Safety of Aqueous Extract of Calea ternifolia Used in Mexican Traditional Medicine

There is a trend to use medicinal plants for primary medical care or as dietary supplements; however, the safety of many of these plants has not been studied. The objective of this work was to determine the toxic effect of the aqueous extract of Calea ternifolia (C. zacatechichi), known popularly as “dream herb” in vivo and in vitro in order to validate its safety. In vivo, the extract had moderate toxicity on A. salina. In vitro, the extract induced eryptosis of 73% at a concentration of 100 μg·mL−1 and it inhibited CYP3A by 99% at a concentration of 375 μg/mL. After administering 8.5 mg/kg of C. ternifolia to rats, we found a reduction in platelets and leukocytes and an increase in urea and the liver enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP). Histological analysis showed spongiform changes in the proximal tubules of renal tissue and a lymphoid infiltrate in liver tissue. This plant is used in the treatment of diabetes, and it is commercialized as a dietary supplement in several countries. Our results show renal and hepatic toxicity; therefore, more profound research on the toxicity of this plant is needed.


Introduction
Because of the current tendency to adopt a more "natural" lifestyle, there is currently great interest in herbal medicine, a medical system that searches for efficient and mildly or nontoxic therapeutic options. Medicinal plants have been used as a cultural inheritance in traditional medicine, but they require scientific validation [1], because they contain active substances that have biological activity and toxicity [2].
Calea ternifolia (syn. C. zacatechichi) (Astereaceae) is a plant native to Mexico and Central America, where it has a long tradition in indigenous culture [3]. It is also called the "dream herb" because it temporarily intensifies the clarity of dreams and significantly influences the central nervous system. For this, C. ternifolia is used in several countries, although it was placed on the prohibited plant list in Poland in 2013 [4]. A more recent study of the neuropharmacological action of the aqueous extract of C. zacatechichi indicates that it has insignificant neuropharmacological effects and also reduces the abdominal pain perception [5]. It is also traditionally used for the treatment of endocrine and gastrointestinal problems [6]. The plant is sold online as a dietary supplement for the treatment of diabetes due to its ability to cause hypoglycemia [7]. Although its mechanism of action is unknown, this activity can be due to compounds that have been isolated from the plant, such as flavonoids [8] and sesquiterpene lactones [9], which have some toxicity. Flavonoids cause deformation, osmotic fragility, and aggregation of erythrocytes [10]. Other flavonoids such as epigallocatechin produce toxicity in rat embryos [11], and quercetin, in addition to being able to capture free radicals and act as an antioxidant, can also carry out pro-oxidant reactions, exerting cytotoxic effects on cells and tissues [12].
The objective of this work was to determine the toxic effect in vivo and in vitro of the aqueous extract of Calea ternifolia (C. zacatechichi), known popularly as "dream herb". Although in this work several of the chemical components of C. ternifolia were identified, we only evaluated and demonstrated the toxicity of the complete aqueous extract.

Evaluation of the Biological Activity of the Extract in
Human Erythrocytes. Human erythrocytes from healthy volunteer donors were obtained to perform the tests described below. All participants gave their written informed consent.

Erythrocyte Cytotoxicity of the Extract by Flow
Cytometry. A sample of peripheral blood with the anticoagulant CDP/ADSOL ™ was collected, and a complete blood count was determined on a Sysmex KX-21N Hematology Analyzer (Sysmex America, Inc., Lincolnshire, IL). A cell adjustment was made to 1 × 10 6 mL − 1 in 5 mL PBS (Cell Pack Phosphate Buffered Saline) and 1 μL of whole blood in 5 mL HANKS′ balanced salt solution (Cat. H-2387 Sigma-Aldrich, Inc., St. Louis, MI). For flow cytometry, a FACS-Calibur E4807 device (Becton Dickinson and Co., Franklin Lakes, NJ) was used. Erythrocytes were suspended at 0.4% [15] and/or 10 5 -10 6 cells·mL − 1 . An eryptosis curve was run using rifampin 50 μg/mL as a positive control and with the extract at concentrations of 1000, 500, 100, and 10 μg/mL.
The sample was centrifuged, and a pellet was obtained that was resuspended in 1 mL HANKS' solution; from this, 100 μg/mL was taken and placed in a fluorescence-activated cell sorting (FACS) tube, 200 μL of HANKS′ solution was added, and the sample was read by flow cytometry.

