Flavanonol Glycosides from the Stems of Myrsine seguinii and Their Neuroprotective Activities

The accumulation of amyloid beta (Aβ) peptides is common in the brains of patients with Alzheimer’s disease, who are characterized by neurological cognitive impairment. In the search for materials with inhibitory activity against the accumulation of the Aβ peptide, seven undescribed flavanonol glycosides (1–7) and five known compounds (8–12) were isolated from stems of Myrsine seguinii by HPLC-qTOF MS/MS-based molecular networking. Interestingly, this plant has been used as a folk medicine for the treatment of various inflammatory conditions. The chemical structures of the isolated compounds (1–12) were elucidated based on spectroscopic data, including 1D and 2D nuclear magnetic resonance (NMR), high-resolution electrospray ionization mass spectrometry (HRESIMS) and electronic circular dichroism (ECD) data. Compounds 2, 6 and 7 showed neuroprotective activity against Aβ-induced cytotoxicity in Aβ42-transfected HT22 cells.


Introduction
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive memory loss and behavioral abnormalities [1]. Due to the increase in the size of the elderly population, the number of patients with AD is currently estimated to be 5.5 million in the United States, and the number is expected to reach over 13.8 million by 2050 [2]. The main pathological features of AD are deposits of extracellular amyloid beta (Aβ) plaques. The insoluble Aβ protein and neurofibrillary tangles of the tau-protein, which are involved in microtubule maintenance in neural cells, ultimately result in memory dysfunction [3]. Aβ peptides composed of 39-42 amino acids are produced by the cleavage of amyloid precursor protein (APP) by β-secretase and γ-secretase [4]. In particular, Aβ 1-40 and Aβ 1-42 , which are more neurotoxic than other Aβ peptides, are excessively produced in cerebral neural cells and accumulate in the form of senile plaques [5]. The accumulated senile plaques form protofibrils, fibrils and plaques by Aβ oligomerization [6], which has been implicated in neuronal dystrophy and synaptic loss through pathways such as neurotoxicity, oxidative stress and inflammatory reactions in both human and mouse models of AD [7][8][9]. The U. S. Food and Drug Administration (FDA) has approved drugs such as tacrine, donepezil, rivastigmine and galantamine (as acetylcholine esterase enzyme inhibitors) and memantine (as an N-methyl-D-aspartate (NMDA) receptor antagonist) for use in AD patients [10]. However, these drugs, which have side effects such as vomiting and hepatotoxicity, have no therapeutic effect and only cause temporary improvement [11]. Galantamine is a naturally occurring plant tertiary alkaloid that acts not only as a reversible inhibitor of acetylcholinesterase but also as an allosteric modulator of nicotinic receptors and is used as a treatment for mild-to-moderate Alzheimer's disease [12]. Natural products are a good source of potential Alzheimer's treatments with relatively few side effects and excellent efficacy.
Myrsine seguinii H. Lév., belonging to the family Primulaceae, occurs as shrubs or trees that can grow up to 2-12 meters in height and is distributed in Southeast Asian countries such as Vietnam and China [13]. The plant mainly grows in tropical and subtropical regions of the world and has a limited distribution in the northern area of Japan [14]. The genus Myrsine comprises approximately 300 species of evergreen shrubs and trees, which have traditionally been used as a folk medicine to treat anti-inflammatory and infectious diseases in Southeast Asian countries [15,16]. M. africana is used as an antitapeworm infection agent and as a treatment for dropsy, colic and dysmenorrhea [17]. In particular, M. seguinii is used in Myanmar as a medicinal plant for treating colds, influenza and headaches, and it has been used to treat inflammatory diseases on Okinawa Island, Japan [18]. The phytochemical components of M. seguinii have been reported to include flavonol glycosides [19], anti-inflammatory prenylated benzoic acid derivatives and myrsinionosides consisting of hydroquinones and p-benzoquinones [20,21]. Inflammation has been reported as a distinctive feature of Alzheimer's patients, along with two other pathological features (β-amyloid and neurofibrillary tangles) and the link between the initial accumulation of β-amyloid and the subsequent development of neurofibrillary tangles has been investigated [22]. Myrsine seguinii is a plant of interest for identifying potential AD therapeutic agents because it has traditionally been used to treat inflammation and is reported to contain chemicals with anti-inflammatory effects.

