Abstract
Context: Acquired immune deficiency syndrome (AIDS) is a severe pandemic disease especially prevalent in poor and developing countries. Thus, developing specific, potent antiviral drugs that restrain infection by human immunodeficiency virus type 1 (HIV-1), a major cause of AIDS, remains an urgent priority.
Objective: This study evaluated 32 extracts and 23 compounds from Vietnamese medicinal plants for their inhibitory effects against HIV-1 ribonuclease H (RNase H) and their role in reversing the cytopathic effects of HIV.
Materials and methods: The plants were air-dried and extracted in different solvent systems to produce plant extracts. Natural compounds were obtained as previously published. Samples were screened for RNase H inhibition followed by a cytopathic assay. Data were analyzed using the Microsoft Excel.
Results and discussion: At 50 μg/mL, 11 plant extracts and five compounds inhibited over 90% of RNase H enzymatic activity. Methanol extracts from Phyllanthus reticulatus and Aglaia aphanamixis leaves inhibited RNase H activity by 99 and 98%, respectively, whereas four extracts showed modest protection against the cytopathic effects of HIV.
Conclusion: The screening results demonstrated that the butanol (BuOH) extract of Celastrus orbiculata leaves, methanol (MeOH) extracts of Glycosmis stenocarpa stems, Eurya ciliata leaves, and especially P. reticulatus leaves showed potential RNase H inhibition and protection against the viral cytopathic effects of HIV-1. Further chemical investigations should be carried out to find the active components of these extracts and compounds as potential anti-HIV drug candidates.
Introduction
Human immunodeficiency virus (HIV) is a pathogenic retrovirus that can cause acquired immune deficiency syndrome (AIDS). After the first cases were identified in the USA in 1981, AIDS spread rapidly, becoming a pandemic disease. The Joint United Nations Programme on HIV/AIDS, UNAIDS, estimated that 33.4 million people suffer from HIV/AIDS worldwide. In Asia, the reported national HIV prevalence is highest in the southeastern countries, although the epidemic trends vary among them. While its prevalence in Cambodia, Myanmar, and Thailand has recently declined, it is increasing in Indonesia and Vietnam. Between 2000 and 2005, the estimated number of HIV sufferers in Vietnam is more than doubled, from 120,000 to 260,000 people (CitationUNAIDS, 2009). Currently, HIV/AIDS is commonly treated with highly active antiretroviral therapy (HAART) (CitationRichman et al., 2009; CitationBroder, 2010). However, the available antiretroviral drugs have fallen short of expectations in many ways, including the need for life-long therapy, the ultimate role of pre-exposure prophylaxis (PrEP), cardiometabolic side effects and other toxicities of long-term therapy, the emergence of drug-resistance and viral genetic diversity, and the continued pace of new HIV-1 infections in many parts of the world (CitationBroder, 2010). Moreover, the rapid development of strains resistant to drugs and the high cost of drugs make them inaccessible to many people in developing countries (CitationRukunga et al., 2002).
HIV reverse transcriptase (RT) is important in the HIV life cycle and is one of the most reliable targets of potential anti-AIDS chemotherapy (CitationHassan Khan & Ather, 2007). This multifunctional enzyme not only exhibits reverse transcription RNA-dependent DNA polymerase (RDDP) and DNA-dependent DNA polymerase (DDDP), but also inherent ribonuclease H (RNase H) activity (CitationHerschhorn & Hizi, 2010). The DNA polymerization and RNase H functions are responsible for converting viral genomic RNA into proviral double-stranded DNA (CitationHerschhorn & Hizi, 2010). Inhibiting RT function interferes with HIV production. RT inhibitors are classified into two broad groups with different inhibitory mechanisms: nucleoside analogs and non-nucleoside inhibitors (CitationCruchaga et al., 2007). Although both classes are useful therapeutically, their use in treating AIDS patients is limited because of the emergence of viral cross-resistance and cellular toxicity. Thus, developing specific, potent antiviral drugs to restrain HIV-1 infection remains an urgent priority.
