Screening of neuraminidase inhibitory activities of some medicinal plants traditionally used in Lingnan Chinese medicines

Background Neuraminidase (NA) is one of the key surface protein of the influenza virus, and has been established as a primary drug target for anti-influenza therapies. This study aimed to screen bioactive herbal extracts from some medicinal plants traditionally used in Lingnan Chinese Medicines by NA activity high-throughput screening assay. Methods One hundred ninety herbal extracts from 95 medicinal plants collected in Guangzhou were screened for their potential inhibitory activities against A (H1N1) influenza neuraminidase, and the most active extracts were further evaluated for their anti-influenza virus activities using virus-induced cytopathic effect (CPE). Results Among the tested 190 herbal extracts, 14 extracts inhibited significantly NA activity (IC50 < 40 μg/mL), and the extracts 1–5, which were obtained from Amomurn villosum Lour, Melaphis chinensis (Bell) Baker, Sanguisorba officinalis and Flos Caryophylli, showed potent inhibitory activity against NA with IC50 values ranging from 4.1 to 9.6 μg/mL. Moreover, the most bioactive extracts 1–5 were found to protect MDCK cells from A (H1N1) influenza virus infection with very low cytotoxicity to the host cells (EC50 values ranged from 1.8 to 14.1 μg/mL, CC50 values ranged from 97.0 to 779.2 μg/mL, SI values ranged from 14 to 438). In addition, quantitative RT-PCR analysis showed that the extracts 1–5 inhibited viral RNA synthesis in a dose-dependent manner. Conclusion We performed in vitro screening of anti-neuraminidase activities of herbal extracts from medicinal plants used in Lingnan Chinese Medicines, and the results indicate that some bioactive extracts are worth further studies to identify the bioactive components responsible for anti-influenza virus activities, to elucidate their modes of action and finally determine their clinical potentials.


Background
Influenza virus causes an acute contagious respiratory tract infection, which is a major contributor to morbidity and mortality among human population. Historically pandemic flu has caused widespread human deaths, most notably the 1918 "Spanish Flu" (A/H1N1) which killed 25-50 million people worldwide [1]. Novel swineorigin influenza A (H1N1 subtype) virus identified in Mexico in 2009 emerges to spread rapidly worldwide via human-human transmission [2] and led to at least 17,798 deaths in 214 countries. Therefore, pandemic influenza A viruses such as the H1N1subtype becomes a serious global public health problem, which calls for more agents of anti-influenza therapies as possible.
Neuraminidase (NA) is an antigenic glycoprotein on the surface of influenza virus, which takes charge of catalyzing the cleavage of neuraminic acid residues to facilitate the detachment from the host cell surface at the end of the viral replication cycle and suppresses their self-aggregation of the virions [3,4]. NA plays a critical role for virus replication and spread in infected tissues during infection, and has been well established as a primary drug target for anti-influenza therapies [5,6]. Some potent NA inhibitors, including oseltamivir, zanamivir, laninamivir and peramivir, have been designed and applied in clinical treatments [7,8]. Unfortunately, resistance to these NA inhibitors has been extensively reported [9][10][11]. Therefore, there is a continuing need for developing novel NA inhibitors as anti-influenza agents. Medicinal plants may be a probable source for the discovery of natural NA inhibitors and might provide leads to develop the NA inhibitors [12].
In order to search for novel anti-influenza agents from natural resources, a library of 190 extracts of 95 medicinal plants traditionally used in Lingnan Chinese Medicines were screened for in vitro inhibitory activity against A (H1N1) influenza virus neuraminidase using highthroughput assay. The most active five extracts (1)(2)(3)(4)(5) were selected to further study their action upon the replication of influenza viruses using cytopathic effect (CPE) reduction assay and quantitative RT-PCR analysis. The results showed that these herbal extracts significantly inhibited the NA activity and the replication of influenza viruses, and exhibited very low cytotoxicity to the host cells.

