Neuraminidase Inhibitor of Garcinia atroviridis L. Fruits and Leaves Using Partial Purification and Molecular Characterization

Neuraminidase (NA) is an enzyme that prevents virions from aggregating within the host cell and promotes cell-to-cell spread by cleaving glycosidic linkages to sialic acid. The best-known neuraminidase is the viral neuraminidase, which present in the influenza virus. Thus, the development of anti-influenza drugs that inhibit NA has emerged as an important and intriguing approach in the treatment of influenza. Garcinia atroviridis L. (GA) dried fruits (GAF) are used commercially as seasoning and in beverages. The main objective of this study was to identify a new potential neuraminidase inhibitor from GA. A bioassay-guided fractionation method was applied to obtain the bioactive compounds leading to the identification of garcinia acid and naringenin. In an enzyme inhibition study, garcinia acid demonstrated the highest activity when compared to naringenin. Garcinia acid had the highest activity, with an IC50 of 17.34–17.53 µg/mL or 91.22–92.21 µM against Clostridium perfringens-NA, and 56.71–57.85 µg/mL or 298.32–304.31 µM against H1N1-NA. Based on molecular docking results, garcinia acid interacted with the triad arginine residues (Arg118, Arg292, and Arg371) of the viral neuraminidase, implying that this compound has the potential to act as a NA enzyme inhibitor.


Introduction
Influenza continues to be a significant public health concern since it causes annual epidemics, and has the potential to spark a global pandemic. Globally, annual seasonal influenza epidemics are predicted to result in approximately 3 to 5 million cases of severe illness, and 290,000 to 650,000 respiratory-related deaths [1]. Among the four genera of influenza viruses (influenza types A, B, C, and D), type A is the most pathogenic group of viruses capable of causing severe respiratory illnesses or death [2]. The 2009 swine flu H1N1 and the highly pathogenic avian flu H5N1 are among influenza A viruses that have posed significant health risks in many parts of the world. The World Health Organization determined swine flu (H1N1) to be a pandemic. There were 94,512 confirmed cases in 123 countries, including 112 cases in Malaysia [3].
The influenza virus (IV) contains two major transmembrane glycoproteins: hemagglutinin (HA), and neuraminidase (NA), whose antigenic and genetic diversity are used to

General Experiments and Spectroscopy
The 1 H and 13 C NMR spectra were recorded at 500 MHz using a BRUKER AVANCE III spectrometer (Rheinstetten, Germany), and the values were reported in parts per million (ppm). Depending on the solubility of the compounds, samples (isolated compounds) were dissolved in deuterated organic solvents. Semi-polar compounds were dissolved in deuterated chloroform (CDCl 3 ), whereas polar compounds were dissolved in 99% D 2 O. The mass spectra were obtained using an Agilent 1100 Series LC-MSD-Trap-VL spectrometer (Agilent Technologies, Avondale, AZ, USA) using electrospray ionization as the ion source type. FTIR and UV spectra were recorded using IR-Prestige-21 (Shimadzu, Kyoto, Japan) spectrometer, and a UV-Vis spectrophotometer (specord-200, Analytical Jena, Germany), respectively. An electrothermal melting point apparatus was used STUART-SMP10 (Cole Parmer, Staffordshire, UK) to obtain melting points. The rotation index was determined using ADP 120 Bellingham (Bellingham & Stanley, Kent, UK).

