2009 Klos et alIn vitro anti-HIV activity of five selected South African medicinal plant extracts

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Journal of Ethnopharmacology 124 (2009) 182188

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Journal of Ethnopharmacology

journa l homepage: www.e lsev ier .com/ locate / je thpharm

In vitro anti-HIV activity of ve selected South African medicinal plant extracts

M. Klosa, M. van de Venterb,, P.J. Milnea, H.N. Traorec, D. Meyerd, V. Oosthuizenb

a Department of Pharmacy, Nelson Mandela Metropolitan University, PO Box 77000, Port Elizabeth 6031, South Africab Department of Biochemistry and Microbiology, Nelson Mandela Metropolitan University, PO Box 77000, Port Elizabeth 6031, South Africac Department of Biochemistry, University of Johannesburg, PO Box 524, Auckland Park, Johannesburg 2006, South Africad Department of Biochemistry, University of Pretoria, Pretoria 0002, South Africa

a r t i c l e i n f o

Article history:Received 13 January 2009Accepted 17 April 2009Available online 3 May 2009

Keywords:Bulbine alooidesCrinum macowaniHIVHypoxis soboliferaLeonotis leonurusTulbaghia violacea

a b s t r a c t

Aim of the study: Five South African medicinal plants, Bulbine alooides (L.) Willd. (Asphodelaceae), Crinummacowani Baker (Amaryllidaceae), Hypoxis sobolifera var. sobolifera (Jacq.) Nel (Hypoxidaceae), Leonotisleonurus (L.) R.Br. (Lamiaceae) and Tulbaghia violacea Harv (Liliaceae) used for the treatment of variousailments, including infectious diseases,were screened for activity against human immunodeciency virus(HIV).Materials and methods: Aqueous and ethanol extracts were tested for inhibitory activity in HIV-1 infectedCEM.NKR-CCR5 cells, and against HIV-1 reverse transcriptase (RT) and HIV-1 protease (PR).Results: In CEM.NKR-CCR5 cells, ethanol extracts of Leonotis leonurus inhibited HIV-1 signicantly (33%reduction in HIV-1 p24, P


M. Klos et al. / Journal of Ethnopharmacology 124 (2009) 182188 183

Hypoxis sobolifera, Leonotis leonurus and Tulbaghia violacea, werestudied to ascertain their potential anti-HIV activity. These plantswere selected based on a literature survey of their ethnomedici-nal usages directly in HIV/AIDS or for symptoms/conditions closelyassociated with this disease.

2. Materials and methods

2.1. Plant extracts

Plant material was collected in one batch from the Nelson Man-dela Metropolitan area (Eastern Cape, South Africa) during themonth of February 2005 and classied by Prof. E. Campbell (BotanyDepartment, Nelson Mandela Metropolitan University). Voucherspecimens were deposited in the NMMU herbarium. The freshplant material was separated into leaves for Leonotis leonurus andunderground parts for Bulbine alooides (roots), Crinum macowani(bulbs), Hypoxis sobolifera (corms) and Tulbaghia violacea (bulbs)and weighed. Plant material was washed before use by rinsing vig-orously with tap water followed by soaking in ethanol (70%, v/v)for 1min and left for approximately 30min for the ethanol to evap-orate. The plant parts were either chopped (in the case of leaves),grated (in the case of roots, bulbs or corms) or homogenized with aWaringblender at lowspeed for 30 s in the case ofCrinummacowanibulbs. Aqueous and ethanol extracts were prepared by macerat-ing the crushed plant material in 200ml deionized water or 95%ethanol in the dark at 4 C or room temperature, respectively. Themarc was left to soak in the menstruum for 3 days and each day thesupernatant was decanted and ltered using Whatman no. 1 paper,and another volume of 200ml fresh solvent added. In the case ofthe aqueous extracts, the total collected ltrate was shell-frozenand lyophilized. Ethanol extracts were dried by rotary evaporationat temperatures of 6065 C to a nal volume of approximately1020ml to which deionized water was added until the ethanolextract component constituted 1020% (v/v) of the nal solutionvolume. This solution was shell-frozen and lyophilized as per theaqueous extracts. Dried extracts were stored in the dark at 4 Cin a dessicant chamber to limit chemical and/or microbiologicaldeterioration.

Before each biological (cellular and enzyme) assay, the requiredamount of the extracts was weighed and reconstituted in DMSOand further diluted to the desired concentration using aseptic tech-niques. DMSO serves to sterilize the extract and once diluted (


184 M. Klos et al. / Journal of Ethnopharmacology 124 (2009) 182188

(FC) reaction to determine the percentage of tannin removed (rel-ative to equivalent tannic acid units) (Julkunen-Tiitto, 1985).

