Chemical Constituents and Neuroprotective Activity of Hypericum hookerianum

Abstract

Objective/Background

The Hypericum hookerianum is widely distributed in Asian countries and prevalently used in traditional herbal medicines for its significant antioxidant, antipyretic, and anticancer properties. To date, very limited reports are available regarding the phytochemical profile, and there are no reports that have studied the neuroprotective potentials of this plant.

Experimental Methods

The aerial parts of H. hookerianum were collected from the Sapa mountainous district, Lao Cai province of Vietnam. The extracts were prepared using methanol, n-butanol, ethyl acetate, and chloroform and fractionated by normal-phase silica gel column chromatography to isolate compounds. The purity of the isolated compounds was analyzed by liquid chromatography combined with mass spectrometry. The structures were studied using nuclear magnetic resonance spectroscopy. All the isolates were evaluated for their neuroprotective activity against the neurotoxicity induced by glutamate in HT-22 hippocampal cells and 6-hydroxydopamine (6-OHDA) in SH-SY5Y neuroblastoma cells. The fluorescent dye calcein-AM image assay was used to measure the cell viability.

Results

Bioassay-guided fractionation of the aerial parts of H. hookerianum led to the isolation of 20 previously reported compounds (1-20), including 3 flavonoids (1-3), 2 xanthones (4 and 5), 1 chalcone (6), 4 lignans (7-0), 1 chromone-C-glycoside (11), 1 cinnamic acid derivative (12), 7 phloroglucinols (13-9), and 1 stilbene (20). The structures of the compounds were determined by spectroscopic methods and compared with the reported data. The extracts and isolated compounds showed neuroprotection in both cell models. The compounds 4-hydroxy-2,6,4′-trimethoxydihydrochalcone (6, EC₅₀ = 1.48 µM) and sesamin (10, EC₅₀ = 2.85 µM) exhibited significant neuroprotective effects in HT-22 and SH-SY5Y cells, respectively.

Conclusion

This is the first report on the neuroprotection of extracts and isolated compounds from H. hookerianum. The results provide information for further studies to develop products for the prevention and treatment of neurological disorders.

Keywords

4-hydroxy-26 4′-trimethoxydihydrochalcone sesamin neuroprotective activity

Introduction

Hypericum hookerianum Wight & Arnott, family Hypericaceae, is widely distributed in Asian countries, including China, Bangladesh, Bhutan, India, Myanmar, Nepal, and Thailand.¹ H. hookerianum has been used in traditional medicine in many countries to treat various diseases owing to its antipyretic, wound healing, antibacterial, and anticancer properties.^1–3 In Vietnam, H. hookerianum is abundant in the northern mountains (Sapa District, Lao Cai Province).⁴ To date, there have been few phytochemical studies of H. hookerianum indicating the presence of phloroglucinols, xanthones, phenolic acids, and triterpenoids.^5–9 The extracts and constituents of H. hookerianum possess antioxidant, anticancer, antiviral, and antibacterial activities.^5–9 The compounds isolated from some species of Hypericum, especially H. perforatum (commonly known as St. John's wort), have been exploited for developing various neuroprotective natural products in many countries.^9,10 However, no studies have examined the neuroprotective effects of H. hookerianum. This paper presents the isolation and evaluation of neuroprotective activity of H. hookerianum extracts and isolated compounds against glutamate and 6-hydroxydopamine (6-OHDA) neurotoxicity in HT-22 and SH-SY5Y cells.

Materials and Methods

General Experimental Procedures

Nuclear magnetic resonance (NMR) (¹H, ¹³C, heteronuclear multiple bond correlation [HMBC], heteronuclear single quantum coherence spectroscopy [HSQC], and correlated spectroscopy [COSY] NMR) experiments were performed using a Bruker AM500 FT-NMR spectrometer and a Bruker Avance NEO 600 NMR spectrometer for ¹H (500 and 600 MHz) and ¹³C (125 and 150 MHz) in dimethyl sulfoxide-d₆, deuterated chloroform (CDCl₃) and methanol-d₄ (CD₃OD) as solvents, with tetramethylsilane as an internal standard. The electrospray ionization-mass spectrometry (ESI-MS) was obtained using liquid chromatography LC/MS-8045 Shimadzu and Agilent 1260 Series Single Quadrupole LC/MS systems. Normal-phase and reversed-phase column chromatography was performed on silica gel (Merck, 63-200 µm) and YMC RP-18 resins (30-50 µm, Fujisilisia Chemical). Precoated silica gel 60_F254 (Merck) and RP-18_F254S (Merck) plates were used for thin-layer chromatography. The compounds were detected by spraying with 10% H₂SO₄ in ethanol.

