Pheophytin a and Chlorophyll a Identified from Environmentally Friendly Cultivation of Green Pepper Enhance Interleukin-2 and Interferon-γ in Peyer’s Patches ex Vivo

Abstract

The oral consumption of capsicum has been reported to increase interleukin (IL)-2 and interferon (IFN)-γ production in Peyer’s patches (PP); however, the active components responsible for these effects have not been completely identified. The beneficial biological effects of green peppers cultivated under environmentally friendly farming conditions (ECP), without the use of chemical pesticides, have rarely been compared with those of green peppers cultivated under conventional farming conditions (CCP). Oral administration of ECP extract significantly induced the production of IL-2 and IFN-γ in concanavalin A-treated cells from PP ex vivo; their levels were much higher than those in the CCP extract-treated group. A comparative analysis of the HPLC profiles indicated a 1.7-fold increase of a peak, named EF-1, at 415 nm in the ECP extract. The major component of EF-1 was identified as pheophytin a, which is a chlorophyll a molecule lacking a central Mg²⁺ ion, as determined from NMR data. Intake of pheophytin a and chlorophyll a significantly increased IL-2 and IFN-γ production, and the percentage of IL-2- and IFN-γ-producing CD4+ T-cells in PP. Taken together, our data suggest that ECPs produce a higher content of pheophytin a than CCPs, and pheophytin a and chlorophyll a are immune-modulating components in green vegetables.

Capsicum is used worldwide as a spice and food vegetable, and it has been used as medicine for various chronic diseases.^1,2) It was reported that the red pepper (the fruit of capsicum) extract contains immune modulators. The oral intake of capsaicin enhances interleukin (IL)-2 and interferon (IFN)-γ production upon Concanavalin A (Con A) stimulation in cells isolated from lymphoid tissue in the intestine.³⁾ Carotenoids such as β-carotene and β-cryptoxanthin were suggested as one of the active components modulating the expression of T helper 1 (Th1) cytokines such as IL-2 and IFN-γ ex vivo.⁴⁾ However, the active components of pepper extract involved in such immune-modulating effects have not been completely identified.

Recent studies have reported that modifying cultivation techniques by using natural biostimulants can affect the nutritional quality of peppers and increase the activities of bioactive components.⁵⁾ The benefits from environmentally friendly farming, which is defined as cultivation by using reduced amounts or without using synthetic chemical pesticides, have been rarely examined.

Peyer’s patches (PP) are macroscopic lymphoid tissues that are distributed throughout the wall of the small and large intestines.⁶⁾ These tissues consist of collections of T-cell areas and large B-cell follicles, which are separated from the intestinal lumen by a single layer of epithelium-containing M-cells, and are infiltrated by a large number of T-cells, B-cells, macrophages, and dendritic cells (DCs). When pathogenic antigens enter through the M-cells, the various immune cells work in unison to promptly antagonize the continuously invading antigens⁷⁾ and also to develop specialized immune responses.⁸⁾ Antigen-presenting cells, including macrophages and DCs that capture antigens, move to the T-cell areas and/or B-cell follicles, where they interact with naïve T-cells, leading to differentiation of the naïve T-cells into CD4+ Th1 cells or Th2 cells.⁹⁾ The differentiated and activated lymphocytes migrate in the blood stream, ultimately accumulating in the mucosa where they protect against exogenous antigens. This process largely depends on the nature of the applied antigens, and is regulated by the production of cytokines: soluble antigens provoke the Ag-specific Th2 immune responses to produce Th2 cytokines, leading to antibody secretion, whereas intracellular pathogens induce the Th1 response, resulting in IL-2 and IFN-γ production, in addition to cell-mediated immunity.¹⁰⁾ Th1-derived IL-2, together with the Th2 cytokine, enhances immunoglobulin A (IgA) production.¹¹⁾ IFN-γ has direct antimicrobial and antitumor activities; promotes the cytotoxic activities of CD8+ T-cells and natural killer cells¹²⁾; and exhibits further regulatory effects on B-cell functions, such as class switching and immunoglobulin production.

