Gastrointestinal diseases are one of the most frequent causes of medical consultation in Mexico and in the world. For this reason they are regarded as public health problems. These diseases affect people of any age or gender, but the most vulnerable are children and the elderly. Globally, these diseases are the leading causes of illness and death in these age groups. The most common agents associated with gastrointestinal infections are the bacteria Escherichia coli, Salmonella typhi, and Shigella spp., parasites such as Giardia lamblia and Entamoeba histolytica, and viruses such as rotavirus and Norwalk virus. Oral-fecal transmission, i.e. ingesting feces-contaminated water or food, is the most common route of infection  .
G. lamblia, the causative agent of giardiasis, is a water-borne intestinal protozoan that infects human and other mammals worldwide   . Approximately 200 million people are affected by giardiasis in tropical and subtropical countries  . Every year, 500,000 new cases of giardiasis are reported  . Over 55% of the Mexican population is seropositive for this parasite  .
Another common gastrointestinal infection is amoebiasis, which is caused by E. histolytica. Around the world, 50 million people per year are infected by E. histolytica, but only 5 million develop the disease, resulting in 100,000 deaths each year  . In this disease, the most common clinical forms are dysentery and amebic liver abscess (ALA)   . In Mexico, 8.4% of the population is seropositive for E. histolytica   . It is estimated that just over one million cases are treated and 1216 deaths per year are caused by this disease  .
Sexually transmitted diseases are the leading cause of acute illness worldwide. Trichomoniasis, caused by the protozoan parasite T. vaginalis, is the most common, curable, sexually transmitted disease, generating more than 170 million cases each year around the world  .
The most common drugs to treat of amoebiasis, giardiasis and trichomoniasis are 5-nitroimidazoles derivatives, such as metronidazole or tinidazole    . A recently reported increase in the resistance to metronidazole and 5-ni- troimidazoles of E. histolytica, G. lamblia and T. vaginalis strains poses a serious problem  , and has caused a heightened interest in the discovery and development of new compounds to treat these diseases  .
Thymus vulgaris is a plant of European origin and belongs to the Lamiaceae family. In the chemical composition of Thymus vulgaris, essential oils are predominant, especially the monocyclic monoterpenes, (thymol and carvacol), as well as other monoterpenes (p-cymene, camphene, limonene, borneol, among others). Besides, Thymus vulgaris contains flavonoids (apigenin and luteonin), methoxylated flavones (cirsilineol, cirsimartina), and some other minor components, such as phenolic acids (caffeic acid and rosmarinic acid), tannins, and triterpenes (ursolic acid and oleanolic acid)  . The latter have been reported in methanol and hexane extracts.
The antiseptic and anti-inflammatory activity of Thymus vulgaris is well- known. In vitro studies have proven high antibiotic activity against Mycobacterum tuberculosis  , cutaneous leishmaniasis  , Bacillus subtilis, Staphylococcus aureus, and Escherichia coli  . Furthermore, Thymus vulgaris has fungicide activity and was able to protect germinating crop seeds from fungal attack  .
The purpose of this study was to investigate the in vitro antiparasitic activity of the Thymus vulgaris methanol extract and of some of its components by verifying their capacity to inhibit the growth of G. lamblia, E. histolytica, and T. vaginalis trophozoites.
2. Materials and Methods
2.1. Chemicals and Stock Solutions
All chemicals were reagent grade. DMSO and the standard drugs were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The other chemicals used in this study were purchased from J. T. Baker (Xalostoc, Edo. de Mexico, Mexico). Sterile bovine serum and PEHPS medium were prepared in our laboratory as described elsewhere  . Metronidazole (Sigma Chemical Co.), a nitroimidazole antibiotic and antiprotozoal agent was dissolved in DMSO to get 500 μg/mL concentration and further diluted adding 500 μL in 10 mL of dimethyl sulfoxide (DMSO), finally this solution was added to media 1:10 v:v to give 12.5 μg/mL final concentration. Ursolic acid and betulinic acid solutions (Sigma Chemical Co.), antiprotozoal pentacylclic triterpenoids, were prepared adding 2 mg of the pure compound (Sigma Chemical Co.) in 2 mL of DMSO and further diluted to 25 μg/mL. Stock solutions were stored at −20˚C until use.
Thymus vulgaris was obtained from local markets and identified at the herbarium of the Faculty of Forestry of the Autonomous University of Nuevo Leon, Mexico (Boucher number 21,344). The aerial parts of the plant were dried in electric oven at 40˚C for 72 h (J.M. Ortiz. Aparatos Eléctricos S.A. de C.V., Monterrey, N.L. México) and ground with the use of an electric mill (Molino del Rey, S.A. de C.V., San Nicolas de los Garza, NL, Mexico).
