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Proving Benefits of Shungite:
Since the time of its discovery, shungite mesmerized the public with its mysterious healing and cleansing properties. Since its popularity and fame was growing, it drew attention of academic community. Everyone was eager to know, what is so special about this unique stone from the North of Russia and why did it possess these properties. For the first time shungite was described in the book “Journey to Lakes Onega, Ladoga and Ilmen” by Nikolai Ozeretskovski which was published in 1792. But back then the stone didn’t even have the name.
The name shungite, which derives from the name of the village Shunga where the stone was found, was given by professor Alexander Aleksandrovich Inostrantsev back in 1885. Professor Inostrantsev wrote the first in-depth scientific work about shungite which later became widely recognized all over Europe and attracted attention. His work was called “The newest member of the group of amorphous carbons” which was published in 1877, and it was followed by “More about shungite” in 1886. The attention to shungite grew substantially after these works were published, and 1907 marked the first time the word “shungite” was described in a dictionary, namely in the Encyclopedic Dictionary of Brockhaus and Efron, the major dictionary of the Russian Empire. But at the time there wasn’t enough academic background to provide comprehensive scientific research in the field of geology, and shungite works were left on the shelves for the time being.
However, the establishment of the new system of higher education in the Soviet Union and the foundation of the Academy of Sciences of the USSR opened new possibilities for the studies of shungite. At the forefront of these studies was, of course, the Academy of Sciences of Karelia, since shungite is exclusive to this region. Such researchers as Pyotr Borisov (“Karelian Shungites”, 1956), V. Sokolov and Y. Kalinin (“Shungites of Karelia and ways of its comprehensive research”, 1975;”Geology of shungitonous volcanogenic sedimentary formations of the Proterozoic period in Karelia”, 1982; “Shungite – the new carbon material”, 1984) and V. Solovov (Radio-shielding properties of composite materials based on shungite rocks and its compounds, 1990) dug deeper into the physical properties of shungite, its composition, its abilities in electromagnetic protection, its origins and possibilities in further application of shungite. These studies helped to create the image of shungite, proved some rumours about it, including its cleansing and shielding properties, and drew attention to further researches, as well as established the Institute of Geology of the Academy of Sciences of Karelia as one of the leading research center in the field in Europe.
With the collapse of Soviet Union and the arrival of the age of globalization, the whole world became interested in studying this mysterious stone from Russia. And arguably the most important study of all was conducted in the US at the beginning of the 1990s. Robert Curl and Richard Smalley of the Rice University and Harold Kroto of the University of Sussex, England, carried out a 10 years long research which resulted in discovery of unique structure of carbon inherent in shungite – fullerenes. For their discovery the three scientists were acknowledged by the Nobel prize in Chemistry in 1996. This discovery created countless possibilities for new inventions in the fields of industry, medicine, energy, new materials and so much more. And it all started when they took a closer look on the piece of shungite from Karelia.
This groundbreaking work was later supplemented by a number of scientific researchers all around the world, including the Russian federation, which solidified the reputation of shungite and proved it to be a reliable material for filtering toxins and neutralizing different kinds of radiation. In turn, it raised the popularity of shungite in water filters and created a buzz around this unique stone. To this we can witness a growing popularity of shungite among common people, who wish to acquire it for personal protection and purification. It is expected to continue to grow as people will learn more and more about this somewhat mysterious mineral through this substantial scientific basis.
The name shungite, which derives from the name of the village Shunga where the stone was found, was given by professor Alexander Aleksandrovich Inostrantsev back in 1885. Professor Inostrantsev wrote the first in-depth scientific work about shungite which later became widely recognized all over Europe and attracted attention. His work was called “The newest member of the group of amorphous carbons” which was published in 1877, and it was followed by “More about shungite” in 1886. The attention to shungite grew substantially after these works were published, and 1907 marked the first time the word “shungite” was described in a dictionary, namely in the Encyclopedic Dictionary of Brockhaus and Efron, the major dictionary of the Russian Empire. But at the time there wasn’t enough academic background to provide comprehensive scientific research in the field of geology, and shungite works were left on the shelves for the time being.
However, the establishment of the new system of higher education in the Soviet Union and the foundation of the Academy of Sciences of the USSR opened new possibilities for the studies of shungite. At the forefront of these studies was, of course, the Academy of Sciences of Karelia, since shungite is exclusive to this region. Such researchers as Pyotr Borisov (“Karelian Shungites”, 1956), V. Sokolov and Y. Kalinin (“Shungites of Karelia and ways of its comprehensive research”, 1975;”Geology of shungitonous volcanogenic sedimentary formations of the Proterozoic period in Karelia”, 1982; “Shungite – the new carbon material”, 1984) and V. Solovov (Radio-shielding properties of composite materials based on shungite rocks and its compounds, 1990) dug deeper into the physical properties of shungite, its composition, its abilities in electromagnetic protection, its origins and possibilities in further application of shungite. These studies helped to create the image of shungite, proved some rumours about it, including its cleansing and shielding properties, and drew attention to further researches, as well as established the Institute of Geology of the Academy of Sciences of Karelia as one of the leading research center in the field in Europe.
With the collapse of Soviet Union and the arrival of the age of globalization, the whole world became interested in studying this mysterious stone from Russia. And arguably the most important study of all was conducted in the US at the beginning of the 1990s. Robert Curl and Richard Smalley of the Rice University and Harold Kroto of the University of Sussex, England, carried out a 10 years long research which resulted in discovery of unique structure of carbon inherent in shungite – fullerenes. For their discovery the three scientists were acknowledged by the Nobel prize in Chemistry in 1996. This discovery created countless possibilities for new inventions in the fields of industry, medicine, energy, new materials and so much more. And it all started when they took a closer look on the piece of shungite from Karelia.
This groundbreaking work was later supplemented by a number of scientific researchers all around the world, including the Russian federation, which solidified the reputation of shungite and proved it to be a reliable material for filtering toxins and neutralizing different kinds of radiation. In turn, it raised the popularity of shungite in water filters and created a buzz around this unique stone. To this we can witness a growing popularity of shungite among common people, who wish to acquire it for personal protection and purification. It is expected to continue to grow as people will learn more and more about this somewhat mysterious mineral through this substantial scientific basis.
Everything You Need to Know About Shungite and EMF Protection
The potential health hazards from cell phone radiation seem to be a pressing issue in a society where billions of people utilize this device 24 hours a day every day for a variety of reasons. And as more people are surrounded by electronic gadgets essentially all the time, the concern about the harmful effects from electromagnetic fields (EMF) continues to increase. But what are EMF’s?
Contents [hide]
The magnetic field is the region that surrounds magnetic material or electrical currents and increases in strength when a current increases. Predictably, the magnetic field strength will quickly reduce the farther it gets from its source. You probably notice the magnetic field in action when permanent magnets pull and repel magnetic materials.
An electric field is an area around a charged particle that displays the direction of a positive charge within the field. The field flows outward in the direction of a positive charge and inward toward a negative charge. You may notice the static electric field when your clothes cling to each other after removing them from the dryer.
Electric fields are generated if a device is turned on or off while magnetic fields are induced only when a current is moving as is the case with power lines. Electric fields are diminished by objects like walls, whereas magnetic fields can pass through any living thing, object or material. Jointly, electric and magnetic fields are called the electromagnetic field or EMF, and the combined forces cause electromagnetic radiation.
What is EMF Radiation?EMF radiation consists of light particles or photons that travel in waves at the speed of light through space and transfer electromagnetic radiant energy. It takes form in gamma rays, X-rays, radio, micro and ultraviolet waves as well as anything used for electrical power and man-made or natural lighting. The National Institute of Environmental Health Science separates EMF’s into two radioactive groups:
Non-ionizing – They have lower frequencies of radiation and include but are not limited to visible light, MRI scans, computers, WIFI, power lines, and microwave ovens.
Ionizing – These have higher levels of radiation that can possibly cause cellular and DNA damage. Examples of ionizing high-frequency radiation include gamma rays, X Rays, and ultraviolet light.
Some people believe non-ionizing radiation is totally safe; however, there have been compelling studies that show that long-term exposure to low-level electromagnetic fields may be detrimental to your health. In fact, more studies are being conducted to determine the relationship between EMFs and cancer.
Is Non-ionizing EMF Radiation Harmful to Your Health?Research has shown that electromagnetic stress causes a vibration within the human body at above average frequencies making it more susceptible to many health issues such as viruses, degenerative diseases, and pollution. In 2010 a cadre of scientists issued a report that outlined the connection between electromagnetic fields and physical health. The report concluded that people who are prone to EMF sensitivity experience an increase in minor medical issues such as fatigue, memory loss, and insomnia as well as more serious problems like increased blood sugar levels and arthritis.
Recently, the International Agency for Research on Cancer which is a part of the World Health Organization released a statement stating that EMFs are “possibly carcinogenic to humans.” If you’re wondering how EMFs can potentially affect your health, this list outlines how electromagnetic frequencies can affect the human body.
READ Does Aluminum Foil Protect You From Electromagnetic Field Radiation?Mood swings and mental instability
Your nervous system is the part of your body that’s most susceptible to the effects of EMF radiation. Memory loss, depression, and a reduced attention span can be signs of a damaged nervous system.
Vulnerable immune system
You will get sick more often, especially with common colds, if your immune system has been compromised by EMF radiation.
Uncommon muscle pain and headaches
Unusual muscles aches, consistent migraines, and an irregular pulse could mean that your muscular and cardiovascular systems have been negatively affected by radiation
Chronic fatigue, anxiousness, and insomnia
Long-term EMF exposure can create erratic hormonal levels which may result in increased fatigue, anxiety, and sleeping problems.
So because electronic devices are an inescapable part of your daily activities, you might be wondering if there’s a functional way to protect yourself from EMF radiation without changing your standard of living. Fortunately, the EMF absorbing stone known as shungite can be a viable solution.
The easiest and most cost-effective way to counteract the effects of electromagnetic radiation is shungite. This EMF absorbing stone is a naturally occurring geomagnetic mineral that known for its absorbing and neutralizing abilities. Shungite is also considered to be a very potent antioxidant that through research has proved to be beneficial to your health.
What is Shungite?Shungite is a shiny stone that is made of mostly carbon which is the same element that diamonds and graphite come from. However, unlike graphite, it has an unusual luminous black color with the higher end stones exhibiting a silver shimmer.
The stone originated two billion years ago and at that time, was made of just pure carbon yet now are stones made from fragments of shungite particles. In its purest form, shungite is composed of small quantities of miniscule hollow carbon particles called fullerenes. Shungite is typically separated into the following three categories based on how much carbon composition exists:
Type I: Elite shungite also known as noble or silver shungiteElite shungite is a black, glasslike mineral with a silver reflect. it has more than 90 percent organic carbon and is believed to possess healing energy. Because it is composed of mostly carbon, elite shungite is very rare, fragile, and difficult to shape which is why it is not polished or cut and is typically sold in its original semicircular shape.
Type II: Petrovsky shungiteThis type of stone was named after the Russian Emperor, Peter the Great and is made of 50 to 75 percent organic carbon. Unlike, elite shungite it can be cut or polished giving it an intense shine. This stone is frequently used to fabricate objects; however, Petrovsky shungite has intense energy stabilizing capabilities that are similar to shungite.
Type III: Black or regular shungiteStructures like pyramids and spheres are often composed of black shungite which is composed of 30 to 50 percent carbon. Because of its solid black color, regular shungite is a favored stone for creating handmade jewelry.
Where Does Shungite Come From?Shungite was discovered in Russia near the Shunga village in the late 1800’s. Peter the Great constructed what would now be considered the first spa in Russia to enjoy the cleansing effect of the stone. Today, the powerful shungite EMF radiation blocking and purifying capability has been recognized by the scientific community.
Shungite is primarily found by Lake Onega in the Karelia region in Russia; however, it has also been found in smaller measures in a coal mine by Chelyabinsk and among the volcanic rocks at Kamchatka. Shungite’s primary source in Karelia produces approximately 300,000,000 tons of the mineral, so there won’t be any shortages of the stone in the near future. Despite Shungite is remote origins, it is very accessible to consumers due to the heightened demand for its purifying and EMF neutralizing capabilities.
How was Shungite Formed?The scientific community hasn’t come to a universal conclusion as to how shungite is formed. But there are many theories on where it comes from with the most dominant being from either an ancient meteorite or an ancient body of water.
READ 2 Types of EMF Exposure and Why You Should Be WorriedOne popular theory is that several billion years ago there were large clusterings of microscopic carbon-based organisms in a prehistoric lake, sea or ocean in what is now North-Western Russia. Over millions of years, the organisms died, and their high carbon deposits formed at the bottom of the sea. As the Earth’s plates shifted through time, the deposits transitioned from the sea to a specific area on land.
The other popular hypothesis is that the shungite deposits came from a meteorite that landed on Earth a long time ago. This theory would account for the high carbon components as well as explain why the mineral is primarily found in one area. Nevertheless, some of shungite’s allure may be due to its curious origins as well as its EMF shielding abilities.
Shungite EMF Protective PropertiesMany studies have concluded that shungite is effective in lessening the effects of electromagnetic sensitivity. So what is the distinctive feature that allows shungite to serve as a shield from EMF radiation? The answer lies in the carbon compound called fullerene.
In 1996, Sir Harold Kroto and two other scientists won a Nobel Prize in chemistry for their discovery of fullerene eleven years earlier. Also known as buckyballs, fullerenes are hollow molecular carbon tubes embedded within shungite. It allows the stone to neutralize electromagnetic radiation, and shungite is the only source where fullerene is found.
Many people consider shungite’s ability to shield the body from EMF radiation as it’s most beneficial characteristic because most of modern society is irrevocably connected and consumed with electronic technology.
Televisions, computers, cell phones, and WiFi all radiate electromagnetic frequencies. Consequently, people who experience a heightened sensitivity to EMFs can benefit from using shungite items as a preventative measure.
How to Use Shungite for EMF ProtectionTo ensure you are receiving the maximum level of protection against the electromagnetic field, it is generally suggested that multiple pieces of shungite be used to block exposure from electronic gadgets that emit electromagnetic frequencies. Basically, you’ll receive more protection from EMF radiation by having more of the shungite stones around you.
In choosing a product that fits best into your lifestyle, it’s prudent to pick a suitable high-quality shungite product as a test case to determine if it is an agreeable fit. Because of their shape and size, shungite pyramids are considered the best source in providing comprehensive EMF protection. Some other popular shungite products include:
Shungite cell phone plates
Shungite cell phone plates
Shungite pendants
Shungite earrings
Shungite bracelets
Some people enjoy using elite shungite nuggets as an EMF stabilizer. These nuggets are often sold in their natural unadulterated state and can be placed in your pocket, by your workspace or next to your bed. Another major shungite benefit is that the stone’s protective qualities last forever only requiring regular cleaning and charging to retain their potency.
Cleaning and Charging ShungiteShungite can be used indefinitely with the proper maintenance. To recharge shungite, rinse the stone for two minutes under warm water, then let the stone sit under full sunlight for three hours, turning once.
Repeat this process whenever you feel the shungite’s absorbing properties are diminishing. For those who use shungite to filter water, the above procedure should be employed monthly to rejuvenate it’s purifying capacity.
To clean the stone, mix warm water with an acidic component such as lemon, and place the shungite in the mixture for several hours. This process removes dirt from the surface of the stone.
Shungite for Water PurificationWater can be purified in a variety of ways to make it safe for consumption. This includes using chlorine, iodine, and ultraviolet as well as boiling the water for filtration. Yet although these approaches destroy bacteria, they also add potentially harmful chemicals to the water and diminish the water quality.
READ Are Your Pets Being Exposed to EMF?Allowing shungite’s stones to sit in water for a few days creates what is known as “shungite water.” Again, this process can be traced to Peter the Great of Russia who gave shungite water to his soldiers and used it in his spa. And now, water testing methods have proven that the shungite mineral can purify water, and it is often found in modern water filtration technology.
