Herbal Medicines: Cytotoxic Effects of Chenopodiaceae Species Used in Argentinian Folk Medicine

Abstract

Chenopodium. sp. (Chenopodiaceae).., common name “Paico,” is a medicinal plant that has been used for centuries as an antirheumatic, anthelmintic, sedative, and analgesic agent and as a spice in food. The main component of the essential oil is ascaridole. The aim of this work was to evaluate the possible cytotoxic effects of aqueous extracts of Chenopodium ambrosioides. L. (ten specimens from different parts of the country), Chenopodium multifidum. L. (three specimens) and to compare them with a nonaromatic Chenopodium. (Chenopodium album.). The extracts [decoction (D) and infusion (I)], which are the usual way of consumption by the population] were assayed by mitotic index in different concentrations (0, 1, 10, 100, and 1000 µg dried plant/mL of culture), on lymphocyte cultures from four healthy donors, as a cytotoxicity biomarker. One-way analysis of variance (ANOVA) test was performed. The results show a statistically significant decrease in all the specimens of C. ambrosioides. and C. multifidum., whereas no effect was observed in the analysis of C. album.. These results suggest a cytotoxic effect of C. ambrosioides. and C. multifidum. aqueous extracts related with the essential oil of the plant.

Keywords:

• Alternative medicine
• ascaridole
• Chenopodium
• cytotoxicity
• essential oil
• genotoxicity
• paico

Introduction

The use of herbal medicines is widespread and growing with as many as 3 of 10 Americans using botanical remedies in a given year. According to the World Health Organization, approximately 75% of the global population (most of the developing world) depends on botanical medicines for their basic health care needs (Akerele, 1985; Duke, 1991). The folk medicine of Argentina employs many herbs to counteract diverse diseases such as catarrh, bronchitis, pneumonia, ulcers, and diarrhea (Ratera & Ratera, 1980; Toursarkissian, 1980; Martinez-Crovetto, 1981).

Members of the Chenopodiaceae family Chenopodium multifidum. L. and Chenopodium ambrosioides. L., popularly known as “Paico” or “Pazote” grow wild in Central and South America. They are one of the plants widely used in popular medicine as vermifuge, emmenagogue, and abortifacient. They are perennial, aromatic plants, more or less pubescent, with lower branched stems often postrated.

The essential oil of paico is a mucous membrane irritant that irritates the gastrointestinal tract, kidneys, and liver. Also, it is a CNS poison. Overdoses of this oil have caused deaths in both man and rats. The main components are monoterpenes, e.g., ascaridole, which is a terpenic peroxide (in average 70% of the essential oil) and has the highest concentration in the seeds of the plant. The primary symptoms during an acute intoxication are gastrointestinal, such as gastroenteritis with diffuse hyperemia at first, and alterations in the CNS later as headache, facial flushing, impaired vision, vertigo, lack of coordination, and paresthesias (Martindale, 1982; Pousset, 1989).

The principal pharmacologic uses described in the literature of Chenopodium. oil are as an anthelmintic, abortifacient, and emmenagogue (Conway & Slocumb, 1979). It is used for the treatment of digestive, respiratory, urogenital, vascular no, and nervous disorders, for metabolic disturbances such as diabetes and hypercholesterolemia, and as a sedative, an antipyretic, and an antirheumatic (De Feo & Senatore, 1993). Also, Kishore et al. (1993) have described fungitoxicity against dermatophytes such as Aspergillus fumigatus. and Cladosporium trichoides..

Aqueous extracts or whole plant preparations are ingested based on their theoretical or demonstrated pharmacologic activity to combat diseases or symptoms. Health outcomes can be tested using standard research methods. Although beliefs of users may differ, both principles of action and methods of evaluation of herbal therapy should be familiar to conventional health care practitioners (Barret, 1999). Although all the ethnopharmacologic properties have been demonstrated for the Chenopodium. oil, there are very few reports about the pharmacologic effects of these extracts (Kliks, 1985).

In the validation of new screening biological biomarkers, three points have to be taken into account: reproducibility, specificity, and sensitivity of the parameters evaluated. The mitotic index may be considered to be a good candidate for cytotoxic effect screening as it appears to be a reproducible, specific, and sensitive biomarker (Rojas et al., 1993).

Considering that in Argentina, aqueous extracts of Chenopodium. sp., such as decoction or infusion, are widely used by the population, the potential cytotoxic effect of these aqueous extracts was tested by mitotic index evaluation in lymphocyte cultures exposed in vitro. to the extracts from four healthy donors.

