Fermented Ginseng Contains an Agonist of Peroxisome Proliferator Activated Receptors α and γ

Abstract

Peroxisome proliferator activated receptor (PPAR) is a nuclear receptor that is one of the transcription factors regulating lipid and glucose metabolism. Fermented ginseng (FG) is a ginseng fermented by Lactobacillus paracasei A221 containing minor ginsenosides and metabolites of fermentation. DNA microarray analysis of rat liver treated with FG indicated that FG affects on lipid metabolism are mediated by PPAR-α. To identify a PPAR-α agonist in FG, PPAR-α transcription reporter assay-guided fractionation was performed. The fraction obtained from the MeOH extract of FG, which showed potent transcription activity of PPAR-α, was fractionated by silica gel column chromatography into 16 subfractions, and further separation and crystallization gave compound 1 together with four known constituents of ginseng, including 20(R)- and 20(S)-protopanaxadiol, and 20(R)- and 20(S)-ginsenoside Rh1. The structure of compound 1 was identified as 10-hydroxy-octadecanoic acid by ¹H- and ¹³C-NMR spectra and by EI-MS analysis of the methyl ester of 1. Compound 1 demonstrated much higher transcription activity of PPAR-α than the other isolated compounds. In addition, compound 1 also showed 5.5-fold higher transcription activity of PPAR-γ than vehicle at the dose of 20 μg/mL. In the present study, we identified 10-hydroxy-octadecanoic acid as a dual PPAR-α/γ agonist in FG. Our study suggested that metabolites of fermentation, in addition to ginsenosides, contribute to the health benefits of FG.

Introduction

The prevalence rates of hyperglycemia, insulin resistance, central obesity, hypertension, and dyslipidemia have been increasing in developed, as well as developing, countries since Dr. Reaven introduced the idea of the metabolic syndrome as syndrome X in 1988.^1
–3 Metabolic syndromes are currently a major worldwide epidemic. Chronic hyperglycemia induces insulin resistance resulting in Type 2 diabetes mellitus (T2DM), which increases the risk of complication of vascular diseases.⁴ To prevent metabolic syndrome and the associated diseases, peroxisome proliferator activated receptor (PPAR) agonists were developed, and their effectiveness to reduce insulin resistance, glycemia, and hypertriglyceridemia has been shown through clinical trials and in vivo and in vitro studies.⁵ PPARs include three isotypes, PPAR-α, PPAR-γ, and PPAR-δ. PPAR-α agonists, known as fibrates, are used clinically for dyslipidemia, resulting in decreased triglyceride levels and increased high density lipoprotein levels. In contrast, PPAR-γ agonists, such as thiazolidinediones, are involved in improving insulin sensitivity.⁶ However, several undesirable side effects, such as edema, heart failure, and bladder cancer, have been reported.^7,8

Ginseng (Panax ginseng, C. A. Meyer, Araliaceae) is one of the most popular herbal medicines in Korea, China, and Japan. Ginseng, as well as its major constituents, including ginsenosides Rb1, Rb2, Rc, Rd, Re, and Rg1, has been shown to possess various biological activities against inflammation, stress, hepatopathy, and metabolic disorders.^9,10,11 Some other contents of ginseng, such as glycans and peptides, also contribute to its wide-ranging benefits.¹² Ginseng and its constituents have demonstrated potential for improving metabolic disorders. In terms of lipid metabolism, ginseng extract containing ginsenosides, proteins, and lipids exerted a hypolipidemic effect in a human study.¹³ In addition, the ginsenoside Rf was reported to regulate lipoprotein metabolism through PPAR-α,¹⁴ while an inhibitory effect of ginseng on PPAR-α functions has also been reported.¹⁵ However, supplementation with Korean red ginseng, which is steamed ginseng roots, resulted in no significant changes in metabolic parameters in a clinical study.¹⁶ Thus, the benefits of ginseng for metabolic disorders has been controversial.

