Biological activity of plant extracts and isolated compounds from Alchornea laxi ﬂ ora : Anti-HIV, antibacterial and cytotoxicity evaluation

This study was designed to assess the cytotoxicity, anti-HIV and antibacterial ef ﬁ cacy of various solvent extracts of roots, stem and leaves of Alchornea laxi ﬂ ora , as well as ﬁ ve compounds isolated from its methanolic stem extract viz.; ellagic acid ( 1 ); 3- O -methyl-ellagic acid ( 2 ), 3- O - β - D -glucopyranosyl- β -sitosterol ( 3 ), 3- O -acetyl- oleanolic acid ( 4 ) and 3- O -acetyl-ursolic acid ( 5 ). The tested crude extracts were prepared from several solvent polarities including: hexane (Hex), chloroform (CHCl 3 ), ethyl acetate (EtOAc), ethanol (EtOH), methanol (MeOH) and water (H 2 O). The anti-HIV properties were assessed on HIV-1 subtype C integrase while the cyto-toxicitywastestedagainstHelacells.Theantibacterialactivitywasstudiedonapanelofpathogensincludinggas- trointestinal, skin, respiratory and urinary-tract infection causing Gram positive bacteria viz.; Bacillus cereus (ATCC 11778), Enterococcus faecalis (ATCC 29212) , Staphylococcus aureus (ATCC 25923) and Staphylococcus saprophyticus (ATCC 15305)] and Gram-negative bacteria, i.e., Escherichia coli (ATCC 25922) , Klebsiella pneumoniae (ATCC 13883) , Moraxella catarrhalis (ATCC 23246). All the tested samples were determined to be non-toxicduetothelowinhibitionsobserved.Themostpotentanti-HIVactivitywasobservedforthemethanolic extractof A.laxi ﬂ ora root (ALR4)with anIC 50 valueof0.21 ng/ml, which was more activethan chicoric acidused as reference drug (6.82 nM). Roots, stem and leaves of A. laxi ﬂ ora extracts exhibited antibacterial activities against most ofthe Gram-positive bacteria with the minimum inhibitory concentrations(MIC) ranging between 50 and 63 μ g/ml. Compounds 1 – 5 displayed antibacterial activities against S. saprophyticus with MIC values as low as 4 μ g/ml. The results inferred from this study demonstrate the potential of A. laxi ﬂ ora root as a source for newanti-HIVdrugsandscienti ﬁ callyvalidatethetraditionaluseof A.laxi ﬂ ora inthetreatmentofgastrointestinal, skin, respiratory and urinary tract related infections. These results reaf ﬁ rm the ethnopharmacological


Introduction
The human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) was first reported in 1981 in the United States of America (USA) (Zhang et al., 2017). It has since become epidemic worldwide. More than 78 million people have suffered from HIV infection and about half of this number have died. HIV/AIDS reportedly remains the leading cause of death in Africa (Global HIV Statistics, 2017). The advances in treatment coupled to multiple campaigns of awareness to prevent HIV/AIDS have significantly slowed down the progression of the disease. Importantly, the antiretroviral therapy significantly impacted the progression of the virus, resulting in the decrease of HIV-related deaths worldwide (Zhang et al., 2017). Additionally, a decrease by 19% of new infections was observed between 2005 and 2014 (Zhang et al., 2017). Due to the emergence of drug-resistance developed by many HIV/AIDS patients and the associated side effects such as a weakened immune system (this exposes the affected individuals to other microbial, viral and parasitic infections), there is a permanent need for the discovery of new treatments for HIV infection. Natural products have been an important source for new drug discovery leads and have led to the development of drugs approved by the USA Food and Drug Administration (FDA) for treatment of several diseases (Newman and Cragg, 2016).
Alchornea laxiflora (Benth.) Pax & K. Hoffm. (Euphorbiaceae) is a shrub widely spread over large parts of Africa. It occurs from eastern Nigeria to Ethiopia and South to the Democratic Republic of Congo (DRC), throughout East Africa to Zimbabwe, Mozambique, north-eastern parts of South Africa as well as Swaziland. It can grow as high as 6 m (Dalziel, 1937;Schmelzer, 2007). Alchornea laxiflora is widely used as a folk medicine in many parts of Africa, as a remedy for numerous diseases ranging from inflammation to heart diseases (Burkill, 1998;Dan et al., 2004;Kayode and Omotoyinbo, 2008). In Cameroon, uses of Alchornea species have been described in the traditional pharmacopoeia to treat a range of microbial infections and parasitic diseases (Adjanohoun et al., 1996;Jiofack et al., 2009). However, A. laxiflora leaves in particular, have been reported to be effective in the treatment of kidney, urinary bladder inflammation and related infectious diseases (Olatunde Farombi et al., 2003;Sofowora, 2008). Furthermore, in the upper Nyong valley forest in Cameroon, A. laxiflora leaves are used to treat dysentery, haemorrhoids and urinary-tract infections (Jiofack et al., 2009) and in Ndop Central sub-division in Cameroon, a decoction (locally known as Mechango or Bambalang) is taken orally for post-partum pain and inflammation (Focho et al., 2009). In Nigeria, A. laxiflora is reportedly used as a topical application to alleviate teething problems in children (Olatunde Farombi et al., 2003). Phytopharmacological studies gleaned from various investigations include the anti-inflammatory (Ogundipe, 1999), hyposensitive and antihistaminic (Kayode and Omotoyinbo, 2008;Sofowora, 2008) properties of leaf extracts.

