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Journal of Ethnopharmacology 124 (2009) 182188

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

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

M. Klos a , M. van de Venter b, , P.J. Milne a , H.N. Traore c , D. Meyer d , V. Oosthuizen b
a

Department of Pharmacy, Nelson Mandela Metropolitan University, PO Box 77000, Port Elizabeth 6031, South Africa Department of Biochemistry and Microbiology, Nelson Mandela Metropolitan University, PO Box 77000, Port Elizabeth 6031, South Africa c Department of Biochemistry, University of Johannesburg, PO Box 524, Auckland Park, Johannesburg 2006, South Africa d Department of Biochemistry, University of Pretoria, Pretoria 0002, South Africa
b

a r t i c l e

i n f o

a b s t r a c t
Aim of the study: Five South African medicinal plants, Bulbine alooides (L.) Willd. (Asphodelaceae), Crinum macowani Baker (Amaryllidaceae), Hypoxis sobolifera var. sobolifera (Jacq.) Nel (Hypoxidaceae), Leonotis leonurus (L.) R.Br. (Lamiaceae) and Tulbaghia violacea Harv (Liliaceae) used for the treatment of various ailments, 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 infected CEM.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 < 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 inhibitory activity was lost upon dereplication for removal of non-specic tannins/polysaccharides. HIV1 PR inhibition was observed for extracts of Hypoxis sobolifera (aqueous), Bulbine alooides (aqueous and ethanol) and Leonotis leonurus (ethanol). Only ethanolic extracts of Bulbine alooides and Leonotis leonurus retained 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 greatest anti-HIV potential in this study through inhibition of HIV-1 PR. 2009 Elsevier Ireland Ltd. All rights reserved.

Article history: Received 13 January 2009 Accepted 17 April 2009 Available online 3 May 2009 Keywords: Bulbine alooides Crinum macowani HIV Hypoxis sobolifera Leonotis leonurus Tulbaghia violacea

1. Introduction It is estimated that HIV has infected 40.3 million people worldwide, with South Africa having the highest prevalence, at 5.5 million HIV-infected people (UNAIDS, 2006). There are two related but distinct types of HIV: HIV-1 and HIV-2 (Fletcher et al., 2002). HIV-1 is the most pathogenic and causes over 99% of HIV infections (Cos et al., 2004). HIV-2 is also known to cause AIDS but is much less prevalent, being present in fewer and isolated geographic locations such as Western Africa. Therefore, most research is done on HIV-1. Current antiretroviral drugs are vitally important to improve the quality and prolong the life of HIV/AIDS patients. However, these drugs have many disadvantages including resistance, toxicity, limited availability, high cost and lack of any curative effect. Thus, it is important to search for improved antiretroviral agents which can be added to or replace the current drugs in the anti-HIV arma-

Abbreviations: BSA, bovine serum albumin; FC, FolinCiocalteau; HIV-1 PR, human immunodeciency virus-1 protease; HIV-1 RT, HIV-1 reverse transcriptase; PBMC, peripheral blood mononuclear cells. Corresponding author. Tel.: +27 41 504 2813; fax: +27 41 504 2814. E-mail address: maryna.vandeventer@nmmu.ac.za (M. van de Venter). 0378-8741/$ see front matter 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.04.043

mentarium. Natural sources, particularly plants, are an excellent source of anti-HIV agents. South Africa has a rich plant biodiversity and a long tradition of medicinal use of plants with approximately 3000 species of plants used as medicines (Van Wyk and Gericke, 2000; Scott et al., 2004). Several of these plants may contain novel anti-HIV compounds. In the past decade, a substantial amount of research has been done worldwide (and a lot more is in progress) to isolate the active leads from plants for preventing transmission of HIV and treatment of AIDS based on ethnopharmacological data (Asres et al., 2001; Vermani and Garg, 2002). Screening plants based on ethnopharmacological data increases the potential of nding novel compounds due to a long history of use (Farnsworth, 1994; Fabricant and Farnsworth, 2001). Even though HIV/AIDS is a relatively new human disease, with minimal ethnobotanical treatments, logical associations of treatments for other likely viral infections (such as hepatitis B) and closely linked disease states or symptoms (wasting, diarrhoea, lymphadenopathy, skin lesions, cough, haemoptysis and genital ulcers) can increase the prospect of nding new plant leads as potential anti-HIV agents (WHO, 1989a,b; Cardellina II and Boyd, 1995; Lewis and Elvin-Lewis, 2003). For the purpose of this study, ve plants commonly used in South Africa in traditional medicines, Bulbine alooides, Crinum macowani,

