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Abstract

Objectives: Natural compounds that can effectively prevent and alleviate allergic symptoms, such as hay fever, are widely sought after from sources such as natural ingredients that can be ingested daily. Coriander extracts have been reported to show anti-allergic activity; however, hot water extracts have not been tested previously. Here, we found that a hot water extract of the aerial parts of coriander inhibited the degranulation of rat basophilic leukemia cell line RBL-2H3 cells and identified the active compounds. Methods: Chromatographic methods were used to isolate compounds from the hot water extract of coriander. The chemical structures were elucidated by analyses of high-resolution electrospray ionization mass spectrum and nuclear magnetic resonance spectral data. The degranulation activity of compounds was evaluated using the β-hexosaminidase release assay. Results: We analyzed the components to identify active compounds. 2S,3S-2-O-caffeoyl-hydroxycitric acid (1), and 2-O-caffeoyl-hydroxycitric acid 6′-O-methyl ester (2) were isolated from the hot water extract of coriander, and both compounds showed degranulation inhibitory activity in RBL-2H3 cells at a concentration below 2.5 mM. The content of compound 1 in the freeze-dried coriander powder was calculated to be 2.0% via HPLC quantification. Conclusions: Compound 1 was considered to be the primary active contributor in the hot water extract of coriander.

Keywords

phenolics coriander RBL-2H3 cell degranulation inhibition caffeoyl hydroxycitric acid

Introduction

Humans are affected by various allergic conditions, such as hay fever, bronchial asthma, and atopic dermatitis. The most well-known mechanism for the onset of allergies, such as hay fever, has long been attributed to type I hypersensitivity (immediate-type hypersensitivity), which involves immunoglobulin E (IgE).¹ Anti-allergic drugs suppress symptoms by disrupting a part of their mechanism of action. However, there are concerns regarding side effects from such medications, such as induced drowsiness and reduced quality of life. Therefore, there is a need to develop functional foods that can safely relieve allergic symptoms. Screening 6 edible plant extracts, we found that water-soluble coriander leaf extract showed an anti-allergic effect in mast cell degranulation assays using RBL-2H3 cells.²

Coriander (Coriandrum sativum L.) is an annual Apiaceae family plant widely cultivated in Africa, Europe, and Asia. It is commonly known as coriander in English and phakchai in Thai; the leaves and seeds are widely used as savory vegetables and seasoning.^3,4 In addition to its use as a seasoning ingredient, it is used in folk medicine in diverse applications, including as a digestive stimulant, antibacterial agent, and for treating airway disorders (bronchitis and cough). In recent years, coriander extracts have shown various physiological activities, including antioxidant,^5–7 antibacterial,^8,9 and anti-inflammatory^10,11 activities. In addition, exposure to coriander extract suppresses the expression of inflammatory mediators in RAW 264.7 cells of lipopolysaccharide-treated mice.¹² Phenolics and carotenoids in coriander are reported to have antioxidant activities.⁴ Furthermore, the ethanolic extract of coriander, which contains isoquercetin and quercetin, showed hepatoprotective activity.¹³ However, it is still unclear which compounds in coriander are responsible for other health functions.

A recent study showed the anti-allergic effects of water-soluble coriander leaf extract in both cultured cells and pollinosis mice.² Furthermore, 8 components have been identified in the methanol-eluate fraction obtained from the water-soluble fraction, which showed degranulation inhibitory activity in RBL-2H3 cells.¹⁴ However, existing in coriander leaves in only small amounts, those components are insufficient to explain the anti-allergic effects of coriander leaves. Active ingredients are expected to present dominantly in the aqueous-eluate fraction and have not yet been isolated. Therefore, in the present study, we isolated the anti-allergic substances from the hot water extract of coriander, determined their structures, and evaluated their activity.

