A Comparative Study of Ascorbic Acid and Capsaicinoid Contents in Red Hot Peppers (Capsicum annum L.) Grown in Southeastern Anatolia Region

Abstract

The aim of this study was to evaluate selected physico-chemical properties, ascorbic acid, and capsaicinoid (capsaicin, dihydrocapsaicin, norhydrocapsaicin, homodihydrocapsaicin, and total capsaicinoid) quantities of red hot pepper populations (Capsicum annum L.) grown in the South-Eastern Anatolia Region. Fresh and sun-dried red hot peppers and red hot pepper seed samples were collected from Kahramanmaraş, pepper of Kahramanmaraş; Gaziantep, pepper of Gaziantep; Kilis, pepper of Kilis; Diyarbakır, pepper of Diyarbakır; and Şanlıurfa, pepper of Şanlıurfa in the months of September through October 2010. The collected samples were analyzed in terms of certain quality parameters and capsaicinoid contents, while ascorbic acid amounts were determined only in fresh samples. All of the examined prameters were found to be significant statistically (P < 0.05). From the obtained results, it might be considered that these red hot pepper samples were suitable for cultivation due to their high-nutritional quality.

Keywords:

• Red hot pepper
• Capsaicinoids
• Ascorbic acid
• HPLC

INTRODUCTION

The genus Capsicum (Family: Solanaceae) commonly known as chilli pepper is a major spice crop. Its popularity stems from the combination of color, taste, and pungency. For a long time, red hot pepper has been known all over the world as a delicious spice with a characteristic smell, taste, and aroma. Besides this property, it is evaluated variously as a pharmaceutical ingredient, various therapeutic purposes, sources of bactericidal agents, natural plant color, and sprays for self-defence.^[1−12]

Pepper is an important nutrient for human diet due to its contained vitamins and antioxidants. In particular, the antioxidant vitamins (vitamins A, C, and E), carotenoids are present in high concentrations in various types of pepper. Ascorbic acid is the biologically active form of dehydroascorbic acid and it prevents some important illness such as cancer, anaemia, diabetics, and cardiovascular diseases. It is fundamentally provided by fruits and vegetables. Fresh pepper contains a higher amount of ascorbic acid than the other fruits and vegetables.^[13–17] In addition, ascorbic acid content of the fresh peppers increases during ripening, but this value decreases during the post-harvesting period. Also, this value might be changed with the cultivar, production practices, maturity at harvest and storage conditions.^[18–20]

A group of alkaloids that are responsible for the pungency and spicy flavor are called as capsaicinoids. Generally, capsaicinoids are formed from the derivative of phenylopropanoid compounds. They are characterized by the high biological activity and their pharmacological, neurological, and dietetic effectiveness.^[21–23]

Although 12 compounds have been identified in peppers, capsaicin and dihydrocapsaicin are the most important of them. In addition to these major capsaicinoids, other minor capsaicinoids such as nordihydrocapsaicin, norcapsaicin, homocapsaicin, homodihydrocapsaicin, nornorcapsaicin, nornornorcapsaicin, and nonivamide are found in hot peppers. Capsaicin and dihydrocapsaicin constitute about 90% of pungency together. In particular, they show antimutagenic, antitumoral, and antioxidant properties. Generally, capsaicin has been recognized as the major component in Capsicum sp. It constitutes 70% of the total capsaicinoids and produces the majority of the pungency (approximately 80–90%).^[9,10,24,25] Capsaicin has important medical values and is effective in the treatment of painful conditions such as: Rheumatic diseases, cluster headaches, painful diabetic neuropathy, and post-herpetic neuralgia, etc. On the other hand, the antibacterial effect of capsaicin on the certain of group bacteria have also been reported.^[23,26–32]

The aim of this study was to quantify the changes in physico-chemical properties, ascorbic acid and capsaicinoid contents of red hot peppers grown in the South-Eastern Anatolia Region. Also, it was tried to determine that the advantages of pepper samples from each other and which one was the best for pepper breeding in terms of quality parameters.

MATERIALS AND METHODS

Materials

Fresh red hot peppers (Capsicum annum L.), sun-dried red hot peppers (Capsicum annum L.), and red hot pepper seed samples (Capsicum annum L.) were used as the materials in this study. The samples were collected from Kahramanmaraş, Gaziantep, Kilis, Diyarbakır, and Şanlıurfa provinces in September and October of the year 2010. The samples were put into polyethylene bags and immediately transported to the laboratory and then stored at –18°C until extraction for analysis.