Here, we show that green peppers cultivated under environmentally friendly farming conditions exhibit an increased amount of pheophytin a, a chlorophyll a lacking a central Mg²⁺ ion, compared to those cultivated under conventional farming conditions. Additionally, oral intake of pheophytin a and chlorophyll a enhanced IL-2 and IFN-γ production in Con A-activated CD4+ T-cells in PP ex vivo. Pheophytin a and chlorophyll a might be active components of green peppers that modulate immunity.

MATERIALS AND METHODS

Cultivation of Green Peppers under Environmentally Friendly Conditions

Environmentally friendly cultivated peppers (ECPs) and conventionally cultivated peppers (CCPs) were separately cultivated according to the manual directed by Chonnam National University (Gwangju, Korea). The fruits of green pepper (Capsicum annuum var. CHEONGYANG) were collected separately from each greenhouse (conventional and environmentally friendly) located in Nampung and Naju (Jeonnam Province, Korea), respectively. There was no significant difference between the 2 areas with regard to the weather, and the same culture conditions were maintained for both. For CCPs, several chemicals, including Ridomil and Deomani (Dongbu Hitek Co., Seoul, Republic of Korea), Ongdalsam (Syngenta Co., Seoul, Republic of Korea), and Hint (Hankook Samgong Co., Suwon, Republic of Korea), were applied as fungicides. A-pam, Bumelang, Mospilan, and Stonet (Syngenta Co.), Setis and Olgami (Dongbu Hitek Co.), and Conido (Bayer CropScience Co., Republic of Korea) were applied as insecticides. For ECPs, only environmentally friendly agents such as Top (Green Biotech Co., Paju, Republic of Korea), Mai (Daeyu Co., Gyeongsan, Republic of Korea), BAC-1 (Chonnam National University, Gwangju, Republic of Korea), Eco (Shinyoung Agro, Goyang, Republic of Korea), Fic (Bioresource, Bucheon, Republic of Korea), Bugpam (Farmskorea Co., Yeoju, Republic of Korea), DipenseM (Dongbu Hitek Co.), Bestop and Bogum (Kyungnong Co., Seoul, Republic of Korea), and Olcatch (Nambo Co., Gyeongju, Republic of Korea) were used for disease control. These agents have been officially registered or are being considered as biocontrol agents or environmentally friendly fertilizers by the Rural Development Administration, Suwon, Republic of Korea. To eliminate sampling bias and differences associated with the harvest time, the peppers were harvested 6 times between May and July, after they were sown in February. During each sampling, 100 g of fresh peppers was collected and freeze dried, and each sample was extracted with 70% ethanol at 60°C for 8 h. Using rotary evaporation and freeze drying, a dried powder for each sample was obtained; approximately 1.2 g of dried powder was harvested from 100 g of fresh peppers. The dry weights were measured for subsequent experiments.

Identification of Pheophytin a from Environmentally Friendly Cultivated Green Peppers

