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Article

Phenolic Constituents from Hypericum aucheri Jaub et. Spach—Isolation, Identification, and Preliminary Evaluation for hMAO-A/B and Neuroprotective Activity

by
Teodor Marinov
1,
Magdalena Kondeva-Burdina
2,
Zlatina Kokanova-Nedialkova
1 and
Paraskev T. Nedialkov
1,*
1
Pharmacognosy Department, Faculty of Pharmacy, Medical University of Sofia, 2 Dunav Str., 1000 Sofia, Bulgaria
2
Department of Pharmacology, Pharmacotherapy and Toxicology, Faculty of Pharmacy, Medical University of Sofia, 2 Dunav Str., 1000 Sofia, Bulgaria
*
Author to whom correspondence should be addressed.
Chemistry 2024, 6(6), 1535-1551; https://doi.org/10.3390/chemistry6060093
Submission received: 27 October 2024 / Revised: 26 November 2024 / Accepted: 28 November 2024 / Published: 30 November 2024
(This article belongs to the Section Biological and Natural Products)
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Abstract

Three new acylated benzophenone O-glycosides named aucherosides A–C (4–6), together with five known compounds such as mangiferin (1), maclurin-6-O-β-D-glucopyranoside (2), 1-O-galloyl-β-D-glucose (3), vanillic acid (7), and 5-hydroxy-2-isopropylchromone-7-O-β-glucoside (8), were isolated from the aerial parts of Hypericum aucheri Jaub. and Spach and identified with spectroscopic methods (1D and 2D NMR, and HRESIMS). Compounds 2, 4–6, 8, and previously isolated from the title plant aucherines A–C (9–11), were tested for hMAO-A and B inhibitory effects and neuroprotection. All tested compounds (1 µM) did not exhibit any inhibitory effect on hMAO-A and showed significant inhibitory activity against the hMAO-B enzyme. Notably, compound 8 demonstrated the strongest hMAO-B inhibition, approaching that of the positive control selegiline. At high concentrations (100 µM), all tested compounds showed no neurotoxic or pro-oxidant effects on rat brain synaptosomes, mitochondria, and microsomes. All tested compounds exhibited good neuroprotective and antioxidant activities in various neurotoxicity models (6-hydroxydopamine-induced neurotoxicity on synaptosomes, tert-butyl hydroperoxide-induced oxidative stress on mitochondria, and non-enzymatic lipid peroxidation on microsomes). The neuroprotective mechanisms of these compounds may include MAO-B inhibition, reactive oxygen species (ROS) scavenging, membrane stabilization, and preservation of reduced glutathione (GSH), the primary nucleophilic ROS scavenger.

Graphical Abstract

1. Introduction

The genus Hypericum belongs to the family Hypericaceae and consists of over 490 species united in 36 sections. Hypericum aucheri Jaub. and Spach is a perennial herb that belongs to sect. Crossophyllum and is distributed in Northeastern Greece (eastern Makedhonia, Thrakia, Thasos), in central and southeastern Bulgaria, as well as in European and northwestern Anatolian Turkey [1]. So far, only a few phytochemical investigations have been done on the composition of the title plant. Some flavonoids, xanthones, a biflavone, and hydroxycinnamic acids were isolated from the ethanol extract of the aerial parts of the title plant [2,3]. Recently, prenylated phloroglucinols and three prenyloxy chromanone derivatives (aucherines A–C) were isolated from dichloromethane extract of H. aucheri [4]. Furthermore, more than 40 compounds, including benzophenones, xanthones, flavonoids, phenolic acids, and chromone, were identified and quantified in ethanol extract by UHPLC-HRMS. The dominant polar phenolic constituents were chlorogenic acid (11.55 mg/g D.W.) and mangiferin (9.13 mg/g D.W.) [5]. In addition, the total content of tannins and flavonoids in H. aucheri were determined as 3.15 ± 0.01 g/100 g and 0.74 ± 0.01 g/100 g D.W., respectively [6].
Simply oxidized benzophenones are considered compounds derived from diphenylmethane. They are a small group of plant metabolites mainly distributed in members of Clusiaceae, Gentianaceae, Hypericaceae, etc., possessing diverse biological activities [7]. So far, from this group only benzophenone O-glycosides isolated from Hypericum thasium [8] and Gentiana verna subsp. pontica [9] were tested for MAO inhibition activity.
Monoamine oxidase (MAO) is a flavin-adenine-dinucleotide—dependent enzyme. The enzyme exists in two different isoforms type A (MAO-A) and B (MAO-B), and they are located at the outer mitochondrial membrane in most mammalian tissue [10]. The two isoenzymes showed different substrate specificity and inhibitor sensitivity. MAO-A catalyzes the oxidative deamination of norepinephrine and 5-hydroxytryptamine (serotonin), while MAO-B showed affinity to 2-phenylethylamine and benzylamine. Furthermore, both isotypes catalyzed the deamination of DA and tyramine [11]. Monoamine oxidase inhibitors were introduced in the 1950s for the treatment of different psychiatric and neurological disorders [12,13,14]. Still, now they are used only as third-line antidepressants (selective MAO-A inhibitors) or as adjuvants for neurodegenerative diseases (selective MAO-B inhibitors) [15].
Parkinson’s disease (PD) is a neurodegenerative disorder that typically affects older individuals and is characterized by the progressive loss of specific neuronal cell populations as well as by the appearance of eosinophilic filamentous inclusions (Lewy bodies) found in the magnocellular neurons of the brainstem nuclei, mainly in the substantia nigra and locus coeruleus [16]. In this disease, the overproduction of reactive oxygen species is increased due to mitochondrial dysfunction and dopamine degradation [17]. In addition, increased levels of basal malondialdehyde were observed, which is an intermediate in the lipid peroxidation process [18]. PD progresses over time. There is currently no cure for this disease, but published reports indicate that therapy with MAO-B inhibitors may delay its progression and protect dopaminergic neurons in the substantia nigra [19,20].
Here, we describe the isolation and structural elucidation of three new acylated benzophenone O-glycosides 4–6 along with five known compounds 1–3, 7, and 8 from the aerial parts of Hypericum aucheri Jaub. and Spach. Compounds 2, 4–6, 8, as well as three previously isolated from the dichloromethane extract of the aerial parts of the title plant prenyloxy chromanone derivatives 9–11 (aucherines A–C) [4], were tested for MAOA/B inhibitory activity and neuroprotection in various neurotoxicity models (6-hydroxydopamine-induced neurotoxicity on synaptosomes; tert-butyl hydroperoxide-induced oxidative stress on mitochondria; and non-enzymatic lipid peroxidation on microsomes). The structures of the isolated and tested compounds are given in Figure 1.