Much like the Russian Czar, many people enjoy the health and wellness benefits of shungite baths. To make these healing baths, place 14 to 17 ounces of shungite stones in hot water for approximately 15 to 20 minutes. After this process, you can savor the regenerative physical and mental effects from this healing bath.
Shungite’s immense antibacterial capacity is one of the reasons it serves as an excellent option for water purification and mineralization. In a case study, bacteria was placed in shungite infused water, and within an hour, the level of bacteria had significantly diminished.
Research shows that shungite removes heavy metal and harmful bacteria from water and enhances its appearance and taste. In addition, many holistic health practitioners suggest using shungite water for a variety of illnesses such as:
Consequently, type I elite shungite is considered the most suitable choice for purifying water because it is composed of the highest percentage fullerene carbon.
Shungite and Geopathic StressGeopathic stress is a somewhat unknown concept that covers the connection between the Earth’s energy and human well-being. It is seen to be a prevailing component in a range of physical and psychological ailments including those that affect the immune system. Geopathic stress is often referred to in the same realm as “sick building syndrome” or “EMF pollution.”
This stress occurs when people spend too much time in areas where the Earth’s energy is disturbed by an unsteady electromagnetic field. This usually stems from living around mineral compounds, electricity lines, formations underground or other geographic stress zones that emit weakened electromagnetic frequencies. So if your home is located in one of these fault lines, it’s important to understand how this can impact your health.
Your general health and chiefly your risk of developing cancer can be drastically reduced when you are protected from geopathic stress. Acclaimed cancer specialist Dr. Hans Nieper found that 92 percent of his patients with cancer were also under geopathic stress. Although geopathic stress does not specifically cause cancer, it does impair the body’s lymphatic system which fights and destroys cancer cells thereby increasing the risk of developing the disease.
Living or working around geopathic stress can also cause chronic insomnia and fatigue which may be minor ailments but can still interfere with a person’s quality of life. However, shungite electromagnetic absorbing stones can reduce the harmful effects from living in a geopathic stress zone.
Checking Shungite AuthenticityYou can verify if Shungite is authentic pretty easily since all authentic shungite stones are electrically conductive. Simply create a streamline of electricity from a battery to a bulb by connecting two wires to each of them, and placing a shungite stone between them. Because of the high carbon level, authentic shungite will conduct electricity by making the bulb lighten.
As you can see, in a modern world where electronic devices and geopathic stress surrounds you, a shungite stone is a simple yet effective way to shield you and your family from EMF radiation. This powerful and attractive stone can be placed beside routers, worn as jewelry or displayed near any electronic gadget. Shungite is a sensible choice in reducing the negative effects of EMF waves to ensure your body and mind stay healthy and balanced.
Contents [hide]
- What is an Electromagnetic Field?
- What is EMF Radiation?
- Is Non-ionizing EMF Radiation Harmful to Your Health?
- What is Shungite?
- Where Does Shungite Come From?
- How was Shungite Formed?
- Shungite EMF Protective Properties
- How to Use Shungite for EMF Protection
- Cleaning and Charging Shungite
- Shungite for Water Purification
- Shungite and Geopathic Stress
- Checking Shungite Authenticity
The magnetic field is the region that surrounds magnetic material or electrical currents and increases in strength when a current increases. Predictably, the magnetic field strength will quickly reduce the farther it gets from its source. You probably notice the magnetic field in action when permanent magnets pull and repel magnetic materials.
An electric field is an area around a charged particle that displays the direction of a positive charge within the field. The field flows outward in the direction of a positive charge and inward toward a negative charge. You may notice the static electric field when your clothes cling to each other after removing them from the dryer.
Electric fields are generated if a device is turned on or off while magnetic fields are induced only when a current is moving as is the case with power lines. Electric fields are diminished by objects like walls, whereas magnetic fields can pass through any living thing, object or material. Jointly, electric and magnetic fields are called the electromagnetic field or EMF, and the combined forces cause electromagnetic radiation.
What is EMF Radiation?EMF radiation consists of light particles or photons that travel in waves at the speed of light through space and transfer electromagnetic radiant energy. It takes form in gamma rays, X-rays, radio, micro and ultraviolet waves as well as anything used for electrical power and man-made or natural lighting. The National Institute of Environmental Health Science separates EMF’s into two radioactive groups:
Non-ionizing – They have lower frequencies of radiation and include but are not limited to visible light, MRI scans, computers, WIFI, power lines, and microwave ovens.
Ionizing – These have higher levels of radiation that can possibly cause cellular and DNA damage. Examples of ionizing high-frequency radiation include gamma rays, X Rays, and ultraviolet light.
Some people believe non-ionizing radiation is totally safe; however, there have been compelling studies that show that long-term exposure to low-level electromagnetic fields may be detrimental to your health. In fact, more studies are being conducted to determine the relationship between EMFs and cancer.
Is Non-ionizing EMF Radiation Harmful to Your Health?Research has shown that electromagnetic stress causes a vibration within the human body at above average frequencies making it more susceptible to many health issues such as viruses, degenerative diseases, and pollution. In 2010 a cadre of scientists issued a report that outlined the connection between electromagnetic fields and physical health. The report concluded that people who are prone to EMF sensitivity experience an increase in minor medical issues such as fatigue, memory loss, and insomnia as well as more serious problems like increased blood sugar levels and arthritis.
Recently, the International Agency for Research on Cancer which is a part of the World Health Organization released a statement stating that EMFs are “possibly carcinogenic to humans.” If you’re wondering how EMFs can potentially affect your health, this list outlines how electromagnetic frequencies can affect the human body.
READ Does Aluminum Foil Protect You From Electromagnetic Field Radiation?Mood swings and mental instability
Your nervous system is the part of your body that’s most susceptible to the effects of EMF radiation. Memory loss, depression, and a reduced attention span can be signs of a damaged nervous system.
Vulnerable immune system
You will get sick more often, especially with common colds, if your immune system has been compromised by EMF radiation.
Uncommon muscle pain and headaches
Unusual muscles aches, consistent migraines, and an irregular pulse could mean that your muscular and cardiovascular systems have been negatively affected by radiation
Chronic fatigue, anxiousness, and insomnia
Long-term EMF exposure can create erratic hormonal levels which may result in increased fatigue, anxiety, and sleeping problems.
So because electronic devices are an inescapable part of your daily activities, you might be wondering if there’s a functional way to protect yourself from EMF radiation without changing your standard of living. Fortunately, the EMF absorbing stone known as shungite can be a viable solution.
The easiest and most cost-effective way to counteract the effects of electromagnetic radiation is shungite. This EMF absorbing stone is a naturally occurring geomagnetic mineral that known for its absorbing and neutralizing abilities. Shungite is also considered to be a very potent antioxidant that through research has proved to be beneficial to your health.
What is Shungite?Shungite is a shiny stone that is made of mostly carbon which is the same element that diamonds and graphite come from. However, unlike graphite, it has an unusual luminous black color with the higher end stones exhibiting a silver shimmer.
The stone originated two billion years ago and at that time, was made of just pure carbon yet now are stones made from fragments of shungite particles. In its purest form, shungite is composed of small quantities of miniscule hollow carbon particles called fullerenes. Shungite is typically separated into the following three categories based on how much carbon composition exists:
Type I: Elite shungite also known as noble or silver shungiteElite shungite is a black, glasslike mineral with a silver reflect. it has more than 90 percent organic carbon and is believed to possess healing energy. Because it is composed of mostly carbon, elite shungite is very rare, fragile, and difficult to shape which is why it is not polished or cut and is typically sold in its original semicircular shape.
Type II: Petrovsky shungiteThis type of stone was named after the Russian Emperor, Peter the Great and is made of 50 to 75 percent organic carbon. Unlike, elite shungite it can be cut or polished giving it an intense shine. This stone is frequently used to fabricate objects; however, Petrovsky shungite has intense energy stabilizing capabilities that are similar to shungite.
Type III: Black or regular shungiteStructures like pyramids and spheres are often composed of black shungite which is composed of 30 to 50 percent carbon. Because of its solid black color, regular shungite is a favored stone for creating handmade jewelry.
Where Does Shungite Come From?Shungite was discovered in Russia near the Shunga village in the late 1800’s. Peter the Great constructed what would now be considered the first spa in Russia to enjoy the cleansing effect of the stone. Today, the powerful shungite EMF radiation blocking and purifying capability has been recognized by the scientific community.
Shungite is primarily found by Lake Onega in the Karelia region in Russia; however, it has also been found in smaller measures in a coal mine by Chelyabinsk and among the volcanic rocks at Kamchatka. Shungite’s primary source in Karelia produces approximately 300,000,000 tons of the mineral, so there won’t be any shortages of the stone in the near future. Despite Shungite is remote origins, it is very accessible to consumers due to the heightened demand for its purifying and EMF neutralizing capabilities.
How was Shungite Formed?The scientific community hasn’t come to a universal conclusion as to how shungite is formed. But there are many theories on where it comes from with the most dominant being from either an ancient meteorite or an ancient body of water.
READ 2 Types of EMF Exposure and Why You Should Be WorriedOne popular theory is that several billion years ago there were large clusterings of microscopic carbon-based organisms in a prehistoric lake, sea or ocean in what is now North-Western Russia. Over millions of years, the organisms died, and their high carbon deposits formed at the bottom of the sea. As the Earth’s plates shifted through time, the deposits transitioned from the sea to a specific area on land.
The other popular hypothesis is that the shungite deposits came from a meteorite that landed on Earth a long time ago. This theory would account for the high carbon components as well as explain why the mineral is primarily found in one area. Nevertheless, some of shungite’s allure may be due to its curious origins as well as its EMF shielding abilities.
Shungite EMF Protective PropertiesMany studies have concluded that shungite is effective in lessening the effects of electromagnetic sensitivity. So what is the distinctive feature that allows shungite to serve as a shield from EMF radiation? The answer lies in the carbon compound called fullerene.
In 1996, Sir Harold Kroto and two other scientists won a Nobel Prize in chemistry for their discovery of fullerene eleven years earlier. Also known as buckyballs, fullerenes are hollow molecular carbon tubes embedded within shungite. It allows the stone to neutralize electromagnetic radiation, and shungite is the only source where fullerene is found.
Many people consider shungite’s ability to shield the body from EMF radiation as it’s most beneficial characteristic because most of modern society is irrevocably connected and consumed with electronic technology.
Televisions, computers, cell phones, and WiFi all radiate electromagnetic frequencies. Consequently, people who experience a heightened sensitivity to EMFs can benefit from using shungite items as a preventative measure.
How to Use Shungite for EMF ProtectionTo ensure you are receiving the maximum level of protection against the electromagnetic field, it is generally suggested that multiple pieces of shungite be used to block exposure from electronic gadgets that emit electromagnetic frequencies. Basically, you’ll receive more protection from EMF radiation by having more of the shungite stones around you.
In choosing a product that fits best into your lifestyle, it’s prudent to pick a suitable high-quality shungite product as a test case to determine if it is an agreeable fit. Because of their shape and size, shungite pyramids are considered the best source in providing comprehensive EMF protection. Some other popular shungite products include:
Shungite cell phone plates
Shungite cell phone plates
Shungite pendants
Shungite earrings
Shungite bracelets
Some people enjoy using elite shungite nuggets as an EMF stabilizer. These nuggets are often sold in their natural unadulterated state and can be placed in your pocket, by your workspace or next to your bed. Another major shungite benefit is that the stone’s protective qualities last forever only requiring regular cleaning and charging to retain their potency.
Cleaning and Charging ShungiteShungite can be used indefinitely with the proper maintenance. To recharge shungite, rinse the stone for two minutes under warm water, then let the stone sit under full sunlight for three hours, turning once.
Repeat this process whenever you feel the shungite’s absorbing properties are diminishing. For those who use shungite to filter water, the above procedure should be employed monthly to rejuvenate it’s purifying capacity.
To clean the stone, mix warm water with an acidic component such as lemon, and place the shungite in the mixture for several hours. This process removes dirt from the surface of the stone.
Shungite for Water PurificationWater can be purified in a variety of ways to make it safe for consumption. This includes using chlorine, iodine, and ultraviolet as well as boiling the water for filtration. Yet although these approaches destroy bacteria, they also add potentially harmful chemicals to the water and diminish the water quality.
READ Are Your Pets Being Exposed to EMF?Allowing shungite’s stones to sit in water for a few days creates what is known as “shungite water.” Again, this process can be traced to Peter the Great of Russia who gave shungite water to his soldiers and used it in his spa. And now, water testing methods have proven that the shungite mineral can purify water, and it is often found in modern water filtration technology.
Much like the Russian Czar, many people enjoy the health and wellness benefits of shungite baths. To make these healing baths, place 14 to 17 ounces of shungite stones in hot water for approximately 15 to 20 minutes. After this process, you can savor the regenerative physical and mental effects from this healing bath.
Shungite’s immense antibacterial capacity is one of the reasons it serves as an excellent option for water purification and mineralization. In a case study, bacteria was placed in shungite infused water, and within an hour, the level of bacteria had significantly diminished.
Research shows that shungite removes heavy metal and harmful bacteria from water and enhances its appearance and taste. In addition, many holistic health practitioners suggest using shungite water for a variety of illnesses such as:
- Diabetes
- Allergies
- Colds
- Heart Disease
- Headaches
- Headaches
- Fatigue
- Fatigue
Consequently, type I elite shungite is considered the most suitable choice for purifying water because it is composed of the highest percentage fullerene carbon.
Shungite and Geopathic StressGeopathic stress is a somewhat unknown concept that covers the connection between the Earth’s energy and human well-being. It is seen to be a prevailing component in a range of physical and psychological ailments including those that affect the immune system. Geopathic stress is often referred to in the same realm as “sick building syndrome” or “EMF pollution.”
This stress occurs when people spend too much time in areas where the Earth’s energy is disturbed by an unsteady electromagnetic field. This usually stems from living around mineral compounds, electricity lines, formations underground or other geographic stress zones that emit weakened electromagnetic frequencies. So if your home is located in one of these fault lines, it’s important to understand how this can impact your health.
Your general health and chiefly your risk of developing cancer can be drastically reduced when you are protected from geopathic stress. Acclaimed cancer specialist Dr. Hans Nieper found that 92 percent of his patients with cancer were also under geopathic stress. Although geopathic stress does not specifically cause cancer, it does impair the body’s lymphatic system which fights and destroys cancer cells thereby increasing the risk of developing the disease.
Living or working around geopathic stress can also cause chronic insomnia and fatigue which may be minor ailments but can still interfere with a person’s quality of life. However, shungite electromagnetic absorbing stones can reduce the harmful effects from living in a geopathic stress zone.
Checking Shungite AuthenticityYou can verify if Shungite is authentic pretty easily since all authentic shungite stones are electrically conductive. Simply create a streamline of electricity from a battery to a bulb by connecting two wires to each of them, and placing a shungite stone between them. Because of the high carbon level, authentic shungite will conduct electricity by making the bulb lighten.
As you can see, in a modern world where electronic devices and geopathic stress surrounds you, a shungite stone is a simple yet effective way to shield you and your family from EMF radiation. This powerful and attractive stone can be placed beside routers, worn as jewelry or displayed near any electronic gadget. Shungite is a sensible choice in reducing the negative effects of EMF waves to ensure your body and mind stay healthy and balanced.
PROFESSORS ARE AVORDED THE NOBEL PRICE FOR FINDING
THAT SHUNGITE IS PROTECTING AGINST EMF RADIATION
The Nobel Prize in Chemistry 1996
Press releaseEnglish
Swedish
9 October 1996
The Royal Swedish Academy of Sciences has decided to award the 1996 Nobel Prize in Chemistry to
Professor Robert F. Curl, Jr., Rice University, Houston, USA,
Professor Sir Harold W. Kroto, University of Sussex, Brighton, U.K., and
Professor Richard E. Smalley, Rice University, Houston, USA,
for their discovery of fullerenes.