Materials and Methods

Plant material

Different species of the Chenopodiaceae family (C. multifidum., C. ambrosioides., and C. album.) were assayed. The specimens were collected from different zones of Argentina (Fig. 1). The characteristics of the 12 specimens collected are described in Table 1. The specimens were identified in the field by Dr. A.A. Gurni, and voucher specimens are kept at the herbarium of the Museum of Pharmacobotany “Juan A. Dominguez” (School of Pharmacy and Biochemistry, University of Buenos Aires).

Figure 1 Different states of the Argentinian country where specimens were collected.

Table 1.. Species of Chenopodiaceae family evaluated (characteristics and place of origin)

Plant^a.	Collection characteristics
Chenopodium ambrosioides.
A.	Tigre, “La Flora” Island (near Luján River), Gurni, s.n., III-1997.
B.	State of Buenos Aires, San Isidro (a), Gurni, s.n.; IV-1997.
C.	State of Buenos Aires, San Isidro (b), Gurni, s.n.; IV-1997.
D.	State of Buenos Aires, Municipio Urbano de la Costa, Santa Teresita, Gurni, s.n., IV-1999
E.	Capital Federal (a), Gurni, s.n., V-1999.
F.	Capital Federal (b), Gurni, s.n., V-1999.
G.	State of Buenos Aires, Núñez, Ciudad Universitaria, Gurni, s.n., IX-1999.
H.	State of Mendoza, Malargüe Dept., Malargüe, Gurni, s.n., III-1998.
I.	State of Jujuy, San Salvador de Jujuy Dept., Ciudad Nieva, Gurni, s.n., III-1999.
Chenopodium multifidum.
J.	State of Chubut, Comodoro Rivadavia, Gurni & Gratti,s.n., III-1996.
K.	State of Chubut, Comodoro Rivadavia, Rada-Tilly (a), Gratti, s.n., 31-I-1999.
L.	State of Chubut, Comodoro Rivadavia, Rada-Tilly (b), Gratti, s.n., 27-III-1999.
Chenopodium album.
M.	State of Buenos Aires, San Isidro Dept., Las Barrancas, Gurni, s.n.; IV-1999.
	As this is a nonaromatic species, it was used as negative control of the essential oil of the Chenopodiaceae family.

^aSites are designated in Figure 1.

Preparation of aqueous extracts (decoction and infusion)

Decoction and infusion (extemporaneous aqueous preparations) were prepared according to Pharmacopeia Argentina.. Both preparations were sterilized through an 0.22-µm filter, and stored at − 20°C.

Lymphocyte cultures and microscopic evaluation

Heparinized blood samples (0.5 mL), obtained from four healthy donors (25–35 years old), were placed in a sterile flask containing 7 mL of HAM F-10 medium, supplemented with 1.5 mL of fetal bovine serum (Gibco) and 0.1 mL of PHA (Gibco-Invitrogen, Argentina). Then, Chenopodium. preparations were added in five different concentrations (0, 1, 10, 100, and 1000 µg/mL of the culture). The concentration of the aqueous extracts was chosen considering a wide range to include the average consumption of herb by the population.

The cultures were incubated for 72 h at 37°C. Two hours before harvesting cells, 0.2 mL of colcemid (10 µg/mL) (Sigma, Argentina) was added to each culture flask. Cells were centrifuged at approximately 800–1000 rpm for 10 min. The supernatant was removed, and 5 mL of a prewarmed (37°C) 0.075 M KCl hypotonic solutions were added. Cells were resuspended and incubated at 37°C for 45 min. The supernatant was removed by centrifugation, and 5 mL of a fixative (methanol:glacial acetic acid, 3:1) was added. The fixative was removed, and the procedure was repeated twice. To make the slides, 5 drops of the fixed cell suspension were laid on clean glass slides and air-dried. Cells were stained following a modified fluorescence plus Giemsa (FPG) technique (Perry & Wolff, 1974). Slides were stained for 20 min in a 0.05% w/v Hoescht 33258 solution, rinsed with, tap water and placed under a near-ultraviolet lamp for 90 min. Then the slides were covered with Sorensen's buffer pH = 6.8, and stained with a 3% Giemsa solution in phosphate buffer (pH = 6.8) for 15 min. The microscopic evaluation was done in an Olympus BHT microscope under 10 × . The mitotic index was calculated as the proportion of metaphase for 2000 cells, in each herb, preparation, donor, and concentration.

