HIV-1 Protease and Reverse Transcriptase Inhibitory Activities of Curcuma aeruginosa Roxb. Rhizome Extracts and the Phytochemical Profile Analysis: In Vitro and In Silico Screening

Human immunodeficiency virus type-1 (HIV-1) infection causes acquired immunodeficiency syndrome (AIDS). Currently, several anti-retroviral drugs are available, but adverse effects of these drugs have been reported. Herein, we focused on the anti-HIV-1 activity of Curcuma aeruginosa Roxb. (CA) extracted by hexane (CA-H), ethyl acetate (CA-EA), and methanol (CA-M). The in vitro HIV-1 protease (PR) and HIV-1 reverse transcriptase (RT) inhibitory activities of CA extracts were screened. CA-M potentially inhibited HIV-1 PR (82.44%) comparable to Pepstatin A (81.48%), followed by CA-EA (67.05%) and CA-H (47.6%), respectively. All extracts exhibited moderate inhibition of HIV-1 RT (64.97 to 76.93%). Besides, phytochemical constituents of CA extracts were identified by GC-MS and UPLC-HRMS. Fatty acids, amino acids, and terpenoids were the major compounds found in the extracts. Furthermore, drug-likeness parameters and the ability of CA-identified compounds on blocking of the HIV-1 PR and RT active sites were in silico investigated. Dihydroergocornine, 3β,6α,7α-trihydroxy-5β-cholan-24-oic acid, and 6β,11β,16α,17α,21-Pentahydroxypregna-1,4-diene-3,20-dione-16,17-acetonide showed strong binding affinities at the active residues of both HIV-1 PR and RT. Moreover, antioxidant activity of CA extracts was determined. CA-EA exhibited the highest antioxidant activity, which positively related to the amount of total phenolic content. This study provided beneficial data for anti-HIV-1 drug discovery from CA extracts.


Introduction
Acquired immunodeficiency syndrome (AIDS) is caused by the infection of the human immunodeficiency virus (HIV). Both HIV-1 and HIV-2 are found in HIV-infected patients, but HIV-1 infection shows more severe clinical pathology and progression of disease than HIV-2 infection [1]. HIV-1 can infect the immune cells and, use the benefit of the host cells to replicate itself, and destroy the host cells. These processes cause immunocompromised hosts, leading to the infection of opportunistic pathogens such as Mycobacterium tuberculosis and finally death [2]. In the HIV-1 life cycle, the virus uses three important enzymes for its replication, including integrase, reverse transcriptase (HIV-1 RT), and protease (HIV-1 PR) [3][4][5]. Thus, the blocking of these enzymes can inhibit viral replication. The enzymatic

HIV-1 Protease Inhibitor Screening
All CA extracts (1 mg/mL) significantly suppressed HIV-1 PR activity compared to DMSO (vehicle control). CA-M exhibited the highest inhibitory activity among all the extracts with the percentage of inhibition at 82.44 ± 2.33%. Interestingly, the percent inhibition of CA-M showed a similar result as that of the positive control Pepstatin A (81.48 ± 1.07%). Besides, CA-EA and CA-H showed inhibition at 67.05 ± 2.19 and 47.61 ± 1.77%, respectively, against HIV-1 PR (Figure 2A). Since, CA-M showed the best HIV-1 PR inhibitory activity, it was selected to test at multiple concentrations. The calculated half-maximal inhibitory concentration of CA-M on HIV-1 PR activity was 0.87 ± 0.02 mg/mL.

HIV-1 Protease Inhibitor Screening
All CA extracts (1 mg/mL) significantly suppressed HIV-1 PR activity compared to DMSO (vehicle control). CA-M exhibited the highest inhibitory activity among all the extracts with the percentage of inhibition at 82.44 ± 2.33%. Interestingly, the percent inhibition of CA-M showed a similar result as that of the positive control Pepstatin A (81.48 ± 1.07%). Besides, CA-EA and CA-H showed inhibition at 67.05 ± 2.19 and 47.61 ± 1.77%, respectively, against HIV-1 PR (Figure 2A). Since, CA-M showed the best HIV-1 PR inhibitory activity, it was selected to test at multiple concentrations. The calculated half-maximal inhibitory concentration of CA-M on HIV-1 PR activity was 0.87 ± 0.02 mg/mL.  . All extracts (1 mg/mL) significantly inhibited both HIV-1 PR and RT compared to DMSO control (One-way ANOVA, Dunnett's test, * p < 0.05). Mean ± SD of at least three independent experiments is presented.

