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- Guayusa (Ilex guayusa, Aquifoliaceae)
- Toxicology
- Safety
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Date:
05-13-2016 | HC# 041661-544
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Re: Guayusa (Ilex guayusa) Extract Is Nontoxic in Rodent Studies and In Vitro Assays
Kapp
RW Jr, Mendes O, Roy S, McQuate RS, Kraska R. General and genetic toxicology of
guayusa concentrate (Ilex guayusa). Int J Toxicol. 2016;35(2):222-242.
Guayusa (Ilex
guayusa, Aquifoliaceae) is a close relative of yerba maté (Ilex paraguariensis) and is
traditionally consumed as a tea or as chewed leaves in the Amazonian region.
Guayusa has been found to contain active compounds known as methylxanthines, including
caffeine, known to have cellular and systemic stimulatory activity. Data on the
safety and adverse effects of guayusa are limited. This study investigated its safety
profile using in vitro and in vivo studies with a concentrated water extract of
guayusa (procured from Runa LLC; Quito, Ecuador).
Guayusa concentrate (GC) was prepared by brewing
leaves in hot water as for tea, at a per weight ratio of 1.3 to 1.6:1 for two to
four hours. In vitro, the Ames test was used to gauge mutagenic activity.
Briefly, this assay measures genetic mutations in Salmonella typhimurium and Escherichia
coli. Water was used as a negative control and several known mutagenic
compounds, such as sodium azide, were employed as positive controls. GC
concentrations of 1.58, 5.0, 15.8, 50, 158, 500, 1580, and 5000 µg/plate were
used, and mutation factor (MF) was calculated as the ratio of mutations
observed in the test group to those in the control group (MF of 2 or 3,
depending upon the strain, is considered mutagenic). A second in vitro test
used to gauge GC toxicity was the chromosomal aberration assay. Human
peripheral blood lymphocytes were incubated with GC for four and 20 hours, and
for four hours with GC and a metabolic activation system mix; two compounds
known to induce chromosomal aberration were used as positive controls, and pure
caffeine was tested for comparison.
Three in vivo tests, both acute and long term, in
rats also were conducted. To determine acute toxicity, female rats between eight
and nine weeks old, said to be more sensitive than males, were studied. GC was
administered orally by gavage at 5000 mg/kg to a single rat. When death did not
occur, the same dosage was given to two other rats. As no mortality occurred,
this study was stopped. Animals were observed daily for 14 days following the
GC ingestion and weighed on days seven and 14; they were then sacrificed and
necropsies were conducted. To determine dosages for a 90-day study, a 14-day
study was conducted in 70 rats (both male and female) at six to seven weeks old,
divided into seven treatment arms, each with five males and five females. Treatments
included a distilled water control, three GC dosages (1200, 2500, and 5000
mg/kg/day), and three caffeine dosages comparable to the caffeine content in
the GC dosages (36, 75, and 150 mg/kg/day). Caffeine dosages were determined
based on expected caffeine content of 3% in GC; all treatments were given by
gavage at 10 ml/kg. Weights and food consumption were recorded at baseline and
on days three, seven, 11, and 14 of the study. Animals were euthanized on day
15. During this experiment, dosing samples were assessed on days one, seven,
and 14 for caffeine and chlorogenic acid content to confirm stability of the preparation.
For the 90-day in vivo experiment, GC dosages of
0, 1200, 2500, and 5000 mg/kg/day and a single caffeine dose of 150 mg/kg/day (the
amount in the 5000 mg/kg/day GC dosage) were chosen. This experiment used 100
rats, both male and female, at eight weeks of age. At baseline and day 81,
rats' eyes were screened. General health was assessed daily and weekly, with
weights and food consumption measured throughout the experiment. At day 86 or 87,
urine and blood samples were taken. At the end of the study, animals were
euthanized, and necropsy and histological analysis were conducted. Dosing
samples and the original GC lot were tested for stability on days one, 43, and
94.
Chemical characterization of the main lot of GC
detected caffeine at 36 mg/ml, chlorogenic acids at 52 mg/ml, and small amounts
of several catechins and theobromine. GC was also analyzed for the presence of
a number of major plant compounds (e.g., delphinidins, beta-sitosterol,
genistein) that were not found. The extract was found to be adequately stable,
and actual caffeine dosages given in all studies were within 15% of the
expected amounts. In the Ames test, no problems such as toxicity,
precipitation, or contamination were seen. The MF did not increase
significantly in any strain at any dose level, so it was determined that GC is
not mutagenic. Also, neither GC nor dose-equivalent concentrations of caffeine
caused chromosomal aberrations in the chromosomal aberration assay.
In the acute toxicity study, rats were hypoactive
and displayed salivation, respiratory, and fecal abnormalities, and hunched
posture. At day three, these adverse effects were gone, and the animals
remained healthy through day 14. It was concluded that the oral median lethal
dose (LD50) of GC is > 5000 mg/kg body weight for female rats. In
the 14-day experiment, no death was observed. Hypoactivity and salivation were seen
in animals given 5000 mg/kg/day of GC and 75 or 150 mg/kg/day of caffeine. In
general, weight loss and decreased food intake occurred in animals consuming either
GC or caffeine. This effect disappeared during the course of the study. Necropsy
did not show any abnormalities associated with GC or caffeine.
During the 90-day in vivo study, no deaths linked
to GC treatment were reported. Three animals died during the study of what were
believed to be unrelated causes (though exact cause of death could not be
determined) and one was sacrificed due to ill health caused by a dental problem.
Hypoactivity and salivation were noted in some animals taking either GC or caffeine.
No animals showed any ocular changes. Across the study, weight and food efficiency
declined in treated groups, but nonsignificantly. According to the histology,
weights of fat pads and several organs in both males and females of the GC and
caffeine groups were significantly decreased as compared to control animals,
and hypertrophy of salivary glands was observed. Urine composition was
unchanged in male rats, with unimportant reduction in protein content in female
rats in the high-dose GC and caffeine groups. Small, dose-dependent changes in
blood measurements (hemoglobin, red blood cell distribution width, and others)
in females, decreased blood triglycerides, and increased cholesterol and liver
function enzymes were noted in rats on both GC and caffeine. The authors review
previous literature reporting most of these biological effects to result from
caffeine consumption.
Overall, this study reports that GC does not show
toxicity in the assays and animals employed here. Adverse effects noted at high
doses were most likely due to the caffeine content of GC, as caffeine was
tested alongside the botanical and caused similar adverse effects. Clarifying
the ideal dosage and adverse effects in a clinical setting is worthy of further
study.
This study was financially supported by Runa LLC.
Three of the authors (RW Kapp Jr, R. Kraska, and RS McQuate) are independent
consultants to Runa LLC.
—Amy C. Keller, PhD
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