Isolation and Purification of Harpagogenin as an Nrf2–ARE Activator from the Tubers of Chinese Artichoke (Stachys sieboldii)

Abstract

Chinese artichoke tuber (Stachys sieboldii Miq.) is used as an herbal medicine as well as edible food. This study examined the effect of the Chinese artichoke extracts on the nuclear factor erythroid 2-related factor 2 (Nrf2)–antioxidant response element (ARE) pathway that induces the expression of antioxidant enzymes to explore its novel characteristics. Hot water extracts exhibited relatively high ARE activity. ARE activity was observed in two fractions when the hot water extracts were separated in the presence of trifluoroacetic acid using HPLC. Conversely, the highly active fraction disappeared when the hot water extracts were separated in the absence of trifluoroacetic acid. These results indicate that acidic degradation produces active ingredients. The structural analysis of the two active fractions identified harpagide, which is an iridoid glucoside, and harpagogenin. In vitro experiments revealed that harpagide was converted into harpagogenin under acidic conditions and that harpagogenin, but not harpagide, had potent ARE activity. Therefore, this study identified harpagogenin, which is an acid hydrolysate of harpagide, as an ARE activator and suggests that Nrf2–ARE pathway activation by Chinese artichoke contributes to the antioxidative effect.

INTRODUCTION

Chinese artichoke (Stachys sieboldii Miq.) is a perennial herbaceous plant that originated in China. Traditional Chinese medicine prescribed Chinese artichoke for many diseases, such as urinary tract infections, colds, cardiac pain, tuberculosis, pneumonia, etc.^1,2) Chinese artichoke tuber (“chorogi” in Japanese or “Crosne” in French) is used as an edible food in Japan and France. The tuber is eaten raw, cooked, or pickled, in salads, soups, and as garnish.

Chinese artichokes have been consumed as a vegetable for centuries, and several studies have reported its health benefits. For example, a Chinese artichoke extracts exhibited a modulating effect of hyaluronidase activity,³⁾ ability to reduce anoxia,¹⁾ and antinephritic activity.⁴⁾ More recently, the extracts have been reported to have a neuroprotective effect and to protect against learning and memory dysfunction.^5–7) The neuroprotective effect may attribute to the antioxidative effect.⁸⁾

Radical-scavenging activities might play an important role in the antioxidative effect of phytochemicals from foods. Polysaccharides extracted from Chinese artichoke tubers present high scavenging activity toward superoxide anion and hydroxyl radicals.²⁾ Besides, antioxidative enzyme upregulation has received considerable attention as another antioxidative effect. Accumulating evidence suggests that many phytochemicals enhance the antioxidant enzyme expression.⁹⁾ The enhanced antioxidant enzyme expression is mainly controlled by the nuclear factor erythroid 2-related factor 2 (Nrf2)–antioxidant response element (ARE) pathway.¹⁰⁾ Accumulating evidence has shown that the activation of the Nrf2–ARE pathway is useful in various cells.¹¹⁾ Unfortunately, the Nrf2–ARE activity of Chinese artichoke tubers remains unknown.

The Chinese artichoke tubers are rich in oligosaccharides, especially stachyose.¹²⁾ Furthermore, the tubers are characterized by secondary metabolites, including iridoid glycosides, such as harpagide, and phenylethanoid glycosides, such as acteoside and stachysoside.¹⁾ However, our knowledge of the pharmacological property of their phytochemicals is limited. The present study has identified a degradation product of harpagide as a novel Nrf2–ARE activator to determine the Nrf2–ARE activity of Chinese artichoke tubers.

MATERIALS AND METHODS

Cell Culture and Luciferase Reporter Analysis

Previously, reporter cells containing a luciferase gene under the control of ARE in the promoter region were generated from rat adrenal pheochromocytoma PC12 cells.¹³⁾ The reporter PC12 cells were maintained in Dulbecco’s modified Eagle medium containing 300 µg/mL of hygromycin B supplemented with 5% fetal calf serum and 10% horse serum. Cell cultures were incubated at 37 °C in a humidified atmosphere of 95% air and 5% CO₂. Equal amounts of Steady-Glo luciferase assay reagent (Promega, Madison, WI, U.S.A.) were added into the cell culture medium 9 h after treatment. The luminescence intensities were detected on a Wallac 1420 ARVOsx Multilabel Plate Reader (PerkinElmer, Inc., Waltham, MA, U.S.A.) to quantify the ARE induction.

