in vitro anti-hiv activity of five selected south african medicinal plant extracts
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Journal of Ethnopharmacology 124 (2009) 182–188
Contents lists available at ScienceDirect
Journal of Ethnopharmacology
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n vitro anti-HIV activity of five selected South African medicinal plant extracts
. Klosa, M. van de Venterb,∗, P.J. Milnea, H.N. Traorec, D. Meyerd, V. Oosthuizenb
Department of Pharmacy, Nelson Mandela Metropolitan University, PO Box 77000, Port Elizabeth 6031, South AfricaDepartment of Biochemistry and Microbiology, Nelson Mandela Metropolitan University, PO Box 77000, Port Elizabeth 6031, South AfricaDepartment of Biochemistry, University of Johannesburg, PO Box 524, Auckland Park, Johannesburg 2006, South AfricaDepartment of Biochemistry, University of Pretoria, Pretoria 0002, South Africa
r t i c l e i n f o
rticle history:eceived 13 January 2009ccepted 17 April 2009vailable online 3 May 2009
eywords:ulbine alooidesrinum macowaniIVypoxis sobolifera
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 immunodeficiency 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 significantly (33%reduction in HIV-1 p24, P < 0.05). HIV-1 RT inhibition (≥50%) was shown for extracts of Bulbine alooides(aqueous and ethanol), Hypoxis sobolifera (aqueous and ethanol) and Leonotis leonurus (aqueous), but
eonotis leonurusulbaghia violacea inhibitory activity was lost upon dereplication for removal of non-specific tannins/polysaccharides. HIV-1 PR inhibition was observed for extracts of Hypoxis sobolifera (aqueous), Bulbine alooides (aqueous andethanol) and Leonotis leonurus (ethanol). Only ethanolic extracts of Bulbine alooides and Leonotis leonurusretained HIV-1 PR inhibition after dereplication with IC50 of 94 �g/ml and 120 �g/ml, respectively.Conclusion: The dereplicated ethanolic extracts of Leonotis leonurus and Bulbine alooides showed the
al in t
greatest anti-HIV potenti. Introduction
It is estimated that HIV has infected 40.3 million people world-ide, with South Africa having the highest prevalence, at 5.5 millionIV-infected people (UNAIDS, 2006). There are two related but dis-
inct types of HIV: HIV-1 and HIV-2 (Fletcher et al., 2002). HIV-1s the most pathogenic and causes over 99% of HIV infections (Cost al., 2004). HIV-2 is also known to cause AIDS but is much lessrevalent, being present in fewer and isolated geographic locationsuch as Western Africa. Therefore, most research is done on HIV-1.
Current antiretroviral drugs are vitally important to improve theuality and prolong the life of HIV/AIDS patients. However, these
rugs have many disadvantages including resistance, toxicity, lim-ted availability, high cost and lack of any curative effect. Thus, it ismportant to search for improved antiretroviral agents which cane added to or replace the current drugs in the anti-HIV arma-
Abbreviations: BSA, bovine serum albumin; FC, Folin–Ciocalteau; HIV-1 PR,uman immunodeficiency virus-1 protease; HIV-1 RT, HIV-1 reverse transcriptase;BMC, peripheral blood mononuclear cells.∗ Corresponding author. Tel.: +27 41 504 2813; fax: +27 41 504 2814.
E-mail address: [email protected] (M. van de Venter).
378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2009.04.043
his study through inhibition of HIV-1 PR.© 2009 Elsevier Ireland Ltd. All rights reserved.
mentarium. Natural sources, particularly plants, are an excellentsource of anti-HIV agents. South Africa has a rich plant biodiversityand a long tradition of medicinal use of plants with approximately3000 species of plants used as medicines (Van Wyk and Gericke,2000; Scott et al., 2004). Several of these plants may contain novelanti-HIV compounds.