Results and Discussion

Inhibitory Effect of the H₂O Extract of Coriander on Degranulation in RBL-2H3 Cells

The hot water extract of coriander (leaves, petioles, and stems) was fractionated using a polystyrene column (MCI gel CHP-20P) to obtain 4 fractions: Frs. 1-4. The H₂O eluate fraction (Fr. 2), which was a large amount of all fractions, was separated by Sephadex LH-20 to yield 6 fractions, Frs. 2-1 to 2-6. The inhibitory effects on degranulation were evaluated using RBL-2H3 cells (Figure 1A and B). Frs. 2-1, 2-4, 2-5, and 2-6 showed degranulation inhibitory activity without cytotoxicity (Figure 1C and D); Fr. 2-5 showed the highest activity between 0.63 mg/mL and 2.5 mg/mL, and it was further purified. Fr. 2-5 was separated into 6 fractions using a µ-Bonda Pak C18 column (Frs. 2-5-1 to 2-5-6). All fractions showed degranulation inhibitory activity between 0.25 mg/mL and 1.0 mg/mL (Figure 2A and B). In Fr. 2-5-1, no major compound, other than sugars, was detected by thin-layer chromatography (TLC). Frs. 2-5-2 and 2-5-3 showed considerable cytotoxicity (Figure 2C). Conversely, Frs. 2-5-4, 2-5-5, and 2-5-6 showed no cytotoxicity (Figure 2D). Further analysis was performed using these fractions, which were purified via preparative high-performance liquid chromatography (HPLC) to isolate compounds 1 (33 mg) and 2 (2.2 mg) (Figure 3).

Figure 1.

Degranulation inhibitory ability and cytotoxicity of the Sephadex LH-20 fractions in RBL-2H3 cells. (A) Degranulation inhibition of fractions of the Sephadex LH-20 fractions (Frs. 2-1∼2-3) in RBL-2H3 cells. (●: Blank, ●: Control, ▴: Fr. 2-1, △: Fr. 2-2, ▴: Fr. 2-3) (B) Degranulation inhibition activity of the Sephadex LH-20 fractions (Frs. 2-4∼2-6) in RBL-2H3 cells. (●: Blank, ●: Control, ◆: Fr. 2-4, ◇: Fr. 2-5, ◆: Fr. 2-6) (C) Cytotoxicity of the Sephadex LH-20 fractions (Frs. 2-1∼2-3) to RBL-2H3 cells. (●: Blank, ●: Control, ▴: Fr. 2-1) (D) Cytotoxicity of the Sephadex LH-20 fractions (Frs. 2-4∼2-6) to RBL-2H3 cells. (●: Blank, ●: Control, ◆: Fr. 2-4, ◇: Fr. 2-5, ◆: Fr. 2-6) The experiment was performed in triplicate, and data are expressed as mean ± SD (n = 3). Dunnett's test was used to assess the statistical significance of differences (*P < .05, **P < .01, and ***P < .001) compared with the control.

Figure 2.

Degranulation inhibitory ability and cytotoxicity of the m-Bonda Pak C₁₈ fractions in RBL-2H3 cells. (A) Degranulation inhibition of fractions of the m-Bonda Pak C₁₈ (Frs. 2-5-1∼2-5-3) in RBL-2H3 cells. (●: Blank, ●: Control, ▴: Fr. 2-5-1, △: Fr. 2-5-2, ▴: Fr. 2-5-3) (B) Degranulation inhibition activity of the m-Bonda Pak C₁₈ fraction (Frs. 2-5-4∼2-5-6) in RBL-2H3 cells. (●: Blank, ●: Control, ▪: Fr. 2-5-4, □: Fr. 2-5-5, ▪: Fr. 2-5-6) (C) Cytotoxicity of the m-Bonda Pak C₁₈ (Frs. 2-5-1∼2-5-3) to RBL-2H3 cells. (●: Blank, ●: Control, ▴: Fr. 2-5-1, △: Fr. 2-5-2, ▴: Fr. 2-5-3) (D) Cytotoxicity of the m-Bonda Pak C₁₈ (Frs. 2-5-4∼2-5-6) to RBL-2H3 cells. (●: Blank, ●: Control, ▪: Fr. 2-5-4, □: Fr. 2-5-5, ▪: 2. (A) Degranulation inhibition of compound 1 Fr. 2-5-6) Experiment was performed in triplicate, and data are expressed as mean ± SD (n = 3). Dunnett's test was used to assess the statistical significance of differences (*P < .05, **P < .01, and ***P < .001) compared with the control.

Figure 3.

The structures of compounds 1 and 2.