G. lamblia 0989-IMSS strain (5 × 103 trophozoites) was cultivated in 5.5 mL TYI-S33 medium in 13 × 100 culture tubes (Pyrex, Corning life Sciences, Corning, New York, NY, USA) at 36.5˚C. The duplication time for G. lamblia trophozoites was 13.1 h (Figure 1).
E. histolytica HM1-IMSS strain (5 × 103 trophozoites) and T. vaginalis GT-15
Figure 1. Growth curve of E. histolytica (a) G. lamlia (b) and T. vaginalis trophozoites (c).
strain (5 × 103 trophozoites) were cultivated in 5.5 mL PEHPS medium  at 36.5˚C. The duplication time for E. histolytica was 21.3 h and for T. vaginalis 11.7 h (Figure 1).
2.4. Preparation of the Methanol Extract
The crude extract was obtained by maceration and mixing with methanol. Briefly, 150 g of the dried, ground, aerial Thymus vulgaris parts were stirred with 500 mL methanol for 72 h on a rotary shaker (PC 622 Corning, New York, NY, USA). The supernatant of crude methanol extract was filtered using Whatman # 1 papers (Whatman International, LTD, Maidstone, England) and air-dried. This extraction was repeated two more times. The pooled filtrates were stored in the dark at 4˚C in the dark until use. Thirty mg of the extract was dissolved in 250 μL dimethylsulfoxide (DMSO), which was further diluted in distilled water to create a stock of 6 mg/mL.
2.5. Fractionation of the Extract
Once the anti-parasitic activity of the methanol extract had been verified (Figure 2), the extract was fractionated by polarity-based partitioning: after a single
Figure 2. Work flows of bio-guided extraction and fractionation process.
hexane:methanol (1:1, v/v) fractionation, and triple ethyl acetate:water:methanol (4:3:1, v/v) and n-butanol:water:methanol (4:3:1, v/v) fractionations, the soluble methanol fraction was recovered. All fractions were allowed to dry completely before being tested for antiprotozoal activity (see below). The best-performing fraction was extracted with chloroform, filtered, and air-dried completely. Both the chloroform-soluble and insoluble fractions were tested for anti-parasitic activity. As only the chloroform-insoluble fraction had significant anti-parasitic activity, it was re-suspended in methanol and filtered. Next, the methanol-soluble portion was three times extracted with n-hexane:methanol (3:1, v/v), whereas the methanol-insoluble part was extracted with acetone, so that four fractions were obtained: 1) a hexane-soluble fraction, 2) a methanol-soluble fraction, 3) an acetone-soluble fraction, and 4) a fraction insoluble in any of the above solvents. The purity of each fraction was determined by thin layer chromatography (TLC), (Figure 3). As the hexane and acetone-soluble fractions produced the same TLC pattern, they were combined before the various fractions were tested again for antiprotozoal activity. Only the mixed hexane/acetone-soluble fraction
Figure 3. Outcomes of thin layer chromatograpy spots represents methanolic extract in the lane a, mixture hexane-acetone extracts in the lane b, and commercial ursolic acid in the lane c.
had antiprotozoal activity. This fraction was characterized by high-performance liquid chromatography (HPLC) coupled to orthogonal acceleration time-of- flight mass spectrometry, mass Spectrometer and the results of data obtained from the database were compared Mascot-Blast Spectrophotometric  .
2.6. Antiprotozoal Assay
The antiprotozoal activity of the methanol extract and its fractions were evaluated as previously described    . Trophozoite suspensions (2 × 104/mL for E. histolytica, 1 × 105/mL for T. vaginalis, and 2 × 105/mL for G. lamblia) in their respective media supplemented with 10% bovine serum were incubated with the various extracts and control solutions at 36.5˚C for 72 h for E. histolytica and 24 h G. lamblia or T. vaginalis. These incubation periods were chosen because of their differential growth kinetic characteristics under standard conditions (Figure 1). Test samples included the crude methanol extract and the pooled hexane/acetone-soluble fraction at 0 - 300 μg/mL. Positive antiprotozoal solutions included two-fold serial dilution series (0 - 25 μg/mL) in appropriate media of ursolic acid and betulinic acid, and metronidazole at 1.25 μg/mL, whereas 5% DMSO served as a negative control. Incubations were stopped by placing the suspensions in ice-cold water for no less than 20 min. The number of trophozoites/mL was determined using a hemocytometer.
