Polyphenolic glycosides isolated from Pogostemon cablin (Blanco) Benth. as novel influenza neuraminidase inhibitors

Background Influenza is historically an ancient disease that causes annual epidemics and, at irregular intervals, pandemics. At present, the first-line drugs (oseltamivir and zanamivir) don’t seem to be optimistic due to the spontaneously arising and spreading of oseltamivir resistance among influenza virus. Pogostemon cablin (Blanco) Benth. (P. cablin) is an important traditional Chinese medicine herb that has been widely used for treatment on common cold, nausea and fever. In our previous study, we have identified an extract derived from P. cablin as a novel selective neuraminidase (NA) inhibitor. Results A series of polyphenolic compounds were isolated from P. cablin for their potential ability to inhibit neuraminidase of influenza A virus. Two new octaketides (1, 2), together with other twenty compounds were isolated from P. cablin. These compounds showed better inhibitory activity against NA. The significant potent compounds of this series were compounds 2 (IC50 = 3.87 ± 0.19 μ mol/ml), 11, 12, 14, 15, 19 and 20 (IC50 was in 2.12 to 3.87 μ mol/ml), which were about fourfold to doubled less potent than zanamivir and could be used to design novel influenza NA inhibitors, especially compound 2, that exhibit increased activity based on these compounds. With the help of molecular docking, we had a preliminary understanding of the mechanism of the two new compounds (1–2)’ NA inhibitory activity. Conclusions Fractions 6 and polyphenolic compounds isolated from fractions 6 showed higher NA inhibition than that of the initial plant exacts. The findings of this study indicate that polyphenolic compounds and fractions 6 derived from P. cablin are potential NA inhibitors. This work is one of the evidence that P. cablin has better inhibitory activity against influenza, which not only enriches the compound library of P. cablin, but also facilitates further development and promises its therapeutic potential for the rising challenge of influenza diseases. Electronic supplementary material The online version of this article (doi:10.1186/s13065-016-0192-x) contains supplementary material, which is available to authorized users.


Background
Influenza can cause serious public health and economic problems, which affects millions of people worldwide. Despite advances in the understanding of molecular and cellular aspects of influenza, the disease remains the major cause of mortality and morbidity among patients with respiratory diseases [1].
Influenza viruses have several proteins that are implicated in virulence: the surface proteins hemagglutinin (HA) and neuraminidase (NA), the polymerase complex (including the PB1, PB2 and PA proteins), and the nonstructural proteins [2]. NA is an antiviral target of high pharmaceutical interest because of its essential role in cleaving sialic acid residues from cell surface glycoprotein and facilitating release of virions from infected cells.
The anti-influenza drugs approved for clinical use are the NA inhibitors (orally administered oseltamivir trade name Tamiflu and inhaled zanamivir trade name Open Access *Correspondence: fisher203@126.com College of Pharmacy, Central South University, Changsha 410013, Hunan, People's Republic of China Relenza). Both of them are sialic acid (Neu5Ac) analogues. Because such inhibitors may be structurally recognized as inhibitors by the cellular NA from the host, this might result in side effects. Therefore, developing novel NA inhibitors to combat influenza virus is desirable.
Natural products, especially those derived from traditional Chinese medicine herbs (TCMH), are still the major source of innovative therapeutic agents for infectious diseases, cancer, lipid disorders and immunomodulation [3]. Pogostemon cablin is an annual herb mostly distributed in the tropical and subtropical regions of Asia. P. cablin has been recorded in Chinese Pharmacopoeia as a traditional herbal medicine for its therapeutic functions, including eliminating heat and dampness, calming nerves, and alleviating fatigue. It is used in traditional Chinese medicine for the treatment of upset stomach, vomiting and diarrhea, headache, and fever [4]. Chemical and pharmacological researches on P. cablin have been carried out in recent years [5]. A number of mono-and sesquiterpenoids [6], triterpenoids and steroids [7], flavonoids [8], alkaloids [9] and phenylpropanoid glycosides [10] have been discovered from the title plant.
P. cablin and polyphenolic compounds present in them have gained a lot of interest due to their beneficial health implications. Dietary polyphenolic compounds, especially phenylpropanoid glycosides, exert antioxidant properties and are better inhibitors of NA of influenza A virus [11]. In our ongoing effort to characterize new natural compounds used in Traditional Chinese Medicine (TCM) herbs with interesting chemical structures and/ or pharmaceutical activities, we studied on the chemical constituents of the aerial parts of P. cablin, which led to the isolation of two new octaketides (1, 2), together with other twenty compounds were isolated from P. cablin. This is the first report that presents compounds 1-9, 11 and 21-22 in this genus.
In a previous study from our research group, several extracts derived from P. Cablin have better inhibitory activity on NA. In extending these studies, we examined the effects of these compounds against NA activity. According to the results obtained, the extracts exhibited better inhibitory activity against NA, and the polyphenolic compounds presents in them are responsible for their biological properties. Our current results imply that these specific plant extracts are a possible source of new natural NA inhibitors (Fig. 1).   [12]. On the basis of above data, the structure of 1 was elucidated as 5, 7-dihydroxy-8-((2R)-2-methylbutan-1-onyl)-methyl phenylacetate.

