Keywords

1 Introduction

The HIV epidemic continues to affect people worldwide. According to the UNAIDS fact sheet of 2018, approximately 36.9 million people were living with HIV in 2017 and since the start of the epidemic, approximately 35.4 million people had died from AIDS related illnesses (UNAIDS 2018).

Antiviral treatment currently involves the use of combination antiretroviral therapy abbreviated as cART (Cihlar and Fordyce 2016). cART usually consists of two nucleotide or nucleoside reverse transcriptase inhibitors and a third drug from another class (Cihlar and Fordyce 2016). Such a combination is known to significantly suppress virus replication leading to substantial improvement in the clinical management of HIV infection by delaying disease progression, prolonging survival and improving the overall quality of life (Cihlar and Fordyce 2016). A total of 25 anti-HIV drugs from 6 different mechanistic classes have reportedly been developed for use since the approval of zidovudine (AZT) in 1987 (Cihlar and Fordyce 2016).

Limitations of existing treatment such as toxicity to the host, development of drug resistant viruses together with the persistence of latent pools of the virus makes lifelong treatment a necessity (Tang and Shafer 2012). AZT and other HIV-drugs originated from anti-cancer investigations of phytochemicals, the latter known for chemical novelty that is higher in natural products than in any other sources (Jadaun et al. 2016). There is ongoing research to support the role of phytochemicals as anti-HIV agents. This chapter focuses on various in-vitro screening assays that are used in the development of phytomedicines for HIV management. Some of the medicinal plants and phytochemicals with potential for use in HIV management are described followed by a description of the replication cycle of the virus as it relates to treatment and some of the associated anti-viral assays that have been used in testing these phytochemicals for HIV enzyme inhibition. The various in-vitro anti-HIV assays that are enzyme and replication based are then discussed.

2 Medicinal Plants in HIV Management

The globe is estimated to have approximately 61,000 tree/shrub species (Qian et al. 2018). Of these, approximately 23,400 species are found in Southern Africa alone (Chingwaru et al. 2015) – making the flora of the region one of the richest in the world. The rich plant diversity in Southern Africa coincides with an unequivocally high HIV burden in the region compared to the rest of the world. The region is also affected by high levels of poverty (Mbirimtengerenji 2007). The Eastern and Southern African region is also home to more than half (53%) of the world’s HIV-infected population (UNAIDS 2018). Despite the fact that conventional HIV drugs have become widely available around the world, their high cost and side-effects/drug resistance have negatively impacted their uptake among the poorer communities particularly those in Southern Africa and parts of Asia (Institute of Medicine 2011). Medicinal plants are popularly used in traditional medicinal practices in communities around the world (Pan et al. 2014). Empirical evidence shows that a number of these medicinal plants help alleviate symptoms of HIV/AIDS (Chingwaru et al. 2015). While Southern Africa is endowed with a rich floral diversity, only a handful of the plants growing in the region have been assessed for anti-HIV activities. The use of plant based medicines in the management of HIV disease is also reported in Indian and Chinese traditional medicine.

Two plants have dominated the traditional management of HIV in Southern Africa, namely Hypoxis hemerocallidea (common name: African potato) and Sutherlandia frutescens (Maroyi 2014). Other plants with a history of use in the management of HIV in Southern Africa and most with in-vitro anti-HIV evidence, include Plectranthus barbatus, Siphonochilus natalensis (Maroyi 2014; Chingwaru et al. 2015), Abrus precatorius (Ma et al. 1998), Erythrina abyssinica (Mohammed et al. 2012), Cassia fistula L. bark (Xu et al. 1996), Bulbine alooides (L.) Willd, Hypoxis sobolifera var. sobolifera (Jacq.) Nel., Leonotis leonurus (L.) R.Br. (Klos et al. 2009) and, Dodonaea angustifolia L.f. (Asres et al. 2001).

In India, HIV patients reportedly use plants such as Boxus sempervirens (Boxwood), Andrographis paniculata (Andrographolide), Azadirachta indica (Neem), Rasagandhi mezhuga, Amukkara chooranum and Nellikkai lehyam in the management of HIV (Fritts et al. 2008).

The gaps that are apparent in the discovery of plant based medicines for use in HIV management require concerted research efforts to ensure safety in the traditional management of the disease. In-vitro assays are an essential platform to improve our understanding of the mechanisms of action of phytomedicines and their levels of safety.