Chemical Analysis

The total solid and ash contents of red hot pepper samples were determined by using the gravimetric method,^[33] crude oil content by the Soxhlet extraction method,^[34] ASTA color value by spectrophotometric method,^[35] and the cellulose content was measured by using a cellulose instrument (FOSS FIBERTEC 2010).^[33]

Ascorbic Acid

The quantification of ascorbic acid content of red hot pepper samples were carried out by high-performance liquid chromatography (HPLC; Agilent HPLC 1200 series Germany) and reversed-phase (250 × 3 mm, 5 mm) ACE brand C18 column (254 nm). For this purpose, 10 g samples were homogenized with 100 mL 6% metaphosphoric acid. After obtained homogenate was respectively filtered from coarse filter paper, passed from 0.45 mM filter and injected to the HPLC.^[36] For elution, the potassium dihydrogen phosphate solution was used. The flow rate (0.5 mL/min) of injection volumes, run time, and temperature were determined as 20 μL, 15 min, and 20°C, respectively.

Separation and Quantification of Capsaicinoids

For this analysis, 2 g crushed red hot pepper sample was extracted with 30 mL acetonitrile and processed in reflux cooler at 80°C for 4 h. Then, the obtained remnant (2 mL) was passed from a 0.45 mM filter and injected to the HPLC.^[37] The separation and quantification of the capsaicinoids was performed by a HPLC method with fluorescence detection (280 nm). For this purpose nucleosil 5 C18 column (250 × 4.6 mm i.d.) and 5 C18 protecting column (4 × 4.6 mm i.d.) was used. For elution, acetonitrile/water/acetic acid (100/100/1) was used at a flow rate of 1.2 mL/min. The injection volume, run time, and temperature were used as 20 μL, 15 min, and 20°C, respectively.^[38]

Statistical Analysis

The study was designed according to the randomised complete block design. The data was analyzed statistically using SPSS statistical software programme version 13 (SPSS Inc., Chicago, IL, USA, 2004).^[39] Analysis of variance (ANOVA) and mean with significant differences were compared by Duncan’s multiple range tests (P < 0.05) and results were presented as the mean ± standard deviation (mean ± SD).

RESULTS AND DISCUSSIONS

The results of total solid, ash, and crude oil amounts, ASTA color values and cellulose contents of the red hot pepper samples are presented in Table 1. At the end of the physico-chemical analysis, some differences were determined among the samples, but these results were as expected. Previously, Bilişli and Erhan,^[40] Galvez et al.,^[41] Başaran,^[42] Choi and Suh,^[43] Doğar,^[44] and Krajayklang et al.^[45] also reported similar findings in red hot peppers.

TABLE 1 The mean values of some physico-chemical properties of the red hot pepper samples and their statistical evaluations^a

	Total solids (%)		Ash (%)		Crude oil (%)		ASTA color value	Cellulose content (%)
Pepper samples	Fresh pepper	Dried pepper	Fresh pepper	Dried pepper	Fresh pepper	Dried pepper	Dried pepper	Dried pepper
PKA	17.2 ± 0.9^c	96.7 ± 0.1^a	1.5 ± 0.10^b	8.3 ± 0.2^b	0.6 ± 0.3^c	5.2 ± 0.4^c	151.5 ± 3.2^a	24.7 ± 0.4^b
PG	19.3 ± 0.3^a	95.0 ± 0.6^b	1.2 ± 0.10^c	7.6 ± 0.2^c	1.7 ± 0.2^a	5.4 ± 0.3^c	128.8 ± 4.6^c	24.4 ± 0.3^b
PKI	18.3 ± 0.9^b	94.6 ± 0.1^c	1.4 ± 0.10^c	8.6 ± 0.2^a	1.5 ± 0.1^b	6.2 ± 0.2^b	140.6 ± 4.7^b	25.3 ± 0.9^a
PD	16.0 ± 0.3^d	90.8 ± 0.3^d	1.7 ± 0.10^a	6.6 ± 0.5^d	1.7 ± 0.1^a	6.6 ± 0.5^a	84.5 ± 5.4^d	22.6 ± 0.5^c
PŞ	9.2 ± 0.5^e	89.8 ± 0.2^e	0.5 ± 0.02^d	8.4 ± 0.2^b	1.5 ± 0.1^b	6.6 ± 0.3^a	47.7 ± 2.5^e	14.0 ± 0.4^d

^a Mean values followed by different letters in the same column are significantly different (P < 0.05).