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Hypoxis sobolifera, Leonotis leonurus and Tulbaghia violacea, were studied to ascertain their potential anti-HIV activity. These plants were selected based on a literature survey of their ethnomedicinal usages directly in HIV/AIDS or for symptoms/conditions closely associated with this disease. 2. Materials and methods 2.1. Plant extracts Plant material was collected in one batch from the Nelson Mandela Metropolitan area (Eastern Cape, South Africa) during the month of February 2005 and classied by Prof. E. Campbell (Botany Department, Nelson Mandela Metropolitan University). Voucher specimens were deposited in the NMMU herbarium. The fresh plant material was separated into leaves for Leonotis leonurus and underground 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 vigorously with tap water followed by soaking in ethanol (70%, v/v) for 1 min and left for approximately 30 min for the ethanol to evaporate. The plant parts were either chopped (in the case of leaves), grated (in the case of roots, bulbs or corms) or homogenized with a Waring blender at low speed for 30 s in the case of Crinum macowani bulbs. Aqueous and ethanol extracts were prepared by macerating the crushed plant material in 200 ml deionized water or 95% ethanol in the dark at 4 C or room temperature, respectively. The marc was left to soak in the menstruum for 3 days and each day the supernatant was decanted and ltered using Whatman no. 1 paper, and another volume of 200 ml fresh solvent added. In the case of the aqueous extracts, the total collected ltrate was shell-frozen and lyophilized. Ethanol extracts were dried by rotary evaporation at temperatures of 6065 C to a nal volume of approximately 1020 ml to which deionized water was added until the ethanol extract component constituted 1020% (v/v) of the nal solution volume. This solution was shell-frozen and lyophilized as per the aqueous extracts. Dried extracts were stored in the dark at 4 C in a dessicant chamber to limit chemical and/or microbiological deterioration. Before each biological (cellular and enzyme) assay, the required amount of the extracts was weighed and reconstituted in DMSO and further diluted to the desired concentration using aseptic techniques. DMSO serves to sterilize the extract and once diluted (<3%, v/v) should have no effect on the biological assays (Houghton and Raman, 1998). The nal concentration of DMSO in each assay did not signicantly affect the enzyme or cells (data not shown). The plants, together with their voucher numbers, abbreviated codes
Table 1 Plant extracts information with yields (w/w) and extracts codes. Plant Voucher specimen number PEU14795 PEU14796 PEU14840 PEU14797 PEU14799 Extraction solvent Aqueous 95% ethanol Aqueous 95% ethanol Aqueous 95% ethanol Aqueous 95% ethanol Aqueousb 95% ethanol

used in later text, plant parts used and yields from extraction are summarised in Table 1. 2.2. Removal of tannins Extracts that had 50% or more inhibitory activity in the enzyme assays had tannins removed by the methods of BSA addition (extracts denoted by the code +BSA e.g. HA+BSA) and physical tannin removal (extracts denoted by the code T e.g. HAT), as described below. 2.3. Tannin adsorption with bovine serum albumin Bovine serum albumin (BSA) was added to assay buffers to a nal assay concentration of 0.2% (w/v) to adsorb possible tannins from crude extracts that showed a 50% or more inhibitory effect in the anti-HIV assays (Devlin et al., 1991; Harnett et al., 2005). The crude extracts that still retained HIV enzyme (RT or PR) inhibitory activity 50% in the presence of BSA were then physically removed of tannins (using polyamide or solvent fractionation) to ensure near to complete removal of tannins and then tested again in the HIV enzyme assay. 2.4. Tannin dereplication from aqueous extracts using polyamide columns Solid phase extraction columns for tannin removal were prepared using modied methods of Collins et al. (1998), Cardellina II and Boyd (1995) and Houghton and Raman (1998). Two grams of polyamide DPA-6S resin (Supelco, USA) was packed into 12 ml polyethylene syringes (12 mm 150 mm) tted with frits. Prior to packing the column the polyamide resin was soaked in 12 ml of deionized water overnight to allow for swelling of the resin and to remove unreacted monomers. The polyamide resin was poured into the column and left for a few minutes to settle and to allow the column to pack rmly. The water was allowed to drain from the column until approximately 12 mm above the surface of the column bed remained. To prevent disturbing the bed surface when applying the sample and during elution, a small ring of Whatman no. 1 lter paper was placed on top of the column. The columns were assayed in triplicate to assess the efcacy of tannin removal. To each column 25 mg of aqueous extract dissolved in 1 ml of deionized water was applied to the column surface and allowed to percolate. The sample was eluted in three fractions: 9 ml water, 10 ml 50% (v/v) aqueous methanol and 10 ml methanol. The ow rate was adjusted to approximately 1 ml/min. These fractions were dried by lyophilization and assayed by the FolinCiocalteau