MUNANA Assays
The MUNANA assay was used to evaluate compounds, extracts, and fractions. MUNANA is a fluorescence-based assay that measures the fluorogenic product 4-methylumbelliferone released by the enzymatic activity of influenza virus NA from the substrate 2 -(4methylumbelliferyl)-D-N-acetylneuraminic acid (MUNANA). MUNANA is a reliable method for assessing the inhibitory effects of NA drugs. MUNANA compares uninhibited NA activity of a virus or bacteria to enzymatic activity after incubation with a range of NI drug concentrations, allowing the determination of IC 50 value as the drug concentration required to reduce NA activity by 50% [28]. Using a micropipette, 25 L of MES buffer was first added to rows A-G and columns 2-12. 50 µL of 1000 µg/mL NA inhibitors were added to the first empty column A-C for one NI and D-F for the second NI. Following that, 25 µL NA was added to each well (row A-G and column 1-12), while 50 µL MES buffer was added to wells in row H (column 1-2) for blank readings. The mixtures were incubated for 30 min in a 37 • C incubator with the plate covered with aluminum foil.
Following incubation, 50 µL of MUNANA substrate was added to each well, whereas 50 µL of NA and MUNANA were added to row H as a positive control (column 3-4). The mixtures were then incubated for an additional hour at 37 • C. After incubation, the reaction was stopped by adding 100 µL of stop solution to each well. Finally, the plate was read using UV excitation on a microplate reader (Turner Biosystem, Sunnyvale, USA).

Data Analysis of Assay
The results were processed using GraphPad Prism v. 5 by fitting experimental data to the logistic graph, which involved percent inhibition over a range of concentrations. The inhibition constant (IC 50 ) was determined by plotting the graph at 50% inhibition using nonlinear regression analysis with the GraphPad Prism software (San Diego, CA, USA). On a semi-log plot, the results were plotted as percent inhibition activity versus inhibitor concentration (nM). The inhibitor concentration was expressed as 0.48 to 250 µg/mL, as the concentration of the inhibitor in the final assay volume.

Molecular Docking
The methods of molecular docking simulation were performed from our previous study [29]. The NA protein of subtype N1 in complex with zanamivir (PDB code: 3B7E) was used as the target. This PDB (3B7E) is NA crystal structures with resolution 1.45 Å from protein virus isolated that recombinant NA from the 1918 influenza virus [30]. Molecular docking simulations were performed with AutoDock 4.2 [31].

Isolation of Compounds from GAF (G. atroviridis Fruits)
The MeOH extract was evaluated against C. perfringens-NA, which resulted in an IC 50 value of 9.43 g/mL as shown in Figure 1. n-Hexane extracts were found to be less active against C. perfringens-NA, whereas EtOAc extracts were observed to be the most active against both C. perfringens-NA and H1N1-NA. Since EtOAc is the most active, it was further fractionated. The fractionation of EtOAc extract resulted in five fractions (GF1: 0.9112 g, GF2: 0.9395 g, GF3: 0.8918 g, GF4: 1.6145 g, and GF5: 0.9011 g).
The assay results revealed that GF2, GF3, and GF4 were active in inhibiting C. perfringens-NA. In contrast, as shown in Figure 2, GF2 was ineffective in inhibiting H1N1-NA.
The crude GAF1 was purified by washing with EtOAc and drying in a desiccator to yield clusters of brownish needle-shaped lactone crystals. The assay results revealed that GF2, GF3, and GF4 were active in inhibiting C. perfringens-NA. In contrast, as shown in Figure 2, GF2 was ineffective in inhibiting H1N1-NA. GF3 precipitate was purified using EtOAC to obtain GF31 (567.5 mg). GF41 was obtained by precipitating GF4 and washing it with hexane-EtOAc, yielding a white powder (1.1102 g) and amorphous yellow solid? GF42 (254.0 mg). GF31, GF41, and GF42 showed the same 1D-NMR data (proton and carbon), thus the compounds were labelled GAF1. The crude GAF1 was purified by washing with EtOAc and drying in a desiccator to yield clusters of brownish needle-shaped lactone crystals.
As illustrated in Figure 3, GAF1 was active against C. perfringens-NA and H1N1-NA. The IC50 values for C. perfringens-NA and H1N1-NA were 17.53 µg/mL or 92.21 µM and 57.85 µg/mL or 304.31 µM, respectively. The assay results revealed that GF2, GF3, and GF4 were active in inhibiting C. perfringens-NA. In contrast, as shown in Figure 2, GF2 was ineffective in inhibiting H1N1-NA. GF3 precipitate was purified using EtOAC to obtain GF31 (567.5 mg). GF41 was obtained by precipitating GF4 and washing it with hexane-EtOAc, yielding a white powder (1.1102 g) and amorphous yellow solid? GF42 (254.0 mg). GF31, GF41, and GF42 showed the same 1D-NMR data (proton and carbon), thus the compounds were labelled GAF1. The crude GAF1 was purified by washing with EtOAc and drying in a desiccator to yield clusters of brownish needle-shaped lactone crystals.