Phytochemical Profile Analysis
Phytochemical constituents in CA-H were analyzed and identified by using GC-MS after comparing the observed mass to the theoretical mass. The major compounds found in CA-H were terpenoids and fatty acids (Supplementary data S1). The chemical profiles of CA-EA and CA-M were investigated by using UPLC-HRMS. We found that CA-EA and CA-M were mainly composed of fatty acids, amino acids, and terpenoids (Supplementary data S2 and S3). As shown in Figure 3, the Venn diagram demonstrated that 38 compounds were detected in both CA-EA and CA-M. Besides, CA-H was composed of 22 unique compounds. All identified compounds found in CA extracts were tabulated in Table 1. of CA-EA and CA-M were investigated by using UPLC-HRMS. We found that CA-EA and CA-M were mainly composed of fatty acids, amino acids, and terpenoids (Supplementary data 2 and 3). As shown in Figure 3, the Venn diagram demonstrated that 38 compounds were detected in both CA-EA and CA-M. Besides, CA-H was composed of 22 unique compounds. All identified compounds found in CA extracts were tabulated in Table 1.

Identified Compound Extract
Lactone

Evaluation of Lipinski s Rule of Five and ADMET Parameters
All identified compounds found in CA extracts were evaluated for their drug-likeness properties. The in silico results indicated that 67 out of 96 compounds complied with Lipinski's rule and showed high GI absorption, with no AMES toxicity, and hepatotoxicity ( Figure 4 and Supplementary data S4). Therefore, these 67 filtered compounds were further studied for their ability to inhibit HIV-1 PR and RT by molecular docking.

Investigations of Antioxidant Constituents and Antioxidant Activity
According to previous study, HIV-1 infection can cause oxidative stress accumulation in the infected cells, leading to immune cell death [22]. Therefore, we would like to evaluate both the antioxidant constituents, including phenols by Folin-Ciocalteu's assay and the antioxidant activity by Trolox equivalent antioxidant capacity (TEAC) assay. For total phenol investigation, the results showed that CA-EA had the highest phenolic content at 118.38 ± 19.53 mM of gallic acid equivalent (GAE)/kg dry weight, followed by CA-M (72.39 ± 7.06 mM GAE/kg dry weight) and CA-H (64.16 ± 2.32 mM GAE/kg dry weight), respectively ( Figure 8A).
Besides, the antioxidant capacity of all CA extracts was determined by measuring ABTS scavenging activity. CA-EA exhibited the highest antioxidant activity with a value of 494.68 ± 24.58 TE mM/kg dry weight, followed by CA-M (371.55 ± 28.86 TE mM/kg dry weight) and CA-H (176.52 ± 20.47 TE mM/kg dry weight) ( Figure 8B). Interestingly, the observed activity indicated that the more total phenolic content provided the higher antioxidant activity of CA extracts.  respectively ( Figure 8A). Besides, the antioxidant capacity of all CA extracts was determined by measuring ABTS scavenging activity. CA-EA exhibited the highest antioxidant activity with a value of 494.68 ± 24.58 TE mM/kg dry weight, followed by CA-M (371.55 ± 28.86 TE mM/kg dry weight) and CA-H (176.52 ± 20.47 TE mM/kg dry weight) ( Figure 8B). Interestingly, the observed activity indicated that the more total phenolic content provided the higher antioxidant activity of CA extracts.