Fermented ginseng (FG), which contains metabolites of ginsenosides produced by fermentation or enzymatic techniques, was shown to have a wide variety of pharmacological activities. FG extracts were shown to exert an antihyperglycemic effect in T2DM mice.¹⁷ However, the active constituents of FG for metabolic disorders have not yet been clarified. We previously reported the protective effect of FG against acetaminophen-induced rat liver injury,¹² in which the transcription activity of PPAR-α indicated a tendency to be increased by FG treatment in rats. This observation suggested the presence of a PPAR-α agonist in FG, which prompted us to perform this investigation. As part of our study on the beneficial health effects of FG, we performed a search for a PPAR-α agonist in FG using bioassay-guided fractionation, which resulted in the isolation of its active constituent. We describe herein the isolation and characterization of this compound.

Materials and Methods

General experimental procedure

Optical rotations were measured with a JASCO DIP-370 digital polarimeter (Jasco, Inc., Easton, MD, USA). Mass spectrometry (MS) was obtained on a Waters LCT PREMIER 2695 (Waters, Milford, MA, USA). 1D and 2D NMR spectra were measured on a Bruker AVANCE-500 instrument using tetramethylsilane (0.03%, v/v) as an internal standard. Column chromatography was performed with silica gel 60 N (63–210 μm, Kanto Chemical Co., Inc., Tokyo, Japan), Sephadex LH-20 (25–100 μm; GE Healthcare, Buckinghamshire, United Kingdom), MCI-gel CHP20P (75–150 μm; Mitsubishi Chemical, Japan), and YMC-gel ODS-A (S-50 μm; YMC Co., Ltd., Tokyo, Japan). Preparative HPLC was performed on JASCO apparatus consisting of a PU-980 prep. pump, UV-970 UV/VIS (at a wavelength of 210 nm), and RI-930 with COSMOSIL Cholester (20 mm i.d. × 250 mm; 5 μm; Nacalai Tesque, Inc., Tokyo, Japan).

Preparation of FG

FG was prepared in the same way as described previously.¹² FG was produced by fermentation of a culture medium, including a ginseng (age 5 years) mixture (15%) and Lactobacillus paracasei A221 (2%, 10¹⁰ colony-forming units [CFU]/mL) for 10 days at 28°C. The fermented culture was powdered by the spray dry method.

Extraction and isolation

FG (1.0 kg) was extracted with MeOH (2.5 L) at room temperature thrice. After removal of the solvent by evaporation, the MeOH extracts (467 g) were partitioned successively between CHCl₃, EtOAc, and H₂O. The CHCl₃-soluble (28.9 g) and EtOAc-soluble (16.3 g) fractions were mixed together based on the results of the luciferase reporter assay and thin-layer chromatography (TLC) examination and subjected to column chromatography over silica gel (CHCl₃: MeOH = 1:0 → 6:4) to give 16 fractions (frs. 1–16). Compound 1 was obtained by the crystallization of fr. 2 from n-hexane-acetone (310 mg). Fraction 3 (3.6 g) was further chromatographed on silica gel n-hexane-EtOAc (7:3 → 1:4) to yield four fractions (frs. 3.1–3.4). Crystallization of fr. 3.1 from acetone furnished 20(R)-protopanaxadiol (PAG020, 328 mg), while 20(S)-protopanaxadiol (PAG021) was obtained from fr. 3.2 by crystallization with EtOAc (334 mg).¹⁸ Silica gel chromatography of fr. 13 (1.3 g) with EtOAc:MeOH:H₂O = 20:1:0.3 gave 20(S)-ginsenoside Rh1 (PAG062, 11 mg) and 20(R)-ginsenoside Rh1 (PAG061, 43 mg).¹⁸ Compound 1 colorless needles; [α]_D 0.0 (c 0.20, MeOH); HRESI-MS m/z 299.2591 [M-H]⁻ (calcd for C₁₈H₃₅O₃, 299.2586); ¹H-NMR (CDCl₃, 500 MHz) δ: 0.86 (3H, t, J = 6.5 Hz), 1.23–1.37 (20H, m), 1.37–1.49 (6H, m), 1.63 (2H, m), 2.33 (2H, t, J = 7.5 Hz), 3.59 (1H, m); ¹³and C-NMR (CDCl₃, 125 MHz) δ: 14.1 (CH₃), 22.6, 24.7, 25.5, 25.6, 29.0, 29.1, 29.2, 29.3, 29.6, 29.6, 29.7, 31.9, 34.0, 37.3, 37.4, 72.1 (C-OH), 179.2 (COOH).