2.5. Tannin dereplication from ethanol extracts using solventfractionation

Removal of tannins from ethanol extracts was performed usinga fractionation method from Houghton and Raman (1998). Driedethanol extracts (50mg) were dissolved in 2ml of methanol. Chlo-roform (8ml) was slowly added and the solution mixed well, toforma chloroform:methanol (4:1)mixture. This solutionwas trans-ferred to a separating funnel and an equal volume of deionizedwater (10ml) was slowly added. The separating funnel was slowlyinverted multiple times to allow for the polyphenols to transferto the aqueous upper phase. The organic lower phase was sepa-rated from the upper aqueous tannin-containing layer and washedwith an equal volume of 1% (w/v) NaCl in water. The organic phase,containing tannin-free extract, was dried under vacuum at roomtemperature and assayed for tannins with the FC reaction.

2.6. Quantitative total polyphenolic determination by theFolinCiocalteau phenol reaction

In order to determine the effectiveness of tannin removal fromthe extracts a modied method of Julkunen-Tiitto (1985) was used.The amount of tannin removed from the extract was estimated byusing a tannic acid standard curve which gave a linear relationshipbetween absorptivity and concentration. The tannic acid standardcurve remained linear below 800g/ml tannic acid. For each assaya new tannic acid standard curve was determined to ensure accu-racy. Tannic acid (Sigma, Missouri, USA) was diluted in 50% (v/v)DMSO and four serial twofold dilutions were made ranging from800 to 100g/ml. Dried extracts before tannin removal and driedextracts after tannin removal were dissolved in 50% DMSO to give800g/ml. To a 2ml reaction tube, 100l of the respective tan-nic acid standard solution or extract solution was added followedby the addition of 200l of FC phenol reagent (Sigma, Missouri,USA). Immediately, 700l of a 20% (w/v) sodium carbonate solu-tion was added and the mixture made to 2ml with water, followedby shaking. Absorption of the solution was read at 690nm. Beforethe samplesweremeasured spectrophotometrically theywere cen-trifuged, if necessary, because of precipitate formation.

2.7. Removal of sulfated polysaccharides

Aqueous tannin-dereplicated extracts, positive for inhibitoryactivity against HIV-1 enzymes, were subjected to a 50% ethanolprecipitation. After ethanol addition, extracts were stirred at 4 Cfor 20min, ltered and dried as described above (Houghton andRaman, 1998). Extracts that had the sulfated polysaccharidesremoved had the addition of PS added to the extract code, e.g.BATPS.

2.8. Viability assay

CEM.NKR-CCR5 cells are a human T-lymphoblastic cell lineobtained through the AIDS Research and Reference program, Divi-sion of AIDS, National Institute of Allergy and Infectious Diseases(NIAID), National Institute of Health (NIH) (MD USA) from DrAlexandra Trkola. These cells were transformed with a retroviralvector to express human CCR5 and show resistance to natural killercell-mediated lysis and do not secrete infectious virus (Trkola etal., 1999). HIV-1 clade C, the most prevalent subtype within theregion of southern Africa, preferentially uses the CCR5 co-receptorfor infection (Hauesser and Mulnger, 2001). The Cell Proliferation

Kit II (XTT) (Roche Diagnostics, Mannheim, Germany) was used forviability determination of CEM.NKR-CCR5 cells.

CEM.NKR-CCR5 cells were maintained and cultured in RPMI1640 containing 2mM l-glutamine (Sigma, Missouri, USA). Sup-plements added included heat inactivated (56 C, 30min) foetalcalf serum (10%, v/v) and antibiotics that consisted of peni-cillin G (10mg/ml), streptomycin sulphate (10mg/ml), fungizone(25g/ml) and gentamicin sulfate (1ng/ml). Cells were culturedat 37 C in a 5% CO2 humidied atmosphere. Acutely HIV-infectedCEM.NKR-CCR5 cells were counted using trypan blue and seededat 1106 cells/ml. To each well of a 96-well plate, 100l of thiscell suspensionwas added giving 5105 cells/ml in the nal 200lwell volume. Extract solutions were prepared by rst mixing withDMSO to surface sterilize the powder, followed by dilution withcomplete medium to a nal concentration of 2mg/ml. Final wellconcentrations tested for each extract corresponded to the CC90concentrations seen in uninfected PBMCs from a 23-year-old maledonor (data not shown). Control wells included a negative con-trol (cells and medium only) and extract/XTT blank controls foreach extract (extract andmediumonly). Testwells included extract,cells and medium. Plates were incubated at 30 C for 7 days. Uponcompletion of the incubation, 50l of a previously prepared XTTworking solutioncontainingN-methyldibenzopyrazinemethyl sul-fate (PMS) and XTT (1:50) was added to all the wells. Plates wereincubated for 4h at 30 C and read at 450nm (reference 690nm).The sameprocedurewas followed for testing theviabilityof extractson uninfected cells, except cells were not infected with HIV.

