Effects of Grilling on Luteolin (3`,4`,5,7-tetrahydroxyflavone) Content in Sweet Green Bell Pepper (Capsicum annuum)
The content of luteloin in green bell pepper (Capsicum annuum) produced in Turkey were determined by RP-HPLC with DAD detection. The luteloin (3`,4`,5,7-Tetrahydroxyflavone) content of green pepper samples were 46.00Â±0.76 mg kg-1 f.w. (average). The alterations of luteloin concentrations with heating process (grilling, kOzleme) and the loss of luteloin amount were also determined. Luteolin contents of grilled peppers were found as 29.96±0.96 mg kg-1 f.w. The method was objective and reproducible for accurate detection of luteloin in green pepper and other pepper varities.
Interest in especially plant food polyphenols has increased owing to their role as antioxidants, antimutagens and scavengers of free radicals and their implication in the prevention of pathologies such as cardiavascular disease and cancer. Epidemiological researches have confirmed a correlation between an increased consumption of phenolic antioxidants and a reduced risk of cardiovascular diseases and certain types of cancer (Hertog et al., 1993; Hertog, 1996; Holman and Katan, 1999; Tokuşoğlu, 2001, 2006).
Several food flavonoids show different kinds of positively biological activity in vivo and in vitro (Rice-Evans et al., 1997; Harborne and Williams, 2000; Tokuşoğlu et al., 2003, 2005a, b).
The phenolic compound luteolin (3',4',5,7-Tetrahydroxyflavone) (Fig. 1) has been shown to have strong antioxidant (Shimoi et al., 1994), anti-inflammatory/antiallergic (Yamamoto et al., 1998) activities, antitumorigenic (Yasukawa et al., 1989) and to be an inhibitor of protein kinase C (Ferriola et al., 1989) and lipoxygenase (Yamamoto et al., 1998). Luteolin monoglucuronide and free luteolin were also detected in human serum after ingestion of luteolin (Shimoi et al., 1998).
Pepper, genus Capsicum, belongs to the great family of tropical plants
Solanaceae (Somos, 1984) and this taxon includes both sweet cultivars eaten
mainly as vegetables and hot cultivars often used as a spice. Green bell peppers
(Capsicum annuum) are considered to be a good sources of various beneficial
phytochemicals, such as carotenoids (β-carotene, lutein and zeaxanthin),
flavonoids and ascorbic acid (vitamin C) (Crosby et al., 2004; Tokuşoğlu,
Especially the yellow, orange and red colors in peppers originate from the
30 different carotenoid pigments produced in the fruit ripening. Quercetin (3,3',4',5,7-penta
hydroxy flavone) which is a food-derived flavonoid has been act anticarcinogen
and antimutagen in vitro and in vivo. Moreover, its strong antioxidant
activity protects LDL from oxidation, reducing the risk of heart disease. Luteolin
(3',4',5,7-Tetrahydroxyflavone) may help prevent breast cancer by reducing excess
estrogen formation (Crosby et al., 2004; Tokuşoğlu, 2006).
Bell peppers are best when they are thick walled and juicy, so they should feel heavy for their size. If a green bell pepper shows streaks of red, it will be slightly sweeter than a totally green one (Crosby et al., 2004; Tokuşoğlu, 2006). Sweet bell peppers are generally consumed as baking or microwaving of whole stuffed peppers with rice and tomato slices.
Research on flavonoid, especially flavonoid aglycons as quercetin, luteolin and flavonoid glycosides, phenolic acid content in pepper fruit is scarce. Flavonoid aglycons have been determined after acid hydrolysis (Hertog et al., 1992; Lee et al., 1995; Howard et al., 2000).
In Turkey, some vegetables is consumed after grilling (közleme) process by using an electrical grill or barbeku at 150-200°C. Grilling process can provide enhanced flavor to some vegetables such as green pepper, aubergine etc.
The objectives of this study was to determine the quantification of luteolin in green bell peppers grown in Manisa and İzmir area in Turkey using Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) with diiod array (DAD) detection and to detect the alterations with grilling effects.
MATERIALS AND METHODS
Chemicals: Luteolin (3',4',5,7-Tetrahydroxyflavone) (Fig. 1) standard was purchased from Sigma Chemical Company, (Poole, Dorset). Absolute ethanol (HPLC grade), methanol (HPLC grade), perchloric acid, phosphoric acid, formic acid, ethyl acetate were purchased from E. Merck co. (Darmstadt, Germany). Sodium acetate and all organic solvents were of analytical grade and obtained from Merck (Darmstadt, Germany). Double-distilled water was purchased from LabScan (Turkey).