Evaluation of NA inhibition activity
NA remains an attractive anti-influenza drug target, while the emergence of viruses resistant to currently available drugs has presented a new challenge. Therefore, compounds 1-22 and fractions 1-7 (Fig. 5) were tested for their inhibitory effects against the influenza virus NA in vitro with the commercial NA inhibitory screening kit. Even though a number of biological activity studies on this plant have been performed, so far only a few anti-influenza virus constituents from P. cablin have been reported. In this study, the half inhibitory concentration (IC 50 ) of compounds 1-22 were evaluated for their inhibitory effects against the influenza virus NA in vitro as a screening system. The NA inhibitory activity experiment results are shown in Tables 2 and 3 (Additional file 2).
Good oral availability can be achieved by right balance between partitioning and solubility properties. To understand the properties of the proposed compounds better, we utilized Molinspiration [26] to predict some properties of the typical compounds (1, 2, 16, 20 and 22) (Table 4), and applied the Lipinski's rule of five [27] to see whether all passed the criteria. Lipinski's rule of five acts as a filter for drug like properties and states that a potential molecule is orally active if it's molecular weight is ⩽500 da, log P ⩽5, number of hydrogen bond acceptors ⩽10, number of hydrogen bond donors ⩽5. Under the Lipinski's rule of five, compounds (1, 2, 16, 20 and 22) presenting mi log P (< 5) suggested that they may all have good oral bioavailability, and compounds 1 and 22

Molecular docking studies
Earlier crystallographic and ensuing SAR studies have revealed that the active site of NA could be divided into four major binding sites [28]. All NA inhibitors on the market or in clinical phases possess strong structural resemblance in those parts, which correspond to the fact that the four pockets are critical for interaction with the active site of NA. The pocket C1 is comprised of positively charged guanidino groups of arginines 118, 292 and 371 and interacts with the carboxylate. In pocket C5, Arg 152 functions as the hydrogen-bond donor. Trp 178 and Ile 222 comprise a small hydrophobic region. In pocket C4, usually a guanidine or an amine group participates in charge-charge interactions and hydrogen bonds to Glu 119, Asp 151, and/or Glu 227. In pocket C6, Glu 276, the side chain of Arg 152, the amidic carbonyl of Trp 178 and Asp 151 form a new hydrophobic binding pocket. Moreover, Glu 277 and Tyr 406 are believed to play a critical role in the catalytic activity of NA [29,30].
From the activity assay results, compounds 1 and 2 showed better inhibitory activities against NA. To provide a further insight on the observed activities, the binding of compounds 1 and 2 in the active site of NA is shown in Fig. 6. we find that the-COOH group of compound 2 interacts with the pocket C4 of NA active site by hydrogen bond with Glu 119 of this subsite, anomeric carbon of glucose binds to the pocket C4 by hydrogen bond interaction with Asp 151, and 5-OH group forms hydrogen bond with Glu 227 of pocket C4.
Moreover, for compound 1, the 7-OH group binds to the pocket C6 by hydrogen bond interaction with Glu 277, the 1-CO-group forms a hydrogen bond with Arg 152 and Arg 292 of pocket C1, and the 5-OH group binds to the pocket C4 by hydrogen bond interaction with Asp151 (Fig. 7). The binding of compound 1 in the active site of NA showed that the three pockets (C1, C4, C6) of the active site of NA were occupied, although not so well as zanamivir, but still can be a lead compound.

General information
Optical rotations were recorded on a Jasco P-2000 automatic digital polarimeter. The 1 H NMR, 13 C NMR, 1 H-1 H  COSY, HSQC and HMBC spectra were recorded on a Bruker AM 500 spectrometer with TMS as the internal standard at 500 MHz and 125 MHz for 1 H and 13 C. The enzyme activity inhibition assay was carried out on a microplate spectrophotometer (Gemini EM; Molecular Devices). Circular dichroism (CD) spectra were recorded on a CD spectrometer (JASCO, J-815-150S, Japan).

Plant material
The aerial part of P. cablin was purchased from Suixi county, Guangdong province, China, in September 2014. The botanical identification was made by Associate Prof. Jin-ping Li. A voucher specimen (NO.GHX140918) was deposited in College of Pharmacy, Central South University.  B3 was chromatographed on a Sephadex LH-20 column (2 × 150 cm) eluted with MeOH system and then was purified by preparative reverse-phase HPLC eluted with 40 % MeOH/H 2 O (+0.2 % formic acid (FA)) to give compound 15 (7 mg, t R = 23 min) and compound 16 (8 mg, t R = 19 min).

Molecular docking
The cocrystal complex of N1 NA in complex with corresponding ligand oseltamivir downloaded from the protein data bank. (PDB ID code 2HU4) [32]. Before docking, the pre-existing ligand was removed out and hydrogen atoms and charges were added. The docking studies were performed using the Surflex-Dock module of Sybyl 8.1, and the maximum number of poses per ligand was set to 10. The active site of the protein was automatically explored and created based on the previous ligand oseltamivir by the Surflex-Dock Protomol Generation Programme, and other parameters were set as default.

Conclusions
The two new compounds (1, 2) and compounds 11, 12, 14, 15, 19 and 20 showed better inhibitory activity against NA in vitro. By comparing with the structures of compound 11, 12, 14, 15, 19 and 20, they all have one caffeoyl, and this is a possible reason that these compounds have better inhibitory activity against NA than other polyphenolic compounds. With the help of molecular docking, we had a preliminary understanding of the mechanism of the two new compounds (1-2)′ NA inhibitory activity. According to the Lipinski's rule of five, compound 1 may be a better lead compound for anti-influenza.
Fractions 6 and polyphenolic compounds isolated from fractions 6 showed higher NA inhibition than that of the initial plant exacts (Tables 2, 3). The findings of this study indicate that polyphenolic compounds and fractions 6 derived from P. cablin are potential NA inhibitors. This work was one of the evidence that P. cablin has better inhibitory activity against influenza, which not only enriches the compound library of P. cablin, but also facilitates further development and promises its therapeutic potential for the rising challenge of influenza diseases.