Chinese Artichoke Extracts Preparation

Chinese artichoke tubers from China were obtained from the local market in Hyogo (Japan). The extraction of Chinese artichoke was performed at the Kameoka Plant of Koshiro Co., Ltd. (Osaka, Japan) and was provided. Chinese artichoke tubers (100 g; wet weight) were chopped up into small pieces and extracted with the solvent (1 L) described below for 1 h. The extraction was filtrated with 150 mesh and evaporated to dryness in a vacuum rotary evaporator. The dried product was pulverized in a mortar.

Purification of the Active Compound

The extracts were dissolved in water and was separated by HPLC with UV detection (Shimadzu, Kyoto, Japan, LC-10A) using a COSMOSIL 5C18-MS-II column (150 × 4.6 mm i.d., with 5 µm particle size) (Nacalai Tesque, Kyoto, Japan) using a flow rate of 1 mL/min (water/acetonitrile mobile phase) at 40 °C. The injected volume was 50 µL and the detection was performed at a 200-nm wavelength. The eluates were collected in volumes of 1 mL for 1 min each. The fractions were evaporated to dryness under nitrogen gas and freeze-dried. The residues were dissolved in water or dimethyl sulfoxide (DMSO).

NMR and MS

Chinese artichoke extracts were cleaned-up via solid-phase extraction before isolation with HPLC for structural analysis. The extracts were applied to a Sep-Pak C18 cartridge (Waters, Milford, MA, U.S.A.) and eluted with water-10% acetonitrile. The structure of the isolated compound was chemically identified by NMR and MS. An AVANCE III 500 spectrometer (Bruker BioSpin, Rheinstetten, Germany) at 500 or 125 MHz in DMSO-d₆ or D₂O was used to record NMR. Chemical shifts for ¹H- and ¹³C-NMR were referenced to tetramethylsilane (0.00 ppm). Assignments were made via ¹H-NMR, ¹³C-NMR, distortionless enhancement by correlation spectroscopy (COSY), nuclear Overhauser effect spectroscopy (NOESY), heteronuclear single-quantum correlation spectroscopy (HSQC), and heteronuclear multiple-bond correlation (HMBC). FAB-MS analysis was performed on a JEOL JMS-700 mass spectrometer (Tokyo, Japan). Electrospray ionization (ESI)-MS analysis was performed on an LCMS-IT-TOF (Shimadzu).

Evaluation of Cell Viability

The lactate dehydrogenase (LDH) release assay was used to determine cell viability. Briefly, 10 µL of culture supernatant was mixed with 90 µL of the LDH substrate mixture (174 mM of lactate, 1.5 mM of nicotinamide adenine dinucleotide, 815 µM of nitroblue tetrazolium, and 13.3 U/mL of diaphorase (Oriental Yeast Co., Ltd., Tokyo, Japan)) in 50 mM of Tris–HCl; pH 8.5. Absorbance was measured at 570 nm after incubation for 20 min at room temperature. Cell viability was calculated as 100% minus the ratio of the activity of the released LDH to total activity; LDH was released by exposure to 6-hydroxydopamine (1 mM, Sigma, St. Louis, MO, U.S.A.) for 48 h.

Statistics

The differences between groups were analyzed using one-way ANOVA and post hoc multiple comparisons using Tukey’s test. Probability values of less than 5% were considered significant. Data are expressed as the mean ± standard error of the mean.

RESULTS

Comparison of Extraction Solvents in ARE Activation

This study primarily aimed to reveal the active ingredients for the Nrf2–ARE pathway in Chinese artichoke extracts. Chinese artichoke tubers were extracted with hot water and 30, 50, or 95% ethanol. The extraction yield was 16.4, 14.9, 16.3, or 5.1% (w/w), respectively (Supplementary Fig. 1). Each extract was subjected to reversed-phase HPLC with a C18 column and a water/acetonitrile solvent system containing 0.1% trifluoroacetic acid (TFA) under a gradient of 1–30% acetonitrile for 30 min (Supplementary Fig. 2). PC12 reporter cells were treated with the Chinese artichoke extracts for 9 h. Each extract concentration-dependently increased luciferase activity (Fig. 1). The extracts with hot water exhibited the highest activity, whereas the extracts with 95% ethanol showed the lowest activity. Therefore, hot water extraction was considered optimal for extracting active ingredients. Sulforaphane is a well-known ARE activator and was used as a positive control.