Plant Material

The aerial parts of H. hookerianum were collected from the Sapa mountainous district, Lao Cai Province, Vietnam, in June 2016. The plant parts were authenticated by botanical expert Do Van Hai of the Institute of Ecology and Biological Research, Vietnam Academy of Science and Technology, and specialists of the Vietnam National Institute of Medicinal Materials. A voucher specimen was deposited at the Institute of Ecology and Biological Research, Vietnam Academy of Science and Technology (No.HH20160619).

Extraction and Isolation

The dried aerial parts of H. hookerianum (5.0 kg) were macerated using 3 volumes of MeOH (25 L) at room temperature for 4 days. The combined extracts were filtered and evaporated under reduced pressure to obtain a green residue (487 g). This crude extract was suspended in distilled H₂O (1 L) and partitioned successively with dichloromethane (CH₂Cl₂), ethyl acetate (EtOAc), and n-butanol. The CH₂Cl₂ extract (80.0 g) was initially fractionated using normal-phase silica gel column chromatography (CC) and eluted with a gradient of n-hexane-EtOAc (1:0, 10:1, 4:1, 2:1, 1:1, v/v; 2.5 L for each ratio) to give 7 fractions (Fr. D1-Fr. D7). The Fraction Fr. D5 was separated by octadecylsilyl (ODS) CC and eluted with MeOH-H₂O (1:2, 1:1, 2:1, 3:1, v/v), to yield 6 subfractions (Fr. D5.1-Fr. D5.6). Compounds 4 (8 mg), 5 (7 mg), 6 (9 mg), and 8 (6 mg) were purified from subfraction Fr. D5.2 by utilizing ODS CC eluting with MeOH-H₂O (1:1, 2:1, v/v). Subfraction Fr. D5.4 was separated by ODS CC (MeOH-H₂O; 2:1, 3:1, v/v) to obtain 9 (12 mg) and 10 (16 mg). Fraction Fr. D2 was chromatographed by ODS CC elution with a gradient system of MeOH-H₂O (2:1, 3:1, 4:1, 5:1, v/v) to give 5 subfractions (Fr. D2.1-Fr. D2.5). Compounds 12 (3 mg), 15 (8 mg), 16 (12 mg), and 19 (11 mg) were obtained from subfraction Fr. D2.1 by ODS CC elution with MeOH-H₂O (2:1, 3:1, v/v). Fraction Fr. D3 was divided into 3 subfractions (Fr. D3.1-Fr. D3.3) using ODS CC and gradient elution with MeOH-H₂O (2:1, 3:1, 4:1, v/v). Subfraction Fr. D3.2 was further subjected to ODS CC using a mobile phase of MeOH-H₂O (3:1, 4:1, v/v), leading to the isolation of compounds 17 (8 mg) and 18 (13 mg).

The EtOAc extract (75 g) was initially fractionated by normal-phase silica gel CC elution with a gradient of CH₂Cl₂-MeOH (1:0, 20:1, 10:1, 5:1, 2:1, 1:1, v/v) to yield 9 fractions (Fr. E1-Fr. E9). Fraction Fr. E1 was subjected to ODS CC using a gradient of MeOH in water to obtain subfractions Fr. E1.1-Fr. E1.5. Subfraction Fr. E1.4 was then purified by ODS CC using gradient conditions (MeOH-H₂O, 1:2 to 2:1, v/v), yielding compounds 1 (18 mg) and 2 (20 mg). Fraction Fr. E5 was fractionated on a Sephadex LH-20 column using MeOH as the eluent to obtain compounds 3 (9 mg), 7 (12 mg), and 11 (7 mg). Fraction Fr. E2 was subjected to 2 purification steps: (1) ODS CC (50%-100% MeOH in water) and (2) normal-phase silica gel CC, using isocratic CH₂Cl₂-acetone; 5:1, v/v to yield compound 20 (3 mg). Fr. E6 was chromatographed by ODS CC eluting with a gradient of MeOH in H₂O (1:2, 1:1, 2:1, v/v) to give 3 subfractions (Fr. E6.1-Fr. E6.3). Subfraction Fr.E6.3 was separated by ODS CC (MeOH-H₂O, 1:1 to 2:1, v/v), resulting in the isolation of compounds 13 (8 mg) and 14 (11 mg).

