Receiver of Lincoln University Scholarship for the Sciences.
Overall Winner of the Canterbury and Westland Science Fair Competition.
Senior Winner of the Canterbury and Westland Science Fair Competition.
The prevalence of Phloridzin in the flesh and skin of Heritage Apples.
By Niamh Peren, 2006
St Margaret’s College
Christchurch, New Zealand
Subject: Biology
Abstract word count: 144
Word count: 3869
Abstract:
Currently there is a lot of public discussion about the food we eat and how it affects us. Antioxidants have been noted for benefiting our health. Such advantages can be found in phloridzin, a type of antioxidant that is found in an apple’s flesh and skin. Phloridzin has the ability to control sugar flow throughout the intestine. After discovering that heritage apples contain a higher level of antioxidants compared with cultivar apples, this study led me to investigate the amount of phloridzin found in a heritage Red Delicious apple. The hypothesis being to find out whether more phloridzin can be found in the flesh or skin.
It was found that phloridzin is predominantly in an apple’s skin, and that if more research could be completed, phloridzin could be made into a juice, which would work as a natural preventative for Type 2 diabetics.
Words: 144
Introduction:
When the body’s cells respire, free radicals are formed. Free radicals create abnormal cells from healthy cells by a process called oxidative stress. Because they are unstable, these abnormal cells damage the tissues that they are in. In turn, oxidative stress is believed to cause premature aging to the body, one result of this can be Type 2 diabetes. A diabetic does not possess the ability to fight free radicals with antioxidants because they have “depleted antioxidant reserves” due to their immune system already using them to help control glucose levels in their blood.
Healthy people maintain antioxidant levels in their bodies without difficulty because they are not being used elsewhere. Antioxidants are thought to inhibit the effects of free radicals by neutralising them and therefore preventing the deterioration of cells. Oxidation neutralises “reactive molecules called free radicals” which are believed to cause cancer, alzheimer’s, thrombotic strokes, asthma, diabetes, ischemic heart disease and premature aging. It is common knowledge that antioxidants are found in plants and they are called beta carotens. They are also known as carotenoids. This study focuses on Phloridzin, a type of antioxidant found in apples.
Phloridzin is a compound that “modulates sugar transport in the intestine and can slow the absorption of sugar” by “blocking the reabsorption of glucose from the kidney tubules producing glycosuria.” In this way it would have a similar function to insulin for a diabetic. Phlordizin comes from an apple tree extract, and is also found in an apples flesh and skin. Phloridzin is a flavonoid known as dihydrochalcones and also a carotenoid. Apples are the only known dietary source of phloridzin compounds.
‘An apple a day keeps the doctor away’ is an old wives tale that we have all heard many times regarding looking after ones health. This saying along with an article published in The Press called “Heritage apples revived” inspired me to take a closer look into the mysteries of a healthy apple. We are dependent on food to survive, and healthy food is said to provoke a healthy body and a healthy lifestyle.
In New Zealand our National Health Board promotes the campaign ‘5+ A Day’, five being the recommended minimum amount of fruit and vegetable servings (400g) to be consumed per person per day. This campaign is trying to stimulate a healthier diet for our people, as there are many health-related problems developing from our present dietary habits. One of the main issues arising in our population is a high level of obesity. Extra weight makes it harder for the body to control glucose levels and produce insulin in the pancreas (due to extra pressure). Therefore an obese person has a greater risk of developing Type 2 diabetes. The government spends an estimated $135 million per year on trying to control and decrease its possibly devastating effects.
An apple is classified as a fruit, thus fitting into the healthy and ‘eat more of’ section in the food pyramid. The United States Apple Association determines that an apple is specifically valuable for a person because of its healthy qualities. If a person snacked on an apple instead of an item from the ‘eat less of’ section in the food pyramid, their digestive system would gain positive components such as “vitamin C, foliate, potassium and boron”, all of which are vital for our digestive system to function. Apples also store high levels of “cinnamic acids, which are chlorogenic and caffeic aids these energy boosting compounds are derived from the plant-based phytonutrient . Apples are “fat free” which would certainly benefit New Zealand’s obesity problem and are “sodium free, cholesterol free, and are an excellent source of fibre” .
