Chromatographical analysis of Phenolic acids in different preparations of pea (Pisum sativum) and chickpea (Cicer arietinum)
P Tiwari, A Singh, U Singh, S Maurya, M Singh
Keywords
co-chromatography., hplc, phenolic acid, pulses
Citation
P Tiwari, A Singh, U Singh, S Maurya, M Singh. Chromatographical analysis of Phenolic acids in different preparations of pea (Pisum sativum) and chickpea (Cicer arietinum). The Internet Journal of Alternative Medicine. 2008 Volume 8 Number 1.
Abstract
Legumes represent one of the most important food components to cover the basic proteins and energy requirements for the health of human beings. The secondary metabolites of legumes reveal valuable medicinal properties. Among the secondary metabolites, phenolic acids have greater importance. Analysis of phenolic acids in pea (
Introduction
Pea (
With the development of various efficient, purification and identification techniques, many bioactive products have been isolated from a number of legume. Phenolics are an important group of natural compounds contributing significantly to the marked pharmacological properties of a number of plants including fruits and legumes 6 . They are important component, either in free or conjugated forms, of almost all the plants in which they are biosynthesized via Shikimic acid or phenylpropanoid pathway 7 . Legumes have been a rich source of various groups of natural products including phenolics. The enzyme especially phenylalanine ammonia lyase (PAL) is not found in animals and humans. This is why animals solely depend on plants for the requirement of phenols in their body. Phenolics have wide therapeutic and pharmacological properties against human and animal diseases and are, therefore considered to be safer in traditional and alternative medical systems over synthetic medicines (Table 1).
Figure 1
Being natural hydrogen-donors, they are strong antioxidants, and natural free-radical scavengers because of their structural characteristics and hydrogen donating properties of the phenol moiety 8 . Their role in the protection of both humans and plants against oxidative stress, which has adverse effects on the tissues, has been reported widely. They also contribute to wound healing and their glycosylated analogs provide a vehicle for the carbohydrate transport and storage, which is relatively osmotically inactive. Individually, phenolics may possess pharmacological uses and find wide application and description in the traditional medical systems.
The present study was undertaken to estimate phenolic acids in two major legumes (chickpea and pea) used regularly as food supplements in India by high performance liquid chromatography (HPLC). The results are presented here.
Materials and Methods
Sample preparation
Two legumes (three varieties of chickpea and sweet pea) were purchased from authentic legume shops. One gram (dry weight) of each legume was weighed and powdered in a pestle-mortar followed by suspending fine crushed sample in 5 ml of ethanol: water (80:20 v/v). These samples were collected in screw-capped specimen tubes (10 ml) and the suspension was subjected to ultrasonication by Branson Sonifiers 450 (Branson Ultrasonic Corps, Danury CT, USA) for 15 min at 4 ° C followed by centrifugation at 7,500 g for 15 min. The clear greenish supernatant was subjected to charcoal treatment to remove pigments from each sample and was then transferred to glass tubes. The residue was re-extracted twice and the supernatant was pooled prior to evaporation under vacuum (Buchi Type Rotavapor). Dried samples were re-suspended in 1.0 ml HPLC grade methanol by vortexing and filtered through membrane filter (pore size 0.45µm, Millipore) and stored at 4 ° C for further analysis by HPLC.
The reagents
Standard phenolic acids, viz., tannic acid, gallic (3, 4, 5- trihydroxybenzoic), vanillic (4-hydroxy-3 methoxybenzoic) cinnamic, caffeic (3,4-dihydroxycinnamic), O-coumaric (4-hydroxycinnamc), ferulic (4-hydroxy-3 methoxycinnamic) and salicylic acids were obtained from Merck, Himedia and Sigma companies. Solvents used during HPLC analysis (e.g. methanol, distilled water etc.) were of the HPLC grade (Merck, Germany).