The percentage of growth inhibition was calculated with respect to untreated controls. The 50% inhibitory concentration (IC50) of each drug was calculated by PROBIT analysis  . Each drug was assayed in triplicate in three independent experiments for each protozoan species, and the mean and 95% confidence limits were calculated.
2.7. Vero Cell Cytotoxicity
Vero cells (African green monkey epithelial kidney cells, ATCC CCL-81) were maintained in complete RPMI media/1 mM pyruvate/10% fetal bovine serum at 37˚C and 5% CO2. To evaluate cytotoxicity, 1 × 105 cells/well were seeded in 96-well plates and incubated for 24 h. Next, the supernatant was replaced with 100 μL of test or control solutions and incubated for another 24 h. Test samples included crude methanol extracts and the hexane/acetone-soluble fractions (9.37 - 300 μg/mL); the positive control was 10% DMSO and the negative control was fresh medium. After the incubation time, the viable cells/well were counted with the use of the trypan blue exclusion method and a hemocytometer. The IC50 was determined by PROBIT   . All assays were performed in triplicate  .
The plant extracts with the most promising antiprotozoal activity were analyzed by TLC on pre-coated silica gel plates with benzene:acetone 9:1 v/v as the mobile phase. The air-dried TLC plates were observed at both 254 nm and 366 nm, and developed by iodine vapors and also exposed to different spraying reagents, in order to determine the Rf values of the observed spots.
2.9. Liebermann-Burchard Test
A drop of the extract was mixed with 1 drop of chloroform and three drops Liebermann-Burchard reagent (sulfuric acid/anhydrous acetic acid/chloroform, 20:1:1 v/v) on a porcelain plate. A change of color to orange, red, blue or green color is indicative of the presence of cholesterol-like compounds.
2.10. Statistical Analysis
For each experiment the mean and standard deviation of three independent experiments performed in triplicate were determined. The IC50 for each protozoa- antiprotozoal extract combination was calculated with the PROBIT trial  . The t student test was performed to compare the means of IC50 of each antiprotozoal extract combination using the statistical software SPSS version 15.0. A p-value < 0.05 was considered as statistically significant.
As 18.22 g dry-weight was obtained from the methanol extract of the aerial parts of 150 g Thymus vulgaris, the production efficiency was 12.14%.
3.1. Antiprotozoal Activity of Thymus Vulgaris Extracts
The 300 µg/mL crude methanol extract of Thymus vulgaris had antiprotozoal activity against G. lamblia (IC50 86.41 ± 2.9 µg/mL) and T. vaginalis (IC50 115.41 ± 2.29 µg/mL), but not against E. histolytica trophozoites (Table 1 and Table 2).
The subsequent fractionation steps were bio-guided by the antiprotozoal assay. Only fractions with antiprotozoal activity underwent further work-up as in-
Table 1. Anti-parasitic activity of Thymus vulgaris extracts.
Table 2. IC 50 antiprotozoal activity and cytotoxicity of the extracts, mixture of fractions and commercial compounds.
dicated in Figure 2. After the first step, the fraction with highest antiprotozoal activity was methanol extract from the ethyl acetate/water/methanol fractionation, causing a ≥ 97% growth inhibition of T. vaginalis and G. lamblia cultures. Next, it was the chloroform-insoluble fraction that caused growth inhibition of about 97% in aforementioned cultures. After the third step, both the hexane and acetone fractions from the chloroform-insoluble, methanol-recovered extract yielded antiprotozoal activity against both species. The mixed hexane-acetone fraction induced had excellent antiprotozoal activity, inducing 96% - 99% growth inhibition (Table 1).
The color change to red in the Liebermann-Burchard test indicated the presence of triterpenes in the mixed hexane-acetone fraction. Further characterization by mass spectrometry chromatography allowed the identification of oleanolic acid, betulinic acid and ursolic acid. These compounds were commercially obtained and their antiprotozoal activity was compared with the mixed hexane-acetone fraction. Only ursolic acid had antiprotozoal activity against both T. vaginalis and G. lamblia. The antiprotozoal activity of ursolic acid was 5 - 14 times stronger than that of the mixed hexane-acetone fraction, which was 1.5 - 2 times stronger than that of the crude methanol extract (Table 2).
Although the crude methanol extract, the hexane-acetone fraction, and commercial ursolic acid were all toxic for Vero cells, the concentration required to achieve an IC50 on Vero cells was 2.2 - 57.8 times higher than the IC50 for the protozoa (Table 2).