2.9. Antiviral assay

The HIV-1 p24 Antigen Assay kit (Beckman Coulter, Miami, FL,USA), an enzyme-linked immunosorbant assay (ELISA), was usedto detect and quantify HIV-1 p24 core protein. At the end of the7 days incubation, culture supernatant (100l) from the HIV-infected CEM.NKR-CCR5 cultures was transferred to the murinemonoclonal-coated 96-well plate for the p24 assay. The protocolwas followed as described by the manufacturer, with absorbancemeasured at 450nm.

2.10. HIV-1 reverse transcriptase assay

The effect of the crude extracts on reverse transcription wastested using a non-radioactive HIV-RT colorimetric ELISA kit fromRoche Diagnostics, Germany. The protocol outlined in the kit wasfollowed, under nuclease-free conditions, using 2ng of enzyme in awell and incubating the reaction for 2h at 37 C. Negative controlsfor the assay included HIV-1 RT with only lysis buffer, HIV-1 RTwith only solvent (2% DMSO) in lysis buffer, and a blank with justABTS. The positive control used was nevirapine (kindly donated byAspen Pharmacare, South Africa), a reverse transcriptase inhibitorused commonly in clinical practice. The HIV-RT inhibition of theplant extracts were measured as a percentage of the inhibition thatoccurred with HIV-1 RT in the presence of no inhibitor in the samesolvent (2% DMSO) as the extracts.

2.11. HIV-1 protease assay

The HIV-1 PR assay was performed using a uorogenic octapep-tide substrate, HIV-FRET(1) (uorescence resonance energytransfer) (AnaSpec Inc., USA) and a recombinant HIV-1 proteasesolution (Bachem, Switzerland). The peptide sequence of HIV-FRET(1) is derived from a natural processing site for HIV-1 PRand has the following structure: 4-(4-dimethylaminophenylazo)-benzoic acid (DABCYL)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-5-[(2-aminoethyl)amino]naphthalene-1 sulfonic acid (EDANS)]. Theprocedure for the continuous uorogenic detection of HIV-1 PR



was adapted from the method of Matayoshi et al. (1990). Theuorogenic substrate was dissolved in DMSO to 1.3mM. Thestock 39M recombinant HIV-1 protease solution was dilutedto a concentration of 222nM with freshly prepared assay buffer(100mM sodium acetate, 1M sodium chloride, 1mg/ml BSA, 1mMEDTA, 1mM dithiothreitol, pH 4.7). To the wells of a 96-well blackmicrotiter plate, 45l of diluted HIV-1 PR (nal concentration was100nM) and 5l of extract or control were added and incubatedat 37 C for 15min. During this incubation, the stock substratewas diluted to 16M by assay buffer and pre-heated to 37 C.The diluted substrate (50l) was added, to initiate the reactionof substrate cleavage by HIV-1 PR, and the microplate shakenat 300 rpm for 1min. The uorescence intensity was measuredkinetically every 30 s over a period of 10min at an excitationwavelength of 355nm and an emission wavelength of 460nm, ata temperature of 37 C, using a Fluoroskan Ascent FL microplatereader (Thermolabsystems). The reaction rates were determinedby the gradient of the initial linear portions (usually the rst510min) of the plot of RFI (relative uorescence intensity) as afunction of time. Negative controls included were HIV-1 PR withonly assay buffer, HIV-1 PR enzyme with DMSO (2%) in assaybuffer and substrate alone. Positive controls included HIV-1 PRwith a general acid-protease inhibitor, acetyl pepstatin (Bachem,Switzerland) or a potent HIV PR specic inhibitor ritonavir (kindlydonated by Aspen Pharmacare, South Africa). The percentageinhibition of HIV-1 PR was calculated as a percentage of a controlwith only the solvent (2% DMSO).