3 Anti-HIV Phytochemicals

A number of plant derived compounds have been identified as promising anti-HIV agents including alkaloids, flavonoids, coumarins, tannins, saponins, phenolics, quinones, and lignans (Kurapati et al. 2016). The remainder of this section provides examples of phytochemicals with anti-HIV activity.

3.1 Alkaloids

Michellamines (A, B and C) are atropisomeric naphthylisoquinoline alkaloid dimers that were isolated from a Cameroonian plant, Ancistrocladus korupensis (Boyd et al. 1994). These alkaloids inhibited HIV-induced cell killing and viral replication in a variety of human cell lines (Boyd et al. 1994). Anti-HIV studies showed that michellamine B was active against a panel of biologically diverse laboratory and clinical strains of HIV-1, including the AZT resistant strain G910-6 and the pyridinone-resistant strain A17; the compound also inhibited several strains of HIV-2 (Boyd et al. 1994). Michellamine D, a homolog of michellamines A-C underwent extensive preclinical evaluation as a potential HIV drug but was found to be highly toxic (Singh et al. 2011). Cepharanthine is a biscoclaurine alkaloid isolated from Stephania cepharantha and was found to suppress HIV-1 LTR-driven gene expression through the inhibition of NF-kB activation (Okamoto et al. 1998). These are just a few of this class of compounds, a lot more alkaloids are reported in the literature with potential inhibitory activity against HIV-1 (Singh et al. 2011).

3.2 Flavonoids

Flavonoids are one of the groups of plant based compounds with known anti-HIV activities, particularly known to inhibit all three HIV enzymes; Reverse transcriptase (RT), protease (PR) and integrase (IN) (Singh et al. 2011). Flavonoids have also been shown to inhibit the attachment of viral gp120 to CD4+ cells as well as reducing viral replication in-vitro (Singh et al. 2011). Flavonoids with potent anti-HIV activity include chalcones, hydroxypanduratin A, quercetin, chrysin, epigallocatechin, thalassionlins A-C, and taxifolin (Singh et al. 2011).

3.3 Coumarins

Calanolide is a popular coumarin which inhibits HIV-1 RT. This compound was first isolated from a tropical tree (Calophyllum lanigerum) in Malaysia (César et al. 2011). The safety and pharmacokinetics of this compound have already been evaluated and it is one of the few plant derived compounds undergoing HIV clinical trials (Saklani and Kutty 2008). Other types of coumarins with reported anti-HIV activity include khellolactone, furanocoumarins, immperatorin, and heraclenol (Singh et al. 2011).

3.4 Terpenes

This is a large group of medicinal compounds that mainly falls under three classes; diterpenes, triterpenes and sesquiterpenes (Singh et al. 2011). These cyclic compounds have been reported to mostly inhibit HIV RT as well as prevent viral replication in cells. Betulinic acid, also known as PA-457 or bevirimat, potently inhibits HIV-1 replication by specifically blocking CA-SP1 cleavage resulting in antimaturation activity (Stoddart et al. 2007). Even though clinical trials of berivimat indicated a significant and clinical reduction of the viral load in infected individuals, a high baseline drug resistance was also revealed (Dang et al. 2013). The high baseline drug resistance poses serious limitations to the clinical potential and further development of this terpene as a potential HIV drug (Dang et al. 2013). New berivimat derivatives are being developed to overcome the drug resistance of the parent compound (Dang et al. 2013). Other terpenes with anti-HIV properties include butenolide-3-epi-litsenolide D2, lactone, lanicilacton C, and limonoid (Singh et al. 2011).

3.5 Phenolic Acids

Several phenolic acids have been highlighted to have anti-HIV properties. Two of these phenolics, dicaffeoyl quinic acid and dicaffeoyl-tartaric, have been identified as potent and selective HIV-1 IN inhibitors (Mcdougall et al. 1998). Other phenolics with anti-HIV activity are phloroglucinol alpha pyrone arzanol which inhibits HIV-1 replication in-vitro (Appendino et al. 2007), and polyphenols geraniin and corilagin that reportedly inhibit HIV-1 RT (Notka et al. 2003).

3.6 Chlorophyll Derivatives

Chlorophyll is a naturally occurring plant pigment known to be highly unstable (Humphrey 2004). The progressive degradation of chlorophyll leads to the formation of similarly colored derivatives (Lanfer-Marquez et al. 2005). Commonly reported chlorophyll derivatives include chlorophylls a and b, pheophytins a and b, and pheophorbide a and b (Lanfer-Marquez et al. 2005). Pheophytin a reportedly inhibits HIV PR in-vitro and also suppressed HIV replication in a chronically infected cell model (Kapewangolo et al. 2017a). Pheophorbide-a has been found to possess anti-HIV effect in-vitro (Zhang et al. 2003).