Structural Elucidation of Compounds 1 and 2

Compound 1 was obtained as a pale-yellow solid, with [α]_D²² + 30.1° (c 0.17, H₂O), and showed an [M-H]⁻ peak at m/z 369.0460 in the negative-ion high-resolution electrospray ionization mass spectrum (HR-ESI-MS), corresponding to the molecular formula C₁₅H₁₄O₁₁. The ¹H nuclear magnetic resonance (NMR) (Table 1), ¹³C NMR, heteronuclear multiple bond correlation (HMBC) spectra (Figure 4A), and optical rotation value were superimposable with those of 2S,3S-2-O-caffeoyl-hydroxycitric acid, which has been previously isolated from corn plants (Zea mays L.).¹⁵ Therefore, compound 1 was identified as 2S,3S-2-O-caffeoyl-hydroxycitric acid.

Figure 4.

Significant heteronuclear multiple bond correlation (HMBC) correlations of compounds 1 and 2. A: compound 1. B: compound 2.

Table 1.

¹H-NMR Spectral Data of Compound 1 in Different Solutions and Reference Compounds.

Comp.	1^a			1^b			1^c			2S, 3S form^b (ref. 15)			2S, 3R form^c (ref. 16)
Position	δ	Coupling	J (Hz)	δ	Coupling	J (Hz)	δ	Coupling	J (Hz)	δ	Coupling	J (Hz)	δ	Coupling	J (Hz)
H-3	7.65	d	16.1	7.64	d	16.0	7.68	d	16.0	7.60	d	16.0	7.63	d	16
H-5	7.08	d	2.3	7.18	d	1.7	7.21	d	1.7	7.13	d	4.0	7.05	d	1
H-9	6.98	dd	2.3, 8.6	7.06	dd	1.7, 8.5	7.10	dd	1.7, 8.1	7.04	dd	4.0, 9.0	6.95	dd	1, 8.5
H-8	6.80	d	8.6	6.88	d	8.5	6.93	d	8.1	6.88	d	9.0	6.75	d	8.5
H-2	6.30	d	16.1	6.29	d	16.0	6.41	d	16.0	6.25	d	16.0	6.33	d	16
H-2'	5.21	s		5.28	s		5.25	s		5.27	s		5.40	s
H-4'a	3.18	d	16.6	3.28	d	16.6	3.21	d	16.6	3.31	d	17.0	2.93	m
H-4'b	3.10	d	16.6	3.15	d	16.6	3.17	d	16.6	3.11	d	17.0	2.93	m

Abbreviation: NMR, nuclear magnetic resonance.

MeOH-d₄

Acetone-d₆

Acetone-d₆, D₂O (1:1) m: multiplet.

Compound 2 was obtained as a pale-yellow solid, with [α]_D²² + 33.1° (c = 0.28, H₂O), and showed an [M-H]⁻ peak at m/z 383.0615 in the negative-ion HR-ESI-MS, corresponding to the molecular formula C₁₆H₁₆O₁₁. The ¹H NMR spectrum of 2 (Table 2) was similar to that of 1 (Table 1), except for the addition of one OCH₃ signal observed downfield at δ_H 3.68 (s, 3H) and δ_C 52.4. These chemical shifts suggest the presence of a methyl ester rather than methyl ether. The position of substitution of the methyl ester was determined by HMBC between δ_H 5.23 of an oxy-bearing methine proton as H-2′ and d_H 3.68 of the methoxy group to δ_C 171.7 as C-6′, the central carbon of hydroxycitric acid (Figure 4B). A diastereoisomeric 2S,3R-2-O-caffeoyl hydroxycitric acid has been isolated from Spondias mombin.¹⁶ The optical rotation value and ¹H NMR spectra of 2 were more consistent with those of 2S,3S-2-O-hydroxycitric acid¹⁵ than those of 2S,3R-2-O-hydroxycitric acid.¹⁶ Furthermore, of these 4 optical isomers, only 2, (2S,3S)- and (2S,3R)-2-hydroxycitric acid, are present in nature.¹⁷ Therefore, the absolute configuration of the hydroxycitric acid moiety in 2 was presumed as 2S,3S-hydroxycitric acid. Therefore, the structure of 2 was deduced as 2S,3S-2-O-caffeoyl-hydroxycitric acid 6′-O-methyl ester. Further studies are required to determine the absolute configuration of 2. The H₂O extract was directly analyzed by HPLC using acetonitrile, despite MeOH being used as the mobile phase, without purification to avoid the assumption of an artifact for the extraction and isolation procedures. Compound 2 was detected as a minor component, together with the primary component of 1 (Figure 5). In the case of 2-O-caffeoylisocitric acid and its 6′-O-methyl ester derived from orchard grass, the levels of these 2 compounds show fluctuation that is possibly related to the effects of changing environmental conditions; 6′-O-methyl ester formation may directly result from methylation of 2-O-caffeoylisocitric acid.¹⁸ Therefore, to our knowledge, 2 represents a novel compound with the 6′-methylester in the hydroxycitric acid moiety.

