Total Phenolic and Flavonoid Content,Lipophilic Components,and Antioxidant Activities of Capsicum annuum Varieties Grown in Omo Nada,Jimma,Ethiopia

Abstract

Objective

This study aimed to determine total phenolic content (TPC), total flavonoid content (TFC), lipophilic components, and antioxidant activities of Capsicum annuum L. varieties grown in Omo Nada, Jimma, Ethiopia.

Methods

Fresh, ripe fruits from three commonly cultivated varieties of C. annuum – Mareko Fana, Kolesha, and Gojeb – were investigated. TPC and TFC were measured using Folin-Ciocalteu and aluminum chloride colorimetry methods in methanol and chloroform extracts, expressed as mg gallic acid equivalents (mg GAE/g) and mg quercetin equivalents (mg QE/g), respectively. Lipophilic components were identified in n-hexane extracts using gas chromatography-mass spectrometry (GC-MS). Antioxidant activities were assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay.

Results

Methanol extracts had the highest TPC and TFC across all varieties, with Gojeb exhibiting the highest values (TPC: 22.98 ± 0.08 mg GAE/g; TFC: 7.67 ± 0.07 mg QE/g). GC-MS analysis identified 80 distinct compounds in n-hexane extracts, with m-xylene and p-xylene (6.68-16.82%), capsaicin (4.22-11.25%), dihydrocapsaicin (2.12-8.24%), vitamin E (5.32-14.72%) and (24R)-stigmast-5 -en-3beta-ol (2.19-6.20%) being among the major components across all varieties. The DPPH assay revealed that methanol extracts of Gojeb had the strongest antioxidant activity with EC₅₀ values of 0.40 ± 0.04 mg/mL. Moreover, the EC₅₀ showed strong negative correlation with TPC ((r = −0.87).

Conclusion

The methanol extracts of C. annuum varieties, especially Gojeb, exhibited high TPC, TFC, and antioxidant activity, highlighting its importance as potential sources of natural antioxidants in food or pharmaceutical industries. Despite their lower antioxidant activities, the n-hexane extracts contain valuable health-promoting compounds. Further research, involving chemical isolation and additional bioanalysis could give more insight about the health benefits of the C. annuum varieties investigated.

Keywords

total phenolic content total flavonoid content lipophilic components antioxidant activity

Introduction

Excessive free radicals, coupled with impaired antioxidant systems, cause oxidative stress¹ and contribute to chronic diseases such as cancer, diabetes, cardiovascular problems and obesity.^2,3 The use of plant based natural products is highly preferred to their synthetic counterparts due to reported side effects and potential toxicity. Consumption of vegetables and fruits is inversely associated with oxidative stress^4,5 as they represent sustainable sources of natural antioxidants, including phenolic compounds, vitamins, carotenoids, terpenoids, alkaloids and fiber.^6,7 This has sparked increased interest in researching the antioxidant activity and bioactive content of plant foods.

The production of various metabolites by plants generally facilitates their chemical adaptation to certain environments, protects them from microbes, insects and herbivores, and promotes their reproduction (as attractants).⁸ It is important to emphasize that human consumption of fruits and vegetables is strongly influenced by compounds responsible for their color, taste and aroma.⁹ Pepper is one of the fruits that is regularly consumed around the world, mainly due to its attractive color, spiciness and aroma.¹⁰ Pepper belongs to the genus Capsicum (family Solanaceae) and has five known domestically grown species: Capsicum annuum, C. acetum, C. chinense, C. frutescens and C. pubescens.¹¹ Among these, C. annuum is one of the most commonly grown species worldwide¹² and in Ethiopia as well.¹³ Ethiopians incorporate pepper into their daily meals, either as a vegetable or as spice products, such as Berbere and Mitmita, which are prepared from ripe peppers. Adults in Ethiopia consume an average of 15 g of red pepper daily with meals,¹³ which is believed to aid digestion and provide health benefits.

Traditionally, peppers are used in various cultures to treat diseases such as arthritis, rheumatism, stomach pain, skin problems and cuts.¹⁴ Studies show that peppers contain capsaicinoids, carotenoids, vitamins and phenolic compounds that prevent oxidative cell damage by interacting with oxygen molecules and neutralizing peroxide radicals.¹¹ This activity reflects the health-promoting properties of pepper fruits and their products included in the daily diet.

The plant, considered a potential source of biologically active compounds, is usually first examined for its antioxidant properties using well-standardized in vitro methods.¹⁵ Studies have shown that the antioxidant activities and chemical content of peppers vary depending on variety (cultivar type), color at maturity stage, and extraction solvents.^16–19 The polarity of extraction solvents is crucial for the analysis of antioxidant activity due to the presence of various compounds. For example, polar extracts of C. annuum fruits provide higher amounts of antioxidant compounds such as phenolic compounds.¹⁶ On the other hand, non-polar solvents are best used for the extraction of lipophilic compounds such as tocopherols, capsaicinoids, terpenoids and carotenoids, which have implications for their nutritional, medicinal and consumer appeal.^18,20

Peppers were introduced to Ethiopia in the seventeenth century and have been a staple of the local diet ever since, consumed both as a vegetable and as a spice. Hot pepper is cultivated in many parts of the country and it is an important source of income for smallholder producers.^13,21 Over time, regional and local varieties have been developed and there has been evidence of genetic diversity and population structure.²² Despite suitable growing conditions, production is also limited by factors such as disease, lack of improved varieties, and soil fertility²¹ which affects the quality of produced peppers.

Literature studies have shown significant differences in the antioxidant activity and active compounds of peppers depending on the species, cultivar type and environmental conditions. Despite significant pepper production and consumption in Ethiopia, studies on the antioxidant activities and chemical contents is lacking. In the Jimma Zone of southwest Ethiopia, C. annuum varieties that include Mareko Fana, Kolesha, and Gojeb are grown in Omo Nada, known for large-volume hot pepper production. This study investigates for the first time the TPC, TFC, lipophilic chemical profiles and DPPH radical scavenging activities of these C. annuum cultivars.

Materials and Methods

Chemicals

Methanol, chloroform, and n-hexane were purchased from Loba Chemie Pvt, Ltd (Mumbai, India). Folin-Ciocalteau reagent (F-CR) (2N) was purchased from Sisco Research Laboratories Pvt Ltd. Additionally, anhydrous sodium carbonate (99.5%) from Blulux, aluminum chloride (96%) from Finkem, potassium acetate (98.15%), gallic acid (99%) and ascorbic acid (99%) from Nice Chemicals Pvt. (Ernakulum), Kerela, India), quercetin (≥95%) from Sigma-Aldrich and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical (85%) from Alpha Chemika were used in this study.