Previous studies have reported the antiparasitic and nutritional activities of Thymus vulgaris   . Thymol and carbachol are Thymus vulgaris-derived essential oils that have been recognized as active agents with analgesic, antifungal, antibiotic, antioxidant, antispasmodic, and insect repellent activities    .
We aimed to extract an antiprotozoal agent from Thymus vulgaris with methanol, because methanol is recognized to be among the most effective solvents to extract compounds from plants    , especially to obtain antibiotic and antiparasitic agents     .
Most antiprotozoal plant extracts are functional at 2 - 100 micrograms/mL  , while extracts that require ≥ 1000 μg/mL to sort effect are considered inactive  . Some researchers encourage that crude extracts with IC50 of about 100 μg/mL should be further investigated as they may contain new, active agents, that could be discovered by bio-guided fractionation using polar solvents that favor compounds with hydroxyl and amine groups   . Applying the principles of this idea, we decided to work up a crude methanol extract with antiprotozoal activity at ≤300 μg/mL that was further fractionated in a multi-step partition process in which a variety of solvent (mixtures) were used and the obtained fractions were tested for antiprotozoal activity. In this way we were able to isolate a mixture of oleanolic acid, ursolic acid, and betulinic acids in the final hexane-acetone fraction. The characterization of these compounds was realized by chromatographic mass spectrometry. Remarkably, there was a similarity between the obtained mass spectrum of the hexane-acetone fraction obtained from Thymus vulgaris and a spectrum obtained from a previously-reported Carya illinoensis extract  . Others have reported that oleanolic acid and ursolic acid, obtained after column chromatography fractionation of a Thymus vulgaris-de- rived methanol extract, had anti-tuberculosis activity  . Antiprotozoal activity against G. lamblia trophozoites has been reported for Thymus zigis subsp. sylvestris-derived essential oil. This essential oil had an IC50 of 185 μg/mL and mainly contained thymol and carvacrol  . Here we present a Thymus vulgaris-de- rived methanol extract that contains oleanolic acid, ursolic acid, and betulinic acids and has anti-protozoal activity against G. lamblia and T. viginalis trophozoites. The antiparasitic activity of ursolic acid had been reported before  , and was confirmed to be the best-performing when commercially obtained oleanolic acid, ursolic acid and betulinic acid were tested in our antiprotozoal assay against T. vaginalis and G. lamblia. As our crude methanol extract (at 300 μg/mL) could not inhibit convincingly inhibit the growth E. histolytica trophozoites, it can be inferred that ursolic acid is not active against this parasite. Behnia et al. have reported positive biological activity against E. histolytica in a water-ethanol extract of Thymus vulgaris with an IC50 of 3 mg/mL  . The different extraction technique and very high concentration of the compound may explain their result. However, bioactivity testing of plant-derived extracts at a concentration beyond a 100 μg/mL is uncommon  . Furthermore, the study by Behnia et al. did not include a cytotoxicity study on mammalian cells  . In our study, the therapeutic index was studied by comparing the IC50 cytotoxicity on Vero cells  and the IC50 for growth inhibition of protozoa species. In our study design, protozoal growth inhibition and Vero cell cytotoxicity were evaluated in separate bioassays, although in antiprotozoal studies co-cultures are common practice. The reasons for our alternative approach were: 1) the protozoa and Vero cells require different culture conditions, and 2) Trichomonas sp. are known to be cytotoxic to Vero cells and other human monolayer cell lines and would interfere in the interpretation of the cytotoxicity results  . The performance of therapeutic indices we found were: commercial ursolic acid ? hexane-acetone fraction > methanol extract. In this assay we did not find any cytotoxic activity on the Vero cells for any of the compounds tested.
Thus, in this paper we report the anti-parasitic activity of the methanol extract of Thymus vulgaris. Anti-parasitic activity was contundent against G. lamblia y T. vaginalis, but insignificant against E. histolytica trophozoites. The mixture of hexane-soluble and acetone-soluble fractions of this methanol extract, which contained ursolic, betulinic, and oleanic acids, had the strongest antiparasitic activity. We provide evidence that ursolic acid was the compound that was mainly responsible for antiprotozoal activity in the methanol extract of Thymus vulgaris.
This study was supported by FIS/IMSS/PROT/G13/1204 and FIS/IMSS/PROT/ G12/1141. Jesus Norberto Garza Gonzalez received a PhD from the Autonomous University of Nuevo León. The authors thank Irene Meester for critically reviewing the manuscript.
Conflict of Interest
There are not conflicts of interest to develop its research or elaborate this manuscript.