Figure 5.

3D HPLC profile of the hot water extract of coriander. Column: Cosmosil AR-II (ϕ4.6 × 250 mm); Solvent: CH₃CN in 0.1% TFA /H₂O in 0.1% TFA, 10/90 (0 min) → 100/0 (30 min); Flow rate: 1.0 mL/min; Column temp.: 40 °C; Detector: PDA (200 nm → 400 nm); Extraction: 6.0 g of dry powders /150 mL hot water, 10 μL injection. Rt 1: 8.4 min, 2: 10.1 min.

Table 2.

¹H-NMR Spectral Data of Compound 2 in Different Solutions.

Solvent	MeOH-d₄			Acetone-d₆			Acetone-d₆, D₂O (1:1)
Position	δ	Coupling	J (Hz)	δ	Coupling	J (Hz)	δ	Coupling	J (Hz)
H-3	7.65	d	16.0	7.63	d	16.0	7.68	d	16.0
H-5	7.08	d	2.3	7.18	d	2.3	7.22	d	1.7
H-9	6.98	dd	2.3, 8.1	7.05	dd	2.3, 8.1	7.11	dd	1.7, 8.6
H-8	6.80	d	8.1	6.88	d	8.1	6.93	d	8.6
H-2	6.30	d	16.0	6.29	d	16.0	6.43	d	16.0
H-2'	5.23	s		5.27	s		5.24	s
H-4'a	3.22	d	16.0	3.28	d	16.0	3.21	br s
H-4'b	3.13	d	16.0	3.15	d	16.0	3.21	br s
OCH₃	3.68	s		3.63	s		3.72	s

Abbreviations: br, broad; NMR, nuclear magnetic resonance.

Quantification of Compound 1

Since the peak of compound 1 was prominent from the analysis results in Figure 5, a quantitative analysis of compound 1 was performed. The content of compound 1 in the freeze-dried coriander powder sample was calculated to be 2.0% via HPLC quantification. Previous studies have shown that flavonoid glycosides represent major components present in vegetative parts of coriander¹⁹; coumarins are one of the characteristic constituents of coriander leaves^14,20; the presence of dihydroxycoumarin glycoside in coriander has been suggested using LC-MS/MS analysis,21 and the contents of dihydroxycoumarin and furanocoumarin glycosides are relatively high.14 However, these compounds were not the predominant components of the hot water extract in our tested coriander sample when compared to the amount of compound 1. The reason for this is unclear. The hot water extract was heated at 100 °C to quench an esterase in coriander, which may have affected the composition. Moreover, MeOH extraction might have been insufficient to dissolve compounds 1 and 2.

Inhibitory Effect of Compounds 1 and 2 on Degranulation

β-Hexosaminidase is present in the granules of mast cells and basophils and is released with histamine when mast cells and basophils are activated.^22–24 The β-hexosaminidase release assay has been used as an alternative to the histamine release assay in several studies.^25,26 β-Hexosaminidase release assays were performed to evaluate the degranulation inhibitory activity of the 2 compounds. Compounds 1 and 2 showed considerable degranulation inhibitory activity from 0.63 to 2.5 mM and 1.25 to 2.5 mM, respectively, in a concentration-dependent manner, without cytotoxicity (Figure 6A and B). The activity did not affect the 6′-methyl ester functional group of 2. Compound 1 did not affect the enzymatic activity of β-hexosaminidase (Table S1). The compound 1-treated cells showed an increase in intracellular β-hexosaminidase and a decrease in extracellular enzyme, indicating that the enzyme remained in the cells without being released to the supernatant after treatment with compound 1 (Table S2). In addition, the total amount of enzyme and the sum of the cell supernatant and cell lysate were the same among blank, control, and compound 1-treated cells. These results indicate that the tested sample inhibits the degranulation without affecting the enzymatic activity of β-hexosaminidase. The anti-allergic effects of 2-O-caffeoyl-hydroxycitric acid derivatives were revealed for the first time in this study.

