Name: Ben Alcini

Date: 12/16/15

Title of Research Project: Does Panax Quinquefolius (American Ginseng) decrease Alanine

Transaminase levels of hepatocytes of adult rats?

Name of Primary Research Advisor: Dr. Najma Javed

Summary of Proposed Research Project:

Background: The liver detoxifies the blood as well as help break down molecules into substances the body can use. Many things can affect the liver, and once damaged there are little ways of treatment. ALT, and amino transferase, is used to detect damage in the liver early on to help either prevent or reverse the damage. These enzyme converts alpha-amino groups into alpha-keto groups. This allows the body to more readily use these substances that is ingested. More often in the present, people are using herbal substances as a “home remedy” for many different types of disease, and most of them that are commonly used do help treat the problems. Ginseng is an herbal supplement that is thought to boost the immune response and aid in digestion and has been proven to aid in blood sugar levels. The purpose of this study is to determine whether or not Panax Quinquefolius (American Ginseng) does benefit those with cirrhosis of the liver. Methods: Ten adult Lewis rats will be randomly placed into two groups, the control group (n=5) of a normal diet and an experimental group (n=5) of which will intake an American Ginseng supplementation into their diets. The experimental rats will receive the ginseng supplementation for 25 days. After the completion of the 25th day, portions of the livers will be removed and broken down into usable samples. A colormetric assay will be performed on these samples along with standards to obtain the ALT OD values for both the control and experimental groups. A two-tailed students t test will be ran on the data collected to show the comparisons between the two groups. All data present will be in the mean +/- SEM format, and the significance of the data will be set at p<0.05. Anticipated Results: A statistically significant reduction in ALT levels will be seen between the control group and the experimental group. This reduction will confirm that American Ginseng can potentially help decrease liver damage with future testing. Conclusions: The reduction of the ALT levels could indicate that American Ginseng can be used to treat liver injuries, upon future testing. This can potentially help in the push for alternative medicines based on further studies and availability of the supplements.

A, Research Question:

Does Panax Quinquefolius (American Ginseng) increase Alanine Transaminase levels of

cirrhotic hepatocytes of adult rats?

B, Null Hypothesis:

Panax Quinquefolius (American Ginseng) does not increase Alanine Transaminase levels of

cirrhotic hepatocytes of adult rats.

C. Background on which research question is based

Cirrhosis of the liver: a more in depth look:

One of the main functions of the liver is to aid in digestion. Alanine Transaminase is an

enzyme produced in the liver that helps allow the liver to perform this function. If one of the

functions of the liver becomes injured or functions improperly, this can lead to many different

health problems. Cirrhosis of the liver is currently the 12 leading cause of death, and the wait for

a liver transplant is one of the longest (Pubmed 2012). The liver processes what you eat and

drink into energy and nutrients for the body to use (liver wellness -2009). The liver is mainly

damaged by what is filtered through it, such as alcohol and viruses. If the liver becomes damaged

in any way, this can cause serious damage to the rest of the body systems. Too much of a

substance can possibly cause damage to the liver, whether it be temporary or chronic. For

example, too much alcohol can cause liver function failure. Other causes of liver disease can be

too much fat accumulating in the liver (mayo clinic disease and conditions – liver disease).

The liver breaks down the fasts and nutrients the body needs into something the body can

use (Pubmed 2012). Mechanistically, if the proteins involved with breaking down the fats that

are sent to the liver, the fat could therefore just be stored in the liver, due to the proteins not

functioning properly. This kind of damage to the liver can cause scar tissue to form either within

or on the liver, termed cirrhosis (mayo clinic staff). Cirrhosis of the liver is difficult to detect,

and go unnoticed till the damage is too great.

Cirrhosis of the liver is the most common disease of the liver, and is one of the most

deadly. It is caused by thickening and inflaming the tissue due to accumulation of fatty deposits,

hepatitis viral infections, and alcohol abuse (Takahiro 1999). Other causes of this disease

include: biliary obstructions, over saturation of vitamin A, and Cystic Fibrosis (Heidelbaugh

2006). Cirrhosis of the liver is an incurable disease, and the only “cure” for now is to remove the

damaged sections of the liver or the liver all together and perform a transplant. Any injury to the

liver can lead to cirrhosis of the liver; therefore treatment for cirrhosis of the liver is very

difficult due to the vast amount of risk factors contributing to the progression of the disease

(Heidelbaugh 2006). It is due to these reasons that cause this disease to be as deadly as it is.

Current treatments for cirrhosis of the liver are only to manage the disease rather than reverse it.