Plant materials
The plant material was collected in bulk from uncultivated farmland on the Elounden Mount in Yaoundé, Cameroon, in January 2010. The species was authenticated by Mr. Victor Nana, a botanist from the National Herbarium of Cameroon in Yaoundé, where a voucher specimen (N°45363/HNC) was deposited.

Extraction of plant materials for biological assessment
All the collected plant materials (leaf, root and stem) were dried individually at ambient temperature and ground into fine powder. Each of the powdered plant materials was soaked in the solvent (10 g plant material/ 50 ml solvent) in an air-tight container at ambient temperature for 72 h. Selected solvents -hexane, chloroform, ethyl acetate, methanol, ethanol and water (in that order) were used for successive extraction of each sample, respectively.

HIV-1 integrase strand transfer reaction assay
The HIV-1 subtype C integrase (CIN) strand transfer inhibition assay was adapted from previously described method (Grobler et al., 2002). Briefly, 20 nM double-stranded biotinylated donor DNA (5′-5 Biotin TEG/ACCCTTTTAGTCAGTGTGGAAAATCTCTAGCA-3′ annealed to 5′ ACTGCTAGAGATTTTCCACACTGACTAAAAG-3′) was immobilised in wells of streptavidin coated 96-well microtiter plates (R&D Systems, USA). Following incubation at room temperature for 40 min and a stringent wash step, 5 μg/ml purified recombinant HIV-1 CIN in buffer 1 (50 mm NaCl, 25 mM Hepes, 25 mM MnCl 2 , 5 mM β-mercaptoethanol, 50 μg/ml BSA, pH 7.5) was added to individual wells. Test samples and chicoric acid were added to individual wells to a final concentration of 20 μM (pure compounds and chicoric acid) and 50 mg/ml (crude extracts). Recombinant HIV-1 subtype C IN was assembled onto the preprocessed donor DNA through incubation for 45 min at room temperature. Strand transfer reaction was initiated through the addition of 10 nM (final concentration) double-stranded FITC-labelled target DNA (5′-TGACCAAGGGCTAATTCACT/36-FAM/− 3′ annealed to 5′-AGTGAATTAGCCCTTGGTCA −/36-FAM/− 3′) in integrase buffer 2 (same as buffer 1, except 25 mm MnCl 2 replaced with 2.5 mm MgCl 2 ). After an incubation period of 60 min at 37°C, the plates were washed using PBS containing 0.05% Tween 20 and 0.01% BSA, followed by the addition of peroxidase-conjugated sheep anti-FITC antibody (Thermo Scientific, USA) diluted 1:1000 in the same PBS buffer. Finally, the plates were washed and peroxidase substrate (Sure Blue ReserveTM, KPL, USA) was added to allow for detection at 620 nm using a Synergy MX (BioTek®) plate reader. Absorbance values were converted to percentage enzyme activity relative to the readings obtained from control wells (enzyme without inhibitor).