Flow Cytometry.
For staining with Annexin V-FITC (Annexin V FITC, cat. 11-8005), the cells were washed with HANKS′ solution and subsequently resuspended in 1 mL of binding buffer at 1-5 × 10 6 ·mL − 1 . From this suspension, 100 μL was taken and 5 μL of annexin V-fluorochrome conjugate was added and incubated at 22-28°C for 10-15 min. The cells were then washed in binding buffer and resuspended in 200 μL of the buffer, kept in the dark at 2-8°C and analyzed by flow cytometry after 4 h.

Mean Corpuscular Volume (MCV).
Initially, the number of cells was adjusted to 1 × 10 6 mL − 1 in phosphate buffer (Cell Pack).
The extract at a concentration of 500 μg was diluted with the cells 1 : 1 (extract : cells) and maintained at 20-25°C for 24 h; later this was read in the automated MCV device Sysmex KX-21 considering 80-97 fL as a normal value [16].
2.6. Thiobarbituric Acid/Malondialdehyde. The aqueous extract at a concentration of 500 μg were assayed for thiobarbituric acid/malondialdehyde reactive substances (TBARS/MDA) to search for lipid peroxidation as a cell damage mechanism, following the method described by Dawn-Linsley et al. [17], using the TBARS Assay Kit item No. 10009055 from Cayman Chemical Co. (http://www. caymanchem.com).

Reactive Oxygen Species.
For the quantification of reactive oxygen species, the Hydrogen Peroxide Cell-Based assay from Cayman Chemical Item Number 600050 was used. A total of 100 μL of cells and 96 μL of each extract (1500, 1000, 750, 500, 250, 100, 10, and 1 μg/mL) were deposited in a 96-well plate and incubated in an atmosphere of 13-15% CO 2 at 37°C for 24 h. They were then centrifuged, and the supernatant was transferred to a black plate adding 10 μL of assay buffer to the extract and standard samples. Catalase was added to the positive control and the reactive enzyme solution to the standard, control, and extracts. Afterwards, they were incubated with constant orbital stirring for 20 min at a temperature of 20-25°C, and fluorescence was measured at 530-590 nm in an ELISA reader (WHY 101) [18].

Toxicity of Extracts against Artemia salina.
The lethality assay on A. salina was performed as described by Leos-Rivas et al. [19], using the same toxicity criteria. Data were collected with IBM SPSS Statistics version 20 with probit regression to calculate LD 50 . The rats were divided into 5 groups of 5 rats each: three groups were given 1.25, 3.75, or 8.5 mg/kg of lyophilized plant extract resuspended in saline. Group 4 was given rifampin 8.5 mg/kg (positive control) and group 5, saline (negative control). Treatments were administered every 24 h for 7 d, and the doses were selected according to the oral reference dose for toxic noncarcinogenic substances [20,21]. After the established time, blood samples were collected by cardiac puncture; one tube contained anticoagulant for hematological analysis and the other tube, without anticoagulant, was sent to biochemical analysis. The rats were immediately sacrificed by dislocation, and tissues and organs were collected for histopathological analysis in 10% buffered formalin.

Histopathological Analysis of the Liver and Kidney.
The organs preserved in 10% buffered formalin were placed on the dissecting plate. The main lobe of the liver and kidney were selected, and the tissue was placed in a formalin capsule for 7 h. The tissue was later placed in a Histokinet (Van Wissel-digital tissue processor) for 12 h with the capsule being ready and embedded in paraffin. Then, the paraffin cubes with the tissue were sectioned using a Spencer 820 Rotary Microtome. The fine slices of paraffin were placed in a flotation bath (Tissue Floating Bath XH-1001) at 52°C. They were deparaffinized for 15-30 min in a Fisher Scientific IsoTemp Incubator at 60°C. Subsequently, they were submerged in concentrated xylol for 10 min for the microscopic observation.

