HPLC Analysis of the Phenolic Profiles in Different Parts of Chilli (Capsicum annum) and Okra (Abelmoschus esculentus L.) Moench
U Singh, A Suman, M Sharma, J Singh, A Singh, S Maurya
U Singh, A Suman, M Sharma, J Singh, A Singh, S Maurya. HPLC Analysis of the Phenolic Profiles in Different Parts of Chilli (Capsicum annum) and Okra (Abelmoschus esculentus L.) Moench. The Internet Journal of Alternative Medicine. 2007 Volume 5 Number 2.
The nature of phenolic acids in the two important vegetable crops chilli (
Vegetables provide substantial amount of nutrients of important for human health because they are particularly important sources of micronutrients and vitamins, e.g., provitamin A, B6, C and E. as well as folic acid. Chilli (
Phytochemical investigations showed that the chief constituents of chilli pericarp are crystalline colorless pungent principle known as capsaicin or capsicutin, a condensation product of 3-Hydroxy-4-methoxybenzylamine and decylenic acid which produces highly irritating vapour on heating 2 . Green chillies are rich in vit. A and C and the seed contains traces of starch. The fruits also contain a fixed oil and nonpungent red coloring matter. Besides these, chilli fruits are reported to contain proteins, fat, minerals, fibres, carbohydrates, calcium, magnesium, riboflavin, oxalic acid and nicotinic acid 2 .
Except for a few report on the presence of the total or individual phenolics, there is a lack of information on the nature of phenolic acids in commercial varieties and plant parts of chilli and okra. Phenolic acids not only provide major quality attributes to the finished products 3 , they also contribute to the astringent taste and thus to overall flavor of the food. Besides, there exist reports showing pronounced physiological and biological properties of a number of phenolic acids, namely, tannic, gallic, caffeic and cinnamaic acids 4,5,6 . It is reported that vegetables constitute an important source of dietary intake of polyphenols and phenolic acids account for almost one third of the total intake of phenolics 7 . This prompted us to study the phenolic acid profile of the two important vegetable crops and results are presented here.
Materials and Methods
Phenolic standards (tannic, gallic, caffeic, vanillic, ferulic, chlorogenic and cinnamic acids) were obtained from Sigma Chemical Co. (St. Louis, MO). Salicylic acid was obtained by Hi Media (Hi Media Laboratories Ltd., Mumbai, India). All the solvents used in this investigation were of HPLC grade.
Freshly harvested plant parts of okra and chilli were collected from the Vegetable Farm, BHU, India and after air drying at room temperature, stored at 4° C. Seeds of okra cultivars namely, BO2, VRO5, IIVRO10 and Prabhani kranti and chilli (green, red, sweet pepper) were obtained from Indian Institute of Vegetable Research, Varanasi (India). All solvents and reagents were of highest analytical grade and were redistilled before use.
Extraction of phenolic acids
One g of freshly harvested plant parts were macerated in paste-mortor followed by suspending fine-crushed sample into 5 ml of ethanol-water (80:20, v/v) in glass tubes. Dry seeds of okra and chilli varieties (1 g) were crushed and suspended in the extraction solvent in a similar way. The suspension was subjected to ultra-sonication (Branson sonifier, USA) at 60 % duty cycles for 25 min at 4°C followed by centrifugation at 7,500 rpm for 15 min. The clear-greenish supernatant of each sample was subjected to charcoal treatment to remove pigments prior to evaporation under vacuo (Buchi Rotavapor Re Type). Dried samples were resuspended in 1.0 ml HPLC grade methanol by vortexing and stored at 4°C for further analysis.
High performance liquid chromatography (HPLC) of the samples was performed with the HPLC system (Shimadzu Corporation, Kyoto, Japan) equipped with two Shimadzu LC-10 ATVP reciprocating pumps, a variable UV-VIS detector (Shimadzu SPD-10 AVP) and a Winchrom integrator (Winchrom). Reverse phase chromatographic analysis was carried out in isocratic conditions using RP C-18 HPLC coloum (250 x 4.6 mm id, particle size 5 µm, Luna 5µ C-18 (2), Phenomenex, USA) at 25°C. Running conditions included injection volume: 5 µl, mobile phase: methanol-0.4 % acetic acid (80:20, v/v), flow rate: 1 ml/min, detection at 290 nm and attenuation response 0.03 AUFS. Samples were filtered through organic solvent compatible membrane filters (Pore size 0.20 µm, Millipore) prior to injection in sample loop. Tannic, gallic, caffeic, vanillic, ferulic, chlorogenic and cinnamic acids were used as internal and external standards. Phenolic compounds present in the samples were identified by comparing retention time (Rt) of the standards and by the co-injection. Contents of phenolic acids were calculated by comparing peak areas of reference compound with those in the samples run under similar elution conditions. Mean relative concentrations of phenolic compounds were statically analyzed by ANOVA and the normality of the results and homogeneity of the variances were tested.