Authentication of M. seguinii H. Lév
In the case of plants used as traditional medicines, special attention should be paid to the authentication of samples, as their intake in the patients may cause severe side effects [24,25]. In the genus Myrsine, which consists of approximately 300 species of evergreen shrubs and trees, the authentication of M. seguinii was attempted based on plant morphology, cross-sectional observations and the DNA barcode technique.
Morphologically, the studied samples were shrubs with a height of 3-4 m or trees with a height up to 10 m. The stems were highly branched and covered by cracked white bark. The branches were 3-5 mm in diameter and were white lenticellate, rugose, reddish puberulent and glabrescent in early stages. The leaves were simple and alternate, with petioles of 2-3 mm. The leaf blade was elliptic to narrowly linear-oblanceolate, 3-7 cm long, 1-2 cm wide, leathery, glabrous, base cuneate and reddish, with an entire margin and acute apex. Lateral veins were apparent, with 20-25 pairs ( Figure 1A). The morphological features closely matched the description of M. seguinii in the Flora of China [26]. A crosssection of a leaf of M. seguinii is illustrated in Figure 1B. The upper and lower epidermis consisted of a single cell layer and covered the entire midrib surface. The upper and lower collenchyma was under the epidermal layer, with straight anticlinal wall cells. Calcium oxalate crystals and secretory schizogenous cavities were distributed in a scattered manner throughout collenchymal tissues. The vascular bundles occupied half of the transverse section area and were covered by 2-4 layers of sclerenchyma. The phloem consisted of 3-5 layers of small cells and covered the xylem. The order of the tissues from the lower to the upper parts of the leaf blade was as follows: lower epidermis, spongy mesophyll, palisade mesophyll and upper epidermis. The image of the stem cross-section shows the secondary structure of the stem of M. seguinii ( Figure 1C). The outermost layer was the cork, which consisted of thick multiple cell layers. This layer was followed by multiple layers of parenchyma cells that showed a variety of shapes and could contain calcium oxalate crystals. The vascular bundles occupied more than half of the transverse section area and were arranged as cascade clusters. Each bundle consisted of secondary phloem, secondary xylem and primary xylem. The sclerenchyma cells covered the secondary phloem in the form of fiber bundles. The primary phloem was not visible. Several secretory schizogenous cavities could be observed inside the pith. Calcium oxalate crystals and secretory schizogenous cavities were distributed in a scattered manner throughout collenchymal tissues. The vascular bundles occupied half of the transverse section area and were covered by 2-4 layers of sclerenchyma. The phloem consisted of 3-5 layers of small cells and covered the xylem. The order of the tissues from the lower to the upper parts of the leaf blade was as follows: lower epidermis, spongy mesophyll, palisade mesophyll and upper epidermis. The image of the stem cross-section shows the secondary structure of the stem of M. seguinii ( Figure 1C). The outermost layer was the cork, which consisted of thick multiple cell layers. This layer was followed by multiple layers of parenchyma cells that showed a variety of shapes and could contain calcium oxalate crystals. The vascular bundles occupied more than half of the transverse section area and were arranged as cascade clusters. Each bundle consisted of secondary phloem, secondary xylem and primary xylem. The sclerenchyma cells covered the secondary phloem in the form of fiber bundles. The primary phloem was not visible.
Several secretory schizogenous cavities could be observed inside the pith. The DNA sequence was compared with published sequences available in GenBank (National Institutes of Health) using the basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information (NCBI) to generate the neighbor-joining (NJ) tree. The results of the DNA sequencing analysis and BLAST analysis showed high similarity with sequences from the genus Myrsine (Table S1). The BLAST analysis of trnH-psbA sequences showed sequence similarity of 99% with M. seguinii distributed in Vietnam, Cambodia and China, and M. umbellate distributed in Ecuador and Brazil. The BLAST analysis of nrITS revealed 98% agreement with M. segunii and M. chatthamica from the Chatham Islands. The experimental sample was grouped with the genus Myrsine in the NJ tree based on matK, rbcL, trnL-trnF and trnH-psbA ( Figure S1). The NJ tree analysis of trnL-trnF showed that the experimental sample was located closest to M. seguinii from Vietnam, Cambodia and China ( Figure S1C). The experimental sample was finally authenticated as M. seguinii H.Lév on the basis of its morphological characteristics and DNA sequencing analysis.

Figure 5.
Effects of compounds 1-12 on the intensity of the green fluorescence induced by pEGFP-C1/Aβ42 plasmid transfection using Lipofectamine in HT22 cells after 10 h of transfection. The transfected cells were continually exposed to the test compounds at 20 μM. After incubation for 24 h, the cells were visualized using fluorescence microscopy.