Once the damage has been done to the tissue, the only way for the liver to reverse it is by re-

growing the tissue once the damaged tissue has been removed.

Alanine Transaminase as an indicator of liver disease:

Alanine Transaminase or ALT is an enzyme produced in the cytosol of hepatocytes

(Kaplan - 2002). This enzyme is very important in digestive metabolic processes. ALT is an

aminotransferase enzyme that catalyzes a reaction that converts an alpha-amino group to an

alpha-keto acid. (Sookoian 2015). This conversion aids in the eventual production of pyruvate

and L-glutamate in the liver (anals.org). The use of a transaminase as a detection enzyme was

first discovered in 1955 by Molander and his colleagues. Molander and his colleagues (1955)

researched the effects and use of serum glutamic oxalacetic transaminase as detection for

hepatocyte injury ( Molander - 1955). Though they did not look at ALT directly, they were able

to determine that transaminases are useful for detecting liver injury.

Alanine Transaminase and its mechanism:

ALT is a type of aminotransferase that converts alpha-amino groups to alpha-keto

groups. When looking at ALT, there are two different forms of which it can be found in: ALT 1

and ALT 2. ALT1 is the form of ALT that creates the end products of pyruvate and glutamate

via a reversible reaction between alanine and 2-oxogluturate (Sookoian 2015).

Scheme 1. ALT reversible reaction

ALT 1 is important due to its role in the metabolism of glucose for the body to use. ALT 1 is

mainly found in the cytosol of the hepatocytes and consists of the majority of the ALT present in

the hepatocyte (Sookoian 2015). ALT 2 is coded by a different gene than ALT 1, but has the

same metabolic processes. ALT 2 catalyzes the reaction of L-alanine to L- glutamate. Unlike

ALT 1, ALT 2 is found in the mitochondrial matrix of the hepatocytes (Sookoian 2015).

ALT is constantly present in circulation in low levels, but when the liver is damaged, the

amount of ALT in the blood is increased. To determine the exact cause of the increased ALT

levels, other tests are ran on the other enzymes and proteins of the liver. The most common way

of which to test for alanine transaminase is by a serum test which detects the levels of ALT in the

serum. Other and more direct methods of testing the levels of ALT consist of tissue samples

from the liver itself. This allows a more accurate reading and determination of whether the liver

is being damaged or not. ALT is the most commonly used marker for liver damage due to it

having increased levels in the serum and tissue prior to the presence of symptoms of the injury.

Effects of Ginseng in disease:

Ginseng is traditionally been used for many years as a therapeutic herb to help with many

different illnesses. Currently there are only three different types or strains of ginseng and they

include: Korean Ginseng, American Ginseng, and Siberian Ginseng. Currently, most research is

being conducted using the Korean Ginseng over the other two types. This is due to the vast

majority of people using ginseng as a therapeutic drug are using the Korean type. Ginseng is

thought to affect the body in many different ways, including: boosting the immune system,

decreasing the number of colds a person gets, and even lowering blood sugar (webmd). Not

much is known about the mechanism for how ginseng is able to induce such an effect onto the

body, but it is still commonly used by many different people and medical practices.

Current studies have shown that American Ginseng of long periods of time can reduce

the levels of fatigue in cancer patients, allowing for the patient to have more energy throughout

the day (Barton 2012). Many studies have been done on the effects of ginseng and its ability to

lower blood sugar levels, testing its usefulness in treating diabetes mellitus. The mechanism

behind this herb is still unknown and current studies are being conducted to determine the extent

of the effects. Though the medical and scientific world does not know much about the

mechanism and effects of ginseng, folk medicine uses it for many different treatments. Ginseng

is used to treat stomach and intestine dysfunctions and to even treat issues with the liver (YQ

2013).

D. Explanation of how background led to your research question.

Currently, the treatments for liver diseases are very few. The most common treatment for

a failed liver is to replace it, and that may take years to accomplish. Studies have shown other

forms of treatment that may seem viable in the future with more testing. Many people today are

trying to use alternative medicine for treatments such as herbs. The mechanism behind the

effects of Ginseng is still unknown, and the full extent of what it does is also unclear. Ginseng

has been shown to reduce GLUT2 levels, which plays a role in glucose transport across the

membrane (Lim 2009). This effect that ginseng has can be correlated to its ability to help

manage type 2 diabetes mellitus. Other studies currently show the effects ginseng has on the

immune system, boosting T helper cell and phagocytosis levels (Kiefer 2003.)