The discovery of carbon atoms bound in the form of a ball is rewardedNew forms of the element carbon – called fullerenes – in which the atoms are arranged in closed shells was discovered in 1985 by Robert F. Curl, Harold W. Kroto and Richard E. Smalley. The number of carbon atoms in the shell can vary, and for this reason numerous new carbon structures have become known. Formerly, six crystalline forms of the element carbon were known, namely two kinds of graphite, two kinds of diamond, chaoit and carbon(VI). The latter two were discovered in 1968 and 1972.
Fullerenes are formed when vaporised carbon condenses in an atmosphere of inert gas. The gaseous carbon is obtained e.g. by directing an intense pulse of laser light at a carbon surface. The released carbon atoms are mixed with a stream of helium gas and combine to form clusters of some few up to hundreds of atoms. The gas is then led into a vacuum chamber where it expands and is cooled to some degrees above absolute zero. The carbon clusters can then be analysed with mass spectrometry.
Curl, Kroto and Smalley performed this experiment together with graduate students J.R. Heath and S.C. OBrien during a period of eleven days in 1985. By fine-tuning the experiment they were able in particular to produce clusters with 60 carbon atoms and clusters with 70. Clusters of 60 carbon atoms, C60, were the most abundant. They found high stability in C60, which suggested a molecular structure of great symmetry. It was suggested that C60 could be a “truncated icosahedron cage”, a polyhedron with 20 hexagonal (6-angled) surfaces and 12 pentagonal (5-angled) surfaces. The pattern of a European football has exactly this structure, as does the geodetic dome designed by the American architect R. Buckminster Fuller for the 1967 Montreal World Exhibition. The researchers named the newly-discovered structure buckminsterfullerene after him.
The discovery of the unique structure of the C60 was published in the journal Nature and had a mixed reception – both criticism and enthusiastic acceptance. No physicist or chemist had expected that carbon would be found in such a symmetrical form other than those already known. Continuing their work during 1985-90, Curl, Kroto and Smalley obtained further evidence that the proposed structure ought to be correct. Among other things they succeeded in identifying carbon clusters that enclosed one or more metal atoms. In 1990 physicists W. Krätschmer and D.R. Huffman for the first time produced isolable quantities of C60 by causing an arc between two graphite rods to burn in a helium atmosphere and extracting the carbon condensate so formed using an organic solvent. They obtained a mixture of C60 and C70, the structures of which could be determined. This confirmed the correctness of the C60 hypothesis. The way was thus open for studying the chemical properties of C60 and other carbon clusters such as C70, C76, C78 and C84. New substances were produced from these compounds, with new and unexpected properties. An entirely new branch of chemistry developed, with consequences in such diverse areas as astrochemistry, superconductivity and materials chemistry/physics.
Background
Many widely diverse research areas coincide in the discovery of the fullerenes. Harold W. Kroto was at the time active in microwave spectroscopy, a science which thanks to the growth of radioastronomy can be used for analysing gas in space, both in stellar atmospheres and in interstellar gas clouds. Kroto was particularly interested in carbon-rich giant stars. He had discovered and investigated spectrum lines in their atmospheres and found that they could be ascribed to a kind of long-chained molecule of only carbon and nitrogen, termed cyanopolyynes. The same sort of molecules is also found in interstellar gas clouds. Kroto’s idea was that these carbon compounds had been formed in stellar atmospheres, not in clouds. He now wished to study the formation of these long-chain molecules more closely.
He got in touch with Richard E. Smalley, whose research was in cluster chemistry, an important part of chemical physics. A cluster is an aggregate of atoms or molecules, something in between microscopic particles and macroscopic particles. Smalley had designed and built a special laser-supersonic cluster beam apparatus able to vaporise almost any known material into a plasma of atoms and study the design and distribution of the clusters. His paramount interest was clusters of metal atoms, e.g. metals included in semiconductors, and he often performed these investigations together with Robert F. Curl, whose background was in microwave and infra-red spectroscopy.
Atoms form clusters
When atoms in a gas phase condense to form clusters, a series is formed where the size of the clusters varies from a few atoms to many hundreds. There are normally two size maxima visible in the distribution curve, one around small clusters and one around large. It is often found that cer-tain cluster sizes may dominate, and the number of atoms in these is termed a “magic number”, a term borrowed from nuclear physics. These dominant cluster sizes were assumed to have some special property such as high symmetry.
Fruitful contact
Through his acquaintanceship with Robert Curl, Kroto learned that it should be possible to use Smalley’s instrument to study the vaporisation and cluster formation of carbon, which might afford him evidence that the long-carbon-chain compounds could have been formed in the hot parts of stellar atmospheres. Curl conveyed this interest to Smalley and the result was that on 1 September 1985 Kroto arrived in Smalley’s laboratory to start, together with Curl and Smalley, the experiments on carbon vaporisation. In the course of the work it proved possible to influence drastically the size distribution of the carbon clusters where, predominantly, 60 appeared as a magic number but also 70 (Fig. 1). The research group now got something else to think about. Instead of long carbon chains, the idea arose that the C60 cluster could have the structure of a truncated (cut off) icosahedron (Fig. 2) since its great stability was assumed to correspond to a closed shell with a highly symmetrical structure. C60 was given a fanciful name, buckminsterfullerene, after the American architect R. Buckminster Fuller, inventor of the geodesic dome. This hectic period ended on 12 September with the despatch of a manuscript entitled C60: Buckminsterfullerene to Nature. The journal received it on 13 September and published the article on 14 November 1985.
Sensational news
For chemists the proposed structure was uniquely beautiful and satisfying. It corresponds to an aromatic, three-dimensional system in which single and double bonds alternated, and was thus of great theoretical significance. Here, moreover, was an entirely new example from a different research tradition with roots in organic chemistry: producing highly symmetrical molecules so as to study their properties. The Platonic bodies have often served as patterns, and hydrocarbons had already been synthesised as tetrahedral, cubic or dodecahedral (12-sided) structures.
Carbon atoms per cluster
Fig. 1.
Using mass spectroscopy it was found that the size distribution of carbon clusters could be drastically affected by increasing the degree of chemical “boiling” in the inlet nozzle to the vacuum chamber. Clusters with 60 and 70 carbon atoms could be produced. (Acc. Chem. Res., Vol. 25, No. 3, 1992)
Fig. 2.
Models of the structures of C60. (Acc. Chem. Res., Vol. 25, No. 3, 1992)Further investigations
To gain further clarity Curl, Kroto and Smalley continued their investigations of C60. They attempted to make it react with other compounds. Gases such as hydrogen, nitrous oxide, carbon monoxide, sulphur dioxide, oxygen or ammonia were injected into the gas stream, but no effect on the C60 peak recorded in the mass spectrometer could be demonstrated. This showed that C60 was a slow-reacting compound. It also turned out that all carbon clusters with an even number of carbon atoms from 40-80 (the interval that could be studied) reacted equally slowly. Analogously with C60 all these should then correspond to entirely closed structures, resembling cages. This was in agreement with Euler’s law, a mathematical proposition which states that for any polygon with n edges, where n is an even number greater than 22, at least one polyhedron can be constructed with 12 pentagons and (n-20)/2 hexagons, which, in simple terms, states that it is possible with 12 pentagons and with none or more than one hexagon to construct a polyhedron. For large n many different closed structures can occur, thus also for C60, and it was presumably the beautiful symmetry of the proposed structure that gave it the preference.
The combination of chemical inertia in clusters with even numbers of carbon atoms and the possibility that all these could possess closed structures in accordance with Euler’s law, led to the proposal that all these carbon clusters should have closed structures. They were given the name fullerenes and conceivably an almost infinite number of fullerenes could exist. The element carbon had thus assumed an almost infinite number of different structures.
C60 and metals
New experiments were rapidly devised to test the C60 hypothesis. Since the C60 structure is hollow, with room for one or more other atoms, attempts were made to enclose a metal atom. A graphite sheet was soaked with a solution of a metal salt (lanthanum chloride, LaCl3) and subjected to vaporisation-condensation experiments. Masspectroscopic analysis of the clusters formed showed the presence of C60La+. These proved to be photoresistant, i.e. irradiation with intense laser light did not remove the metal atoms. This reinforced the idea that metal atoms were captured inside the cage structure.
The possibility of producing clusters with a metal atom enclosed gave rise to what was termed the “shrink-wrapping” experiment. Ions of one and the same size or at least similar sizes were gathered in a magnetic trap and subjected to a laser pulse. It then turned out that the laser beam caused the carbon cage to shrink by 2 carbon atoms at a time: at a certain cage size, when the pressure on the metal atom inside became too great, the fragmentation ceased. The shell had then shrunk so that it fitted exactly around the metal atom. For C60Cs+ this size was at C48Cs+, for C60K+ it was at C44K+ and for C60+ at C32+.
Further strong evidence gave rise to new chemistry
At the end of the 1980s, strong evidence was available that the C60 hypothesis was correct. In 1990 the synthesis of macroscopic quantities of C60 through carbon arc vaporisation between two graphite electrodes permitted the attainment of full certainty – the whole battery of methods for structure determination could be applied to C60 and other fullerenes and completely confirmed the fullerene hypothesis. As opposed to the other forms of carbon the fullerenes represent well-defined chemical compounds with in some respects new properties. A whole new chemistry has developed to manipulate the fullerene structure, and the properties of fullerenes can be studied systematically. It is possible to produce superconducting salts of C60, new three-dimensional polymers, new catalysts, new materials and electrical and optical properties, sensors, and so on. In addition, it has been possible to produce thin tubes with closed ends, nanotubes, arranged in the same way as fullerenes. From a theoretical viewpoint, the discovery of the fullerenes has influenced our conception of such widely separated scientific problems as the galactic carbon cycle and classical aromaticity, a keystone of theoretical chemistry. No practically useful applications have yet been produced, but this is not to be expected as early as six years after macroscopic quantities of fullerenes became available.
Further reading
Jim Baggott, Perfect Symmetry: The Accidental Discovery of Buckminsterfullerene, Oxford University Press, 1994, IX + 315 pp.
Hugh Aldersey-Williams, The Most Beautiful Molecule: An Adventure in Chemistry, Aurum Press, London, 1995, IX + 340 pp.
Robert F. Curl and Richard E. Smalley, Probing C60, Science, 18 Nov. 1988 Vol. 242.
Harold Kroto, Space, Stars, C60, and Soot, Science, 25 Nov. 1988 Vol. 242.
H.W. Kroto, A.W. Allaf, and S.P. Balm, C60: Buckminsterfullerene, American Chemical Society, 1991.
Richard E. Smalley, Great Balls of Carbon; The Story of Buckminsterfullerene, The Sciences, March/April 1991.
The All-Star of Buckyball; Profile: Richard E. Smalley, Scientific American, September 1993.
Rudy M. Baum, Commercial Uses of Fullerenes and Derivatives Slow to Develop, News Focus, Nov. 22, 1993 C&EN.
Hargittai, Istv(SIGMA)n, Discoverers of Buckminsterfullerene, The Chemical Intelligencer, Springer-Verlag, New York, 1995.
Robert F. Curl Jr., was born in 1933 in Alice, Texas, USA: Ph.D. in chemistry in 1957 at University of California, Berkeley, USA. Curl has since 1958 worked at Rice University, where he has been a professor since 1967.
Professor Robert F. Curl Jr.
Department of Chemistry
Rice University
P.O. Box 1892
Houston, TX 77251, USA
Sir Harold W. Kroto was born in 1939 in Wisbech, Cambridgeshire, UK. He obtained his Ph.D. in 1964 at the University of Sheffield, UK. In 1967 he moved to the University of Sussex, where he still works. In 1985 he became Professor of Chemistry there and in 1991 Royal Society Research Professor.
Professor Sir Harold W. Kroto
School of Chemistry and Molecular Sciences
University of Sussex
Brighton, Sussex BN1 9QJ, UK
Richard E. Smalley was born in 1943 in Akron, Ohio, USA. Ph.D. in chemistry 1973 at Princeton University, USA. Professor of Chemistry at Rice University since 1981 and also Professor of Physics at the same university since 1990. Member of the National Academy of Sciences in the USA and other bodies.
Professor Richard E. Smalley
Department of Chemistry
Rice University
P.O. Box 1892
Houston, TX 77251, USA
To cite this section
Press releaseEnglish
Swedish
9 October 1996
The Royal Swedish Academy of Sciences has decided to award the 1996 Nobel Prize in Chemistry to
Professor Robert F. Curl, Jr., Rice University, Houston, USA,
Professor Sir Harold W. Kroto, University of Sussex, Brighton, U.K., and
Professor Richard E. Smalley, Rice University, Houston, USA,
for their discovery of fullerenes.
The discovery of carbon atoms bound in the form of a ball is rewardedNew forms of the element carbon – called fullerenes – in which the atoms are arranged in closed shells was discovered in 1985 by Robert F. Curl, Harold W. Kroto and Richard E. Smalley. The number of carbon atoms in the shell can vary, and for this reason numerous new carbon structures have become known. Formerly, six crystalline forms of the element carbon were known, namely two kinds of graphite, two kinds of diamond, chaoit and carbon(VI). The latter two were discovered in 1968 and 1972.
Fullerenes are formed when vaporised carbon condenses in an atmosphere of inert gas. The gaseous carbon is obtained e.g. by directing an intense pulse of laser light at a carbon surface. The released carbon atoms are mixed with a stream of helium gas and combine to form clusters of some few up to hundreds of atoms. The gas is then led into a vacuum chamber where it expands and is cooled to some degrees above absolute zero. The carbon clusters can then be analysed with mass spectrometry.
Curl, Kroto and Smalley performed this experiment together with graduate students J.R. Heath and S.C. OBrien during a period of eleven days in 1985. By fine-tuning the experiment they were able in particular to produce clusters with 60 carbon atoms and clusters with 70. Clusters of 60 carbon atoms, C60, were the most abundant. They found high stability in C60, which suggested a molecular structure of great symmetry. It was suggested that C60 could be a “truncated icosahedron cage”, a polyhedron with 20 hexagonal (6-angled) surfaces and 12 pentagonal (5-angled) surfaces. The pattern of a European football has exactly this structure, as does the geodetic dome designed by the American architect R. Buckminster Fuller for the 1967 Montreal World Exhibition. The researchers named the newly-discovered structure buckminsterfullerene after him.
The discovery of the unique structure of the C60 was published in the journal Nature and had a mixed reception – both criticism and enthusiastic acceptance. No physicist or chemist had expected that carbon would be found in such a symmetrical form other than those already known. Continuing their work during 1985-90, Curl, Kroto and Smalley obtained further evidence that the proposed structure ought to be correct. Among other things they succeeded in identifying carbon clusters that enclosed one or more metal atoms. In 1990 physicists W. Krätschmer and D.R. Huffman for the first time produced isolable quantities of C60 by causing an arc between two graphite rods to burn in a helium atmosphere and extracting the carbon condensate so formed using an organic solvent. They obtained a mixture of C60 and C70, the structures of which could be determined. This confirmed the correctness of the C60 hypothesis. The way was thus open for studying the chemical properties of C60 and other carbon clusters such as C70, C76, C78 and C84. New substances were produced from these compounds, with new and unexpected properties. An entirely new branch of chemistry developed, with consequences in such diverse areas as astrochemistry, superconductivity and materials chemistry/physics.
Background
Many widely diverse research areas coincide in the discovery of the fullerenes. Harold W. Kroto was at the time active in microwave spectroscopy, a science which thanks to the growth of radioastronomy can be used for analysing gas in space, both in stellar atmospheres and in interstellar gas clouds. Kroto was particularly interested in carbon-rich giant stars. He had discovered and investigated spectrum lines in their atmospheres and found that they could be ascribed to a kind of long-chained molecule of only carbon and nitrogen, termed cyanopolyynes. The same sort of molecules is also found in interstellar gas clouds. Kroto’s idea was that these carbon compounds had been formed in stellar atmospheres, not in clouds. He now wished to study the formation of these long-chain molecules more closely.