Apples available in supermarkets have been designed to appeal to consumers’ tastebuds and their visual appreciation, not necessarily for their healthy components. This usually implies that the sugar content is high and the actual healthy components low. Minerals and antioxidants found in popular cultivar apples have been reduced because taste is the main priority. Meaning that the cultivar apple, which is likely to have been sprayed is not an absolute health intensifier. The alternative solution to these cultivar apples is the organic heritage apple. If apples are bred organically, antioxidants find it easier to enter the human body because they are not diminished, unlike modern cultivated apples that are often rinsed or peeled before eaten (this would remove some of the phloridzin). If fruit is sprayed during cultivation then washing or peeling is necessary to remove the chemical products.
Phenolics are compounds that provide a plant with their colour, taste and protection and phloridzin is part of the phenolic structure of an apple. They are a class of phytonutrients, which act as a plant’s immunity. In humans, phenolics help to prevent cell damage that creates diabetes. To compare heritage apples to cultivar apples, Mark Christensen’s research for the New Zealand Tree Crops Association provided the necessary information. He found that the total phenolics in an apples skin were: 543.5μg per cm2 for the cultivar apple Pacific Rose , and for the heritage apple Red Delicious 872.4μg per cm2 . This presents a considerable difference.
Flavonoids also provide plants with immunity from disease, and because of their taste are readily consumed by animals. The total amount of flavonoids found within a cultivar Pacific Rose apple is 4.1μg per grams of flesh weight , and 4.5μg per grams of flesh weight in heritage apple Red Delicious . The difference of flavonoids found in the flesh of a cultivar and heritage apple do not differ as greatly as the total phenolics. However, heritage apples have higher levels of both phenolics and flavonoids and so the heritage apple and its antioxidant phloridzin content will be further investigated.
The Red Delicious heritage apple variety has a profound “antioxidant punch”, according to both New Zealand scientist Mark Christensen and Canadian scientist Rong Tsao. Both have been studying apples separately. The heritage apple chosen to be investigated was the Red Delicious because it was one of the few heritage varieties commonly available in New Zealand, and because it showed some of the highest levels of both phenolics and flavonoids compared with the other heritage apples researched by Mark Christensen. Red Delicious supposedly originated from a seedling rootstock and was discovered in America in about 1870. It contains large amounts of phloridzin. If the healthy benefits of eating heritage apples becomes well publicised then this may encourage the public to introduce heritage apples into their diets to benefit from their antioxidants.
There is a shortage of knowledge concerning heritage apples. After contacting Wendy Davis of the USApple Board, I was surprised to find that she had never heard of heritage apples and was not quite sure what the term ‘heritage’ apples implied, “Can you please explain?” In New Zealand, Central Otago Orchardists have removed heritage apples from their orchards because the demand for them died away during the 1960’s. As a result heritage apples are now relatively unknown, even to the professionals, and so people are not aware of the increased antioxidant levels in these apples and how they could benefit their health.
To test the level of the beneficial antioxidant compound, phloridzin in Red Delicious apples, I carried out an investigation at my school and also at the Medical Toxicology Laboratory in Christchurch’s Public Hospital. The equipment needed was not available anywhere else.
Aim:
To find out where the highest level of phloridzin can be found in heritage Red Delicious apples (skin or flesh); to educate people against peeling, and research the significance of phloridzin in helping those with type 2 diabetes.
Hypothesis:
The antioxidant phloridzin in Red Delicious Heritage apples, will be higher in the skin, and according to Mark Christensen’s for New Zealand Tree Crops Association it covers more μg per cm2 on the skin, compared to flesh. It could be beneficial to Type 2 diabetes sufferers because it controls sugar flow in the intestine. It could therefore be made into an extract for people with Type 2 diabetes to drink as a control measure for diabetic symptoms.