DNA microarray data analysis

DNA microarray data were obtained from a hepatoprotective study of FG according to the procedure described in our previous report.¹² Rat liver samples of four groups (9 weeks Slc: Wistar, SPF male) were obtained in the animal experiments. Rats (n = 3) with an oral pretreatment of FG (50 mg/kg) for eight consecutive days or without FG pretreatment were administered acetaminophen (APAP: 500 mg/kg) to induce liver injury. The liver samples of these two groups were used for the DNA microarray analysis. Another two groups, vehicle-treated or FG-treated rats both without APAP administration, were also prepared for the microarray analysis. Median values of measured gene expressions in each group were calculated and compared among the four groups; these values were expressed as fold changes relative to the vehicle-treated rat sample. A relative fold change value more than 5-fold was considered to be significant.

Plasmid construction for reporter assay

To measure the transcription activities of PPAR-α and -γ, pcDNA3.1-GAL4-PPAR-α, pcDNA3.1-GAL4-PPAR-γ, and upstream activating sequence (UAS) reporter plasmids were constructed as follows. Full-length rat's PPAR-α or mouse's PPAR-γ was subcloned into the pcDNA3.1 His-V5 vector (Invitrogen, Carlsbad, CA, USA), where the GAL4 gene was inserted.¹⁹ Four copies of the UAS sequence were subcloned into pGL3-luciferase reporter vector (Promega Corporation, Madison, WI, USA). pSV-β-galactosidase expression vector was obtained from Promega Corporation.

Cell culture and transfection

CV-1 cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 2 mM l-glutamine, and 50 μg/mL penicillin and streptomycin. After overnight incubation of the cells at 37°C in a humidified atmosphere of 5% CO₂-95% air, three plasmids containing UAS reporter plasmid and β-galactosidase expression vector with GAL4-PPAR-α or GAL4-PPAR-γ plasmid were cotransfected into CV-1 cells by TransFast™ (Promega Corporation). At 48 h after cotransfection, each fraction obtained by column chromatography of the FG extract was added to medium at doses of 5 or 20 μg/mL and the cells were further incubated for 24 h. WY14643 (Calbiochem) and troglitazone (Sigma, St. Louis, MO, USA) were added at the dose of 10 μM as PPAR-α and PPAR-γ agonist, respectively. Cells were collected by passive lysis buffer (Promega Corporation) and incubated for 1 h under room temperature. The luciferase activities of the lysates were measured using a Luciferase Assay Kit (Promega Corporation) with Luminescencer JNR II (ATTO, Tokyo, Japan) and were normalized with the β-galactosidase activities.

Complex of PPAR-α with coactivator

The binding between PPAR-α and CREB binding protein (CBP) known as a coactivator of PPAR-α was measured using an ELISA method (Fujikura Kasei Co., Ltd., Tokyo, Japan). CBP peptides were coated into 96-well plates for 1 h at room temperature. The recombinant PPAR-α was mixed with each compound 1 at the dose of 0.005, 0.05, 0.5, 5, 50, and 500 μM and the mixture was incubated in the plates for 1 h at room temperature. The plates were washed with wash buffer thrice and incubated with an HRP coupled secondary antibody for 30 min at room temperature. Tetramethylbenzenes were added to detect the complexes and 450 nm absorbance was measured. WY14643 was used as a positive control.