2. Materials and Methods

2.1. General Experimental Procedures

Optical rotations were measured at 20 °C temperature on an Autopol VI (Rudolph Research Analytical, Hackettstown, NJ, USA) polarimeter equipped with a TempTrol™ 100 mm sample cell. UV spectra were recorded in a wavelength range of 190–500 nm in LC/MS grade MeOH on a Libra S70 UV/Vis spectrophotometer (Biochrom, Cambridge, UK). 1H, 13C and 2D NMR spectra were measured at 300 K on a Bruker Biospin GmbH (Rheinstetten, Germany) AVANCE NEO 400 (operating at 400.13 MHz for 1H and 100.61 MHz for 13C) with an iTBO BBF/H/F probe or on an AVANCE NEO 600 (operating at 600.18 MHz for 1H and 150.92 MHz for 13C) with the Prodigy BBO cryoprobe. All spectra were recorded in CD3OD (Methanol-d4 99.8% Deutero GmbH) and compared with signals of the residue solvent (δH 3.31 for 1H and δC 49.15 for 13C). Shift values (δH and δC) are always given in ppm and J values in Hz. The standard pulse sequence and phase cycling were used for MQF-COSY, HSQC, HMBC, and NOESY experiments. The NMR data were processed using Bruker TopSpin 4.2.0 software. HR-ESI-MS and MS/MS spectra were obtained in negative ion mode on a Q Exactive Plus (ThermoFisher Scientific, Inc., Bremen, Germany) mass spectrometer with a heated HESI-II source. The MS data were processed using FreeStyle™ 1.8 SP2 computer software. Semi-preparative (SP) HPLC was performed on a Waters (Milford, MA, USA) Breeze 2 high-pressure binary gradient system consisting of a pump model 1525EF, manual injector 7725i, and a UV detector model 2489. Separations were achieved on a semi-preparative HPLC column Kromasil C18 (250 × 10 mm, 5 μm) purchased from Eka Chemicals AB (Bohus, Sweden) at 5 mL min−1.

2.2. Plant Material

The aerial parts of Hypericum aucheri Jaub. et Spach were collected from a wild habitat near Momchilgrad (Kardzali District, Bulgaria) in July 2015 and was authenticated by P. Nedialkov. A voucher specimen (SOM-Co-1344) is kept in the herbarium of the Institute of Biodiversity and Ecosystem Research (IBER) at the Bulgarian Academy of Sciences (BAS).

2.3. Extraction and Isolation

The powdered air-dried aerial parts of Hypericum aucheri (171.4 g) were subsequently percolated with CH2Cl2 (5 L) and then with MeOH (7 L) at room temperature. The crude MeOH extract was evaporated in a vacuum. The resulting residue (40.78 g) was suspended in hot H2O (100 mL) and, after cooling was subjected to CC on Diaion HP-20 (65 × 150 mm) with MeOH [30% (m/m), 60% (m/m) and 100%] to give three fractions (A; B and C). The pale-yellow crystals of compound 1 that appeared in fraction A were removed by centrifugation (4000 rpm, 10 min). Subsequently, the supernatants of fraction A (6.84 g) and fraction B (8.60 g) were chromatographed on MCI gel column (30 × 500 mm) with MeOH-H2O mixtures (10:90 → 70:30) to give 14 and 16 subfractions, respectively (A1-14; B1-16). Subfractions A2 (703 mg), B3 (538 mg), and B6 (522 mg) were submitted to the Sephadex LH20 column to give 16 (A2.1-16), 28 (B3.1-28), and 30 (B6.1-30) subfractions. Subfraction A2.12 (132 mg) and A2.13 (73 mg) were pooled and followed by isocratic semi-preparative HPLC with MeOH-0.05% TFA (7:93, 5 mL/min) mobile phase to give pure compound 2 (135 mg) and 3 (2 mg). While SP-HPLC (MeOH-0.05%TFA, 32:68, 5 mL/min) of B3.13 (76 mg) give pure compound 4 (24 mg), 5 (38 mg), 6 (4.5 mg), and 7 (1.5 mg). In addition, subfraction B6.9 (44 mg) was subjected to SP-HPLC eluted isocratically with MeOH-0.05% TFA (37:63, 5 mL/min) to give compound 8 (14 mg). The isolation and identification of compounds 9–11 was given elsewhere [4].

2.4. Acid Hydrolysis of Compounds (4–6)

One mg of each compound (4–6) was hydrolyzed with 80% TFA (500 µL) in a boiling water bath (100 °C) for 1 h 40 min. The mixtures were subjected to SPE on a Phenomenex (Torrance, CA, USA) Strata C18-E (55 μm, 70 Å, 200 mg, 3 mL) cartridge. The sorbent was eluted with dd H2O (5 × 500 μL), and the resulting solutions were evaporated to dryness. The sugar derivatization and liquid chromatography analysis were carried out following Tanaka et al.’s method with minor adjustments. The sugars were heated in a water bath (100 °C, 1 h) with a 100 µL solution of l-cycteine methyl ester in pyridine (C = 12.5 mg/mL), then 100 µL phenyl isothiocyanate in pyridine (C = 12.5 µL/mL) was added and heated under the same conditions. Then, the derivatives were analyzed directly on the Kromasil C18 column by UHPLC-HR-ESI-MS. Finally, the configurations were determined as d-glucose by comparing their retention times (tR = 16.42 min) with those of standard d- (tR = 16.33 min) and l-glucose (tR = 14.99 min) derivatives.

2.5. Spectral Data of the New Compounds (4–6)

2.5.1. Aucheroside A (4)

[ α ] D 20 : –2.14 (c 0.1110, MeOH); UV (MeOH) λmax (log ε): 230 (4.44), 283 (3.99), 317 (3.98) nm; 1H and 13C NMR data (Table 1 and Table 2); HR-ESI-MS: m/z 527.1199 [M − H]− (calcd. for C26H23O12, 527.1184); MS/MS (NCE=40): m/z 151.0024 (100), 261.0404 (76), 109.0280(71), 405.0829 (70), 107.0124 (28).