Method:
EQUIPMENT:
X15 Red Delicious apples (organic)
X1 plug (8mm by 1.5mm)
X1 scalpel
Mortar and Pestle
Blender
Solvent - Ethanol:Water:Formic Acid (80:20:1)
Fridge (4 degrees)
Centrifuge
X9 Centrifuging Tubes
X15 Squeezers
X15 Testubes
100mL Measuring cylinder
Preparation of Samples for HPLC Machine Testing
John Bostock of J.B. Apples, Hastings, New Zealand, donated two trays of Organic Red Delicious Apples to this experiment. The apples had to be organic so that the phloridzin would be at its natural level and not affected by sprays or by needing to be washed due to sprays. From a total of fifteen chosen apples, they were divided into three replicate groups of five. For each group of five apples, four plugs were taken by a five point star-like scheme from the upper poles of each apple. A plug consisted of a 8mm long and 1.5mm in diameter cylinder. The volume of each plug is approximately equivalent because the ends of the plug are removed. To separate flesh and skin, a scalpel blade is used to cut between them, thus the human eye must approximate its volume. The skins are kept so the level original levels of phloridzin can be measured. The skins are ground with a mortar and pestle. The plugs for each flesh group are combined in a blender to make a single sample. The group flesh samples are then mixed with 50mL of solvent (this is repeated using the skins). To extract the polyphenol antioxidants, in particular phloridzin, 50mL of a solvent made to the ratio of ethanol (80) : water (20) : formic acid (1) is added to the samples. Its acidic strength is equal to a bee sting. Samples are then stored in the fridge at 4degrees celcius for 24 hours to secure the polyphenol antioxidant levels. After 24hours the samples are centrifuged to allow the liquid to be distilled from the apple solid. Repeat the process with 5mLs of solvent and continue centrifuging and distilling until all the apple solids have been removed. All samples must then be kept in a freezer until analysed by the HPLC Machine to prevent evaporation and reverse-phase occurring.
High Performance Liquid Chromatography (HPLC) Machines:
The HPLC machine is used in the Medical Toxicology Laboratory in Christchurch’s Public Hospital to separate the components in blood samples. An HPLC machine performs advanced light chromatography, perfect for separating the compounds found within apples. It is a very sensitive and expensive device, which is why the apple samples had to be centrifuged numerous times to refine the product. At the laboratory the samples were centrifuged again at 60knots for 6minutes, as the school’s centrifuge only operated at 45watts. This was just in case any apple solids, which are far larger than blood components, were still present.
An HPLC machine works by pumping a solvent and the sample (which is injected) through a column. The column is in the shape of a long pipe, and it is “crowded with a tangle of ropey molecules. Tiny molecules from the sample are able to pass through the column quickly, but bigger molecules take more time”. The detector “shines a light on a window within the machine which transmits the light through the sample… the electric “eye” can detect light shining through the window and the sample, and saves that information as an electric signal.” The times that molecules separate are collected and recorded by the HPLC machine. An attached computer graphs the data indicating the times and quantity of individual groups of molecules, when they separate from the sample.
Results:
Here is the standard of phloridzin tested by an HPLC Machine. It was tested so the following samples’ results could be comparable. Phloridzin molecules come off the standard after 22.459 minutes. The amount of phloridzin produced was 494.51746mg/L.
Heritage Apples: Red Delicious
Graph 1: Phloridzin levels in flesh of red Delicious Apples sample 1.
16.92098mg/L of phloridzin was found in this sample.
Graph 2: Phloridzin levels in flesh of Red Delicious Apples sample 2.
16.94834mg/L of phloridzin was found in this sample.
Graph 3: Phloridzin levels in flesh of Red Delicious apple sample 3.
15.78933mg/L of phloridzin was found in this sample.
The average level of phloridzin found in the flesh of a Red Delicious apple is 16.553mg/L (rounded to 3 decimal places).
Graph 1: Phloridzin levels in skin of Red Delicious apples sample 1.
100.47052mg/L of phloridzin was found in this sample.
Graph 2: Phloridzin levels in skin of Red Delicious apples sample 2.
122.45026mg/L of phloridzin was found in this sample.
Graph 3: Phloridzin levels in skin of Red Delicious apples sample 3.
83.16047mg/L of phloridzin was found in this sample.
The average level of phloridzin found in the skin of a Red Delicious apple is 98.694mg/L (rounded to 3 decimal places).
Conclusion:
The level of phloridzin was greater in the skin than the flesh of the heritage Red Delicious apple. This is shown in the average of phloridzin in both the skin and flesh. The skin has an average of 98.694mg/L of phloridzin, whereas the flesh only has an average of 16.553mg/L. The results showed that the skin of a Red Delicious apple must be consumed if a person truly wants to benefit from its antioxidants. All samples were measured in mg/L, and can therefore be compared with one another. Mark Christensen’s research was measured in both μg per cm2 and μg per grams of flesh weight. These results therefore had to be converted into mg/L for a comparison to be made.
