Anti-Influenza Virus Activity and Chemical Components from the Parasitic Plant Cuscuta japonica Choisy on Dimocarpus longans Lour.

Dodder (Cuscuta spp.) is a parasitic weed damaging many plants and agricultural production. The native obligate parasite Cuscuta japonica Choisy (Japanese dodder) parasitizes Dimocarpus longans Lour., Ficus septica Burm. F., Ficus microcarpa L.f., Mikania micrantha H.B.K. and Melia azedarach Linn, respectively. Five Japanese dodders growing on different plants exhibit slightly different metabolites and amounts which present different pharmacological effects. Among these plants, a significant antiviral activity against influenza A virus (IAV) was found in Japanese dodder parasitizing on D. longans Lour. (CL). To further explore methanol extract components in Japanese dodder (CL), four undescribed aromatic glycosides, cuscutasides A–D (compounds 1–4) were isolated, together with twenty-six known compounds 5–30. The chemical structures of 1–4 were elucidated using a combination of spectroscopic techniques. The eighteen isolated compounds were evaluated for antiviral activity against IAV activity. Among them, 1-monopalmitin (29) displayed potent activity against influenza A virus (A/WSN/1933(H1N1)) with EC50 2.28 ± 0.04 μM and without noteworthy cytotoxicity in MDCK cells. The interrupt step of 29 on the IAV life cycle was determined. These data provide invaluable information for new applications for this otherwise harmful weed.


Introduction
Influenza A virus (IAV) is a highly contagious, epidemic etiology respiratory illness that has affected 3-5 million population annually worldwide with high mortality and morbidity [1]. This virus has developed multiple strategies to evade host immune defenses including continuous genetic changes through mutation and re-assortment [2]. The influenza viral life-cycle is well known. The viral ion channel protein M2 and viral releasing neuraminidase are the clinical therapeutic targets present time. However, most currently circulating IAV's are resistant to M2 inhibitors, neuraminidase inhibitors are the only class of recommended anti-IAV treatments to date [3]. It is still unmet for anti-IAV agents. Plants have long been thought a potential source of anti-viral drugs. With the development of new influenza strains resistant to current commercially antiviral drugs, a wider range of influenza antiviral compounds is needed [4].
Cuscuta japonica Choisy (Japanese dodder) is a unique kind of holoparastic vine from the Convolvulaceae family. Dodder is completely dependent on the host plant for nourishment to survive, so its phytochemical constituents depend on the type of host [5]. When Cuscuta sp. parasitizes a medicinal plant such as Taxus brevifolia and grows thereon and can produce secondary metabolites lead to a pharmaceutical component (camptothecin) [6]. Dodder is a parasitic weed and spreads rapidly, afflicting many commercial crops, ornamentals and native plants by virtually decimating or killing them [7]. Herbicides are able to control the parasitic weed and are surely phytotoxic to the host plants.
There are thus advantages in utilizing this harmful weed as a source of valuable products, including increased effective management and reduced negative impacts on the environment.
Cuscuta is a genus of about 170 species of threadlike, yellowish, orange or reddish parasitic plants distributed mainly in North and South America. A few species are found in Asia and Europe. The C. chinensis Lam., C. japonica Choisy, and Cuscutae Semen seeds are a well-known traditional medicinal material, commonly used for improving sexual function, tonifying the livers and kidneys, reducing urination, treating aches and weakness in the loins and knees, and treating pharyngitis [8]. Investigation into the phytochemistry of Cuscuta spp. seeds has so far led to the isolation of many compounds, consisting of flavonoids, alkaloids, steroids, fatty acids, lignans [7], aromatics [9], resin glycosides [10] and polysaccharides [7]. These compounds exhibit a range of pharmacological effects such as hepatoprotective, anti-osteoporotic, immune regulation, neuroprotection, antioxidative, anti-aging, cytotoxic, renoprotective, reproductive system, prevention of abortion, antimutagenic effect, antidiabetic, cardioprotective, antidepressant, and anti-inflammatory activities [7]. However, the parasitic plant C. japonica growing onto a plethora of different plants has only been the subject of rare studies concerning host-parasite relationships and phytochemical constituents. In this work we analyzed the chemical fingerprint in five Japanese dodders (C. japonica) parasitizing on different plants using HPLC, thus evidencing slight differences.

Results
The chemical constituent patterns of five Japanese dodder parasitizing on different plants, Among them, the most active extract from Japanese dodder (CL) was further subjected to solvent partitioning and three layers were obtained, including ethyl acetate (CLE), n-butanol (CLB), and water layers (CLW). The CLE further showed stronger anti-IAV activity at 50 µg/mL without cytotoxicity against MDCK cells at 100 µg/mL (Figure 2b). To understand the source of the active compound in the CLE extract, we were isolated by employing diaion HP-20, Sephardex LH-20, silica gel, and C 18 column chromatography and further purified by RP-HPLC to obtain 30 compounds, including four new compounds 1-4 ( Figure 3) and twenty-six known compounds.