2.5.2. Aucheroside B (5)

[ α ] D 20 : –7.37 (c 0.1065, MeOH); UV (MeOH) λmax (log ε): 229 (4.36), 283 (3.96), 318 (3.96) nm; 1H and 13C NMR data (Table 1 and Table 2); HR-ESI-MS: m/z 527.1197 [M−H]− (calcd. for C26H23O12, 527.1184); MS/MS (NCE = 40): m/z 261.0403 (100), 151.0023 (82), 109.0279 (47), 107.0123 (24), 405.0833 (2).

2.5.3. Aucheroside C (6)

[ α ] D 20 : –7.76 (c 0.0352, MeOH); UV (MeOH) λmax (log ε): 228 (4.55), 284 (4.12), 315 (4.11) nm; 1H and 13C NMR data (Table 1 and Table 2); HR-ESI-MS: m/z 527.1191 [M−H]− (calcd. for C26H23O12, 527.1184); MS/MS (NCE = 40): m/z 151.0024 (100), 261.0404 (98), 109.0279 (74), 405.0828 (51), 107.0123 (30).
Spectral data can be reviewed in the Supplementary Materials.

2.6. Determination of Human Recombinant MAO-A and MAO-B Enzyme Activity

The activity of recombinant human MAO-A (hMAO-A) and MAO-B (hMAO-B) was determined fluorometrically. Tyramine hydrochloride was used as substrate. The activity was determined by measuring the production of H2O2 using the Amplex Red (N-acetyl-3,7-dihydroxyphenoxazine) test in the presence of horseradish peroxidase [21,22]. Working solutions of the test substances, reagents, and hMAO-A or hMAO-B enzymes were prepared in the reaction buffer according to the manufacturer’s instructions. Pure working solutions of the enzymes in reaction buffer, enzyme solutions containing hydrogen peroxide, and pure reaction buffer were used as controls. The test substances and positive controls were applied at a final concentration of 1 µM. The enzymes and the tested compounds or positive controls were placed in a 96-well plate, with eight replicates per substance or positive control. The plate was incubated in the dark at 37 °C for 30 min. Fluorometric measurements were then performed using a Synergy 2 Microplate Reader at two wavelengths: 570 nm and 690 nm.

2.7. In Vitro Neuroprotection Activity

2.7.1. Animals

In the experiments, a total of 30 animals were used. These animals were procured from the Bulgarian Academy of Sciences National Breeding Center in Sofia, Bulgaria, and Slivnitsa, Bulgaria. The animals were kept in plexiglass cages under standard conditions, with free access to water and food. They were also exposed to a 12-h light and 12-h dark regime, and the temperature was maintained between 20–25 °C. The animals’ food was withheld for 12 h before each study. The procedures used in this study agreed with the European Communities Council Directive 2010/63/EU for animal experiments and Ordinance No. 15, which outlines the minimum requirements for the protection and welfare of experimental animals (SG No. 17, 2006). The experiments with animals were approved by the Bulgarian Food Safety Agency with Permission No. 273, valid until 20.07.2025.

2.7.2. Preparation, Isolation, and Incubation of Rat Brain Synaptosomes and Mitochondria

To isolate synaptosomes and mitochondria, a subcellular fractionation method involved a Percoll (a colloidal silica solution) gradient, as described by Taupin et al. [23] with small modifications. Briefly, a brain homogenate was prepared and centrifuged at 1000× g for 5 min at +4 °C. The supernatant was then collected and subjected to a second centrifugation process, which led to synaptosomes and mitochondria being used for further experiments. The supernatants obtained from the two rounds of centrifugation were combined and divided equally into four tubes. The tubes were then subjected to three more rounds of centrifugation at 10,000× g for 20 min each, at a temperature of +4 °C. The supernatants of the final two centrifugations were used to isolate synaptosomes and mitochondria according to the three-step protocol: (1). A 90% stock solution of Percoll was prepared; (2). Stock solutions were diluted to 16% and 10%, and 4 mL of each was placed into six test tubes; (3). 4 mL of 7.5% Percoll was added to the residue obtained after the last centrifugation. The centrifugation was carried out at +4 °C for 20 min at 15,000× g, forming three layers. The bottom, middle, and top layers contain mitochondria, lipids, and synaptosomes (at the 16% to 10% Percoll limit). Each layer from the tubes was collected using a glass pipette and transferred into a separate tube. Buffer B + glucose was added to it. The mixtures were centrifuged at 10,000× g for 20 min at +4 °C. Thus, the isolation buffer is exchanged with the incubation buffer. After centrifugation, the pellet with the synaptosomes was mixed and made up with buffer B + glucose. Synaptosomes and mitochondria were incubated with the tested compounds (2, 4–6, and 8–11) at concentrations of 100 µM and 50 µM for 1 h.

2.7.3. In Vitro Dopamine Model of Neurotoxicity

This in vitro model closely resembles the neurodegenerative processes that primarily occur in Parkinson’s disease (PD). The metabolism of 6-OHDA results in the creation of reactive quinones (p-quinone), which produce reactive oxygen species (ROS). These reactive metabolites and ROS can damage both pre- and post-synaptic membranes in the brain, ultimately resulting in neuronal cell damage [24]. Synaptosomes were incubated with 6-OHDA (150 μM) for 1 h.

2.7.4. MTT Assay to Assess the Viability of Synaptosomes

Synaptosomes’ viability was assessed by an MTT test adhering to the procedure described by Mungarro-Menchaca et al. [25] with some minor modifications. After 1 h of incubation with the substances and toxic agent, synaptosomes were centrifuged on a microcentrifuge for 1 min at 15,000× g. The pellet was mixed gently with buffer B + glucose, and the supernatant where 6-OHDA was discarded to prevent oxidation of MTT was centrifuged again at 15,000× g for 1 min. After the second wash, buffer B + glucose was added to the pellet. 60 µL of MTT solution was added to the washed synaptosomes. The plates were incubated with the MTT solution at 37 °C for 10 min. After incubation, the samples were centrifuged at 15,000× g for 2 min. The excess liquid was removed, and a DMSO solution was used to dissolve the formed formazan crystals. After dissolution, the amount of formazan was measured spectrophotometrically at λ = 580 nm.