Ascorbic Acid Contents

In this study, ascorbic acid content was determined only in the fresh red hot peppers. As seen in Table 2, the highest mean ascorbic acid value was found in pepper of Gaziantep (PG; 248.04 ± 32.3 mg/100 g) and followed by pepper of Kilis (PKI; 213.8 ± 19.0 mg/100 g), pepper of Diyarbakır (PD; 186.2 ± 10.4 mg/100 g), pepper of Şanlıurfa (PŞ; 151.3 ± 11.6 mg/100 g), and pepper of Kahramanmaraş (PKA; 142.8 ± 20.1 mg/100 g), respectively. Similar results were also reported by Gahungu et al.,^[10] Perucka and Materska,^[16] Martinez, et al.^[19] However, Topuz and Özdemir^[46] found lower values in five Capsicum annuum cultivars grown in Turkey.

TABLE 2 The mean ascorbic acid contents of fresh red hot pepper samples and their statistical evaluations^a

Pepper samples	Ascorbic acid (mg/100 g)
PKA	142.80 ± 20.1^d
PG	248.04 ± 32.3^a
PKI	213.80 ± 19.0^b
PD	186.20 ± 10.4^c
PŞ	151.30 ± 11.6^d

^a Mean values followed by different letters in the same column are significantly different (P < 0.05).

The determined quantity differences among the samples were probably due to the several factors including soil, climate, growing conditions, cultivar, production practices, fruit ripening, post-harvest handlings, and storage conditions.^[6,17,19,47] When other studies were observed, ascorbic acid contents of the investigated red hot pepper samples were found to be higher than the other studies carried out in Turkey.^[37] In contrast, the values were similar to the results of a study conducted in India by Deepa et al.^[48]

As seen in Table 2, there were statistically significant differences (P < 0.05) among the red hot pepper samples in terms of ascorbic acid contents (Table 2). According to statistical evaluations the ascorbic acid values of PKA and PŞ showed a similar trend with respect to statistical evaluations. Conversely, PG, PKI, and PD showed differences from each other and the other samples statistically (P < 0.05). The obtained results were also similar to Kumar and Tata,^[20] Galvez et al.,^[41] Sevgican,^[49] and Kuşcu’s^[50] findings. Also, Osuna-Garcia et al.^[15] and Deepa et al.^[48] reported that the ascorbic acid contents of fresh pepper samples showed a variation depending on the cultivar.

Capsaicinoid Contents of Red Hot Peppers

In this study, the fresh red hot peppers, dried red hot peppers, and red hot pepper seed samples were investigated in terms of capsaicinoids. At the end of the analysis, capsaicin, dihydrocapsaicin, norhydrocapsaicin, homohydrocapsaicin, and total capsaicinoid compounds were determined in these peppers. However, capsaicin and dihydrocapsaicin were found to be higher values than the other capsaicinoids. Similarly, Perucka and Materska,^[23] Gnayfeed et al.,^[51] Dong,^[52] and Perucka and Oleszek^[53] reported that the capsaicin, dihydrocapsaicin, and nordihydrocapsaicin were the major capsaicinoids in different Capsicum annuum varieties. Vera-Guzman^[54] also reported that the C. annuum had higher capsaicin content (4.9 to 142 μg mL⁻¹) than dihydrocapsaicin (1.5 to 65.5 μg mL⁻¹). When fresh red hot peppers were examined in terms of their capsaicinoid contents, these values of peppers were found quite high and capsaicin, dihydrocapsaicin, norhydrocapsaicin, and total capsaicinoid levels of samples had a statistically significant differences (P < 0.05) (Table 3). Similarly, some authors reported that the hot peppers were a better source of capsaicinoids than the sweet ones.^[16]

TABLE 3 The mean capsaicinoid contents of fresh red hot pepper samples and their statistical evaluations^a