Code BA BE CA CE HA HE LA LE TA TE

Plant part used

Initial weight of fresh plant extract (g) 158.69 154.45 859.47 789.16 123.65 125.93 99.49 111.90 81.90 48.63

Yield (%, w/w)a 3.55 4.17 3.09 2.09 5.62 2.38 1.21 3.42 1.68 2.71

Bulbine alooides Crinum macowani Hypoxis sobolifera

Roots Bulbs Corms Leaves Bulbs

Leonotis leonurus Tulbaghia violacea

a

Yield represents the percentage recovery of dried extract per weight as compared with the original fresh plant material. For TA extracts only an 1 day maceration extraction was used to avoid degradation of Tulbaghia violacea compounds in aqueous solution (Kubec et al., 2002; Motsei et al., 2003).
b

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(FC) reaction to determine the percentage of tannin removed (relative to equivalent tannic acid units) (Julkunen-Tiitto, 1985). 2.5. Tannin dereplication from ethanol extracts using solvent fractionation Removal of tannins from ethanol extracts was performed using a fractionation method from Houghton and Raman (1998). Dried ethanol extracts (50 mg) were dissolved in 2 ml of methanol. Chloroform (8 ml) was slowly added and the solution mixed well, to form a chloroform:methanol (4:1) mixture. This solution was transferred to a separating funnel and an equal volume of deionized water (10 ml) was slowly added. The separating funnel was slowly inverted multiple times to allow for the polyphenols to transfer to the aqueous upper phase. The organic lower phase was separated from the upper aqueous tannin-containing layer and washed with an equal volume of 1% (w/v) NaCl in water. The organic phase, containing tannin-free extract, was dried under vacuum at room temperature and assayed for tannins with the FC reaction. 2.6. Quantitative total polyphenolic determination by the FolinCiocalteau phenol reaction In order to determine the effectiveness of tannin removal from the extracts a modied method of Julkunen-Tiitto (1985) was used. The amount of tannin removed from the extract was estimated by using a tannic acid standard curve which gave a linear relationship between absorptivity and concentration. The tannic acid standard curve remained linear below 800 g/ml tannic acid. For each assay a new tannic acid standard curve was determined to ensure accuracy. Tannic acid (Sigma, Missouri, USA) was diluted in 50% (v/v) DMSO and four serial twofold dilutions were made ranging from 800 to 100 g/ml. Dried extracts before tannin removal and dried extracts after tannin removal were dissolved in 50% DMSO to give 800 g/ml. To a 2 ml reaction tube, 100 l of the respective tannic acid standard solution or extract solution was added followed by the addition of 200 l of FC phenol reagent (Sigma, Missouri, USA). Immediately, 700 l of a 20% (w/v) sodium carbonate solution was added and the mixture made to 2 ml with water, followed by shaking. Absorption of the solution was read at 690 nm. Before the samples were measured spectrophotometrically they were centrifuged, if necessary, because of precipitate formation. 2.7. Removal of sulfated polysaccharides Aqueous tannin-dereplicated extracts, positive for inhibitory activity against HIV-1 enzymes, were subjected to a 50% ethanol precipitation. After ethanol addition, extracts were stirred at 4 C for 20 min, ltered and dried as described above (Houghton and Raman, 1998). Extracts that had the sulfated polysaccharides removed had the addition of PS added to the extract code, e.g. BATPS. 2.8. Viability assay