Fig. 1. Effect of Chinese Artichoke Extracts on ARE-Dependent Transcriptional Activity

Chinese artichoke tubers (100 g; wet weight) were extracted with indicated solvents. PC12 reporter cells were treated with the Chinese artichoke extracts or sulforaphane (3 µM) for 9 h. The concentrations of each extract were converted to wet weight. ARE-dependent transcriptional activity was determined by luciferase assay. n = 3. ** p < 0.01, *** p < 0.001, compared with control.

Bioactivity-Guided Purification of the ARE Activator from Chinese Artichoke

The Chinese artichoke extracts in hot water was subjected to reversed-phase HPLC with a C18 column and a water/acetonitrile solvent system containing 0.1% TFA under a gradient of 1–30% acetonitrile for 30 min (Fig. 2A). TFA is frequently used as a mobile phase additive for reversed-phase HPLC. Fraction 11 exhibited a markedly elevated luciferase activity (Fig. 2B). The eluted active fraction 11 was collected and subjected to the second purification step. Two peaks (A and B) showed luciferase activities corresponding to fractions 11–12 and 18, respectively, in the second purification by HPLC under an isocratic condition of 3% acetonitrile containing 0.1% TFA (Figs. 2C, D). The fraction was repeatedly subjected to HPLC under the same conditions (an isocratic condition of 3% acetonitrile containing 0.1% TFA) because fractions 11–12 still seemed to contain several components. Surprisingly, peak B was again detected from fractions 11–12 (peak A) of the second step (Fig. 2E). These results indicate that peak A produces peak B during the second purification step. The reason may be ingredient decomposition in the evaporation to dryness process.

Fig. 2. Chromatographic Purification of Active Fraction from Hot Water Extracts of Chinese Artichoke in the Presence of TFA

A: HPLC chromatogram of the first purification step of Chinese artichoke hot water extracts. The black line indicates UV absorption (200 nm) and the gray line indicates the concentration of acetonitrile (1–30%) containing 0.1% TFA. The eluate of 10–11 min surrounded by a broken line corresponds to fraction 11. B: Effects of first HPLC fractions of Chinese artichoke extracts on ARE activity. PC12 reporter cells were treated with each fraction of Chinese artichoke or sulforaphane (3 µM) for 9 h. C: HPLC chromatogram of fraction 11 from the first purification step. The black line indicates UV absorption (200 nm) and the gray line indicates the acetonitrile (3%) concentration containing 0.1% TFA. The eluates of 10–12 min and 17–18 min surrounded by a broken line correspond to s A and B, respectively. D: Effects of second HPLC fractions of Chinese artichoke extracts on ARE activity. PC12 reporter cells were treated with each fraction of Chinese artichoke or sulforaphane (3 µM) for 9 h. E: HPLC chromatogram of peak A from the second purification step. The black line indicates UV absorption (200 nm) and the gray line indicates the concentration of acetonitrile (3%) containing 0.1% TFA. The concentration of each fraction was converted to wet weight. n = 3. ** p < 0.01, *** p < 0.001, compared with control.

Fractionation and ARE Activity of Chinese Artichoke Extracts without TFA

The hot water extracts of Chinese artichoke were subjected to HPLC under a gradient of 1–30% acetonitrile without TFA for 30 min to avoid ingredient decomposition (Fig. 3A). None of the fractions showed a markedly elevated luciferase activity, including fraction 11, which exhibited the activity in the presence of TFA (Fig. 3B). However, the eluted fraction 11 was collected and subjected to the second purification step by HPLC under an isocratic condition of 3% acetonitrile without TFA. Peak A was observed from 11 to 12 min but with no peak at the time (17–18 min corresponding to peak B (Fig. 3C). A markedly elevated luciferase activity was not shown in any fractions in the second purification step (Fig. 3D).

