Four New Polyprenylated Acylphloroglucinols from Hypericum perforatum L.

Hyperforatums A–D (1–4), four new polyprenylated acylphloroglucinols, together with 13 known compounds were isolated and identified from the aerial parts of Hypericum perforatum L. (St. John’s wort). Their structures were confirmed with a comprehensive analysis comprising spectroscopic methods, including 1D and 2D NMR, HRESIMS, and electronic circular dichroism (ECD) calculations. Hyperforatum A featured an unusual chromene-1,4-dione bicyclic system, and hyperforatums B and C were two rare monocyclic PPAPs with five-membered furanone cores. Compound 1 exhibited a moderate inhibition effect on NO production in BV-2 microglial cells stimulated by LPS.


Introduction
The genus Hypericum is a large family, consisting of approximately 500 species [1].Plants of this genus are widely distributed throughout the world and some of them are used as folk medicinal herbs [2].H. perforatum (St.John's wort) is extensively used to treat mild to moderate mental depression in many countries [3].In addition, the extracts of H. perforatum showed anti-neurodegenerative disease, antitumor, and antimicrobial activities [4][5][6].Chemical researches studying this plant revealed the presence of diverse PPAPs [7][8][9], flavonoids [10], phenolic acids, and so on [11].Now, more than 1100 polycyclic polyprenylated acylphloroglucinols (PPAPs) have been isolated and identified from the genus Hypericum [12], but complex and novel carbon skeletons of PPAPs are consistently found from this plant, for example, hyperfols A and B [9], hyperforen A [13], hyperforones A-J [14].Moreover, these PPAPs demonstrated significant neuroprotective effects, especially against Alzheimer's disease.Thus, the discovery of intricate PPAPs is essential as they are the leading compounds for the treatment of Alzheimer's disease.
The planar structure of compound 1 was established by interpreting its 2D NMR data (Figure 2  .Furthermore, a methoxy group was located at C-1 due to the HMBC correlation.Thus, the planar construction of compound 1 was finally built (Figure 2).
The planar structure of compound 1 was established by interpreting its 2D NMR data (Figure 2 S6).Furthermore, a methoxy group was located at C-1 due to the HMBC correlation.Thus, the planar construction of compound 1 was finally built (Figure 2).The relative configuration of compound 1 was elucidated by the NOESY data (Figure 3).The NOESY correlations of H-2/H-15b, H-2/H-4, H-2/H2-26b, H-5a/H-21a, H-5a/H-31b, and H-31b/H3-OCH3 indicated that H-2 and the isoprenyl group at C-3 and C-8 were αoriented; moreover, the isoprenyl group at C-4 and C-6 and the methoxy group were in the same β-orientation (Figure S7).From the above analysis, the relative configuration of compound 1 was determined to be 1R*, 2S*, 3R*, 4S*, 6S*, 8R*.Moreover, the absolute configuration of (1R, 2S, 3R, 4S, 6S, 8R)-1a was determined using the calculated ECD data, showing good agreement with the experimental data (Figure 4).The relative configuration of compound 1 was elucidated by the NOESY data (Figure 3).The NOESY correlations of H-2/H-15b, H-2/H-4, H-2/H 2 -26b, H-5a/H-21a, H-5a/H-31b, and H-31b/H 3 -OCH 3 indicated that H-2 and the isoprenyl group at C-3 and C-8 were α-oriented; moreover, the isoprenyl group at C-4 and C-6 and the methoxy group were in the same β-orientation (Figure S7).From the above analysis, the relative configuration of compound 1 was determined to be 1R*, 2S*, 3R*, 4S*, 6S*, 8R*.Moreover, the absolute configuration of (1R, 2S, 3R, 4S, 6S, 8R)-1a was determined using the calculated ECD data, showing good agreement with the experimental data (Figure 4).The relative configuration of compound 1 was elucidated by the NOESY data (Figure 3).The NOESY correlations of H-2/H-15b, H-2/H-4, H-2/H2-26b, H-5a/H-21a, H-5a/H-31b, and H-31b/H3-OCH3 indicated that H-2 and the isoprenyl group at C-3 and C-8 were αoriented; moreover, the isoprenyl group at C-4 and C-6 and the methoxy group were in the same β-orientation (Figure S7).From the above analysis, the relative configuration of compound 1 was determined to be 1R*, 2S*, 3R*, 4S*, 6S*, 8R*.Moreover, the absolute configuration of (1R, 2S, 3R, 4S, 6S, 8R)-1a was determined using the calculated ECD data, showing good agreement with the experimental data (Figure 4).Hyperforatum B (2) was purified as a colorless oil.The molecular formula of C36H56O6 was confirmed by its HRESIMS data (m/z: [M + Na] + calcd.607.3969; found 607.3958), indicating 9 degrees of unsaturation (Figure S18).1).These deviations might have resulted from the differences in the configurations of the isoprenyl group at C-4.When the orientation of the isoprenyl group was changed at the C-4 position, the two large groups attached at the ends of the C-4 position had steric hindrance effects, which might have led to a significant difference of chemical shifts around the C-4 position.The deduction cannot be confirmed by the key NOESY correlations because the isoprenyl group was located in a flexible side chain.The NOESY correlations of H-8/H-5a indicated that the stereochemistry of C-8 was α-oriented (Figures 3 and S17).The absolute configuration of compound 2 was unable to be calculated due to the chiral center being on a flexible chain.Moreover, the chemical shifts of H 2 -21 and H-22 also changed significantly (Table 1).These deviations might have resulted from the differences in the configurations of the isoprenyl group at C-4.When the orientation of the isoprenyl group was changed at the C-4 position, the two large groups attached at the ends of the C-4 position had steric hindrance effects, which might have led to a significant difference of chemical shifts around the C-4 position.The deduction cannot be confirmed by the key NOESY correlations because the isoprenyl group was located in a flexible side chain.The NOESY correlations of H-8/H-5a indicated that the stereochemistry of C-8 was α-oriented (Figure 3 and Figure S17).The absolute configuration of compound 2 was unable to be calculated due to the chiral center being on a flexible chain.The molecular formula of hyperforatum C (3) was deduced as C 27 H 44 O 3 based on its HRESIMS data (m/z: [M + Na] + calcd 439.3183, found 439.3177), suggesting 6 unsaturation sites (Figure S28).The 1 H NMR spectrum showed the presence of 3 olefinic protons (δ H 5.11, 2H, overlap; 4.93, 1H, t, J = 7.2 Hz), 7 singlet methyls (δ H 1.03-1.69,s), and an isopropyl (δ H 2.69, 1H, m; 1.22, d, 3H, J = 6.8 Hz; 1.20, d, 3H, J = 6.8 Hz).The 13 C NMR data of compound 3 displayed 27 carbons assigned to 9 methyls, 5 methylenes, 6 methines (4 olefinic), and 7 nonprotonated carbons (1 carbonyl, 4 olefinic, 2 oxygenated).The above analysis suggested that compound 3 should be a monocyclic PPAP (Figures S21-S23).The HMBC interactions from  S26).Compound 3 might be obtained with a rearrangement of monocyclic polyprenylated acylphloroglucinols (MPAPs).The relative configuration of compound 3 could not be determined by the NOESY correlations because its chiral center was located in the flexible chain.Meantime, ECD calculations were quite challenging in the determination of the absolute configuration of compound 3. Unfortunately, its crystal failed to be obtained after the solvent conditions were changed multiple times.
There are many reports on the anti-Alzheimer's effects of H. perforatum [14,32].Chronic inflammation is an important cause of the development of Alzheimer's disease's pathogenesis [33].The production of nitric oxide (NO) in LPS-stimulated microglial cells is used as a cellular model to evaluate the effects of anti-neuroinflammation.Since we ended up with insufficient quantities of compounds 2, 3, and 4 to complete activity evaluation, we assessed the biological activity of compound 1 only.Consequently, compound 1 significantly inhibited NO production at 40 µM (Figure 5).However, its anti-inflammatory mechanisms need to be further explored.