2.7.5. Determination of Reduced Glutathione (GSH) in Isolated Brain Synaptosomes

The reduced GSH in isolated brain synaptosomes was measured using the protocol described in the literature [26] with small modifications. After precipitation of the proteins with trichloroacetic acid, the thiol groups in the supernatant were determined by DTNB, which produced a yellow-colored compound that absorbs light at λ = 412 nm. After incubation, synaptosomes were centrifuged at 4000× g for 3 min. The supernatant was removed, and the pellet was taken for GSH determination. It was treated with 5% trichloroacetic acid, left for 10 min on ice, and then centrifuged at 8000× g for 10 min (20 °C). The supernatant was taken for GSH determination and frozen at −20 °C. Immediately before measurement, the samples were neutralized with 5 N NaOH.

2.7.6. Tert-Butyl Hydroperoxide-Induced Oxidative Stress

Isolated brain mitochondria were incubated with 75 µM tert-butyl hydroperoxide (t-BuOOH) according to the procedure described by Karlsson et al. [27].

2.7.7. Determination of Malondialdehyde (MDA) Production in Brain Mitochondria

The malondialdehyde (MDA) production in brain mitochondria was established according to the Shirani et al. procedure with small modifications [28]. Briefly, 0.3 mL of 0.2% thiobarbituric acid and 0.25 mL of sulfuric acid (0.05 M) were added to the mitochondria, and the mixture was boiled for 30 min. Then, the tubes were placed on ice, and 0.4 mL of n-butanol was added to each. The tubes were centrifuged at 3500× g for 10 min. The amount of MDA was determined spectrophotometrically at 532 nm.

2.7.8. Determination of GSH Level in Brain Mitochondria

The GSH level in brain mitochondria was established according to the procedure described by Shirani et al. with small modifications [28]. Briefly, after the mitochondria were incubated with the substances and t-BuOOH, the reaction was stopped with 5% trichloroacetic acid, and each sample was homogenized with the acid and left on ice. After centrifugation of the homogenate at 6000× g, a 0.04% solution of DTNB was added to the supernatant to give it a yellow color, the determination being spectrophotometric at 412 nm.

2.7.9. Isolation of Brain Microsomes

The brain microsomes were isolated by following the described protocol [29] with some minor modifications. The rat brains were homogenized in nine volume parts of 0.1 M Tris buffer containing: 0.1 mM Dithiothreitol, 0.1 mM Phenylmethylsulfonyl fluoride, 0.2 mM EDTA, 1.15% KCl, and 20% (v/v) glycerol (pH 7.4). The resulting homogenate was centrifuged twice at 17,000× g for 30 min. The supernatants from the two centrifugations were pooled and centrifuged twice at 100,000× g for 1 h. The pellet was frozen in 0.1 M Tris buffer.

2.7.10. Iron/Ascorbate-Induced Lipid Peroxidation (LPO)

Non-enzyme-induced lipid peroxidation was induced with 20 μM ferrous sulfate solution and 0.5 mM ascorbic acid solution. The protocol was described elsewhere [30].

2.7.11. Determination of MDA in Brain Microsomes

The MDA levels in brain microsomes were measured following the protocol established by Mansuy et al. [30] with small modifications. When microsome incubation along with the substances and toxic agent ended, the reaction was stopped with 0.5 mL of 20% trichloroacetic acid followed by 0.5 mL of 0.67% thiobarbituric acid. The ongoing reactions were associated with forming a colored complex between the malondialdehyde formed and thiobarbituric acid. The determination of MDA was spectrophotometric at 535 nm. A molar extinction coefficient of 1.56 × 105 M−1 cm−1 was used for the calculation.

2.8. Statistical Methods

The experiments on isolated brain synaptosomes, mitochondria, and microsomes were statistically analyzed using the non-parametric Mann–Whitney method at significance levels p < 0.05, p < 0.01, and p < 0.001 with the “MEDCALC” program. The statistical processing of hMAO-A and hMAO-B activity results was performed using GraphPad Prism 5.0 software.

3. Results and Discussion

3.1. Identification of the Isolated Compounds

The structures (Figure 1) of known compounds were determined as mangiferin 1, maclurin-6-O-β-d-glucopyranoside 2 [31,32], 1-O-galloyl-β-d-glucose 3, vanillic acid 7, and 5-hydroxy-2-isopropylchromone-7-O-β-glucoside 8 [33] by comparing these with reported NMR data and with authentic samples. Excluding 1, all other compounds were isolated for the first time for the title species. Furthermore, 2 was previously found only in species of the genera Gentiana and Garcinia [7]. The isolation of this compound from a representative of the genus Hypericum is reported here for the first time. A detailed procedure for isolation and identification of compounds 9–11 is given in the literature [4].
Compound 4 was isolated as a pale-yellow amorphous solid. High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) in negative mode revealed a deprotonated molecule [M − H]− at m/z 527.1199 (calculated mass for C26H23O12 is 527.1184), corresponding to the molecular formula C26H24O12 with 15 degrees of unsaturation. Total acid hydrolysis of 4 gave d-glucose as a sugar moiety. The MS2 spectrum of compound 4 showed a fragment ion at m/z 405.08 (Figure 2), resulting from a neutral loss of 122 Da, typical for esterified benzoic acid. Cleavage of the glycosidic bond, resulting in the loss of benzoylated hexose [M−C13H14O6−H]−, produced a product ion at m/z 261.04, corresponding to the deprotonated molecule of the aglycone. This ion further fragmented at the carbonyl group and aromatic ring B bond, yielding fragments at m/z 151.00 and 109.03. A subsequent loss of CO2 from the former fragment produced an ion at m/z 107.01, consistent with the postulated fragmentation pathway of maclurin [34].
The 13C-NMR spectrum of compound 4 (Table 1) showed signals for three oxygen-bearing aromatic carbons at δC 162.4, 159.7, and 158.6, a quaternary carbon at δC 110.8, and two aromatic methine carbons at δC 98.4 and 96.1. The latter two signals exhibited cross-peaks in the HSQC experiment with doublets at δH 6.09 and 6.29 in the 1H-NMR spectrum (Table 2). These signals are characteristic of a mono-substituted phloroglucinol ring. Additionally, a quaternary carbon signal at δC 132.8, two oxygen-bearing aromatic carbon signals at δC 152.2 and 146.1, and three aromatic methine carbons at δC 125.1, 117.9, and 115.8, were observed in the 13C-NMR spectrum of compound 4. The latter three signals correlated in the HSQC experiment with the double doublet at δH 7.25, and the doublets at δH 7.31 and 6.79, respectively. These signals are typical for a mono-substituted catechol ring. The first two signals strongly correlated with the carbonyl at δC 197.7 in the HMBC experiment (Figure 3).
Moreover, the 13C-NMR spectrum of compound 4 revealed six carbon signals at δC 102.3, 76.3, 75.8, 75.0, 72.7, and 62.3, corresponding to the glucopyranose unit. In the HSQC experiment, the former signal exhibited a cross-peak with a doublet at δH 4.97 (H-1″), with a large coupling constant (J = 7.8 Hz) characteristic of an anomeric proton in the β-configuration. These NMR spectral data closely resemble those previously published for maclurin-6-O-β-d-glucopyranoside 2 [31,32]. Finally, signals in the 13C-NMR spectrum at δC 167.4, 131.3, 134.6, 130.9, and 129.7, corresponding to a carbonyl, a quaternary carbon, and three aromatic methines, respectively, were assigned to a benzoyl moiety. In the HMBC experiment, the carbonyl signal exhibited a cross-peak with the double doublet at δH 5.03 (H-4″) from the glucose moiety, unequivocally indicating the esterification position of the benzoyl group. Thus, the structure of compound 4 was established as maclurin-6-O-(4″-benzoyl)-β-d-glucopyranoside. This new natural compound was given the trivial name aucheroside A.
Compound 5 was isolated as a pale-yellow amorphous solid and showed an identical molecular formula as well as very similar MS2, 1H-NMR, and 13C-NMR spectra to those of compound 4 (Table 1 and Table 2). The only difference was the position of the benzoyl group on the sugar moiety, which was determined unequivocally by HMBC (Figure 3) correlation between δH 4.68 (H-6a″) and δH 4.33 (H-6b″) and the carbonyl group of the benzoyl unit at δC 168.2 (C=O). Thus, the structure of compound 5 was established as maclurin-6-O-(6″-benzoyl)-β-D-glucopyranoside. This new natural compound was given the trivial name aucheroside B.
Compound 6 was also isolated as a pale-yellow amorphous solid and exhibited spectral data nearly identical to compounds 4 and 5 (Table 1 and Table 2). However, the HMBC experiment revealed a correlation between the sugar proton at δH 5.21 (H-3″) and the benzoyl carbonyl signal at δC 168.2 (C=O), indicating that esterification of the benzoyl group occurred at the C-3″ position of the sugar moiety. Therefore, compound 6 was identified as maclurin-6-O-(3″-benzoyl)-β-D-glucopyranoside. This new natural compound was given the trivial name aucheroside C.