Apparatus and Instruments

A double beam UV-vis spectrophotometer (SPECORD 200 PLUS, Analytikjena, Germany) and GC-MS (Agilent 8890 GC coupled with 5977B single quadrupole mass spectrometer detector (MSD) and an Agilent G4513A auto sampler (Agilent Technologies, USA) were the main instruments used.

Sample Collection and Preparation

Fruits of three C. annuum cultivars (Gojeb, Kolesha and Mareko Fana) were collected from Omo Nada farm and brought to the Postgraduate Laboratory of Analytical Chemistry, Jimma University. Samples were cleaned, air-dried in the shade at room temperature, ground, and stored until extraction. Figure 1 shows the dried fruits of the C. annuum varieties.

Figure 1.

Dried fruits of C. annuum varieties cultivated in Omo Nada (local names: Gojeb (a), Kolesha, (b), and Mareko Fana (c)).

Determination of Total Phenolic Content and Total Flavonoid Content

Extraction Procedure

Methanol and chloroform were used to prepare extracts for the determination of TPC and TFC. Briefly, the powdered samples (10 g each) were transferred to two amber bottles and filled with 100 mL of methanol and chloroform separately. Each of the mixtures was shaken for 30 min and stored at room temperature for 24 h. The resulting infusions were filtered with Whatman no.1 filter paper and the remaining residues were re-extracted with an equivalent volume of the same solvent used previously. The filtrates were combined and reduced to a quarter of their original volume using a rotary evaporator at 40 °C. These concentrates were dried at room temperature in dark and the extraction yield was calculated as the percentage of dry extract per weight of dried fruit powder.²³ Finally, 0.05 g of dry extract each pepper varieties was dissolved in 50 mL of methanol (95% v/v) to obtain 1 mg/mL solutions. These solutions were used to determine TPC and TFC.

Determination of TPC

The Folin-Ciocalteu assay described by Molole et al²³ was used with a slight modification. Briefly, 0.5 mL of gallic acid standards (0-0.125 mg/mL), samples (1 mg/mL), or blank (95% methanol) was mixed with 2 mL of F-CR (diluted 1:10 in distilled water). After 3 min, 4 mL of Na₂CO₃ (7.5%) was added and mixed well. The absorbance of the resulting blue-colored solution was measured at 760 nm after incubation for 30 min at room temperature.

Determination of TFC

The TFC was determined using the aluminum chloride colorimetric procedure reported by Al-Owaisi et al²⁴ with slight changes. First, 0.05 g of quercetin was dissolved in 50 mL of methanol (95% v/v) and diluted to obtain calibration standards (0.005 to 0.065 mg/mL). Then, 0.5 mL of the standards, blank, or C. annuum extracts (1 mg/mL) was mixed with 0.1 mL of aluminum chloride (10%), 0.1 mL of 1 mol/L potassium acetate, and 2.8 mL of distilled water in a test tube and incubated for 30 min at room temperature. The absorbance of the reaction mixture was measured at 432 nm. Finally, the TFC was calculated from the quercetin calibration equation (y = 0.0091-0.002, R²= 0.999) and expressed as milligrams of quercetin equivalent per gram of dry extract (mg QE/g).

Gas Chromatography – Mass Spectroscopy (GC–MS) Analysis of Lipophilic Components

An Agilent 8890A gas chromatograph coupled with 5977B mass selective detector (MSD) was used to examine lipophilic components of C. annuum varieties. First, 10 g of each of the pulverized fruit samples were extracted with 100 mL n-hexane using soxhlet apparatus for 12 h. The extracts obtained were filtered and the solvent was reduced using a rotary evaporator. Separation of the constituents of these extracts was carried out on an HP-5MS fused silica capillary column (30 m × 0.25 mm, internal diameter; 0.25 µm film thickness) coated with a 25% diphenyl-95% dimethyl siloxane phase. Helium (He) was used as the carrier gas. The flow rate of the gas was 1 mL/min. To carry out the experiment, 1 µL of sample or blank was injected in splitless mode, with the injection port temperature adjusted to 250 °C. The oven temperature was first set at 70 °C for 2 min and then ramped up at a rate of 5 °C/min to 220 °C. The temperature of the transfer line of the mass spectrometer detector (MSD) was 300 °C. To determine the mass spectra, we operated the MSD in electron ionization (EI) mode, with an ionization energy of 70 eV. The mass spectra were scanned in the range of 10–400 m/z. The temperature of the ion source was 290 °C, and that of the MS quadrupole was 150 °C. All the peaks were identified based on 70% and above mass spectral matching from the libraries. The mass spectra were detected in 75 min.

DPPH Radical Scavenging Activity

The antioxidant activity of all the three extracts was determined using the DPPH free radical scavenging assay following the previously modified method²³ but with some slight changes. Briefly, 1 mL of each crude extract and ascorbic acid solutions of 1.0, 0.80, 0.60, 0.40, 0.20, and 0.10 mg/mL were separately mixed with 2 mL of 0.04 mg/mL DPPH in test tubes and shaken vigorously. Similarly, the control sample was prepared by mixing 1 mL of methanol with 2 mL of 0.04 mg/mL DPPH solution. Absorbance of these solutions was recorded at 517 nm after 30 min of incubation in the dark. The percentage of DPPH free radicals remaining in the mixtures was then calculated using equation 1.

% of remaining DPPH = \frac{A_{sample}}{A_{control}} \times 100

(1)

where A_control= absorbance of the control and A_sample= absorbance of the sample.

Finally, the effective concentration of the extracts needed to scavenge 50% of the initial DPPH concentration (EC₅₀) was determined from the plot of the remaining % DPPH versus concentration of extracts.

Statistical Analyses

All experiments and measurements were performed in triplicate and the results were expressed as means with standard deviation (M ± SD). All descriptive statistical analysis were performed using Graph Pad Prism version 8.0.2. In addition, analysis of variance (ANOVA) with Tukey's multiple comparison test (P < 0.05) was performed to compare the means.

Results

Extraction Yield, Total Phenolics and Flavonoids Contents

The extraction yield varied depending on the pepper variety and extraction solvent type used (Table 1). Methanol provided the highest extraction yield (26.77-40.97%) for all pepper varieties, while chloroform provided the lowest yield (5.33-7.23%). In each case, the Mareko Fana variety gave the highest yield, while the Kolesha variety gave the lowest yield.

Table 1.