He got in touch with Richard E. Smalley, whose research was in cluster chemistry, an important part of chemical physics. A cluster is an aggregate of atoms or molecules, something in between microscopic particles and macroscopic particles. Smalley had designed and built a special laser-supersonic cluster beam apparatus able to vaporise almost any known material into a plasma of atoms and study the design and distribution of the clusters. His paramount interest was clusters of metal atoms, e.g. metals included in semiconductors, and he often performed these investigations together with Robert F. Curl, whose background was in microwave and infra-red spectroscopy.
Atoms form clusters
When atoms in a gas phase condense to form clusters, a series is formed where the size of the clusters varies from a few atoms to many hundreds. There are normally two size maxima visible in the distribution curve, one around small clusters and one around large. It is often found that cer-tain cluster sizes may dominate, and the number of atoms in these is termed a “magic number”, a term borrowed from nuclear physics. These dominant cluster sizes were assumed to have some special property such as high symmetry.
Fruitful contact
Through his acquaintanceship with Robert Curl, Kroto learned that it should be possible to use Smalley’s instrument to study the vaporisation and cluster formation of carbon, which might afford him evidence that the long-carbon-chain compounds could have been formed in the hot parts of stellar atmospheres. Curl conveyed this interest to Smalley and the result was that on 1 September 1985 Kroto arrived in Smalley’s laboratory to start, together with Curl and Smalley, the experiments on carbon vaporisation. In the course of the work it proved possible to influence drastically the size distribution of the carbon clusters where, predominantly, 60 appeared as a magic number but also 70 (Fig. 1). The research group now got something else to think about. Instead of long carbon chains, the idea arose that the C60 cluster could have the structure of a truncated (cut off) icosahedron (Fig. 2) since its great stability was assumed to correspond to a closed shell with a highly symmetrical structure. C60 was given a fanciful name, buckminsterfullerene, after the American architect R. Buckminster Fuller, inventor of the geodesic dome. This hectic period ended on 12 September with the despatch of a manuscript entitled C60: Buckminsterfullerene to Nature. The journal received it on 13 September and published the article on 14 November 1985.
Sensational news
For chemists the proposed structure was uniquely beautiful and satisfying. It corresponds to an aromatic, three-dimensional system in which single and double bonds alternated, and was thus of great theoretical significance. Here, moreover, was an entirely new example from a different research tradition with roots in organic chemistry: producing highly symmetrical molecules so as to study their properties. The Platonic bodies have often served as patterns, and hydrocarbons had already been synthesised as tetrahedral, cubic or dodecahedral (12-sided) structures.
Carbon atoms per cluster
Fig. 1.
Using mass spectroscopy it was found that the size distribution of carbon clusters could be drastically affected by increasing the degree of chemical “boiling” in the inlet nozzle to the vacuum chamber. Clusters with 60 and 70 carbon atoms could be produced. (Acc. Chem. Res., Vol. 25, No. 3, 1992)
Fig. 2.
Models of the structures of C60. (Acc. Chem. Res., Vol. 25, No. 3, 1992)Further investigations
To gain further clarity Curl, Kroto and Smalley continued their investigations of C60. They attempted to make it react with other compounds. Gases such as hydrogen, nitrous oxide, carbon monoxide, sulphur dioxide, oxygen or ammonia were injected into the gas stream, but no effect on the C60 peak recorded in the mass spectrometer could be demonstrated. This showed that C60 was a slow-reacting compound. It also turned out that all carbon clusters with an even number of carbon atoms from 40-80 (the interval that could be studied) reacted equally slowly. Analogously with C60 all these should then correspond to entirely closed structures, resembling cages. This was in agreement with Euler’s law, a mathematical proposition which states that for any polygon with n edges, where n is an even number greater than 22, at least one polyhedron can be constructed with 12 pentagons and (n-20)/2 hexagons, which, in simple terms, states that it is possible with 12 pentagons and with none or more than one hexagon to construct a polyhedron. For large n many different closed structures can occur, thus also for C60, and it was presumably the beautiful symmetry of the proposed structure that gave it the preference.
The combination of chemical inertia in clusters with even numbers of carbon atoms and the possibility that all these could possess closed structures in accordance with Euler’s law, led to the proposal that all these carbon clusters should have closed structures. They were given the name fullerenes and conceivably an almost infinite number of fullerenes could exist. The element carbon had thus assumed an almost infinite number of different structures.
C60 and metals
New experiments were rapidly devised to test the C60 hypothesis. Since the C60 structure is hollow, with room for one or more other atoms, attempts were made to enclose a metal atom. A graphite sheet was soaked with a solution of a metal salt (lanthanum chloride, LaCl3) and subjected to vaporisation-condensation experiments. Masspectroscopic analysis of the clusters formed showed the presence of C60La+. These proved to be photoresistant, i.e. irradiation with intense laser light did not remove the metal atoms. This reinforced the idea that metal atoms were captured inside the cage structure.
The possibility of producing clusters with a metal atom enclosed gave rise to what was termed the “shrink-wrapping” experiment. Ions of one and the same size or at least similar sizes were gathered in a magnetic trap and subjected to a laser pulse. It then turned out that the laser beam caused the carbon cage to shrink by 2 carbon atoms at a time: at a certain cage size, when the pressure on the metal atom inside became too great, the fragmentation ceased. The shell had then shrunk so that it fitted exactly around the metal atom. For C60Cs+ this size was at C48Cs+, for C60K+ it was at C44K+ and for C60+ at C32+.
Further strong evidence gave rise to new chemistry
At the end of the 1980s, strong evidence was available that the C60 hypothesis was correct. In 1990 the synthesis of macroscopic quantities of C60 through carbon arc vaporisation between two graphite electrodes permitted the attainment of full certainty – the whole battery of methods for structure determination could be applied to C60 and other fullerenes and completely confirmed the fullerene hypothesis. As opposed to the other forms of carbon the fullerenes represent well-defined chemical compounds with in some respects new properties. A whole new chemistry has developed to manipulate the fullerene structure, and the properties of fullerenes can be studied systematically. It is possible to produce superconducting salts of C60, new three-dimensional polymers, new catalysts, new materials and electrical and optical properties, sensors, and so on. In addition, it has been possible to produce thin tubes with closed ends, nanotubes, arranged in the same way as fullerenes. From a theoretical viewpoint, the discovery of the fullerenes has influenced our conception of such widely separated scientific problems as the galactic carbon cycle and classical aromaticity, a keystone of theoretical chemistry. No practically useful applications have yet been produced, but this is not to be expected as early as six years after macroscopic quantities of fullerenes became available.
Further reading
Jim Baggott, Perfect Symmetry: The Accidental Discovery of Buckminsterfullerene, Oxford University Press, 1994, IX + 315 pp.
Hugh Aldersey-Williams, The Most Beautiful Molecule: An Adventure in Chemistry, Aurum Press, London, 1995, IX + 340 pp.
Robert F. Curl and Richard E. Smalley, Probing C60, Science, 18 Nov. 1988 Vol. 242.
Harold Kroto, Space, Stars, C60, and Soot, Science, 25 Nov. 1988 Vol. 242.
H.W. Kroto, A.W. Allaf, and S.P. Balm, C60: Buckminsterfullerene, American Chemical Society, 1991.
Richard E. Smalley, Great Balls of Carbon; The Story of Buckminsterfullerene, The Sciences, March/April 1991.
The All-Star of Buckyball; Profile: Richard E. Smalley, Scientific American, September 1993.
Rudy M. Baum, Commercial Uses of Fullerenes and Derivatives Slow to Develop, News Focus, Nov. 22, 1993 C&EN.
Hargittai, Istv(SIGMA)n, Discoverers of Buckminsterfullerene, The Chemical Intelligencer, Springer-Verlag, New York, 1995.
Robert F. Curl Jr., was born in 1933 in Alice, Texas, USA: Ph.D. in chemistry in 1957 at University of California, Berkeley, USA. Curl has since 1958 worked at Rice University, where he has been a professor since 1967.
Professor Robert F. Curl Jr.
Department of Chemistry
Rice University
P.O. Box 1892
Houston, TX 77251, USA
Sir Harold W. Kroto was born in 1939 in Wisbech, Cambridgeshire, UK. He obtained his Ph.D. in 1964 at the University of Sheffield, UK. In 1967 he moved to the University of Sussex, where he still works. In 1985 he became Professor of Chemistry there and in 1991 Royal Society Research Professor.
Professor Sir Harold W. Kroto
School of Chemistry and Molecular Sciences
University of Sussex
Brighton, Sussex BN1 9QJ, UK
Richard E. Smalley was born in 1943 in Akron, Ohio, USA. Ph.D. in chemistry 1973 at Princeton University, USA. Professor of Chemistry at Rice University since 1981 and also Professor of Physics at the same university since 1990. Member of the National Academy of Sciences in the USA and other bodies.
Professor Richard E. Smalley
Department of Chemistry
Rice University
P.O. Box 1892
Houston, TX 77251, USA
To cite this section
EFFECT OF MAGNETIC IMPURITIES IN BASED ON SHUNGITE ELECTROMAGNETIC ABSORBERS ON ITS SHIELDING PROPERTIES
EFFECT OF MAGNETIC IMPURITIES IN BASED ON SHUNGITE ELECTROMAGNETIC ABSORBERS ON ITS SHIELDING PROPERTIES Pukhir H.A.1 , Mahmoud M.Sh. 2 1 MSc, post-graduate student of the information security department of Belarussian State University of Informatics and Radioelectronics, P. Brovka St., 6, Minsk, 220013, Republic of Belarus, +375172900174, e-mail: [email protected] 2 MSc, post-graduate student of the information security department of Belarussian State University of Informatics and Radioelectronics, P. Brovka St., 6, Minsk, 220013, Republic of Belarus, +375172900174, e-mail: [email protected] Abstract The effect of ferromagnetic impurities in electromagnetic based shungite absorbers is studied. The presence of the particles magnetic powder in composite structure influences on attenuation and reflection characteristics of shielding material has been established. It is found that exact quantity of magnetic inclusions in relation to the shungite powder are principal for reflection coefficient and has effect on electromagnetic radiation attenuation. The compound with powders of shungite and nickel-zinc ferrite in different percentages has investigated. The dependence of the shielding characteristics of the composite is shown. The use of these composite materials for creation of electromagnetic shielding constructions are considered. 1. Introduction Using of electromagnetic resources is inalienable part of modern society. Today we cannot imagine our life without most electronic devises. Some of them help us to keep our living. But we obtain some ecological problems apart from useful effect of electromagnetic energy. High levels of electromagnetic radiation are not habitat. That is why we must constrain electromagnetic radiation emission. Application of electromagnetic shields is the main technique to decrease negative effect from electromagnetic radiation on biological forms. The main purpose for scientists in this scientific area is to create shielding materials and constructions that have high efficiency in wide range of frequency, low cost and are suitable for any applications. Nowadays powdery carbonaceous materials are widely used in designs to create screens of electromagnetic radiation with reduced weight and size characteristics and corrosion resistant. In [1], [2] it is shown the efficiency of use for these purposes schungite minerals. In this case, the actual task is reducing the proportion of energy reflected from the surface shields electromagnetic radiation. The aim of this paper investigation is determination of electromagnetic radiation attenuation features by shungite powder with ferromagnetic impurities and establishing of shielding properties depending on compound of composite. 2. Experiment To increase the efficiency of the screen, which is a composite material based on schungite we may add magnetic inclusions for the introduction of additional magnetic losses. This will also reduce the proportion of energy reflected from the shielding screen. Additional attenuation of electromagnetic radiation makes a presence in the absorber slurry components [3] [4]. We used shungite, consisting of 68% of the silicates in the form of silicon oxide and 29% of globular graphite-like carbon, powdered magnetic materials - ferrites, water, knitted fabric and the binder. Powdered shungite with a particle diameter of 10 microns was mixed with powdered NiZn-ferrite consisting of similar sized particles as a percentage shungite / magnetic powder, 90:10, 80:20, 70:30, 60:40, 50:50, respectively. To obtain a homogeneous structure in the resulting powder mixture was added a small amount of water to obtain a homogeneous viscous mass. Sealing of the samples was carried out using polyethylene. To fix the powder on a knitted structure matrix was used binder, for example, acrylic dye. For the experiment samples with size 5 cm × 5 cm. The sample thickness was about 3 cm. 978-1-4244-6051-9/11/$26.00 ©2011 IEEE Measurement of the shielding characteristics of the samples, sealed with polyethylene, is performed immediately after sample preparation. The samples, sealed in a textile binder matrix, incubated for two days prior to curing. To measure the performance of screening used a panoramic measuring attenuation and voltage standing wave ratio (VSWR). For the samples studied experimentally obtained values of the transfer, which in magnitude is equal to the weakening of EMR, and voltage standing wave ratio, translated into the reflection coefficient in the frequency range 8 ... 11.5 GHz. The relative error is less than 5%. Before measurement of attenuation, samples water saturation was estimated by weighing. 3. Results and discussion Studies have measured the shielding characteristics suggest that the presence in the shungite shielding material magnetic inclusions affect the reflectivity of the screen. A significant reduction in reflectivity (Fig. 1b) for the sample sealed with polyethylene in the composition of which 70% shungite and 30% NiZn-ferrite. Viewed sample shows the lowest reflectance in the presence of a metal reflector behind the sample in the area from 8 to 9.5 GHz (Fig. 1c). For samples number 1 and number 4 is characterized by the resonance phenomenon to reduce the reflection coefficient on the section from 9,5 to 10,5 GHz. The studied materials provide attenuation to 25 dB (Fig. 1a) in the measured range and show the relative stability of performance across the measured frequency range. 0 5 10 15 20 25 30 8 8,5 9 9,5 10 10,5 11 11,5 Frequency, GHz Attenuation, dB №5 №4 №3 №2 №1 a) -8 -6 -4 -2 0 8 8,5 9 9,5 10 10,5 11 11,5 Frequency, dB Reflection coefficient, dB №5 №4 №3 №2 №1 -25 -20 -15 -10 -5 0 8 8,5 9 9,5 10 10,5 11 11,5 Frequency, GHz Reflection coefficient, dB №5 №4 №3 №2 №1 b) c) Figure 1 - Frequency response attenuation (a), reflectance (b) and reflection coefficient in the presence of a metal reflector behind the sample (a) in the range 8 ... 11.5 GHz samples, sealed polyethylene with the following percentages schungite and magnetic powders: № 1 - 50:50, № 2 - 60:40, № 3 - 90:10, № 4 - 80:20, № 5 - 70:30. 0 2 4 6 8 10 12 8 8,5 9 9,5 10 10,5 11 11,5 Frequency, GHz Attenuatioon, dB 13/1 11/1 12/1 -10 -8 -6 -4 -2 0 8 8,5 9 9,5 10 10,5 11 11,5 Frequency, GHZ Reflection coefficient, dB 13/1 11/1 12/1 a) b) -18 -16 -14 -12 -10 -8 -6 -4 -2 0 8 8,5 9 9,5 10 10,5 11 11,5 Frequency, GHz Reflection coefficient, dB 13/1 11/1 12/1 c) Figure 2 - Frequency response attenuation (a), reflectance (b) and reflection coefficient in the presence of a metal reflector behind the sample (a) in the range 8 ... 11.5 GHz on samples of knitted matrix with a binder, and the following percentages schungite and magnetic powder : № 11 / 1 - 70:30, № 12 / 1 - 50:50, № 13 / 1 - 90:10. After analyzing the frequency dependence of the transmission and reflection of the samples, sealed in a textile binder matrix can also be argued to reduce the reflectance in the range 8 ... 10 GHz for the sample with percentages shungite / magnetic powder is 70:30 (Fig. 2b). The test samples provide attenuation of about 15 dB (Fig. 2a). Reducing attenuation due to the presence of less water fractions in the bulk of the composite compared with the samples sealed polyethylene. Given the uncertainty of the measurements obtained shielding characteristics is stable over the entire frequency range. 5. Conclusion Reducing the reflection coefficient for samples with a certain ratio of powder components can be explained by selection of the values of magnetic and dielectric components of the composite, which reduces the value of the wave resistance of the medium screen. Additional attenuation due to absorption of electromagnetic radiation of a composite material is achieved by introducing water-based components. Thus, by studying the magnetic and dielectric properties of the components of the absorbers of electromagnetic radiation on the basis of powder schungite can simulate the shielding design with the required values of the parameters of attenuation and reflection. Also it allows you to select the optimal composition of the filler screen of electromagnetic radiation on the basis of the required operating conditions of the electromagnetic shields. 7. References 1. T. Borbot'ko, U. Kalinin, N. Kolbun, e. Kryshtopova, L. Lynkov, “Carbon-containing minerals and their applications” Minsk, Bestprint, 2009 – 156 p. 2. T. Borbot'ko, E. Krishtopova, L. Lynkov, “Effect of binders on the shielding properties of composite materials from powdered schungite” Doklady BSUIR, N6, 2007, pp.3–7. 3. H. Pukhir, “Formation of EMI shielding materials based on composite media with dielectric losses” Proc. of "International scientific-technical conference on 45 years of MRTI-BSUIR" Conference, Minsk, 2009, pp. 195–196. 1. E. Ukrainec, N. Kolbun, “Shielding properties of multilayer structures of electromagnetic shields on the basis of materials with small-sized inclusions of metals and liquids” Doklady BSUIR, N 4, 2003, p.35.