Kit II (XTT) (Roche Diagnostics, Mannheim, Germany) was used for viability determination of CEM.NKR -CCR5 cells. CEM.NKR -CCR5 cells were maintained and cultured in RPMI 1640 containing 2 mM l-glutamine (Sigma, Missouri, USA). Supplements added included heat inactivated (56 C, 30 min) foetal calf serum (10%, v/v) and antibiotics that consisted of penicillin G (10 mg/ml), streptomycin sulphate (10 mg/ml), fungizone (25 g/ml) and gentamicin sulfate (1 ng/ml). Cells were cultured at 37 C in a 5% CO2 humidied atmosphere. Acutely HIV-infected CEM.NKR -CCR5 cells were counted using trypan blue and seeded at 1 106 cells/ml. To each well of a 96-well plate, 100 l of this cell suspension was added giving 5 105 cells/ml in the nal 200 l well volume. Extract solutions were prepared by rst mixing with DMSO to surface sterilize the powder, followed by dilution with complete medium to a nal concentration of 2 mg/ml. Final well concentrations tested for each extract corresponded to the CC90 concentrations seen in uninfected PBMCs from a 23-year-old male donor (data not shown). Control wells included a negative control (cells and medium only) and extract/XTT blank controls for each extract (extract and medium only). Test wells included extract, cells and medium. Plates were incubated at 30 C for 7 days. Upon completion of the incubation, 50 l of a previously prepared XTT working solution containing N-methyl dibenzopyrazine methyl sulfate (PMS) and XTT (1:50) was added to all the wells. Plates were incubated for 4 h at 30 C and read at 450 nm (reference 690 nm). The same procedure was followed for testing the viability of extracts on 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 used to detect and quantify HIV-1 p24 core protein. At the end of the 7 days incubation, culture supernatant (100 l) from the HIVinfected CEM.NKR -CCR5 cultures was transferred to the murine monoclonal-coated 96-well plate for the p24 assay. The protocol was followed as described by the manufacturer, with absorbance measured at 450 nm. 2.10. HIV-1 reverse transcriptase assay The effect of the crude extracts on reverse transcription was tested using a non-radioactive HIV-RT colorimetric ELISA kit from Roche Diagnostics, Germany. The protocol outlined in the kit was followed, under nuclease-free conditions, using 2 ng of enzyme in a well and incubating the reaction for 2 h at 37 C. Negative controls for the assay included HIV-1 RT with only lysis buffer, HIV-1 RT with only solvent (2% DMSO) in lysis buffer, and a blank with just ABTS. The positive control used was nevirapine (kindly donated by Aspen Pharmacare, South Africa), a reverse transcriptase inhibitor used commonly in clinical practice. The HIV-RT inhibition of the plant extracts were measured as a percentage of the inhibition that occurred with HIV-1 RT in the presence of no inhibitor in the same solvent (2% DMSO) as the extracts. 2.11. HIV-1 protease assay

CEM.NKR -CCR5 cells are a human T-lymphoblastic cell line obtained through the AIDS Research and Reference program, Division of AIDS, National Institute of Allergy and Infectious Diseases (NIAID), National Institute of Health (NIH) (MD USA) from Dr Alexandra Trkola. These cells were transformed with a retroviral vector to express human CCR5 and show resistance to natural killer cell-mediated lysis and do not secrete infectious virus (Trkola et al., 1999). HIV-1 clade C, the most prevalent subtype within the region of southern Africa, preferentially uses the CCR5 co-receptor for infection (Hauesser and Mulnger, 2001). The Cell Proliferation

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

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was adapted from the method of Matayoshi et al. (1990). The uorogenic substrate was dissolved in DMSO to 1.3 mM. The stock 39 M recombinant HIV-1 protease solution was diluted to a concentration of 222 nM with freshly prepared assay buffer (100 mM sodium acetate, 1 M sodium chloride, 1 mg/ml BSA, 1 mM EDTA, 1 mM dithiothreitol, pH 4.7). To the wells of a 96-well black microtiter plate, 45 l of diluted HIV-1 PR (nal concentration was 100 nM) and 5 l of extract or control were added and incubated at 37 C for 15 min. During this incubation, the stock substrate was diluted to 16 M by assay buffer and pre-heated to 37 C. The diluted substrate (50 l) was added, to initiate the reaction of substrate cleavage by HIV-1 PR, and the microplate shaken at 300 rpm for 1 min. The uorescence intensity was measured kinetically every 30 s over a period of 10 min at an excitation wavelength of 355 nm and an emission wavelength of 460 nm, at a temperature of 37 C, using a Fluoroskan Ascent FL microplate reader (Thermolabsystems). The reaction rates were determined by the gradient of the initial linear portions (usually the rst 510 min) of the plot of RFI (relative uorescence intensity) as a function of time. Negative controls included were HIV-1 PR with only assay buffer, HIV-1 PR enzyme with DMSO (2%) in assay buffer and substrate alone. Positive controls included HIV-1 PR with a general acid-protease inhibitor, acetyl pepstatin (Bachem, Switzerland) or a potent HIV PR specic inhibitor ritonavir (kindly donated by Aspen Pharmacare, South Africa). The percentage inhibition of HIV-1 PR was calculated as a percentage of a control with only the solvent (2% DMSO). 2.12. Statistical analysis Signicance determinations were obtained by applying a twotailed unpaired t-test. All results with P < 0.05 were considered signicant. 3. Results 3.1. Tannin dereplication The results of tannin dereplication (relative to tannic acid) from the aqueous and ethanol extracts, that showed 50% inhibition in the reverse transcriptase and/or protease assays, are summarised in Table 2. The polyamide/fractionation tannin dereplication methods removed tannins to between 3 and 5% (w/w) except for BE where tannins could not be reduced to less than 9.5% using the fractionation method. 3.2. Viability and antiviral assays The extracts were tested at concentrations corresponding to their respective CC90 s (i.e. yielding 90% viable cells after a 48 h expoTable 2 Tannin content of plant extracts relative to tannic acid standard before and after tannin dereplication. Average tannic acid in extracts (%, w/w) Initial tannic acidc Tannic acida Tannic acidb HAa BAa BEb LEb
a b