Evaluation:
Parts of my experiment were valid, but in hindsight I can see ways to improve my experiment. For these reasons my experiment was a fair test; the same sized samples were used, the same testing technique was applied to each sample, the same equipment was used and washed properly in between each sample to prevent contamination and statistical analysis allowed me to provide validity. Three samples were taken from groups of five apples to test their skin and flesh, the samples provided three repetitions. A larger sample size would have provided me with more accurate results, however this could not be done due to time restraints with using the Medical Laboratory equipment. A possible error that I may have made was due to human error in separating the skin from the flesh, because I sliced them manually. Although I was careful, this may not have been 100% accurate for all fifteen apples.
The apples were limited to one orchard and one batch of apples, to ensure that they were grown in the same environmental conditions, both biotic and abiotic. If the Red Delicious apples were gathered from different locations that experienced different seasonal variations, then it could be determined whether or not the environment has any influence on the phloridzin levels. A larger range of samples and a greater range of apple varieties could have formalised a relationship between heritage and cultivar apples and the levels of phloridzin that they contain. I presumed phloridzin levels would be lowered by washing the skin of Red Delicious apples, but this should have been determined experimentally by comparing washed and unwashed apples. It would also have been nice to be able to test the general publics response to eating heritage apples by having the opportunity to let them sample these apples at a supermarket. This would have helped determine whether or not consumers would eat them.
The graphs from the HPLC machine show that phloridzin molecules come off the apple samples between 22.266 and 22.459 minutes. The labelled peak within that timeframe is therefore phloridzin (re: the standard, which comes off as stated in results, at 22.459minutes). Due to the samples being from different Red Delicious apples, it was possible that the phloridzin molecules tested on the HPLC machine would come off within a time range, as had happened previously when the laboratory had been testing other samples. These apple samples were however finely distilled from centrifuging, so it was not the case.
The HPLC machine shows the greatest difference of phloridzin levels between the skin samples. They vary between 83.16047mg/L and 122.45026mg/L for the heritage Red Delicious apples. Although this difference is very large, it reinforces nature’s influence on these organic apples. The differencial between the skin samples becomes less important when compared with the difference of phloridzin levels between the skin and flesh samples. The flesh samples contained levels of phloridzin ranging from 15.78933mg/L to 16.94834mg/L. The smallest difference therefore between the levels of phloridzin on the apples skin and flesh was 67.37114mg/L, which is a considerable difference. This proves that there is more phloridzin in the skin of a Red Delicious apple.
This investigation and related research has led me to link the use of phloridzin, the antioxidant, with a possibility for controlling Type 2 diabetes. It is conceivable that phloridzin could be used for medicinal purposes, because of its ability to control sugar flow throughout the intestine. If more time and resources were available, I would have liked to research how much phloridzin is required to benefit a person. If I knew the amount of phloridzin needed to modulate sugar flow throughout the intestine, and how long phloridzin lasted in the intestine before it needed to be boosted, like an insulin injection, I think that I could make a juice which would work as a natural modulator for diabetes.
Discussion:
To make a juice, I would need to investigate how long phloridzin lasts as an antioxidant in juice form, and how much phloridzin is required to make a difference as a beneficial antioxidant. That information, would determine the quantity and how many times a day a person would have to drink the heritage apple juice to gain its antioxidant benefits. It would also be interesting to find out whether or not sprayed heritage apples have lower levels of phloridzin on the skin through having to be washed. I presumed that washing off the sprays and possibly the spray itself would affect the level of phloridzin in my experiment. If a certain spray did not affect the level of phloridzin, then it is likely to be more cost effective to use a heritage apple that has been sprayed, as the product yield would be greater.
If a juice is able to be produced, it may be able to regulate the glucose levels in the body of a type 2 diabetes person. Whether or not the phloridzin is stable as a juice would indicate whether the juice would need to be made everyday or whether it could be manufactured and stored. Once this has been determined, I would know whether or not phloridzin can be administered as a juice as opposed to being offered as a fruit only.
Diabetes is an illness that is derived from too much sugar in the blood. Although glucose is essential for the brain and body to function, a person with Type 1 diabetes can either go into a coma or hypo-coma if they have too much sugar content. The reason for this inability to handle too much or too little glucose is because the pancreas does not produce enough insulin. People who do not have diabetes make insulin naturally. Insulin works by controlling the levels of glucose in the blood. Contemporary insulin is fast acting and active at all times, whereas it used to act in short bursts and release itself slowly into the body. Phlordizin could help diabetics control their blood sugar levels; its qualities may be able to prevent large amounts of glucose entering the system at once. There are two categories for diabetes, Type 1 and Type 2.