4-Hydroxy-2,6,4′-trimethoxydihydrochalcone (6) was evident as a pale yellow amorphous powder; ¹H and ¹³C NMR data, see Supplemental Table S1; ESI-MS m/z: 339.0 [M + Na]⁺.

Neuroprotective Activity Against Glutamate-Induced Toxicity in HT-22 Cells

HT-22 mouse hippocampal neuronal cells obtained from the Korean Cell Line Bank (Seoul, South Korea) were cultured in DMEM (Dulbecco's Modified Eagle Medium) high-glucose media with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (100 U/mL) at 37°C in an atmosphere of 5% CO₂. HT-22 cells (3000 per well) were seeded in 96-well plates and incubated for 24 h. Subsequently, the cells were pretreated with various concentrations of the samples for 2 h, followed by the addition of glutamate (5 mM). After 12 h incubation, the medium was removed and 100 µL of 0.5 µM calcein AM solution prepared in DPBS (Dulbecco's Phosphate Buffered Saline) was added. Live cells were imaged with the operetta high content screening system (Perkin Elmer, USA) using GFP channel (ex/em, 488 nm/514 ± 15), and counted using Harmony 3.5 software (Perkin Elmer, USA).

Neuroprotective Activity Against 6-OHDA-Induced Toxicity in SH-SY5Y Cells

SH-SY5Y neuroblastoma cells purchased from the Korean cell line bank (Seoul, South Korea) were cultivated in DMEM containing 10% FBS and 1% penicillin/streptomycin (100 U/mL) at 37°C under 5% CO₂. SH-SY5Y cells (50,000 per well) were seeded in 96-well plates and incubated for 24 h. Subsequently, the cells were treated with various concentrations of the samples for 2 h, followed by the addition of 6-OHDA (50 μM). After 24 h incubation, the medium was removed and 100 µL of 0.5 µM calcein AM solution prepared in DPBS was added. The live cells were imaged with the operetta high content screening system (Perkin Elmer, USA), and counted using Harmony 3.5 software (Perkin Elmer, USA).^11,12

Statistical analysis

GraphPad Prism software (version 6.0) was used for the statistical analysis and construction of bar graphs. The statistical significance of the compound treatment groups was determined using one-way analysis of variance. The P-values < .05 indicated statistical significance.

Results

Neuroprotective Activities of H. hookerianum Extracts

The neuroprotective effects of H. hookerianum extracts were assessed using a glutamate-induced cell death in HT-22 murine hippocampal cell model. HT-22 cells were treated with glutamate (5 mM) in either the absence or presence of 3 concentrations (5.56, 16.67, and 50 μg/mL) of the extracts. Cell viability was reduced to approximately 50% after exposure to 5 mM glutamate for 12 h. Interestingly, the reduction in cell viability induced by glutamate was not observed in the extract co-treated wells, except in the n-butanol fraction at 50 μg/mL, which showed cytotoxicity in HT-22 cells (Supplemental Figure S2). The data show that the H. hookerianum extracts strongly protect the HT-22 cells from glutamate-induced cell death. The dichloromethane and ethyl acetate fractions were chosen for further phytochemical investigation due to their strong neuroprotective effects.

Phytochemical Investigation

Using chromatographic methods, 20 known compounds were isolated from dichloromethane and ethyl acetate fractions of the methanol extract of H. hookerianum aerial parts. The structures of these isolates were identified as quercetin (1),¹³ (-)-epicatechin (2),¹³ quercitrin (3),¹³ 3-hydroxy-2,4-dimethoxyxanthone (4),¹⁴ neriifolone A (5),¹⁵ and 4-hydroxy-26,4′-trimethoxydihydrochalcone (6) (Supplemental Table S1 and Figure S1), isolariciresinol 9′-O-β-D-glucopyranoside (7),¹⁶ isocubein (8),¹⁷ piperitol (9),¹⁸ sesamin (10),¹⁸ 5,7-dihydroxy-2-(1-methylpropyl) chromone-8-β-D-glucopyranoside (11),¹⁹ ethyl-4-methoxy-trans-cinnamate (12),²⁰ multifidol glucoside (13),²¹ 2-(2-methylbutyryl)phloroglucinol 1-O-(6′′-O-β-D-apiofuranosyl)-β-D-glucopyranoside (14),²² chipericumin D (15),²³ uralione D (16),²⁴ hypercohin K (17),²⁵ uraloidin A (18),²⁶ furohyperforin (19),²⁷ and piceatannol (20)²⁸ by spectroscopic analyses and comparison with literature data (Figure 1).