In the past decade, a substantial amount of research has beendone worldwide (and a lot more is in progress) to isolate the activeleads from plants for preventing transmission of HIV and treatmentof AIDS based on ethnopharmacological data (Asres et al., 2001;Vermani and Garg, 2002). Screening plants based on ethnopharma-cological data increases the potential of finding novel compoundsdue to a long history of use (Farnsworth, 1994; Fabricant andFarnsworth, 2001). Even though HIV/AIDS is a relatively new humandisease, with minimal ethnobotanical treatments, logical associa-tions of treatments for other likely viral infections (such as hepatitisB) and closely linked disease states or symptoms (wasting, diar-rhoea, lymphadenopathy, skin lesions, cough, haemoptysis and
genital ulcers) can increase the prospect of finding new plant leadsas potential anti-HIV agents (WHO, 1989a,b; Cardellina II and Boyd,1995; Lewis and Elvin-Lewis, 2003).For the purpose of this study, five plants commonly used in SouthAfrica in traditional medicines, Bulbine alooides, Crinum macowani,
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ypoxis sobolifera, Leonotis leonurus and Tulbaghia violacea, weretudied to ascertain their potential anti-HIV activity. These plantsere selected based on a literature survey of their ethnomedici-al usages directly in HIV/AIDS or for symptoms/conditions closelyssociated with this disease.
. Materials and methods
.1. Plant extracts
Plant material was collected in one batch from the Nelson Man-ela Metropolitan area (Eastern Cape, South Africa) during theonth of February 2005 and classified by Prof. E. Campbell (Botanyepartment, Nelson Mandela Metropolitan University). Voucher
pecimens were deposited in the NMMU herbarium. The freshlant material was separated into leaves for Leonotis leonurus andnderground parts for Bulbine alooides (roots), Crinum macowanibulbs), Hypoxis sobolifera (corms) and Tulbaghia violacea (bulbs)nd weighed. Plant material was washed before use by rinsing vig-rously with tap water followed by soaking in ethanol (70%, v/v)or 1 min and left for approximately 30 min for the ethanol to evap-rate. The plant parts were either chopped (in the case of leaves),rated (in the case of roots, bulbs or corms) or homogenized with aaring blender at low speed for 30 s in the case of Crinum macowani
ulbs. Aqueous and ethanol extracts were prepared by macerat-ng the crushed plant material in 200 ml deionized water or 95%thanol in the dark at 4 ◦C or room temperature, respectively. Thearc was left to soak in the menstruum for 3 days and each day the
upernatant was decanted and filtered using Whatman no. 1 paper,nd another volume of 200 ml fresh solvent added. In the case ofhe aqueous extracts, the total collected filtrate was shell-frozennd lyophilized. Ethanol extracts were dried by rotary evaporationt temperatures of 60–65 ◦C to a final volume of approximately0–20 ml to which deionized water was added until the ethanolxtract component constituted 10–20% (v/v) of the final solutionolume. This solution was shell-frozen and lyophilized as per thequeous extracts. Dried extracts were stored in the dark at 4 ◦Cn a dessicant chamber to limit chemical and/or microbiologicaleterioration.
Before each biological (cellular and enzyme) assay, the requiredmount of the extracts was weighed and reconstituted in DMSOnd further diluted to the desired concentration using aseptic tech-
iques. DMSO serves to sterilize the extract and once diluted (<3%,/v) should have no effect on the biological assays (Houghton andaman, 1998). The final concentration of DMSO in each assay didot significantly affect the enzyme or cells (data not shown). Thelants, together with their voucher numbers, abbreviated codesable 1lant extracts information with yields (w/w) and extracts codes.
lant Voucher specimennumber
Extraction solvent C
ulbine alooides PEU14795Aqueous B95% ethanol B
rinum macowani PEU14796Aqueous C95% ethanol C
ypoxis sobolifera PEU14840Aqueous H95% ethanol H
eonotis leonurus PEU14797Aqueous LA95% ethanol LE
ulbaghia violacea PEU14799Aqueousb TA95% ethanol T
a Yield represents the percentage recovery of dried extract per weight as compared witb For TA extracts only an 1 day maceration extraction was used to avoid degradation of
003).
acology 124 (2009) 182–188 183
used in later text, plant parts used and yields from extraction aresummarised in Table 1.
2.2. Removal of tannins
Extracts that had 50% or more inhibitory activity in the enzymeassays had tannins removed by the methods of BSA addition(extracts denoted by the code ‘+BSA’ e.g. ‘HA+BSA’) and physicaltannin removal (extracts denoted by the code ‘−T’ e.g. ‘HA−T’), asdescribed below.