Each ethanol extract (10 mg/mL) was analyzed using a high-performance liquid chromatography (HPLC) system (Waters 996 PDA HPLC system; Waters Corp., Milford, MA, U.S.A.) equipped with a diode array detector and a 150 mm×4.6 mm Cosmosil 5C18-MS-II column (particle size of 5 µm; Nacalai Tesque, Inc., Kyoto, Japan) at a flow rate of 0.7 mL/min. The solvents constituting the mobile phase were 0.1% trifluoroacetic acid (TFA) in water (solvent A) and 0.1% TFA in methanol (solvent B). The gradient program was as follows: 0–70 min, linear gradient solvent system from 0% B to 100% B; 70–100 min, 100% B. The HPLC chromatograms of the ECP and CCP extracts at 415 nm, 320 nm, 275 nm, and 210 nm were compared. To isolate the EF-1 fraction, the freeze-dried peppers (dry weight, 100 g) were extracted with acetone (2 L), and the residue (2 g) was subjected to silica gel column chromatography (70–230 mesh; Merck & Co., Whitehouse Station, NJ, U.S.A.) and eluted with 9% ethyl acetate in n-hexane. The fraction containing EF-1 was concentrated, and the residue (290 mg) was purified by normal-phase HPLC (Luna 10 µm silica 250×10 mm column, Phenomenex, Inc., Torrance, CA, U.S.A.) by using 20% ethyl acetate in n-hexane at a flow rate of 10 mL/min to yield compound EF-1 (19 mg). The structure of EF-1 was elucidated based on extensive spectroscopic analyses (¹H-NMR, correlation spectroscopy, heteronuclear single-quantum correlation spectroscopy (HSQC), and heteronuclear multiple bond correlation spectroscopy (HMBC)) and comparison to an authentic pheophytin a sample (C5753; Sigma-Aldrich Corp., St. Louis, MO, U.S.A.).¹³⁾ NMR spectra were recorded in CDCl₃ using a Varian Unity Plus 500 MHz NMR spectrometer (Varian, Inc., Palo Alto, CA, U.S.A.).

Reagents

Con A, capsaicin, sodium dodecyl sulfate (SDS), chlorophyll a, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) were purchased from Sigma-Aldrich. All antibodies were obtained from BD Biosciences (San Diego, CA, U.S.A.). Pheophytin a was generated from chlorophyll a according to a previously reported method.¹³⁾ Chlorophyll a (10 mg) was dissolved in 100 µL of acetone, diluted by adding 250 µL of methanol, and adjusted to pH 2.5 by adding 62.5 µL of 1 N HCl. The reaction solution was stirred for 3 h at an ambient temperature, and then stored under refrigeration for 24 h. Pheophytin a (5 mg), generated as the reaction precipitate, was obtained as a dark bluish pigment by filtration.

Animal and Test Sample Treatments

C57BL/6J mice (male, 6 weeks of age; n=10–12/group) received an oral dose of CCP extract or ECP extract (both 100 mg/kg/day), capsaicin (10 mg/kg/day), pheophytin a (1, 5, or 10 mg/kg/day), chlorophyll a (1, 5, or 10 mg/kg/day), or EF-1 (10 mg/kg/day) for 4 d. CCP extract, ECP extract, capsaicin, chlorophyll a, and EF-1 were suspended in 5% ethanol in 0.1% methylcellulose solution. The control group received an equal volume of vehicle instead of test sample. The mice were sacrificed to obtain the small intestines 2 d after the last treatment. The animal experiments complied with the University Animal Care and Use Committee guidelines at Daejeon University.

Cell Culture

The single PP cell suspension was obtained from the small intestine according to previously described methods.³⁾ Briefly, the PP were dissected from the small intestine and washed twice with ice-cold phosphate-buffered saline (PBS). The single PP cell was liberated by grinding the PP on a cell dissociation sieve (100 mesh screen, Sigma) and filtering through a 70-µm cell strainer (BD Bioscience). The cells were washed twice with ice-cold PBS and cultured (3×10⁶ cells/mL) in RPMI 1640 medium supplemented with 10% fetal bovine serum (Gibco-Invitrogen, Carlsbad, CA, U.S.A.) and 100 µg/mL of penicillin-streptomycin (Lonza, Walkersville, MD, U.S.A.) at 37°C in a 5% CO₂ humidified-air atmosphere.

MTT Assay

PP cells were plated at 10⁴ cells per well in 96-well plates. The cells were grown in the presence of test samples at 37°C in a 5% CO₂ incubator. After 24 h of incubation with the test sample, the viable cells were stained with 10 µL of MTT solution (5 mg/mL) for 4 h at 37°C. Formazan crystals were dissolved in detergent (10% SDS in 0.05 N HCl) for 18 h, and the absorbance was measured at 590 nm using an enzyme-linked immunosorbent assay (ELISA) microplate reader (Molecular Devices, Sunnyvale, CA, U.S.A.).