Fig. 1. Effects of extracts on the viability of HIV-1 infected (A) CEM.NKR -CCR5 cells and on HIV-1 p24 antigen levels (B). All results are the percent difference relative to untreated cells. Results in (A) are from XTT viability determinations. For (B), supernatants from the infected cells were tested for HIV-1 p24 levels using an ELISA based assay. Error bars represent SEM and n = 10 and 3 for (A) and (B), respectively. Asterisks represent signicant values as determined by the Students t-test (*P < 0.05). Extracts were tested at the following nal 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 signicant inhibitory effect on the 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 antigen levels, can be seen in Fig. 1B. The results are represented in percent difference for ease of comparison. The extracts of HA, HE, LA, TA and TE stimulated growth in infected CEM.NKR -CCR5 cells, while only extract BA had a significant inhibitory effect on these cells (P < 0.05). The only signicant decrease 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 infected cells without extract (Fig. 1B). 3.3. HIV-1 reverse transcriptase assay The results for the average percentage HIV-1 RT inhibition can be seen in Fig. 2. The percentage inhibition of controls and extracts were calculated relative to uninhibited HIV-1 RT in 2% DMSO, after the average blank reading was subtracted from each absorbance. All the extracts showed some degree of signicant HIV-1 RT inhibition but those which showed 50% HIV-1 RT inhibition were the extracts of BA, BE, HA, HE and LA. Only HA retained its activity in the presence of BSA (55.3 3.0%) and thus was the only extract to be removed of tannins by a polyamide column. The tannin depleted HA fraction (HA-T) lost its activity to 10.1 6.1% and therefore this

After dereplicationc 1.11 1.45 4.80 4.66 9.46 3.65 0.02 1.12 3.01 1.97 3.20 0.31

Average removal of tannins (%, w/w) 98.89 98.55 75.71 41.01 39.40 68.96

100 100 19.76 1.75 7.90 0.17 15.61 0.37 11.76 0.34

Tannins removed by polyamide column. Tannins removed by fractionation with chloroform:methanol (4:1) and water. c Results are the average (SD) of three separate tannin dereplication steps (each tannin dereplication method was followed by a FC reaction done in duplicate).

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Fig. 2. HIV-1 RT inhibition by various plant extracts. The +BSA code represents fractions with BSA added and the T represents fractions with tannins removed. The data represent the mean HIV-1 RT inhibition (relative to an untreated control with solvent only) of various plant extracts, each tested at 0.2 mg/ml in the nal reaction volume. Extracts showing 50% inhibition were tested again in the presence of 0.2% BSA to adsorb tannins. The control, nevirapine was tested at 0.05 mg/ml in triplicate. The RT inhibition of all aqueous and ethanol crude extracts are the mean of three separate experiments (n = 7). In the case of fractions with BSA added the results are an average of two separate experiments (n = 5). The HA-T column is the average of a single experiment performed in triplicate. Error bars represent SEM and asterisks represent P values as determined by a two-tailed unpaired t-test (*P < 0.05).