Figure 6.

Degranulation inhibition and cytotoxicity of compound 1 and 2. (A) Degranulation inhibition of compound 1 and 2 in RBL-2H3 cells. (●: Blank, ●: Control, ▪: compound 1, □: compound 2) (B) Cytotoxicity of compound 1 and 2 to RBL-2H3 cells. (●: Blank, ●: Control, ▪: compound 1, □: compound 2) Experiment was performed in triplicate, and data are expressed as mean ± SD (n = 3). Dunnett's test was used to assess the statistical significance of differences (**P < .01 and ***P < .001) compared with the control.

A rudimentary structure-activity relationship study on degranulation activity was conducted using commercially available caffeic and hydroxycitric acid. Hydroxycitric acid was insoluble in DMSO and buffer and could not be examined. Caffeic acid inhibited degranulation (Figure 7), indicating that the caffeic acid structure of compounds 1 and 2 contributes to the activity. The role of the hydroxycitric acid moiety in the degranulation of compounds 1 and 2 may be due to various factors, such as the carboxylic acid–induced ionophore and the increase in water solubility of caffeic acid ester. However, further studies are needed in this regard.

Figure 7.

Degranulation inhibition of caffeic acid in RBL-2H3 cells. (●: Blank, ●: Control, 〇: Caffeic acid) The experiment was performed in triplicate. Data are expressed as mean ± SD (n = 3). Dunnett's test was used to assess the statistical significance of differences (**P < .01 and ***P < .001) compared with the control.

Previous studies on the water-soluble fraction of coriander reported that tryptophan, phenylalanine, dihydroxycoumarin glucoside, (3S)-3-methyl-6-hydroxyisocoumarin 8-O-β-D-glucopyranoside, quercetin glucuronide, and rutin contribute to the inhibition of degranulation.14 Although these components showed almost the same activity range compared to compounds 1 and 2, the yield of compound 1 was an order of magnitude higher; thus, it is inferred to be the active component.

Conclusions

The hot water extract of coriander inhibited degranulation in RBL-2H3 cells. Compounds 1 and 2 were isolated from the aqueous extract of coriander in this study. Their chemical structures were determined to be 2S,3S-2-O-caffeoyl-hydroxycitric acid and 2-O-caffeoyl-hydroxycitric acid 6′-O-methylester, respectively, and their isolation from coriander has been described for the first time. Compounds 1 and 2 showed moderate inhibition of degranulation. Quantitative analysis of 1 in dried coriander powder showed that it was the component with the highest content of more than 2.0%, excluding primary metabolites, such as sugars. Thus, compound 1 was considered to be the primary active contributor to the degranulation inhibitory activity of the hot water extract of coriander. The mechanism of action and in vivo anti-allergy activity of compound 1 should be evaluated in future studies. The findings show that coriander extracts may represent natural anti-allergy agents that can help relieve allergic conditions.

Experimental

General Experimental Procedures

Electrospray ionization-mass spectrometric (MS) analysis was performed on a maXis II mass spectrometer (Bruker Co.). MeOH (Fujifilm Wako Pure Chemical Co.) was used as the solvent for LC/MS. MeOH in 5 mM dibutylammonium acetate (Ion-Pair Reagent for LC-MS; Tokyo Chemical Industry Co., Ltd) was added at a 0.1 mL/min flow rate. NMR spectra were recorded using a JEOL ECA 500 NMR spectrometer (JEOL). ¹H-NMR spectra were recorded in MeOH-d₄, Acetone-d₆, and Acetone-d₆, D₂O (1:1) solutions. The ¹³C-NMR spectra were recorded in MeOH-d₄ solution. The chemical shifts (δ) are reported in parts per million (ppm) and J values in Hz, using MeOH-d₄ for ¹H-NMR (3.31 ppm) and ¹³C-NMR (49.0 ppm) as an internal standard, and using Acetone-d₆ for tetramethyl silane (0 ppm) as an internal standard. P-1020 (JASCO Co.) was used as the optical rotation measurement device. The UV spectrum was measured using a V-750 spectrophotometer (JASCO Co.). Preparative HPLC was performed using a SHIMADZU LC-20AT pump, SHIMADZU RID-20A detector, Sugai U-620 column heater, and column of X-Select CHS Prep C₁₈ (5 mm, ϕ10.0 × 250 mm; Waters Co.) with a flow rate of 2.0 mL/min and column temperature of 40 °C. Flash chromatography was performed on a µ-Bonda Pak C₁₈ column (Radial-Pak Cartridge ϕ25 × 100 mm × 2; Waters Co.). TLC was performed on pre-coated silica gel 60 F₂₅₄ (Merck Ltd), and detection was achieved by spraying with 10% H₂SO₄ followed by heating. Column chromatography was performed on MCI gel CHP20P (Mitsubishi Chemical Co.) and Sephadex LH-20 columns (GE Healthcare Bioscience Co.).

