Protective Effect of Beta-Sitosterol against Ethanol Toxemia

Journal of Undergraduate Research at Journal of Undergraduate Research at

Minnesota State University, Mankato Minnesota State University, Mankato

Volume 17 Article 5

2017

Protective Effect of Beta-Sitosterol against Ethanol Toxemia Protective Effect of Beta-Sitosterol against Ethanol Toxemia

Dakota Cooper

Minnesota State University, Mankato

, [email protected]

April Boucher-Zamzo

Minnesota State University, Mankato

, [email protected]

Follow this and additional works at: https://cornerstone.lib.mnsu.edu/jur

Part of the Pharmacology Commons, and the Toxicology Commons

Recommended Citation Recommended Citation

Cooper, Dakota and Boucher-Zamzo, April (2017) "Protective Effect of Beta-Sitosterol against Ethanol

Toxemia,"

Journal of Undergraduate Research at Minnesota State University, Mankato

: Vol. 17, Article 5.

DOI: https://doi.org/10.56816/2378-6949.1203

Available at: https://cornerstone.lib.mnsu.edu/jur/vol17/iss1/5

This Article is brought to you for free and open access by the Journals at Cornerstone: A Collection of Scholarly

and Creative Works for Minnesota State University, Mankato. It has been accepted for inclusion in Journal of

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Collection of Scholarly and Creative Works for Minnesota State University, Mankato.

Protective Effect of Beta-Sitosterol against Ethanol Toxemia

Dakota Cooper, April Boucher-Zamzo

Abstract:

Herbal medications have no indications of interactions with alcohol on their labels. There

has been at least one clinical finding that saw palmetto with active ingredient beta-sitosterol used

for enlarged male prostates caused pancreatic damage. This study with 20 male mice

hypothesized that ethanol would increase the toxicity of beta-sitosterol. It was found that mice

dosed by gavage with 0.1 ml of 95 proof ethanol died starting at the second dose and increased to

100% by the sixth dose (unexpected based on literature). 60-microgram injections of beta-

sitosterol prevented deaths in 80% of the mice past the sixth dose opposite to what was expected.

Beta-sitosterol failed to protect the liver, enlarged the pancreas, and protected the kidneys from

ethanol induced damage.

Introduction:

Large quantities of herbal medications of unknown content claim medical properties,

these are available over-the-counter or on the Internet. Saw Palmetto (Serenoa repens), a plant, is

sold to older men with enlarged prostates (benign prostatic hyperplasia or BPH) or anticipating

BPH that contains this herb or one of its purified active ingredient. One of the ingredients of Saw

Palmetto is beta-sitosterol which has been shown to be pharmacologically active (1). However,

they are not warned about possible toxicity/side effects.

A published clinical study of a 65 year old male with a variety of health problems and

heavy alcohol use developed pancreatitis (inflammation and dangerous deterioration of

pancreatic cells) one week after treatment with Saw Palmetto (2). The doctors did not believe

alcohol was the leading cause of pancreas damage as indicated by very high blood (and urine)

sugar levels which indicated the insulin producing cells of the pancreas were damaged. This is

confusing because alcohol intake also causes pancreatic and liver damage (3). The hypothesis of

the study is that mice given ethanol and beta-sitosterol will have decreased liver and pancreas

function as determined by increased urine glucose (pancreatic damage) or urine bilirubin (causes

jaundice color that indicates liver damage [4]).

Cooper and Boucher-Zamzo: Protective Effect of Beta-Sitosterol against Ethanol Toxemia

Published by Cornerstone: A Collection of Scholarly and Creative Works for Minnesota State University, Mankato, 2017

Materials and Methods:

Twenty male mice of the strain C57/Bl6 ages 6-13 weeks were weighed and separated

into four age-balanced groups of five mice. The groups of mice were then placed into cages that

had water and food freely available. The animal protocols were approved by the Minnesota State

University Institutional Animal Care and Use Committee (IACUC) approval number 16-06

Mice dosed twice per week on Mondays and Wednesdays with 0.1 ml 0.849% NaCl (oral

saline by curved gavage needle), 0.1 ml sterile corn oil i.p. (abdominal injection) as the control;

0.1 ml Saline, 0.1 ml 60 µg Beta-Sitosterol in corn oil, 0.1 ml 0.425% NaCl plus 95 proof

ethanol (oral ethanol), i.p. corn oil; and 0.1 ml oral ethanol, 0.1 ml i.p. beta-sitosterol. These

groups were selected to give clarity on how the compounds, ethanol and beta-sitosterol,

interacted individually and in combination. Mice euthanized after week eight when one control

mouse died. Oral dosing was based on a previous study that was investigating deleterious effects

of ethanol (5). Abdominal dosing by injection was based on a similar study in mice (6).

Urine collected on Fridays and analyzed with VWR urine test strips. Initially the urine

was analyzed for glucose (pancreas damage), ketones (near death or ketoacidosis from pancreas

damage), bilirubin, urobilinogen (liver damage and kidney damage) and protein (kidney damage

prior to death). After mice started to die from the treatments, the urine analysis was expanded to

test for pH (acidosis), and leukocytes (white blood cells in urine) in addition to the original five

compounds. Urine was collected from the mice by spinning the by the tail and then picking the

mice up by the tail and scruff. The mice were then placed over a funnel and gentle pressure was

applied to the lower abdomen.

Mice dissected immediately after euthanization or frozen after death, weighed and

dissected following thawing and organ data collected. Organ analysis was performed on the

liver, intestines, pancreas, kidney and heart. Organs were weighed and inspected for condition.

Two-way ANOVA was run using SYSTAT 9.0® software with P<0.05 accepted as

statistically different.

