled water in a bean:water ratio of 100:1 (w/v) at room temperature for 24 h. The conditioned black beans were placed on trays for drying at 60 °C for 6 h in an oven (Electrolux, EOB31003X, Spain). Later, the seed coats were removed using a mechanical seed decorticator (Square D, SCDGE 4364, India). The decortication time was 90 s. The mixture of decorticated beans was separated by three sieves into cotyledons (2.81 mm), seed coats (1.00 mm), a mixture of fine particles of cotyledons, hilum and seed coats (0.251 mm), and a pan that collected the finest particles. Subsequently, the seed coats were milled with a coffee grinder (Krups GX4100, Mexico). Design of Experiments for Flavonoids and Saponins Extraction A randomized, three-factor, three-level Box–Behnken design (Table 1) was carried out to optimize the extraction of flavonoids and saponins. Temperature (°C), stirring time (h), and methanol–water proportion (%) were varied. The extractions were performed with 70, 80 or 90 % methanol in water (v/v)
using a mass-solvent ratio of 1:10 (w/v) at three different temperatures (25, 35 or 45 °C). The mixture was stirred for 1, 2, or 3 h at 250 rpm and left for one additional hour to allow sedimentation. The supernatant was recovered and vacuum filtered through a Whatman filter paper No. 1. The resulting extracts were concentrated in a rotary evaporator to remove methanol. The bath temperature was set at 60 °C, and pressure in the vacuum pump at a range of −70 to −90 kPa. Once methanol was removed, the concentrated extracts were lyophilized and the resulting freeze-dried powder stored at −80 °C until analysis. Flavonoids and Saponins Quantification and Identification Flavonoids and saponins were quantified in a high pressure liquid chromatograph coupled with UV-Visible detector and an evaporative light scattering detector, HPLC-UV–VISELSD (1200 Series, Agilent Technologies, Santa Clara, CA), respectively. The HPLC was equipped with a Zorbax SB-Aq (3×150 mm, 3.5 μm) column and data generated through Agilent ChemStation software. Separation conditions and identification of flavonoids and saponins were performed as previously described [16]. Briefly, HPLC-MS-TOF (Model G1969A Agilent 1100 Santa Clara, CA) was used with the same chromatographic conditions of the HPLC-UV–VISELSD method. Mass spectra were collected using ESI in positive mode under the following conditions: m/z range, 100–1400; nitrogen gas; gas temperature, 250 °C; drying gas flow rate, 13 L/min; nebulizer. pressure, 50 psig; capillary voltage, 4000 V; and fragment voltage, 70 V. Extracted ion chromatograms were obtained considering accurate mass obtained for compounds or their adducts with Na or K with an error range of 0.01 units using the Analyst QS 1.1 software (Applied Biosystems, Carlsbad, CA). The three main glycosylated flavonoids were quantified with the chromatogram obtained at 360 nm using a calibration curve of the corresponding aglycone standard (Sigma, St. Louis, MO) and expressed in mg/100 g. On the other hand, saponins were quantified with the ELSD chromatogram using a calibration curve of soyasaponin I (Sigma, St. Louis, MO) and expressed as equivalents in mg/100 g. Inhibition of in Vitro Micellar Solubility of Cholesterol The in vitro micellar solubility of cholesterol of each extract was measured according to the method before described [19], with some modifications previously suggested [20]. Each freeze-dried extract or stigmasterol (Sigma, St. Louis, MO), used as control, was added to the micellar mix in a concentration of 5 mg/ml. The mixture was incubated at 37 °C for 24 h and centrifuged at 100,000 g for 60 min at 37 °C. The supernatant was collected for the determination of cholesterol
Plant Foods Hum Nutr Table 1 Independent variables and levels for Box-Behnken design to optimize the extraction of flavonoids and saponins associated to black bean seed coat Independent variables
Temperature (°C) Extraction time (h) Methanol used in system (%)
Symbol
X1 X2 X3
Levels −1
0
1
25 1 70
35 2 80
45 3 90
A randomized, three-factor, three-level Box–Behnken design was carried out with three replicates of each point with 46 experiments in total
concentration. Cholesterol concentrations were determined using HPLC-UV–VIS (1200 Series, Agilent Technologies,
Fig. 1 Surface plots of total flavonoid content (TFC) and total saponin content (TSC) in extracts obtained in the Box-Behnken design. Plots were hold in the optimal value for temperature (upper), time (middle) and
Santa Clara, CA) equipped with a Luna C8 column (250× 4.6 mm i.d.; Phenomenex, Torrance, CA). Mobile phase consisted of (A) acetonitrile and (B) 55 % methanol and 45 % water (acidified with 1 % formic acid). The elution gradient for B was as follows: 0–7 min, 0 % isocratic (with a flow rate of 0.6 ml/min); 7–15 min, 0–15 % (0.6–1.2 ml/ min); 15–20 min, 15–80 % (1.2–1.5 ml/min); 20–50 min, 80– 100 % (flow rate: 1.5 ml/min). The column temperature was maintained at 30 °C and cholesterol was quantified at 205 nm. Data was expressed as inhibition of cholesterol micellar solubility (%) obtained as [Cs / (2−Co)]×100, where Cs is cholesterol concentration in supernatants with the tested extracts or control and Co is cholesterol concentration in supernatant without disruptor that was substracted from the 2 μg/ml of the cholesterol used in the system.
solvent composition (bottom) for the extraction of flavonoids (a) and saponins (b) associated to black bean seed coats
Plant Foods Hum Nutr Table 2 Composition of Flavonoid-rich and saponin-rich extracts obtained from black bean seed coat with the optimal conditions found in the Box-Behnken design Identified compounds Flavonoids Myricetin 3-O-glucoside Quercetin 3-O-glucoside Kaempferol 3-O-glucoside TFCc Saponins Phaseoside I Soyasaponin Af Deacetyl soyasaponin Af Soyasaponin Ba Soyasaponin αg Soyasaponin βg Soyasaponin γg TSCd
Flavonoid-rich extracta (mg/100 g)
Saponin-rich extractb (mg/100 g)
178.01±4.18 1424.19±23.03 8.94±0.02 1611.14±27.21
74.71±3.18 712.86±6.97 4.87±0.03 792.44±12.41
1.01±0.21 11.84±0.35 1.25±0.02 2.68±0.32 10.23±1.13 4.13±1.06 1.75±0.08 32.90±2.35
1.97±2.03 17.01±0.13 3.14±0.08 4.01±0.29 15.09±1.02 6.02±1.04 2.42±0.10 49.66±3.83
a
The optimal conditions estimated for the model were temperature of 45 °C; Time of extraction of 3 h; and 80 % of aqueous-methanol as solvent system
b
The optimal conditions estimated for the model were temperature of 25 °C; Time of extraction of 3 h; and 85 % of aqueous-methanol as solvent system c
TFC=total flavonoid content, significant difference at P