Herein, we investigated the inhibitory effects of 32 extracts and 23 compounds from Vietnamese medicinal plants for their inhibitory effects against HIV-1 RNase H and their role in reversing the cytopathic effects of HIV.
Material and methods
Preparation of plant samples
Plants were collected in different geographical zones throughout Vietnam and were identified by Prof. Ngo Van Trai, at the National Institute of Medicinal Materials (NIMM), Ministry of Health, Hanoi, Vietnam (). Voucher specimens were deposited in the NIMM herbarium. The samples were dried in the shade and ground into a powder. Then, 20 g of each sample were extracted three times ultrasonically in 100 mL of different solvents at room temperature and filtered. The filtered solutions were combined and vacuum-dried to produce the extracts. Additionally, compounds from several plants, including the plant-based synthetic compound, indirubin-3′-oxime, were obtained as described previously (CitationCuong et al., 2006, Citation2010b; CitationCuong & Tuan, 2006; CitationNhut et al., 2007). Plant extracts and natural products were dissolved in dimethyl sulfoxide (DMSO) for the bioassays.
Table 1. RNase H inhibitory and HIV-1 cytopathic effects of Vietnamese medicinal plant extracts.
Preparation of oligonucleotides
The oligonucleotides 5′-GAU CUG AGC CUG GGA GCU-fluorescein-3′ and 5′-dabcyl-AGC TCC CAG GCT CAG ATC-3′ were synthesized and provided as the annealed RNA/DNA hybrid by TriLink Biotechnologies (San Diego, CA). The oligonucleotides 5′-(rA)22-fluorescein-3′ and 5′-dabcyl-(dT)22-3′ were products of Dharmacon (Lafayette, CO) and Midland Certified Reagent Co. (Midland, TX), respectively. The hybrid heteroduplex was formed by mixing 5′-(rA)22-fluorescein-3′ and 5′-dabcyl-(dT)22-3′ dissolved in 50 mM Tris, pH 8.0, containing 60 mM KCl, in a ratio of 1:1.2 followed by heating at 90°C for 5 min and slow cooling to room temperature. Aliquots of stock hybrid heteroduplex were stored at −20°C until use. Recombinant wild-type p66/p51 HIV-1 RT was overexpressed and purified as described (CitationFletcher et al., 1996).
Spectroscopic measurements
Details of the RNase H FRET assay have been previously described (CitationParniak et al., 2003). Inhibition assays in 96-well microplates were carried out using a SpectraMax Gemini XS dual-scanning microplate spectrofluorometer (Molecular Devices, Sunnyvale, CA). Assays in 384-well microplates were performed using a Victor2V multilabel plate reader (Perkin-Elmer Life Sciences, Boston, MA).
Microplate assay of RNase H activity
The assays were conducted in a total volume of 100 μL containing 50 mM Tris, pH 8.0, 60 mM KCl, and 5 mM MgCl2, with final concentrations of 0.25 μM RNA/DNA hybrid and 1.0 nM recombinant p66/p51 HIV-1 RT. Stock solutions of the RNA/DNA hybrid and HIV-1 RT were diluted appropriately immediately before use. Reactions were initiated by adding HIV-1 RT and ran for 30 min at 37°C. Reactions were quenched by the addition of 50 μL of 0.5 M EDTA at pH 8.0. The fluorescence intensity of each well was assessed using excitation and emission wavelengths of 490 and 528 nm, respectively, with the cutoff filter set to 515 nm. To assess the effect of the inhibitors, 1 μL of inhibitor in DMSO was added to the microplate well before adding the substrate and RT solutions (CitationParniak et al., 2003).