Sampling: Five different group of green bell peppers with same maturity were prepared for analysis. Three group of green peppers (Capsicum annuum) were purchased from 3 different greengrocers in the local markets at a period of their most frequent consumption and two group of samples were collected from growing area in İzmir and Manisa, Turkey. (Each 5 kg as five units and harvesting season: 2005 fall). The edible parts of samples were used. After purchasing, the samples were immediately cleaned and scrubbed well due to the some bell peppers had waxed and the some samples had possible soiled etc. The seeds of pepper samples were removed and samples chopped into small pieces. Then, pieces of pepper samples were squeezed with blender and homogenized under N2 atmosphere. Final samples stored at -18°C prior to subsequent analysis.
Proximate analysis: The moisture content was determined by drying a
representative 2 g sample at 100-105°C for 40 h Total ash was determined
by incineration of a representative 2 g sample at 450°C for 30 h. Total
lipids of samples were determined according to described method by Tokuşoğlu
and Ünal (2003) regarding edible algae lipids. Crude protein was determined
by Kjeldahl protein units (Gerhardt incineration apparatus and Gerhardt Vapodest
20 distillation apparatus) and protein was calculated as nitrogen (%)x6.25 (AOAC,
1990). Available carbohydrate was determined by the anthrone spectrophotometric
method (Osborne, 1986). All proximate chemical determinations were done in triplicate.
The energy content of the green bell pepper was determined by multiplying the
values obtained for crude protein, total carbohydrate and total lipid by 4.00,
3.75 and 9.00, respectively and summing the results. Final results were multiplied
with 4.18 and energy content was obtained as kJoule. Dietary fiber content of
peppers was determined based on AOAC (1990) whereas ascorbic acid levels were
determined according to Yıldırım et al. (2002).
Heating process (grilling, közleme): Electrical grill was heated to 150°C before process. Green bell peppers were grilled during 7-8 min at 150°C by using electrical grill.
Extraction conditions for luteolin analysis: Twenty five milliliter of acidified ethanol containing 1% perchloric acid was added to same volume of sample extract. To this extract, 2 mL of 1.2 M perchloric acid was added in order to obtain aglycon form of lutein. Final extract was dissolved with 5 mL of ethyl acetate and reaction mixture was stirred at ice bath for ½ h. Final extract was filtered through a 0.5 μm (Acrodisc) filter prior to filtration 10 μL of filtrate was injected into the HPLC.
HPLC conditions: Green pepper samples were analyzed by RP-HPLC using UV detection. Five micrometer Inertsil ODS-3V column (150x4.6 mm I.D.) [Phenomenex, CAL, USA] using methanol/phosphate buffer (20 mM, pH 3.0)/formic acid (55: 40: 5 v/v/v) as mobile phase with a flow rate of 1.0 mL min-1. UV Absorbance detector was set 254 nm., detector sensitivity was 0.05 AUFS and column oven was set at 35°C. The samples were injected as 10 μL. All determinations were performed from 3 separate extractions and each was injected in triplicate (n = 3).
Analytical precision of method: The standard were dissolved to 500 μg
mL-1 with methanol (HPLC grade). After the preparation of stock solutions,
they were stored at -28°C. For calibration, all standards were prepared
as 25 μL/1 mL methanol from above-mentioned stock solutions. Calibration
curves of this thorough the origin and their R2-values was 0.9999.
The quantitative amounts of luteolin in green pepper samples were determined
using these curves. The linearity plotting at 254 nm was (y = 187.762 x + 30.855)
(R2 = 0.9999). The assay recoveries for luteolin ranged from 98.96
to 99.99%. The inter-day precision (RSD) for luteolin was 45.2±0.72 mg
kg-1 f.w. sample. The analytical parameters of luteolin in Table
1. The Detection Limits (DL) of luteolin was 0.45 mg kg-1 while
the Limits Of Quantification (LOQ) was 0.05 μg mL-1.
|| The analytical parameters of luteolin
Determination of total flavonoids: Total flavonoids were analyzed according
to procedure described by Zhishen (1999). One milliliter of extract was placed
in a 10 mL volumetric flask. Five milliliter of bidistilled and 0.3 mL of NaNO2
were added and mixed. About 0.6 mL of 10% AlCl3.6H2O was
added after 5 min. Two millilitre of 1 M NaOH was added 5 min later and the
solution mixture, vigorously. The absorbance of final solution was measured
at 510 nm, immediately. Total flavonoid contents were calculated using a standard
calibration curve, prepared from (+) catechin.