Extraction yield, TPC, and TFC of Methanol and Chloroform Extracts of C. annum Varieties.

C. annuum Variety	Extraction Solvent	Extraction yield (%/g dry sample)	TPC (mg GAE/g)	TFC (mg QE/g)
Gojeb	Methanol	35.97 ± 0.25	22.98 ± 0.08	7.67 ± 0.07
Gojeb	Chloroform	6.67 ± 0.21	14.63 ± 0.09	4.32 ± 0.19
Kolesha	Methanol	26.77 ± 0.10	15.18 ± 0.13	5.5 ± 0.052
Kolesha	Chloroform	5.33 ± 0.15	12.03 ± 0.03	4.73 ± 0.32*
Mareko Fana	Methanol	40.97 ± 0.28	18.50 ± 0.42	5.73 ± 0.02
Mareko Fana	Chloroform	7.23 ± 0.25	13.85 ± 0.74	4.72 ± 0.07*

* Values followed by an asterisk are not significantly different (P > 0.05)

The TPC of C. annuum extracts was determined employing the Folin-Ciocalteu assay using Gallic acid calibration curve (y = 0.00981x + 0.023, R² = 0.999) and the results were expressed as mg of gallic acid equivalents per g of dry extract (mg GAE/g). The results showed remarkable variability with both extraction solvents and pepper variety. The highest TPC was found in methanol extracts, ordered as follows: Kolesha (4.63 ± 0.09 mg GAE/g) < Mareko Fana (18.50 ± 0.42 mg GAE/g) < Gojeb (22.98 ± 0.08 mg GAE/g). In comparison, the TPC obtained in chloroform extracts were lower but showed similar trend as in methanol, ranging from 12.03 ± 0.03 mg GAE/g (Kolesha) to 14.63 ± 0.09 mg GAE/g (Gojeb) (Table 1). The TFC was calculated from a quercetin calibration equation (y = 0.0091× - 0.002, R² = 0.999) and expressed as milligrams of quercetin equivalents per gram of dry extract (mg QE/g). The TFC was low and showed slight variation with respect to both the extraction solvents and pepper varieties. It was in the range of 5.5 ± 0.05 to 7.67 ± 0.07 mg QE/g, and 4.32 ± 0.19 to 4.73 ± 0.32 mg QE/g for methanol and chloroform extracts, respectively. There was no significant statistical difference in TFC between Kolesha and Mareko Fana chloroform extracts (Table 1).

Lipophilic Chemical Components

The n-hexane extracts (lipophilic) from C. annuum fruit samples were obtained by Soxhlet extraction and their chemical composition was analyzed by GC-MS. The chromatograms presented in Figure 2 reveal that the three different varieties exhibit similarities in peak shapes and positions, particularly among the major components. Mass spectrometric analysis identified a total of 80 different compounds across the samples (Table 2). Specifically, 41, 47 and 43 compounds were detected in Gojeb, Kolesha, Mareko Fana varieties, respectively. Among these compounds, 16 were common to all three varieties. Moreover, some of the compounds detected were shared by two of the three varieties, while others were only detected in one of them. For example, m-xylene and p-xylene (6.68-16.82%), capsaicin (4.22-11.25%), dihydrocapsaicin (2.12-8.24%), vitamin E (5.32-14.72%), and (24R)-stigmast-5 -en-3beta-ol (2.19-6.20%) were among the main components common to all the three varieties. On other hand, 1-ethynylcyclopentanol (18.39%), and urs-12-en-3-ol, (3beta)- (6.12%) were the most abundant components only in the Mareko Fana, while 9-octadecenoic acid (Z)- was highly abundant in Gojeb (34.18%) and Kolesha (57.84%) varieties only (Table 2). The results also showed that chemical groups such as ketones, esters, alcohols, terpenoids, and alkaloids were detected in large numbers.

Figure 2.

Chromatograms of n-hexane extracts of C. annuum varieties: Gojeb (a), Kolesha (b), and Mareko Fana (c).

Table 2.

Detected Compounds in n-Hexane Extracts of C. annuum Varieties.