4 HIV Life Cycle and Phytochemical Targets for Therapy

HIV infects cells of the immune system, mostly CD4+ T helper cells (Martínez-Bonet et al. 2015), and the infection ends up destroying the host’s ability to effectively fight off the virus due to a compromised defense system. The replication cycle of HIV, commonly referred to as the life cycle of HIV, consist of two main steps. These are; (1) the entry and integration step, and (2) translation and budding step. Understanding the life cycle of HIV has led to the development and improvement of antiretroviral drugs with potential for controlling or completely eliminating the virus (Kirchhoff 2013). Key aspects of the life cycle that have been explored for treatment are virus fusion, reverse transcription, integration steps consisting of 3′ processing and the strand transfer (ST) steps as well as protein maturation steps (Pommier et al. 2005). Current HIV therapy targets different steps of the HIV cycle, however, complete eradication of the virus is compromised by the existence of reservoirs that harbor latent or dormant HIV (Schwartz et al. 2017) which is reactivated upon treatment interruption (Martínez-Bonet et al. 2015). The different stages of the HIV replication cycle are explained below.

4.1 Viral Entry and Integration

This step occurs at the surface of the cell and involves the virion bearing two copies of ribonucleic acid (RNA) binding to the CD4+ receptor and chemokine co-receptors, CCR5 or CXCR4. This is mediated by gp120/gp41 protein complex found on the surface of the viral envelop (Meanwell and Kadow 2003). A series of processes involving conformational changes and eventually fusion, with the viral core being inserted into the host cell followed by uncoating and the release of viral contents within the cytoplasm of these cells then follows (Chan and Kim 1998). The viral RNA is reverse transcribed by reverse transcriptase to a complementary deoxyribonucleic acid (cDNA) strand, which is subsequently transported into the nucleus as the pre-integration complex (PIC). Within the nucleus, the cDNA is integrated into the host DNA catalysed by the viral enzyme, integrase. Integration of the viral DNA into the host allows for the production of HIV proteins during the normal gene expression process.

4.2 Translation and Budding

The transcription step of the viral DNA results in viral genomic RNA and subsequently translation of viral protein which are processed and assembled in the cytoplasm by HIV protease. HIV protease further catalyses the maturation of viral particles via proteolytic processing into infectious virions. The virions consist of viral proteins and of two single stranded unspliced viral RNA which then bud off from the cells. As the virus replicates and makes new copies of itself, the course of infection in the infected individual is the gradual loss and destruction of naive and memory CD4+ T cells leading to AIDS which is the final stages of the infection course (Vidya Vijayan et al. 2017).

4.3 HIV Phytochemical Targets

Phytochemicals with anti-HIV properties are widely reported. Classes of phytochemicals targeting different steps of the HIV cycle include alkaloids, flavonoids, coumarins, saponins, terpenes and phenolics (Kurapati et al. 2016). Unique anti-HIV agents of plant origin exist and the most notable one is possibly Bevirimat, a novel inhibitor of HIV isolated from a Chinese herb, Syzygium claviflorum, which targets maturation of newly formed HIV particles. The bioactivity of anti-HIV plant extracts could be justified as promising therapy for HIV since they have a high potential of targeting more than one stage in the HIV cycle due to the presence of various phytochemicals in the extract.

5 In-vitro HIV Enzyme Models for Screening Phytochemicals for Anti-HIV Activity

Specific HIV enzyme-based assays have been established to screen for potential anti-HIV activity in-vitro. The principles behind these assays are described here. It should be noted that there are no techniques specific for investigating the anti-HIV potential of natural products and assays described here have been used to determine in-vitro anti-HIV activity of both synthetic and natural drug agents.