HPLC analysis of the aqueous extract was performed using a photodiode array detector SPD-M10A, interface box SCL-10A, dual HPLC Pump LC-10AD, column oven CTO-10AC, and degasser DGU-14A (SHIMADZU Co.). Cosmosil AR-II (5 μm, 250 × 4.6 mm; Nacalai Tesque) was used as the HPLC column. The solvent comprised acetonitrile with 0.1% trifluoroacetic acid (TFA)/H₂O with 0.1% TFA, used as follows: 10/90 (vol/vol) (0 min) → 100/0 (30 min) → 10/90 (31 min) → 10/90 (40 min). The flow rate was set to 1.0 mL/min. The column temperature was 40 °C. The detection wavelength was 200 to 400 nm.

Dulbecco's modified Eagle's medium (DMEM), penicillin, streptomycin, fetal bovine serum (FBS), bovine serum albumin (BSA), mouse anti-dinitrophenyl (DNP) monoclonal IgE, and DNP–human serum albumin (HSA) conjugates were purchased from Sigma-Aldrich (St. Louis). Unless otherwise noted, all other chemicals were purchased from Fujifilm Wako Pure Chemicals or Nacalai Tesque.

Plant Material

Coriandrum sativum L. (taxonomical form: CRUISER) was collected on November 23, 2019, from a farm in Gunma, Japan. Fresh coriander (leaves, petioles, and stems) was blanched in hot water at 80 °C for 20 s, drained, and then frozen at −40 °C. The frozen material was freeze-dried (FDU-1200 in a dry chamber, EYELA Co.) for 48 h. The freeze-dried material was ground in a hammer mill and sieved through a 48-mesh sieve. A voucher specimen (CS2019G) was deposited in the Natural Product Laboratory of the Faculty of Pharmaceutical Sciences, Sojo University.

Extraction and Isolation

To a powder of freeze-dried coriander (leaves, petioles, and stems) (48.59 g), distilled H₂O (1.0 L) was added, followed by extraction using sonication (Ultrasonic Cleaner: US-105 (200 W), SND Co.) for 4 h (60 min × 4) at 50 °C. This process was performed 4 times. The mixed extracts were concentrated under reduced pressure to obtain the H₂O extract (23.63 g). This was dissolved in H₂O (150 mL) and heated at 100 °C for 10 min to evaluate its thermal stability. After cooling the solution to room temperature (20 °C-25 °C), it was subjected to separation using an MCI gel (ϕ50 × 160 mm) and successively eluted with H₂O, 50% MeOH, and 100% MeOH (each 1.0 L) to yield 4 fractions. The first fraction (2.19 g) had passed through the MCI gel, the second fraction (18.99 g) was an eluate of H₂O, the third fraction (1.04 g) was an eluate of 50% MeOH, and the fourth fraction (0.21 g) was an eluate of MeOH. Part of the second fraction (2.52 g) was loaded onto a Sephadex LH-20 column (ϕ30 × 1000 mm, eluted with 50% MeOH), collected into test tubes (each 20 mL), and divided based on the results of TLC observation to obtain 6 fractions: Fr. 2-1 to 2-6. The fraction relatively rich in aromatic compounds (Fr. 2-5, 367 mg) was applied to a µ-Bonda Pak C₁₈ column [ϕ25 × 100 mm × 2, and eluted with a H₂O-MeOH gradient (H₂O containing 10%, 20%, and 30% MeOH; 150 mL of each gradient solution)] to yield 6 fractions (Frs. 2-5-1 to 2-5-6). The 30% MeOH eluate fractions, Frs. 2-5-4 to 2-5-6, were combined (75 mg) and purified using preparative HPLC [X-Select CHS Prep C₁₈ (ϕ10 × 250 mm, eluting with a 40% MeOH in 0.1% TFA)] to yield compounds 1 (33 mg) and 2 (2.2 mg).