Cytotoxic activity
This was adopted from our previously described method (Mbosso Teinkela et al., 2018). Briefly, to assess the overt cytotoxicity, samples were incubated at 25 μg/ml for extracts and 20 μM for pure compounds in 96-well plates containing HeLa cells (human cervix adenocarcinoma), maintained in a culture medium made of Dulbecco's Modified Eagle's Medium (DMEM) with 5 mM L-glutamine (Lonza) and supplemented with 10% fetal bovine serum (FBS) and antibiotics (penicillin/ streptomycin/fungizone -PSF) for 24 h. The number of cells surviving drug exposure were counted using the resazurin based reagent and resorufin fluorescence was quantified (Excitation560/Emission590) in a multiwell plate reader.

Single concentration screening
The percentage of cell viability was calculated at a fixed concentration of 20 μM for pure isolated compounds or 25 μg/ml for natural plant extracts. Experiments were performed in triplicate wells, and the standard deviation (SD) was derived. For comparative purposes, Emetine (which induced cell apoptosis) was used as a positive control drug standard at a concentration of 10 μM. Samples were tested for HIV-1 integrase at a concentration of 20 μM in the case of pure compounds and 25 μg/ml for crude extracts and Chicoric acid was used as positive control for HIV-1 integrase at a concentration of 20 μM.

Dose response
The IC 50 (50% inhibitory concentration) values of tested extract/ compound exhibiting a low percentage viability or low integrase activity were determined from the resulting dose-response curve by nonlinear regression using Prism 5 program (Version 5.02, Graph Pad Software, Inc). IC50 values for cytotoxicity were not determined due to the low inhibition observed by the preliminary single concentration screening.

Antibacterial activity
The antibacterial activity of the crude extracts and pure compounds was evaluated by the micro-dilution assay (Siwe Noundou et al., 2016) against four Gram-positive bacteria i.e. B. cereus ATCC 11778, E. faecalis ATCC 29212, S. aureus ATCC 25923 and S. saprophyticus ATCC 15305, as well as four Gram-negative bacterial strains i.e. E. coli ATCC 25922, K. pneumoniae ATCC 13883, M. catarrhalis ATCC 23246 and P. mirabilis ATCC 43071. All bacterial strains were confirmed pure from stock cultures and maintained in the Pharmaceutical Microbiology Laboratories at the University of Witwatersrand, Johannesburg, South Africa. The stock solutions of samples (32 mg/ml for crude extracts and 1 mg/ml for isolated compounds) were prepared as follows: Acetone was used for the extracts i.e. Hex, CHCl 3 and EtOAc and compounds 4 and 5 while a solution of DMSO/H 2 O (5:95, v/v) was used for EtOH and MeOH extracts and compounds 1, 2 and 3. The aqueous extracts were used as are. All the above stock solutions were placed in duplicate (using adjacent wells) and further serially diluted (2-fold dilution) with sterile water in a 96-well microtitre plate to obtain a 1 mg/ml and 0.031 mg/ml solution in the final row for the extracts and isolated compounds respectively. To the above dilutions in the well plates, equal volumes (100 μl) of bacterial suspension yielding approximately an inoculum size of 1 × 10 6 colony forming units (CFU/ml) were added. The plates were sealed with sterile seals and incubated at 37°C for 24 h. Thereafter, 0.04% (w/v) p-iodonitrotetrazolium (INT) was added to each well and the plates were kept at ambient temperature for 6 h. The results were recorded visually under a light source. All well plate experiments were carried out in triplicate (2 × 3). Sterile broth containing bacterial suspension was used to monitor the viability of the test organism, while ciprofloxacin hydrochloride (0.01 μg/ml) was used as the positive antibacterial control. Acetone or DMSO were used as negative control. The final concentration of acetone or DMSO in the well had no effect on the bacterial growth.