5.1 HIV Reverse Transcriptase Activity Assays

The RT enzyme of HIV is a heterodimer consisting of 66- and 51-kDa subunits (Fields et al. 1996) and it is involved in converting viral RNA to cDNA. This multifunctional enzyme is involved in RNA-dependent and DNA-dependent polymerisation, strand displacement synthesis and strand transfer, and degrades the RNA strand in the RNA/DNA hybrid (Schultz and Champoux 2008). It performs these functions through its polymerase function (for which there are two classes of inhibitors, the nucleoside reverse transcriptase and non-nucleoside reverse transcriptase inhibitors) and an RNase H function which is unique to the C terminus of the p66 subunit (Su et al. 2010). The polymerase function requires either RNA or DNA as the template making use of a host transfer RNA (tRNA) as primer (tRNAlys3) (Sarafianos et al. 2009). The RNase H activity is required for processing the tRNA primer to begin minus-strand DNA synthesis and degradation of viral RNA during synthesis followed by preparation of the polypurine tract DNA-RNA hybrid which serves as the primer for positive strand DNA synthesis (Schultz and Champoux 2008). All these processes result in the copying of a single stranded RNA to a double stranded DNA (Sarafianos et al. 2009; Schultz and Champoux 2008).

5.1.1 Targeting the Polymerase Domain

The assay provides a quantitative measure of RT activity and takes advantage of the ability of RT to synthesise DNA, starting from the template/primer hybrid poly (A) x oligo (dT)15. Digoxigenin and biotin labelled nucleotides are incorporated into the same DNA molecule as it is freshly synthesized by RT. The detection and quantification of newly synthesized DNA follows a sandwich ELISA protocol where the biotin-labelled DNA binds to the surface of streptavidin-coated microplate modules. An antibody to digoxigenin, conjugated to peroxidase (anti-DIG- POD) is then added and binds to the digoxigenin-labelled nucleotides. Finally, a peroxidase substrate, 2, 2′-azino-di-[3-ethylbenzthiazoline sulfonate (6)] diammonium salt crystals (ABTS), is added. The peroxidase enzyme catalyzes the cleavage of the substrate to produce a coloured reaction product. The absorbance of the samples is determined using a microplate (ELISA) reader, and is directly correlated to the level of RT activity in the sample (Kapewangolo et al. 2017a). Other assay variations which make use of the same or similar principles have been reported (Suzuki et al. 1993). These assays have moved from the previous unsafe isotope-based assays which required special equipment and tedious procedures (Kuno et al. 1999). Screening for HIV anti-RT activity using the method described here can be done both as direct enzyme assays or using compound treated cells supernatant in a replication assay. Examples of plant products that have reportedly shown anti-HIV activity as a result of inhibiting the polymerase domain of HIV include the flavonoid myricetin (Pasetto et al. 2014) and crude plant extracts of Hoodia gordonii (Kapewangolo et al. 2016a) and Sceletium tortuosum (Kapewangolo et al. 2016b).

5.1.2 RNAse H Activity Assays

Drugs that target the RNase H function of RT are important since they offer alternatives which focus on the RNase H binding function of RT. Unfortunately, assays that target RNase H are cumbersome and costly, not sensitive and make use of radiolabelled substrates, as such, this target has not been widely explored (Wu et al. 2017). An isotope based anti- RNase H activity study on the effect of nine South African medicinal plants against HIV-1 was assessed by measuring the degree of degradation of the 3H-labelled RNA strand in a RNA/DNA hybrid by RT in the presence of the test substance (Bessong et al. 2005). Of the nine plants, Combretum molle demonstrated the highest inhibition effect on the RNAse activity (Bessong et al. 2005). Wu et al. (2017) recently reported a label free fluorescence assay for rapid detection of RNase H activity based on Tb3+-induced G-quadruplex conjugates (Wu et al. 2017). The assay makes use of an RNA:DNA hybrid probe which in the presence of RNase H, can be cleaved to produce a single-strand G-rich oligonucleotide. The ssG rich oligonucleotide is then subsequently induced by Tb3+ to form a stable G-quadruplex structure leading to an enhancement in Tb3+ fluorescence. This quick, rapid and much safer method is a positive move from the traditional techniques.