Statistical analysis

For the reporter assay, significant difference was determined by Student's t-test. P < .01 was considered to be statistically significant.

Results

Downregulated gene expressions of APAP-treated rat livers and the effects of FG

DNA microarray analyses were conducted on the APAP-treated livers. Six genes, including Stac3, Bhmt1, Ca3, Fads1, Scd1, and Fmo1, were downregulated more than 5-fold (Table 1). Stac3 gene expression in the APAP-treated rat liver samples was downregulated 15.5-fold compared to that of the control samples. All of the downregulated genes were recovered in the FG-pretreated rat samples, with Stac3 gene expression 9.7-fold higher than in the samples without the FG pretreatment.

Table 1.

The Downregulated Genes More Than 5-Fold by APAP-Administration and Alterated by FG Pretreatment With or Without APAP Treatment

	Accession No.	Gene name	Symbol	APAP (A)	FG+APAP (B)	FG (C)	A/B
Downregulated Genes	NM_001130558	SH3 and cysteine-rich domain 3	Stac3	−15.5	−1.6	1.1	9.7
	NM_030850	Betaine—homocysteine S-methyltransferase 1	Bhmt1	−11.5	−2.6	1.4	4.4
	NM_019292	Carbonic anhydrase 3	Ca3	−6.7	−3.7	1.0	1.8
	NM_053445	Fatty acid desaturase 1	Fads1	−6.4	−2.9	1.1	2.2
	NM_139192	Acyl-CoA desaturase 1	Scd1	−6.0	−2.3	−1.3	2.7
	NM_012792	Hepatic flavin containing monooxygenase 1	Fmo1	−5.3	−2.9	1.7	1.8

The gene expressions in the rat livers were analyzed by DNA microarray. (A) The fold changes were calculated by comparison between gene expressions of vehicle-treated rats and APAP-treated rats. (B) The fold changes were calculated by comparison between gene expressions of vehicle-treated rats and APAP-treated rats pretreated with FG. (C) The fold changes were calculated by comparison between gene expressions of vehicle-treated rats and FG pretreated rats.

FG, fermented ginseng.

PPAR agonist activity of fractions from the MeOH extract of FG

Luciferase reporter assay indicated that the transcription activities of PPAR-α for the CHCl₃- and EtOAc-soluble fractions were 1.7- and 1.3-fold higher compared with the vehicle control, respectively (Table 2). In contrast, the H₂O-soluble fraction did not show PPAR-α transcription activity (data not shown). TLC examination of the CHCl₃- and EtOAc-soluble fractions showed the presence of similar compounds. Therefore, both fractions were mixed together and subjected to silica gel column chromatography to give 16 fractions. Compound 1, obtained from fr. 2 by crystallization, showed 12.6-fold higher transcription activity than the control at 5 μg/mL (Table 2). Four ginsenoside metabolites were also isolated from the combined fraction. PAG020, PAG021, PAG061, and PAG062 did not exert PPAR-α transcription activities. Compound 1 exerted 5.5-fold higher PPAR-γ activity at 20 μg/mL, although it did not show transcriptional activity of PPAR-γ at 5 μg/mL (Table 3). To clarify whether compound 1 interacts with PPAR-α directly, detection for the complex of PPAR-α and the coactivator by the ELISA method was performed. Compound 1 behaved as an agonist of PPAR-α forming complex of the CBP. EC₅₀ of compound 1 and WY14643 1 was 2.1 and 4.1, respectively (Fig. 1).

FIG. 1.

PPAR-α agonist activity for compound 1 and WY14643 measured by an ELISA method. Coactivator peptides were coated on 98-well plates. Compound 1 and WY14643 were added at the dose of 0.005, 0.05, 0.5, 5, 50, and 500 μM together with recombinant PPAR-α. The complex of the coactivator and PPAR-α was detected on the absorbance at 450 nm. PPAR, peroxisome proliferator activated receptor.

Table 2.