HIV-1 Integrase inhibitory activity
Most of the root extracts of A. laxiflora (ALR series) inhibited the activities of HIV-1 Integrase ( Fig. 1a and Table 1). The methanolic root extract (ALR4) exhibited noteworthy HIV-1 Integrase inhibitory activity with an IC 50 value of 0.21 ng/ml, chicoric acid was taken as a reference (IC 50 = 6.82 μM). The ethanolic root extract (ALR5) also displayed marked HIV-1 Integrase inhibitory activity with an IC 50 value of 67.07 μg/ml, while the ethyl acetate root fraction (ALR3) IC 50 value was found to be 6.034 μg/ml. The cytotoxicity studies results (Table 1) showed that none of these extracts were cytotoxic. The results of our findings are in agreement with previous reports on the anti-HIV efficacy on A. laxiflora. In fact, Buhner, (2012), reported that A. laxiflora was found to be strongly active against HIV-1 and HIV-2 in vitro, more so than Azidothymidine (AZT) (no IC 50 value shown nor the part of the plant used). AZT is the first drug approved by the US FDA in the fight against AIDS (Zhang et al., 2017). A closely related species of the same genus namely Alchornea cordifolia was also found to be active against HIV-1 and HIV-2 (Ayisi and Nyadedzor, 2003).
The methanolic extract of the stem of A. laxiflora (ALS4) inhibited the activities of HIV-1 Integrase by 91.75% (Table 1), but the IC 50 was not determined. The activity of isolated compounds from ALS4 (Fig. 2) on HIV-1 Integrase was also investigated ( Fig. 1b and Table1). While all the isolated compounds were found to be non-cytotoxic (Table 1), some of these compounds inhibited the activities of HIV-1 Integrase (Table 1). Ellagic acid (1) displayed the best anti-HIV-1 Integrase activity with IC 50 value of 90.23 μM. The IC 50 values of 3-O-methylellagic acid (2) and 3-O-acethyl of oleanolic acid (4) were N 100 μM. This result follows the same trend as observed in the literature. To this instance, ellagic acid isolated from Lagerstroemia speciosa L. was found to be non-toxic and to inhibit HIV-1 activity with an IC 50 value of 73 μg/ml (Nutan et al., 2013). This result is the first report on the identification of compounds that can be responsible for the anti-HIV activity of A. laxiflora. It is therefore suggested that ellagic acid can be a potential agent for the development of novel drugs against HIV-1.

Antibacterial activity of solvent extracts
The extracts of three plant parts (leaves, stems and roots) of A. laxiflora prepared in the order of solvent polarity (i.e. hexane (Hex), chloroform (CHCl 3 ), ethyl acetate (EtOAc), Ethanol (EtOH), methanol (MeOH) and water (H 2 O) are depicted in Table 2.
The antibacterial observations include inhibitory effects on many of the selected pathogens representing gastrointestinal, skin, respiratory and urinary tract infections. Methanol extracts of all three plant parts were found to be the most active when compared to other extracts. The best results for the leaf extracts were observed for the EtOAc, MeOH and EtOH extracts against Klebsiella pneumoniae (MIC 63 μg/ml, for each of the three extracts), a Gram-negative strain associated with respiratory ailments (Peleg and Hooper, 2010). Staphylococcus saprophyticus, a Gram-positive urinary tract pathogen (Hovelius and Mardh, 1984), was particularly susceptible to most extracts (MIC ranging between 63 and 250 μg/ml) and this is in agreement with one of the most commonly listed traditional uses for Alchornea laxiflora, in the treatment of diarrhoea. However, the activity of all extracts on Proteus mirabilis appeared to be weak compared to S. saphrophyticus. It would be prudent to consider a study on other urinary tract pathogens which could provide further information on the wider use of A. laxiflora extracts. The root extracts showed higher activity against B. cereus and E. faecalis at MICs of 63 μg/ml and on S. aureus at 50 μg/ml. The stem extracts of ethanol and methanol showed significant MIC values on S. saprophyticus at 63 μg/ml. However, the EtOAc, CHCl3 and Hex extracts showed MICs of about 250 μg/ml. The leaf extracts exhibited highest MICs of above 100 μg/ml for activity on B. cereus and E. faecalis and significant in the case of the respiratory pathogen K. pneumoniae and the urinary pathogen S. saprophyticus. According to Bueno, (2012), plant extracts displaying MIC values below or equal to 100 μg/ml are considered to show noteworthy antimicrobial activity (Bueno, 2012). Based on the above statement, we could consider the A. laxiflora plant extract concentrations showing significant MICs below 100 μg/ml as having significant antimicrobial activity. This is the first report on the comparative antibacterial study of A. laxiflora different parts.