Antioxidant and Anti-Inflammatory Effects of Shungite against Ultraviolet B Irradiation-Induced Skin Damage in Hairless MiceOxid Med Cell Longev. 2017; 2017: 7340143.
Published online 2017 Aug 13. doi: 10.1155/2017/7340143
PMCID: PMC5574306
PMID: 28894510Antioxidant and Anti-Inflammatory Effects of Shungite against Ultraviolet B Irradiation-Induced Skin Damage in Hairless MiceMa. Easter Joy Sajo, 1 Cheol-Su Kim, 2 Soo-Ki Kim, 2 Kwang Yong Shim, 3 Tae-Young Kang, 4 , * and Kyu-Jae Lee 1 , 5 , *
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Go to:AbstractAs fullerene-based compound applications have been rapidly increasing in the health industry, the need of biomedical research is urgently in demand. While shungite is regarded as a natural source of fullerene, it remains poorly documented. Here, we explored the in vivo effects of shungite against ultraviolet B- (UVB-) induced skin damage by investigating the physiological skin parameters, immune-redox profiling, and oxidative stress molecular signaling. Toward this, mice were UVB-irradiated with 0.75 mW/cm2 for two consecutive days. Consecutively, shungite was topically applied on the dorsal side of the mice for 7 days. First, we found significant improvements in the skin parameters of the shungite-treated groups revealed by the reduction in roughness, pigmentation, and wrinkle measurement. Second, the immunokine profiling in mouse serum and skin lysates showed a reduction in the proinflammatory response in the shungite-treated groups. Accordingly, the redox profile of shungite-treated groups showed counterbalance of ROS/RNS and superoxide levels in serum and skin lysates. Last, we have confirmed the involvement of Nrf2- and MAPK-mediated oxidative stress pathways in the antioxidant mechanism of shungite. Collectively, the results clearly show that shungite has an antioxidant and anti-inflammatory action against UVB-induced skin damage in hairless mice.
Go to:1. IntroductionUltraviolet (UV) radiation often causes various skin diseases [1]. At short-term UV exposure, it could suppress immune function, and at chronic exposure, it could lead to photoaging and/or carcinoma [2, 3]. Skin damage induced by UV irradiation includes photosensitivity, erythema, and DNA damage resulting in invisible changes of cell and gene level [4–6]. These involve alterations in immune response such as increased mast cells, outburst of cytokines by keratinocytes, and suppressed levels of Langerhans cells [7–9]. It is generally known that the skin is the first line of defense in our immune system that causes it to be one of the primary candidates and targets of oxidative stress [10, 11]. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have a major participation in the pathogenesis of UV-induced skin damage by both direct DNA damage and indirect ROS-mediated oxidative damage [12–14]. Furthermore, the immune dysfunction and ROS would aggravate the skin barrier structure and function, consequently leading to photoaging [15]. With these reports, targeting ROS-induced cellular damage or immune dysfunction in skin barrier is a strategic move to prevent UV-induced skin damage. A plethora of antioxidant agents in different forms are readily available such as cosmetics, inhalant, and foods to reduce UV-induced skin damage [16–20]. However, convenient treatments or manipulations of UV-induced skin injury are still limited.
According to scholars, even if shungite has been around for billions of years, the present comprehension of this promising mineral is underway. Scientists have shown interest in the study of carbon from shungite rocks for over two centuries focusing mainly in the structural, chemical, and geological investigations [21]. Shungite rocks contain one of the oldest noncrystal carbon found to be originated in a village named Shunga in the Karelian shore of the Lake Onega (Russia) [21]. The carbon in shungite rocks penetrates in nearly all rocks of the region over an area in excess of 9000 km2 [21]. Shungite rocks are divided into five types based on the carbon percentage (1–98 wt.%) [22]. Type I is the rocks that occur in shungite deposits containing the highest amount of carbon (96–98 wt.%) with traces of other elements (0.1–0.5% H, 0.6–1.5% O, 0.7–1.0% N, and 0.2–0.4% S) [23]. On the other hand, type III is the most common one with 30 wt.% of carbon. In the ancient times, shungite is said to be related to allergies, skin diseases, diabetes mellitus, stomatitis, periodontal disease, hair loss, cosmetic flaws, and many other diseases [21]. It is characterized by high reactivity in thermal processes, high absorption, catalytic properties, electrical conductivity, and chemical stability. Shungite particles, regardless of their size, have bipolar properties [21]. By the end of the twentieth century, scientists had partially explained the reasons for the beneficial effect of shungite. As it turned out, this mineral is mainly composed of carbon, much of which is represented by the spherical molecules of fullerenes. Eventually, the revelation of fullerene in shungite rocks provided a new impetus to the exploration of shungite [24].
In this study, we explored the therapeutic in vivo effects of shungite against UVB-induced skin damage comparing the antioxidant power of mineral-rich shungite and mineral-less shungite with commercially available fullerene C60. We hypothesize that shungite might have possible antioxidant and anti-inflammatory effects against ultraviolet B- (UVB-) induced skin damage in hairless mice. To verify this hypothesis, we investigated the physiological skin parameters, immune-redox profiling, and oxidative stress-related signaling of mineral-rich and mineral-less shungite-treated hairless mice.
Go to:2. Materials and Methods2.1. MiceEight-week-old male hairless mice were obtained from Orient Bio Inc. (Seongnam, Republic of Korea) and were maintained at 22 ± 2°C and 40–60% humidity under a cycle of 12 : 12 hr light dark. The mice were acclimatized for one week and randomly assigned into six groups: the non-UVB-irradiated normal control group (NC), and the five UVB-irradiated groups: no treatment group (UV), fullerene-treated group (PC), olive oil-treated group (OIL), mineral-rich shungite-treated group (MRS), and mineral-less shungite-treated group (MLS). About 200 μL of treatment compounds (200 μg/mL) was topically administered using the hands with gloves applying the same pressure all throughout the dorsal side of the mouse. At the end of the experiment, mice were euthanized through inhalant anesthetics, CO2 gas for 20 seconds, and then the mice were decapitated. The animal use and protocol for this experiment was approved by the Institutional Animal Care and Use Committee (IACUC), Yonsei University Wonju Campus (YWC-151113-1).
2.2. UVB ExposureThe experimental mice were placed in a plastic cage and were irradiated using TL 40 W/12RS Philips, an unfiltered fluorescent sun lamps (Amsterdam, Netherlands) emitting rays of 290–320 nm. The distance from the lamps to the dorsal skin surface of the mice was 20 cm. The intensity of UVB lamp was 0.75 ± 0.10 mW/cm2 using YK-34 UV, a UV light meter by Lutron Electronics Inc. (Taipei, Taiwan), and the total cumulative dose energy of UVB irradiated was 2700 mJ/cm2. The mice were irradiated for 15 mins in 2 consecutive days.
2.3. Chemical and Shungite Suspension PreparationNatural shungite stone was purchased from Karelian Shungite Factory (Republic of Karelia, Russia). Natural shungite has 2.3–2.4 g/cm3 of specific gravity, 0.5% of porosity, 1000–1500 kgf/cm2 of compressed strength, and 1100–1200 kg/m3 of density and is composed of 28% carbon, 57% SiO2, 4.3% Al2O3, 2.8% FeO, 1.5% K2O, 1.5% S, 1.2% MgO, 0.3% CaO, 0.2% TiO2, 0.2% Na2O, and 3% H2O crystal. Mineral-rich shungite (MRS) and mineral-less shungite (MLS) were produced by MST Technology Ltd. (Incheon, Republic of Korea) through the treatment processes such as grinding of stone, treatment of alkaline solution and acidic solution, washing and filtration, treatment of boron (B), and high-temperature treatment process. The property and components of shungite vary according to the pulverized particle size of stone, mixing proportion of alkaline solution, acidic solution, and boron-containing compound with shungite powder, concentration of treatment solutions, and heating time and temperature. MRS and MLS powder used as an experimental material were refined to increase carbon ratio (MRS: 86.4%, NLS: 99.4% by energy dispersive X-ray spectroscopy (Missouri, USA)) using different treatment processes, and fullerene-like carbons such as C55, C60, C70, C74, C93, and C112 were mainly detected by MALDI-TOF mass spectrometry (Shimadzu Corp., Kyoto, Japan). Carbon powder were mixed with olive oil from Sigma-Aldrich Co. LLC. (Darmstadt, Germany) as a vehicle for the topical treatment on the skin.
Manufactured buckminsterfullerene C60 was purchased from Vaughter Wellness (London, United Kingdom) with 99.8% purity. Mineral-rich shungite was treated with KOH as an alkaline treatment with high temperature. Mineral-less shungite was treated with HNO3 and high-temperature (3000°C) treatment of mineral-rich shungite. These mixtures were sonicated using a vortex and sonicator until all of the solutions were mixed vigorously.
2.4. Characterization of Mineral-Less ShungiteComposition and visualization of shungite with mineral-less were analyzed by Energy-dispersive X-ray (EDX) spectroscopy. The mineral percent composition includes 86.43% carbon, 0.18% sodium, 1.33% magnesium, 3.17% silicon, 1.09% sulfur, 0.22% chlorine, 0.95% potassium, 5.33% calcium, 1.06% iron, and 0.2% copper.
2.5. Physiological Analysis of the Skin SurfaceSkin condition of the mice was assessed before and after 7-day treatment with a device for skin diagnostics, Aramo TS Device (Seongnam, Republic of Korea). The device is a comprehensive system for noninvasive, optical analysis of several dimensionless parameters of the skin: moisture, elasticity, sebum porosity, smoothness (evenness), discolourations (pigmentation), and wrinkles. The measurements were taken in very specific points located on the dorsal side of the mice, and the results were the difference before and after treatment to assess the improvement.
2.6. Immune Profile
2.6.1. White Blood Cell (WBC) and Its Differential Count AnalysisBlood was collected from the retro-orbital plexus in tubes coated with anticoagulant and was mixed with an automatic mixer for 5 min. Thereafter, WBC and its differential members such as lymphocytes, monocytes, neutrophil, eosinophil, and basophil were measured using an automatic blood analyser by HEMAVET HV950 FS, Drew Scientific Inc. (Texas, USA).
2.6.2. Serum and Skin Lysate Inflammatory CytokinesSerum and skin lysate inflammatory cytokines including IL-1β, IL-6, IL-10, IL-17, IL-KC, and TNF-α were analyzed using Bio-Plex Cytokine Assay by Bio-Rad (California, USA) according to the manufacturer's instructions. Standard curves for each adipokine and cytokine were generated by using the reference concentrations provided in the kits. Mean fluorescence intensity was acquired on a Luminex technology by Bio-Rad's Bio-Plex 200 system Multiplex Bead Array System™ (California, USA) and analyzed with associated software using a 5-parameter logistic method.
2.7. Redox Profile
2.7.1. Intracellular Reactive Oxygen Species (ROS) DetectionROS/RNS detection kit by Enzo Life Sciences Inc. (New York, USA) was used according to the manufacturer's instructions to determine effects of shungite compounds on oxidative stress and superoxide in serum and skin lysates. Twenty-five (25) μL sample was loaded, and detection solution was added to each well. The plate was read in the DTX-800 multimode microplate reader by Beckman Counter Inc. (California, USA) using a filter set of 485/20 excitation and 528/20 emission to detect oxidative species.
2.7.2. NO AssayThe nitrite (NO2−) present in the blood serum and skin lysates of mice was detected using the Griess reagent by Promega Corp. (Madison, USA). Briefly, 50 μL of the serum was mixed with an equal volume of Griess reagent in a 96-well microtiter plate and incubated at room temperature for 15 min. The absorbance was read at 540 nm using a DTX-880 multimode microplate reader by Beckman Counter Inc. (California, USA). The NO2− concentration was calculated by comparison with the representative NO2− standard curve generated by serial two-fold dilutions of sodium nitrate.
2.7.3. Antioxidant Endogenous Enzyme ActivitiesThe activity of superoxide dismutase (SOD), glutathione peroxidase (GPx), and myeloperoxidase (MPO) in serum and skin lysates was measured using the Biovision kit (California, USA). The crude skin lysate was centrifuged at 14,000 rpm for 15 min. at 4°C, and the debris was discarded. Skin lysate was then checked for protein concentration using Pierce™ BCA Protein Assay Kit by Thermo Scientific (Illinois, USA). Finally, the normalized concentrations of cell lysates were used to measure the activities of different antioxidant enzymes (SOD, GPx, and MPO) according to the manufacturer's instruction.
2.8. Western Blot AnalysisBriefly, the prepared skin lysate with the normalized protein concentration was equally loaded and separated by electrophoresis on SDS-polyacrylamide gels. Ten (10) percent separating gel and 5% stacking gel were prepared with the following components. 10% separating gel (10 mL) consisted of 4.0 mL dH2O, 3.3 mL 30% acrylamide mix, 2.5 mL 1.5 M Tris (pH 8.8), 100 μL 10% SDS, 100 μL 10% APS, and 4 μL TEMED. 4% stacking gel (5 mL) consist of 2.7 mL dH2O, 670 μL 30% acrylamide mix, 500 μL 1.0 M Tris (pH 6.8), 40 μL 10% SDS, 40 μL 10% APS, and 4 μL TEMED. The 15 μL ~ 20 μL of the sample with sample loading buffer with optimized concentration was loaded in the gel. Then, it will be run in 30 mA, 70–80 v. The PVDF membranes were blocked with 5% nonfat skim milk at room temperature for 2 hr and were incubated with the following primary antibodies: phospho-JNK and phospho-p38, ERK, Nrf2, and β-actin (dilution: 1 : 2000; Cell Signaling Technology, Massachusetts, USA) in Tris-buffered saline/tween 20 (1X TBST) containing 5% bovine serum albumin overnight in 4°C. The secondary antibody used was anti-rabbit (dilution: 1 : 2000; Cell Signaling Technology), and then it was incubated at room temperature for 2 hr. Specific protein bands were visualized by the enhanced chemiluminescence (ECL Pierce Biotechnology) using UVP Biospectrum 600 Imaging System (UVP, LLC, Upland, CA, USA). β-Actin (dilution: 1 : 2000, Cell Signaling Technology) was used as loading controls for the total protein content.
2.9. Data Management and AnalysisStatistical analysis was carried out using analysis of variance (ANOVA) followed by subsequent multiple comparison test (Dunnett's multiple comparison test) using GraphPad Prism version 7.0 software packages (California, USA). Differences were considered significant at ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.