Standard extraction preparation
Dried powdered plants (100 g) were extracted with ethyl acetate (EtOAc, 250 mL × 3) and methanol (MeOH, 250 mL × 3) by ultrasound wave at 40 kHz and 400 W at 45°C for 30 min, the filtrates were evaporated under vacuum at 45°C to give the EtOAc and MeOH extracts, respectively. A total of 190 herbal extracts were obtained. A stock solution for each extract was prepared by dissolution to dimethyl sulfoxide (DMSO), 50 mg of each extract was suspended in 1 ml of DMSO ensuing stock concentration of 50 μg/μL. The solutions were filtered by using 0.22 μm filters, and stored at − 20°C. The concentration of DMSO in test dilutions was restricted to no more than 0.5% (v/v) to minimize potential effects of the solvent on enzyme activity and cell growth.
In vitro screening of plant extracts for NA activity Inhibition of influenza virus NA activity was determined by a standard fluorimetric method [13,14] using4methylumbelliferyl-α-D-N-acetyl-neuraminate (MUNANA) (Sigma) as substrate, in 96-well microplates. The reaction mixture containing the extracts or compounds, and NA enzyme in MES buffer (32.5 mM) and calcium chloride (4 mM, pH 6.5) was incubated for 60 min. After incubation, the reaction was terminated by adding NaOH (34 mM). Fluorescence intensity (M) was quantified with excitation wavelength at 360 nm and emission wavelength at 450 nm.
Percentage inhibition was calculated relative to a blank reaction mixture (solvent control) containing virus NA and solvent (% Inhibition = [1-(M extract /M control )] × 100). The 50% inhibitory concentration (IC 50 ) was defined as the concentration of NA inhibitor necessary to reduce NA activity by 50% relative to a blank reaction mixture. IC 50 values displayed represent the mean of three individual determinations each performed in triplicate assays. Zanamivir (Sigma) was used as the reference compound.

Cytotoxicity assay
The cytotoxicity of medicinal plant extracts was determined with the MTT (Sigma) method as described previously [15]. Briefly, different concentrations of the extracts and compounds were added to each well of a 96well culture plate containing a confluent cell monolayer in triplicate, blank medium was used as the control. After incubation at 37°C in an atmosphere of 5% CO 2 for 72 h, 12 μL of MTT solution (5 mg/ml in phosphate buffered saline) was added to each well. The plate was further incubated at 37°C for 3 h to allow formation of formazan product. After removing the medium, 100 μL of DMSO was added to dissolve the formazan crystals. After 15 min, the contents of the wells were homogenized on a microplate shaker. The optical densities (OD) were then determined by measuring absorbance with a microplate spectrophotometer at a wavelength of 540 nm and a reference wavelength of 620 nm. The median cytotoxic concentration (CC 50 ) was calculated as the concentration of the constituent that reduced the viable cells to 50% of the untreated control. The maximal non-cytotoxic concentration (MNCC) was defined as the maximal concentration of the sample that did not exert a cytotoxic effect and resulted in more than 90% viable cells.

CPE reduction assay
The anti-viral activity of the extracts was measured by a virus-induced cytopathic effect (CPE) reduction assay as described previously [14,16]. Briefly, 100 μL of virus suspension of 200 tissue culture infective dose (TCID 50 /mL) was added to each well of a 96well culture plate containing confluent a MDCK cells monolayer. After incubation at 37°C for 2 h, the virus solution was removed, and 100 μL of serial dilutions of the extracts and ribavirin were added to each well of the 96-well culture plates, using the maximal noncytotoxic concentration (MNCC) as the highest concentration. The plates were incubated at 37°C in a humidified 5% CO 2 atmosphere for 48 h, and then the CPE was assessed. The virus-induced CPE was scored as follows: 0 = no CPE, 1 = 0-25% CPE, 2 = 25%-50% CPE, 3 = 50%-75% CPE, and 4 = 75%-100% CPE. Apart from test group, there were control group (treated with FBS-free medium instead of extracts and virus) and model group (treated with FBS-free medium and virus instead of extracts and virus). The CPE inhibition ratios were calculated using the equation: CPE inhibition % = 100 -[(OD test -OD control ) *100/ (OD model -OD control )]. The OD test , OD model , and OD control mean the optical density of test group, model group, and control group, respectively. At least three independent experiments with three parallel experiments were performed to determine the mean and SD value.
Measurement of viral RNA synthesis by quantitative and reverse transcription PCR (qPCR) A549 cells were grown in RPMI1640 to about 90% confluence and were infected with influenza virus A/PR/8/34 (H1N1) influenza virus at 100 TCID 50 , followed by administration of test extracts for 5 h. To determine the expression level of hemagglutinin (HA) gene mRNA of influenza virus, cells were harvested and the total RNA was extracted by TRIzol (Invitrogen) according to the manufacture's instruction. The primer sequences which were designed by Primer-BLAST from NCBI for quantitative real-time PCR of influenza virus were 5'-CCTGCTCGAAGACAGCCA-CAACG-3′ (sense) and 5'-TTCCCAAGAGCCATCCGG CGA-3′ (antisense). The GAPDH were used as internal control of cellular RNAs, with primer sequence of 5′-TGC TCCGAAGGGTGGCCCTTA-3′(sense) and 5′-TGCGT GTTTCCAGAGCCGTGC-3′(antisense). The total RNA was reverse transcribed into cDNA using the TransScript First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). The cDNA was used as template for realtime PCR conducted by SsoFast EvaGreen PCR 2 × master mix (Bio-Rad) using CFX 96 Realtime PCR system (Bio Rad location) according to the manufacture's protocol. The data was analyzed using the mode for normalised expression (2 -ΔΔCq ).