2.3. Tannin adsorption with bovine serum albumin
Bovine serum albumin (BSA) was added to assay buffers to afinal assay concentration of 0.2% (w/v) to adsorb possible tanninsfrom crude extracts that showed a 50% or more inhibitory effect inthe anti-HIV assays (Devlin et al., 1991; Harnett et al., 2005). Thecrude extracts that still retained HIV enzyme (RT or PR) inhibitoryactivity ≥50% in the presence of BSA were then physically removedof tannins (using polyamide or solvent fractionation) to ensure nearto complete removal of tannins and then tested again in the HIVenzyme assay.
2.4. Tannin dereplication from aqueous extracts using polyamidecolumns
Solid phase extraction columns for tannin removal were pre-pared using modified methods of Collins et al. (1998), CardellinaII and Boyd (1995) and Houghton and Raman (1998). Two gramsof polyamide DPA-6S resin (Supelco, USA) was packed into 12 mlpolyethylene syringes (12 mm × 150 mm) fitted with frits. Prior topacking the column the polyamide resin was soaked in 12 ml ofdeionized water overnight to allow for swelling of the resin andto remove unreacted monomers. The polyamide resin was pouredinto the column and left for a few minutes to settle and to allowthe column to pack firmly. The water was allowed to drain fromthe column until approximately 1–2 mm above the surface of thecolumn bed remained. To prevent disturbing the bed surface whenapplying the sample and during elution, a small ring of Whatmanno. 1 filter paper was placed on top of the column.
The columns were assayed in triplicate to assess the efficacy oftannin removal. To each column 25 mg of aqueous extract dissolved
in 1 ml of deionized water was applied to the column surface andallowed to percolate. The sample was eluted in three fractions: 9 mlwater, 10 ml 50% (v/v) aqueous methanol and 10 ml methanol. Theflow rate was adjusted to approximately 1 ml/min. These fractionswere dried by lyophilization and assayed by the Folin–Ciocalteauode Plant part used Initial weight of freshplant extract (g)
Yield (%, w/w)a
ARoots
158.69 3.55E 154.45 4.17
ABulbs
859.47 3.09E 789.16 2.09
ACorms
123.65 5.62E 125.93 2.38
Leaves99.49 1.21
111.90 3.42
Bulbs81.90 1.68
E 48.63 2.71
h the original fresh plant material.Tulbaghia violacea compounds in aqueous solution (Kubec et al., 2002; Motsei et al.,
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FC) reaction to determine the percentage of tannin removed (rel-tive to equivalent tannic acid units) (Julkunen-Tiitto, 1985).
.5. Tannin dereplication from ethanol extracts using solventractionation
Removal of tannins from ethanol extracts was performed usingfractionation method from Houghton and Raman (1998). Dried
thanol extracts (50 mg) were dissolved in 2 ml of methanol. Chlo-oform (8 ml) was slowly added and the solution mixed well, toorm a chloroform:methanol (4:1) mixture. This solution was trans-erred to a separating funnel and an equal volume of deionizedater (10 ml) was slowly added. The separating funnel was slowly
nverted multiple times to allow for the polyphenols to transfero the aqueous upper phase. The organic lower phase was sepa-ated from the upper aqueous tannin-containing layer and washedith an equal volume of 1% (w/v) NaCl in water. The organic phase,
ontaining tannin-free extract, was dried under vacuum at roomemperature and assayed for tannins with the FC reaction.
.6. Quantitative total polyphenolic determination by theolin–Ciocalteau phenol reaction
In order to determine the effectiveness of tannin removal fromhe extracts a modified method of Julkunen-Tiitto (1985) was used.he amount of tannin removed from the extract was estimated bysing a tannic acid standard curve which gave a linear relationshipetween absorptivity and concentration. The tannic acid standardurve remained linear below 800 �g/ml tannic acid. For each assaynew tannic acid standard curve was determined to ensure accu-
acy. Tannic acid (Sigma, Missouri, USA) was diluted in 50% (v/v)MSO and four serial twofold dilutions were made ranging from00 to 100 �g/ml. Dried extracts before tannin removal and driedxtracts after tannin removal were dissolved in 50% DMSO to give00 �g/ml. To a 2 ml reaction tube, 100 �l of the respective tan-ic acid standard solution or extract solution was added followedy the addition of 200 �l of FC phenol reagent (Sigma, Missouri,SA). Immediately, 700 �l of a 20% (w/v) sodium carbonate solu-
ion was added and the mixture made to 2 ml with water, followedy shaking. Absorption of the solution was read at 690 nm. Beforehe samples were measured spectrophotometrically they were cen-rifuged, if necessary, because of precipitate formation.