Discussion
In this study, the studied plant was authenticated as M. seguinii H. Lév based on its morphological characteristics and DNA sequencing analysis. The misidentification of plants can result in toxic responses in patients who ingest the plants [24]. The expressed plant phenotype is described as a feature of the genotype that is modified in response to Figure 5. Effects of compounds 1-12 on the intensity of the green fluorescence induced by pEGFP-C1/Aβ 42 plasmid transfection using Lipofectamine in HT22 cells after 10 h of transfection. The transfected cells were continually exposed to the test compounds at 20 µM. After incubation for 24 h, the cells were visualized using fluorescence microscopy.

Discussion
In this study, the studied plant was authenticated as M. seguinii H. Lév based on its morphological characteristics and DNA sequencing analysis. The misidentification of plants can result in toxic responses in patients who ingest the plants [24]. The expressed plant phenotype is described as a feature of the genotype that is modified in response to different environmental conditions [31]. Since the authentication of this plant was difficult, analyses of morphological characteristics, leaf and stem cross-sections, and the five DNA barcode sequences (matK, rbcL, trnH-psbA, trnL-trnF and nrITS) were performed. Seven undescribed flavanonols (1-7) along with five known compounds (8)(9)(10)(11)(12) were isolated by MS/MS molecular networking from the stem of M. seguinii. Compounds 1-7 were 3,3 ,4 ,5,7-pentahydroxyflavanones with one rhamnose and a variety of attached benzoyl groups, including galloyl, 3,4-dihydroxybenzoyl, vanilloyl, 4-hydroxybenzoyl and transferuloyl groups. Flavanonol glycosides with aromatic groups are uncommon derivatives that can be isolated from plants. In particular, M. seguinii is unique in that flavanonol glycosides with various functional groups can be isolated from this single plant species.
Compounds 2, 6 and 7 showed significant neuroprotective activities against Aβinduced cytotoxicity in Aβ 42 -transfected HT22 cells. The occurrence of AD is known to involve deposits of insoluble Aβ proteins and neurofibrillary tangles of tau-protein.
Oxidative stress and inflammation are among the results of the production of these proteins [22,32]. Flavanonol glycosides from leaves of Engelhardia roxburghiana were found to have anti-inflammatory properties in an in vitro assay. These derivatives exert inhibitory effects on the mRNA expression of IL-1β and Cox-2 depending on the presence of galloyl or coumaroyl moieties [33]. The stereoisomers of astilbin based on the C-2 and C-3 configurations could affect the capacities of different antioxidants in the DPPH radical system and ABTS + radical scavenging and anti-inflammatory effects by stimulating IL-1β, IL-6 and NO in RAW264.7 cells [34]. These results also suggest that the observed neuroprotective effects can be attributed to various aromatic groups, such as galloyl, hydroxybenzoyl and trans-feruloyl groups, and show different protective effects against Aβ-induced cytotoxicity depending on the type of substituent. Future research should focus on the structural specificity of the biological activity of phytochemicals.

General Experimental Procedures
Optical rotations were measured on a JASCO P-2000 polarimeter (JASCO International Co. Ltd., Tokyo, Japan). IR data were collected using a Nicolet 6700 FT-IR spectrometer (Thermo Electron Corp., Waltham, MA, USA). ECD spectra were recorded using Chirascan Plus (Applied Photophysics Ltd., Surrey, UK). UHPLC systems (Ultimate 3000, Thermo Scientific, Milan, Italy) coupled to a Waters Xevo G2 QTOF MS spectrometer (Waters, Co., Milford, MA, USA) were used to generate HRESIMS values and perform LC-MS/MS analysis. Semipreparative HPLC was performed using a Gilson HPLC system with a UV/VIS-155 detector and a 321 pump. An RS Tech Optima Pak C18 column (10 × 250 mm, 10 µm) was used as the HPLC column. All solvents employed for extraction and isolation were of analytical grade. The 1D ( 1 H and 13 C) and 2D (HSQC, HMBC and 1 H-1 H COSY) NMR spectra were collected using JEOL 400 MHz (JEOL Ltd., Tokyo, Japan), Bruker Avance 500 MHz and Bruker Avance 800 MHz NMR spectrometers (Bruker, Billerica, MA, USA). Diaion TM HP-20 ion exchange resin and GE Healthcare Sephadex TM LH-20 (18-111 µm) were employed for column chromatography. Thin layer chromatography was performed with silica gel 60 F 254 and RP-18 F 254 plates.

