Antiprotozoal and antimycobacterial activities of Persea americana seeds

Background Persea americana seeds are widely used in traditional Mexican medicine to treat rheumatism, asthma, infectious processes as well as diarrhea and dysentery caused by intestinal parasites. Methods The chloroformic and ethanolic extracts of P. americana seeds were prepared by maceration and their amoebicidal, giardicidal and trichomonicidal activity was evaluated. These extracts were also tested against Mycobacterium tuberculosis H37Rv, four mono-resistant and two multidrug resistant strains of M. tuberculosis as well as five non tuberculosis mycobacterium strains by MABA assay. Results The chloroformic and ethanolic extracts of P. americana seeds showed significant activity against E. histolytica, G. lamblia and T. vaginalis (IC50 <0.634 μg/ml). The chloroformic extract inhibited the growth of M. tuberculosis H37Rv, M. tuberculosis MDR SIN 4 isolate, three M. tuberculosis H37Rv mono-resistant reference strains and four non tuberculosis mycobacteria (M. fortuitum, M. avium, M. smegmatis and M. absessus) showing MIC values ≤50 μg/ml. Contrariwise, the ethanolic extract affected only the growth of two mono-resistant strains of M. tuberculosis H37Rv and M. smegmatis (MIC ≤50 μg/ml). Conclusions The CHCl3 and EtOH seed extracts from P. americana showed amoebicidal and giardicidal activity. Importantly, the CHCl3 extract inhibited the growth of a MDR M. tuberculosis isolate and three out of four mono-resistant reference strains of M. tuberculosis H37Rv, showing a MIC = 50 μg/ml. This extract was also active against the NTM strains, M. fortuitum, M. avium, M. smegmatis and M. abscessus, with MIC values <50 μg/ml.


Background
Persea americana Mill. (Lauraceae) is an edible fruit commonly known as aguacate (avocado) that grows throughout the tropics. The seeds (crude or toasted) are employed in traditional Mexican medicine to treat skin rashes, diarrhea, and dysentery caused by helminths and amoebas, for the cure of infectious processes caused by fungi and bacteria, as well as for the treatment of asthma, high blood pressure, and rheumatism [1][2][3][4][5]. The seeds of P. americana used alone or mixed with other species, such as Psidium guajava, Mentha piperita or Ocimum basilicum, are mainly employed for the treatment of diarrhea [4].
Interestingly, the hypolipemic effect of the MeOH extract obtained from P. americana seeds has been demonstrated in male rats with induced hypercholesterolemia [10,11]. This extract reduced total cholesterol levels, triglycerides and Low density lipoprotein (LDL); on the other hand, increased the levels of High density lipoprotein (HDL). The same effect was described for the aqueous extract, which also reduced blood pressure both in normal rats and those with high blood pressure; in addition, it exerted a hypoglycemic effect on rats and rabbits with diabetes [12][13][14][15]. The aqueous extract showed a median Lethal dose (LD 50 ) = 10 g/kg in rats when it was administered orally. Importantly, it did not alter the hematological parameters nor the levels of Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), albumin, and creatinine in male and female rats were treated for 28 days [16].
The hexanic and MeOH seed extracts of P. americana have been described to have a Minimum inhibitory concentration (MIC) of <1.25 μg/ml against Candida ssp., Cryptococcus neoformans and Malassezia pachydermatis. These extracts were also active against Artemia salina, with Lethal concentration (LC 50 ) values of 2.37 and 24.13 mg/ml, respectively. They were also active against Aedes aegypti larvae with LC 50 values of 16.7 and 8.9 mg/ml, respectively [17]. On the other hand, the MeOH extract from P. americana leaves inhibited completely the growth of M. tuberculosis H37Ra (MIC = 125 μg/ml) and H37Rv (MIC = 62.5 μg/ml); furthermore, the hexane fraction inhibited the growth of both mycobacteria with MIC = 31.2 μg/ml [18]. In addition, the EtOH extract was active against both Gram-positive and -negative bacteria (with the exception of Staphylococcus epidermis and Escherichia coli) with MIC = 500 μg/ml [19]. Regarding the bacterial activity of P. americana (var Hass and Fuerte), the acetone seed extract exhibited moderate activity against Bacillus cereus, Staphylococcus aureus and Listeria monocytogenes [9]. The trypanomicidal activity of the MeOH extract from P. americana seeds has been also tested [20]. It showed moderate activity when was evaluated at the concentration range of 250-500 μg/ml. In the case of the aqueous seed extract, it had a slight anti-Giardia duodenalis (syn G. lamblia) activity, inducing 23% of mortality at 4 mg/ml [21].
An important antioxidant activity (AOA) of the MeOH extract of P. americana seeds and leaves has been described by different methods [8,[22][23][24]. Besides, AOA has been reported in the 100% EtOAc, 70% acetone and 70% MeOH of the peel, pulp and seed extract [9].
Up to now, the activity of the chloroformic (CHCl 3 ) and ethanol (EtOH) extracts obtained from P. americana seeds against anaerobic protozoan and M. tuberculosis H37Rv strains with different level of drug resistance has not yet been evaluated. Therefore, herein, the activity of both extracts was tested against the anaerobic protozoa Giardia lamblia, Entamoeba histolytica and Trichomonas vaginalis. In addition, their antimycobacterial activity was evaluated against four mono-resistant reference strains of M. tuberculosis H37Rv, two MDR M. tuberculosis clinical isolates and five non-tuberculosis mycobacterium (NTM).