S. No	Name of Compounds	Formula	Peak area (%)
S. No	Name of Compounds	Formula	Gojeb	Kolesha	Mareko Fana
1	m-xylene and p-xylene	C₈H₁₀	7.88	6.68	16.82
2	cis/trans-5-Phenyl-2-tetrahydro furyl methyl 2'-pyridyl sulfide	C₁₆H₁₇NOS	0.11	0.1	0.37
3	3-(Methoxy amino)-4-methyl-1- phenyl-pentan-1-one	C₁₃H₁₉NO₂	-	0.18	0.38
4	Benzene,(2-nitro-2-propenyl)-	C₉H₉NO₂	-	0.07	-
5	(+-)-trans-p-Menthan-1,8-dien-5- acetate	C₁₂H₁₈O₂	0.12	0.07	-
6	1-Butoxyoctane	C₁₂H₂₆O	0.16	-	0.34
7	Bicyclo [4.1.0] hept-3-ene-2-thiol, 3,7,7-trimethyl-, [1S-(1alpha,2alpha, 6alpha)]-	C₁₀H₁₆S	0.23	0.20	-
8	S-[3-(4-Methylphenyl)-1-methylthio- 3-oxopropenyl]-S, S-dimethylsulfinylinium perchlorate	C₁₃H₁₇Cl O₅S₂	-	-	0.35
9	25,8-Trimethylnonane	C₁₂H₂₆	-	0.45	-
10	1,3-Dimethyl-3-propylazetidine-2,4-dione	C₈H₁₃NO₂	0.13	-	-
11	2,2-dimethyl propyl bis(fluoranyl)-(4-methyl phenyl) methane sulfonate	C₁₃H₁₈F₂O₃S	0.07	0.06	-
12	Phenol, 2-(1-phenylethyl)-	C₁₄H₁₄O	0.15	0.13	1.19
13	(4E)-2-hydroxy-5,9-dimethyl-3-deca-4,8-dienone	C₁₂H₂₀O₂	-	0.06
14	trans-1,5-Dimethyl-1,5-diphosphacyclooctane- 1-oxide	C₈H₁₈OP₂	-	-	0.51
15	trans-beta-Ionone	C₁₃H₂₀O	-	0.31
16	2(4H)-Benzofuranone,5,6,7,7a-tetrahydro- 4,4,7a-trimethyl-, (R)-	C₁₁H₁₆O₂	1.33	1.04	1.87
17	1,2,2-Trimethyl-1-propyl phenylchorophosphonothioate	C₁₂H₁₈ClOPS	-	0.18	-
18	(2R)-2-(2-iodanylethyl) oxirane	C₄H₇IO	-	-	0.12
19	1-(benzyl amino)-3,3-dimethyl-butan-2-ol	C₁₃H₂₁NO	-	0.13	-
20	6-Hydroxy-4,4,7a-trimethyl-5,6,7,7a-tetrahydrobenzofuran- 2(4H)-one	C₁₁H₁₆ O₃	1.29	0.75	2.07
21	5-Anilino-2,3-dimethylhexan-3-ol	C₁₄H₂₃NO	-	-	0.15
22	2-Heptanone, 6-methyl-	C₈H₁₆O	0.32	0.22	0.45
23	3-(4-tert-butylphenyl) furan-2,5-dione	C₁₄H₁₄O₃	0.69	0.43	1.05
24	Cyclohexene, 1-butyl-4-methylene-	C₁₁H₁₈	-	-	0.64
25	8-methylcapric acid methyl ester	C₁₂H₂₄O₂	0.29
26	(Z)-2-((2-Methylthio) styrylthio) aniline	C₁₅H₁₅NS₂	-	-	0.34
27	Dibutyl phthalate	C₁₆H₂₂O₄	0.30	0.07	-
28	Decanoic acid, 2,8-dimethyl-, methyl ester	C₁₃H₂₆O₂	0.20	-	0.74
29	Pentanoic acid,4,4-dimethyl-5-oxo-	C₇H₁₂O₃	-	-	0.07
30	1,2-Tetradecanediol	C₁₄H₃₀O₂	0.16	-	-
31	(E) and (Z)-2-Octylidenecyclopropanecarbaldehyde	C₁₂H₂₀O	0.16	-	0.48
32	2-Fluoro-11-tetradecyn-1-ol	C₁₄H₂₅FO	-	0.17	-
33	Phytol	C₂₀H₄₀O	0.91	0.81	2.66
34	Octadecanoic acid, 8-methyl-, methyl ester	C₂₀H₄₀O₂	0.24		0.49
35	9-Octadecenoic acid (Z)-	C₁₈H₃₄O₂	34.18	57.84	-
36	1-Ethynylcyclopentanol	C₇H₁₀O	-	-	18.39
37	6-oxabicyclo [3.1.0] hexane-2,4-diol	C₅H₈O₃	-	0.41	0.16
38	3-(Hydroxymethyl)pentane nitrile	C₆H₁₁NO	-	-	0.12
39	2,2,5,5-Tetradeuteriomethoxycyclohexane	C₇H₁₀D₄O	-	0.20	-
40	2-(4-Pentenyl)-4-methyl-2,5-dihydrofuran	C₉H₁₄O	-	0.12
41	3-Hydroxyoct-4-en-2-one	C₈H₁₄O₂	0.31	-	-
42	Methaneselenoic acid, O-(1,1-diethylpropyl) ester	C₈H₁₆OSe	-	0.19	-
43	(E,5S)-3,5-dimethylhept-3-en-1-yne	C₉H₁₄	0.21	0.15	0.35
44	(2'S)-2-[(2R,5S)-5'-Hydroxymethyl-tetrahydrofuran-2'- yl])-propan-1-ol	C₈H₁₆O₃	-	-	0.23
45	2-tert-Butoxytetrahydrofuran	C₈H₁₆O₂	0.13	0.11	-
46	Nordhydrocapsaicin	C₁₇H₂₇NO₃	2.61	-	-
47	n-Cetyl thiocyanate	C₁₇H₃₃NS	0.63	0.18	-
48	7-ethyl-7-borabicyclo [3.3.1] nonane	C₁₀H₁₉B	-	-	0.25
49	i-Propyl-3-(phenylamino)-2-(phenylseleno)-3(phenyl) propanoate	C₂₄H₂₅NO₂Se	-	-	0.47
50	(E)-2-amino-8-octadecene-1,3,4-triol	C₁₈H₃₇NO₃	0.27	-	-
51	(E,4S,6S,8S)-2,4,6,8-tetramethyl-2-undecenoic acid	C₁₅H₂₈O₂	-	0.17	-
52	Capsaicin	C₁₈H₂₇NO₃	11.25	4.22	5.51
53	Dihydrocapsaicin	C₁₈H₂₉NO₃	8.24	2.12	2.39
54	(+-) 2-exo-Hydroxycineole	C₁₀H₁₈O₂	-	0.11
55	4-Hydroxy-3-(4-fluorobenzylamino) cyclobutenone	C₁₁H₁₀FNO₂	-	-	0.07
56	9,12-Octadecadienoicacid(Z,Z)-,2-hydroxy-1- (hydroxymethyl)ethyl ester	C₂₁H₃₈O₄	-	4.60	-
57	3a,4,5,6,7,7a-Hexahydro-3H-indene-3a-carbaldehyde	C₁₀H₁₄O	0.13	0.29	0.54
58	1-(2,3-dimethylbut-3-en-2-yl)-4-methoxy-benzene	C₁₃H₁₈O	0.79	-	-
59	2,4,7-Metheno-3H-cyclopenta[a]pentalen-3-one, decahydro-6a-d-	C₁₂H₁₃DO	-	-	0.82
60	Supraene	C₃₀H₅₀	2.41	1.27	2.10
61	7-Methoxy-1,2,3,4-tetrahydro-6-isoquinolinol	C₁₀H₁₃NO₂	1.48	1.31	2.10
62	Tritriacontane	C₃₃H₆₈	-	0.55	-
63	Hexadecane, 2,6,10,14-tetramethyl-	C₂₀H₄₂	0.76	-	-
64	3-iodanyl-4-(5-nitrothiophen-2-yl) butan-2-one	C₈H₈INO₃S	-	-	0.84
65	3-Ethyl-1-naphthol	C₁₂H₁₂O	0.15	-	0.40
66	2,4-Dimethylnaphthalen-1-ol	C₁₂H₁₂O	-	0.34	-
67	4-Hydroxy-3-methyleneoctan-2-one	C₉H₁₆O₂	-	-	0.41
68	tert-butyl-dimethyl-[(2R,3R)-2-vinyltetrahydropyran-3-yl] oxy-silane	C₁₃H₂₆O₂Si	0.21	-	-
69	2H-Benzimidazol-2-one, 5-acetyl-1,3-dihydro-6-methyl-	C₁₀H₁₀N₂O₂	1.04	0.68	-
70	2,4-Octadienoicacid,6-methyl-,1,2,6,7,8,8a-hexahydro-7- hydroxy-1,8a-dimethyl-6-oxo-2-naphthalenyl ester,[1R- [1alpha,2alpha(2E,4E,6S*),7beta,8a alpha]]-	C₂₁H₂₈O₄	-	-	1.36
71	1-(hydroxymethyl)-7-methoxy-1,2,3,4-tetrahydroisoquinolin- 8-ol	C₁₁H₁₅NO₃	-	-	0.65
72	1,4-Dimethoxy-2,5-bis(trideuteriomethyl)benzene	C₁₀H₈D₆O₂	-	0.14	-
73	Vitamin E	C₂₉H₅₀O₂	8.74	5.32	14.72
74	Campesterol	C₂₈H₄₈O	1.72	0.97	-
75	(2R)-2-(2-iodanylethyl) oxirane	C₄H₇IO	-	0.14	-
76	(24R)-Stigmast-5-en-3beta-ol	C₂₉H₅₀O	3.84	2.19	6.20
77	Olean-12-en-3-ol, (3beta)-	C₃₀H₅₀O	2.32	1.22	3.41
78	Urs-12-en-3-ol, (3beta)-	C₃₀H₅₀O	-	-	6.12
79	alpha-Amyrin	C₃₀H₅₀O	3.62	2.08	-
80	(2R,3S)-2-[(Z)-oct-2-enyl]-3-undecyl-oxirane	C₂₁H₄₀O	-	0.96	-