Pepper samples	Capsaicin (mg/kg)	Dihydrocapsaicin (mg/kg)	Norhydrocapsaicin (mg/kg)	Homohydrocapsaicin (mg/kg)	Total capsaicinoid (mg/kg)
PKA	68.0 ± 16.8^a	35.5 ± 8.7^bc	12.20 ± 4.99^a	1.6 ± 1.15^a	106.20 ± 26.0^b
PG	43.0 ± 11.3^b	29.5 ± 7.7^cd	1.60 ± 0.45^d	ND	74.03 ± 19.1^c
PKI	75.1 ± 23.0^a	49.4 ± 19.2^a	3.70 ± 1.41^bc	0.1 ± 0.20^c	128.40 ± 42.3^a
PD	50.0 ± 16.0^b	43.1 ± 14.0^a^b	3.10 ± 0.80^cd	0.3 ± 0.20^bc	96.40 ± 30.7^b
PŞ	41.4 ± 11.3^b	25.3 ± 9.6^d	5.07 ± 1.90^b	0.6 ± 0.40^b	72.30 ± 21.6^c

^a Mean values followed by different letters in the same column are significantly different (P < 0.05).

The capsaicin, dihydrocapsaicin, and total capsaicinoid values of fresh red hot pepper samples were analyzed, the highest mean values were found in PKI and the lowest mean amounts were determined in the PŞ. Gahungu et al.^[10] also reported similar capsaicin and dihydrocapsaicin contents in fresh pepper samples. As the norhydrocapsaisin amount of peppers was examined, the highest value was found in PKA and followed by PŞ, PKI, PD, and PG, respectively. Similarly, the homohydrocapsaisin content of peppers ranged between samples PG and PKA.

As seen in Table 3, capsaicin values of the PG, PD, PŞ, and PKA, PKI showed a similar trend with respect to statistical evaluations and these two groups of sample were found completely different statistically (P < 0.05). Homohydrocapsaicin contents of the PG and PKI were found to be similar, while these samples differed from other samples at the P < 0.05 level. Otherwise, the dihydrocapsaicin and norhydrocapsaisin values of all fresh red hot pepper samples showed statistically significant differences (P < 0.05). Also, the total capsaicinoid contents of PKA and PD were similar, and the PG and PŞ showed similarity in terms of statistical evaluations, and the differences between PKI and others were significant statistically (P < 0.05) (Table 3). Similar variations were previously reported by Nwokem et al.^[8] and Sanatombi and Sharma^[55] on the capsaicinoid contents of different pepper samples.

The capsaicin, dihydrocapsaicin, norhydrocapsaicin, homohydrocapsaicin, and total capsaicinoid amounts of dried red hot pepper samples showed statistically significant (P < 0.05) differences (Table 4). When Tables 3 and 4 were analyzed, the statistical properties of dried and fresh red hot pepper samples showed the same properties, although capsaicinoid contents of dried peppers were higher than fresh samples. This situation might be explained by the increasing of capsaicinoid content with the increment of dry matter content during the drying process. When taken into account of capsaicin, dihydrocapsaicin, and total capsaicinoid contents, the highest mean values were in PD, while the lowest mean values were in PŞ. However, the lowest mean norhydrocapsaicin and homohydrocapsaicin contents were determined in PG, but the highest mean values were found in PŞ.

TABLE 4 The mean capsaicinoid contents of dried red hot pepper samples and their statistical evaluations^a

Pepper samples	Capsaicin (mg/kg)	Dihydrocapsaicin (mg/kg)	Norhydrocapsaicin (mg/kg)	Homohydrocapsaicin (mg/kg)	Total capsaicinoid (mg/kg)
PKA	395.0 ± 143.9^a	295.6 ± 114.00^bc	19.1 ± 6.2^a	2.5 ± 1.07^a	712.3 ± 264.5^b
PG	368.3 ± 39.4^b	239.4 ± 27.04^cd	15.1 ± 1.4^d	ND	623.0 ± 67.5^c
PKI	450.3 ± 59.2^a	304.1 ± 47.30^a	19.8 ± 2.6^bc	1.8 ± 0.50^c	774.8 ± 108.0^a
PD	524.0 ± 43.7^b	405.4 ± 32.40^a^b	27.3 ± 2.7^cd	3.2 ± 0.70^bc	964.0 ± 80.2^b
PŞ	158.2 ± 34.6^b	163.2 ± 36.60^d	36.5 ± 9.5^b	4.9 ± 1.20^b	363.0 ± 78.8^c

^a Mean values followed by different letters in the same column are significantly different (P < 0.05). Note: ND: Not determined.