.7. Removal of sulfated polysaccharides
Aqueous tannin-dereplicated extracts, positive for inhibitoryctivity against HIV-1 enzymes, were subjected to a 50% ethanolrecipitation. After ethanol addition, extracts were stirred at 4 ◦Cor 20 min, filtered and dried as described above (Houghton andaman, 1998). Extracts that had the sulfated polysaccharidesemoved had the addition of ‘−PS’ added to the extract code, e.g.A−T−PS.
.8. Viability assay
CEM.NKR-CCR5 cells are a human T-lymphoblastic cell linebtained through the AIDS Research and Reference program, Divi-ion of AIDS, National Institute of Allergy and Infectious DiseasesNIAID), National Institute of Health (NIH) (MD USA) from Drlexandra Trkola. These cells were transformed with a retroviral
ector to express human CCR5 and show resistance to natural killerell-mediated lysis and do not secrete infectious virus (Trkola etl., 1999). HIV-1 clade C, the most prevalent subtype within theegion of southern Africa, preferentially uses the CCR5 co-receptoror infection (Hauesser and Mulfinger, 2001). The Cell Proliferationacology 124 (2009) 182–188
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 2 mM l-glutamine (Sigma, Missouri, USA). Sup-plements added included heat inactivated (56 ◦C, 30 min) foetalcalf serum (10%, v/v) and antibiotics that consisted of peni-cillin G (10 mg/ml), streptomycin sulphate (10 mg/ml), fungizone(25 �g/ml) and gentamicin sulfate (1 ng/ml). Cells were culturedat 37 ◦C in a 5% CO2 humidified atmosphere. Acutely HIV-infectedCEM.NKR-CCR5 cells were counted using trypan blue and seededat 1 × 106 cells/ml. To each well of a 96-well plate, 100 �l of thiscell suspension was added giving 5 × 105 cells/ml in the final 200 �lwell volume. Extract solutions were prepared by first mixing withDMSO to surface sterilize the powder, followed by dilution withcomplete medium to a final concentration of 2 mg/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 and medium only). Test wells included extract,cells and medium. Plates were incubated at 30 ◦C for 7 days. Uponcompletion of the incubation, 50 �l of a previously prepared XTTworking solution containing N-methyl dibenzopyrazine methyl sul-fate (PMS) and XTT (1:50) was added to all the wells. Plates wereincubated for 4 h at 30 ◦C and read at 450 nm (reference 690 nm).The same procedure was followed for testing the viability of 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 (100 �l) 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 450 nm.
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 2 ng of enzyme in awell and incubating the reaction for 2 h 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 fluorogenic octapep-tide substrate, HIV-FRET(1) (fluorescence 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 fluorogenic detection of HIV-1 PR
pharmacology 124 (2009) 182–188 185
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Fig. 1. Effects of extracts on the viability of HIV-1 infected (A) CEM.NKR-CCR5 cellsand on HIV-1 p24 antigen levels (B). All results are the percent difference relative tountreated cells. Results in (A) are from XTT viability determinations. For (B), super-natants from the infected cells were tested for HIV-1 p24 levels using an ELISA basedassay. Error bars represent SEM and n = 10 and 3 for (A) and (B), respectively. Aster-
M. Klos et al. / Journal of Ethno
as adapted from the method of Matayoshi et al. (1990). Theuorogenic substrate was dissolved in DMSO to 1.3 mM. Thetock 39 �M recombinant HIV-1 protease solution was dilutedo a concentration of 222 nM with freshly prepared assay buffer100 mM sodium acetate, 1 M sodium chloride, 1 mg/ml BSA, 1 mMDTA, 1 mM dithiothreitol, pH 4.7). To the wells of a 96-well blackicrotiter plate, 45 �l of diluted HIV-1 PR (final concentration was
00 nM) and 5 �l of extract or control were added and incubatedt 37 ◦C for 15 min. During this incubation, the stock substrateas diluted to 16 �M by assay buffer and pre-heated to 37 ◦C.