Results
The chemical constituent patterns of five Japanese dodder parasitizing on different plants, D. longans Lour (CL), F. septica Burm. F. (CF), F. microcarpa L. f. (CFM), M. micrantha H. B. K. (CM) and M. azedarach Linn. (CMA), respectively, detected by HPLC-DAD analyses were slightly different ( Figure 1). The MeOH extract (100 μg/mL) of Japanese dodder parasitizing on D. longans Lour (CL) showed anti-IAV activity with no cytotoxicity in Madin-Darby canine kidney (MDCK) cells ( Figure  2a). Among them, the most active extract from Japanese dodder (CL) was further subjected to solvent partitioning and three layers were obtained, including ethyl acetate (CLE), n-butanol (CLB), and water layers (CLW). The CLE further showed stronger anti-IAV activity at 50 μg/mL without cytotoxicity against MDCK cells at 100 μg/mL (Figure 2b). To understand the source of the active compound in the CLE extract, we were isolated by employing diaion HP-20, Sephardex LH-20, silica gel, and C18 column chromatography and further purified by RP-HPLC to obtain 30 compounds, including four new compounds 1-4 ( Figure 3) and twenty-six known compounds.

Discussion
Comparative profiling of metabolites by HPLC-DAD in five Japanese dodders after attachment to different hosts revealed several metabolic changes and amounts. Japanese dodders parasitizing on different plants exhibited distinct anti-virus activation depending on the host. The investigation of Japanese dodder parasitizing on D. longans Lour. (CL) and its EtOAc fractions (CLE) showed it can effectively inhibits IAV. In this work, four new compounds 1-4 and twenty-six known compounds 5-30 were isolated from Japanese dodder (CL). It is interesting to note that 1-monopalmitin (29), a simple glycerol esterified fatty acid which has a role as a plant metabolite, was found to be one of anti-IAV (A/WSN/1933(H1N1)) substances from the whole plant Japanese dodder (CL). Moreover, the two fatty acids 1-monopalmitin (29) and 1-α-linolenoylglycerol (30) were more potent than betulinic acid which was used as a positive control. The fully saturated (C19:0) fatty acid, 1-monopalmitin (29) showed higher antiviral activities against IAV than the unsaturated (C21:3) fatty acid 30. The current results suggest that fully saturated fatty acids play a crucial role in the anti-influenza virus activity that was observed.

HPLC-DAD Analysis
Five methanol crude extracts, EtOAc layer (CLE) and isolated compounds were diluted in MeOH (conc. 1.0 mg/mL), respectively. All samples were filtered through a filter cartridge (pore size of 0.22 µm) prior to analyses. The HPLC profiles of crude extracts, CLE fraction and the isolated compounds were performed on a RP-18 column at a flow rate of 1.0 mL/min and detected at 254 and 280 nm. The injection volume was 5 µL. The separation was performed using a mixture of 0.1% trifluoroacetic acid (TFA) water solution (A) and methanol (B) with the gradient elution: 0-120 min from 5% B to 55% B.

Anti-Inflluenza Virus Assay
The anti-IAV activity was displayed using plaque reduction assay as described previously [34]. In brief, the five crude extracts (CL, CF, CFM, CM and CMA), three fractions (CLE, CLW, and CLW), compounds (1-4, 9, 10, 13, 15-17, 19, 22-24, and 26-30) cytotoxicity were determined by MTS cell viability assay at the concentration of 100 µM of each samples in MDCK cells in the first. MDCK cells were cultured in DMEM, supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) and 1% penicillin and streptomycin (PS; HyClone, Marlborough, MA, USA) at 37 • C with 5% CO 2 incubation. The non-cytotoxic samples were selected to the following plaque reduction assay. MDCK cells (2 × 10 6 cells/well) were pre-seeded in a six-well plate at 37 • C overnight. The cell monolayer cells was then infected with influenza virus (A/WSN/1933 (H1N1), 100 PFU/well) with 25 µM of each samples and the plaque forming assay was performed. The anti-IAV activity of each sample was calculated using the plaque number compared with that of the virus infected control. The most effective compound 29, was used to further perform the dose dependent plaque reduction assay. The 50% and 90% effective concentrations (EC 50 and EC 90 ) were defined as the concentration required for 50% and 90% inhibition of virus infection, respectively.

Time-Of-Addition Assay
To determine the stage of at which 1-monopalmitin (29) inhibits IAV infection, three time points, including post-treatment, pre-treatment and mix-treatment, were used as previous study [34]. In brief, for the stage of post treatment, the viruses infected the cells for 1 h and the cells were overlaid with 0.3% agarose gel containing the compound for 48 h. The effect of 1-monopalmitin inhibits IAV at post-infection stage was determined using the virus plaque reduction assay. For the stage of Pre-treatment, the cells were pretreated with 1-monopalmitin for 1 h followed by virus infection of the pre-treated cells for another 1 h. After the virus removed, plaque reduction assay were performed. For the stage of Mix-treatment, the viruses were pretreated with 1-monopalmitin for 1 h, and then the mixture of compound and viruses was added to the cells for another 1 h. After the mixture remove, plaque reduction assay were performed.

Conclusions
Bioactivity-guided isolation of Japanese dodder (CL) let to the isolation and identification of four new compounds 1-4 and twenty-six known compounds 5-30. The structures were established via extensive spectroscopic investigations, including 1D and 2D NMR, UV, IR and HRESIMS techniques. The eighteen isolated compounds were evaluated for antiviral activity against IAV activity. 1-Monopalmitin (29) exhibits potential to be developed as a promising agent against influenza virus infection. This study is important as it explain the chemical and biological diversity of the whole plant Japanese dodder parasitizing on D. longans Lour. Furthermore, the results provide invaluable information for the new application of the harmful weed.