Statistical analysis
Statistical analysis was performed using the Student's unpaired t-test. The results were presented as mean ± S.D. (n = 3). * p < 0.05 and ** p < 0.001 indicate a statistically significant difference as compared to the untreated control.

Results
NA has been validated as one of the most important targets to screen the drugs of anti-influenza virus. We first examined the ability of 190 organic extracts from 95 medicinal plants to inhibit NA activity by in vitro screening assay. Zanamivir was used as a positive control, its IC 50 value to NA inhibition was 0.05 μg/mL. 14 extracts were found to effectively inhibit the NA activity at the concentration of 40 μg/mL. Among them, 5 extracts exhibited potent inhibition of NA activity, 9 extracts exhibited moderate NA inhibitory activity with IC 50 values ranged from 4.1 to 37.3 μg/mL. The    showed a moderate inhibitory activity on NA with the IC 50 values ranging from 20.3 to 37.3 μg/mL. These results demonstrated that these plant extracts possessed significant inhibitory activities against influenza virus NA and the most active extracts 1-5 were then selected to further study their effects on the replication of influenza virus. To validate whether these extracts 1-5 that exhibited NA inhibitory activity could protect host cells from influenza virus A (H1N1) infections, the CPE reduction assay was carried out in MDCK cells. The human influenza virus A/PR/8/34 (H1N1) strain was used to infect MDCK cells. Cells were incubated in the presence or absence of the extracts 1-5, after 48 h of incubation, their CPE reduction activity on virus multiplication was then examined. As shown in Table 2, the extracts 1-5 could protect MDCK cells from the infection of influenza virus A (H1N1), exhibited a drastic reduction of influenza virus-induced CPE. The EC 50 values of the extracts 1-5 ranged from 1.8 to 14.1 μg/mL, similar to the results obtained in NA assays. Among the five extracts, the MeOH extract (2) from the fruits of Amomurn villosum had excellent CPE activity with very low EC 50 values of 1.8 μg/mL, this is comparable to that of the positive compound ribavirin (3.2 μg/mL). The viability of MDCK cells incubated in the presence or absence of the extracts was evaluated by MTT assay, the CC 50 values of the extracts 1-5 was found to be from 97.0 to 779.2 μg/mL, suggesting that the extracts protected significantly host cells from influenza virus infection and did not exhibit considerable cytotoxicity against MDCK cells. The maximal non-cytotoxic concentration (MNCC) of the extracts 1-5 were found to be from 30 to 300 μg/mL in MDCK cells. Their therapeutic selective index (SI) in MDCK cells ranged from  To further examine whether the protective effect of the extracts1-5 is related with the inhibition of influenza viral replication, total RNA was extracted and subjected to quantitative reverse-transcription PCR in the A/H1N1 virus-infected A549 cells. Our results showed that treatment with the extracts 1-5 for 5 h resulted in a substantial reduction in viral RNA expression level in a dose-dependent manner (Fig. 1). All extracts 1-5 at the high concentration (30 μg/mL) had significant inhibitory effects on viral RNA expression as compared with untreated control, even more powerful than ribavirin (Fig. 1). The extracts 2-5 at medium concentration (10 μg/mL) also demonstrated significant inhibitory effects on viral RNA synthesis. Interestingly, the extracts 3 and 4 at low concentration of 3 μg/mL still significantly inhibited RNA synthesis of influenza viruses. These data indicate that the extracts 1-5 could inhibit significantly the replication of influenza viruses in cultures by RT-PCR analysis, which validated their anti-influenza viral activity obtained by CPE reduction assay.

