12 January - 20 June 2016

Toxicology of ethanolic extract of Stevia rebaudiana Bertoni leaves investigated

A study published in Regulatory Toxicology and Pharmacology by researchers from the National Centre for Food Safety Risk Assessment, Beijing, China, has sought to assess the toxicity of ethanolic extracts of Stevia rebaudiana Bertoni leaves via a series of in vitro and in vivo tests.

A study published in Regulatory Toxicology and Pharmacology by researchers from the National Centre for Food Safety Risk Assessment, Beijing, China, has sought to assess the toxicity of ethanolic extracts of Stevia rebaudiana Bertoni leaves via a series of in vitro and in vivo tests.

 Zhang et al. note that aside from steviol and its glycosides, stevioside and rebaudioside A, several other products, including flavonoids and vitamins, can be isolated from the leaves of Stevia rebaudiana Bertoni.  A previous study by Lopez et al. (2016) has indicted that Stevia rebaudiana ethanolic extract shows “better anti-oxidant properties and anti-proliferative effects in tumour cells” than stevioside. The researchers note that this, along with the anti-hyperglycaemic, anti-hypertensive and other effects of stevioside and related compounds, suggests stevia may have a further role to play in food additives and supplements. However, as toxicological studies on stevia ethanolic extracts are very limited, they aimed to “evaluate the toxicity of ethanolic extract of Stevia rebaudiana Bertoni leaves through a battery of in vitro and in vivo tests”.

Initially, Zhang et al sought to determine genotoxicity by performing three tests. Bacterial reverse mutation assay was conducted using histidine-deficient strains of Salmonella typhimurium, mouse bone marrow micronucleus assay was performed using 50 mice given two doses of Stevia extract at 2500, 5000 and 10000 mg/kg body weight while mouse sperm malformation assay was conducted using 35 mice given doses of 2500, 5000, 10000 mg/kg body weight daily of 5 days. While for all three tests, the positive control groups showed increases in revertant colonies, micronuclei formation and sperm malformation respectively, the stevia extract treatment groups showed no such increases compared to the control. Zhang et al state that taken together, their tests indicated that “Stevia rebaudiana Bertoni leaves extract showed no genotoxicity under multiple in vitro and in vivo tests”.

Zhang et al. then performed a 90-day feeding experiment in order to assess “sub-chronic oral toxicity”.  Eighty Sprague-Daley rats, equally gender split, were divided into 4 groups: a control and those given stevia extract at dietary concentrations of either 1.04%, 2.08% or 3.12%. Zhang et al note that these are equivalent to safety factors of 100, 200 and 300 times the recommended daily intake of extract (500mg/day) for an adult of approximately 60kg. Body weights and food consumption were recorded weekly, while blood was taken at the mid-point and end of the study. At the end of the study organs including liver, kidney spleen heart and testes or ovaries) were weighed, recorded and examined.

Zhang et al. note that “no mortality or treatment-related adverse clinical appearances were found” during the study. They also note that no significant differences in food consumption, body weight or absolute and relative (to body weight) organ weight were found between the treatment and control groups. The researchers indicate that “sporadic significant changes of several blood parameters and leukocyte differential count” was found in female rats in the middle doses group but this was not repeated in the low or high doses groups. Female rats in the high dose group showed “significant reduction of cholesterol, total protein and albumin” compared to the control, suggested to be due to the lipid- lowering properties of polyphenols.

In discussion, Zhang et al. indicate that their results demonstrate that “Stevia rebaudiana leaves ethanolic extract has no adverse effects in the genotoxicity study and sub-chronic oral toxicity study” at dosage levels up to 300 times that of the recommended intake and suggest this provides supportive evidence for the safety of this extract which may have uses in functional foods and nutritional supplements beyond sweetener.

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