he diluted substrate (50 �l) was added, to initiate the reactionf substrate cleavage by HIV-1 PR, and the microplate shakent 300 rpm for 1 min. The fluorescence intensity was measuredinetically every 30 s over a period of 10 min at an excitationavelength of 355 nm and an emission wavelength of 460 nm, attemperature of 37 ◦C, using a Fluoroskan Ascent FL microplate
eader (Thermolabsystems). The reaction rates were determinedy the gradient of the initial linear portions (usually the first–10 min) of the plot of RFI (relative fluorescence intensity) as aunction of time. Negative controls included were HIV-1 PR withnly assay buffer, HIV-1 PR enzyme with DMSO (2%) in assayuffer and substrate alone. Positive controls included HIV-1 PRith a general acid-protease inhibitor, acetyl pepstatin (Bachem,
witzerland) or a potent HIV PR specific inhibitor ritonavir (kindlyonated by Aspen Pharmacare, South Africa). The percentage
nhibition of HIV-1 PR was calculated as a percentage of a controlith only the solvent (2% DMSO).
.12. Statistical analysis
Significance determinations were obtained by applying a two-ailed unpaired t-test. All results with P < 0.05 were consideredignificant.
. Results
.1. Tannin dereplication
The results of tannin dereplication (relative to tannic acid) fromhe aqueous and ethanol extracts, that showed ≥50% inhibition inhe reverse transcriptase and/or protease assays, are summarisedn Table 2. The polyamide/fractionation tannin dereplication meth-ds removed tannins to between 3 and 5% (w/w) except for BEhere tannins could not be reduced to less than ∼9.5% using the
ractionation method.
.2. Viability and antiviral assays
The extracts were tested at concentrations corresponding toheir respective CC90s (i.e. yielding 90% viable cells after a 48 h expo-
able 2annin content of plant extracts relative to tannic acid standard before and afterannin dereplication.
Average tannic acid in extracts (%, w/w) Average removal oftannins (%, w/w)Initial tannic acidc After dereplicationc
annic acida 100 1.11 ± 0.02 98.89annic acidb 100 1.45 ± 1.12 98.55Aa 19.76 ± 1.75 4.80 ± 3.01 75.71Aa 7.90 ± 0.17 4.66 ± 1.97 41.01Eb 15.61 ± 0.37 9.46 ± 3.20 39.40Eb 11.76 ± 0.34 3.65 ± 0.31 68.96
a Tannins removed by polyamide column.b Tannins removed by fractionation with chloroform:methanol (4:1) and water.c Results are the average (±SD) of three separate tannin dereplication steps (each
annin dereplication method was followed by a FC reaction done in duplicate).
isks represent significant values as determined by the Student’s t-test (*P < 0.05).Extracts were tested at the following final concentrations (�g/ml): BA, 2; BE, 60; CA,0.6; CE, 0.4; HA, 190; HE, 25; LA, 150; LE, 100; TA, 200; TE, 200.
sure) on PBMCs isolated from a healthy donor (results not shown).These extract concentrations had no significant inhibitory effect onthe viability of uninfected CEM.NKR-CCR5 cells (results not shown).The effect on infected CEM.NKR-CCR5 cells can be seen in Fig. 1A.The amount of HIV-1 inhibition, as determined by viral p24 antigenlevels, can be seen in Fig. 1B. The results are represented in percentdifference for ease of comparison.
The extracts of HA, HE, LA, TA and TE stimulated growth ininfected CEM.NKR-CCR5 cells, while only extract BA had a signif-icant inhibitory effect on these cells (P < 0.05). The only significantdecrease in viral p24 levels was observed with the extract of LE,with a decrease of 33.0 ± 3.9% (P < 0.05) as compared to infectedcells without extract (Fig. 1B).
3.3. HIV-1 reverse transcriptase assay
The results for the average percentage HIV-1 RT inhibitioncan be seen in Fig. 2. The percentage inhibition of controls andextracts were calculated relative to uninhibited HIV-1 RT in 2%DMSO, after the average blank reading was subtracted from eachabsorbance.