Statistical analysis: Statistical analysis was performed using Student's t-test. The differences between the all groups with a value of p<0.01 was considered significant.
RESULTS AND DISCUSSION
The proximate chemical composition, dietary fiber, vitamin C (ascorbic acid) and energy values of green bell peppers grown in country (as per cent) as shown in Table 2 (p<0.01). Dry matter (%) was found as 9.92±0.24 (Table 2). Total fat, total protein and total available carbohydrates of peppers were 0.33±0.08, 0.99±0.03 and 10.63±0.05 g, respectively (p<0.01). Energy values were determined as 46.79±1.03 cal (195.58±4.29 kjoule) (Table 2). Dietary fiber content and vitamin C (ascorbic acid) levels of peppers were found as 2.76±0.10 g and 133.00±1.06 mg (Table 2).
Luteloin was perfectly separated with the HPLC-DAD procedure used and Fig. 2 shows the baseline separation of luteloin in green bell peppers (Fig. 2). Peak identity was confirmed when peak retention times (avg. RT 17.2 min) were identical to that of the pure standard in mobile phase. Table 3 shows the luteloin content in sweet green bell samples. In present study, the luteloin concentration ranged 18.53-48.96 mg kg-1 (n = 12).
||The proximate chemical composition, dietary fiber amounts,
vitamin C levels and energy values of analyzed green bell peppers (p<0.01)
|Data are based on dry wt. (%), Mean±SD (n = 3)
||The luteolin composition of raw and grilled (köz) green
bell peppers (p<0.01)
|Values are means of triplicate determinations. All analysis
Andrea et al. (2003) determined 10.7 mg kg-1 f.w. of luteolin
and 20.1 mg kg-1 of total flavonoids in green bell peppers grown
in Hungary. Luteloin content of some green bell peppers in our research was
in accordance with the studies described by Andrea et al. (2003).
Lee et al. (2005) determined that the developments of flavonoids in pepper (Capsicum sp.) depends on the genetic variety and environmental variation. The researchers found that especially cultivar effects were significant for luteolin and some phytochemicals at each location including a greenhouse at College Station, Texas, USA. According to their study, Fidel (37 μg g-1) and Banana Supreme (21.5 μg g-1) were the best sources of luteolin. Luteloin data in our study was higher than the study described by Lee et al. (2005).
Luteolin contents in field-grown fruits may also be affected by a wide scale of environmental influences containing temperature, soil, nutritional condition of plant and light levels (Tokuşoğlu, 2006).
Arabi Paola et al. (2004) detected the flavonoids in vegetable foods commonly consumed in Brasil. Based on their findings, luteolin was found in green pepper as 2.1±0.2 mg/100 g f.w. (Capsicum annuum L. var. annuum) at second semester of 2001 and as 1.2±0.2 mg/100 g f.w. at first semester of 2002. Total flavonoid was 6.2 mg/100 g f.w. at second semester of 2001 while 3.0 mg/100 g f.w. at first semester of 2002. It has been showed that seasonal variations affected the lutein concentration of green peppers in the study described by Arabi Paola et al. (2004).
Our luteolin concentration findings are in agreement with the study reported by Arabi Paola et al. (2004).
Ioku et al. (2005) indicated that luteolin content was 8.15±4.10
mg/100 g of f.w. green pepper in Japan. This level was higher than our data.
Based on their findings, flavonoid contents were increased in October to December
and the tendency to decrease in May to June.
|| Luteolin in green bell pepper (Capsicum annuum)
Miean and Mohamed (2001) determined the 33.0 mg kg-1 of luteolin
in green chili and Crosby et al. (2004) detected that several paprika
and cayenne types have recently been confirmed as excellent sources of the flavonoid
compounds quercetin and luteolin, with levels between 260 and 400 ppm.
Luteolin contents of grilled peppers were found as 29.96±0.96 mg kg-1 f.w. and total phenolic substances were 92.83±1.74 mg kg-1 f.w. (p<0.01) (Table 3). No study could be found concerning luteoin concentration and total phenolic levels in grilled green peppers. In this context, this data gave important results.
It may be concluded that Turkish green bell peppers are a good source of luteolin. It should be utilize the good candidate parents for the breeding program to develop novel pepper varieties with increased health benefits and enhanced beneficial phytochemicals such as luteolin. The study regarding genetic varietal effects of green bell peppers is also in progress.
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