Plant material
P. americana seeds were obtained from the town of Ario de Rosales in the state of Michoacan, Mexico in August 2009. This material was then dried at room temperature and under conditions of darkness; the material was then ground. The plant was botanically identified by Abigail Aguilar, M.Sc., and a voucher specimen was deposited at the Herbarium of the Instituto Mexicano del Seguro Social, Mexico (IMSSM) with code number 14256.

Preparation of extracts
The dry and powdered plant material (1.364 kg) was macerated three times with CHCl 3 analytical reagent -AR-(J.T. Baker) at room temperature for 7 days. The extract was filtered and concentrated to dryness under low pressure at 40°C. The plant material was later macerated with EtOH AR grade (J.T. Baker) three times for 7 days and the solvent was eliminated under reduced pressure until the solvent-free extract was obtained.

Antiprotozoal activity evaluation
For this assay, the E. histolytica strain HM1-IMSS and the T. vaginalis GT9 strain were cultured in a TYI-S-33 modified medium supplemented with 10% calf serum; G. lamblia strain IMSS:0989:1 was maintained in a TYI-S-33 medium supplemented with 10% calf serum and bovine bile. In vitro susceptibility assays were performed according to the method previously described [25,26]. Briefly, 5 × 10 4 trophozoites of G. lamblia were incubated for 48 h at 37°C with increasing concentrations of the EtOH and CHCl 3 extracts of P. americana seeds using Dimethyl sulfoxide (DMSO) as a suitable solvent. After incubation, G. lamblia trophozoites were washed and subcultured for an additional 48 h in a fresh medium alone. For E. histolytica and T. vaginalis, 6 × 10 3 trophozoites were incubated for 72 h at 37°C with increasing concentrations of the samples tested. Metronidazole was included as a positive control; parasites without treatment but with the highest DMSO concentration used for sample dilutions were included as a negative control. G. lamblia, T. vaginalis and E. histolytica trophozoites were counted and the 50% Inhibitory concentration (IC 50 ) was calculated by Probit analysis. The experiments were carried out in triplicate and repeated at least twice. The antimycobacterial activity was carried out by means of the Microplate alamar blue assay (MABA) previously described [26,27]. EtOH and CHCl 3 extracts (10 mg) were solubilized in 500 μL of DMSO and from these stock solutions, several dilutions were prepared to achieve concentrations between 200 and 3.13 μg/ml. The maximum DMSO concentration used in the MABA assay does not affect mycobacterial growth, as it was previously reported [28]. Extracts that presented MIC <100 μg/ml were considered to have good antimycobacterial activity. Rifampicin and isoniazid at 0.06 μg/mL were included as a positive control for the H37Rv strain, for the MDR clinical isolates and for NTM; the same drugs were used but at concentrations of 100 and 3.1 μg/ml, respectively.