5.2 HIV Protease Activity Assays

These assays make use of quantitative techniques such as fluorescence and high performance liquid chromatography. One such fluorescence technique involves the use of a fluorogenic HIV PR substrate 1; Arg-glu-(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Lys-(DABCYL)-Arg (Sigma Aldrich, Missouri USA). This substrate is a synthetic peptide Tyr-Pro cleavage site for HIV PR as well as two covalently modified amino acids for the detection of cleavage (Matayoshi et al. 1990). One of the modifications involves the attachment of the fluorophore 5-(2-aminoethylamino)-1-naphthalene sulfonate (EDANS) to the glutamic residue. The other modification is the addition of an acceptor chromophore 4′-dimethylaminoazobenzene-4-caboxylate (DABCYL) to the lysine residue. The modified amino acids are on opposite sides of the cleavage site. Spatial orientation and overlap of the DABCYL absorbance with the EDANS emission permits resonance energy transfer between the two moieties and quenching of the EDANS fluorescence at 490 nm occurs. However, when HIV PR cleaves the peptide, the DABCYL group is no longer proximal to the fluorophore and emission at 490 nm cannot be detected. A drug that inhibits HIV PR therefore prevents this cleavage thus allowing quenching to occur such that the EDANS fluorescence signal is diminished. Models of such an assay are used for high throughput screening in 96-well format. Medicinal plants with inhibition effect on HIV-1 PR include Ocimum labiatum and the isolated bioactive compound pheophytin a (Kapewangolo et al. 2017a), a popular commercial plant Hoodia gordonii (Apocynaceae) (Kapewangolo et al. 2016a), Sceletium tortuosum (Kapewangolo et al. 2016b) and Bulbine frutescens (Shikalepo et al. 2018). Polyphenols have diverse multi-target against HIV which includes inhibition of HIV PR (Andrae-marobela et al. 2013).

5.3 HIV Integrase Activity Assays

Unlike the other two HIV enzymes, RT and PR, HIV IN doesn’t have a mammalian equivalent making it a suitable target for HIV therapy. HIV IN integrates the viral DNA into the host genome in two steps. The first step involves the removal of two nucleotides from the viral cDNA and the second step which is referred to as DNA strand transfer involves the integration of the cDNA into the host genome (Goldgur et al. 1999). Direct HIV IN assays have been developed with inhibitors such as phytochemicals/crude plant extracts targeting the 3′-processing (removal of nucleotides) and subsequent DNA strand transfer recombination reaction. One such method is a non-radioactive assay used to quantitatively measure the effects of anti-viral agents on HIV-1 IN activity in a 96-well plate format (Kapewangolo et al. 2016a). In this assay, a full-length recombinant HIV-1 integrase protein is loaded onto the HIV-1 LTR U5 donor substrate (DS) DNA containing an end-labeled biotin. Inhibitors are added to the enzyme reaction with subsequent addition of a different double stranded target substrate (TS) DNA containing a 3′-end modification. The HIV-1 integrase cleaves the terminal two bases from the exposed 3′-end of the HIV-1 LTR DS DNA and then catalyzes a strand-transfer recombination reaction to integrate the DS DNA into the TS DNA. Detection of the reaction products is done colorimetrically using an HRP-labeled antibody directed against the TS 3′-end modification. In addition to other anti-HIV properties, flavonols such as quercetin and kaempferol reportedly inhibit HIV IN (Andrae-marobela et al. 2013). Medicinal plants tested for HIV IN inhibition include Bulbine frutescens (Shikalepo et al. 2018) and Sceletium tortuosum (Kapewangolo et al. 2016b).

6 HIV Replication Assay Models for in-vitro Anti-HIV Screening

HIV primarily infects CD4+ T cells and causes AIDS as a result of the depletion of this subpopulation of cells, which is crucial for an effective immune system (Rambaut et al. 2004). Most importantly, the virus infects cells that express the CD4 receptor and chemokine co-receptors, CCR5 and CXCR4, allowing for viral entry (Alkhatib 2009).

Some anti-HIV assays aimed at studying HIV replication focusing on inhibition of replication by potential drugs including phytochemicals are the simple prescreening methods such as the tetrazolium dye based assays, 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) and 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT), the p24 expression assay, syncytium formation assay and luciferase activity detection assays. These assays are performed on human T cells including both primary peripheral blood mononuclear cells (PBMCs) and continuous cell lines such as CEM-SS, MT4, H9 and C8166 (Rege et al. 2015). These inexpensive 96 well format assays facilitate the estimation of the 50% cytotoxic concentrations (CC50s) and effective concentrations (EC50) of potential drugs albeit with limitations such as no information on the mechanism of action of the potential compound.

6.1 Tetrazolium Dye Based Assays

The MTT and XTT assays involve dyes whose reduction to formazan is used to estimate cell viability and proliferation by spectrophotometric measurements. Considering that several T cell lines are susceptible to the lytic effect of HIV replication, these dyes have been employed in measuring both this cytolytic effect and hence anti-HIV activity as well as the cytotoxic effect of potential drugs (Rege et al. 2015).