Fig. 3. HIV-1 protease inhibition by various plant extracts. The data represent the average percentage HIV-1 PR inhibition (relative to an untreated control with solvent only) from the various plant extracts from two separate experiments each done in triplicate. All extracts were tested at 0.2 mg/ml, and the two controls were tested at 20 ng/ml and 2 M for ritonavir and pepstatin, respectively. Error bars represent SEM (n = 6 and 3 for whole extracts and tannin dereplicated extracts, respectively) and asterisks represent P values as determined by the two-tailed unpaired t-tests (*P < 0.05).

extract was not further fractionated since its HIV-1 RT inhibitory activity was found to be too low. 3.4. HIV-1 protease assays The results for the average percentage HIV-1 PR inhibition can be seen in Fig. 3. The percentage inhibition was calculated relative to 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 of tannins and, for those where activity still remained, removed of polysaccharides followed by testing again for inhibition of HIV1 PR. Since BSA was already present in the protease assay buffer at 0.1%, tannin adsorption would already have taken place. Therefore no further BSA was added to the assay and tannin removal via

polyamide/fractionation methods was performed in the next step with extracts showing greater than 50% PR inhibition. With HA-T, activity was lost to 22.8% HIV-1 PR inhibition. This was similar to the loss of activity of HA in the HIV-1 RT assay once tannins were removed, therefore showing the non-specicity of tannins and how they can inhibit many enzymes. Activity was also lost from BA, but it still retained some inhibitory activity at 59.1%. However, since this was an aqueous extract, polysaccharides were removed by ethanol precipitation and the activity was decreased to 42.4%. BE and LE still retained activity after tannin dereplication at 74.5 and 63.9%, respectively. Therefore since these extracts may contain HIV-1 PR inhibitors that are not due to tannins, but possibly due to novel compound(s), a doseresponse curve was performed with these fractions to determine the IC50 . Doseresponse curves were calculated using the GraphPad Prism software (GraphPad Software Inc., California, USA) (Fig. 4). The doseresponse curves gave similar IC50 values of 94.3 and 120.6 g/ml for BE and LE extracts, respectively.

Fig. 4. HIV-1 PR doseresponse curves for ritonavir (A) and tannin-dereplicated extracts of BE and LE (B). Results are the average of two separate experiments (n = 6) with error bars representing SEM.

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4. Discussion and conclusions None of the current substances with antiviral activity against HIV are without toxicities and resistance and hence there is a strong need to improve the current antiretroviral armamentarium. A potential source of novel compounds for HIV is from medicinal plants or other natural products. In order to nd such potential anti-HIV agents from medicinal plants, we have screened various medicinal plants commonly used in South African traditional medicine. In this paper, we report the importance of tannin/polysaccharide dereplication from plant extracts and the in vitro anti-HIV activity of ethanolic and aqueous extracts from ve South African medicinal plants. 4.1. Tannin/polysaccharide dereplication Tannins are polyphenols widely found in plants and most HIVinhibitory effects attributed to plant extracts are due to tannins (Cardellina II et al., 1993). Tannins are non-specic because they crosslink with many proteins, inhibiting a large range of biological systems and enzymatic pathways (Cardellina II et al., 1993; Chung et al., 1998; Houghton and Raman, 1998; Cowan, 1999). Due to this non-specicity, tannins are known to cause possible liver damage, have carcinogenic potential and have anti-nutritional activity. On the other hand, their high potential to bind proteins has been demonstrated in some studies to result in specic in vitro activities like antiviral, antimutagenic, anticarcinogenic, and immunomodulatory, arguing for sufcient selectivity for a potential therapeutic use (Chung et al., 1998). In vivo tannins are not as effective as an in vitro assay will suggest since they will be too highly bound to serum albumin and therefore have a low bioavailability in the body (Ballick, 1994). However, tannins may be the only compounds present that are responsible for a reputed or observed biological effect in a plant extract, and have attracted considerable interest as bioactive compounds in their own right. Nevertheless, if one is interested in discovering novel types of molecules, elimination of these compounds at an early stage is desirable (Houghton and Raman, 1998). In general, the polyamide/fractionation tannin dereplication methods removed tannins to between 3 and 5% (w/w) (Table 2). The only exception was for BE where tannins could not be reduced to less than 9.5% using the fractionation method. This may be due to the FC reagent reacting with unknown non-specic substances in the BE extract therefore over-estimating the tannin content. Indeed, the FC phenol reagent most likely over assumed the tannin content in all the extracts because of minor non-specic reactivity. Interference of the FC reagent can occur with carbohydrates, glucose, ascorbic acid, proteins and non-tannin phenols which could have been present in the BE extract (Julkunen-Tiitto, 1985). It must also be noted that some small polyphenolics may not be retained by the polyamide column or pass into the aqueous phase with the fractionation method, and therefore the possibility of some small polyphenolic molecules in the BE extract also exists (Collins et al., 1998). Sulfated polysaccharides have consistently shown activity in anti-HIV assays with aqueous extracts and it is believed to function in destabilizing the glycoprotein complex and/or inhibiting reverse transcriptase (Greene and Peterlin, 2002). Many compounds from this class have already been described extensively in the literature as having anti-HIV activity (Baba et al., 1988). Like tannins, in vivo effectiveness is low since polysaccharides are poorly absorbed from the gastrointestinal tract because of the fact that polysaccharides break down to monosaccharides on oral administration (Ballick, 1994). As for tannin dereplication, if one wishes to discover novel inhibitors from a plant extract, dereplication of polysaccharides is needed.