DPPH Radical Scavenging Activity

The antioxidant activity of C. annuum cultivars was assessed using the DPPH radical scavenging assay, and the EC₅₀ values are presented in Table 3. The results shows that the radical scavenging activity varied significantly with extraction solvents and pepper varieties. Methanol extracts of all peppers exhibited the highest free radical scavenging activity (EC₅₀, 0.40 - 1.4 mg/mL) than chloroform (EC₅₀, 0.82 - 1.52 mg/mL) and n-hexane (EC₅₀, 2.66 - 2.73 mg/mL) extracts. The DPPH radical scavenging activity was in order of: Gojeb > Mareko Fana > Kolesha for both methanol and chloroform extracts. The EC₅₀ values of n-hexane extracts were not significantly different (P > 0.05) across the pepper varieties studied.

Table 3.

DPPH Radical Scavenging Activity.

Sample	EC₅₀ (mg/mL)
Sample	Methanol	Chloroform	n-hexane*
Gojeb	0.40 ± 0.04	0.82 ± 0.02	2.70 ± 0.22
Kolesha	1.4 ± 0.02	1.52 ± 0.04	2.73 ± 0.57
Mareko Fana	0.92 ± 0.01	1.36 ± 0.06	2.66 ± 0.06
Ascorbic Acid	0.067 ± 0.02

*, Values followed by an asterisk are not significantly different (P > 0.05)

Discussion

As the consumption of fruits and vegetables are recommended for prevention against chronic diseases caused by oxidative cell damage, studying the antioxidant activities of fruits and vegetables has become interesting. Mostly used as a spice, pepper is a widely consumed fruit in many countries. Their antioxidant activity, as well as chemical contents vary with species (varieties), ecotype (environmental conditions), and extraction solvents.^17,18 C. annuum is grown widely in Ethiopia with genetic variation observed in capsicum germplasms across the region.²² With focus on antioxidant activity, phenolics content (TPC and TFC), GC-MS analysis of lipophilic extracts, and free radical scavenging activities of fully ripened fruits of three common varieties of C. annuum grown in Omo Nada, Jimma (Southern Ethiopia) were investigated.

Methanol and chloroform were used in a two-step process to extract phenols and flavonoids. Initially, shaking for 30 minat room temperature improved the contact between the solvents and the samples. This was followed by a 24-h soaking period in the dark to ensure stable extraction. The re-extraction step also contributed to the high extraction yields observed (Table 1). The extraction yield for methanol, ranging from 26.77% to 40.97%, was notably higher than that for chloroform, which ranged from 5.33% to 7.23% for all C. annuum varieties. This difference could be attributed to the higher amount of polar molecules in methanol extracts. Previous study associated the extraction of solids in polar solvents with the significant amount of highly soluble sugars in C. annuum fruits.²⁰ It is also worth noting that consumers tend to prefer peppers rich in fiber. Results showed high percentages of extractable solids particularly in Mareko Fana (40.97 ± 0.28%) and Gojeb (35.97 ± 0.25%) varieties. Dietary soluble fiber from fruits has shown significant prebiotic effects²⁵ and is inversely associated with the risk of chronic diseases such as cancer, type 2 diabetes, cardiovascular disease, and mortality.²⁶

The TPC of the studied C. annuum cultivars followed the same trend for both methanol and chloroform in the order: Kolesha < Mareko Fana < Gojeb cultivars (Table 1), with the variation in both extraction solvents and pepper variety are shown. The higher TPC in methanol extracts compared to that observed in chloroform extracts could be due to the higher polarity of methanol. Previous studies also highlight TPC variability with solvents and pepper species. Red pepper methanol extract had a TPC (22.76 mg GAE/g) comparable to that of methanol extract of Gojeb variety (22.98 ± 0.08 mg GAE/g), while ethanol extract had a lower TPC (17.1 mg GAE/g).¹⁶ Another study found a higher TPC (25.538 mg GAE/g) in C. annuum pericarp.¹⁷ The TPC (1.10-6.82 mg GAE/g DW) obtained in Tunisian cultivars¹⁰ was lower compared to the TPC of C. annuum varieties currently investigated. A diet rich in polyphenols protects against cardiovascular disease, cancer, diabetes and obesity, while ingredients higher in phenolic compounds extend the shelf life of foods.^27,28

Flavonoids are another polyphenol class that exhibit anticancer, antioxidant, anti-inflammatory, antiviral, neuroprotective, and cardio-protective effects.²⁹ The TFC of the C. annuum varieties in methanol extracts showed similar trend to that of TPC (Table 1). For chloroform extracts, however, the TFCs of Mareko Fana and Kolesha did not differ significantly but they were slightly higher than that observed in Gojeb cultivar (Table 1). The TFC values obtained here were higher than those reported in another study on C. annuum fruits (0.204 to 0.962 mg QE/g).³⁰

Apart from the phenolics and flavonoids typically extracted with polar solvents, peppers also contain many compounds whose bioactivity is linked to their lipophilicity.²⁰ GC-MS is most commonly used for analysis of volatile and lipophilic components of plant extracts. This analysis highlights both the diversity and overlap in the lipophilic chemical profiles of these C. annuum varieties. For instance, m-xylene and p-xylene, capsaicin, dihydrocapsaicin, Vitamin E, and (24R)-Stigmast-5-en-3beta-ol were among the major components detected. Meanwhile, 9-octadecenoic acid (Z)- was highly abundant in the Gojeb and Kolesha varieties, while 1-ethynylcyclopentanol, and urs-12-en-3-ol, (3beta)- were most prevalent in Mareko Fana (Table 2). In a previous study, the n-hexane fraction of C. annuum cultivars showed a high content in vitamin E, β-caryophyllene, neophytadiene, α-humulene, certain fatty acids, and methyl esters such as palmitic acid and linoleic acid.³⁰ Another study showed that alcohols and hydrocarbons were abundant, with 1-Decanol, 2-octyl- and docosanoic acid, docosyl ester as the major components.¹⁹ The variation in the chemical contents of C. annuum could be due to the cultivars types and environmental conditions.