Fig. 3. Chromatographic Purification of Chinese Artichoke Hot Water Extracts in the Absence of TFA

A: HPLC chromatogram of the first purification step of Chinese artichoke hot water extracts. The black line indicates UV absorption (200 nm) and the gray line indicates the acetonitrile (1–30%) concentration without TFA. The eluate of 10–11 min surrounded by a broken line corresponds to fraction 11. B: Effects of first HPLC fractions of Chinese artichoke extracts on ARE activity. PC12 reporter cells were treated with each fraction of Chinese artichoke or sulforaphane (3 µM) for 9 h. C: HPLC chromatogram of fraction 11 from the first purification step. The black line indicates UV absorption (200 nm) and the gray line indicates the acetonitrile (3%) concentration without TFA. The eluates of 11–12 min surrounded by a broken line correspond to peak A. D: Effects of second HPLC fractions of Chinese artichoke extracts on ARE activity. PC12 reporter cells were treated with each fraction of Chinese artichoke or sulforaphane (3 µM) for 9 h. n = 3. ** p < 0.01, *** p < 0.001, compared with control.

Production of Peak B from Peak A under Acidic Conditions

Peak A was incubated under acidic conditions to confirm the decomposition of peak A to peak B. The eluted peak A of the second purification step without TFA was collected. The purified peak A was incubated in water containing 0.1% TFA (pH: 2.0) at 40 °C for 1–2 h. The reaction solution was subjected to HPLC under an isocratic condition of 3% acetonitrile without TFA. Peak A gradually decreased and peak B increased in a time-dependent manner (Fig. 4A). The conversion of peak A into peak B was also enhanced in a temperature-dependent manner (Figs. 4B, C) although excess acid hydrolysis resulted in peak B degradation (Fig. 4C). The first fluid (pH: 1.2) following the Japanese Pharmacopoeia disintegration and dissolution test was used as an artificial gastric juice equivalent. Peak A was promptly converted into peak B in the artificial gastric juice (pH: 1.2) at 37 °C (Fig. 4D).

Fig. 4. Acid Hydrolysis of Peak A to Peak B

(A–C): The purified peak A was incubated in 0.1% TFA (pH 2.0) at 40 (A), 60 (B), and 80 (C) °C for indicated periods. D: The purified peak A was incubated in the first fluid (0.2% NaCl, 0.26% HCl, pH 1.2) following to the Japanese Pharmacopoeia disintegration and dissolution test at 37 °C for indicated periods. Subsequently, the amounts of peak A and peak B were measured by HPLC without TFA.

Structural Analysis of Peak A from Chinese Artichoke

The collection by HPLC without TFA obtained 15 mg of peak A from 50 g wet weight of Chinese artichoke tubers. Positive low-resolution FAB-MS (DMSO/glycerol) gave the molecular ion [M + Na]⁺ at m/z 387 and the fragment ion at m/z 167. Negative low-resolution MS (DMSO/glycerol) gave the molecular ion [M–H]⁻ at m/z 363. Furthermore, positive high-resolution MS (DMSO/glycerol) found the base peak at m/z [M + Na]⁺ 387.1272 (Calcd for C₁₅H₂₄O₁₀Na: 387.1262). Supplementary Figs. 3A and B show ¹H- and ¹³C-NMR spectrum. This compound was estimated to be harpagide (C₁₅H₂₄O₁₀, Mw = 364) by two-dimensional NMR (2D-NMR) spectroscopy (COSY, NOESY, HSQC, and HMBC). The ¹H-NMR analysis demonstrated identical chemical shifts and peak patterns with the isolated compound in commercially available harpagide (data not shown). The optical rotation of the peak A was [α]_D²⁹=−152.7 (c = 0.83, MeOH) and that of commercially available harpagide was [α]_D²⁹=−171.1 (c = 0.83, MeOH). These results confirmed that peak A from Chinese artichoke was harpagide (Fig. 5A).

Fig. 5. Chemical Structures of Peak A and Peak B

A: Three-dimensional structure of harpagide (peak A). The relative stereostructure of peak A was confirmed by ¹H–¹H COSY, NOESY, HMBC, and HSQC experiments. B: Three-dimensional structure of harpagogenin (peak B). The relative stereostructure was confirmed by ¹H–¹H COSY, HMBC, and HSQC experiments.