2.12. Statistical analysis

Signicance determinations were obtained by applying a two-tailed unpaired t-test. All results with P



Fig. 2. HIV-1 RT inhibition by various plant extracts. The +BSA code representsfractions with BSA added and the T represents fractions with tannins removed.The data represent the mean HIV-1 RT inhibition (relative to an untreated controlwith solvent only) of various plant extracts, each tested at 0.2mg/ml in the nalreactionvolume. Extracts showing50% inhibitionwere tested again in thepresenceof 0.2% BSA to adsorb tannins. The control, nevirapine was tested at 0.05mg/ml intriplicate. The RT inhibition of all aqueous and ethanol crude extracts are the meanof three separate experiments (n=7). In the case of fractions with BSA added theresults are an average of two separate experiments (n=5). The HA-T column is theaverage of a single experiment performed in triplicate. Error bars represent SEM andasterisks represent P values as determined by a two-tailed unpaired t-test (*P



4. Discussion and conclusions

None of the current substances with antiviral activity againstHIV are without toxicities and resistance and hence there is astrong need to improve the current antiretroviral armamentarium.A potential source of novel compounds for HIV is from medicinalplants or other natural products. In order to nd such poten-tial anti-HIV agents from medicinal plants, we have screenedvarious medicinal plants commonly used in South African tra-ditional medicine. In this paper, we report the importance oftannin/polysaccharide dereplication from plant extracts and the invitro anti-HIV activity of ethanolic and aqueous extracts from veSouth African medicinal plants.

4.1. Tannin/polysaccharide dereplication

Tannins are polyphenols widely found in plants and most HIV-inhibitory effects attributed to plant extracts are due to tannins(Cardellina II et al., 1993). Tannins are non-specic because theycrosslink with many proteins, inhibiting a large range of biologicalsystems and enzymatic pathways (Cardellina II et al., 1993; Chunget al., 1998; Houghton and Raman, 1998; Cowan, 1999). Due tothis non-specicity, tannins are known to cause possible liver dam-age, have carcinogenic potential and have anti-nutritional activity.On the other hand, their high potential to bind proteins has beendemonstrated in some studies to result in specic in vitro activitieslike antiviral, antimutagenic, anticarcinogenic, and immunomodu-latory, arguing for sufcient selectivity for a potential therapeuticuse (Chung et al., 1998). In vivo tannins are not as effective asan in vitro assay will suggest since they will be too highly boundto serum albumin and therefore have a low bioavailability in thebody (Ballick, 1994). However, tannins may be the only compoundspresent that are responsible for a reputed or observed biologicaleffect in a plant extract, and have attracted considerable interestas bioactive compounds in their own right. Nevertheless, if oneis interested in discovering novel types of molecules, eliminationof these compounds at an early stage is desirable (Houghton andRaman, 1998).

In general, the polyamide/fractionation tannin dereplicationmethods removed tannins to between 3 and 5% (w/w) (Table 2).The only exception was for BE where tannins could not be reducedto less than 9.5% using the fractionation method. This may be dueto the FC reagent reactingwith unknownnon-specic substances intheBEextract therefore over-estimating the tannin content. Indeed,the FC phenol reagent most likely over assumed the tannin contentin all the extracts because of minor non-specic reactivity. Inter-ference of the FC reagent can occur with carbohydrates, glucose,ascorbic acid, proteins and non-tannin phenols which could havebeen present in the BE extract (Julkunen-Tiitto, 1985). It must alsobe noted that some small polyphenolics may not be retained bythe polyamide column or pass into the aqueous phase with thefractionation method, and therefore the possibility of some smallpolyphenolic molecules in the BE extract also exists (Collins et al.,1998).

Sulfated polysaccharides have consistently shown activity inanti-HIV assays with aqueous extracts and it is believed to functionin destabilizing the glycoprotein complex and/or inhibiting reversetranscriptase (Greene and Peterlin, 2002). Many compounds fromthis class have already been described extensively in the literatureas having anti-HIV activity (Baba et al., 1988). Like tannins, in vivoeffectiveness is low since polysaccharides are poorly absorbed fromthe gastrointestinal tract because of the fact that polysaccharidesbreak down to monosaccharides on oral administration (Ballick,1994). As for tannin dereplication, if one wishes to discover novelinhibitors from a plant extract, dereplication of polysaccharides isneeded.

Polysaccharides were only removed from the BA-T extract asthis was the only aqueous tannin-dereplicated extract that showedinhibitory activity of HIV-1 protease (Fig. 3). The activity of theextract was then decreased after polysaccharide removal. This lossin activity after polysaccharides were removed indicates that somepolysaccharides (together with tannins previously removed) weremost likely responsible for most of BAs inhibitory activity.