Compounds detected in the C. annuum varieties have been reported to possess valuable health benefits. Capsaicinoids, natural alkaloids in chili peppers, provide a strong flavor and health benefits, including anti-cancer and anti-inflammatory properties. The most common are capsaicin and dihydrocapsaicin, with capsaicin being more abundant in many capsicum species.¹¹ Among these, capsaicin offers numerous health benefits, including pain relief, anticancer properties, blood sugar regulation, treatment for hypertension and heart disease, antimicrobial and and anti-obesity effects.³¹

Tetrahydroisoquinoline alkaloids, including 7-methoxy-1,2,3,4-tetrahydro-6-isoquinolinol and 1-(hydroxymethyl)-7-methoxy-1,2,3,4-tetrahydroisoquinolin-8-ol, were detected in this study (Table 2) probably for the first time. Isoquinoline alkaloids have antimicrobial, antibacterial, and antitumor activities.³² Vitamin E is another important compound with anti-inflammatory, antimicrobial, radical scavenging, and antispasmodic properties.³³

Triterpenoids, particularly pentacyclic triterpenoids, including α-amyrin, olean-12-en-3-ol, and urs-12-en-3-ol, are present in all cultivars. These compounds offer health benefits such as antioxidants, anti-inflammatory, anti-cancer, and anti-microbial properties.³⁴ Cultivars of C. annuum also contain sterols, including (24R)-Stigmast-5-en-3beta-ol and campesterol (Table 2), which can reduce cholesterol and inflammation while bolstering the immune system.²⁹ The Mareko Fana cultivar also contains phytol, an acyclic diterpene alcohol, with antioxidant, anti-inflammatory, anti-schistosomiasis, and antiallergic properties.³⁵

Bioactive actinidioides including 2(4H)-Benzofuranone,5,6,7,7a-tetrahydro-4,4,7a-trimethyl-, (R)-, (dihydroactinidioide) and 6-hydrroxy-4,4,7a-trimethyl-5,6,7,7a-tetrahydrobenzofuran-2 (4H) - one (loliolide) were also detected (Table 21). Loliolide, a carotenoid metabolite, exhibit analgesic, antidiabetic, antibacterial, and antifungal activities and was also detected in oleoresin of C. annuum fruits.³⁶ Loliolide has been related to the antioxidant activity and acetylcholinesterase inhibition.³⁷

Plants with a high concentration of fatty acids and fatty acid esters have been found to have remarkable antioxidant and antimicrobial activities.³⁸ The chemical analysis of C. annuum n-hexane extracts in this study revealed that unsaturated fatty acid, 9-Octadecenoic acid (Z)- present at higher percentage in Gojeb (34.18%) and Kolesha (57.84%) while 9,12-Octadecadienoic acid (Z,Z)-,2-hydroxy-1-(hydroxymethyl) ethyl ester (4.6%) was among the abundant compounds in Mareko Fana. The presence of these compounds suggests that consuming these pepper fruits could provide various health benefits.

Free radical reaction is the main reason for oxidative damage of living cells and food deterioration. Capacities of C. annuum extracts to capture free radicals were examined using DPPH radical scavenging assay. The chloroform and n-hexane extracts were considerably less effective radical scavengers than the methanol extracts, with the chloroform being more active than the n-hexane extracts for all the three cultivars (Table 3). The results showed variation within pepper varieties for methanol and chloroform extracts only with the lowest EC₅₀ values found for Gojeb varieties. Peppers fruits contain antioxidants including phenolic acids, flavonoids, capsaicinoids, and vitamin E among other compounds. In this study, the EC₅₀ values decreased with increasing TPC or TFC, and a statistical analysis revealed negative correlations between EC₅₀ and both TPC (r = −0.87) and TFC (r = −0.69). This is due to the ability of phenolic compounds to neutralize DPPH free radicals by either transferring electrons or hydrogen atoms.²³ Despite the detection of numerous antioxidant compounds by GC-MS analysis in n-hexane extracts, the low radical scavenging activities observed contrast with previous findings correlating DPPH scavenging with phenolic and flavonoid contents in polar solvents.^16,20 The EC₅₀ values recorded in the current study were relatively higher compared to those reported in previous research (EC₅₀= 131.29 to 217.63 µg/mL).³⁹ This difference may be partially attributed to color variation in C. annuum fruits. Additionally, environmental factors (such as high temperatures and cold climates), farming practices, and genetic factors can contribute to variation in antioxidant activities and phenolic contents of C. annuum.^40–42 A study by Kim et al demonstrated that variations in phenolic acid content of C. annuum peppers were influenced by both the year and variety.⁴¹ Shotorbani et al also reported high free radical scavenging effects of sweet bell peppers (C. annuum) at high temperature.⁴² Although such study has not been reported for peppers grown in Ethiopia, genetic variability has been reported since its introduction in seventeenth century.²² Generally consumption of peppers with high antioxidant activities and phenolic contents have important effects on prevention against against cardiovascular disease, cancer, diabetes and obesity.⁴³

Limitation of the Study

Although significant phenolic contents (TPC and TFC) and DPPH radical scavenging activity were recorded in methanol extracts, either isolation studies or GC-MS identification of chemical profiles were not carried out. Further investigation involving chemical isolation and additional in vivo and in vitro biological activities analysis could be conducted to get information about more health benefits of the C. annuum varieties investigated.