All the extracts showed some degree of significant HIV-1 RT inhi-
bition but those which showed ≥50% HIV-1 RT inhibition were theextracts of BA, BE, HA, HE and LA. Only HA retained its activity inthe presence of BSA (55.3 ± 3.0%) and thus was the only extract tobe removed of tannins by a polyamide column. The tannin depletedHA fraction (HA-T) lost its activity to 10.1 ± 6.1% and therefore this
186 M. Klos et al. / Journal of Ethnopharmacology 124 (2009) 182–188
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 0.2 mg/ml in the finalreaction volume. Extracts showing ≥50% inhibition were tested again in the presenceof 0.2% BSA to adsorb tannins. The control, nevirapine was tested at 0.05 mg/ml intriplicate. The RT inhibition of all aqueous and ethanol crude extracts are the meanoraa
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Fig. 3. HIV-1 protease inhibition by various plant extracts. The data represent theaverage percentage HIV-1 PR inhibition (relative to an untreated control with solventonly) from the various plant extracts from two separate experiments each done intriplicate. All extracts were tested at 0.2 mg/ml, and the two controls were testedat 20 ng/ml and 2 �M for ritonavir and pepstatin, respectively. Error bars representSEM (n = 6 and 3 for whole extracts and tannin dereplicated extracts, respectively)
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f three separate experiments (n = 7). In the case of fractions with BSA added theesults are an average of two separate experiments (n = 5). The HA-T column is theverage of a single experiment performed in triplicate. Error bars represent SEM andsterisks represent P values as determined by a two-tailed unpaired t-test (*P < 0.05).
xtract was not further fractionated since its HIV-1 RT inhibitoryctivity was found to be too low.
.4. HIV-1 protease assays
The results for the average percentage HIV-1 PR inhibition cane seen in Fig. 3. The percentage inhibition was calculated relativeo uninhibited HIV-1 PR in 2% DMSO.
The crude extracts that revealed inhibition of HIV-1 PR activity≥50%) were HA, BA BE and LE. These active fractions were removed
f tannins and, for those where activity still remained, removedf polysaccharides followed by testing again for inhibition of HIV-PR. Since BSA was already present in the protease assay buffert 0.1%, tannin adsorption would already have taken place. There-ore no further BSA was added to the assay and tannin removal via
ig. 4. HIV-1 PR dose–response curves for ritonavir (A) and tannin-dereplicated extractsrror bars representing SEM.
and asterisks represent P values as determined by the two-tailed unpaired t-tests(*P < 0.05).
polyamide/fractionation methods was performed in the next stepwith extracts showing greater than 50% PR inhibition. With HA-T,activity was lost to 22.8% HIV-1 PR inhibition. This was similar tothe loss of activity of HA in the HIV-1 RT assay once tannins wereremoved, therefore showing the non-specificity of tannins and howthey can inhibit many enzymes. Activity was also lost from BA, but itstill retained some inhibitory activity at 59.1%. However, since thiswas an aqueous extract, polysaccharides were removed by ethanolprecipitation and the activity was decreased to 42.4%.
BE and LE still retained activity after tannin dereplication at 74.5and 63.9%, respectively. Therefore since these extracts may containHIV-1 PR inhibitors that are not due to tannins, but possibly dueto novel compound(s), a dose–response curve was performed with
these fractions to determine the IC50. Dose–response curves werecalculated using the GraphPad Prism® software (GraphPad Soft-ware Inc., California, USA) (Fig. 4). The dose–response curves gavesimilar IC50 values of 94.3 and 120.6 �g/ml for BE and LE extracts,respectively.of BE and LE (B). Results are the average of two separate experiments (n = 6) with
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. Discussion and conclusions
None of the current substances with antiviral activity againstIV are without toxicities and resistance and hence there is a
trong need to improve the current antiretroviral armamentarium.potential source of novel compounds for HIV is from medicinal
lants or other natural products. In order to find such poten-ial anti-HIV agents from medicinal plants, we have screenedarious medicinal plants commonly used in South African tra-itional medicine. In this paper, we report the importance ofannin/polysaccharide dereplication from plant extracts and the initro anti-HIV activity of ethanolic and aqueous extracts from fiveouth African medicinal plants.