Structural Analysis of Peak B from Chinese Artichoke

Peak B was obtained from peak A by decomposition in water containing 0.1% TFA at 80 °C for 20 min. Positive ESI-MS (DMSO) analysis gave the molecular ion [M + H]⁺ at m/z 167.0696 (Calcd for C₉H₁₁O₃: 167.0703). Negative ESI-MS (DMSO) analysis gave the molecular ion [M–H]⁻ at m/z. 165.0563 (Calcd. for C₉H₉O₃: 165.0557). ¹H- and ¹³C-NMR spectrum were shown in Supplementary Figs. 4A and B. This compound was estimated to be harpagogenin (C₉H₁₀O₃, Mw = 166) by 2D-NMR spectroscopy (COSY, HSQC, and HMBC). Harpagogenin has been reported as an acid hydrolysate of harpagide derived from Devil’s Claw (Harpagophytum procumbens).¹⁴⁾ The NMR spectrum of peak B isolated from Chinese artichoke demonstrated identical chemical shifts and peak patterns with the previously reported one of harpagogenin. Peak B was the (E)-geometric isomer following the chemical shifts and coupling constants of H-6 and H-7 (Fig. 5B)

Activation of ARE-Dependent Transcription by Harpagide and Harpagogenin

PC12 reporter cells were treated with harpagide and harpagogenin to validate the ARE activity of two isolated compounds. Whereas harpagide did not affect ARE activity, harpagogenin increased luciferase activity in a concentration-dependent manner (Figs. 6A, B). Harpagogenin demonstrated a lesser efficacy in ARE activation than sulforaphane.¹⁵⁾ Rather, the activation by harpagogenin decreased at a concentration of ≥200 µM (Fig. 6B). That was because harpagogenin at ≥100 µM exhibited cytotoxicity (Fig. 6C).

Fig. 6. Biological Properties of Compounds Isolated from Chinese Artichoke

A: Effect of harpagide on ARE activity. B: Effect of harpagogenin on ARE activity. PC12 reporter cells were treated with harpagide, harpagogenin, or sulforaphane for 9 h. n = 3. C: Effect of harpagogenin on cell viability. PC12 cells were treated with harpagogenin for 48 h. n = 4. * p < 0.05, *** p < 0.001, compared with control.

DISCUSSION

The present study demonstrated that harpagogenin, which is an acid hydrolysate of harpagide, possessed ARE activity. Chinese artichoke contained harpagide, but not harpagogenin. Oral intake of Chinese artichoke may produce harpagogenin because harpagide is suggested to be converted to harpagogenin in gastric juices.

Our results indicated that Chinese artichoke extracts can activate the Nrf2–ARE pathway. Especially, hot water extraction for active ingredients was more effective. However, any of the acid-free fractions of hot water extracts did not exhibit intensive ARE activity (Fig. 3B). This finding suggests that hot water extracts of Chinese artichoke do not include harpagogenin which was a responsible active ingredient. Therefore, the ARE activity of hot water extracts might result from a sum of low activity of acid-free fractions. The harpagide-containing fraction under acidic conditions was contaminated with harpagogenin. Hence, harpagide was converted into harpagogenin under acidic conditions.

Nrf2–ARE activators generally have several common structural characteristics.^16,17) Among them, harpagogenin has two α, β-unsaturated carbonyl moieties for the reaction of a Michael acceptor with a nucleophile. Cysteines of Kelch-like ECH-associated protein 1 (Keap1), which is an Nrf2 suppresser, were especially reactive to electrophilic reagents.¹⁷⁾ Harpagogenin may significantly covalently adduct with Keap1 although mechanisms for ARE activation of harpagogenin remain unresolved. ARE activation of harpagogenin demonstrated lower potency than sulforaphane but had similar efficacy.¹⁵⁾ However, high reactive compounds induce non-specific reactions. In fact, Fig. 6C showed the cytotoxicity of harpagogenin and Fig. 6B showed the lesser efficacy in ARE activation than sulforaphane. In addition, supplementary Fig. 5 showed that the stability of harpagogenin was relatively poor. Although these findings may limit the usefulness of harpagogenin as an ARE activator, further biological activity studies are needed.