ELISA

PP cells (2×10⁶ cells/mL) were cultured with or without 5 µg/mL Con A for 72 h at 37°C in a 5% CO₂ incubator. IL-2 and IFN-γ levels were measured using an ELISA kit (BD Biosciences) according to the recommendations of the respective manufacturer.

Flow Cytometry Analysis

For intracellular staining, PP cells were fixed and permeabilized using BD Cytofix/Cytoperm™ Plus (BD Biosciences) and stained with antibodies specific to anti-mouse CD4-fluorescein isothiocyanate (FITC), IL-2-PE, and IFN-γ-APC in staining buffer (PBS containing 1% fetal bovine serum and 0.01% sodium azide) for 30 min on ice. The cells were analyzed using flow cytometry and CellQuest software (BD Biosciences).

Statistical Analysis

All data are expressed as mean±standard deviation. A one-way analysis of variance (SPSS, Inc., Chicago, IL, U.S.A.) was used to analyze the differences between the control group and the experimental groups. Duncan’s multiple comparison tests were used to compare the mean values of the treatments. p-values <0.05 were considered to be statistically significant.

RESULTS

Comparison of the Effects of CCP and ECP on IL-2 and IFN-γ Production in PP ex Vivo

To evaluate whether ECPs have more potent effects on IL-2 and IFN-γ production than do CCPs, mice were orally administered the CCP or ECP extract (100 mg/kg/day) for 4 d. Two days later, PP cells were isolated and cultured, and the levels of the cytokines in the cell culture supernatant were measured. As shown in Fig. 1, IL-2 and IFN-γ production was induced by stimulation with Con A (5 µg/mL). IL-2 production was strongly increased in the presence of Con A for 72 h in the ECP extract-treated group compared to the vehicle control, resulting in a 530% increase. Whereas the CCP extract showed a slight, but significant, increase in IL-2 production (Fig. 1A), which was slightly higher than the values obtained by capsaicin (3–10 mg/kg/day) administration (data not shown).^3,4) There was a 230% and 520% increase in IFN-γ production in the CCP- and ECP extract-treated groups, respectively, compared to the control (Fig. 1B). These data indicate that the ECP extract has more potent effects on IL-2 and IFN-γ production than does the CCP extract.

Fig. 1. Effects of CCPs and ECPs on IL-2 and IFN-γ Production in PP Cells ex Vivo

Mice were orally treated with the extracts of CCP, ECP, or vehicle once a day for 4 consecutive days. The isolated PP cells were cultured in the absence or presence of Con A (5 µg/mL) for 72 h. The levels of (A) IL-2 and (B) IFN-γ in the culture supernatant of the PP cells were determined by ELISA. The data represent mean±S.D. of triplicate determinations. The data presented are representative of 3 independent experiments (one-way ANOVA: ^# p<0.05 vs. CCP, * p<0.05, ** p<0.01 vs. control).

Identification of Pheophytin a from Environmentally Friendly Cultivated Green Pepper