3.2. Effects of the Compounds 2, 4–6, 8–11 on Human Recombinant MAO-A/MAO-B Enzyme (hMAO-A/hMAO-B)

The tested compounds (2, 4–6, 8–11) and the positive controls were evaluated at a single concentration of 1 µM. Chlorgyline and selegiline were selected for positive controls in hMAO-A and hMAO-B tests, respectively. Experiments were run according to the published protocols [21,22] with small modifications. The results are graphically presented in Figure 4. When administered alone at a tested concentration, compounds 2, 4–6, and 8–11 showed no statistically significant MAO-A inhibitory effect compared to the control (Figure 4A). Only the classical MAO-A inhibitor chlorgyline significantly decreased enzyme activity by 55% compared to the control (pure hMAO-A). In contrast, all tested compounds statistically significantly inhibited hMAO-B compared to the control (pure hMAO-B) (Figure 4A). Compounds 2, 6, and 9 reduced the hMAO-B activity by 40%, while 4, 5, 10, and 11 reduced it by 35%. Compound 8 demonstrated the most prominent inhibitory effect on hMAO-B, reducing enzyme activity by 50%, comparable to the effect of selegiline, a classical MAO-B inhibitor, which decreased enzyme activity by 55%.

3.3. Neuroprotective Effects of the Compounds 2, 4–6, 8, 9–11 on Isolated Rat Brain Synaptosomes, Mitochondria, and Microsomes

3.3.1. Effects of the Compounds 2, 4–6, 8, 9–11 on Isolated Rat Brain Synaptosomes

To establish the toxicity of the tested compounds 2, 4–6, 8, 9–11, and the positive control silybin, a relatively high (100 µM) concentration was chosen. When administered alone, these compounds showed no neurotoxic effects on isolated rat brain synaptosomes. None of the tested compounds practically altered the key biomarkers that define the functional and metabolic status of the synaptosomes, specifically synaptosome viability as measured by the MTT assay (Figure 5A) and the level of reduced glutathione (GSH) (Figure 5B).
Historically, various models have been developed to study the specific structures of the nervous system. Over 30 years ago, the introduction of 6-hydroxydopamine (6-OHDA) and several other synthetic dopamine (DA) analogs marked a significant advancement in studying catecholaminergic neurotoxic compounds. 6-OHDA, structurally similar to DA and norepinephrine (NE), is used in research due to its affinity for dopamine and NE transporters, allowing it to enter and damage neurons. Once inside, it generates reactive species that harm neuronal structures. Its effects are seen in the peripheral and central nervous systems, affecting various organs and causing neuronal degeneration in multiple phases. In the central nervous system, moderate doses of 6-OHDA deplete NE and DA, leading to long-term behavioral changes and severe physical symptoms in experimental animals, including aphagia, adipsia, and seizures [24].
In this model of neurotoxicity, all examined compounds, at a concentration of 50 µM, exhibited significant neuroprotective and antioxidant activity compared to the control (toxic agent). Administered alone at a concentration of 150 µM, 6-OHDA significantly decreased synaptosome viability and GSH levels by 50% compared to the control (non-treated synaptosomes) (Figure 6). When combined with 6-OHDA, all compounds and Silybin, at a concentration of 50 µM, preserved synaptosome viability and GSH levels as follows: (1). Compounds 2, 9–11, and silybin preserved synaptosome viability at 70%, while 4–6 and 8 preserved it at 60%, compared to the control (pure 6-OHDA) (Figure 6A); (2). Compounds 2, 4–6, and 8 preserved GSH levels at 70%, while 9–11 and silybin preserved them at 80%, compared to the control (pure 6-OHDA) (Figure 6B).