4.2. Anti-HIV activity

The results described in this study indicate that the tannin-dereplicated ethanol extracts of Bulbine alooides and Leonotisleonurus possess anti-HIV properties of possible therapeutic inter-est, through HIV-1 PR inhibition, with an IC50 of 94.3 and120.6g/ml, respectively (Fig. 4B). While these are weak inhibitoryIC50 values, compared to the ritonavir control which had an IC50of 5.3ng/ml (Fig. 4A), it must be remembered that the crude plantextracts are highly impure compared to the pure ritonavir. It maybe that the amount of active compound extracted is a very smallpercentage of the extract. Therefore, assuming that there is a sin-gle inhibitor responsible for HIV-1 PR inhibition, concentrations1001000 times less than the IC50 seen for the extracts may bethe actual inhibitory concentration (Houghton and Raman, 1998).

The crude LE extract, at a concentration of 100g/ml, was theonlyextract to showsignicantHIV inhibitionby reduction inHIV-1p24 levels of 33.0% (Fig. 1B). LEmay possibly have caused decreasedHIV-1 p24 levels in the CEM.NKR-CCR5 cells via inhibition of HIVprotease since the concentration usedwas in the range of its IC50 forthis enzyme (Fig. 4B). The reduction in HIV-1 p24 levels observedwith BEwas statistically not signicant (P>0.05). It should be notedthat the concentration of BE used in this experiment was 60g/mland was therefore lower than the IC50. Higher concentrations ofBE may decrease the p24 levels signicantly, but at the same timemaybe too toxic to infected CEM.NKR-CCR5 cells. The same concen-trations of BE and LE exposed to PBMCs from a healthy donor for48h, were found to be non-toxic (data not shown). Further studies,at a greater range of concentrations, may be needed on these twoextracts to try and nd the concentration at which cytotoxicity islowest and HIV-1 inhibition is highest in CEM.NKR-CCR5 cells.

It must be noted that the fractions of HA, HE, LA, TA and TEsignicantly enhanced cell proliferation in HIV-infected CEM.NKR-CCR5 cells (Fig. 1A) by 44.2, 21.0, 14.5, 34.3 and 18.6%, respectively(Fig. 1A). This increased T-cell proliferation, however, cannot be dueto viral inhibition, as p24 viral antigen levels did not correspond-ingly decrease (Fig. 1B).

In the HIV-1 RT assay, the extracts of BA, BE, HA, HE and LAshowed 50% HIV-1 RT inhibition, and thus were re-tested in thepresence of 0.2% BSA (Fig. 2). Only the extract of HA retainedits activity in the presence of BSA (55.3%). However, after tannindereplication using polyamide columns, inhibition of HA was lostto 10.1% (HA-T). Therefore, further consideration of this extractwas not necessary since its HIV-1 RT inhibitory activity could beattributed to the non-selective tannin compounds.

No known HIV inhibitory compounds exist in the extracts ofBulbine alooides or Leonotis leonurus. This study reports, for the rsttime, that in vitroanti-HIVactivity exists in theseextracts. Itmustbenoted that Leonotis leonurus is used traditionally for hepatitis treat-ment and it has been found that plants used for hepatitis treatmentoften exhibit one or more anti-HIV activities. This includes somespecies from the Lamiaceae family (Lewis and Elvin-Lewis, 2003).In addition, HIV-1 PR inhibitors are usually lipid soluble agentsso it is possible that the ethanol extracted more of the HIV-1 PRinhibitor(s) from BE and LE than the aqueous extraction (Hoggardand Owen, 2003). Therefore with a more lipid soluble extract, suchas chloroform, more of the unknown protease inhibitor(s) may beextracted.



In conclusion, this study shows the importance of includ-ing dereplication procedures for tannins/polysaccharides duringscreening processes of plant materials since this will allow fornovel antiretroviral drug discovery. This study revealed that thetannin-dereplicated ethanol extracts of Leonotis leonurus and Bul-bine alooides have mild HIV-1 PR inhibitory activity in vitro. Furtherpurication of the tannin-dereplicated active extract fractions ofthe leaves and roots of Leonotis leonurus and Bulbine alooides,respectively, will allow more conclusive data regarding the poten-tial of a potent novel HIV inhibitor being present in these extracts.

Acknowledgment

The authors wish to acknowledge the support of this studyby the National Research Foundation (NRF) of South Africa (GUN:2069228).

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2009 Klos et alIn vitro anti-HIV activity of five selected South African medicinal plant extracts