We then compared the individual HPLC chromatograms of each extract to identify the compound responsible for the effects of the ECP extract on cytokine production. The chromatograms of ECP and CCP extracts were almost identical to each other. However, one peak at 415 nm with a retention time of 96.1 min, named EF-1, was much larger in the ECP extract than in the CCP extract: the peak area of EF-1 in the ECP extract was 1.7-fold larger than in the CCP extract (Fig. 2A). HPLC analysis indicated that the ECP extract contained approximately 1.2 mg of EF-1 fraction per gram of the total extract, whereas the CCP extract contained approximately 0.68 mg of EF-1. As capsaicin in peppers was also reported to enhance IL-2 and IFN-γ in PP, we tried to detect the capsaicin peaks in HPLC of ECP and CCP. However, no peak was detected in HPLC chromatograms of ECP and CCP (data not shown). Next, we investigated the biological activities of EF-1 on the production of IL-2 and IFN-γ by oral gavage of the fraction to animals. The EF-1-administered group (10 mg/kg/day) showed increased levels of IL-2 and IFN-γ with Con A stimulation of up to 230% and 250%, respectively (Figs. 2B, C). By comparing the EF-1 NMR spectra with those of the authentic EF-1 sample, we identified the molecule as pheophytin a (Fig. 3), a chlorophyll a lacking a central Mg²⁺ ion. Additionally, the peak detected at 415 nm with a 74.5 min retention time was identified as chlorophyll a when compared with the authentic compound (Fig. 2A). The amounts of chlorophyll a in CCP and ECP extracts were 1.36 mg/g and 1.48 mg/g, respectively. These data indicate that ECPs has a higher content of pheophytin a than CCPs.

Fig. 2. Effects of EF-1 on IL-2 and IFN-γ Production in PP Cells ex Vivo

(A) HPLC comparison between the extracts of CCP and ECP. The arrowhead (↓) indicates EF-1, and the arrowhead (▼) indicates chlorophyll a. The mice were orally administrated EF-1 (10 mg/kg) or vehicle once a day for 4 consecutive days. The isolated PP cells were cultured in the absence or presence of Con A (5 µg/mL) for 72 h. The levels of (B) IL-2 and (C) IFN-γ in the culture supernatant of PP cells were determined by ELISA. The data represent the mean±S.D. of triplicates. The data presented are representative of 3 independent experiments (one-way ANOVA: * p<0.05 vs. control).

Fig. 3. Chemical Structure of EF-1

Effects of Pheophytin a and Chlorophyll a on IL-2 and IFN-γ Production in PP ex Vivo

To confirm that pheophytin a enhances cytokine production, we evaluated the effects of chemically produced pheophytin a from chlorophyll a on IL-2 and IFN-γ ex vivo. We also assessed the effects of chlorophyll a because pheophytin a can be generated from chlorophyll a during the extraction and purification process.¹⁴⁾ As shown in Table 1, Con A stimulation significantly induced IL-2 and IFN-γ production (approximately 123 and 88 pg/mL, respectively). Consistent with the EF-1 fraction, pheophytin a-administered groups showed increased levels of IL-2 and IFN-γ in a dose-dependent manner (Table 1). Oral gavage of pheophytin a (10 mg/kg) significantly induced the production of IL-2 and IFN-γ, resulting in increases of 180% and 230%, respectively. The cytokine levels induced by pheophytin a were comparable to those of EF-1 and capsaicin, a positive control in this study.³⁾ Chlorophyll a elicited higher levels of cytokines in PP cells than were induced by pheophytin a. Administration of chlorophyll a significantly upregulated IL-2 levels by approximately 180% (5 mg/kg) and 330% (10 mg/kg). IFN-γ levels were increased to 380% of the control, even at the dose of 1 mg/kg. These results suggest that administration of pheophytin a and chlorophyll a induce the immune response, enhancing IL-2 and IFN-γ production in PP ex vivo.

Table 1. Effects of Pheophytin a and Chlorophyll a on IL-2 and IFN-γ Productions in Cultured PP Cells ex Vivo

Mice treatments		ELISA
Sample	Concentration (mg/kg of body weight)	IL-2 (pg/mL)	IFN-γ (pg/mL)
Normal	–	3.9±0.82	8.35±1.18
Control	Con A (5 µg/mL)	122.9±6.41	87.57±4.08
Capsaicin	10	237.64±13.38*	206.71±12.04*
Pheophytin a	1	124.45±11.64	174.51±11.7
	5	216.24±12.45	191.62±13.63
	10	230.95±9.02*	202.26±13.5*
Chlorophyll a	1	186.75±3.87	334.72±9.36**
	5	238.09±11.91*	343.77±9.05**
	10	403.44±13.83**	384.19±12.55**