Discussion
Although HIV-1 infection has been found for many years, this infection is still uncurable due to the complexity of the virus. Patients who are infected with HIV-1 cannot eradicate the viruses from their bodies and need to control the number of viruses by continuously taking anti-retroviral drugs. Now, many effective antiviral drugs are available but the side effects from using these drugs are also found. The typical adverse effects of antiretroviral drugs are hepatotoxicity [23], mitochondrial toxicity [6], hypersensitivity, and lipodystrophy [24]. The development of highly effective drugs with low adverse effects is a critical study for the better life of HIV-1 infected patients. Natural products have been investigated for their pharmaceutical activities and used as a scaffold for novel drug development [25,26].
Herein, we focused on inhibitory effects of CA extracts and their derived compounds against HIV-1 enzymes: PR and RT. Our results showed that CA extracts served as potential HIV-1 inhibitors. The in vitro screening assays demonstrated that all CA extracts (1 mg/mL) showed significant inhibition against both enzyme activities. Among all extracts, CA-M showed the strongest inhibition against HIV-1 PR activity. Interestingly, the effect of CA-M on HIV-1 PR inhibition was comparable to the effect of Pepstatin A (1 mM). A previous study on HIV-1 PR inhibitory activity, Alpinia galanga, a plant in Zingiberaceae, displayed that the methanol extract inhibited HIV-1 PR more effectively than the aqueous extract [27]. Yu YB et al. demonstrated that alnustic acid methyl ester, a triterpenoid isolated from methanol extract of Alnus firma could exhibit inhibition against HIV-1 PR [28]. Hydroxypanduratin A and panduratin A isolated from methanol extract of Boesenbergia