In the case of MTT (developed by Mosmann 1983), an insoluble blue product is produced and requires a solubilisation step using solvents (Mosmann 1983). Solvents such as acidified isopropanol in a 1:9 ratio (1 part 1 M HCl and 9 part of isopropanol) and DMSO have been used. The product obtained from XTT reduction is water soluble and brownish. An additional coupling reagent, phenazine methosulfate (PMS) is used in the XTT assays to enhance formazan formation. For both assays, spectrophotometric readings are taken within 4–24 h of treatment at one of different wavelengths of the absorbance spectrum; MTT (540–600 nm and 650–690 background) and XTT (450–500 nm and 630–690 nm background). MTT has reportedly been used in assessing the antiviral and cytotoxic effect of phytochemicals (Liu et al. 2018). The selectivity index of these products for HIV is measured from dividing the CC50 by the EC50.

The use of the dynamic impedance based real time, label-free cell electronic sensing analyser for measuring cell proliferation, cytotoxicity start time, cell recovery, and cell response patterns (Fonteh et al. 2015), is a useful alternative to MTT and XTT albeit much more expensive. The system is applicable in research applications in drug development, toxicology, cancer, medical microbiology and virology. Other cell viability and proliferation assays such as the annexin/PI apoptosis assay and the caboxyfluoreiscenin succinimidyl ester assays which are flow cytometry based are alternatives which are more specific but with low throughput and more costly. These are not discussed here.

6.2 Syncytium Formation Cell Counting Assay

Syncytium results from cell fusion induced by HIV. Microscopy has been used to determine syncytium formation by counting the giant multinucleated cells under an inverted microscope at 100x magnification (Rashed et al. 2013). This method does not allow for quantification of syncytium nor does it provide an estimate of the number of cells involved in cell fusion (Wünschmann and Stapleton 2000). Wunchsmann and Stapleton (2000) described a technique that involved staining of the DNA with propidium iodide to determine cell size and fluorescence parameters using flow cytometry. Using this technique, the authors were able to distinguish between syncytium forming and non syncytium forming cells. The assay showed that a decrease in cell numbers in HIV infected MT-2 cell lines was as a result of decrease in syncytium formation than viral cell death making it an inexpensive assay for use in place of p24 ELISA (Wünschmann and Stapleton 2000). Syncytium inhibition studies have been reported for flavonoids such as scutellarin (Zhang et al. 2005).

6.3 p24 Antigen Assay

The p24 antigen is the main structural protein of HIV whose detection serves as an early diagnosis for HIV infection (Xia et al. 2015). The assay is performed by collecting cell free supernatant from compounds treated cells following incubation for a desired number of period usually 4 days post treatment which is optimal for viral production (Rege et al. 2015). An ELISA assay is then performed for detection of p24 activity. Plant derived compounds have been shown to be activators of latent virus, resulting in p24 increase in culture supernatant (Kapewangolo et al. 2017b) as well as inhibitors of the HIV replicative process resulting in decreases in p24 antigen production (Pasetto et al. 2014).

6.4 Luciferase Activity Assay for Viral Infectivity Measuremen

This is an HIV replication assay performed using cells such as the TZM-bl cell line. These cells were generated from JC.53 cells by introducing separate integrated copies of the luciferase and β-galactosidase genes under the control of the HIV-1 promoter (Platt et al. 1998) and are highly sensitive to infection with diverse isolates of HIV-1. These assays make use of molecularly cloned pseudoviruses designed to undergo a single round of infection readily detectable in genetically engineered cell lines that contain a tat-responsive reporter gene such as luciferase e.g. the TZM-bl cell line (Montefiori 2004).

In assays that involve TZM-bl cell line, the cells are seeded overnight followed by the addition of virus pretreated with varying concentration of phytochemicals and incubated for 4 h. Cells are then washed to remove unbound virus and further incubated for 48 h. The cells are washed with PBS, lysed and the lysate treated with a substrate followed by measurement of luciferase activity using a fluorimeter. The results are expressed as percentage inhibition and IC50 is calculated. Flavonoids have been shown to inhibit HIV activity as a result of inhibiting the luciferase activity (Pasetto et al. 2014).

7 Conclusion

Constituents of medicinal plants continue to hold promise as potential anti-HIV drug agents. In-vitro anti-HIV assays are important to improve our understanding of the biology of HIV, the mechanisms of action of plant based compounds, biochemical reactions with implications towards inhibition of HIV and safety of such medicines. While the in-vitro assays are important, they cannot be used alone without the need for animal/clinical studies. The current drive for marketing phytochemicals is drawn from the ongoing popular use of medicinal plants in the traditional management of HIV/AIDS.