3.3.2. Effects of the Compounds 2, 4–6, 8–11 on Isolated Rat Brain Mitochondria

One of the main ways that neurotoxic compounds induce apoptosis or necrosis is through oxidative stress [35]. An often-used method to cause oxidative stress in experimental in vitro models is the toxic agent tert-butyl hydroperoxide (t-BuOOH). The mechanisms of its action are as follows: (1). It forms reactive free radicals that compromise membrane integrity and induce lipid peroxidation; (2). It alters glutathione activity by decreasing glutathione reductase activity and increasing glutathione peroxidase activity, decreasing GSH levels; (3). It increases intracellular calcium, thus stimulating apoptosis; (4). It oxidizes sulfhydryl groups (-SH) of mitochondrial enzymes, leading to a blockage of cellular respiration [27]; (5). It causes mitochondrial calcium uptake, which leads to the mitochondrial generation of reactive oxygen species (ROS) and decreases the mitochondrial membrane potential, thereby blocking cellular respiration and causing cell death [35].
Administered alone at a concentration of 100 µM, compounds 2, 4–6, 8–11, and silybin did not exhibit any neurotoxic effects on isolated rat brain mitochondria. These compounds did not alter key biomarkers that characterize the functional and metabolic status of mitochondria—specifically, malondialdehyde (MDA) production (Figure 7A) and the level of reduced glutathione (GSH) (Figure 7B).
In this neurotoxicity model, all tested compounds at a concentration of 50 µM demonstrated significant neuroprotective and antioxidant activity compared to the control (toxic agent). When administered alone at a concentration of 75 µM, t-BuOOH significantly increased MDA production by 198% and decreased GSH levels by 50% compared to the control (non-treated mitochondria) (Figure 8). When combined with t-BuOOH, all compounds and silybin at a concentration of 50 µM preserved GSH levels and reduced MDA production. Specifically, compounds 4–6 and 2 reduced MDA production by 27%; compound 8 by 25%; compounds 9 and 10 by 28%; and compound 11 and silybin by 29%, compared to the control (pure t-BuOOH) (Figure 8A). Regarding GSH levels, compounds 2, 4–6, and 8 preserved 60% of GSH levels; while compounds 9–11 and silybin preserved 80% of GSH levels, compared to the control (pure t-BuOOH) (Figure 8B).

3.3.3. Effects of the Compounds 2, 4–6, and 8–11 on Isolated Rat Brain Microsomes

Microsomes are heterogeneous, vesicle-like fragments, ranging from 20–200 nm in diameter, formed in vitro from parts of the endoplasmic reticulum during the fragmentation of eukaryotic cells. These structures are not present in healthy, living cells. Microsomes can be concentrated and separated from other cell organelles through repeated centrifugation. They have a red-brown color due to the presence of heme and serve as an in vitro model for analyzing the metabolic activity of cytochrome P450 (CYP) enzymes. Microsomes are commonly used in experiments to study new drugs’ stability and metabolic profiles during the drug-discovery and synthesis processes. These systems have been integrated into advanced applications using automated incubation processes and microsomal fractionation. They can store many enzymes involved in phase I and II biotransformation, allowing for the creation of a repository to study genetic polymorphism. Additionally, microsomes are utilized as a model for lipid membranes [36].
When administered alone at a high concentration (100 µM), compounds 2, 4–6, 8–11, and silybin did not alter malondialdehyde (MDA) production on isolated rat brain microsomes (Figure 9), which is evidence that they did not exhibit any pro-oxidant effects.
During respiration in aerobic organisms, reactive oxygen species (ROS), such as superoxide anion and hydrogen peroxide, are continuously generated. At physiological concentrations, ROS are essential for normal cellular functions. However, their overproduction can harm the cell, leading to oxidative stress and subsequent damage to lipids, proteins, and DNA. Unsaturated fatty acids in the cell membrane are particularly vulnerable to ROS overproduction. Lipid hydroperoxides can alter membrane density and disrupt membrane protein functions. Additionally, they can undergo iron-dependent one-electron reduction and oxidation, forming epoxy peroxide radicals. These radicals initiate a chain reaction of lipid peroxidation, producing cellular toxins such as the reactive aldehydes 4-hydroxynonenal and malondialdehyde. These end-products further damage proteins, DNA, and other macromolecules, exacerbating cellular damage [35].
In the non-enzyme lipid peroxidation neurotoxicity model, all examined compounds exhibited significant antioxidant activity at a concentration of 50 µM compared to the iron/ascorbic acid. Without the enzyme, lipid peroxidation increased MDA production by 350% compared to the control (non-treated microsomes) (see Figure 10). At the same concentration, all tested compounds and silybin effectively reduced MDA production: compounds 2 and 5 by 53%; compounds 8 and 11 by 49%; compounds 4, 6, and 10 by 51%; compound 9 by 48%; and silybin by 54% compared to the iron/ascorbic acid.
Based on the conducted experiments, it can be suggested that the possible mechanisms of neuroprotection of the investigated compounds may be: (1). Stabilization of the cell membrane by reducing the production of MDA, a classical biomarker of lipid peroxidation; (2). Neutralization of free radicals by maintaining the level of GSH, which is the main nucleophile responsible for scavenging free radicals and reactive metabolites.

4. Conclusions

Three new acylated benzophenone O-glycosides 4–6, named aucherosides A–C, along with five known compounds, including mangiferin 1, maclurin-6-O-β-d-glucopyranoside 2, 1-O-galloyl-β-d-glucose 3, vanillic acid 7, and 5-hydroxy-2-isopropylchromone-7-O-β-glucoside 8, were isolated from the aerial parts of Hypericum aucheri Jaub. and Spach and identified using spectroscopic methods (1D and 2D NMR, and HRESIMS). Compounds 2, 4–6, 8, and three previously isolated phenyloxy chromanone derivatives 9–11 (aucherines A–C) were tested for MAO-A and MAO-B inhibitory activity and in various neurotoxicity models, including 6-hydroxydopamine-induced neurotoxicity on synaptosomes, tert-butyl hydroperoxide-induced oxidative stress on mitochondria, and non-enzymatic lipid peroxidation on microsomes.
All tested compounds (1 µM) showed no hMAO-A enzymatic activity but exhibited statistically significant inhibitory activity against hMAO-B. Compound 8 demonstrated the most prominent inhibitory effect on hMAO-B, reducing enzyme activity by 50%, compared to the positive control selegiline (55%). The other tested compounds reduced hMAO-B activity by 35–40%.
At a concentration of 50 µM, all tested compounds displayed statistically significant neuroprotective activities on isolated rat brain synaptosomes using a 6-hydroxydopamine in vitro model. Combined with 6-OHDA, all compounds and silybin preserved synaptosome viability and GSH levels. Compounds 9–11 exhibited the most prominent neuroprotective and antioxidant effects, similar to the positive control silybin. They preserved synaptosome viability at 70% and GSH levels at 80%.
In a tert-butyl hydroperoxide-induced oxidative stress model on isolated rat brain mitochondria, all tested compounds (50 µM) demonstrated significant neuroprotective and antioxidant activity. Aucherines A–C 9–11 showed the highest activity, preserving 80% of GSH levels and reducing MDA production by 28–29%, compared to the positive control silybin, which showed 80% and 29% values, respectively.
In a model of non-enzyme-induced lipid peroxidation on isolated rat brain microsomes, all tested compounds (50 µM) exhibited significant antioxidant activity. They effectively reduced MDA production by 48–53%, which was very close to the positive control silybin (54%).
The neuroprotective mechanisms of all tested compounds may include MAO-B inhibition, ROS scavenging, membrane stabilization, and preservation of GSH, the primary nucleophilic ROS scavenger.