Discussion
Although HIV-1 infection has been found for many years, this infection is still uncurable due to the complexity of the virus. Patients who are infected with HIV-1 cannot eradicate the viruses from their bodies and need to control the number of viruses by continuously taking anti-retroviral drugs. Now, many effective antiviral drugs are available but the side effects from using these drugs are also found. The typical adverse effects of antiretroviral drugs are hepatotoxicity [23], mitochondrial toxicity [6], hypersensitivity, and lipodystrophy [24]. The development of highly effective drugs with low adverse effects is a critical study for the better life of HIV-1 infected patients. Natural products have been investigated for their pharmaceutical activities and used as a scaffold for novel drug development [25,26].
Herein, we focused on inhibitory effects of CA extracts and their derived compounds against HIV-1 enzymes: PR and RT. Our results showed that CA extracts served as potential HIV-1 inhibitors. The in vitro screening assays demonstrated that all CA extracts (1 mg/mL) showed significant inhibition against both enzyme activities. Among all extracts, CA-M showed the strongest inhibition against HIV-1 PR activity. Interestingly, the effect of CA-M on HIV-1 PR inhibition was comparable to the effect of Pepstatin A (1 mM). A previous study on HIV-1 PR inhibitory activity, Alpinia galanga, a plant in Zingiberaceae, displayed that the methanol extract inhibited HIV-1 PR more effectively than the aqueous extract [27]. Yu YB et al. demonstrated that alnustic acid methyl ester, a triterpenoid isolated from methanol extract of Alnus firma could exhibit inhibition against HIV-1 PR [28]. Hydroxypanduratin A and panduratin A isolated from methanol extract of Boesenbergia pandurata possessed anti-HIV-1 PR activity [29]. These data indicate that the methanol extract of CA rhizomes might composed of phytochemical active compound(s) that potentially inhibit HIV-1 PR activity.
The hallmark characteristic of HIV-1 infection is the depletion of immune cells, especially CD4 + T-cells. A previous study demonstrated that the decrease of the CD4 + T-cells was triggered by the HIV-1 envelope glycoproteins, leading to oxidative stress accumulation and cell death [22]. Treatment of antioxidative agent, N-acetylcysteine, in the HIV-1 infected patients could up-regulate glutathione level and total antioxidant status, whereas lipid peroxidation was reduced. [30]. These data suggest that the control of HIV-1 load and antioxidant status are important keys for the treatment of people who live with HIV-1. Our current results showed that CA extracts exhibited not only anti-HIV-1 but also antioxidant activities. Moon-ai W and colleagues reported that crude homogenate and ammonium sulfate cut fraction of CA rhizomes contained high superoxide dismutase activity which indicated the antioxidative effect [31]. Moreover, Zingiberaceae plants such as Curcuma longa, Zingiber officinale, and Kaempferia parviflora exhibited antioxidant activity by scavenging 1,1diphenyl-2-picrylhydrazyl (DPPH) and 2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) free radicals [32,33]. Phytochemical constituents in CA extracts were analyzed using GC-MS and UPLC-HRMS techniques. The compounds in CA-H extract were identified by GC-MS; terpenoids and fatty acids were mostly found in this fraction. In addition, CA-EA and CA-M extracts were analyzed by UPLC-HRMS. These extracts were mainly composed of fatty acids, amino acids, and terpenoids. A previous study reported the presence of a high amount of sugars, organic acids, alkanes, fatty acids, and terpenoids in methyl tert-butyl ether, methanol, and chloroform extracts of CA rhizomes [34]. Similarly, our finding discovered α-terpineol, oleic acid, palmitic acid, and linoleic acid as in the previous study [34]. Interestingly, our previous study reported that linoleic acid isolated from Auricularia polytricha, an edible mushroom, could inhibit HIV-1 PR activity [12].
HIV-1 RT plays multifunctional activities, including DNA polymerase and RNase H activities. The DNA polymerase generates cDNA from viral RNA template while the RNase H degrades viral RNA. The two enzymatic activities of HIV-1 RT cooperate to convert the viral RNA into cDNA in the reverse transcription step of the HIV-1 replication cycle [5]. Therefore, interferences of these HIV-1 RT active sites can block the viral replication process. In our current study, we evaluated the possibility of identified compounds from CA extracts on inhibition of both DNA polymerase and RNase H activities. Computer-aid molecular docking by AutoDock 4.2 software demonstrated that 3β,6α,7α-trihydroxy-5β-cholan-24-oic acid; 27-nor-5β-cholestane-3α,7α,12α,24,25-pentol; xanthumin; prostaglandin F1a alcohol; QH2; prostaglandin H1; and lactone of PGF-MUM potentially bound DNA polymerase domain of HIV-1 RT. On the contrary, the results from AutoDock Vina exhibited that none of candidates provided the better binding affinity than the reference molecule, as shown in Table 3. The different algorithms of AutoDock 4.2 and AutoDock Vina may cause the different predicted binding energies [40,41]. AutoDock Vina is an improved accuracy of molecular docking program developed from AutoDock 4.2 [42]. Nevertheless, arglabin and Nevirapine showed almost the same binding energy value. Interestingly, the docking results of arglabin were relevant in both molecular docking platforms. Besides, these results supported our in vitro screening for polymerase activity of HIV-1 RT inhibition by CA extract. We also found that CA extracts moderately suppressed HIV-RT activity ( Figure 2B).
This study provided the promising active compounds that might inhibit HIV-1 PR and RT activities. These data will be an interesting idea for anti-HIV drug development from plant-based sources. Moreover, this zingiber is commonly used in Eastern traditional medicine, knowing of novel medicinal property is a value-added of this herb. Besides, other herbs that contain these ingredients may also provide the similar biological properties, especially the plants in Zingiberaceae family. Furthermore, the effects of individual compounds on HIV-1 PR and RT inhibitions remain to be elucidated.
To summarize, methanol extract of CA exhibited strong anti-HIV-1 PR and mild inhibition of HIV-1 RT. Moreover, we reported the possible active components in the CA extract that might have anti-HIV-1 PR and RT activities. We suggest that CA could be a beneficial source of natural products with anti-retroviral properties against HIV-1. Furthermore, the determination of anti-HIV-1 effects of the proposed compounds should be additionally studied under suitable in vitro/in vivo conditions.