Conclusion

This study provides the TPC, TFC, lipophilic chemical contents and antioxidant activities of Capsicum annuum L. varieties grown in Omo Nada, Jimma, Ethiopia. The results indicate that the Gojeb variety exhibits the highest total phenolic and flavonoid content, particularly in methanol extracts, which also demonstrated the highest antioxidant activity with the lowest EC₅₀ values in the DPPH assay. Despite their low antioxidant activities, GC-MS analysis revealed a number of compounds with vital health benefits in lipophilic extracts. In general, 80 different chemical compounds were detected with 16 being common to all the varieties. Compounds such as m-xylene and p-xylene, capsaicin, dihydrocapsaicin, Vitamin E, and (24R)-Stigmast-5-en-3beta-ol were abundant across all varieties. Furthermore, ketones, esters, alcohols and terpenoids were predominant chemical groups. This study underscores the importance of these local pepper varieties as potential sources of natural antioxidants in food or pharmaceutical industries.

Footnotes

Acknowledgments

The authors would like to thank Jimma University, Jimma, Ethiopia, for providing materials, and facilities required to carry out this work.

Author Contributions

Zemani Kebu: Conceptualization, Formal Analysis, Investigation, Methodology, Resource, Validation, Writing - Original Draft Preparation, Data Curation.

Abera Gure: Conceptualization, Methodology, Supervision, Writing - Review & Editing.

Guyo Jilo Molole: Conceptualization, Formal Analysis, Investigation, Methodology, Validation, Visualization, Writing - Original Draft Preparation, Writing - Review & Editing.

Consent to Participate

Not applicable

Consent for Publication

Not applicable

Data Availability

All datasets obtained during the current study are included in the manuscript.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical Considerations

Not applicable

Funding

This study was funded by Jimma University via M.Sc. Students’ Research Fund.

ORCID iD

Guyo Jilo Molole

Statements and Declarations

Not applicable

References

Jomova

Raptova

Alomar

, et al. Reactive oxygen species, toxicity, oxidative stress, and antioxidants: chronic diseases and aging. Arch Toxicol. 2023;97(10):2499‐2574. doi:https://doi.org/10.1007/s00204-023-03562-9

Singh

Kukreti

Saso

Kukreti

. Oxidative stress: a key modulator in neurodegenerative diseases. Molecules. 2019;24(8):1583. doi:https://doi.org/10.3390/molecules24081583

Niemann

Rohrbach

Miller

Newby

Fuster

Kovacic

. Oxidative stress and cardiovascular risk: obesity, diabetes, smoking, and pollution: part 3 of a 3-part series. J Am Coll Cardiol. 2017;70(2):230‐251. doi:https://doi.org/10.1016/j.jacc.2017.05.043

Zhang

Gan

, et al. Antioxidant phytochemicals for the prevention and treatment of chronic diseases. Molecules. 2015;20(12):21138‐21156. doi:https://doi.org/10.3390/molecules201219753

Arias

Feijoo

Moreira

. Exploring the potential of antioxidants from fruits and vegetables and strategies for their recovery. Innovat Food Sci Emerging Technol. 2022;77(2022):102974. doi:https://doi.org/10.1016/j.ifset.2022.102974

Jideani

AIO

Silungwe

Takalani

Omolola

Udeh

Anyasi

. Antioxidant-rich natural fruit and vegetable products and human health. Int J Food Properties. 2021;24(1):41‐67. doi:https://doi.org/10.1080/10942912.2020.1866597

Kumar

Nirmal

Kumar

, et al. Major phytochemicals: recent advances in health benefits and extraction method. Molecules. 2023;28(2):887. doi:https://doi.org/10.3390/molecules28020887

Jiménez-Viveros

Núñez-Palenius

Fierros-Romero

Valiente-Banuet

. Modification of light characteristics affect the phytochemical profile of peppers. Horticulturae. 2023;9(1):72. doi:https://doi.org/10.3390/horticulturae9010072

Abbas

Zhou

O’Neill Rothenberg

Alam

Wang

. Aroma components in horticultural crops: chemical diversity and usage of metabolic engineering for industrial applications. Plants (Basel). 2023;12(9):1748. doi:https://doi.org/10.3390/plants12091748

10.

Lahbib

Dabbou

Bok

Pandino

Lombardo

Gazzah

. Variation of biochemical and antioxidant activity with respect to the part of Capsicum annuum fruit from Tunisian autochthonous cultivars. Ind Crops Prod. 2017;104(2017):164‐170. doi:https://doi.org/10.1016/j.indcrop.2017.04.037

11.

Mendes

N de S

Gonçalves

ÉCB de A.

The role of bioactive components found in peppers. Trends Food Sci Technol. 2020;99(2020):229‐243. doi:https://doi.org/10.1016/j.tifs.2020.02.032

12.

García-Gaytán

Gómez-Merino

Trejo-Téllez

Baca-Castillo

García-Morales

, The chilhuacle chili (Capsicum annuum L.) in Mexico: description of the variety, its cultivation, and uses. Int J Agronomy. 2017;2017(104):e5641680. doi:https://doi.org/10.1155/2017/5641680

13.

Gobie

. A seminar review on red pepper (Capsicum) production and marketing in Ethiopia. Cogent Food Agric. 2019;5(1):1647593. doi:https://doi.org/10.1080/23311932.2019.1647593

14.

Hernández-Ortega

Ortiz-Moreno

Hernández-Navarro

, et al. Antioxidant, antinociceptive, and anti-inflammatory effects of carotenoids extracted from dried pepper (Capsicum annuum L.). BioMed Res Int. 2012;2012:524019. doi:https://doi.org/10.1155/2012/524019

15.

Munteanu

Apetrei

. Analytical methods used in determining antioxidant activity: a review. Int J Mol Sci. 2021;22(7):3380. doi:https://doi.org/10.3390/ijms22073380

16.

Salamatullah

M, Hayat

Mabood Husain

, et al. Effects of different solvents extractions on total polyphenol content, HPLC analysis, antioxidant capacity, and antimicrobial properties of peppers (red, yellow, and green (Capsicum annum L.)). Evid Based Complement Alternat Med. 2022;2022:7372101. doi:https://doi.org/10.1155/2022/7372101

17.

Zamljen

Jakopič

Hudina

et al.

Influence of intra and inter species variation in chilies (Capsicum spp.) on metabolite composition of three fruit segments. Sci Rep. 2021;11:4932. doi:https://doi.org/10.1038/s41598-021-84458-5

18.

Bae

Jayaprakasha

Jifon

Patil

. Variation of antioxidant activity and the levels of bioactive compounds in lipophilic and hydrophilic extracts from hot pepper (Capsicum spp.) cultivars. Food Chem. 2012;134(4):1912‐1918. doi:https://doi.org/10.1016/j.foodchem.2012.03.108

19.