Discussion
In the course of our screening of NA inhibitors for influenza virus A (H1N1), a total of 190 extracts of 95 medicinal plants traditionally used in Lingnan Chinese Medicines were submitted to in vitro screening for their NA inhibitory activities. Among of them, the organic extracts 1-5, obtained from Melaphis chinensis, Amomurn villosum, Sanguisorba officinalis and Flos Caryophylli, were found to significantly inhibit the NA activity (IC 50 < 10 μg/mL, Table 1) and the replication of influenza virus in a dose-dependent manner (Fig. 1), and exhibited very low cytotoxicity to the host cells with the high selective index (SI) values ranging 14 to 438 (Table 2). Therefore, these Chinese herb extracts might contain bioactive components responsible for anti-influenza virus activity at non-toxic concentration and they could be a promising source of natural NA inhibitors.
It was demonstrated previously that the aqueous extracts of barks, leaves and galls of Melaphis chinensis have anti-influenza virus activity and some compounds such as gallotannins isolated from M. chinensis are responsible for the anti-influenza virus effect [17]. The presence of such compounds in our EtOAc and MeOH extracts of galls of M. chinensis may explain the biological activities seen in our screenings.
Flos Caryophylli also known as cloves, is considered acrid, warm and aromatic in Traditional Chinese Medicines for the treatment of stomachache, diarrhea and dental pain Fig. 1 Dose-dependent inhibitory effect of the extracts 1-5 on viral RNA synthesis. A549 cells were infected with 100 TCID 50 influenza H1N1 viruses and treated with different concentrations of the extracts 1-5 (3, 10 or 30 μg/mL) and the DMSO (0.03%) for 5 h. The total RNA was extracted and followed by qPCR analysis. To quantify the changes in gene expression, the 2 -ΔΔC(q) method was used to calculate relative changes which were normalized to the GAPDH gene and the untreated control (model group, which was set to 1). Value calculated as Mean ± SD of three independent tests, with * p < 0.05 and ** p < 0.001, respectively [18]. It was reported that the hot water extract of Flos Caryophylli have been shown to have anti-herpes virus, anti-hepatitis C virus and anti-cytomegalovirus activities in vitro and in vivo, and compounds such as ellagitannin and eugeniin were identified as the bioactive components with anti-virus properties [19]. In the present study, the MeOH extract of Flos Caryophylli showed IC 50 value of 9.1 μg/mL towards NA and EC 50 value of 14.1 μg/mL against influenza virus. In our latest phytochemical study on the MeOH extract of Flos Caryophylli [14], a bioassay-guided isolation led to identification of ten flavonoids, seven tannins and two chromones as NA inhibitors with IC 50 values ranging from 8.4 to 94.1 μM. These polyphenolic constituents were found to protect MDCK cells from A(H1N1) influenza infections (EC 50 = 1.5-84.7 μM) with very low cytotoxicity to the host cells (CC 50 = 374.3-1266.9 μM)), with selective index (SI) ranging from 7 to 297 [14].
The roots of S. officinalis (Rosaceae) are well-known Chinese herbs officially listed in the Chinese Pharmacopeia and have been used for the treatment of bleeding, diarrhea and burns. Early chemical studies showed that S. officinalis synthesize a variety of secondary metabolites, particularly polyphenols, triterpenoids, saponins and flavonoids with specific biological activities such as anti-asthmatic, antibacterial, anti-cancer and anti-inflammation [20][21][22][23][24][25]. A variety of flavonoids, saponins and polyphenols isolated from medicinal plant have been studied extensively and exhibited anti-influenza activities [12]. The MeOH extract of S. officinalis showed strong activities towards NA (IC 50 : 5.1 μg/mL) and against influenza virus (EC 50 : 8.1 μg/mL). The anti-influenza activity may be due to the presence of flavonoids and polyphenols in the MeOH fraction.
The fruits of A. villosum (Zingiberaceae) were consumed widely as popular cooking spices in East Asian countries and have been traditionally used as a medicine to treat various digestive disorders [26]. The volatile oils of the fruits of A. villosum were shown to be the major components and suggested to be responsible for the different biological activities such as analgesic, antioxidation and anti-inflammation [27]. In this study, the MeOH extract of the fruits of A. villosumwas shown to significantly inhibit NA activities (IC 50 : 4.9 μg/mL) and protect the host cells from CPE damage (EC 50 : 1.8 μg/ mL) without cytotoxicity, and its therapeutic selective index (SI) is 439 in MDCK cell culture.
In this study, we limit our study on EtOAc and MeOH extracts of medical plants since bioassay-guided isolation of neuraminidase inhibitors in aqueous extracts remains a challenging task for us. However, this may decrease the risk of false-positive results in the enzyme-based screening caused by some interfering components present within aqueous extracts. Future study will try to improve the screening methods on aqueous extracts that may also contain active components with anti-neuraminidase activity.

Conclusion
We carried out the in vitro screening of antineuraminidase activity of 190 herbal extracts from 95 medicinal plants traditionally used in Lingnan Chinese Medicines. Among the tested extracts, 5 extracts, obtained from Amomurn villosum, Melaphis chinensis, Sanguisorba officinalis and Flos Caryophylli, showed potent NA inhibitory activity. Comprehensive literature survey revealed that no study has been reported on the effects of the organic extracts of A. villosum and S. officinalis on antiinfluenza virus activities and small-molecule NA inhibitors from these extracts have not been chemically identified yet. Further studies are underway to isolate bioactive components of these extracts by bioassay-guided fractionation, and to explore their antiviral mechanisms and finally determine their clinical potentials.