Plant Material and Extract Preparation
The rhizomes of Curcuma aeruginosa Roxb. (CA) were collected from the Chatuchak market, Bangkok, Thailand (13.7995 • N, 100.5492 • E). The rhizomes were dried and blended into powders. The powders were extracted using the maceration technique. Firstly, the plant material (200 g) was macerated in methanol (500 mL) and incubated for 72 h. Subsequently, the methanol extract was collected, filtered, and then evaporated using a rotary evaporator to get crude extract (12.14 g). After that, the crude extract was fractionated by hexane (200 mL). The hexane solution was collected and evaporated to obtain a hexane extract (CA-H, 0.22 g). Next, the residue from hexane extraction was fractionated by ethyl acetate (200 mL). The ethyl acetate soluble liquor was collected and concentrated by rotary evaporation to make ethyl acetate extract (CA-EA, 5.18 g). Finally, the residue from ethyl acetate extraction was extracted with methanol (100 mL) to obtain a methanol extract (CA-M, 1.24 g). The schematic of extraction is shown in Figure 1.

HIV-1 Protease Inhibitor Screening
HIV-1 protease inhibitory activity of the sample (1 mg/mL) was measured using a fluorometric HIV-1 protease inhibitor screening kit (Biovision Inc. USA) according to the manufacturer's recommendation. One millimolar of Pepstatin A and 1% (v/v) of DMSO were utilized as a known HIV-1 PR inhibitor and vehicle control, respectively. The fluorescence was measured in a kinetic mode 330 (excitation) and 450 nm (emission) for 90 min at 37 • C using the PerkinElmer EnSpire plate reader (Perkin-Elmer).

HIV-1 Reverse Transcriptase Inhibitor Screening
The extracts at 1 mg/mL were tested for HIV-1 RT inhibitor using an HIV-1 reverse transcriptase assay, colorimetric kit (Roche, Germany) according to manufacturer's protocol. Nevirapine (200 µM), a HIV-1 RT inhibitory drug was used as a positive control. DMSO (1%, v/v) was determined as a vehicle control. The color reactions were measured at wavelength 405 nm and 490 nm as a reference wavelength by using an EnSpire plate reader (Perkin-Elmer).

Phytochemical Profile Analysis
The CA-H extract was subjected to Gas Chromatography-Mass Spectrometry (GC-MS) analysis for the identification of phytochemical profile. While CA-EA and CA-M extracts were analyzed for their chemical profiles by Ultra Performance Liquid Chromatography-High Resolution Mass Spectroscopy (UPLC-HRMS, Agilent Technologies, Santa Clara, CA, USA) analysis (Institute of Chemical Technology, India). The compounds were identified by comparing an observed mass to the theoretical mass available in the public database.

Evaluation of Lipinski's Rule of Five and ADMET Parameters
To determine drug-likeness prediction, Lipinski's rule of five and absorption property of the phytochemicals were evaluated using the SwissADME online program (http://www. swissadme.ch, accessed on 17 August 2021 [36]. Besides, toxicity parameters were predicted by submitting the SMILES files of the small molecules to the pkCSM online database (http://biosig.unimelb.edu.au/pkcsm/prediction, accessed on 17 August 2021 [47]. The compounds that passed Lipinski's rule and were predicted to have high gastrointestinal (GI) absorption and nontoxicity were further studied for in silico molecular docking.

Molecular Docking Analysis
HIV-1 protease (PDB: 5KR0) [48] and HIV-1 RT (PDB: 3QIP) [49] were obtained from the Protein Data Bank. The proteins were prepared using AutoDockTools-1.5.6. The water molecules and the original ligands: Amprenavir, Nevirapine, and 5,6-dihydroxy-2-[(2-phenyl-1H-indol-3-yl) methyl] pyrimidine-4-carboxylic acid (P4Y) were removed. Moreover, all missing hydrogen atoms were added in the protein structures. Then the file format was converted to PDBQT before molecular docking analysis. Energy minimization for the ligands was done using BIOVIA Discovery Studio 2020 software. All candidate ligands were docked against HIV-1 PR and RT by using AutoDock 4.2 [13]. For HIV-1 PR, the grid box was set to 60 × 60 × 60 xyz points of size and 0.375 Å of spacing at the center of HIV-1 PR. Moreover, two active sites of HIV-1 RT, including polymerase and RNase H active sites, were studied. The parameters of grid boxes for polymerase and RNase H active sites were set to 40 × 40 × 40 xyz points of size, 0.375 Å of spacing, and grid center at 9.831 × 13.983 × 17.1 xyz; and 35 × 35 × 35 xyz points of size, 0.375 Å of spacing, and grid center at 0.144 × 74.854 × 34.626 xyz, respectively. The top 10 compounds provided the highest binding affinity were further run on another molecular docking platform, AutoDock Vina program [42] to support the AutoDock 4.2 results. Hydrogen bond interactions of the docked structures were further analyzed using the Discovery Studio Visualizer.