Results and discussion
By means of the maceration process, 88.7 g of the CHCl 3 extract and 77.2 g of the EtOH extract from P. americana seeds were obtained with an average yield of 6% with respect to the plant material's dry weight. A preliminary phytochemical analysis by Thin layer chromatography (TLC) of the CHCl 3 extract led to the detection of the presence of β-sitosterol, phytol and palmitic acid. On the other hand, catechin and epicatechin were detected in the EtOH extract by TLC. All compounds were identified by comparison of the Retention factor (R f ) with their commercial reference. A previous study by Rodríguez-Carpena et al. [9] reported a high concentration of catechin and epicatechin in 100% EtOAc, 70% acetone and 70% MeOH extracts obtained from P. americana seeds and peel.
It is interesting to note that, the reference drug, metronidazole (IC 50 = 0.210 μg/ml) showed only three and two times higher anti-Giardia activity than the CHCl 3 and EtOH extracts. However, in the case of E. histolytica, the CHCl 3 and EtOH extracts were seven and six times less potent than metronidazole (IC 50 = 0.060 μg/ml). For  T. vaginalis, these extracts showed weak activity, being 16 (CHCl 3 ) and 17 (EtOH) times less potent than metronidazole (CI 50 = 0.037 μg/ml).
In traditional Mexican medicine, P. americana seeds are used to treat diarrhea [1][2][3][4]. It is shown here that P. americana EtOH and CHCl 3 seed extracts are indeed responsible for the activity against two anaerobic parasites that cause diarrhea: G. lamblia, and E. histolytica. The in vitro and in vivo giardicidal activity of epicatechin isolated from plants such as Rubus coriifolius and Heliantemum glomeratus has been reported [29,30]. In this regard, epicatechin was detected in P. americana EtOH seed extract by TLC analysis; hence, this compound may be responsible for the giardicidal activity observed in this study. Further studies are required to define the active compound(s) responsible for the antiprotozoal activity of the CHCl 3 extract.
It is worth considering the fact that the EtOH and CHCl 3 extracts constitute potential sources of compounds that can be employed as prototype molecules for the development of novel antiprotozoal agents as an alternative treatment of clinical isolates with metronidazole resistance.
The results of the antimycobacterial activity of the EtOH and CHCl 3 extracts determined by MABA assay are presented in Table 2. It is important to note that the CHCl 3 extract inhibited the growth of M. tuberculosis To date, the hexanic and MeOH extracts obtained from the stems and leaves of P. americana have been reported to inhibit the growth of M. tuberculosis H37Rv and M. tuberculosis H37Ra strains [18,31]. The reported antimycobacterial activity of the MeOH extracts was attributed to the presence of lignans [31] and the MIC was slightly lower than the CHCl 3 extract tested in this study.
Since the presence of MDR and extensively drugresistant (XDR) M. tuberculosis cases is increasing rapidly and current chemotherapy is prolonged, poorly effective, expensive and is accompanied by severe side effects [26]; it is necessary to have recourse to an alternative treatment against these strains or that can even aid in and/or shorten the currently available that could have a different mechanism of action. Therefore, the high activity exhibited by the P. americana CHCl 3 seed extract against mono-resistant strains of M. tuberculosis H37Rv, MDR clinical isolates and NTM that is described in this study, it is of great interest.
From the clinical point of view, NTM are becoming relevant, because of the so-called mycobacteriosis and are currently recognized as important pathogens associated with both immune-deficient and immune-competent patients. The mycobacteria tested in this study are representative of the most common NTM isolated from pulmonary cases (M. abscessus and M. avium) or associated with soft tissue infections (M. fortuitum and M. chelonae) [32]. Although M. smegmatis is a poor pathogenic bacterium, it was included in the NTM group because it is widely employed in the determination of the antimycobacterial activity of new compounds [32,33]. In the case of NTM, the majority is naturally resistant to some of the first-line anti-TB drugs such as INH and RIF; thus, effective drugs against NTM are scarce than those for TB, emphasizing the urgency of finding novel active compounds that could be used in the treatment of the NTM group. Based on our results, P. americana seeds may be a source for potential moieties (molecules) against NTM. We are currently conducting the isolation and identification of the active compounds responsible for the antimycobacterial activity observed with the CHCl 3 extract.

Conclusions
Herein, to the best of our knowledge, the activity of the CHCl 3 and EtOH seed extracts from P. americana against two intestinal parasites that cause diarrhea: E. histolytica, and G. lamblia has been demonstrated. In addition, based on our results, CHCl 3 seed extract may be a source for potential moieties (molecules) against M. tuberculosis drug-resistant species as well as NTM.
Further studies are required for the identification of the active compounds responsible for the antiprotozoal and antimycobacterial activity observed with the EtOH and CHCl 3 seed extracts from P. americana. manuscript. C-G collected the P. americana seeds and contributed to the preparing the CHCl 3 and EtOH extracts. JL-H and RR-N evaluated the antimycobacterial activity from CHCl 3 and EtOH extracts. The antiprotozoal activity from both extracts was determined by AT and LY-M, who also contributed to the manuscript preparation. All authors have read and approved the final manuscript.