HIV-1 cytopathic assay
Extracts were dissolved in DMSO at 20 mg/mL, and diluted 1:200 into the assay plates, yielding a final top concentration of 100 μg/mL, with eight 2-fold dilutions to a low dose of 0.78 μg/mL. Samples were tested in triplicate dose–response format using HIV-1RF in CEM-SS cells by a previously published method (CitationWeislow et al., 1989). In brief, these are microtiter assays, which quantitate the ability of a compound to inhibit HIV-1-induced cell killing via syncytium formation. Cytoprotection and compound cytotoxicity are measured with the CellTiter 96 Reagent (Promega, Madison, WI) 6 days after infection. This reagent contains the tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS), and the electron-coupling agent phenazine ethosulfate in a colorless stable solution, which upon reduction by viable cells forms a colored solution with absorbance at 490 nm. Antiviral and toxicity data are reported as the concentration of compound required to inhibit 50% virus-induced cell killing (50% effective concentration [EC50]) and the concentration of compound required to reduce cell viability by 50% (IC50). All data are derived from triplicate tests with the variation of the mean averaging 10%.
Statistical analysis
Statistical analysis was performed using Microsoft Excel. The data are the mean of triplicate experiments.
Results and discussion
The ability of the 32 plant extracts to inhibit RNase H is summarized in . Of the samples examined, the Aglaia aphanamixis Pellegr. (Meliaceae), Bousingonia mekongense Pierre ex Pl. (Apocynaceae), Camellia sinensis (L.) O.Ktze, Eurya annamensis Gagn., Eurya ciliata Merr. (Theaceae), Fissistigma polyanthoides (DC.) Merr., Goniothalamus gracilipes Ban. (Annonaceae), and Phyllanthus reticulatus Poir. (Euphorbiaceae) extracts had over 90% inhibitory effect on RNase H at a concentration of 50 μg/mL. The methanol extracts of P. reticulatus leaves and stems inhibited RNase H the most, by 99 and 96%, respectively. The remaining samples exhibited moderate but significant inhibitory activity of 56–89%. As far as our knowledge, with the exception of C. sinensis, none of plant extracts in this study have been reported as an anti-HIV agent using RNase H inhibition or cytopathic assay. Neither A. aphanamixis nor G. gracilipes have been reported previously in phytochemical or biological studies. E. ciliata and E. annamensis contain triterpene fatty acid esters and flavonoids, including apigenin, chrysoeriol, and quercitrin, which exhibit considerable monoamine oxygenase (MAO) inhibitory activity (CitationCuong et al., 2006; CitationCuong & Tuan, 2006). Chrysoeriol from E. ciliata was also found to enhance the proliferation and differentiation of osteoblastic MC3T3-E1 cells (CitationTai et al., 2009). From the leaves and twigs of B. mekongense, scopoletin, ursolic acid, oleanolic acid, and α- and β-amyrin were isolated (CitationCuong et al., 2005). In folk remedies, P. reticulatus is used for a variety of ailments, including smallpox, syphilis, asthma, diarrhea, and gum bleeding. It is also claimed to have antidiabetic activity in tribal areas (CitationKumar et al., 2008). Phyllanthus species have been found to contain bioactive alkaloids, flavonoids, lignan, phenol, and terpenes (CitationLam et al., 2007). Previous reports have demonstrated its antiplasmodial activity (CitationOmulokoli et al., 1997) and antidiabetic activity (CitationKumar et al., 2008). However, there are no reports on its inhibitory effect against RNase H.
For further evaluation, plant extracts that inhibited RNase H by >75% in the enzymatic assay were tested in a cellular model of HIV-1 cytopathicity. Dose–response curves were constructed for each sample at eight concentrations ranging from 0.78 to 100 μg/mL to obtain their EC50 and IC50 values. Of the 16 extracts tested, only four showed significant protection against the viral cytopathic effect. These were the BuOH extract of Celastrus orbiculata leaves, and the MeOH extracts of Glycosmis stenocarpa stems, E. ciliata leaves, and P. reticulatus leaves ().