Compound 1 [2S,3S-2-O-caffeoyl-hydroxycitric acid]

A pale yellow solid; [α]_D²⁵ = + 30.1° (c 0.17, H₂O); UV λ_max log ε 3.99 (331 nm); ¹H NMR: see Table 1; ¹³C NMR δ 174.6 (C-5′), 173.2 (C-6′), 170.0 (C-1′), 167.9 (C-1), 149.8 (C-7), 148.5 (C-3), 146.8 (C-6), 127.6 (C-4), 123.4 (C-9), 116.6 (C-8), 115.3 (C-5), 113.7 (C-2), 77.4 (C-3′), 77.0 (C-2′), 41.0 (C-4′); HR-ESI-MS m/z 369.0460 [M - H]⁻ (calculated for C₁₅H₁₃O₁₁, 369.0463).

Compound 2 [2-O-caffeoyl-hydroxycitric acid 6′-O-methylester]

A pale yellow solid; [α]_D²⁵ = + 33.1° (c 0.28, H₂O); UV λmax log ε 3.75 (332 nm); ¹H NMR: see Table 2; ¹³C NMR δ 174.5 (C-5′), 171.7 (C-6′), 169.9 (C-1′), 167.8 (C-1), 149.9 (C-7), 148.5 (C-3), 146.8 (C-6), 127.6 (C-4), 123.4 (C-9), 116.6 (C-8), 115.3 (C-5), 113.7 (C-2), 77.4 (C-3′), 77.0 (C-2′), 52.4 (-OCH₃), 41.2 (C-4′); HR-ESI-MS m/z 383.0615 [M - H]⁻ (calculated for C₁₆H₁₅O₁₁, 383.0620).

Quantification of Compound 1

Boiling H₂O (15 mL) was added to a sample of freeze-dried coriander (1.0 g) following extraction via sonication for 1 h at 50 °C. The extraction was repeated 3 times, and the combined filtrate was mixed to a total volume of 50.0 mL. An aliquot of the sample (10 μL) was injected 5 times into the HPLC system. Compound 1 was quantified using MeOH with 0.1% TFA/H₂O with 0.1% TFA as follows: 5/95 (vol/vol) (0 min) → 100/0 (30 min) → 100/0 (35 min) → 5/95 (36 min) → 5/95 (45 min). The flow rate was set to 1.0 mL/min. Ultraviolet detection was performed at 330 nm (Rt = 14.1 min). A calibration curve was generated from 0.025 to 1.0 mg/mL (y = 1,494,870x – 91,707, R² = 0.9996; y: area, x: concentration).

Cell Culture

Rat basophilic leukemia RBL-2H3 cells were obtained from the American Type Culture Collection and cultured in DMEM supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% FBS at 37 °C in a humidified 5% CO₂ to 95% air atmosphere.

β-Hexosaminidase Release Assay

A β-hexosaminidase fluorescence assay kit (Cell Biolabs Inc.) was used in this study. RBL-2H3 cells suspended in DMEM containing 10% FBS and 50 ng/mL of anti-DNP IgE were seeded into a 96-well culture plate (Corning Inc.) at 4 × 10⁴ cells/well and cultured for 24 h. After washing with modified Tyrode's (MT) buffer (135 mM NaCl, 20 mM HEPES, 5.6 mM glucose, 5 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, and 0.05% BSA, pH 7.4) 2 times, the anti-DNP IgE-sensitised cells were treated with 120 μL of MT buffer containing various concentrations of either the sample or distilled H₂O as a blank and control for 10 min. Next, 10 μL of DNP–HSA solution diluted in MT buffer at a concentration of 0.625 μg/mL was added to each well, and the plate was incubated for 30 min. MT buffer (10 µL) was added to blank-treated cells. After incubation, 50 μL of the supernatant was transferred to a 96-well black-bottom microplate. Subsequently, 50 μL of the substrate was added to each well, and the plate was incubated at 37 °C for 15 min. After incubation, 100 μL of neutralization buffer was added to each well, and the absorbance was measured at an excitation wavelength of 340 nm and an emission wavelength of 465 nm using an Infinite 200 PRO plate reader (Tecan Group Ltd).