Analysis of Parameters Evaluated in Male Wistar Rat
Blood after Exposure to Treatments. The twenty-five experimental animal models were weighed before the assay and distributed in cages in 5 × 5 experimental units. After the established time, blood samples with an anticoagulant were collected and hematological parameters were analyzed using conventional methods.
2.9.5. Induction of CYP450 by the Extract. CYP450 induction was performed by the kinetic enzyme method with the commercial kit VIVID ® CYP450 RED (cat. no. P2856) [22]. Ketoconazole 10 μM (as a positive inhibitor) (Sigma-Aldrich Cat. No. UC280), rifampin 10 μM (Rifadin, as an inducer), and 2X buffer diluted with HPLC grade water were placed in a 96-well microplate with 100 μL of different concentrations of the extract and 100 μL of reaction buffer and as a target 200 μL of buffer. A total of 50 μL of 5 nM baculosomes was added to each well, the plate was incubated for 10 min at a temperature of 18-25°C, and a pre-reading was performed. The Vivid-NADP + substrate was prepared, and 10 μL was added to each well and immediately read in an an ELISA (WHY 101) reader at 520 nm.
2.9.6. Statistical Analysis. All the experiments were performed in triplicate, and the data were expressed as mean ± standard deviation (SD). The median lethal doses (LD 50 ) were determined using a Probit regression with the SPSS version 20 software. The data were analyzed against negative control using comparison of pairs of treatment means (t-test), and statistical significance was established as P � 0.01 for a confidence interval of 99%.

Results
The collected plants were identified at the UAAAN Torreon Unit with registration number CRB-Agroecology 002/012, obtaining a yield of 17% (p/p) of the aqueous extract. In the characterization of the C. ternifolia extract by HPLC (Figure 1), 30 compounds were found, and the retention time of three of them corresponded to the standards used: rutin (56.652), glycoside, quercetin (53.409), and epicatechin (34.661). Table 1 shows the results obtained with the aqueous extract of C. ternifolia that caused an induction of eryptosis of 73% at a concentration of 100 μg·mL − 1 ; the positive control (rifampin) caused an induction of 76% at a concentration of 25 μg·mL − 1 . There was a slight increase in the MCV with regard to normal values (>97 fL). The TBARS/ MDA values were between 7.5 and 8.6 μM greater than normal range (18.6-39.4 μM). In the determination of H 2 O 2 , mean values were 14 μM in comparison with 4.0 μM for the control (without exposure to the extract). The aqueous extract of C. ternifolia had mild toxicity against A. salina.
In the experiment with the extract of C. ternifolia administered orally to male Wistar rats only at a dose of 8.5 mg/kg, spongiform changes were found in the proximal tubules of renal tissue ( Figure 2) and a lymphoid infiltrate was present in liver tissue (Figure 3) after exposure to the extract in an assay performed in triplicate on the animal model.
The blood values of the animals under study before and after the administration of the aqueous extract of C. ternifolia at 8.5 mg/kg are shown in Table 2.
In these, platelets and leukocytes decreased and urea and liver enzymes (ALT, AST, and ALP) increased. Table 3 shows the results of CYP3A4 inhibition of human baculosomes by the extract at different concentrations.