Figure 1.

Chemical structures of isolated compounds (1-20).

Except for phloroglucinol (furohyperforin 19), the other compounds are described in H. hookerianum for the first time. This is also the first report on the discovery of compounds 5–10, 12–14, and 20 in the genus Hypericum. The results provide new information on the phytochemical profile of the genus Hypericum and H. hookerianum in particular.

Compound 6 was isolated as a pale yellow amorphous powder. Its molecular formula, C₁₈H₂₀O₅, was deduced from the positive ESI-MS ion peak at m/z 339.0 [M + Na]⁺, in combination with the ¹H- and ¹³C NMR data. The ¹H NMR spectrum of 6 (Supplemental Table S1) contained four aromatic protons with an AA′XX′ pattern at δ_H 6.85 (2H, d, J = 8.4 Hz) and 7.93 (2H, d, J = 8.4 Hz), and a singlet signal at δ_H 6.13 (2H, s) of the symmetrically substituted aromatic ring. Furthermore, 2 methylene groups at δ_H 2.97 and 3.03 and 3 methoxy groups at δ_H 3.77 (6H, s) and 3.81 (3H, s) were also found in the ¹H NMR spectrum. The ¹³C NMR spectrum of 6 displayed 18 carbon signals assigned to 3 methyl groups, 2 methylenes, 6 methines, and 7 quaternary carbons (ketone group at δ_C 199.6), based on HSQC, suggesting that compound 6 could be a dihydrochalcone. The HMBC of H-2′ (δ_H 7.93) and 4′-OCH₃ (δ_H 3.81) to C-4′ (δ_C 159.7), H-2′ (δ_H 7.93), and H-8 (δ_H 3.03) to C-9 (δ_C 199.6) indicated that the methoxy group was linked to C-4′ of ring A of the dihydrochalcone moiety. The 2 remaining methoxy groups were connected to C-2 and C-6, whereas a hydroxyl group was attached to C-4 of ring B. This deduction was demonstrated by the HMBC interactions of H-3/H-5 (δ_H 6.13), 2-OCH₃/4-OCH₃ (δ_H 3.77), H-7 (δ_H 2.97) to C-2/C-6 (δ_C 158.9), and H-3/H-5 (δ_H 6.13) to C-4 (δ_C 159.8), along with the MS data. From the above analysis, compound 6 was determined to be 4-hydroxy-2,6,4′-trimethoxydihydrochalcone (Supplemental Figure S1). To date, compound 6 has been reported in only 2 published papers, including a phytochemical investigation of Soymida febrifuga (Meliaceae) and a synthesis study.^29,30 However, the NMR data for the compound (6) were not provided in these previous studies. This is the first report of the NMR spectral data for 4-hydroxy-2,6,4′-trimethoxydihydrochalcone (6).

Neuroprotective Activities of Isolated Compounds

All isolates were screened for neuroprotective activity against glutamate-induced neuronal toxicity in HT-22 cells at 3 concentrations (5.56, 16.67, and 50 μM). N-acetyl-L-cysteine (NAC, 1 mM) was used as positive control. As shown in Supplemental Figure S3, cell viability decreased to approximately 40% that of the control after treatment with 5 mM glutamate. NAC (1 mM) recovered the viability to 98.0%. Among the isolates, compounds 1, 6, 11, and 12 exhibited strong neuroprotective effects at least at one concentration, with a cell survival > 70%. Consequently, these 4 compounds were further examined for their neuroprotective activity using a 6-point serial concentration to determine EC₅₀ values. Compound 6 displayed the strongest neuroprotective potential, with an EC₅₀ value of 1.48 µM. The EC₅₀ values of compounds 1, 11, and 12 were 9.70, 26.78, and 35.70 µM, respectively (Supplemental Figure S4). The neuroprotective activities of compounds 6, 11, and 12 are reported for the first time.