Results:

Journal of Undergraduate Research at Minnesota State University, Mankato, Vol. 17 [2017], Art. 5

https://cornerstone.lib.mnsu.edu/jur/vol17/iss1/5

DOI: 10.56816/2378-6949.1203

There was no statistical difference in the body weights of the treatment groups as

determined by two-way ANOVA and seen in Table 1.

Table 1. Mean+SEM body weights of treatment groups.

Treatment Groups

Body Weights

Saline and corn oil

25.456±0.890

Saline and beta-sitosterol

26.100±1.069

Ethanol and corn oil

23.273±2.398

Ethanol and beta-sitosterol

25.042±0.506

Mortality was seen in the two treatment groups that were dosed with ethanol following

the second dose. In the ethanol treatment group, ethanol and corn oil, two mice died after dosing

and one mouse died in the ethanol and beta-sitosterol group as seen in Figure 1. Mortality

increased after each subsequent dose in the ethanol treatment group until all mice were dead,

after dose six. One mouse in the control group died in week eight whereupon the study was

terminated.

Figure 1. Mortality of treatment groups based on dose number. * denotes different from control p<.05 (LSD posthoc

following significant effect of ethanol and beta-sitosterol effects). Ethanol mortality unexpected based on published

study results (3).

Following dissection the intestines were weighed and visually inspected for appearance

and condition. Statistical analysis of the intestinal weights indicated a statistical difference

between groups treated with ethanol and groups not treated with ethanol as seen in Figure 2.

Cooper and Boucher-Zamzo: Protective Effect of Beta-Sitosterol against Ethanol Toxemia

Published by Cornerstone: A Collection of Scholarly and Creative Works for Minnesota State University, Mankato, 2017

Visual inspection of the intestines revealed distension of the intestines in groups treated with

ethanol.

The livers of the mice in the ethanol treatment groups were visually dark red in color with

dark spots throughout the tissue. There was a statistical difference between groups treated with

ethanol and groups not treated with ethanol as determined by a two-way ANOVA and as seen in

Figure 2.

Figure 2. Ethanol showed expected negative effects on liver (damage) and intestinal weights (distended but lacking

brush border) compared to control mice. * denotes p<.05 compared to control (ethanol effect alone). Error bars =

SEM.

Visually there was no noticeable difference in pancreases other than size, which is

accounted for based on the weight data obtained. Statistical analysis showed a statistical

difference in the weights of pancreases in groups treated with beta-sitosterol compared to groups

not given beta-sitosterol, as seen in Figure 3.

Journal of Undergraduate Research at Minnesota State University, Mankato, Vol. 17 [2017], Art. 5

https://cornerstone.lib.mnsu.edu/jur/vol17/iss1/5

DOI: 10.56816/2378-6949.1203

Figure 3. Beta-sitosterol increased pancreas size. * denotes p<.05 (beta-sitosterol effects alone).Error bars = SEM.

Ethanol reduced kidney weight. This result was not as pronounced in the beta-sitosterol

and ethanol treatment group. Statistical analysis showed an interaction between ethanol and beta-

sitosterol, as seen in Figure 4.

Figure 4. Interactions of Ethanol and Beta-sitosterol on the kidneys. * denotes p<.05 different from control and

ethanol and beta-sitosterol dosed mice (ethanol*beta-sitosterol interaction term meaning beta-sitosterol prevented

kidney damage). Error bars=SEM

Cooper and Boucher-Zamzo: Protective Effect of Beta-Sitosterol against Ethanol Toxemia

Published by Cornerstone: A Collection of Scholarly and Creative Works for Minnesota State University, Mankato, 2017

Discussion:

Ethanol binge-like drinking caused intestine and liver damage and kills mice as expected.

The mechanism of intestinal distension was not investigated by this study. The mice which were

treated with ethanol showed signs of liver damage, which is consistent with current literature.

Beta-sitosterol caused an enlargement of the pancreas. This is expected if it represents

hypertrophy due to damaging inflammation. This contradicts previous studies which indicated

beta-sitosterol as an anti-inflammatory compound (7). However, the vast majority of studies

involving beta-sitosterol have been focused on the prostrate and it is currently unknown if the

anti-inflammatory property extends to other organs as well. As glucose administration is the only

method to prevent alcohol toxemia in humans due to hypoglycemia, pancreatitis may explain

why mice were protected from ethanol. This result needs future experiments to check blood

glucose and insulin levels as the urinalysis was inconclusive except for higher protein and

ketones in dying mice.

Beta-sitosterol mitigated the kidney damage by the ethanol. This was determined through

statistical analysis. This is unexpected but may be a key factor in mice surviving ethanol.

Phytohormones, such as beta-sitosterol, have been shown to prevent renal dysfunction (7,8). This

was not the focus of this study. Future studies are needed to investigate the mechanism of kidney

protection.

Decreased mortality was seen in the beta-sitosterol and ethanol treatment compared to the

ethanol treatment. This result was interesting as increased mortality was expected. This result

shows that data reveals unknown mechanisms.

Acknowledgements:

As researchers we thank our mentor, Dr. Steven Mercurio, for guiding us through the

research process. Brent Pearson for instructions on handling the animals and the Animal care

facilities team for project support. Flevis Waindim for the heart and kidney data. The Minnesota

State University Mankato Undergraduate Research Center for funding.

Journal of Undergraduate Research at Minnesota State University, Mankato, Vol. 17 [2017], Art. 5

https://cornerstone.lib.mnsu.edu/jur/vol17/iss1/5

DOI: 10.56816/2378-6949.1203

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Cooper and Boucher-Zamzo: Protective Effect of Beta-Sitosterol against Ethanol Toxemia

Published by Cornerstone: A Collection of Scholarly and Creative Works for Minnesota State University, Mankato, 2017