Go to:3. Results and Discussion3.1. The Effects of Shungite on the Physiological Skin Surface of the MiceThis study aims to examine whether topical application of MRS and MLS could attenuate the related skin damage caused by UVB irradiation in hairless mice compared to the pure fullerene C60 positive control. We also compared the experimental groups (MRS and MLS groups) to non-UVB-irradiated group, negative control UVB-irradiated only group, and UVB + olive oil-treated group. We used two experimental groups (MRS is close to the natural shungite that has the most mineral present while the MLS is with least mineral material) to explore the effects of the two different types and also to rule out the effects of other minerals present in the material. On the other note, olive oil was used in the experiment because it has been used as a solubilizing excipient or vehicle in our positive control fullerene C60. Studies have shown that olive oil is the most biocompatible organic solvent for C60 fullerene [25]. Olive oil has been used as an alternative for the toxic glycerol or toluene. In addition, olive oil is one of the most effective and safe solubilizing excipients easily available [26]. However, since olive oil itself is already known to have its antioxidant effects and was also found to have protective effect after UVB exposure [27], it is essential to compare its effect with the olive oil only treatment with the experimental groups which used olive oil as a vehicle.
To verify the clinical evaluation of the skin, we checked different skin parameters and we found that the skin moisture and elasticity of the shungite-treated (MRS and MLS) mice had improved compared to the NC and UV groups, comparable to PC (Figure 1). Furthermore, the roughness, pigmentation, and wrinkle had been significantly reduced and showed improvement after 7 days of topical application of shungite. Pore size of MRS and MLS groups did not show significant reduction, but it has improved compared to the PC. These results show clinical evidences of the antioxidant and anti-inflammatory effects of MRS and MLS in the outward skin surface.
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Figure 1Mineral-rich and mineral-less shungite-treated groups improved physiological skin parameters. Shown are the effects of mineral-rich shungite (MRS) and mineral-less shungite (MLS) on the mouse skin moisture (a), elasticity (b), roughness (c), pore size (d), pigmentation (e), and wrinkle (f). Control groups include non-UVB-irradiated group (NC), UVB-irradiated only group (UV), UVB + fullerene C60-treated positive control group (PC), and UVB + olive oil-treated group (OIL), while experimental groups include UVB + mineral-rich shungite-treated group (MRS) and mineral-less shungite-treated group (MLS). Statistical analysis was carried out using analysis of variance (ANOVA) followed by subsequent multiple comparison test (Tukey's multiple comparisons test) using GraphPad Prism version 5.0. Values are mean ± SD, n = 9. Differences were considered significant at ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.
3.2. Immune Profiling
3.2.1. Total WBC and Its Differential CountTotal WBC counts were used to evaluate the overview of the severity of inflammation response. Table 1 showed the total WBC count suppression of MRS and MLS compared to UV and PC. The altered WBC counts may be indicative of direct or indirect effects of the compound treatment on cellular toxicity and proliferation. The recruitment of total WBC and differential counts are highest in PC followed by MLS then MRS. Overall, the suppressed count of the total WBC in the negative control group (UV) was rescued by the shungite-treated groups as well as their differential counts on both MRS and MLS groups after 7 days of topical application of shungite.
Table 1Slight suppression of total white blood cells (WBC) and their differential counts in the mineral-rich shungite-treated group. Shown are the effects of mineral-rich shungite (MRS) and mineral-less shungite (MLS) on the total WBC (a) and their differential counts including neutrophil (b), lymphocyte (c), monocyte (d), basophil (e), and eosinophils (f). Control groups include non-UVB-irradiated group (NC), UVB-irradiated only group (UV), UVB + fullerene C60-treated positive control group (PC), and UVB + olive oil-treated group (OIL), while experimental groups include UVB + mineral-rich shungite-treated group (MRS) and mineral-less shungite-treated group (MLS). Statistical analysis was carried out using analysis of variance (ANOVA) followed by subsequent multiple comparison test (Tukey's multiple comparisons test) using GraphPad Prism version 5.0. Data were expressed as mean ± SD, n = 9.
Units in K/mLNCUVPCOILMRSMLSTotal WBC3.16 ± 0.833.098 ± 0.914.25 ± 1.943.54 ± 2.223.032 ± 0.663.76 ± 1.26
Neutrophil1.12 ± 0.351.326 ± 0.761.59 ± 0.861.31 ± 0.861.03 ± 0.281.41 ± 0.44
Lymphocyte1.97 ± 0.622.094 ± 0.632.34 ± 0.961.99 ± 1.411.82 ± 0.422.13 ± 0.82
Monocyte0.13 ± 0.0710.15 ± 0.900.22 ± 0.110.18 ± 0.130.13 ± 0.040.17 ± 0.07
Eosinophil0.02 ± 0.0130.041 ± 0.0580.076 ± 0.130.05 ± 0.060.034 ± 0.0840.033 ± 0.044
Basophil0.007 ± 0.00480.011 ± 0.0170.027 ± 0.0480.014 ± 0.0170.019 ± 0.0360.013 ± 0.016
3.2.2. Proinflammatory Cytokine Levels in Serum and Skin Lysates of Shungite-Treated UVB-Induced MiceFurther evidence showed that shungite might mediate inflammation response that can influence immunological homeostasis. To delineate this proposition, we measured the serum (Figure 2) and skin lysate (Figure 3) cytokine profiling to gauge the inflammatory cytokine balance. The overall trend of the serum levels of IL-1β, IL-6, IL-10, IL-KC, and TNF-α concentration in shungite-treated mice was lower than that of all the control groups except for IL-17. The UV group showed slightly higher than the NC group. Comparing cytokine levels of serum, MLS showed lower level of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) as well as the anti-inflammatory cytokine (IL-10) than the PC group (Figure 2). Similar trend was observed in skin lysate levels of IL-1β, IL-6, IL-10, IL-KC, and TNF-α, except IL-17 concentrations in shungite-treated (Figure 3). In shungite-treated group, lower level of proinflammatory cytokines (IL-1β, TNF-α, and IL-6) and regulatory cytokines in skin keratinocytes such as IL-17 and IL-KC is highly compatible with the redox profiling results. Cumulative evidence showed that UVB leads to a shift from the production of proinflammatory cytokines such as IL-1β, IL-12, IL-8, TNF-α, and IFN-γ to the production of anti-inflammatory cytokines such as IL-4, IL-10, and IL-13 [28, 29]. Breakdown of pro- and anti-inflammatory cytokines leads to various diseases such as atopic dermatitis, rheumatoid arthritis, and psoriasis. As observed in the results, a similar trend was seen in shungite-treated group in IL-1β and TNF-α. This may be ascribed to the synergism between two cytokines. On the other hand, the role of IL-6 in UVB-induced skin immune responses is to regulate the release and production of other proinflammatory cytokines [30]. The mechanism which is regulated by IL-17 was not noticeable in our results. IL-KC appears to have the key role in immunosuppressive effects of UVB as it mediates IL-10 which is known to be an anti-inflammatory cytokine [31]. Based on cytokine profiling, shungite treatment restored the inflammatory cytokine imbalance evoked by UV irradiation. UVB irradiation on the skin would induce alteration of antigen-presenting cells including Langerhans cells and imbalance of a cell-mediated immune response. IL-10 is known as an important immunoregulatory cytokine to downregulate inflammatory responses and regulate differentiation and proliferation of several immune cells such as T cells, B cell, natural killer (NK) cells, mast cells, and granulocytes [32]. In that context, this immunomodulation of shungite might justify the antioxidant effect and potential clinical therapeutic usage for skin and oxidative stress disorders [33].
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Figure 2Serum proinflammatory cytokine levels in mineral-rich and mineral-less shungite groups showed immunomodulatory effect. Shown are the serum cytokine concentrations of IL-1β (a), IL-6 (b), IL-10 (c), IL-17 (d), IL-KC (e), and TNF-α (f) determined using multiplex assay in shungite-treated UVB-induced mice. Control groups include non-UVB-irradiated group (NC), UVB-irradiated only group (UV), UVB + positive control fullerene C60-treated group (PC), and UVB + olive oil-treated group (OIL), while experimental groups include UVB + mineral-rich shungite-treated group (MRS) and mineral-less shungite-treated group (MLS). Statistical analysis was carried out using analysis of variance (ANOVA) followed by subsequent multiple comparison test (Tukey's multiple comparisons test) using GraphPad Prism version 5.0. All values are represented as mean ± SD (n = 9), ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 versus each treatment.
Figure 3Skin lysate proinflammatory cytokine levels in mineral-rich shungite (MRS) and mineral-less shungite (MLS) groups showed immunomodulatory effect. Shown are the skin lysate cytokine concentrations of IL-1β (a), IL-6 (b), IL-10 (c), IL-17 (d), IL-KC (e), and TNF-α (f) determined using multiplex assay in shungite-treated UVB-induced mice. Control groups include non-UVB-irradiated group (NC), UVB-irradiated only group (UV), UVB + positive control fullerene C60-treated group (PC), and UVB + olive oil-treated group (OIL), while experimental groups include UVB + mineral-rich shungite-treated group (MRS) and mineral-less shungite-treated group (MLS). Statistical analysis was carried out using analysis of variance (ANOVA) followed by subsequent multiple comparison test (Tukey's multiple comparisons test) using GraphPad Prism version 5.0. All values are represented as mean ± SD, n = 9, ∗p < 0.05, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 versus each treatment.
3.3. Redox Profiling
3.3.1. Effects of Shungite on the Levels of Total Intracellular ROS and Nitric Oxide (NO) in Mouse Serum and SkinThe generation of intracellular ROS and superoxide of both skin lysates in MRS and MLS had reduced levels compared to the high levels of ROS in UV group. However, the reduction of ROS/RNS was not that dramatical in the circulating serum (Figure 4). On the other hand, the NO levels in serum and skin lysates in shungite-treated groups were not significant but a lower trend had been observed in the serum NO levels in shungite-treated group (Figure 5). NO plays a pivotal role in macrophage-mediated cytotoxicity by acting as an effector molecule. NO is a reactive molecule that reacts with ROS to produce reactive nitrogen species; moreover, it is recognized as a mediator and regulator of immune responses. NO has various physiological and pathophysiological responses depending on its relative concentration.
Figure 4Reduction of total intracellular reactive oxygen/nitrogen (ROS/RNS) and superoxide dismutase (SOD) species in mouse serum and skin lysate of shungite-treated groups. Shown are the effects of shungite in the total ROS/RNS in serum (a) and skin lysates (b) as well as the SOD concentration in serum (c) and skin lysates (d) as determined by an ROS/RNS detection kit by Enzo Life Sciences Inc. Control groups include non-UVB-irradiated group (NC), UVB-irradiated only group (UV), UVB + fullerene C60-treated positive control group (PC), and UVB + olive oil-treated group (OIL), while experimental groups include UVB + mineral-rich shungite-treated group (MRS) and mineral-less shungite-treated group (MLS). Statistical analysis was carried out using analysis of variance (ANOVA) followed by subsequent multiple comparison test (Tukey's multiple comparisons test) using GraphPad Prism version 5.0. Data were expressed as mean fluorescence ± SD, n = 9. Differences were considered significant at ∗p < 0.05 versus each treatment.
Figure 5Slight increase of nitric oxide (NO) levels in mouse serum and skin lysates of shungite-treated groups. Shown are the NO levels in serum (a) and skin lysates (b) that were determined using Greiss reagent. Control groups include non-UVB-irradiated group (NC), UVB-irradiated only group (UV), UVB + fullerene C60-treated positive control group (PC), and UVB + olive oil-treated group (OIL), while experimental groups include UVB + mineral-rich shungite-treated group (MRS) and mineral-less shungite-treated group (MLS). Statistical analysis was carried out using analysis of variance (ANOVA) followed by subsequent multiple comparison test (Tukey's multiple comparisons test) using GraphPad Prism version 5.0. Data were expressed as mean ± SD, n = 9.
3.3.2. Serum and Skin Lysate Scavenging Enzyme ActivityConsistent with the total ROS/RNS result, the activities of ROS-scavenging enzymes such as intracellular GPx and SOD levels were increased. Levels of myeloperoxidase activities in shungite-treated groups had decreased in serum and skin lysates, and this could be to compensate for other enzymes dissociating hydrogen peroxide (Figure 6).
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Figure 6Antioxidant enzyme activities of serum and skin lysates in shungite-treated mice groups: glutathione peroxidase (GPx), superoxide dismutase (SOD), and myeloperoxidase (MPO) levels. Shown are the antioxidant biological enzyme activities such GPX in serum (a) and skin lysates (d), SOD in serum (b) and skin lysates (e), and MPO in serum (c) and skin lysates (f) of shungite-treated UVB-induced mice. Control groups include non-UVB-irradiated group (NC), UVB-irradiated only group (UV), UVB + fullerene-treated positive control group (PC), and UVB + olive oil-treated group (OIL), while experimental groups include UVB + mineral-rich shungite-treated group (MRS) and mineral-less shungite-treated group (MLS). Statistical analysis was carried out using analysis of variance (ANOVA) followed by subsequent multiple comparison test (Tukey's multiple comparisons test) using GraphPad Prism version 5.0. Data were expressed as mean ± SD, n = 9, ∗p < 0.05, ∗∗p < 0.01 versus each treatment groups.
3.4. Involvement of Nrf2 and p-ERK, p-p38 MAPK, and p-JNK Signaling Pathways in the Antioxidant Effect of Shungite against UVB-Induced Oxidative StressTo fully elucidate the underlying molecular mechanism of antioxidant effect mediated by shungite, we hypothesized those different pathways that can lead to inflammatory. To prove our hypothesis, we determined whether the skin lysate of the shungite-treated group can induce the phosphorylation of Nrf2, p-p38 MAPK, and p-JNK. Figure 7 showed that there was a marked increase in phosphorylation of Nrf2, p-p38 MAPK, and p-JNK. This result indicates that topical application of shungite can induce phosphorylation of Nrf2, p-p38 MAPK, and p-JNK. This study also found out that Nrf2 and MAPK proteins, p-ERK and p-JNK, are involved in the antioxidant effect of shungite against UVB-induced oxidative stress. Nrf2 is a transcription factor that regulates expression of many detoxification or antioxidant enzymes. It is plausible that fullerene transiently increases the intracellular level of ROS and/or activates p38 MAPK signaling pathway, which may possibly lead to facilitating the dissociation of Nrf2 from Keap. The resultant Nrf2/ARE activation induced phase II detoxification or antioxidant enzyme, thereby potentiating cellular defence capacity against cell death.
Figure 7Nrf2 and MAPK proteins, P-ERK and p-JNK, are involved in the antioxidant effect of shungite against UVB-induced oxidative stress. Signaling cascade controlling cellular responses were measured for protein expression. Mineral-rich shungite (MRS) and mineral less shungite (MRS) induced phosphorylation of p-ERK (a), p-JNK (b) and Nrf2 (c), and β-actin (d) signaling pathway. Control groups include non-UVB-irradiated group (NC), UVB-irradiated only group (UV), UVB + positive control fullerene-treated group (PC), and UVB + olive oil-treated group (OIL), while experimental groups include UVB + mineral-rich shungite-treated group (MRS) and mineral-less shungite-treated group (MLS). Statistical analysis was carried out using analysis of variance (ANOVA) followed by subsequent multiple comparison test (Tukey's multiple comparisons test) using GraphPad Prism version 5.0. All values are represented as mean ± SD, n = 9, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 versus each treatment.