Isolation of Compounds from GAL (G. atroviridis Leaves)
The MUNANA assay was performed to evaluate both extracts to confirm their activity against C. perfringens-NA and H1N1-NA, as shown in Figure 4a. The MeOH extract of GAL demonstrated significant inhibition of C. perfringens-NA with an IC50 value of 36.17 g/mL and the EtOAC extract exhibited inhibitory activity against both NA (38.39 g/mL), and thus was isolated further to yield four fractions (GALF1, GALF2, GALF3, and GALF4).  From the four fractions obtained, fractions GALF2 and (1.7772 g) and GALF3 (2.0258 g) showed good NA inhibition on both C. perfringens and H1N1, and GALF2 was further isolated to yield GAL1 (8.9 mg) and GAL2 (22.6 mg) ( Figure 5). GAL2 has the same Rf as GAF1 (0.45, BuOH-CH3COOH-H2O 4:1:5) and showed inhibition against H1N1-NA with a maximum inhibitory concentration of 63.7% or an IC50 of 56.71 µg/mL, as shown in

Isolation of Compounds from GAL (G. atroviridis Leaves)
The MUNANA assay was performed to evaluate both extracts to confirm their activity against C. perfringens-NA and H1N1-NA, as shown in Figure 4a. The MeOH extract of GAL demonstrated significant inhibition of C. perfringens-NA with an IC 50 value of 36.17 g/mL and the EtOAC extract exhibited inhibitory activity against both NA (38.39 g/mL), and thus was isolated further to yield four fractions (GALF1, GALF2, GALF3, and GALF4).

Isolation of Compounds from GAL (G. atroviridis Leaves)
The MUNANA assay was performed to evaluate both extracts to confirm their activity against C. perfringens-NA and H1N1-NA, as shown in Figure 4a. The MeOH extract of GAL demonstrated significant inhibition of C. perfringens-NA with an IC50 value of 36.17 g/mL and the EtOAC extract exhibited inhibitory activity against both NA (38.39 g/mL), and thus was isolated further to yield four fractions (GALF1, GALF2, GALF3, and GALF4).   From the four fractions obtained, fractions GALF2 and (1.7772 g) and GALF3 (2.0258 g) showed good NA inhibition on both C. perfringens and H1N1, and GALF2 was further isolated to yield GAL1 (8.9 mg) and GAL2 (22.6 mg) ( Figure 5). GAL2 has the same Rf as GAF1 (0.45, BuOH-CH 3 COOH-H 2 O 4:1:5) and showed inhibition against H1N1-NA with a maximum inhibitory concentration of 63.7% or an IC 50 of 56.71 µg/mL, as shown in Figure 6b.   The IR spectra at wavenumber 3376 cm −1 revealed a broad absorption band identified as the carboxylic acid hydroxyl group. Additionally, it was associated with the presence of a carbonyl group at 1734 cm −1 , while C-O-C ether was detected with a broad signal at 1232 cm −1 . However, this structure lacked a conjugated system of alkenes or an aromatic group, as evidenced by the absence of a weak stretching vibration at 2800-2900 cm −1 .  The IR spectra at wavenumber 3376 cm −1 revealed a broad absorption band identified as the carboxylic acid hydroxyl group. Additionally, it was associated with the presence of a carbonyl group at 1734 cm −1 , while C-O-C ether was detected with a broad signal at 1232 cm −1 . However, this structure lacked a conjugated system of alkenes or an aromatic group, as evidenced by the absence of a weak stretching vibration at 2800-2900 cm −1 .
The chemical shift at 175.86 ppm in the 2D-HMBC spectrum, as shown in Table S1 and Figure 8, was identified as a carbonyl carbon, which correlated with three-bond correlations with H-4a, H-4b, and H-2 protons. The chemical shift at δ 170.16 ppm was identified as lactone carbonyl of C-5 by correlation (HMBC) between H-2 proton (δ 4.92, s) and H-4a (δ 2.72) and H-4b (δ 3.26). The C-2 carbonyl of carboxylic acid group was predicted to have a chemical shift at δ 175.86 ppm. The other carbonyl at δ 172.77 ppm was assigned to the other carbonyl of carboxylic acid group (C-1 ), which was confirmed by its correlation with H-2 protons (4.92). (s, 1H). The positive-ESI mass spectrum's molecular ion peak at m/z 191 [M + H] + indicated that this compound had the molecular formula C 6 H 6 O 7 . Compared to previous studies [24,32,33], GAF1 had the same structure as (2S,3S)-tetrahydro-3-hydroxy-5-oxofuran-2,3-dicarboxylic acid or garcinia acid, as illustrated in Figure 8. The IR spectra at wavenumber 3376 cm −1 revealed a broad absorption band id as the carboxylic acid hydroxyl group. Additionally, it was associated with the p of a carbonyl group at 1734 cm −1 , while C-O-C ether was detected with a broad s 1232 cm −1 . However, this structure lacked a conjugated system of alkenes or an a group, as evidenced by the absence of a weak stretching vibration at 2800-2900 cm The 13 [24,32,33], GAF1 had the same structure as (2S,3S)-t dro-3-hydroxy-5-oxofuran-2,3-dicarboxylic acid or garcinia acid, as illustrated in 8.  The NMR data of GAL2 also showed similar spectra as GAF1, and is interpreted as garcinia acid. This was also in accordance with the data reported by Polavarapu et al. [33]. This means garcinia acid can be obtained from the fruits and leaves of G. atroviridis.
Two signals at As shown in Figure 9, the spectroscopic data were consistent with the literature [26,27] for naringenin or 2S-5,7,4 -trihydroxyflavanone, and the NMR data are listed in Table S2. 6.80 (J = 9.0) in ring B were assigned to H-2′ or H-6′ and H-3′or H-5′, respectively. NMR spectrum data were not observed by the 1 H-NMR signals, despite the pres the phenolic group indicated by the UV and IR spectrum.