Mice were treated orally with various concentrations of capsaicin, pheophytin a, or chlorophyll a once a day for 4 consecutive days, and the PP cells were collected 6 d after the first treatment and cultured with Con A (5 µg/mL) for 72 h. Levels of IL-2 and IFN-γ were determined by ELISA. The data represent the mean±S.D. of three independent experiments. * p<0.05, ** p<0.01 vs. control (one-way ANOVA).

Effects of Pheophytin a and Chlorophyll a on IL-2- and IFN-γ-Producing T-Cells ex Vivo

Because T-cells are the major cells that produce IL-2 and IFN-γ under Con A stimulation, we investigated CD4+ T-cell populations by intracellular staining and flow cytometry analysis. Data from intracellular staining showed that the percentage of IL-2- and IFN-γ-producing CD4+ T-cells was significantly increased in mice treated with pheophytin a (10 mg/kg; Figs. 4A, f and 4B, o), and the levels were very similar to those with capsaicin (10 mg/kg; Figs. 4A, c and 4B, l). Chlorophyll a increased the level of IL-2-producing CD4+ cells under Con A stimulation, resulting in 1.8–2.6-fold increases at all concentrations (Fig. 4A), and the values were higher than those observed with the use of capsaicin. The percentage of IFN-γ-producing CD4+ T-cells was also increased by 2-fold compared to that of the control (Figs. 4B, r). Because CD8+ T cells produce IFN-γ, we measured the percentage of IFN-γ-producing CD8+ T-cells. However, there were no significant changes between groups (data not shown). Taken together, our data suggest that pheophytin a and chlorophyll a induce IL-2 and IFN-γ production in activated CD4+ T-cells in PP after oral intake.

Fig. 4. Effects of Pheophytin a and Chlorophyll a on IL-2- and IFN-γ-Producing CD4+ T-Cells

Mice were orally treated with various concentrations of capsaicin, pheophytin a, or chlorophyll a once a day for 4 consecutive days. PP cells were collected and cultured in the absence or presence of Con A (5 µg/mL) for 48 h. The cells were stained with (A) anti-CD4-FITC and anti-IL-2-PE or (B) anti-CD4-FITC and anti-IFN-γ-APC. The fluorescence intensities were analyzed by flow cytometry. The data represent the mean±S.D. of triplicates. The data presented are representative of 3 independent experiments (one-way ANOVA test: * p<0.05, ** p<0.01 vs. control).

DISCUSSION

Chlorophyll a is a pigment found in almost all green vegetables, and pheophytin a is converted from chlorophyll a by heating and/or weak acid treatment during cooking.^14,15) There is increasing evidence that chlorophyll a and pheophytin a have various biological activities. Recently, chlorophyll a and pheophytin a were reported to have antioxidant activities in vitro. Pheophytin a, identified from the brown alga Saccharina japonica, suppressed nitric oxide production and inducible nitric oxide synthase (iNOS) expression in lipopolysaccharide (LPS)-stimulated RAW294.7 cells.¹⁶⁾ Chlorophyll a and pheophytins exhibit anti-inflammatory effects and inhibitory effects on tumor necrosis factor (TNF)-α gene expression, which might be mediated by the suppression of nuclear factor-kappa B (NF-κB) activation.¹⁷⁾ Additionally, chlorophyll derivatives belonging to the porphyrin family have cytotoxic effects on tumor cells,¹⁸⁾ and both pheophytin and pheophorbide prevent mutagenesis in vitro and inhibit tumor cell growth.¹⁹⁾