The highest mean capsaicin and total capsaicinoid values of red hot pepper seeds were found in PKA, and the lowest mean values were found in PŞ. Otherwise, the highest mean value of dihydrocapsaicin was found in PKI and the lowest mean value was determined in PKA. The norhydrocapsaicin values ranged between PŞ, PK, PKI, PD, and PG, respectively, and the highest mean homohydrocapsaisin value was determined in PD and the lowest mean values were found in PKA and PG (Table 5). According to statistical evaluations, the capsaicin contents of PG and PD pepper seed samples were similar to each other but these samples showed differences from other samples statistically (P < 0.05). As seen in Table 5, the dihdrocapsaicin contents of samples PKA and PŞ and samples PG and PD showed a similar trend with respect to statistical evaluations. On the other hand, these samples showed statistically significant (P < 0.05) differences from each other and the PKI. The norhydrocapsaicin contents of PKA and PŞ were similar, although these samples differed from other samples statistically (P < 0.05) and homohydrocapsaicin values of PKA, PG, and PKI showed a similar trend with respect to statistical evaluations, although PD and PŞ differed from each other and the other samples (P < 0.05) statistically. Also, the total capsaicinoid contents of PG and PD were similar, but the differences between these samples and others were significant statistically (P < 0.05; Table 5).

TABLE 5 The mean capsaicinoid contents of red hot pepper seed samples and their statistical evaluations^a

Pepper samples	Capsaicin (mg/kg)	Dihydrocapsaicin (mg/kg)	Norhydrocapsaicin (mg/kg)	Homohydrocapsaicin (mg/kg)	Total capsaicinoid (mg/kg)
PKA	266.6 ± 104.4^a	34.8 ± 8.7^c	11.9 ± 4.9^a	ND	489.6 ± 190.3^a
PG	97.5 ± 41.8^c	64.6 ± 33.5^b	3.1 ± 2.0^c	ND	165.3 ± 76.0^c
PKI	156.0 ± 54.9^b	108.6 ± 34.2^a	6.5 ± 2.8^b	0.5 ± 0.4^c	270.7 ± 90.1^b
PD	87.4 ± 22.0^c	66.5 ± 16.9^b	4.7 ± 1.3^bc	1.4 ± 0.6^a	156.5 ± 41.6^c
PŞ	23.9 ± 5.4^d	28.6 ± 5.7^c	12.0 ± 6.2^a	1.1 ± 0.7^b	65.6 ± 15.6^d

^a Mean values followed by different letters in the same column are significantly different (P < 0.05). Note: ND: Not determined.

The obtained results also showed that capsaicin and dihydrocapsaicin were the major capsaicinoids in red hot peppers and and the change of their capsaicinoid contents were connected to the differences of samples. These results were similar to Poyrazoğlu et al.^[6] and Gahungu et al.’s^[10] findings; but Ziino et al.^[56] reported higher results from them. Consequently, the highest mean capsaicin, dihydrocapsaicin, norhydrocapsaicin, homohydrocapsaicin, and total capsaicinoid levels were determined in dried red hot pepper samples and followed by red hot pepper seed samples and fresh red hot pepper samples, respectively. This situation could be explained by the increase in dry matter during sun-drying. Similar results were also reported by Kumar and Tata,^[20] Perucka and Materska,^[23] and Perucka and Oleszek.^[54]

CONCLUSIONS

In this study, red hot pepper samples were analyzed in terms of certain parameters. The ascorbic acid content was determined only in fresh red hot peppers, physico-chemical properties were analyzed in fresh and dried red hot peppers, while capsaicin, dihydrocapsaicin, norhydrocapsaicin, homohydrocapsaicin, and total capsaicinoid levels were analysed in fresh-dried red hot peppers and red hot pepper seed samples. According to the results, red hot pepper samples produced in different provinces showed statistically significant differences (P < 0.05) in terms of all investigated parameters. Generally, the ascorbic ascorbic acid values of investigated samples were quite high. On the other hand, dried red hot pepper samples had the highest quantities of capsaicinoid and followed by red hot pepper seed samples and fresh red hot peppers, respectively. Consequently, the quality parameters of the samples showed differences with the soil, climate, growing conditions, and properties of pepper samples (fresh, dry, or seed).