Neolignans and Norlignans from Insect Medicine Polyphaga plancyi and Their Biological Activities

Abstract Ten neolignans or norlignans (1–10) including eight new compounds were isolated from the whole bodies of Polyphaga plancyi Bolivar. Their structures were identified by spectroscopic data. Compounds 3, 4, 8, and 9 are racemates indicated by chiral HPLC analysis. Chiral separation followed by ECD calculations allowed to clarify the absolute configurations of all the antipodes. All the new compounds were evaluated for their biological properties toward extracellular matrix in rat renal proximal tubular cells, human cancer cells (K562, A549, and Huh7), EV71, ROCK2, JAK3, DDR1, and coagulation. Graphic Abstract Electronic supplementary material The online version of this article (10.1007/s13659-020-00262-0) contains supplementary material, which is available to authorized users.

Keywords Polyphaga plancyi · Insect medicine · Lignans · Renal protection · Anticoagulant activity In honor of Professor Jun Zhou.
Hong-Jie Zhu and Te Xu have contributed equally to this work.

Introduction
Insects are a special group of existence on the earth. Facing the changing environment, they have strong adaptability, productivity and continuity. They seem small and insignificant, but the largest number of living beings on the planet, and their presence has created a huge molecular libraries [1,2]. The tenacious vitality of insects is highly likely to be related to their unique molecular mechanisms [2], which provides us a confidence to track their bioactive molecules.
In China, the insect Polyphaga plancyi Bolivar has been used for promoting blood circulation [3]. Whereas, so far there have few reports about its chemical composition [4]. Previous studies revealed the significance of structurally novel non-peptide small molecules (NPSMs) [5][6][7][8][9][10][11], inspiring our further interest on insect chemistry. As far as the title species was concerned, eight new compounds and two known compounds belonging to neolignans and norlignans were isolated in this study (Fig. 1). Chiral HPLC separation was used to afford optically active isomers. Subsequent absolute configuration clarification was aided by electronic circular dichroism (ECD) calculations. In addition, the biological activities of all the new compounds were assessed using multiple assays.
Plancyin D (4) has the molecular formula C 19 H 20 O 6 (10 degrees of unsaturation), based on analysis of its HREIMS, 13 C NMR, and DEPT spectra. The 1 H and 13 C NMR data of 4 resemble those of 3, differing in that the additional existence of a methyl in 4. The HMBC correlation of H-10′ (δ H 3.63, s)/C-9′ (δ C 175.2) indicates the connection position of the methyl group as shown. Thus, the planar structure of 4 was determined. For the structure like 3, it is challenging to clarify the stereochemistry at C-8. Separation of 4 by chiral HPLC afforded (+)-4 and (−)-4, whose absolute configurations were further determined by ECD calculations and comparison (Supplementary material) with the data of 3, allowing (+)-4 to be 8S.
Plancyin E (5) has the molecular formula C 17 H 18 O 4 (9 degrees of unsaturation), based on analysis of its HREIMS and NMR data. Inspection of these NMR data disclosed that the structure of 5 extremely resemble that of 3, differing in that a carboxyl group of C-8 is absent gaining support from the HMBC correlations of H-8/C-1, C-4′, C-6′ and the chemical shift of C-8 (δ C 39.0). Therefore, the structure of 5 was constructed.
Proteins or peptides are commonly considered to be the active substances in the insects. However, the chemical profiling and biological role of nonpeptidal small molecules in the insects remains largely unknown. In this study, ten lignan derivatives were characterized. It is evident that compounds 5, 6, and 9 are diverse norlignans and there exist four types of connection pattern for the ten lignans, adding new facets for the insect derived natural products.