.1. Tannin/polysaccharide dereplication
Tannins are polyphenols widely found in plants and most HIV-nhibitory effects attributed to plant extracts are due to tanninsCardellina II et al., 1993). Tannins are non-specific because theyrosslink with many proteins, inhibiting a large range of biologicalystems and enzymatic pathways (Cardellina II et al., 1993; Chungt al., 1998; Houghton and Raman, 1998; Cowan, 1999). Due tohis non-specificity, tannins are known to cause possible liver dam-ge, have carcinogenic potential and have anti-nutritional activity.n the other hand, their high potential to bind proteins has beenemonstrated in some studies to result in specific in vitro activities
ike antiviral, antimutagenic, anticarcinogenic, and immunomodu-atory, arguing for sufficient selectivity for a potential therapeuticse (Chung et al., 1998). In vivo tannins are not as effective asn in vitro assay will suggest since they will be too highly boundo serum albumin and therefore have a low bioavailability in theody (Ballick, 1994). However, tannins may be the only compoundsresent that are responsible for a reputed or observed biologicalffect in a plant extract, and have attracted considerable interests bioactive compounds in their own right. Nevertheless, if ones interested in discovering novel types of molecules, eliminationf these compounds at an early stage is desirable (Houghton andaman, 1998).
In general, the polyamide/fractionation tannin dereplicationethods removed tannins to between 3 and 5% (w/w) (Table 2).
he only exception was for BE where tannins could not be reducedo less than ∼9.5% using the fractionation method. This may be dueo the FC reagent reacting with unknown non-specific substances inhe BE extract therefore over-estimating the tannin content. Indeed,he FC phenol reagent most likely over assumed the tannin contentn all the extracts because of minor non-specific reactivity. Inter-erence of the FC reagent can occur with carbohydrates, glucose,scorbic acid, proteins and non-tannin phenols which could haveeen present in the BE extract (Julkunen-Tiitto, 1985). It must alsoe noted that some small polyphenolics may not be retained byhe polyamide column or pass into the aqueous phase with theractionation method, and therefore the possibility of some smallolyphenolic molecules in the BE extract also exists (Collins et al.,998).
Sulfated polysaccharides have consistently shown activity innti-HIV assays with aqueous extracts and it is believed to functionn destabilizing the glycoprotein complex and/or inhibiting reverseranscriptase (Greene and Peterlin, 2002). Many compounds fromhis class have already been described extensively in the literatures having anti-HIV activity (Baba et al., 1988). Like tannins, in vivoffectiveness is low since polysaccharides are poorly absorbed from
he gastrointestinal tract because of the fact that polysaccharidesreak down to monosaccharides on oral administration (Ballick,994). As for tannin dereplication, if one wishes to discover novelnhibitors from a plant extract, dereplication of polysaccharides iseeded.acology 124 (2009) 182–188 187
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 BA’s 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 and120.6 �g/ml, respectively (Fig. 4B). While these are weak inhibitoryIC50 values, compared to the ritonavir control which had an IC50of 5.3 ng/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, concentrations100–1000 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 100 �g/ml, was theonly extract to show significant HIV inhibition by reduction in HIV-1p24 levels of 33.0% (Fig. 1B). LE may possibly have caused decreasedHIV-1 p24 levels in the CEM.NKR-CCR5 cells via inhibition of HIVprotease since the concentration used was in the range of its IC50 forthis enzyme (Fig. 4B). The reduction in HIV-1 p24 levels observedwith BE was statistically not significant (P > 0.05). It should be notedthat the concentration of BE used in this experiment was 60 �g/mland was therefore lower than the IC50. Higher concentrations ofBE may decrease the p24 levels significantly, but at the same timemay be too toxic to infected CEM.NKR-CCR5 cells. The same concen-trations of BE and LE exposed to PBMCs from a healthy donor for48 h, 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 find 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 TEsignificantly 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 firsttime, that in vitro anti-HIV activity exists in these extracts. It must benoted 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 agents
so 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.
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88 M. Klos et al. / Journal of Ethno
In conclusion, this study shows the importance of includ-ng dereplication procedures for tannins/polysaccharides duringcreening processes of plant materials since this will allow forovel antiretroviral drug discovery. This study revealed that theannin-dereplicated ethanol extracts of Leonotis leonurus and Bul-ine alooides have mild HIV-1 PR inhibitory activity in vitro. Furtherurification of the tannin-dereplicated active extract fractions ofhe leaves and roots of Leonotis leonurus and Bulbine alooides,espectively, will allow more conclusive data regarding the poten-ial of a potent novel HIV inhibitor being present in these extracts.
cknowledgment
The authors wish to acknowledge the support of this studyy the National Research Foundation (NRF) of South Africa (GUN:069228).
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