5. Patents

It should be noted that the investigations described here did not result in any patent.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/chemistry6060093/s1, Figures S1–S21: 1D and 2D NMR, UV, and HRESIMS spectra of the new compounds.

Author Contributions

Conceptualization, P.T.N., and M.K.-B.; Methodology, P.T.N., M.K.-B., T.M., and Z.K.-N.; Software, P.T.N., T.M., and M.K.-B.; Validation, P.T.N., M.K.-B., and Z.K.-N.; Formal analysis, T.M., and Z.K.-N.; Investigation, P.T.N., T.M., M.K.-B., and Z.K.-N.; Resources, P.T.N., and M.K.-B.; Data curation, P.T.N., and Z.K.-N.; Writing—original draft preparation, P.T.N., T.M., and M.K.-B.; Writing—review and editing, P.T.N., and Z.K.-N.; Visualization, T.M., and M.K.-B.; Supervision, P.T.N., and M.K.-B.; Project administration, Z.K.-N., P.T.N., and M.K.-B.; Funding acquisition, Z.K.-N., P.T.N., T.M., and M.K.-B. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the European Union-NextGenerationEU, through the National Recovery and Resilience Plan of the Republic of Bulgaria, grant number BG-RRP-2.004-0004-C01.

Data Availability Statement

Data are contained within the article and in Supplementary Materials.