Morphology and DNA Sequencing Analysis of M. seguinii
The sample was authenticated by matching its morphological features with available taxonomic descriptions of M. seguinii H. Lév. Photographs were obtained with a Canon EOS 60D camera and Canon 100 mm f2.8 IS Macro lens (Canon Inc. Tokyo, Japan). An EZ4 stereo microscope was used to analyze the features of M. seguinii leaf and stem cross-sections. Genomic DNA was extracted from a branch from which the outer skin was removed using a G-spinII Plant Genomic DNA extraction kit (Invitrogen, Seoul, Korea). The extracted genomic DNA was amplified by PCR using primers targeting four chloroplast genomic markers (matK, rbcL, trnH-psbA and trnL-trnF) and the nuclear genome marker ITS region. The amplified PCRs products were purified using a PCR quick-spin TM PCR Purification kit (Intron, Seongnam, Korea) and the DNA sequences were determined with the primers used for PCR. The DNA sequence was modified using Geneious 11.1.2 to determine the final DNA sequence.

LC-MS/MS Analysis and Molecular Networking
LC-MS/MS analysis was carried out by using UHPLC systems (Ultimate 3000, Thermo Scientific, Milan, Italy) coupled to a Waters Xevo G2 QTOF MS spectrometer (Waters, Co., Milford, MA, USA) with an Acquity UPCL BEH C18 column (2.1 × 100 mm, 1.7 µm). The mobile phase was composed of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The gradient program was as follows: 0-20 min, linear gradient of 10% to 90% B; 20-22 min, isocratic at 100% B; 22-24 min, return to the initial conditions. The flow rate was 0.3 mL/min, and the injection volume was 2 µL during the acquisition of negative polarity. The MS/MS analysis was performed in data-dependent scan mode,

Cell Culture and Cell Viability Assay
HT22 immortalized mouse hippocampal neuronal cells at 70% confluence were cultured in DMEM (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin for 48 h. Then, the HT22 cells were seeded in 96-well plates at 3000 cells/well. After incubation for 24 h, the test compounds (20 µM) were added to the cells for 24 h and cell viability was measured via the MTT (Sigma, St Louis, MO, USA) reagent method. A 20 µL aliquot of MTT solution (2 mg/mL) was injected into each well, and the plate was incubated for 3 h in the dark. After the resultant formazan crystals were dissolved in DMSO, the absorbance was measured with a microplate reader (VersaMaxTM, Randor, PA, USA) at 570 nm.

Cytotoxicity Assay of Aβ 1-42 -Transfected HT22 Cells
HT22 cells were seeded in 96-well plates at 3000 cells/well and incubated at 37 • C with 5% CO 2 for 3 h [35,36]. The cells in each well were transfected with 0.2 µg of the pEGFP-C1/Aβ 42 plasmid (originating from Professor Junsoo Park, Yonsei University, Seoul, Korea) with Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). After transfection for 10 h, the tested compounds dissolved in the medium were added to the seeded cells, and the cells were incubated for 24 h. Then, 20 µL of MTT solution (2 mg/mL) was added, and the cells were incubated for 3 h in the dark. The cells were subsequently washed with phosphate-buffered saline (PBS) (Takara, Kusatsu, Japan), and 100 mL of DMSO was added to solubilize formazan. The absorbance at 570 nm was measured with a microplate reader (VersaMaxTM, Randor, PA, USA). Fluorescence imaging was conducted by using a fluorescence microscope (Olympus ix70, Tokyo, Japan).

Statistical Analysis
Data were evaluated as the mean ± SD of three independent experiments. Data were processed by analysis of variance (ANOVA) which was conducted using SPSS Statistics 23 (SPSS, Inc., Chicago, IL, USA). Statistically significant p values were established at * p < 0.05, ** p < 0.01, *** p < 0.001.

Conclusions
The sample studied herein was authenticated as M. seguinii H. Lév on the basis of its morphological features and the DNA barcode technique. Seven new flavanonol glycosides with various conjugated aromatic groups and five known flavanonol glycosides were isolated by LC-MS/MS molecular networking, and their neuroprotective effects against Aβ 42 -induced cytotoxicity were measured. Among the isolated compounds, compounds 2, 6 and 7 showed potential neuroprotective activity. Compound 2, with a galloyl group and a cis-configuration at the C-2 and C-3 positions, showed the strongest protective activity. These results suggested that flavanonol glycosides from M. seguinii could be good candidates for use in AD treatment.