Statistical analysis

Statistical analysis was performed using two-way analysis of variance (ANOVA) randomized block design for each biomarker (Graphpad Instat).

Results

The results are presented in Tables 2-5 considering (a) Species of Chenopodium.: C. ambrosioides., and C. multifidum.; (b) type of preparation evaluated: decoction and infusion. Elsewhere, C. album. is included as negative control of the essential oil in all of them.

Table 2.. Mitotic index in lymphocyte cell cultures exposed to infusion of C. ambrosioides.

	Mitotic index (%)
	Negative control of extract	1 µg/mL	10 µg/mL	100 µg/mL	1000 µg/mL
C. album. (Negative control essential oil)	5.32±0.25	4.69±0.12	4.89±0.12	4.t3±0.18	4.70±0.62
C. ambrosioides.
A.	5.04±0.13	4.23±0.12^a.	4.51±0.17^a.	4.32±0.14^a.	3.96±0.15^a.
B.	4.95±0.35	4.39±0.32^b.	3.84±0.29^b.	3.18±0.36^b.	2.75±0.36^b.
C.	5.32±0.25	4.58±0.11^a.	3.40±0.30^a.	2.74±0.10^a.	2.13±0.24^a.
D.	5.54±0.18	4.43±0.12^a.	3.55±0.25^a.	2.81±0.22^a.	2.10±0.26^a.
E.	4.70±0.11	3.97±0.22^a.	3.10±0.27^a.	2.30±0.11^a.	1.55±0.10^a.
F.	4.70±0.11	3.72±0.21^a.	3.27±0.37^a.	2.56±0.26^a.	1.67±0.17^a.
G.	4.70±0.11	3.50±0.13^a.	3.07±0.24^a.	2.65±0.24^a.	2.17±0.21^a.
H.	4.84±0.32	3.84±0.26^a.	3.42±0.30^a.	2.53±0.11^a.	2.53±0.22^a.
I.	4.90±0.35	4.13±0.22^a.	4.28±0.18^a.	3.17±0.20^a.	1.85±0.18^a.

^a.p < 0.0001.

^b.p < 0.005.

Table 3.. Mitotic index in lymphocyte cell cultures exposed to infusion of C. multifidum.

	Mitotic index (%)
	Negative control of extract	1 µg/mL	10 µg/mL	100 µg/mL	1000 µg/mL
C. album. (Negative control essential oil)	5.32±0.25	4.69±0.12	4.89±0.12	4.13±0.18	4.70±0.62
C. multifidum.
J.	6.28±0.51	5.88±0.47^c.	5.05±0.79^c.	4.91±0.91^c.	3.92±0.66^c.
K.	4.90±0.35	5.07±0.29^b.	4.13±0.31^b.	2.87±0.20^b.	2.02±0.88^b.
L.	4.90±0.35	4.90±0.35^a.	4.47±0.43^a.	2.95±0.13^a.	1.93±0.16^a.

^a.p < 0.0001.

^b.p < 0.0005.

^c.p < 0.01.

Table 4.. Mitotic index in lymphocyte cell cultures exposed to decoction of C. ambrosioides.

	Mitotic index (%)
	Negative control of extract	1 µg/mL	10 µg/mL	100 µg/mL	1000 µg/mL
C. album. (Negative control essential oil)	5.55±0.18	5.27±0.17	5.21±0.06	5.09±0.22	5.06±0.04
C. ambrosioides.
A.	5.05±0.13	3.66±0.25^a.	3.19±0.10^a.	3.15±0.24^a.	2.23±0.19^a.
B.	5.23±0.30	4.38±0.18^c.	3.59±0.29^c.	3.30±0.06^c.	3.23±0.21^c.
C.	5.53±0.18	4.89±0.30^a.	3.91±0.43^a.	2.61±0.27^a.	2.03±0.18^a.
D.	5.53±0.18	4.49±0.24^a.	3.71±0.22^a.	2.84±0.35^a.	2.31±0.05^a.
E.	4.70±0.11	3.59±0.19^a.	3.15±0.24^a.	2.90±0.20^a.	2.13±0.12^a.
F.	4.70±0.11	4.33±0.13^a.	3.70±0.04^a.	2.80±0.08^a.	1.76±0.14^a.
G.	4.70±0.11	3.83±0.02^a.	3.17±0.12^a.	2.33±0.15^a.	2.00±0.14^a.
H.	4.95±0.35	3.60±0.18^b.	3.37±0.18^b.	3.24±0.24^b.	2.94±0.19^b.
I.	4.90±0.35	4.10±0.11^a.	3.31±0.10^a.	2.56±0.09^a.	1.60±0.29^a.