The use of Ginseng as an alternative treatment can be possible once more studies have

been conducted. The use of ginseng as a treatment of liver disease is just one possibility for this

“folk” medicine. Currently, three treatments for any liver diseases are either to remove the

injured parts, treat the symptoms if it’s an viral infection, or to replace the liver entirely

(webmd). ALT is one of the few markers for possible early detection for injuries in the liver.

ALT levels increase in the blood when the liver is damaged in some way, therefore if ginseng

has the positive effect that it is thought to have the levels of ALT should be decreased in some

way. With studies on the effects of ginseng, such as its ability to boost the immune response,

there may be more possible ways to treat liver injuries.

E. Anticipated Results of the research project.

Due to different strains of Ginseng and their different mechanisms, data obtained by Lim

et al (2009). was used to determine American Ginseng results. Though the strains of ginseng can

be said to be similar, the mechanisms they use are quite different. What is anticipated to occur is

a drop in ALT levels in the cirrhotic liver and an increase in the ALT levels of the cirrhotic liver

with Ginseng. The anticipated results are shown below.

Table 1: Mean +/- SE reduced ALT levels measured in IU/L after introduction of American Ginseng into diet. Data marked with * show significant difference compared to control. Data marked with a show significant difference compared to Sham group. The significance is set at p<0.05.

Control Sham group Ginseng with no cirrhosis

Cirrhosis with Ginseng

Alanine Transaminase

IU/L

71.3 ± 22.6

N=5

42.062 ±13.087 *

N=5

73.4 ± 19.5

N=5

55.08 ± 15.96*a

N=5

Control Data source: Lim [2009]. Sham Data Source: Watson [1989].

F. Significance of Project

Currently, treatments for any type of liver disease are limited, and other treatments other

than just replacing the organ are in desperate need. ALT and other aminotransferases are great

markers for early detection of liver injures (Molander 1955). The use of the aminotransferases

has grown in the recent years, and the mechanisms behind them are still not fully understood.

Other studies, such as this one, look into alternative methods of treatment for these diseases and

others alike. Herbal supplementation has grown in interest in recent years and shows a lot of

promise. Ginseng for example has been shown to not only aid in blood sugar levels but also in

psychological methods (Kiefer 2003).

This study looks into the possible aid that American Ginseng can bring to the liver. By

looking at ALT levels, the amount of damage to the liver or possible damage due to the ginseng

can be measured. Increased levels of ALT would indicate that the liver is being damaged in

some way, and in hopes of this study, the ALT levels will decrease to some degree. This could

potentially be a step forward into looking into different types of herbal supplementation as a

treatment for many different diseases or as a coping mechanism for the symptoms.

G. Contemplated method of approach to problem.

1.) Rationale for approach to be used:

For the purpose of this study, the experimental protocol for tissue sampling by Abcam

Company will be followed. This protocol allows for the use of both the serum and tissue ALT

testing, in which the tissue ALT testing will be used. For the purposes of this study, and the

availability of the equipment, the method of which the ALT is measured in by the optical density

(OD).

The data collected via this method will be processed in two different ways. The first way

is based on the standard curve that the protocol kit requires and the other is just a straight

comparison of the control group and the experimental group; making this an alteration to the

previously mentioned protocol (abcam company). The dosage level determined for this study

was based on the amount that is consumed on average, scaled to a rat. This study has to be done

in an animal model to carefully investigate the effect of Ginseng on liver disease.

2. Experimental Design

The rats will be separated into four groups for testing. The groups being tested are as

follows: a control with a healthy liver and no ginseng treatment, a sham control (just receiving

Ginseng), experimental with cirrhosis of the liver, and the final group being an experimental with

cirrhosis of the liver and ginseng treatment. Each group will have an n of 5. The rats chosen for

each group will be chosen at random. The experimental groups and the sham group will be put

on a diet that differs from the control group by ingesting American Ginseng. This diet will

continue for 3 months and then the livers of the rats will be harvested. Not all of the liver will be

removed for this study; the desired results can be seen with taking the maximum amount aloud of

the liver. This will allow the rats to survive the study with the least amount of liver possible,

except for the experimental groups, due to the induction of the cirrhosis. ALT levels in each

group will be examined and compared.

3. Tentative Time Table

Approximate Dates What will be done

Fall 2015 Formation of research proposal for PHYS 585 and form research committee, as well as obtain a research advisor for the project.

Spring 2016 Finalize research proposal and obtain all necessary forms and permission from research advisor and committee.

Summer 2016 Gather rats and supplies for the study. Perform the study with 3 month American ginseng diet. At the end of the 3 months, take portion of liver for analysis

Fall 2016 Process data collected from study and form a final research paper or thesis on study.