Discussion

Oxidative stress is the primary cause of neuronal cell death and is implicated in many acute brain injuries and chronic neurodegenerative diseases, such as ischemic stroke, Alzheimer's disease, and Parkinson's disease.^31–33 Glutamate is an excitatory neurotransmitter in the mammalian central nervous system. However, excessive glutamate in the central nervous system induces excitotoxicity and oxidative stress, leading to neuronal loss.³⁴ Glutamate-induced neurotoxicity results in ROS accumulation by inhibiting cysteine uptake into neuronal cells through the cysteine/glutamate antiporter.³⁵ Glutamate-induced neurotoxicity is a common model of oxidative stress-induced cell death. Previous studies have demonstrated that extracts of H. hookerianum possess strong antioxidant properties in different in vitro and in vivo models.^1–3,36 Therefore, the neuroprotective effect of isolated extracts on HT-22 cells may involve their antioxidative activity.

In addition to the glutamate-induced HT-22 cell model, all isolates were screened for their neuroprotective properties at 3 concentrations (5.56, 16.67, and 50 μM) against 6-OHDA-mediated neurotoxicity in SH-SY5Y cells. As displayed in Supplemental Figure S5, 50 µM 6-OHDA decreased cell viability to approximately 36% after 24 h of treatment. Compounds 9, 10, and 15 showed neuroprotection at least at one concentration by increasing cell viability to 70%, whereas the other compounds exhibited either weak or no effects. Compounds 9, 10, and 15 were further evaluated for their neuroprotective effects using a series of 6 serial dilutions (Supplemental Figure S6). Compound 10 exhibited better neuroprotective activity with an EC₅₀ value of 2.85 µM. The EC₅₀ values of compounds 9 and 15 were 26.18 and 47.08 µM, respectively (Supplemental Figure S6). In this study, the neuroprotective effect of compounds 9, 10 and 15 against 6-OHDA-induced toxicity in SH-SY5Y cells were evaluated for the first time. These data clearly show the neuroprotective effect of the isolated compounds against 6-OHDA in SH-SY5Y cells.

Parkinson's disease is a progressive neurodegenerative disorder characterized by progressive loss of dopaminergic neurons. The neurotoxin 6-OHDA has a similar structure to dopamine and is widely used to selectively damage dopaminergic neurons in vitro and in vivo.^37,38 Once in neurons, 6-OHDA accumulates and undergoes non-enzymatic auto-oxidation promoting the formation of ROS. The SH-SY5Y is a dopaminergic neuronal cell line of human neuroblastoma and it has been utilized in in vitro assays to determine the effect of protective and therapeutic agents for Parkinson's disease.^38,39 The present results implicate 2 furofuran lignans, 9 and 10, as potential therapeutic candidates that need to be investigated further (Supplemental Figure S6). Notably, despite possessing a strong neuroprotective effect against glutamate toxicity in HT-22 cells, compounds 1, 6, and 11 exhibited weak activity against 6-OHDA-induced SH-SY5Y cell damage. Further studies are required to determine the mechanisms of action of these compounds.

Conclusion

Twenty known compounds (1-20) were isolated from the aerial parts of H. hookerianum. Ten of these isolated compounds (5–10, 12–14, and 20) are novel for Hypericum species. Except for phloroglucinol furohyperforin (19), the remaining compounds have not been isolated previously from H. hookerianum.

In this study the NMR spectral data of 4-hydroxy-2,6,4′-trimethoxydihydrochalcone (6) are provided for the first time. The extracts and isolated compounds 1, 6, and 11 showed neuroprotective activity against glutamate-induced HT-22 cell damage, while 2 furofuran lignans, 9 and 10, attenuated 6-OHDA-mediated neurotoxicity in SH-SY5Y cells. The findings provide new information on the phytochemical profile of H. hookerianum and their neuroprotective properties.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X231164818 - Supplemental material for Chemical Constituents and Neuroprotective Activity of Hypericum hookerianum

Supplemental material, sj-docx-1-npx-10.1177_1934578X231164818 for Chemical Constituents and Neuroprotective Activity of Hypericum hookerianum by Vu Duy Hong, Van Tuan Vu, Baskar Selvaraj, Nguyen Manh Tuyen, Nguyen Minh Khoi, Nguyen Duy Thuan, Phuong Thien Thuong and Jae Wook Lee in Natural Product Communications

Footnotes

Declaration of Conflicting Interests

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

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by research grants NRF-2019K1A3A1A82113697, KIST intramural grants (2Z06661 and 2Z06803), and annual funding for R&D of Vietnam-Korea Institute of Science and Technology (VKIST).

Supplemental Material

Supplemental material for this article is available online.

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