Plant Materials
Air-dried aerial portions of H. perforatum were collected in August 2018 from Shangluo City, China.The plant was identified by Pro.Zhen-hai Wu.The sample (no.20180805HPL) was preserved at Shaanxi Key Laboratory of Natural Products and Chemical Biology, Northwest A&F University.

Plant Materials
Air-dried aerial portions of H. perforatum were collected in August 2018 from Shangluo City, China.The plant was identified by Pro.Zhen-hai Wu.The sample (no.20180805HPL) was preserved at Shaanxi Key Laboratory of Natural Products and Chemical Biology, Northwest A&F University.

Cell Culture
Microglial BV-2 cells from China Center for Type Culture Collection (Wuhan, China) were cultivated in DMEM (Gibco, New York, NY, USA) containing 10% FBS (Gibco) and antibiotics (100 U/mL streptomyces and penicillin) in humidified incubators under 5% CO 2 at 37 • C.

Measurement of Nitric Oxide (NO) Production
BV-2 cells were seeded in 96-well plates (2 × 10 5 cells/mL) overnight.The cells were treated with LPS (2 µg/mL) and various concentrations of hyperforatum A (1) (10, 20, 40 µM) for 24 h, with S-Methylisothiourea (SMT) as the positive control.The production of NO was measured in cell supernatants with a Griess reagent.The absorbance was recorded at 540 nm using a microplate reader.The MTT method was applied to determine the cell viability after incubation using the test compound.

Statistical Analysis
All data were presented as mean ± SD and analyzed with GraphPad Prism 9.0 software.The significant differences between different groups were performed using one-way ANOVA multiple comparisons.

Conclusions
The phytochemical components of the PPAPs and terpenoids were investigated from the aerial parts of H. perforatum.Seventeen secondary metabolites, including five PPAPs and nine terpenoids, were isolated and identified from the title plants.This study reported two unusual carbon cores of PPAPs, of which hyperforatum A was a chromene-1,4-dione bicyclic system, and hyperforatum B and C possessed rare monocyclic features.The new compound, hyperforatum A (1), displayed a moderate inhibitory capacity on LPS-induced NO production.

Figure 4 .
Figure 4. Calculated and experimental ECD spectra of compound 1.