High Performance Liquid Chromatographic (HPLC) analysis
High performance liquid chromatography (HPLC) of the samples was performed on HPLC system (Shimadzu Corporation, Kyoto, Japan) equipped with two Shimadzu LC-10 ATVP reciprocating pumps, a variable UV-VIS detector (Shimadzu SPD-10 AVP UV-VIS) and Rheodyne model 7725 in factor with a loop size of 20 µl, and integrator and CLASS-VP software for data recording and processing (Shimadzu). Reverse phase chromatographic analysis was carried out in isocratic conditions using C-18 reverse phase HPLC column {(250 X 4.6 mm i.d., particle size 5 µm) Luna 5 µ C-18 (2), Phenomenex, Torrance USA at 25 ° C. Running conditions included mobile phase methanol-0.4% acetic acid (80:20, v/v), flow rate 1.0 ml / min, injection volume 5 µl and detection at 290 nm 9 . Sample was injected thrice in the sample loop and the means of the peak areas of individual compounds were taken for quantification. Tannic, caffeic, vanillic, ferulic, cinnamic and salicylic acids were used as internal and external standards. Phenolic compounds present in the sample were identified by comparing retention time (Rt.) of the standards tannic (TA, Rt.2.94 min), gallic (GA, Rt. 3.10 min), caffeic (Caf-A, Rt. 3.48 min), vanillic (VA, Rt. 3.76 min), ferulic (FA, Rt. 4.02 min), O-coumaric (O-Cou-A Rt. 4.64 min) cinnamic (CA, Rt. 6.67 min) and salicylic acids (SA, Rt. 7.86 min). These phenolic acids were identified by co-injection of internal and external standards for their confirmation. Amounts of individual compounds were calculated by comparing peak areas of reference compounds with those in the samples run under similar elution conditions.
Quantitative estimation of phenolic acids and preparation of standard curve
Phenolic acid standards were accurately weighted (1 mg/ml) and dissolved in HPLC grade methanol. Concentrations ranging from 1 µg/ml to 10 µg/ml were prepared by serial dilution. Quantitative estimation of phenolic acids was conducted by reverse phase C-18 column under similar running conditions as has been described for the analysis of the samples. The analysis was carried out in triplicate and the detection was monitored at 290 nm. Calibration curves were plotted showing a linear correlation and peak areas for phenolic acids separately.
Results
Several phenolic acids in both the legumes (pea and chickpea) were detected (Tables 2a, b and 3).
Figure 2
Figure 3
Figure 4
UDL** = under detectable level
* Sample No. 1 Immature seed (green pea) crushed in 80% ethanol water
2 Oven dried (60 ° C/24h)
3 Dried mature seed crushed in 80% ethanol
4 15 minutes boiled mature seed
4 (a, b) Mature seed soaked in distilled water for 24 hours
(a) – Extract (b) – Seed
5 Immature seed soaked in distilled water for 24 hours
6 Extract of immature seed (15 minutes boiled)
7 Extract of mature seed (15 minutes boiled)
8 Seed of boiled immature seed
9 Fried immature seed with salt
10 Fried immature seed without salt
11 Flour of pea
Analysis revealed that maximum amount of tannic acid (43.416 µg/g) was detected in dried mature sweet pea seeds crushed in 80% ethanol followed by flour of sweet pea (16.20 µg/g) in dried mature sweet pea seeds crushed in 80% ethanol followed by fried Radhey variety of chickpea without salt (5.170 µg/g), avarodhi variety of chickpea with salt (5.053 µg/g), fried immature sweet pea seed without salt (4.842 µg/g) and fried immature sweet pea seeds with salt (3.004 µg/g). In remaining sweet pea preparations the amount was less than 3.0 µg/g. Gallic acid was maximum in dried mature sweet pea seeds (204.59 µg/g), followed by flour of sweet pea (44.94 µg/g), 15 min boiled mature sweet pea seeds (21.99 µg/g), extract of immature sweet pea seeds soaked in distilled water for 24 h (20.948 µg/g), fried immature sweet pea seeds without salt (19.949 µg/g), avarodhi variety of chickpea dried seeds crushed in 80% ethanol (11.626 µg/g), kabuli variety of chickpea seeds boiled in distilled water (14.54 µg/g), avarodhi variety of chickpea dried seeds crushed in 80% ethanol (11.626 µg/g). In remaining legume preparations the amount was less than 11.50 µg/g. Ferulic acid was maximum in extract of mature sweet pea soaked in distilled water for 24 h (30.405 µg/g), followed by radhey variety of dried chickpea seeds crushed in 80% ethanol (10.268 µg/g) followed by 15 min boiled mature sweet pea seeds (8.966 µg/g). In remaining legume preparations, the amount was less than 5.0 µg/g. Similarly, cinnamic acid was seen only in extract of avarodhi variety of boiled chickpea seeds in distilled water (0.085 µg/g). Similarly cinnamic acid