Transcription Activities of PPAR-α in the Treatment of FG-Fractions and Isolated Compounds

		Luciferase activity ratio (μg/mL)
	Yield (g)	5	20
Vehicle	—	1.0 ± 0.2
CHCl₃-soluble fraction	28.9	1.7 ± 0.2^a	—
EtOAc-soluble fraction	16.3	1.3 ± 0.2	—
Vehicle	—	1.0 ± 0.1
Fraction number
1	2.3	3.2 ± 0.8^a	9.1 ± 0.7^a
2	5.7	11.7 ± 1.0^a	17.8 ± 1.3^a
3	3.6	1.5 ± 0.2	3.2 ± 0.4^a
4	3.8	1.9 ± 0.3^a	7.4 ± 1.0^a
5	2.8	2.0 ± 0.3^a	3.6 ± 0.2^a
6	1.2	1.7 ± 0.2^a	3.2 ± 0.4^a
7	1.0	1.2 ± 0.1	1.8 ± 0.2^a
8	1.5	1.4 ± 0.2	2.2 ± 0.7
9	6.9	1.1 ± 0.2	1.6 ± 0.2^a
10	1.9	1.2 ± 0.1	1.6 ± 0.1^a
11	0.8	1.6 ± 0.1^a	1.4 ± 0.1^a
12	1.9	1.5 ± 0.2^a	1.6 ± 0.2
13	1.3	1.2 ± 0.3	1.7 ± 0.3
14	1.5	1.4 ± 0.3	1.7 ± 0.1^a
15	0.5	1.1 ± 0.1	1.4 ± 0.1^a
16	2.1	1.4 ± 0.1^a	0.6 ± 0.3
Compound 1	0.7	12.6 ± 1.8^a	13.3 ± 1.2^a
20(R)-protopanaxadiol (PAG020)	0.3	1.1 ± 0.1	1.4 ± 0.0^a
20(S)-protopanaxadiol (PAG021)	0.3	1.5 ± 0.2	1.2 ± 0.7
20(R)-ginsenoside Rh1 (PAG061)	0.1	1.5 ± 0.2	1.4 ± 0.2
20(S)-ginsenoside Rh1 (PAG062)	0.5	1.5 ± 0.2	1.5 ± 0.2
WY14643 (10 μM)		15.7 ± 0.5^a

The ratio of luciferase activity was indicated mean value ± SD (n = 3).

P < .01 compared with vehicle controls.

PPAR, peroxisome proliferator activated receptor; SD, standard deviation.

Table 3.

Transcription Activities of PPAR-γ in the Treatment of Compound 1

	PPAR-γ: luciferase activity ratio (μg/mL)
	5	20
Vehicle	1.0 ± 0.2
Compound 1	1.2 ± 0.3	5.5 ± 0.5^a
Troglitazone (10 μM)	4.6 ± 0.9^a

The ratio of luciferase activity was indicated mean value ± SD (n = 3).

P < .01 compared with vehicle controls.

Structural analyses

Compound 1 (1) was obtained in the form of optically inactive colorless needles, and its molecular formula was determined as C₁₈H₃₅O₃ by HRESI-MS (m/z 299.2591 [M-H]⁻, calcd for C₁₈H₃₅O₃, 299.2586). The ¹H- and ¹³C-NMR spectra of 1 showed the presence of a terminal methyl [δ_H 0.86 (3H, t, J = 6.5 Hz); δ_C 14.1], an oxygen-bearing methine [δ_H 3.59 (1H, m); δ_C 72.1], and a carboxyl group (δ_C 179.2), together with 15 methylene groups. These spectral data suggested 1 to be a monohydroxy-octadecanoic acid. Since the position of the hydroxy group could not be determined by NMR spectral examination, the EI-MS fragment of the methyl ester of 1 (1a) was analyzed and compared with that of methyl 12-hydroxy-stearate. The EI-MS of 1a revealed peaks at m/z 201 and 172 due to α-cleavage, together with a prominent base peak at m/z 169 due to the loss of MeOH from the fragment at m/z 201. These fragment peaks were well correlated with those (m/z 229, 200, and 197) observed in the EI-MS of methyl 12-hydroxy-stearate. Each peak of methyl 12-hydroxy-stearate was 28 mass units larger than those found in 1a. This observation indicated the position of the hydroxy group in 1 to be C-10. From these observations, the structure of 1 was assigned as 10-hydroxy-octadecanoic acid.