Total Phenolic Content Measurement
The total phenolic content in the extracts was assessed by Folin-Ciocalteu's method [50]. Briefly, the extract at 1 mg/mL was mixed with the Folin-Ciocalteu's phenol reagent (10%, v/v) in a 96-well plate, followed by incubation in dark for 20 min. Next, 7.5% (w/v) of sodium carbonate (Na 2 CO 3 ) was added and further incubated for 20 min. The absorbance at 760 nm was measured by using a microplate reader (BioTek Synergy Mx). Gallic acid was employed as the standard compound. The results were represented as mM of gallic acid equivalent (GAE)/kg dry weight.

Antioxidant Activity Measurement
The antioxidant activity of the extracts was detected by Trolox equivalent antioxidant capacity (TEAC) assay, following the previous study of Panthong S et al. [51]. All CA extracts (1 mg/mL) were determined for ABTS radical scavenging capacity comparing to Trolox, a vitamin E analogue. The results were reported in Trolox equivalent (TE) mM/kg dry weight.

Statistical Analysis
All values were expressed as the mean ± standard deviation (SD) of triplicated experimental analysis. Statistical significance was analyzed by one-way ANOVA, Dunnett's test using SPSS 16.0 software. p < 0.05 was considered significant.

Conclusions
Our current study demonstrated a novel biological activity of Curcuma aeruginosa Roxb. (CA) extracts on HIV-1 inhibition by suppressing HIV-RT and HIV-1 PR activities. In vitro screenings of CA extracts (1 mg/mL) showed that all CA extracts exhibited moderate inhibition against HIV-1 RT activity ranging from 64.97 to 76.93%. Interestingly, the methanolic fraction (CA-M) provided the highest HIV-1 PR inhibitory activity at 82.44%. Besides, CA-EA exhibited the highest antioxidant effect, followed by CA-M and CA-H, respectively. The compositions of phenolic compounds present in CA extracts were related to the radical scavenging activity of the extracts. Furthermore, phytochemical profiling showed that CA extracts were mainly composed of fatty acids, amino acids, and terpenoids. Additionally, in silico virtual screening indicated that dihydroergocornine, 3β,6α,7α-trihydroxy-5β-cholan-24-oic acid, and 6β,11β,16α,17α,21-Pentahydroxypregna-1,4-diene-3,20-dione-16,17-acetonide provided dual enzymatic inhibition on HIV-1 PR and RT. This study obtained beneficial data for anti-HIV-1 drug development from C. aeruginosa Roxb. extracts.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/ 10.3390/ph14111115/s1, Figure S1: Chromatograms of chemical constituents presented in CA-H, Table S1: Phytochemical constituents in CA-H identified by GC-MS, Figure S2: Chromatograms of chemical constituents presented in CA-EA, Table S2: Phytochemical constituents in CA-EA identified by UHPLC-HRMS, Figure S3: Chromatograms of chemical constituents presented in CA-M, Table S3: Phytochemical constituents in CA-M identified by UHPLC-HRMS, Table S4: Prediction of Lipinski's rule of five, GI absorption, and toxicity parameters of identified compounds from CA extracts, Table  S5: Molecular docking results of CA-identified compounds at the active site of HIV-1 PR, Table  S6: Molecular docking results of CA-identified compounds at the DNA polymerase active site of HIV-1 RT, Table S7