^a.p < 0.0001.

^b.p < 0.001.

^c.p < 0.01.

Table 5.. Mitotic index in lymphocyte cell cultures exposed to decoction of C. multifidum.

	Mitotic index (%)
	Negative control of extract	1 µg/mL	10 µg/mL	100 µg/mL	1000 µg/mL
C. album. (Negative control essential oil)	5.55±0.18	5.27±0.17	5.21±0.06	5.09±0.22	5.06±0.04
C. multifidum.
J.	6.40±0.20	6.01±0.60^a.	5.06±0.72^a.	4.62±0.77^a.	3.82±0.53^a.
K.	4.90±0.35	4.70±0.12^a.	4.00±0.64^a.	2.74±0.09^a.	1.43±0.12^a.
L.	4.90±0.35	4.80±0.08^a.	3.45±0.06^a.	2.88±0.16^a.	1.79±0.29^a.

^a.p. < 0.0001.

When the effect of the aqueous extracts of C. multifidum. on lymphocyte cultures was analyzed, a decrease in mitotic index (MI) was found for decoction and infusion treatments. Our results also show an inhibition of the MI for decoction as dose-related (r = − 0.86) in the three specimens analyzed, and it could be interpreted as cellular death (Rojas et al., 1993) related to the cytotoxicity effect of the aqueous extract (Tables 3 and 5).

When potential cytotoxicity of the aqueous extracts of the nine specimens of C. ambrosioides. on lymphocyte cultures was analyzed through mitotic index evaluation, a significant decrease was found. These results acquired a larger significance for decoction than for infusion (Tables 2 and 4).

On the other hand, C. album. did not induce any change in the mitotic index values of lymphocyte cell cultures exposed to the aqueous extracts (Tables 2 to 5).

Discussion

In the current work, our results show a decrease in the MI values in lymphocyte cultures exposed in vitro. to the aqueous extracts of the nine specimens of C. ambrosioides. and the three specimens of C. multifidum. (Tables 2 to 5). These results could be related to active principles present in the aqueous extracts that could induce cellular death or a delay in any stage of the cell proliferation cycle.

Mitotic (MI) and Replication indexes (RI) are used as indicators of adequate cell proliferation biomarkers. RI is a measure of the relative number of generations during a specified time in culture by the use of bromodeoxyuridine (this method identifies cells that have performed different numbers of DNA replications in culture). MI measures the proportion of cells in the M-phase of the cell cycle and its inhibition could be considered as cellular death or a delay in the cell proliferation kinetics (Rojas et al., 1993). A cytotoxic effect of both medicinal herbs was observed, evidenced by the decrease of the MI (Tables 2 to 5). In previous works, we concluded that neither C. multifidum. nor C. ambrosioides. induced any change in replication index values (Gadano et al., 20002002), so it could be concluded that the aqueous extracts of C. ambrosioides. and C. multifidum. induced cellular death. Other evaluation of the potential cytotoxic effect was done with a nonaromatic species of the same family (C. album.). These results show no effect in the MI values (Tables 2 to 5). Our results suggest that the active principles of the essential oil of the aromatic species (C. ambrosioides. and C. multifidum.) are involved in the cytotoxic effect induced.

These results suggest that C. ambrosioides. specimens and C. multifidum. specimens induce cytotoxic damage in the experimental conditions assayed. Considering that the same effect was induced (with statistically similar magnitude) and they were collected in a widespread area of Argentina, we can conclude that the active principles responsible for the cytotoxic effects are independent of the place where the herb grows, as they are characteristics of the species and they are constituents of the essential oil of the plant.

Genetic biomarker determination would help to estimate the potential toxicity of medicinal herbs in order to establish a regulation of medicinal plant consumption, which could be considered an important way of protecting public health. Physicians should educate themselves and their patients about the efficacy and adverse interactions of herbal agents and the limitations of our current present knowledge of them (Akerele, 1985; Duke, 1991).

Acknowledgments

We are very grateful to Patricia Pedraglio who contributed to improvement of the English of the manuscript. This present work was supported by grants from UBACYT (B-034) and the Alberto J. Roemmers Foundation.