3.5. ConclusionThis study confirms the hypothesis that shungite, a natural fullerene, has antioxidant properties as it reduced the intracellular ROS production and enhanced the antioxidant enzyme activities (GPx, SOD, and MPO) in vivo. It is the first note to show several mechanisms and evidences of shungite's antioxidant effects including the improvement of several skin test parameters (moisture, elasticity, roughness, pore size, pigmentation, and wrinkle) and recovery of total WBC. Most importantly, shungite counterbalanced ROS/antioxidant paradigm as shown by the reduction of ROS/RNS levels and superoxide levels in both serum and skin lysate levels. Consistently, the activities of ROS-scavenging enzymes such as GPx and SOD levels were increased. In line, the inflammatory markers such as the cytokine levels of IL-1β, IL-6, IL-10, Il-17, IL-KC, and TNF-α in mice serum and skin lysates were lower than those in the NC group. Last, we found that Nrf2 and MAPK proteins, p-ERK and p-JNK, were involved in the antioxidant effect of shungite against UVB-induced oxidative stress. Given these, our data verify that mineral-rich shungite and mineral-less shungite were effective in the overall screening of its antioxidant and anti-inflammatory properties compared to the other control treatments. Further, MLS has a stronger antioxidant and anti-inflammatory effects than MRS. Synthesizing these, we rationally infer that shungite could be a one potential agent in such other diseases as well and can also be an alternative to the known applications of pure fullerene. However, the comparison of shungite-treated groups to other potent agents on other skin damage and immune- and oxidative-related diseases such as psoriasis or atopic dermatitis was not part of the study, but could be one of the future study targets. Since the processibility of pure fullerene is very challenging and very expensive, this study suggests that natural mineral shungite, as a novel antioxidant, could provide a new therapeutic insight against oxidative- and inflammatory-related diseases.
Go to:AcknowledgmentsThis work was supported by the Yonsei University Research Fund of 2016.
Go to:Conflicts of InterestThe authors have no conflict of interest to declare.
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LINK TO BOOK ABOUT SHUNGITE
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Published online 2017 Aug 13. doi: 10.1155/2017/7340143
PMCID: PMC5574306
PMID: 28894510Antioxidant and Anti-Inflammatory Effects of Shungite against Ultraviolet B Irradiation-Induced Skin Damage in Hairless MiceMa. Easter Joy Sajo, 1 Cheol-Su Kim, 2 Soo-Ki Kim, 2 Kwang Yong Shim, 3 Tae-Young Kang, 4 , * and Kyu-Jae Lee 1 , 5 , *
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Go to:AbstractAs fullerene-based compound applications have been rapidly increasing in the health industry, the need of biomedical research is urgently in demand. While shungite is regarded as a natural source of fullerene, it remains poorly documented. Here, we explored the in vivo effects of shungite against ultraviolet B- (UVB-) induced skin damage by investigating the physiological skin parameters, immune-redox profiling, and oxidative stress molecular signaling. Toward this, mice were UVB-irradiated with 0.75 mW/cm2 for two consecutive days. Consecutively, shungite was topically applied on the dorsal side of the mice for 7 days. First, we found significant improvements in the skin parameters of the shungite-treated groups revealed by the reduction in roughness, pigmentation, and wrinkle measurement. Second, the immunokine profiling in mouse serum and skin lysates showed a reduction in the proinflammatory response in the shungite-treated groups. Accordingly, the redox profile of shungite-treated groups showed counterbalance of ROS/RNS and superoxide levels in serum and skin lysates. Last, we have confirmed the involvement of Nrf2- and MAPK-mediated oxidative stress pathways in the antioxidant mechanism of shungite. Collectively, the results clearly show that shungite has an antioxidant and anti-inflammatory action against UVB-induced skin damage in hairless mice.
Go to:1. IntroductionUltraviolet (UV) radiation often causes various skin diseases [1]. At short-term UV exposure, it could suppress immune function, and at chronic exposure, it could lead to photoaging and/or carcinoma [2, 3]. Skin damage induced by UV irradiation includes photosensitivity, erythema, and DNA damage resulting in invisible changes of cell and gene level [4–6]. These involve alterations in immune response such as increased mast cells, outburst of cytokines by keratinocytes, and suppressed levels of Langerhans cells [7–9]. It is generally known that the skin is the first line of defense in our immune system that causes it to be one of the primary candidates and targets of oxidative stress [10, 11]. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have a major participation in the pathogenesis of UV-induced skin damage by both direct DNA damage and indirect ROS-mediated oxidative damage [12–14]. Furthermore, the immune dysfunction and ROS would aggravate the skin barrier structure and function, consequently leading to photoaging [15]. With these reports, targeting ROS-induced cellular damage or immune dysfunction in skin barrier is a strategic move to prevent UV-induced skin damage. A plethora of antioxidant agents in different forms are readily available such as cosmetics, inhalant, and foods to reduce UV-induced skin damage [16–20]. However, convenient treatments or manipulations of UV-induced skin injury are still limited.
According to scholars, even if shungite has been around for billions of years, the present comprehension of this promising mineral is underway. Scientists have shown interest in the study of carbon from shungite rocks for over two centuries focusing mainly in the structural, chemical, and geological investigations [21]. Shungite rocks contain one of the oldest noncrystal carbon found to be originated in a village named Shunga in the Karelian shore of the Lake Onega (Russia) [21]. The carbon in shungite rocks penetrates in nearly all rocks of the region over an area in excess of 9000 km2 [21]. Shungite rocks are divided into five types based on the carbon percentage (1–98 wt.%) [22]. Type I is the rocks that occur in shungite deposits containing the highest amount of carbon (96–98 wt.%) with traces of other elements (0.1–0.5% H, 0.6–1.5% O, 0.7–1.0% N, and 0.2–0.4% S) [23]. On the other hand, type III is the most common one with 30 wt.% of carbon. In the ancient times, shungite is said to be related to allergies, skin diseases, diabetes mellitus, stomatitis, periodontal disease, hair loss, cosmetic flaws, and many other diseases [21]. It is characterized by high reactivity in thermal processes, high absorption, catalytic properties, electrical conductivity, and chemical stability. Shungite particles, regardless of their size, have bipolar properties [21]. By the end of the twentieth century, scientists had partially explained the reasons for the beneficial effect of shungite. As it turned out, this mineral is mainly composed of carbon, much of which is represented by the spherical molecules of fullerenes. Eventually, the revelation of fullerene in shungite rocks provided a new impetus to the exploration of shungite [24].
In this study, we explored the therapeutic in vivo effects of shungite against UVB-induced skin damage comparing the antioxidant power of mineral-rich shungite and mineral-less shungite with commercially available fullerene C60. We hypothesize that shungite might have possible antioxidant and anti-inflammatory effects against ultraviolet B- (UVB-) induced skin damage in hairless mice. To verify this hypothesis, we investigated the physiological skin parameters, immune-redox profiling, and oxidative stress-related signaling of mineral-rich and mineral-less shungite-treated hairless mice.
Go to:2. Materials and Methods2.1. MiceEight-week-old male hairless mice were obtained from Orient Bio Inc. (Seongnam, Republic of Korea) and were maintained at 22 ± 2°C and 40–60% humidity under a cycle of 12 : 12 hr light dark. The mice were acclimatized for one week and randomly assigned into six groups: the non-UVB-irradiated normal control group (NC), and the five UVB-irradiated groups: no treatment group (UV), fullerene-treated group (PC), olive oil-treated group (OIL), mineral-rich shungite-treated group (MRS), and mineral-less shungite-treated group (MLS). About 200 μL of treatment compounds (200 μg/mL) was topically administered using the hands with gloves applying the same pressure all throughout the dorsal side of the mouse. At the end of the experiment, mice were euthanized through inhalant anesthetics, CO2 gas for 20 seconds, and then the mice were decapitated. The animal use and protocol for this experiment was approved by the Institutional Animal Care and Use Committee (IACUC), Yonsei University Wonju Campus (YWC-151113-1).
2.2. UVB ExposureThe experimental mice were placed in a plastic cage and were irradiated using TL 40 W/12RS Philips, an unfiltered fluorescent sun lamps (Amsterdam, Netherlands) emitting rays of 290–320 nm. The distance from the lamps to the dorsal skin surface of the mice was 20 cm. The intensity of UVB lamp was 0.75 ± 0.10 mW/cm2 using YK-34 UV, a UV light meter by Lutron Electronics Inc. (Taipei, Taiwan), and the total cumulative dose energy of UVB irradiated was 2700 mJ/cm2. The mice were irradiated for 15 mins in 2 consecutive days.
2.3. Chemical and Shungite Suspension PreparationNatural shungite stone was purchased from Karelian Shungite Factory (Republic of Karelia, Russia). Natural shungite has 2.3–2.4 g/cm3 of specific gravity, 0.5% of porosity, 1000–1500 kgf/cm2 of compressed strength, and 1100–1200 kg/m3 of density and is composed of 28% carbon, 57% SiO2, 4.3% Al2O3, 2.8% FeO, 1.5% K2O, 1.5% S, 1.2% MgO, 0.3% CaO, 0.2% TiO2, 0.2% Na2O, and 3% H2O crystal. Mineral-rich shungite (MRS) and mineral-less shungite (MLS) were produced by MST Technology Ltd. (Incheon, Republic of Korea) through the treatment processes such as grinding of stone, treatment of alkaline solution and acidic solution, washing and filtration, treatment of boron (B), and high-temperature treatment process. The property and components of shungite vary according to the pulverized particle size of stone, mixing proportion of alkaline solution, acidic solution, and boron-containing compound with shungite powder, concentration of treatment solutions, and heating time and temperature. MRS and MLS powder used as an experimental material were refined to increase carbon ratio (MRS: 86.4%, NLS: 99.4% by energy dispersive X-ray spectroscopy (Missouri, USA)) using different treatment processes, and fullerene-like carbons such as C55, C60, C70, C74, C93, and C112 were mainly detected by MALDI-TOF mass spectrometry (Shimadzu Corp., Kyoto, Japan). Carbon powder were mixed with olive oil from Sigma-Aldrich Co. LLC. (Darmstadt, Germany) as a vehicle for the topical treatment on the skin.
Manufactured buckminsterfullerene C60 was purchased from Vaughter Wellness (London, United Kingdom) with 99.8% purity. Mineral-rich shungite was treated with KOH as an alkaline treatment with high temperature. Mineral-less shungite was treated with HNO3 and high-temperature (3000°C) treatment of mineral-rich shungite. These mixtures were sonicated using a vortex and sonicator until all of the solutions were mixed vigorously.
2.4. Characterization of Mineral-Less ShungiteComposition and visualization of shungite with mineral-less were analyzed by Energy-dispersive X-ray (EDX) spectroscopy. The mineral percent composition includes 86.43% carbon, 0.18% sodium, 1.33% magnesium, 3.17% silicon, 1.09% sulfur, 0.22% chlorine, 0.95% potassium, 5.33% calcium, 1.06% iron, and 0.2% copper.
2.5. Physiological Analysis of the Skin SurfaceSkin condition of the mice was assessed before and after 7-day treatment with a device for skin diagnostics, Aramo TS Device (Seongnam, Republic of Korea). The device is a comprehensive system for noninvasive, optical analysis of several dimensionless parameters of the skin: moisture, elasticity, sebum porosity, smoothness (evenness), discolourations (pigmentation), and wrinkles. The measurements were taken in very specific points located on the dorsal side of the mice, and the results were the difference before and after treatment to assess the improvement.
2.6. Immune Profile
2.6.1. White Blood Cell (WBC) and Its Differential Count AnalysisBlood was collected from the retro-orbital plexus in tubes coated with anticoagulant and was mixed with an automatic mixer for 5 min. Thereafter, WBC and its differential members such as lymphocytes, monocytes, neutrophil, eosinophil, and basophil were measured using an automatic blood analyser by HEMAVET HV950 FS, Drew Scientific Inc. (Texas, USA).
2.6.2. Serum and Skin Lysate Inflammatory CytokinesSerum and skin lysate inflammatory cytokines including IL-1β, IL-6, IL-10, IL-17, IL-KC, and TNF-α were analyzed using Bio-Plex Cytokine Assay by Bio-Rad (California, USA) according to the manufacturer's instructions. Standard curves for each adipokine and cytokine were generated by using the reference concentrations provided in the kits. Mean fluorescence intensity was acquired on a Luminex technology by Bio-Rad's Bio-Plex 200 system Multiplex Bead Array System™ (California, USA) and analyzed with associated software using a 5-parameter logistic method.
2.7. Redox Profile
2.7.1. Intracellular Reactive Oxygen Species (ROS) DetectionROS/RNS detection kit by Enzo Life Sciences Inc. (New York, USA) was used according to the manufacturer's instructions to determine effects of shungite compounds on oxidative stress and superoxide in serum and skin lysates. Twenty-five (25) μL sample was loaded, and detection solution was added to each well. The plate was read in the DTX-800 multimode microplate reader by Beckman Counter Inc. (California, USA) using a filter set of 485/20 excitation and 528/20 emission to detect oxidative species.
2.7.2. NO AssayThe nitrite (NO2−) present in the blood serum and skin lysates of mice was detected using the Griess reagent by Promega Corp. (Madison, USA). Briefly, 50 μL of the serum was mixed with an equal volume of Griess reagent in a 96-well microtiter plate and incubated at room temperature for 15 min. The absorbance was read at 540 nm using a DTX-880 multimode microplate reader by Beckman Counter Inc. (California, USA). The NO2− concentration was calculated by comparison with the representative NO2− standard curve generated by serial two-fold dilutions of sodium nitrate.
2.7.3. Antioxidant Endogenous Enzyme ActivitiesThe activity of superoxide dismutase (SOD), glutathione peroxidase (GPx), and myeloperoxidase (MPO) in serum and skin lysates was measured using the Biovision kit (California, USA). The crude skin lysate was centrifuged at 14,000 rpm for 15 min. at 4°C, and the debris was discarded. Skin lysate was then checked for protein concentration using Pierce™ BCA Protein Assay Kit by Thermo Scientific (Illinois, USA). Finally, the normalized concentrations of cell lysates were used to measure the activities of different antioxidant enzymes (SOD, GPx, and MPO) according to the manufacturer's instruction.
2.8. Western Blot AnalysisBriefly, the prepared skin lysate with the normalized protein concentration was equally loaded and separated by electrophoresis on SDS-polyacrylamide gels. Ten (10) percent separating gel and 5% stacking gel were prepared with the following components. 10% separating gel (10 mL) consisted of 4.0 mL dH2O, 3.3 mL 30% acrylamide mix, 2.5 mL 1.5 M Tris (pH 8.8), 100 μL 10% SDS, 100 μL 10% APS, and 4 μL TEMED. 4% stacking gel (5 mL) consist of 2.7 mL dH2O, 670 μL 30% acrylamide mix, 500 μL 1.0 M Tris (pH 6.8), 40 μL 10% SDS, 40 μL 10% APS, and 4 μL TEMED. The 15 μL ~ 20 μL of the sample with sample loading buffer with optimized concentration was loaded in the gel. Then, it will be run in 30 mA, 70–80 v. The PVDF membranes were blocked with 5% nonfat skim milk at room temperature for 2 hr and were incubated with the following primary antibodies: phospho-JNK and phospho-p38, ERK, Nrf2, and β-actin (dilution: 1 : 2000; Cell Signaling Technology, Massachusetts, USA) in Tris-buffered saline/tween 20 (1X TBST) containing 5% bovine serum albumin overnight in 4°C. The secondary antibody used was anti-rabbit (dilution: 1 : 2000; Cell Signaling Technology), and then it was incubated at room temperature for 2 hr. Specific protein bands were visualized by the enhanced chemiluminescence (ECL Pierce Biotechnology) using UVP Biospectrum 600 Imaging System (UVP, LLC, Upland, CA, USA). β-Actin (dilution: 1 : 2000, Cell Signaling Technology) was used as loading controls for the total protein content.
2.9. Data Management and AnalysisStatistical analysis was carried out using analysis of variance (ANOVA) followed by subsequent multiple comparison test (Dunnett's multiple comparison test) using GraphPad Prism version 7.0 software packages (California, USA). Differences were considered significant at ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.
Go to:3. Results and Discussion3.1. The Effects of Shungite on the Physiological Skin Surface of the MiceThis study aims to examine whether topical application of MRS and MLS could attenuate the related skin damage caused by UVB irradiation in hairless mice compared to the pure fullerene C60 positive control. We also compared the experimental groups (MRS and MLS groups) to non-UVB-irradiated group, negative control UVB-irradiated only group, and UVB + olive oil-treated group. We used two experimental groups (MRS is close to the natural shungite that has the most mineral present while the MLS is with least mineral material) to explore the effects of the two different types and also to rule out the effects of other minerals present in the material. On the other note, olive oil was used in the experiment because it has been used as a solubilizing excipient or vehicle in our positive control fullerene C60. Studies have shown that olive oil is the most biocompatible organic solvent for C60 fullerene [25]. Olive oil has been used as an alternative for the toxic glycerol or toluene. In addition, olive oil is one of the most effective and safe solubilizing excipients easily available [26]. However, since olive oil itself is already known to have its antioxidant effects and was also found to have protective effect after UVB exposure [27], it is essential to compare its effect with the olive oil only treatment with the experimental groups which used olive oil as a vehicle.