Harpagogenin has been already reported as an acid hydrolysate of harpagide.¹⁴⁾ Excess hydrolysis reaction induces degradation of harpagogenin. From Fig. 4D, it was calculated that the yield of harpagogenin under the condition (pH 1.2, at 37 °C) for 2 h was 46.6%. Zhang et al.¹⁸⁾ reported that a hydrolyzed product of harpagide with β-glucosidase was different from harpagogenin. Therefore, harpagogenin may be produced in the stomach, but not in the intestine, when harpagide is taken orally. It was calculated that 15 mg of harpagide was obtained from 50 g wet weight of Chinese artichoke. Assuming a gastric residence time of 2 h, about half of harpagide is degraded to give harpagogenin. If the stomach volume is 1 L after eating 50 g of Chinese artichoke, the concentration of harpagogenin in the stomach is about 20 µM. Therefore, it is possible that eating Chinese artichoke activates the Nrf2–ARE pathway in gastric epithelial cells, although it is unclear whether harpagogenin is absorbed into the body.

Because harpagogenin possibly and easily binds amino and thiol groups, it may have biological activities other than ARE activation. Harpagogenin is not normally found in plants and is produced from harpagide in special environments. Therefore, there are very few papers on its biological activity. Anti-inflammatory, analgesic and spasmolytic effects have been reported, but details are unknown.^19,20) Intraperitoneal harpagogenin administration exhibited an anti-inflammatory effect in the granuloma pouch test.¹⁹⁾ To our knowledge, these are the only reports on the pharmacological property of harpagogenin. The relationship between Nrf2 and anti-inflammatory action has been often mentioned,²¹⁾ thus the anti-inflammatory effect of harpagogenin may be attributed to Nrf2–ARE pathway activation. Conversely, harpagide did not influence the tumor necrosis factor-α synthesis in lipopolysaccharide-stimulated monocytes.²²⁾

In conclusion, Nrf2–ARE pathway activation participates in the antioxidative effect of Chinese artichoke extracts. The present study identified harpagogenin as a responsible active ingredient. However, further investigations are required for the evaluation of the biological property of harpagogenin. Therefore, the intake of Chinese artichokes may reduce oxidative stress and contribute to human health.

Acknowledgments

This work was supported by a Grant from Nitto Pharmaceutical Industries, Ltd. (Kyoto, Japan).

Conflict of Interest

This study was funded by Nitto Pharmaceutical Industries, Ltd. (Kyoto, Japan).

Data Availability

The data that support the findings of this study are available from the corresponding author, upon reasonable request.

Supplementary Materials

This article contains supplementary materials.