Additionally, 23 compounds from several plants including the plant-based synthetic compound (indirubin-3′-oxime) from previous works were also tested in the enzymatic assay. At the concentration of 50 μg/mL, almost half of these samples showed potent inhibition of activity with a range of 49–97% (). Of these, several anionic polysaccharide compounds, epigallocatechin-3-gallate (EGCG), and indirubin-3′-oxime exhibited higher effect with inhibition values over 80% at the tested concentration. EGCG is the most abundant catechin in green tea, and is also a potent antioxidant that may have therapeutic properties for many disorders including cancer (CitationKatiyar et al., 2007). There has been research investigating the benefit of EGCG from green tea in the treatment of HIV infection. It has been shown to reduce plaques related to AIDS-related dementia as well as to block glycoprotein 120 (gp120). The fucoidan compounds isolated from the seaweeds Sargassum polycystum, and Sargassum kuetzingii also had high RNase H inhibitory activity. Generally, fucoidans were isolated from ground wet or frozen seaweed by either cold (20–25°C) or hot (60–70°C) extraction with 0.4% HCl, hydrophobic chromatography, and precipitation in 60–80% ethanol. Nowadays, pharmaceutical research has been done on fucoidans, which are now being marketed as nutraceuticals and food supplements. Other reports indicate that fucoidan can induce apoptosis in human lymphoma cell lines and inhibit hyperplasia in rabbits (CitationAisa et al., 2004).
Table 2. RNase H inhibitory effect of some natural compounds.
Finally, the active compound indirubin-3′-oxime is considered a member of a new compound class for treating cancer, particularly leukemia and other immunological diseases (CitationKagialis-Girard et al., 2007). Indirubin-3′-oxime was synthesized with a condensation reaction between hydroxylamine and indirubin, an indole-type alkaloid that could be easily isolated from the leaves of several plants, such as Polygonum tinctorium (Polygonaceae), Isatis indigotica (Brassicaceae), Indigofera suffruticosa (Fabaceae), Indigofera tinctoria (Fabaceae), and Strobilanthes cusia (Acanthaceae) (Cuong et al., Citation2010a,Citationb). Recently, indirubin-3′-oxime was found to induce cell cycle arrest and apoptosis in Hep-2 human laryngeal carcinoma cells (CitationKameswaran and Ramanibai, 2009). In the effects on RNase H activity, indirubin-3′-oxime exhibited inhibition of 82% at the concentration of 50 μg/mL. Based on this result, indirubin-3′-oxime and its derivatives may have potential as cancer and antiviral disease drugs.
Conclusion
Fifty-five plant samples including extracts, organic compounds, and a plant-based synthetic compound from Vietnamese medicinal plants were screened for their inhibitory effects against HIV-1 RT RNase H activity. At 50 μg/mL, the methanol extracts P. reticulatus and A. aphanamixis leaves demonstrated the strongest inhibitory activities, of 99 and 98%, respectively. Second, the CHCl3 extract of B. mekongense whole plant, the EtOAc extract of C. sinensis leaves, MeOH extract of E. annamensis leaves and E. ciliata leaves, EtOH extract of G. gracilipes leaves, MeOH extracts of F. polyanthoides leaves and stem barks also showed highly inhibitory effects against RNase H activity with inhibition values >90%. Natural compounds, including EGCG from C. sinensis, fucoidans from seaweeds S. kuetzingii, S. polycystum, sulfated galactan from Gelidiella acerosa, and the synthetic compound, indirubin-3′-oxime, inhibited RNase H activity with the inhibition values over 90%. Four of the plant extracts including the BuOH extract of C. orbiculata leaves, the MeOH extracts of G. stenocarpa stems, E. ciliata leaves, and especially P. reticulatus leaves showed potential inhibition of RNase H and protection against the viral cytopathic effect of the HIV-1. Further chemical investigations should be carried out to find the active components of these extracts and compounds as potential anti-HIV drug candidates.
Acknowledgements
We would like to thank the Ministry of Science and Technology, Vietnam, for financial support in form of the Vietnam–Korea International Collaboration Project (No. 30/823/2007/HD-NDT). This work was supported partly by Priority Research Centers Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009-0093815), Republic of Korea. This research was also supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.
Declaration of interest
The authors report no conflicts of interest.
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