Ahmad

Alqathama

Aldholmi

, et al. Gas chromatography-mass spectrometry (GC-MS) metabolites profiling and biological activities of various Capsicum annum cultivars. Plants (Basel). 2022;11(8):1022. doi:https://doi.org/10.3390/plants11081022

20.

Chilczuk

Marciniak

Kontek

Materska

. Diversity of the chemical profile and biological activity of Capsicum annuum L. extracts in relation to their lipophilicity. Molecules. 2021;26(17):5215. doi:https://doi.org/10.3390/molecules26175215

21.

Bekele

. Opportunities and potential of hot pepper (Capsicum annuum L.) production in Ethiopia. Eur J Agric Forest Res. 2022;10:14-20(2). https://doi.org/10.37745/ejafr.2013/vol9n21420

22.

Solomon

Han

Lee

Jang

Kang

. Genetic diversity and population structure of Ethiopian Capsicum germplasms. PLOS ONE. 2019;14(5):e0216886. doi:https://doi.org/10.1371/journal.pone.0216886

23.

Molole

Gure

Abdissa

. Determination of total phenolic content and antioxidant activity of Commiphora mollis (Oliv.) Engl. resin. BMC Chem. 2022;16(1):48. doi:https://doi.org/10.1186/s13065-022-00841-x

24.

Al-Owaisi

Al-Hadiwi

Khan

. GC-MS analysis, determination of total phenolics, flavonoid content and free radical scavenging activities of various crude extracts of Moringa peregrina (Forssk.) Fiori leaves. Asian Pac J Trop Biomed. 2014;4(12):964‐970. doi:https://doi.org/10.12980/APJTB.4.201414B295

25.

, et al. Physicochemical characteristics and biological activities of soluble dietary fibers isolated from the leaves of different quinoa cultivars. Food Res Int. 2023;163:112166. doi:https://doi.org/10.1016/j.foodres.2022.112166

26.

Partula

Deschasaux

Druesne-Pecollo

, et al. Associations between consumption of dietary fibers and the risk of cardiovascular diseases, cancers, type 2 diabetes, and mortality in the prospective NutriNet-Santé cohort. Am J Clin Nutr. 2020;112(1):195‐207. doi:https://doi.org/10.1093/ajcn/nqaa063

27.

Bié

Sepodes

Fernandes

PCB

Ribeiro

MHL

. Polyphenols in health and disease: gut microbiota, bioaccessibility, and bioavailability. Compounds. 2023;3(1):40‐72. doi:https://doi.org/10.3390/compounds3010005

28.

Nardini

. Phenolic compounds in food: characterization and health benefits. Molecules. 2022;27(3):783. doi:https://doi.org/10.3390/molecules27030783

29.

Vezza

Canet

de Marañón

Bañuls

Rocha

Víctor

. Phytosterols: nutritional health players in the management of obesity and its related disorders. Antioxidants. 2020;9(12):1266. doi:https://doi.org/10.3390/antiox9121266

30.

Tundis

. Antioxidant and hypoglycaemic activities and their relationship to phytochemicals in Capsicum annuum cultivars during fruit development.Food Sci Technol. 2013; 53(1):370-377. doi:10.1016/j.lwt.2013.02.013

31.

Azlan

Sultana

Huei

Razman

. Antioxidant, anti-obesity, nutritional and other beneficial effects of different chili pepper: a review. Molecules. 2022;27(3):898. doi:https://doi.org/10.3390/molecules27030898

32.

Dembitsky

Gloriozova

Poroikov

. Naturally occurring plant isoquinoline N-oxide alkaloids: their pharmacological and SAR activities. Phytomedicine. 2015;22(1):183‐202. doi:https://doi.org/10.1016/j.phymed.2014.11.002

33.

Mujeeb

Bajpai

Pathak

. Phytochemical evaluation, antimicrobial activity, and determination of bioactive components from leaves of aegle marmelos. BioMed Res Int. 2014;2014:1-11. doi:https://doi.org/10.1155/2014/497606

34.

Ghante

Jamkhande

. Role of pentacyclic triterpenoids in chemoprevention and anticancer treatment: an overview on targets and underling mechanisms. J Pharmacopuncture. 2019;22(2):55‐67. doi:https://doi.org/10.3831/KPI.201.22.007

35.

de Moraes

de Oliveira

Costa

, et al. Phytol, a diterpene alcohol from chlorophyll, as a drug against neglected tropical disease schistosomiasis mansoni. PLoS Negl Trop Dis. 2014;8(1):e2617. doi:https://doi.org/10.1371/journal.pntd.0002617

36.

Sharma

Fuloria

Alam

, et al. Chemical composition and antimicrobial activity of oleoresin of Capsicum annuum fruits. Mindanao J Sci Technol. 2021;19(1):29‐43.

37.

Dawra

El Rayess

El Beyrouthy

, et al. Biological activities and chemical characterization of the Lebanese endemic plant Origanum ehrenbergii Boiss. Flavour Fragrance J. 2021;36(3):339‐351. doi:https://doi.org/10.1002/ffj.3646

38.

Gıdık

. Antioxidant, antimicrobial activities and fatty acid compositions of wild berberis spp. By different techniques combined with chemometrics (PCA and HCA). Molecules. 2021;26(24):7448. doi:https://doi.org/10.3390/molecules26247448

39.

Choi

Kim

Han

. Physicochemical properties and antioxidant activity of colored peppers (Capsicum annuum L.). Food Sci Biotechnol. 2023;32(2):209. doi:https://doi.org/10.1007/s10068-022-01177-x

40.

Rácz

Czégény

Kutyáncsánin

Nagy

Hideg

Csepregi

. Fight against cold: photosynthetic and antioxidant responses of different bell pepper cultivars (Capsicum annuum L.) to cold stress. Biologia Futura. 2023;74(3):327‐335. doi:https://doi.org/10.1007/s42977-023-00182-3

41.

Kim

Lee

, et al. Influence of genetic and environmental factors on the contents of carotenoids and phenolic acids in red pepper fruits (Capsicum annuum L.). Appl Biol Chem. 2021;64(1):85. doi:https://doi.org/10.1186/s13765-021-00657-8

42.

Shotorbani

Jamei

Heidari

. Antioxidant activities of two sweet pepper Capsicum annuum L. varieties phenolic extracts and the effects of thermal treatment. Avicenna J Phytomed. 2013;3(1):25.

43.

Boswell

Stoll

Carr

Nagel

Vitha

Mabbott

. An advanced, interactive, high-performance liquid chromatography simulator and instructor resources. J Chem Educ. 2013;90(2):198‐202. doi:https://doi.org/10.1021/ed300117b