REFERENCES

• 1) Yamahara J, Kitani T, Kobayashi H, Kawahara Y. Studies on Stachys sieboldii MIQ. II. Anti-anoxia action and the active constituents. Yakugaku Zasshi, 110, 932–935 (1990).
• 2) Feng K, Chen W, Sun L, Liu J, Zhao Y, Li L, Wang Y, Zhang W. Optimization extraction, preliminary characterization and antioxidant activity in vitro of polysaccharides from Stachys sieboldii Miq. tubers. Carbohydr. Polym., 125, 45–52 (2015).
• 3) Takeda Y, Fujita T, Satoh T, Kakegawa H. On the glycosidic constituents of Stachys sieboldi MIQ. and their effects on hyaluronidase activity. Yakugaku Zasshi, 105, 955–959 (1985).
• 4) Hayashi K, Nagamatsu T, Ito M, Hattori T, Suzuki Y. Acetoside, a component of Stachys sieboldii MIQ, may be a promising antinephritic agent: effect of acteoside on crescentic-type anti-GBM nephritis in rats. Jpn. J. Pharmacol., 65, 143–151 (1994).
• 5) Harada S, Tsujita T, Ono A, Miyagi K, Mori T, Tokuyama S. Stachys sieboldii (Labiatae, Chorogi) protects against learning and memory dysfunction associated with ischemic brain injury. J. Nutr. Sci. Vitaminol. (Tokyo), 61, 167–174 (2015).
• 6) Ravichandran VA, Kim M, Lee SJ, Jeong ES, Cha YS. Protective effects of Stachys sieboldii MIQ extract in SK-N-SH cells and its memory ameliorative effect in mice. J. Food Biochem., 41, e12411 (2017).
• 7) Ravichandran VA, Kim M, Han SK, Cha YS. Stachys sieboldii extract supplementation attenuates memory deficits by modulating BDNF-CREB and its downstream molecules, in animal models of memory impairment. Nutrients, 10, 917 (2018).
• 8) Venditti A, Frezza C, Celona D, Bianco A, Serafini M, Cianfaglione K, Fiorini D, Ferraro S, Maggi F, Lizzi AR, Celenza G. Polar constituents, protection against reactive oxygen species, and nutritional value of Chinese artichoke (Stachys affinis Bunge). Food Chem., 221, 473–481 (2017).
• 9) Dinkova-Kostova AT, Talalay P. Direct and indirect antioxidant properties of inducers of cytoprotective proteins. Mol. Nutr. Food Res., 52 (Suppl. 1), S128–S138 (2008).
• 10) Motohashi H, Yamamoto M. Nrf2-Keap1 defines a physiologically important stress response mechanism. Trends Mol. Med., 10, 549–557 (2004).
• 11) Tsuruta Y, Katsuyama Y, Okano Y, Ozawa T, Yoshimoto S, Ando H, Masaki H, Ichihashi M. Possible involvement of dermal fibroblasts in modulating Nrf2 signaling in epidermal keratinocytes. Biol. Pharm. Bull., 46, 725–729 (2023).
• 12) Yin J, Yang G, Wang S, Chen Y. Purification and determination of stachyose in Chinese artichoke (Stachys Sieboldii Miq.) by high-performance liquid chromatography with evaporative light scattering detection. Talanta, 70, 208–212 (2006).
• 13) Izumi Y, Matsumura A, Wakita S, Akagi K, Fukuda H, Kume T, Irie K, Takada-Takatori Y, Sugimoto H, Hashimoto T, Akaike A. Isolation, identification, and biological evaluation of Nrf2–ARE activator from the leaves of green perilla (Perilla frutescens var. crispa f. viridis). Free Radic. Biol. Med., 53, 669–679 (2012).
• 14) Vanhaelen M, Vanhaelen-Fastré R, Elchami AA, Fontaine J. Biological analysis of Harpagophytum procumbens D.C. I. Preparation and structure of harpagogenin. J. Pharm. Belg., 36, 38–42 (1981).
• 15) Izumi Y, Kataoka H, Inose Y, Akaike A, Koyama Y, Kume T. Neuroprotective effect of an Nrf2–ARE activator identified from a chemical library on dopaminergic neurons. Eur. J. Pharmacol., 818, 470–479 (2018).
• 16) Talalay P, De Long MJ, Prochaska HJ. Identification of a common chemical signal regulating the induction of enzymes that protect against chemical carcinogenesis. Proc. Natl. Acad. Sci. U.S.A., 85, 8261–8265 (1988).
• 17) Holtzclaw WD, Dinkova-Kostova AT, Talalay P. Protection against electrophile and oxidative stress by induction of phase 2 genes: the quest for the elusive sensor that responds to inducers. Adv. Enzyme Regul., 44, 335–367 (2004).
• 18) Zhang L, Feng L, Jia Q, Xu J, Wang R, Wang Z, Wu Y, Li Y. Effects of β-glucosidase hydrolyzed products of harpagide and harpagoside on cyclooxygenase-2 (COX-2) in vitro. Bioorg. Med. Chem., 19, 4882–4886 (2011).
• 19) Eichler O, Koch C. Antiphlogistic, analgesic and spasmolytic effect of harpagoside, a glycoside from the root of Harpagophytum procumbens DC. Arzneimittelforschung, 20, 107–109 (1970).
• 20) Fontaine J, Elchami AA, Vanhaelen M, Vanhaelen-Fastré R. Biological analysis of Harpagophytum procumbens D.C. II. Pharmacological analysis of the effects of harpagoside, harpagide and harpagogenine on the isolated guinea-pig ileum. J. Pharm. Belg., 36, 321–324 (1981).
• 21) Thimmulappa RK, Scollick C, Traore K, Yates M, Trush MA, Liby KT, Sporn MB, Yamamoto M, Kensler TW, Biswal S. Nrf2-dependent protection from LPS induced inflammatory response and mortality by CDDO-Imidazolide. Biochem. Biophys. Res. Commun., 351, 883–889 (2006).
• 22) Fiebich BL, Heinrich M, Hiller KO, Kammerer N. Inhibition of TNF-alpha synthesis in LPS-stimulated primary human monocytes by Harpagophytum extract SteiHap 69. Phytomedicine, 8, 28–30 (2001).