In this study, we demonstrated two major findings. First, we found for the first time that oral intake of pheophytin a and chlorophyll a induced IL-2 and IFN-γ production in activated CD4+ T cells in PP ex vivo. Chlorophyll a showed stronger effects than pheophytin a. A low dose of chlorophyll a significantly induced IL-2 and IFN-γ production, whereas pheophytin a produced cytokines at 10 mg/kg (Table 1), indicating that chlorophyll a and pheophytin a may be immune-modulating components of green vegetables such as pepper. The actual mechanism by which chlorophyll a intake induces cytokine production ex vivo remains to be elucidated. Based on the observation that treatments in vitro did not enhance cytokine production in the presence of Con A (data not shown), it is likely that chlorophyll a and pheophytin a might not directly increase cytokine production in PP cells, although this interpretation must be regarded with caution, because the in vitro system does not completely replicate ex vivo conditions. Other cells would be direct targets of the compounds. Our recent data indicate that intake of chlorophyll a increased the CD11b+ cell population, which is mainly expressed in dendritic cells in PP; moreover, cytokine-producing CD11b+ cells were also increased in PP both in vivo and in vitro. It is plausible that the ex vivo cytokine production from activated T-cells may result from recalling the interaction between T-cells and CD11b+ cells activated by oral administration of chlorophyll a. Additionally, it is worth noting that there was a slight lag between the increase in IFN-γ production by chlorophyll a (Table 1) and IFN-γ-producing CD4+ T-cells (Fig. 4B), especially at the lower doses. We assumed that the difference might be caused by IFN-γ-producing CD11b+ cells (data not shown). Further studies on the target cells and molecules of chlorophyll a and pheophytin a in the intestinal immune system are definitely needed.

Taken together, we suggest that ingestion of fresh green vegetables containing chlorophyll a can induce IL-2 and IFN-γ production in activated T-cells under various stimuli in the intestine, implying that the green food diet would efficiently enhance immune activity against invading pathogens and abnormal cells. Indeed, many clinical studies have shown that an increased consumption of green vegetables and fruits is associated with immune-enhancing and immune-modulating activities, resulting in antiviral, antibacterial, anticancer, and anti-allergic effects.^20–24) Therefore, further studies to clarify the roles of chlorophylls and pheophytins in the immune response are definitely needed to understand immune dysregulation and disease status, such as in infection and cancer.

The second significant finding of our present study is that compared with CCPs, ECPs contain a higher content of pheophytin a, a biologically active molecule. We assume that it was generated as a result of the increased chlorophyll a content and the extraction process, because pheophytin a was not detected in raw green peppers according to two reports analyzing ingredients of peppers.^25,26) Unfortunately, as we did not measure the components using raw green peppers in this study, this will be the subject of our future research. It is also worth asking why the chlrorophyll a and/or pheophytin a content is higher in ECPs than in CCPs. It is known that biostimulants not only improve antioxidant activity²⁷⁾ but also show positive effects from their phenolic, vitamin C, and pigment components.⁵⁾ The fungicides and insecticides used for cultivating green peppers might have an influence on the production of biologically active compounds such as pheophytins. Pesticides change the nutrient content of plant food. Alteration of plant-commensal microorganism communities and soil conditions by pesticides suggests that environmentally friendly cultivated crops produce different amounts of nutrients or biologically active components than conventionally cultivated crops.^28,29) However, further investigation on the mechanism by which ECPs increase the production of an active immune stimulator, pheophytin a, is required.

The initial goal of our study was to compare the immune-enhancing effects of CCP and ECPs. However, from the results of a comparison of the activities of Th1 cytokines such as IL-2 and IFN-γ between ECPs and CCPs in PPs, we found that ECPs possess a higher level of EF-1 that contains pheophytin a that stimulates immune-mediated activities similarly to chlorophyll a in PP ex vivo. Our study suggests that pheophytin a and chlorophyll a from green vegetables, such as green peppers, have great potential as immune modulators in the intestine.

Acknowledgements

This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korea government (MSIP) (No. 2012–0002497 and 2011–0030702) and the Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

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