Discussion
The extract yield obtained from the plant was 1.7%. There are no previous reports although it has been found that the family Asteraceae has a yield of 8-10% when latex is extracted from its root [23] and aerial parts [24]. In the chemical study of C. ternifolia, rutin and quercetin were found, and there are previous reports of the presence of these flavonoids from Escandón-Rivera [9].
Some drugs such as ipratropium bromide and albuterol sulfate, which are used for obstructive lung disease, produce eryptosis [25]. Plant components that cause this same problem have been reported [26]. These natural components have antimicrobial, antiparasitic, anti-inflammatory, anticarcinogenic, cardiotonic, immunosuppressive, anti-infertility, neuroprotective, and antiatherosclerotic properties. One example of these is plumbagin (5-hydroxy-2-methyl-1.4-naphthoquinone). This phenolic compound induces eryptosis and/or apoptosis by mitochondrial depolarization, inhibiting the protein family Bcl-2. In this study, we found   that C. ternifolia possesses phenolic compounds and that exposure of erythrocytes to the extract caused the induction of eryptosis. The polyphenolic components showed minimal hemolytic activity on human erythrocytes [27]. In this study, C. ternifolia extract showed a slight increase with regard to normal values. In another study, the membrane of human erythrocytes exposed to radiation in the presence of quercetin was analyzed, and it was found that the concentration of TBARS increases in the presence of this compound, in addition to causing hemolysis [28]. In this study, something similar was observed with the presence of the C. ternifolia extract, which contains quercetin (increased values of TBARS/MDA).
A concentration greater or equal to 50 μM of pure compounds is considered cytotoxic for animals, plants, and bacteria. However, in human blood, the cytotoxic concentration is ∼35 μM [29]. Other authors report that it is near zero [30]. The aqueous extract of C. ternifolia used in this study has low toxicity and had weak toxicity against A. salina at 777 μg·mL − 1 . According to the following criteria, DL 50 > 1000 μg·mL − 1 is nontoxic; ≥500 and ≤1000 μg·mL − 1 is weakly toxic; and <500 μg·mL − 1 is toxic [31]. No previous studies of the toxicity of this plant were found.
There is research on the effects of phenolic compounds on cell function. Among these compounds, quercetin has been associated with oral toxicity [32]. The extract of C. ternifolia at a concentration of 8.5 mg/kg produced spongiform changes in the proximal tubules of renal tissue and the presence of a lymphoid infiltrate in liver tissue. Mossoba et al. [33], in a study of cell and mitochondrial function in human cells of the proximal tubule of the kidney (HK-2), reported elevated levels of nephrotoxicity biomarkers in C. ternifolia. Singh et al. [34], in studies of E. alba of the family Astereaceae, which contains flavonoids, reported similar results in the liver with adverse biochemical and histological effects at a concentration of 2,500 mg/kg. They observed that the oral administration of an aqueous extract of E. alba in female rats produced harmful effects on biochemical parameters and increased transaminase levels after liver damage due to induced membrane permeability; in a similar study alteration was found [33]. Later, the same author evaluated the aqueous extract of Phyllanthus niruri leaves and found liver damage at greater doses [35].
The group of enzymes that metabolize xenobiotics are called P450 cytochromes (CYPs), in particular, the enzyme CYP3A4. Inhibition of the activity of CYPs causes toxic effects in the body, and induction increases in its expression, affecting the metabolism, elimination and efficacy of drugs. Both processes can be evaluated and quantified with the modulation of CYP3A4 [36].
In recent studies, it has been found that the ligands of CYP450 interact with organic molecules [37]. In the study performed, the response of CYP450 to exposure of the study plant was observed.
The phytochemical components found in plants are organic substances that are metabolized in the liver with the possibility of inhibiting or inducing the expression of CYP3A4 in mammals. The characteristic of this enzyme is that the substrate occupying the active site leads to an inappropriate decrease in water and a decrease in the displacement of activated oxygen in water; thus, hydration is considered to be highly dependent on the ligand of some CYP isoforms. In addition, CYPs that metabolize drugs are characterized by a low degree of substrate selectivity [38]. CYP3A4 is involved in the oxidation of substrates and is responsible for the metabolism of drugs and phytochemical components, such as quercetin, a substance that is found in plants and is used in traditional medicine. This substance is considered an inhibitor of this enzyme, and also, it is necessary to evaluate the risks and benefits of its use as a natural product [32]. The function of these inhibitors leads to an increase in the effects that result from the consumption  of their phytochemical components since they are not metabolized in the liver or in any other place in the body. Phenolic components, such as rutin, epicatechin, and chlorogenic acid, have also been found in the study plant. As for quercetin, epicatechin, and rutin, it has been shown that they have the potential to attenuate radiation and cytogenic damage [39]. In this study, an assay was performed to determine the induction of CYP3A4, where activity was observed. Therefore, it can be considered that phenolic compounds act as antioxidants, but can also carry out prooxidant reactions, exerting cytotoxic effects on cells and tissues as well as the induction and inhibition of CYP450. It is also possible to consider that there is inhibition of CYP3A4 when there is exposure to the studied plant extract.
In this in vitro study, the aqueous extract of C. ternifolia showed moderate toxicity against A. salina, alterations in human erythrocytes, and inhibition of CYP3A4. In vivo results indicate high levels of biomarkers for nephrotoxicity and hepatotoxicity, possibly due to the chemical compounds, such as flavonoids, found in this study.

Conclusions
Based on the study conducted, C. ternifolia should be used for short periods of time because it has low toxicity against A. salina and produces undesirable effects in the liver and kidney of Winstar rats at 8.5 mg/kg. In traditional medicine, this plant is usually used for long periods of time, such as in the control of hiperglycemia. It is important to continue with the studies to identify the chemical components responsible for the biological activity, toxicity, and mechanism of action.

Data Availability
The data used to support the findings of this study are included within the article.

Conflicts of Interest
The authors declare that there are no conflicts of interest regarding the publication of this paper.