Acknowledgments

T. Marinov acknowledges the contract D-192/2022 with the Medical Science Council at the Medical University of Sofia for aiding the isolation of the new compounds.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. The structures of compounds isolated from the aerial parts of Hypericum aucheri.
Figure 1. The structures of compounds isolated from the aerial parts of Hypericum aucheri.
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Figure 2. A plausible fragmentation pattern of compound 4.
Figure 2. A plausible fragmentation pattern of compound 4.
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Figure 3. Key HMBC correlations of compounds 4–6.
Figure 3. Key HMBC correlations of compounds 4–6.
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Figure 4. Effects of the compounds 2, 4–6, 8, 9–11, and the positive control, administered alone at concentration 1 µM: (A). on hMAO-A enzyme activity with chlorgyline as a positive control and (B). on hMAO-B with selegiline as a positive control. ** p < 0.01; *** p < 0.001 vs. control (pure hMAO-A/B).
Figure 4. Effects of the compounds 2, 4–6, 8, 9–11, and the positive control, administered alone at concentration 1 µM: (A). on hMAO-A enzyme activity with chlorgyline as a positive control and (B). on hMAO-B with selegiline as a positive control. ** p < 0.01; *** p < 0.001 vs. control (pure hMAO-A/B).
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Figure 5. Toxic effects of the tested compounds 2, 4–6, 8, 9–11, and the positive control silybin, administered alone at a concentration of 100 µM: (A). on synaptosomes viability; (B). on GSH level at isolated rat brain synaptosomes.
Figure 5. Toxic effects of the tested compounds 2, 4–6, 8, 9–11, and the positive control silybin, administered alone at a concentration of 100 µM: (A). on synaptosomes viability; (B). on GSH level at isolated rat brain synaptosomes.
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Figure 6. Effects of the tested compounds 2, 4–6, 8, 9–11, and the positive control silybin, at a concentration of 50 µM, in combination with 6-OHDA at a concentration of 150 µM: (A). on synaptosomes viability; (B). on GSH level at isolated rat brain synaptosomes. *** p < 0.001 vs. control (non-treated synaptosomes); ++ p < 0.01 vs. control (pure 6-OHDA).
Figure 6. Effects of the tested compounds 2, 4–6, 8, 9–11, and the positive control silybin, at a concentration of 50 µM, in combination with 6-OHDA at a concentration of 150 µM: (A). on synaptosomes viability; (B). on GSH level at isolated rat brain synaptosomes. *** p < 0.001 vs. control (non-treated synaptosomes); ++ p < 0.01 vs. control (pure 6-OHDA).
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Figure 7. Effects of the tested compounds 2, 4–6, 8–11, and the positive control silybin, administered alone at a concentration of 100 µM: (A). on MDA production at isolated rat brain mitochondria; (B). on GSH level at isolated rat brain mitochondria.
Figure 7. Effects of the tested compounds 2, 4–6, 8–11, and the positive control silybin, administered alone at a concentration of 100 µM: (A). on MDA production at isolated rat brain mitochondria; (B). on GSH level at isolated rat brain mitochondria.
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Figure 8. Effects of the tested compounds 2, 4–6, 8–11, and the positive control silybin, at concentration 50 µM, in combination with t-BuOOH at a concentration of 75 µM: (A). on MDA production at isolated rat brain mitochondria; (B). on GSH level at isolated rat brain mitochondria. ***—p < 0.001 vs. control (non-treated mitochondria). +—p < 0.05 vs. control (pure t-BuOOH).
Figure 8. Effects of the tested compounds 2, 4–6, 8–11, and the positive control silybin, at concentration 50 µM, in combination with t-BuOOH at a concentration of 75 µM: (A). on MDA production at isolated rat brain mitochondria; (B). on GSH level at isolated rat brain mitochondria. ***—p < 0.001 vs. control (non-treated mitochondria). +—p < 0.05 vs. control (pure t-BuOOH).
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Figure 9. Effects of the tested compounds 2, 4–6, 8–11, and silybin, administered alone at a concentration 100 µM, on MDA production at isolated rat brain microsomes.
Figure 9. Effects of the tested compounds 2, 4–6, 8–11, and silybin, administered alone at a concentration 100 µM, on MDA production at isolated rat brain microsomes.
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Figure 10. Effects of the tested compounds 2, 4–6, 8–11, and silybin, at a concentration of 50 µM, in conditions of non-enzyme lipid peroxidation with 20 μM ferrous sulfate and 0.5 mM ascorbic acid on MDA production at isolated rat brain microsomes. ***—p < 0.001 vs. control (non-treated mitochondria). +++—p < 0.001 vs. control (pure iron/ascorbate).
Figure 10. Effects of the tested compounds 2, 4–6, 8–11, and silybin, at a concentration of 50 µM, in conditions of non-enzyme lipid peroxidation with 20 μM ferrous sulfate and 0.5 mM ascorbic acid on MDA production at isolated rat brain microsomes. ***—p < 0.001 vs. control (non-treated mitochondria). +++—p < 0.001 vs. control (pure iron/ascorbate).
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Table 1. 13C-NMR spectral data of compounds 4, 5 (100.61 MHz), and 6 (150.92 MHz) in CD3OD 1.
Table 1. 13C-NMR spectral data of compounds 4, 5 (100.61 MHz), and 6 (150.92 MHz) in CD3OD 1.
Carbon No.δC, Mult
456
Aglycone unit
1110.8, C110.5, C110.8, C
2159.7, C159.8, C159.7, C
398.4, CH98.2, CH98.4, CH
4162.4, C162.5, C162.4, C
596.1, CH95.9, CH96.0, CH
6158.6, C158.6, C158.6, C
1′132.8, C132.9, C132.8, C
2′117.9, CH117.7, CH118.0, CH
3′146.1, C146.0, C146.0, C
4′152.2, C152.0, C152.1, C
5′115.8, CH115.7, CH115.8, CH
6′125.1, CH125.0, CH125.0, CH
C=O197.7, C197.8, C197.7, C
d-glucopyranose
1″102.3, CH101.8, CH102.2, CH
2″75.0, CH74.7, CH73.2, CH
3″75.8, CH77.9, CH79.5, CH
4″72.7, CH71.8, CH69.4, CH
5″76.3, CH75.7, CH78.2, CH
6″62.3, CH265.6, CH262.3, CH2
Benzoyl unit
1′″131.3, C131.3, C131.8, C
2′″ and 6′″130.9, CH130.9, CH130.9, CH
3′″ and 5′″129.7, CH129.8, CH129.6, CH
4′″134.6, CH134.4, CH134.3, CH
C=O167.4, C168.2, C168.0, C
1 Assignments and multiplicities were based on HSQC and HMBC experiments. Chemical shifts were given in ppm and were referenced to the solvent’s residual signal at δC 49.15.
Table 2. 1H-NMR spectroscopic data of compounds 4, 5 (400.13 MHz) and 6 (600.18 MHz) in CD3OD 1.
Table 2. 1H-NMR spectroscopic data of compounds 4, 5 (400.13 MHz) and 6 (600.18 MHz) in CD3OD 1.
PositionδH (J in Hz)
456
36.09 d (2.0)6.08 d (2.0)6.08 d (2.0)
56.29 d (2.0)6.28 d (2.0)6.28 d (2.0)
2′7.31 d (2.1)7.27 d (2.0)7.28 d (2.1)
5′6.79 d (8.3)6.73 d (8.3)6.78 d (8.3)
6′7.25 dd (2.1, 8.3)7.19 dd (8.3, 2.0)7.22 dd (2.1, 8.3)
1″4.97 d (7.8)4.91 d (7.7)5.02 d (7.7)
2″3.27 dd (7.8, 9.3)3.14 dd (8.9, 7.7)3.40 dd (9.5, 7.7)
3″3.75 t (9.3)3.42 dd (9.0, 8.9)5.21 dd (9.5, 9.4)
4″5.03 dd (9.2, 9.6)3.35 dd (9.0, 8.9)3.64 dd (9.7, 9.4)
5″3.72 m3.72 m3.52 m
6″3.67 dd (2.4, 12.3);
3.58 dd (5.5, 12.3)		4.68 dd (11.8, 2.0);
4.33 dd (11.8, 7.3)		3.89 dd (12, 2);
3.74 dd (12, 5)
2′″ + 6′″8.05 m8.04 m8.05 m
3′″ + 5′″7.48 m7.47 m7.47 m
4′″7.62 m7.60 m7.59 m
1 Assignments were based on COSY, HSQC, and HMBC experiments. Chemical shifts were given in ppm and were referenced to the solvent’s residual signal at δH 3.31.
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MDPI and ACS Style

Marinov, T.; Kondeva-Burdina, M.; Kokanova-Nedialkova, Z.; Nedialkov, P.T. Phenolic Constituents from Hypericum aucheri Jaub et. Spach—Isolation, Identification, and Preliminary Evaluation for hMAO-A/B and Neuroprotective Activity. Chemistry 2024, 6, 1535-1551. https://doi.org/10.3390/chemistry6060093

AMA Style

Marinov T, Kondeva-Burdina M, Kokanova-Nedialkova Z, Nedialkov PT. Phenolic Constituents from Hypericum aucheri Jaub et. Spach—Isolation, Identification, and Preliminary Evaluation for hMAO-A/B and Neuroprotective Activity. Chemistry. 2024; 6(6):1535-1551. https://doi.org/10.3390/chemistry6060093

Chicago/Turabian Style

Marinov, Teodor, Magdalena Kondeva-Burdina, Zlatina Kokanova-Nedialkova, and Paraskev T. Nedialkov. 2024. "Phenolic Constituents from Hypericum aucheri Jaub et. Spach—Isolation, Identification, and Preliminary Evaluation for hMAO-A/B and Neuroprotective Activity" Chemistry 6, no. 6: 1535-1551. https://doi.org/10.3390/chemistry6060093

APA Style

Marinov, T., Kondeva-Burdina, M., Kokanova-Nedialkova, Z., & Nedialkov, P. T. (2024). Phenolic Constituents from Hypericum aucheri Jaub et. Spach—Isolation, Identification, and Preliminary Evaluation for hMAO-A/B and Neuroprotective Activity. Chemistry, 6(6), 1535-1551. https://doi.org/10.3390/chemistry6060093

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