Glucose is a product of the digestion of carbohydrate. The intestine is the organ where glucose is mainly absorbed and this is where phloridzin acts to control sugar flow. Glycogen is a product that will stay in a person’s liver and be used for instant energy, but if there is excess sugar then that will become fat. A diabetic tests their glucose levels frequently. The average and safe levels of glucose range between four and eight mmol/litre. Hypoglycaemia occurs when blood sugar is less than four mmol/L. This is a very serious condition and a diabetic in this circumstance must immediately eat something containing lots of glucose. The aim is to raise the glucose level in the person’s blood. Fruit, like an apple, cannot be used in this situation because it contains too many fructose sugars and not enough glucose. Fructose is harder to digest and therefore it takes longer to absorb.
The antioxidant phloridzin could not help a person with Type 1 diabetes because the illness can only be controlled by insulin and not by natural preventatives. Type 1 diabetics have the extreme version of the illness. Their body does not produce any, or only little amounts of insulin. It is common for their immune system to have destroyed or be destroying their pancreas, making it impossible for them to produce insulin. A person with Type 1 diabetes is usually in their youth, and they would die if they did not receive insulin. 10% of New Zealanders have diabetes which is a staggering amount of people relying on pigs to manufacture insulin!
People with Type 2 diabetes are insulin deficient due to lifestyle choices. They can therefore sometimes be treated by weight-loss and by regular physical activity. Since apples have high levels of antioxidants, such as phloridzin which “modulate sugar transport in the intestine and can slow the absorption of sugar” it could be used as an alternative product to treat their diabetes. People with Type 2 diabetes take medication to stimulate the insulin production levels in their pancreas, since their bodies do not produce enough or are resistant to insulin. Phloridzin may be adequate to control the transport of sugar so that Type 2 diabetes sufferers do not require additional medication.
A person with diabetes must control their “blood glucose, cholesterol, blood pressure and other cardiovascular risks” If a person does not take care of themselves then they are liable for “heart attack, stroke, eye problems and kidney disease” To use phloridizin for its advantages would be beneficial to New Zealand because 1800 New Zealanders are spending $90 a month for Avandia, a diabetic drug.
It has become popular in society for people to drink juice, so a healthy and useful juice containing valuable antioxidants, especially phloridzin, would be welcomed into an already accepting market. To make the drink extra beneficial for the consumer, other minerals, vitamins and plant extracts such as vitamin e (another antioxidant that has been associated with helping diabetes) could be added to the heritage apple extract juice (containing phloridzin), to ensure that a greater cross section of peoples needs could be met.
Apples have traditionally been used by the British to create ciders. By connecting the knowledge that “Our bodies produce natural antioxidants, but diabetics have depleted antioxidant reserves and their levels of Vitamin C are especially low ”, a juice could be formed for Type 2 diabetics, and it could act like a natural preventative.
In this case, mixing apples with a citrus fruit profits the juice. Once an apple is cut, it browns easily and quickly within several minutes. This browning is called “enzymatic oxidation” and under normal atmospheric conditions it can now be prolonged. By applying a natural product called Ascorbic Acid, found in lemons, limes, feijoas, grapefruit and oranges, the browning can be slowed by five weeks. The apples must be kept at low temperatures between five and ten degrees celcius , for this to work. USDA researcher, George Buta said it “slows down microbial decay and therefore the associated changes in organic acids and sugars during storage” For the juice to work, another area of interest would need to be investigated; whether the citrus fruit or strong acid affected phloridzin and its action in the intestine.
From the research gathered and experimental work completed, questions have arisen which are yet to be answered. The hypothesis was to find out whether phloridzin was more common in the flesh or skin of the heritage apple, Red Delicious. The results showed a great difference between the flesh and skin, and that phloridzin levels are higher in the skin. Phloridzin and its glucose controlling qualities were also recognised, and the idea that these apples could be processed to create a valuable and healthy juice for Type 2 diabetics was also discussed.
Bibliography of works cited only:
PLEASE CONTACT ME IF YOU WOULD LIKE TO SEE REFERENCES.
Acknowledgements:
Toxicology Laboratory at Christchurch’s Public Hospital
John Bostock of J.B Apples, Hastings, New Zealand
St Margaret’s College, Christchurch, New Zealand