was detected only in extract of avarodhi variety of boiled chickpea seeds soaked in distilled water (0.085 µg/g), followed by radhey variety of chickpea seeds soaked in distilled water for 24 h (0.082 µg/g), extract of mature sweet pea seeds soaked in distilled water for 24 h (0.0643 µg/g), while other preparations of legume showed very little amount of cinnamc acid. The caffeic acid was maximum in extract of 15 min boiled mature sweet pea seeds (5.836 µg/g) followed by extract of mature sweet pea soaked in distilled water for 24 h (2.328 µg/g), radhey variety of chickpea seeds soaked in distilled water for 24 h (2.087 µg/g), while in other preparations caffeic acid was found in traces. Vanillic acid was maximum in extract of immature sweet pea seeds soaked in distilled water for 24 h (10.113 µg/g ) followed by kabuli variety of fried chickpea seeds without salt (1.664 µg/g), kabuli variety of chickpea seeds soaked in distilled water for 24 h (1.642 µg/g), while in other preparations vanillic acid was found in traces. The salicylic acid was maximum in radhey variety of chickpea dried seeds crushed in 80% ethanol (4.102 µg/g) followed by kabuli seeds fried with salt (2.146 µg/g) while in other preparations it was found in traces. O-coumaric acid was found only in chickpea but not in sweet pea. It was maximum in avarodhi chickpea seeds soaked in distilled water for 24 h (2.085 µg/g) while in other preparations it was found in traces (Tables 2b, c).
Discussion
Legumes are the most important food category that have been extensively used as stable food to cover basic proteins and energy requirements throughout the history of human civilization. Besides adding nutrient, legumes also possess valuable medicinal properties because of the presence of several potential bioactive secondary metabolites, viz., enzyme inhibitors, lectins, phylates, oxalates, polyphenols, phytosterols and saponins that prevent humans from various diseases, viz., mainly coronary heart disease and diabetes 10 . The legumes under investigation are traditionally known to have pronounced effect on human health. Phenolics constitute and important group of natural products contributing significantly to the medicinal value of a number of plants including legumes.
Chlorogenic and isochlorogenic acids produce anti-lipoxygenase and anti-cycloxygenase activity and have been suggested to possess anti-inflammatory property 11 . Chlorogenic acid has also been found to significantly inhibit HVS-1 replication without any cytotoxicity 12 . Caffeic acid is the most prominent cinnamate that provides protection against genotoxic agents. It has been demonstrated to possess anti-carcinogenic properties in experimental animals 13 . It has also been reported that caffeic acid in the form of an extract of the artichoke (rich in chlorogenic and caffeic acids) can be used to lower serum cholesterol level in human beings [[[14.15]]] . Several reports indicate that the presence of pre-existing and inducible phenolics acids in chickpea (
HPLC analysis of
Cinnamic and hydroxycinnamic acids are rather more abundant and diverse groups of phenolics with higher dietary intake 10 . In terms of dietary load, total cinnamic acid intake in different populations ranges up to 1000 mg/day. Cinnamic acid and analogs provide natural protection against infections caused by pathogenic microorganisms 19 . 4-propoxycinnamic acid residue shows antimalarial acctivity 20 . Ferulic acid is present as natural dietary supplements with pronounced anti-inflammatory and antioxidant activity and is a pharmacological agent used as photoprotectants in skin lotions 21 . Gallic acid has anti-inflammatory and cytoxic property against all cancer cell lines studied
Review of literature reveals that very little work has been done on the estimation of individual phenolic acids in pea and chickpea. Phenolic compounds of seed coats of white and coloured variety of pea and their antioxidant activity have been studied by Transisca and Ciska 28 . They found protocatechuic, genticic and vanillic acids in coloured seed coat while ferulic and coumaric acids in the white seed coat. Wang et al., 29 estimated total phenolics in field pea. Cherit et al., 30 found that gallic, cinnamic, ferulic and chlorogenic acids were associated with the protection of chickpea from fungal attributes through induced resistance. Recently, Dilis and Trichopoulon 31 , estimated total phenolics in pulses. However, the detailed phenolic acid analysis in different preparation of three varieties of chickpea and one of pea are being reported for the first time.