Biological Evaluation
To explore the mechanism underlying the antifibrotic effect of the compounds, we first examined whether the compounds affected TGF-β1-induced activation of the marker genes in NRK-52e cells (40 μM, data not shown). As presented in Fig. 7, compound 1 reduces three marker genes, especially for fibronectin and α-SMA in TGF-β1-induced NRK-52e cells (Fig. 7a). Compound 7 significantly inhibits three marker genes (Fig. 7b). Besides, Western bolt assay shows that compounds 1 and 7 decrease the protein expression with a dose-dependent manner in TGF-β1induced NRK-52e cells (Fig. 8a, b). Next, we assessed the expression of Smad2/3 phosphorylation in NRK-52e cells.

Insect Material
The specimen of Polyphaga plancyi was purchased from Henan Province, China, in November 2014, and identified by Prof. Da-Rong Yang at Kunming Institute of

Biological Evaluation for EV71
Compounds 1-9 were evaluated for their EV71 inhibitory activities in vitro. VERO cells were plated into 384-well plate at a suitable density and allowed to adhere prior to addition of varying concentrations of drugs. Then the cells were covered with enteroviruses EV71 which was diluted at fresh culture medium. After that, the cells were incubated at 37 °C for a further 72 h. Then discard the old medium, add fresh medium containing CCK-8, and incubated in 37 °C for 2 h. The A450 was then measured with an Envision Plate Reader (PerkinElmer).

Anticoagulant Assay
The prothrombin time (PT) and thrombin time (TT) was determined with a coagulometer (TECO MC-4000, Germany). All the reagents were purchased from TECO GmbH (Germany). PT reagent, TT reagent, and normal human plasma was reconstituted in 4 mL (or 1 mL for plasma) of distilled water, according to the instructions of the manufacturer. The compounds were dissolved to 2 mM in DMSO at various concentrations. Then use 20 mM Tris-HCl pH 7.4 (including 5% Tween 80) diluted to 200 µM to be measured. For the PT and TT assays, 5 μL samples (or 10 μL for TT) were mixed with 45 μL (or 90 μL for TT) of normal human plasma and incubated for 2 min at 37 °C; 100 μL of PT (or 50 μL of TT) reagent was then added and the clotting time was recorded.

Platelet Aggregation Assay
Turbidometric measurements of platelet aggregation inhibition were performed in a Chronolog Model 700 Aggregometer (Chronolog Corporation, Havertown, PA, USA) according to Born's method [17,18]. The present study was approved by the Research Ethics Committee of Kunming Institute of Botany, Chinese Academy of Sciences. The blood from the rabbits by ear central arter puncture, were anticoagulated with 3.8% sodium citrate (9:1, v/v). Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared shortly after blood collection by spinning the sample at 180 g for 10 min at 22 °C. The PRP was carefully removed and the remaining blood centrifuged at 2400×g for 10 min to obtain PPP. The centrifuge temperature was maintained at 22 °C. Platelet counts were adjusted by the addition of PPP to the PRP to achieve a count of 250 × 10 9 /L. Platelet aggregation studies were completed within 3 h of preparation of PRP. Immediately after preparation of PRP, 250 μL was transferred into each of the test tubes, with 250 μL PPP set as a control. Before addition of inducers, the compounds were incubated with PRP at 37 °C for 5 min. Final concentration of agonist was: arachidonic acid (AA) 0.5 mM as positive reference. Percentage inhibition by the compounds was calculated according to the formula: where A: maximum change of turbidity in DMSO added, B: maximum change of turbidity in sample added.

Computational Methods
Molecular Merck force field (MMFF) and DFT/TDDFT calculations were performed with Spartan'14 software package (Wavefunction Inc., Irvine, CA, USA) and Gaussian 09 program package [19], the conformational search generated low-energy conformers within a 10 kcal/mol energy was finished by software Conflex 7. Geometry optimizations of compounds 3, 8, and 9 were carried out at the DFT/B3LYP/6-311G (d, p) level. The calculated ECD spectra were determined by using Gaussian 09 software employing the TDDFT-B3LYP functional and the 6-311G (d, p) basis sets. ECD calculations further were conducted at the B3LYP SCRF (PCM)/6-311G (d,p) level in MeOH.

Compliance with Ethical Standards
Conflicts of interest The authors declare that they have no conflict of interest.
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