Binding Interaction of Isolated Compound from G. atroviridis
The result of bioassay-guided isolation of GA is presented in Table 1. Molecula ing simulation was conducted on GAF1 and GAL1 against NA. As shown in Fig  GAF1 and GAL1 interacted well with the active site of NA.

Binding Interaction of Isolated Compound from G. atroviridis
The result of bioassay-guided isolation of GA is presented in Table 1. Molecular docking simulation was conducted on GAF1 and GAL1 against NA. As shown in Figure 10, GAF1 and GAL1 interacted well with the active site of NA.
The two carboxylic acid moieties present in the garcinia acid structure may play an important role in its activity against NA. As shown in Figure 10a, the carboxylic acid group at C-2 docked close to the arginine triad with strong hydrogen bonds (2-3 Å) and interacted with Tyr406 at a distance of 2.7-2.8 Å. However, no hydrophobic interaction was observed as it moved away from the hydrophobic residues (Ileu222, Arg224, Ser246, and Glu276).
Naringenin interacted with the arginine triad via hydrogen bonding (with Arg118 and Arg371) and cation-pi interactions (with Arg292), as illustrated in Figure 10b. Arg224 also formed a cation-pi interaction with naringenin's ring C, but it docked away from the hydrophobic residues Ile222, Glu276, and Ser246, resulting in a less favorable interaction with the NA binding site.   The two carboxylic acid moieties present in the garcinia acid structure may play an important role in its activity against NA. As shown in Figure 10a, the carboxylic acid group at C-2 docked close to the arginine triad with strong hydrogen bonds (2-3 Å) and interacted with Tyr406 at a distance of 2.7-2.8 Å. However, no hydrophobic interaction