Polysaccharides were only removed from the BA-T extract as this was the only aqueous tannin-dereplicated extract that showed inhibitory activity of HIV-1 protease (Fig. 3). The activity of the extract was then decreased after polysaccharide removal. This loss in activity after polysaccharides were removed indicates that some polysaccharides (together with tannins previously removed) were most likely responsible for most of BAs inhibitory activity. 4.2. Anti-HIV activity The results described in this study indicate that the tannindereplicated ethanol extracts of Bulbine alooides and Leonotis leonurus possess anti-HIV properties of possible therapeutic interest, through HIV-1 PR inhibition, with an IC50 of 94.3 and 120.6 g/ml, respectively (Fig. 4B). While these are weak inhibitory IC50 values, compared to the ritonavir control which had an IC50 of 5.3 ng/ml (Fig. 4A), it must be remembered that the crude plant extracts are highly impure compared to the pure ritonavir. It may be that the amount of active compound extracted is a very small percentage of the extract. Therefore, assuming that there is a single inhibitor responsible for HIV-1 PR inhibition, concentrations 1001000 times less than the IC50 seen for the extracts may be the actual inhibitory concentration (Houghton and Raman, 1998). The crude LE extract, at a concentration of 100 g/ml, was the only extract to show signicant HIV inhibition by reduction in HIV-1 p24 levels of 33.0% (Fig. 1B). LE may possibly have caused decreased HIV-1 p24 levels in the CEM.NKR -CCR5 cells via inhibition of HIV protease since the concentration used was in the range of its IC50 for this enzyme (Fig. 4B). The reduction in HIV-1 p24 levels observed with BE was statistically not signicant (P > 0.05). It should be noted that the concentration of BE used in this experiment was 60 g/ml and was therefore lower than the IC50 . Higher concentrations of BE may decrease the p24 levels signicantly, but at the same time may be too toxic to infected CEM.NKR -CCR5 cells. The same concentrations of BE and LE exposed to PBMCs from a healthy donor for 48 h, were found to be non-toxic (data not shown). Further studies, at a greater range of concentrations, may be needed on these two extracts to try and nd the concentration at which cytotoxicity is lowest and HIV-1 inhibition is highest in CEM.NKR -CCR5 cells. It must be noted that the fractions of HA, HE, LA, TA and TE signicantly 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 due to viral inhibition, as p24 viral antigen levels did not correspondingly decrease (Fig. 1B). In the HIV-1 RT assay, the extracts of BA, BE, HA, HE and LA showed 50% HIV-1 RT inhibition, and thus were re-tested in the presence of 0.2% BSA (Fig. 2). Only the extract of HA retained its activity in the presence of BSA (55.3%). However, after tannin dereplication using polyamide columns, inhibition of HA was lost to 10.1% (HA-T). Therefore, further consideration of this extract was not necessary since its HIV-1 RT inhibitory activity could be attributed to the non-selective tannin compounds. No known HIV inhibitory compounds exist in the extracts of Bulbine alooides or Leonotis leonurus. This study reports, for the rst time, that in vitro anti-HIV activity exists in these extracts. It must be noted that Leonotis leonurus is used traditionally for hepatitis treatment and it has been found that plants used for hepatitis treatment often exhibit one or more anti-HIV activities. This includes some species 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 PR inhibitor(s) from BE and LE than the aqueous extraction (Hoggard and Owen, 2003). Therefore with a more lipid soluble extract, such as chloroform, more of the unknown protease inhibitor(s) may be extracted.