Measurement of Cell Viability

Cytotoxicity was evaluated using Cell Count Reagent (Nacalai Tesque) based on WST-8, following the manufacturer's instructions. Anti-DNP IgE-sensitized cells were treated with various concentrations of sample and stimulated with DNP-has, as described above. After washing with DMEM, fresh medium containing 5% Cell Count Reagent was added to each well, and the plate was incubated at 37 °C for 20 to 40 min. After incubation, the absorbance was measured at 450 nm using an iMark microplate reader (Bio-Rad Laboratories).

Statistical Analysis

Experiments were performed in triplicate, and the data are expressed as the mean ± standard error of mean. Statistical analyses were performed using GraphPad Prism version 8.43 (GraphPad Software). One-way analysis of variance followed by Dunnett's multiple comparison test was used to assess statistical significance. Values of *P < .05, **P < .01, and ***P < .001 were considered significant.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X231183603 - Supplemental material for Isolation and Quantification of 2-O-Caffeoyl-hydroxycitric Acid, an Active Component in Hot Water Extracts of Coriander (Coriandrum sativum L.), Which Inhibits Degranulation of RBL-2H3 Cells

Supplemental material, sj-docx-1-npx-10.1177_1934578X231183603 for Isolation and Quantification of 2-O-Caffeoyl-hydroxycitric Acid, an Active Component in Hot Water Extracts of Coriander (Coriandrum sativum L.), Which Inhibits Degranulation of RBL-2H3 Cells by Tsuyoshi Ikeda, Takuya Sugahara, Honoka Kanamitsu, Keisuke Nakashima, Hiroyuki Onda, Nanami Yoshino, Kosuke Nishi and Momoko Ishida in Natural Product Communications

Footnotes

Acknowledgments

The authors express our appreciation to Dr Daisuke Nakano, Prof. Junei Kinjo, and Mr Hiroshi Hanazono of Fukuoka University for assistance with ESI TOF MS. The authors also sincerely thank Ms. Kasumi Gondo, Ms. Nanako Ishizaka, and Ms. Kaho Sakai of Sojo University for their collaboration in the extraction and isolation of the compounds.

Authors Contribution

Supervision, T. S.; conceptualization, T. I., T. S., H. O., and M. I.; data curation, H. K., K. N.; Investigation, T. I., H. K., K. N., and M. I.; Writing – original draft, T. I. and M. I.; Writing – review & editing, T. S., H. O., N. Y., and K. N.; Project administration, H. O. and N. Y. All authors have read and agreed to the published version of the manuscript.

Declaration of Conflicting Interests

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

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Supplemental Material

Supplemental material for this article is available online.

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Supplementary Material

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Isolation and Quantification of 2- O -Caffeoyl-hydroxycitric Acid,an Active Component in Hot Water Extracts of Coriander ( Coriandrum sativum L.),Which Inhibits Degranulation of RBL-2H3 Cells

Abstract

Keywords

Introduction

Results and Discussion

Inhibitory Effect of the H2O Extract of Coriander on Degranulation in RBL-2H3 Cells

Structural Elucidation of Compounds 1 and 2

Quantification of Compound 1

Inhibitory Effect of Compounds 1 and 2 on Degranulation

Conclusions

Experimental

General Experimental Procedures

Plant Material

Extraction and Isolation

Compound 1 [2S,3S-2-O-caffeoyl-hydroxycitric acid]

Compound 2 [2-O-caffeoyl-hydroxycitric acid 6′-O-methylester]

Quantification of Compound 1

Cell Culture

β-Hexosaminidase Release Assay

Measurement of Cell Viability

Statistical Analysis

Supplemental Material

sj-docx-1-npx-10.1177_1934578X231183603 - Supplemental material for Isolation and Quantification of 2-O-Caffeoyl-hydroxycitric Acid, an Active Component in Hot Water Extracts of Coriander (Coriandrum sativum L.), Which Inhibits Degranulation of RBL-2H3 Cells

Footnotes

Acknowledgments

Authors Contribution

Declaration of Conflicting Interests

Funding

Supplemental Material

References

Supplementary Material

Inhibitory Effect of the H₂O Extract of Coriander on Degranulation in RBL-2H3 Cells