Antibacterial activity of isolated compounds
The five isolated compounds (Fig. 2) were screened for antibacterial activity against the same eight pathogens as the extracts (Table 2).

Gastrointestinal (GI) pathogens
All the compounds showed an MIC of 125 μg/ml against B. cereus; compounds 1, 2 and 3 exhibited an MIC of 63 μg/ml against E. faecalis, whereas, MICs of 125 μg/ml were observed for compounds 4 and 5 against E. faecalis. The minimum inhibitory concentration against E. coli was observed at 63 μg/ml for all compounds.

Skin pathogen
All the five compounds displayed an MIC of 125 μg/ml against S. aureus.

Respiratory pathogens
All the five compounds exhibited an MIC of b31 μg/ml against Gram-negative K. pneumoniae, compounds 4 and 5 displayed antibacterial activity with MIC value of 16 μg/ml against M. catarrhalis; compounds 1 and 3 exhibited an MIC of 125 μg/ml against M. catarrhalis while compound 2 showed an MIC of 250 μg/ml against the same pathogen.

Urinary pathogens
Compounds 3-5 were the most active with an MIC value of 4 μg/ml against Gram-positive S. saprophyticus. Compounds 4 and 5 were the most active against P. mirabilis whith MIC value of 63 μg/ml each.

Conclusions
In continuation of the quest for discovery of effective anti-HIV and antimicrobial compounds, screening of herbal extracts and compounds is indeed an expedient procedure to discover new entities that may be used in developing future drugs. In this study, the isolated compounds did not display significant anti-HIV integrase activity. The most potent anti-HIV activity was observed for the methanolic extract of Alchornea laxiflora root (ALR4) with an IC 50 of 0.21 ng/ml. This is an indication that further investigations are required to isolate and identify putative new anti-HIV integrase from the roots of A. laxiflora. Our next objective is therefore to isolate and identify the anti-HIV integrase compounds from the roots of A. laxiflora. The five isolated compounds exhibited significant activity with MIC values ranging between 4 and 63 μg/ml against all the bacteria except B. cereus. 3-O-β-D-glucopyranosyl-β-sitosterol, 3-O-acetyl-oleanolic acid and 3-O-acetyl-ursolic acid displayed the highest antibacterial activity (MIC value of 4 μg/ml) against S. saprophyticus. The roots seem to be the most active plant component of A. laxiflora as the root extracts (MIC as low as 50 μg/ml) were more active as compared to the stem and leaves extracts. The highest antibacterial activities were observed for the medium polarity extracts (EtOH, MeOH, EtOAc and CHCl 3 ). The antibacterial activity of A. laxiflora against gastro-intestinal (B. cereus, E. faecalis and E. coli), skin (S. aureus), respiratory (K. pneumoniae and M. catarrhalis) and urinary (P. mirabilis and S. saprophyticus) pathogens is indeed scientifically demonstrated. The isolated phytosteroid (3-O-β-D-glucopyranosyl-β-sitosterol) and triterpenoids (3-O-acetyl-oleanolic acid and 3-O-acetyl-ursolic acid) showed more potent antibacterial activity than the phenolic compounds (ellagic acid and its methyl derivative). These isolated compounds are reported here for the first time to be responsible for antibacterial activities of A. laxiflora. It is important to note that the roots seem to be the most anti-HIV and antibacterial active component of A. laxiflora. The overall results provide evidence that A. laxiflora as well as some of its isolated components (3-O-β-D-glucopyranosyl-β-sitosterol, 3-O-acetyl-oleanolic acid and 3-O-acetyl-ursolic acid) might be potential sources of new anti-HIV and antimicrobial drugs.