Discussion
The two compounds isolated from G. atroviridis were found to be active against NA. GAF1 and GAL2 have been identified as garcinia acid, while GAL1 has been identified as naringenin. Garcinia acid was found to be more active than naringenin, with IC 50 values of between 17.34-17.53 µg/mL against C. perfringens-NA and 56.71-57.85 µg/mL against H1N1-NA. Garcinia acid and its derivatives have been shown to have biological activities including antifungal [35] and anti-atherosclerosis [36], besides being used as an active ingredient to aid in weight loss [37] or anti-obesity [38] via appetite suppression and inhibition of fat production. They form most of the content in GA's fruits, and are also known to be present in the root. In addition to garcinia acid, atrovirisidone, naringenin, and 3,8"-bi-naringenin were also previously reported to be isolated from the root, while atroviridin was isolated from the stem bark [39]. Garcinia acid obtained from the fruits (GAF1) and leaves (GAL2) of G. atroviridis has previously been isolated from Garcinia cambogia, Hisbiscus cannabinus [23], Garcinia indica, and Garcinia atroviridis [40]. Although other pharmacological activities of garcinia acid have been described in the literature, its activity against NA or as anti influenza has never been reported.
Naringenin, on the other hand, is a flavanone derivative with a skeleton similar to xanthone. Citrus and grapefruit are the most common sources of this compound [41]. It was also discovered in the bark of G. atroviridis. Naringenin has been shown to have potent antioxidant [42], anti-inflammatory [43,44], antiandrogenic [45], estrogenic [46], monoamine oxidase (MAO)-inhibitor, [41] anticancer-antitumor [47], and anti-dyslipidaemia [44,46] properties. In this study, naringenin was found to have weak activity against NA, with IC 50 values of 107 µg/mL or 393.38 µM for C. perfringens-NA and 123 µg/mL or 452.20 µM for H1N1-NA. This is consistent with Liu et al. (2008) [48] who reported that this compound has a weak NA inhibitory activity with an IC 50 value greater than 100 µM. However, the authors did not explore the binding interaction in detail. This will be explained further below.
Based on the molecular docking results, the binding free energy of garcinia acid (−8.31 kcal/mol) and naringenin (−8.51 kcal/mol) against NA-H1N1 (3BE7) did not differ much. Both garcinia acid and naringenin interacted with important amino acids via hydrogen bonding, as can be seen in their interaction with the triad arginine residues. These triad arginine residues interacted with the carboxylate of the sialic acid substrate from the virus [49], providing a structural basis in the development of potent inhibitors. However, there was no hydrophobic interactions between garcinia acid and the lipophilic pocket of NA that consisted of several amino acids including Glu276, Ala246, Arg224, and Ile222 [50]. Naringenin has a strong hydrophobic interaction due to the formation of a pi-pi cation interaction between its aromatic ring and the hydrophobic residue of NA (orange line) as shown in Figure 10b. This interaction is believed to confer the inhibitory activity of these two compounds against NA [51]. NA inhibition activity of garcinia acid was contributed favourably by the carboxylic acid groups. However, the absence of hydrophobic interaction may be the reason for its weak activity. On the other hand, the lack of carboxylic acid moiety in naringenin can be considered as a significant drawback, thus seem to contribute towards the reduction of the activity reduces the activity of naringenin against NA. This is because of the important interaction that the carboxylic acid moiety contributed in the interaction of a ligand with Arg371, which is considered as the most important residue among the arginine triads that interact with the carboxylic acid moieties of NA inhibitors [52].

Conclusions
Bioassay-guided fractionation yielded garcinia acid and naringenin from G. atroviridis L. fruit and leaves, respectively. Both have inhibitory activity against the NA enzymes. Garcinia acid has a good inhibitory activity against C. perfringens-NA, with an IC 50 of 17.34-17.53 µg/mL, and 56.71-57.85 µg/mL against H1N1-NA. Garcinia acid was found to form a strong ionic interaction with triad arginine based on molecular docking study. The findings in this study provides insight into the ability of G. atroviridis to inhibit neuraminidase of influenza virus.

Supplementary Materials:
The following are available online, Figure S1: Spectroscopy data of GAF1, Figure S2: Spectroscopy data of GAL1, Table S1: NMR data of Garcinia Acid and Table S2: NMR data of Naringenin.