Results and Discussion
HPLC analysis of different parts of okra (
Gallic acid was found to be the major phenolic acid in all parts of green chilli. It is evident from Fig. 1 A and B that the highest concentration of the compound was detected in chilli collar (38.46 µg/g fresh wt) that was almost 19.42 per cent of the total phenolic composition as observed by the HPLC analysis. Significant amount of this phenolic acid also detected in other parts such as pulp, leaves, roots and seed but in stem, it was minimum (3.67 µg/g fresh wt).
Agronomic and genetic varietal differences in chilli (
A number of phenolic acids, namely, tannic, caffiec, ferulic, chlorogenic and cinnamic acids were detected in different parts of green chilli (Table 1). Except in fruits and seeds, which contained 80.89 and 50.97 µg/g of tannic acid, respectively, this compound could not be traced in other parts. Caffeic acid was highest in collar region (83.64 µg/g) followed by root (64.82 µg/g), pulp (15.97 µg/g) and stem (0.46 µg/g). Ferulic acid was recorded in almost all parts of green chilli except in pulp, but the concentration did not exceed above 1.00 µg/g fresh wt in any case. Chlorogenic acid was found only in pulp (0.83 µg/g) while cinnamic acid in pulp (0.54 µg/g) as well as seed (0.12 µg/g). Per cent amount of these phenolics varied significantly in the plant parts (Table 1). In chilli seeds, tannic acid constituted almost 53.76 per cent of the total phenolics while as high as 75.45 and 67.03 per cent of caffeic acid was present in root and collar region of chilli, respectively.
Table 2 shows the amount of various phenolic acids in different varieties of chilli. Seeds of red chilli contained as high as 105.79 µg/g tannic acid followed by 50.97 µg/g in seed of green chilli which constituted almost 81.11 and 53.76 per cent of the total phenolics, respectively. Caffeic acid was highest in sweet pepper seed (39.49 µg/g) followed by black pepper seed (30.39 µg/g) and red chilli pulp (25.18 µg/g). Interestingly, vanillic acid in trace (0.064 µg/g) was detected only in the fruit of sweet pepper (0.094 µg/g). Amount of ferulic acid ranged from as high as 2.39 µg/g in sweet pepper fruit to as low as 0.27 µg/g in green chilli seed. Chlorogenic acid was traced only in red chilli pulp (0.84 µg/g) and sweet pepper seed (0.46 µg/g) while in all other varieties and plant parts, it was absent. On the other hand, cinnamic acid was constantly present in all varieties with varying amount and its amount in total phenolic also varied significantly (Table 2).
There exist certain reports on the phytochemical investigation of green chilli. Presence of caffeic acid in the fruits of selected pepper cultivars was reported by Haword
Concentration of tannic, gallic, ferulic and chlorogenic acids also varied significantly in various parts of okra (Fig. 2). The amount of gallic acid was significantly lower in all plant parts as compared to ferulic, chlorogenic and tannic acids. Maximum content of gallic acid was recorded in okra seed (2.13 µg/g fresh wt) followed by root (0.55 µg/g) while in other parts it was below 0.15 µg/g. Okra leaf and stem were rich in tannic acid (12.19 and 104.42 µg/g, respectively) while ferulic acid was maximum (10.96 µg/g) in stem tissues followed by leaf, root, pulp and seed. Chlorogenic acid was maximum in okra pulp (9.62 µg/g) followed by the leaf (9.15 µg/g). In stem and seed only minor amount of chlorogenic acid was observed, whereas it could not be traced in root (Fig. 2).
Among the three phenolics, gallic acid was the major constituent in the seeds of all the varieties. Maximum content of the compound (8.12 µg/g) was recorded in BO2 variety with 66.9 per cent followed by Prabhani kranti, VRO6, IIVRO10 and VRO5. Ferulic acid content was quite low as it reached only 0.43 µg/g in variety BO2 while in all other varieties, the amount was quite less. The per cent composition of ferulic acid varied form as high as 11.68 per cent of the total phenolics in variety VRO5 to as less as 2.22 per cent in Prabhani kranti. Chlorogenic acid content was found to be even less than ferulic and gallic acids. Maximum content of chlorogenic acid was recorded in the VRO6 variety (0.32 µg/g). The compound, however, could not be traced in varieties VRO5 and IIVRO10. Estimation of total phenolics in okra seeds has seen reported by Suportra and Mukharji 11 but phenolic compounds have not been estimated from various parts of okra. Sharma and Rai 12 studied the antagonism between absiscic and phenolic compound in rooting of
Vegetables are on the of the important source of dietary intake of polyphenols 7 . That phenolic acids play a vital role not only in maintaining taste, flavor and quality of the vegetables, but are also able to add important medicinal values to the foods is well documented 3 . Reports on the selective cytotoxicity of gallic acid 9 and antimicrobial activities of gallic and tannic acids 4 indicate medicinal significance of these phenolic acids. Interestingly, these compounds were significantly reported in this study. In view of the above facts, these results themselves point out vital significance of these phenolic acids in the two important vegetable crops, chilli and okra.
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