4.) Statistical analysis of data:

For the purpose of this study, all optical density measurements recorded will be processed

by MINITAB software. Statistical tests will be ran to determine significance and compare the

groups. The tests that will be ran include the one-way ANOVA along with Tukey as the

secondary test. Data presented will be in mean +/- SEM format and statistical significance will

be set at p<0.05 between the control groups and experimental groups, expressed as IU/L (Lim

2009).

5.) Experimental methodology

Adult Lewis Rats will be obtained and used throughout the course of this study, and

housed under normal conditions. These conditions include: normal 12 hour dark cycle and have

access to water continuously. For the purposes of this study, alterations to protcols provided by

abcam and Seglen, found in Methods of Cell Biology, were made. This study requires multiple

procedures prior to actual analysis of the data. The control group and cirrhosis induce group in

this study will receive a normal diet and will have access to water continuously. The sham group

along with the experimental cirrhotic rats with ginseng treatment will be provided American

Ginseng via oral gavage.

A. Inducing Cirrhosis of the liver

For this study, cirrhosis of the liver must be induced in two of the experimental groups. In

order to do this, methods similar to that of Wardi et al (2001) will be performed. TAA,

thioacetamide, will be used to induce cirrhosis of the liver. Approximately 200g/mL of TAA will

be intraperitoneally injected into the two different groups of rats. This process will span over the

course of 12 weeks, twice a week. At the end of the twelve week period, in order to determine if

cirrhosis of the liver has been induced; liver biopsies will be performed. To perform the biopsy,

methods in Corbin et al (2003) will be followed. The rats will be put under light anesthia and

restrained to a surgical table. The fossa between the right side of the xiphoid process and inferior

to the ridge of the right costal margin will be found and then the site will be cleaned. A small, 10

mm, incision will be made and a biopsy gun vertically inserted and used. Once the tissue has

been extracted, pressure will be applied to the incision site and then cleaned and closed. The rats

will then be returned to their cage for recovery. The tissue will then be prepared for scoring.

B. Obtaining Liver Tissue Sample

For this study, tissue sample of the rat livers will be taken. Alterations to the protocols

provided by Seglen et al. were made for this study. The rats will be anesthetized by

intraperitoneal injection of inaction. Once unconscious has been confirmed, the rat will be taped

down to a surgical table. An incision along the midline on the rat’s abdomen will be made, all the

way up to but not into the thoracic cavity. The portal and caval veins will be located (Prescott-

1976). Once located, two loose ties will be placed on each vein; one close to the liver and the

other further away. These ties are to hold the cannula in place. Prior to removing the liver, it

must be perfused with saline. To accomplish this, small incisions in both the portal and caval

veins are made, and the cannula is inserted into the incisions. The liver is then flushed with

saline via the cannulation. In this study, the oxygenator that is typically used in this protocol is

omitted.

Once the livers have been perfused, some of the livers will be removed from the

abdominal cavity and washed with PBS (abcam). The amount needed for this study is at least 50

mg ( abcam). Not all of the liver will be used. To prepare the samples for testing, the collected

tissue must be suspended in ALT assay buffer and homogenized via Dounce Homgenizer (10-15

passes). Homgenate will be centrifuged at 4o at top speed for 5 minutes to remove any insoluble

material. Supernant is collected once centrifuge is completed and kept on ice. This will be the

sample that is tested for ALT levels.

B. ALT testing

ALT levels will be measured using the ALT testing kit provided by abcam. For this

study, a standard curve will be created for interpretation of the measurements collected. The

standards used in this study will be plated to equal 20 microliter dilutions and the samples will be

ran in duplicate, also at 20 microliters. For this test to perform correctly, reaction mixes must be

created. This study will perform the colormetric assay to measure the OD values of the samples

and the reaction mix will be made accordingly to his assay (abcam). The samples with reaction

mix will be measured at 570nm.

6.) Anticipated Problems

Liver perfusion is extremely difficult to perform, and takes time (Google book cel bio).

The amount of time needed to remove a part of the liver from each rat and create a usable sample

takes time. The time lapse between each rat can possibly alter the results shown, and/or allow

more time for contamination. Other small possible problems could be the base level of the rats

being vastly different from one another, causing the data collected to be thrown off. For this

study to be successful it is important to keep the possibility of contamination extremely low and

for the amount of time it takes to prepare each tissue sample low.

7.) Ethical aspects of the proposed research project:

Research for this study must be approved prior to execution by the Animal Care and Use

Committee of Ball State University. All rats will be treated humanely and all measures to reduce

pain will be taken. Due to the liver being able to function with some partially removed, all rats

for this study will not be killed for a complete liver harvest.

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