Discussion

In the DNA microarray analyses, Stac3 gene expression was drastically downregulated by APAP-induced liver injury, and FG pretreatment attenuated the alteration. In contrast, FG pretreatment without APAP administration did not alter Stac3 expression. Thus, the results implied that FG exerted a beneficial effect on APAP-induced liver injury. The Stac3 gene was reported to be associated with lipid metabolism and to interact with PPAR-α.^20,21 Furthermore, the other altered genes, Fads1, Scd1, and Fmo1, which are listed in Table 1, are also known to regulate lipid metabolisms through PPAR-α activity.^22
–24 Therefore, FG was suggested to contain an active constituent stimulating PPAR-α.

To identify the PPAR agonist in the FG, we attempted to isolate the compound by PPAR-α transcription reporter assay-guided fractionation. The MeOH extract of FG was partitioned successively among CHCl₃, EtOAc, and H₂O to give CHCl₃-, EtOAc-, and H₂O-soluble fractions. The transcription activities of PPAR-α of the CHCl₃- and EtOAc-soluble fractions were 1.7- and 1.3-fold higher compared with the vehicle control, respectively. Since TLC examination showed the presence of similar compounds in the CHCl₃- and EtOAc-soluble fractions, they were mixed together and fractionated by silica gel column chromatography to give 16 fractions. Among these, the highest transcription activity of PPAR-α was found in fr. 2 at the doses of both 5 and 20 μg/mL. Compound 1 was obtained by crystallization from fr. 2, whose capability of interaction with PPAR-α was assessed by an ELISA method. The result indicated that PPAR-α formed a complex with CBP by compound 1, confirming compound 1 to be the PPAR agonist in the FG.

The estimated PPAR agonist activity for the combined CHCl₃ and EtOAc fraction based on the isolated amounts was similar to those of the CHCl₃ and EtOAc fractions. Thus, compound 1 was suggested to contribute most of the PPAR agonist activity of the combined CHCl₃ and EtOAc fraction. Furthermore, compound 1 exerted 5.5-fold higher transcription activity of PPAR-γ than the control at the dose of 20 μg/mL, although it did not show transcription activity at the dose of 5 μg/mL. The results of this study indicate that compound 1 is the major PPAR-α agonist with weak PPAR-γ agonist activity in FG. Korean red ginseng and ginsenosides were shown to improve insulin sensitivity,^25,26 and ginsenosides Rb1 and Re have been reported to be PPAR-γ agonists.^27,28 Therefore, compound 1 appeared to be an additional PPAR-γ agonist in FG.

The structure of compound 1 was classified as 10-hydroxy-octadecanoic acid by extensive spectroscopic analysis, including ¹H- and ¹³C-NMR spectra, and by EI-MS analysis of the methyl ester of 1. Monohydroxy-unsaturated fatty acids were known to exert PPAR-α- and PPAR-γ-agonist activity.²⁹ These data suggest that 10-hydroxy-octadecanoic acid in FG exerts PPAR-α- and PPAR-γ-agonist activity. In addition, Lactobacillus plantarum is known to produce 10-hydroxy-octadecanoic acid from linoleic acid.³⁰ Therefore, 10-hydroxy-octadecanoic acid is considered to be a fermentation product of L. paracasei A221.

In the present study, we identified 10-hydroxy-octadecanoic acid as an agonist of PPAR-α and -γ in FG. Not only the ginsenoside metabolites produced by L. paracasei A221 fermentation in FG but also the secondary metabolite of L. paracasei A221 contribute to the wide-ranging pharmacological effects of FG.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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