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188 M. Klos et al. / Journal of Ethnopharmacology 124 (2009) 182188 Fabricant, D.S., Farnsworth, N.R., 2001. The value of plants used in traditional medicine for drug discovery. Environmental Health Perspectives 109, 6975. Farnsworth, N.R., 1994. Ethnopharmacology and drug development. Ethnobotany, drug development and biodiversity conservation-exploring the linkages. In: Ethnobotany and the Search for New Drugs, Ciba Foundation Symposium, vol. 185. John Wiley and Sons, Chichester, pp. 4259. Fletcher, C.V., Kakuda, T.N., Collier, A.C., 2002. In: Dipiro, J.T., Talbert, R.L., Yee, G.C., Matzke, G.R., Wells, B.G., Posey, L.M. (Eds.), PharmacotherapyA Pathophysiologic Approach, 5th edition. Mcgraw-Hill Medical Publishing Division, United States of America, pp. 21512174. Greene, W.C., Peterlin, B.M., 2002. Charting HIVs remarkable voyage through the cell: basic science as a passport to future therapy. Nature Medicine 8, 673680. Harnett, S.M., Oosthuizen, V., van de Venter, M., 2005. Anti-HIV activities of organic and aqueous extracts of Sutherlandia frutescens and Lobostemon trigonus. Journal of Ethnopharmacology 96, 113119. Hauesser, D.P., Mulnger, L., 2001. HIV in Southern Africa: a need for the development of a vaccine with cross-clade activity. Journal of Young Investigators 4, 6170. Hoggard, P.G., Owen, A., 2003. The mechanisms that control intracellular penetration of the HIV protease inhibitors. Journal of Antimicrobial Chemotherapy 51, 493496. Houghton, P.J., Raman, A., 1998. Laboratory Handbook for the Fractionation of Natural Extracts. Chapman and Hall, London/Weinheim/New York/Tokyo/Melbourne/Madras. Julkunen-Tiitto, R., 1985. Phenolic constituents in the leaves of northern willows: methods for the analysis of certain phenolics. Journal of Agricultural and Food Chemistry 22, 213217. Kubec, R., Velisek, J., Musah, R.A., 2002. The amino acid precursors and odor formation in society garlic (Tulbaghia violacea). Phytochemistry 60, 2125. Lewis, W.H., Elvin-Lewis, M.P.F., 2003. Medical Botany: Plants affecting Human Health, 2nd edition. John Wiley and Sons, New Jersey, pp. 97129. Matayoshi, E.D., Wang, G.T., Krafft, G.A., Erickson, J., 1990. Novel uorogenic substrates for assaying retroviral proteases by resonance energy transfer. Science 247, 954958. Motsei, M.L., Lindsey, K.L., van Staden, J., Jger, A.K., 2003. Screening of traditionally used South African Plants for antifungal activity against Candida albicans. Journal of Ethnopharmacology 86, 235241. Scott, G., Springeld, E.P., Coldrey, N., 2004. A pharmacognostical study of 26 South African plant species used as traditional medicines. Pharmaceutical Biology 42, 186213. Trkola, A., Matthews, J., Gordon, C., Ketas, T., Moore, J.P., 1999. A cell line-based neutralization assay for the primary human immunodeciency virus type 1 isolates that use either CCR5 or CXCR4 coreceptor. Journal of Virology 73, 89668974. United Nations Programme on HIV/AIDS (UNAIDS), 2006. Report on the global AIDS epidemic, Available online: http://www.unaids.org/en/HIV data/ 2006GlobalReport/default.asp. Van Wyk, B., Gericke, N. (Eds.), 2000. Peoples Plants: A Guide to Useful Plants of South Africa, rst ed. Briza Publications, South Africa, pp. 1012. Vermani, K., Garg, S., 2002. Herbal medicines for sexually transmitted diseases and AIDS. Journal of Ethnopharmacology 80, 4966. World Health Organisation (WHO), 1989a. In vitro screening of traditional medicines for anti-HIV activity: memorandum from a WHO meeting. Bulletin of the World Health Organization 87, 613618. World Health Organisation, Geneva (WHO), 1989b. Report of a WHO Informal Consultation on Traditional Medicine and AIDS: In Vitro Screening for Anti-HIV Activity. Global Programme on AIDS and Traditional Medicine Programme, pp. 117.

In conclusion, this study shows the importance of including dereplication procedures for tannins/polysaccharides during screening processes of plant materials since this will allow for novel antiretroviral drug discovery. This study revealed that the tannin-dereplicated ethanol extracts of Leonotis leonurus and Bulbine alooides have mild HIV-1 PR inhibitory activity in vitro. Further purication of the tannin-dereplicated active extract fractions of the leaves and roots of Leonotis leonurus and Bulbine alooides, respectively, will allow more conclusive data regarding the potential of a potent novel HIV inhibitor being present in these extracts. Acknowledgment The authors wish to acknowledge the support of this study by the National Research Foundation (NRF) of South Africa (GUN: 2069228). References
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