To verify the clinical evaluation of the skin, we checked different skin parameters and we found that the skin moisture and elasticity of the shungite-treated (MRS and MLS) mice had improved compared to the NC and UV groups, comparable to PC (Figure 1). Furthermore, the roughness, pigmentation, and wrinkle had been significantly reduced and showed improvement after 7 days of topical application of shungite. Pore size of MRS and MLS groups did not show significant reduction, but it has improved compared to the PC. These results show clinical evidences of the antioxidant and anti-inflammatory effects of MRS and MLS in the outward skin surface.
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Figure 1Mineral-rich and mineral-less shungite-treated groups improved physiological skin parameters. Shown are the effects of mineral-rich shungite (MRS) and mineral-less shungite (MLS) on the mouse skin moisture (a), elasticity (b), roughness (c), pore size (d), pigmentation (e), and wrinkle (f). Control groups include non-UVB-irradiated group (NC), UVB-irradiated only group (UV), UVB + fullerene C60-treated positive control group (PC), and UVB + olive oil-treated group (OIL), while experimental groups include UVB + mineral-rich shungite-treated group (MRS) and mineral-less shungite-treated group (MLS). Statistical analysis was carried out using analysis of variance (ANOVA) followed by subsequent multiple comparison test (Tukey's multiple comparisons test) using GraphPad Prism version 5.0. Values are mean ± SD, n = 9. Differences were considered significant at ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.
3.2. Immune Profiling
3.2.1. Total WBC and Its Differential CountTotal WBC counts were used to evaluate the overview of the severity of inflammation response. Table 1 showed the total WBC count suppression of MRS and MLS compared to UV and PC. The altered WBC counts may be indicative of direct or indirect effects of the compound treatment on cellular toxicity and proliferation. The recruitment of total WBC and differential counts are highest in PC followed by MLS then MRS. Overall, the suppressed count of the total WBC in the negative control group (UV) was rescued by the shungite-treated groups as well as their differential counts on both MRS and MLS groups after 7 days of topical application of shungite.
Table 1Slight suppression of total white blood cells (WBC) and their differential counts in the mineral-rich shungite-treated group. Shown are the effects of mineral-rich shungite (MRS) and mineral-less shungite (MLS) on the total WBC (a) and their differential counts including neutrophil (b), lymphocyte (c), monocyte (d), basophil (e), and eosinophils (f). Control groups include non-UVB-irradiated group (NC), UVB-irradiated only group (UV), UVB + fullerene C60-treated positive control group (PC), and UVB + olive oil-treated group (OIL), while experimental groups include UVB + mineral-rich shungite-treated group (MRS) and mineral-less shungite-treated group (MLS). Statistical analysis was carried out using analysis of variance (ANOVA) followed by subsequent multiple comparison test (Tukey's multiple comparisons test) using GraphPad Prism version 5.0. Data were expressed as mean ± SD, n = 9.
Units in K/mLNCUVPCOILMRSMLSTotal WBC3.16 ± 0.833.098 ± 0.914.25 ± 1.943.54 ± 2.223.032 ± 0.663.76 ± 1.26
Neutrophil1.12 ± 0.351.326 ± 0.761.59 ± 0.861.31 ± 0.861.03 ± 0.281.41 ± 0.44
Lymphocyte1.97 ± 0.622.094 ± 0.632.34 ± 0.961.99 ± 1.411.82 ± 0.422.13 ± 0.82
Monocyte0.13 ± 0.0710.15 ± 0.900.22 ± 0.110.18 ± 0.130.13 ± 0.040.17 ± 0.07
Eosinophil0.02 ± 0.0130.041 ± 0.0580.076 ± 0.130.05 ± 0.060.034 ± 0.0840.033 ± 0.044
Basophil0.007 ± 0.00480.011 ± 0.0170.027 ± 0.0480.014 ± 0.0170.019 ± 0.0360.013 ± 0.016
3.2.2. Proinflammatory Cytokine Levels in Serum and Skin Lysates of Shungite-Treated UVB-Induced MiceFurther evidence showed that shungite might mediate inflammation response that can influence immunological homeostasis. To delineate this proposition, we measured the serum (Figure 2) and skin lysate (Figure 3) cytokine profiling to gauge the inflammatory cytokine balance. The overall trend of the serum levels of IL-1β, IL-6, IL-10, IL-KC, and TNF-α concentration in shungite-treated mice was lower than that of all the control groups except for IL-17. The UV group showed slightly higher than the NC group. Comparing cytokine levels of serum, MLS showed lower level of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) as well as the anti-inflammatory cytokine (IL-10) than the PC group (Figure 2). Similar trend was observed in skin lysate levels of IL-1β, IL-6, IL-10, IL-KC, and TNF-α, except IL-17 concentrations in shungite-treated (Figure 3). In shungite-treated group, lower level of proinflammatory cytokines (IL-1β, TNF-α, and IL-6) and regulatory cytokines in skin keratinocytes such as IL-17 and IL-KC is highly compatible with the redox profiling results. Cumulative evidence showed that UVB leads to a shift from the production of proinflammatory cytokines such as IL-1β, IL-12, IL-8, TNF-α, and IFN-γ to the production of anti-inflammatory cytokines such as IL-4, IL-10, and IL-13 [28, 29]. Breakdown of pro- and anti-inflammatory cytokines leads to various diseases such as atopic dermatitis, rheumatoid arthritis, and psoriasis. As observed in the results, a similar trend was seen in shungite-treated group in IL-1β and TNF-α. This may be ascribed to the synergism between two cytokines. On the other hand, the role of IL-6 in UVB-induced skin immune responses is to regulate the release and production of other proinflammatory cytokines [30]. The mechanism which is regulated by IL-17 was not noticeable in our results. IL-KC appears to have the key role in immunosuppressive effects of UVB as it mediates IL-10 which is known to be an anti-inflammatory cytokine [31]. Based on cytokine profiling, shungite treatment restored the inflammatory cytokine imbalance evoked by UV irradiation. UVB irradiation on the skin would induce alteration of antigen-presenting cells including Langerhans cells and imbalance of a cell-mediated immune response. IL-10 is known as an important immunoregulatory cytokine to downregulate inflammatory responses and regulate differentiation and proliferation of several immune cells such as T cells, B cell, natural killer (NK) cells, mast cells, and granulocytes [32]. In that context, this immunomodulation of shungite might justify the antioxidant effect and potential clinical therapeutic usage for skin and oxidative stress disorders [33].
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Figure 2Serum proinflammatory cytokine levels in mineral-rich and mineral-less shungite groups showed immunomodulatory effect. Shown are the serum cytokine concentrations of IL-1β (a), IL-6 (b), IL-10 (c), IL-17 (d), IL-KC (e), and TNF-α (f) determined using multiplex assay in shungite-treated UVB-induced mice. Control groups include non-UVB-irradiated group (NC), UVB-irradiated only group (UV), UVB + positive control fullerene C60-treated group (PC), and UVB + olive oil-treated group (OIL), while experimental groups include UVB + mineral-rich shungite-treated group (MRS) and mineral-less shungite-treated group (MLS). Statistical analysis was carried out using analysis of variance (ANOVA) followed by subsequent multiple comparison test (Tukey's multiple comparisons test) using GraphPad Prism version 5.0. All values are represented as mean ± SD (n = 9), ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 versus each treatment.
Figure 3Skin lysate proinflammatory cytokine levels in mineral-rich shungite (MRS) and mineral-less shungite (MLS) groups showed immunomodulatory effect. Shown are the skin lysate cytokine concentrations of IL-1β (a), IL-6 (b), IL-10 (c), IL-17 (d), IL-KC (e), and TNF-α (f) determined using multiplex assay in shungite-treated UVB-induced mice. Control groups include non-UVB-irradiated group (NC), UVB-irradiated only group (UV), UVB + positive control fullerene C60-treated group (PC), and UVB + olive oil-treated group (OIL), while experimental groups include UVB + mineral-rich shungite-treated group (MRS) and mineral-less shungite-treated group (MLS). Statistical analysis was carried out using analysis of variance (ANOVA) followed by subsequent multiple comparison test (Tukey's multiple comparisons test) using GraphPad Prism version 5.0. All values are represented as mean ± SD, n = 9, ∗p < 0.05, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 versus each treatment.
3.3. Redox Profiling
3.3.1. Effects of Shungite on the Levels of Total Intracellular ROS and Nitric Oxide (NO) in Mouse Serum and SkinThe generation of intracellular ROS and superoxide of both skin lysates in MRS and MLS had reduced levels compared to the high levels of ROS in UV group. However, the reduction of ROS/RNS was not that dramatical in the circulating serum (Figure 4). On the other hand, the NO levels in serum and skin lysates in shungite-treated groups were not significant but a lower trend had been observed in the serum NO levels in shungite-treated group (Figure 5). NO plays a pivotal role in macrophage-mediated cytotoxicity by acting as an effector molecule. NO is a reactive molecule that reacts with ROS to produce reactive nitrogen species; moreover, it is recognized as a mediator and regulator of immune responses. NO has various physiological and pathophysiological responses depending on its relative concentration.
Figure 4Reduction of total intracellular reactive oxygen/nitrogen (ROS/RNS) and superoxide dismutase (SOD) species in mouse serum and skin lysate of shungite-treated groups. Shown are the effects of shungite in the total ROS/RNS in serum (a) and skin lysates (b) as well as the SOD concentration in serum (c) and skin lysates (d) as determined by an ROS/RNS detection kit by Enzo Life Sciences Inc. Control groups include non-UVB-irradiated group (NC), UVB-irradiated only group (UV), UVB + fullerene C60-treated positive control group (PC), and UVB + olive oil-treated group (OIL), while experimental groups include UVB + mineral-rich shungite-treated group (MRS) and mineral-less shungite-treated group (MLS). Statistical analysis was carried out using analysis of variance (ANOVA) followed by subsequent multiple comparison test (Tukey's multiple comparisons test) using GraphPad Prism version 5.0. Data were expressed as mean fluorescence ± SD, n = 9. Differences were considered significant at ∗p < 0.05 versus each treatment.
Figure 5Slight increase of nitric oxide (NO) levels in mouse serum and skin lysates of shungite-treated groups. Shown are the NO levels in serum (a) and skin lysates (b) that were determined using Greiss reagent. Control groups include non-UVB-irradiated group (NC), UVB-irradiated only group (UV), UVB + fullerene C60-treated positive control group (PC), and UVB + olive oil-treated group (OIL), while experimental groups include UVB + mineral-rich shungite-treated group (MRS) and mineral-less shungite-treated group (MLS). Statistical analysis was carried out using analysis of variance (ANOVA) followed by subsequent multiple comparison test (Tukey's multiple comparisons test) using GraphPad Prism version 5.0. Data were expressed as mean ± SD, n = 9.
3.3.2. Serum and Skin Lysate Scavenging Enzyme ActivityConsistent with the total ROS/RNS result, the activities of ROS-scavenging enzymes such as intracellular GPx and SOD levels were increased. Levels of myeloperoxidase activities in shungite-treated groups had decreased in serum and skin lysates, and this could be to compensate for other enzymes dissociating hydrogen peroxide (Figure 6).
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Figure 6Antioxidant enzyme activities of serum and skin lysates in shungite-treated mice groups: glutathione peroxidase (GPx), superoxide dismutase (SOD), and myeloperoxidase (MPO) levels. Shown are the antioxidant biological enzyme activities such GPX in serum (a) and skin lysates (d), SOD in serum (b) and skin lysates (e), and MPO in serum (c) and skin lysates (f) of shungite-treated UVB-induced mice. Control groups include non-UVB-irradiated group (NC), UVB-irradiated only group (UV), UVB + fullerene-treated positive control group (PC), and UVB + olive oil-treated group (OIL), while experimental groups include UVB + mineral-rich shungite-treated group (MRS) and mineral-less shungite-treated group (MLS). Statistical analysis was carried out using analysis of variance (ANOVA) followed by subsequent multiple comparison test (Tukey's multiple comparisons test) using GraphPad Prism version 5.0. Data were expressed as mean ± SD, n = 9, ∗p < 0.05, ∗∗p < 0.01 versus each treatment groups.
3.4. Involvement of Nrf2 and p-ERK, p-p38 MAPK, and p-JNK Signaling Pathways in the Antioxidant Effect of Shungite against UVB-Induced Oxidative StressTo fully elucidate the underlying molecular mechanism of antioxidant effect mediated by shungite, we hypothesized those different pathways that can lead to inflammatory. To prove our hypothesis, we determined whether the skin lysate of the shungite-treated group can induce the phosphorylation of Nrf2, p-p38 MAPK, and p-JNK. Figure 7 showed that there was a marked increase in phosphorylation of Nrf2, p-p38 MAPK, and p-JNK. This result indicates that topical application of shungite can induce phosphorylation of Nrf2, p-p38 MAPK, and p-JNK. This study also found out that Nrf2 and MAPK proteins, p-ERK and p-JNK, are involved in the antioxidant effect of shungite against UVB-induced oxidative stress. Nrf2 is a transcription factor that regulates expression of many detoxification or antioxidant enzymes. It is plausible that fullerene transiently increases the intracellular level of ROS and/or activates p38 MAPK signaling pathway, which may possibly lead to facilitating the dissociation of Nrf2 from Keap. The resultant Nrf2/ARE activation induced phase II detoxification or antioxidant enzyme, thereby potentiating cellular defence capacity against cell death.
Figure 7Nrf2 and MAPK proteins, P-ERK and p-JNK, are involved in the antioxidant effect of shungite against UVB-induced oxidative stress. Signaling cascade controlling cellular responses were measured for protein expression. Mineral-rich shungite (MRS) and mineral less shungite (MRS) induced phosphorylation of p-ERK (a), p-JNK (b) and Nrf2 (c), and β-actin (d) signaling pathway. Control groups include non-UVB-irradiated group (NC), UVB-irradiated only group (UV), UVB + positive control fullerene-treated group (PC), and UVB + olive oil-treated group (OIL), while experimental groups include UVB + mineral-rich shungite-treated group (MRS) and mineral-less shungite-treated group (MLS). Statistical analysis was carried out using analysis of variance (ANOVA) followed by subsequent multiple comparison test (Tukey's multiple comparisons test) using GraphPad Prism version 5.0. All values are represented as mean ± SD, n = 9, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 versus each treatment.
3.5. ConclusionThis study confirms the hypothesis that shungite, a natural fullerene, has antioxidant properties as it reduced the intracellular ROS production and enhanced the antioxidant enzyme activities (GPx, SOD, and MPO) in vivo. It is the first note to show several mechanisms and evidences of shungite's antioxidant effects including the improvement of several skin test parameters (moisture, elasticity, roughness, pore size, pigmentation, and wrinkle) and recovery of total WBC. Most importantly, shungite counterbalanced ROS/antioxidant paradigm as shown by the reduction of ROS/RNS levels and superoxide levels in both serum and skin lysate levels. Consistently, the activities of ROS-scavenging enzymes such as GPx and SOD levels were increased. In line, the inflammatory markers such as the cytokine levels of IL-1β, IL-6, IL-10, Il-17, IL-KC, and TNF-α in mice serum and skin lysates were lower than those in the NC group. Last, we found that Nrf2 and MAPK proteins, p-ERK and p-JNK, were involved in the antioxidant effect of shungite against UVB-induced oxidative stress. Given these, our data verify that mineral-rich shungite and mineral-less shungite were effective in the overall screening of its antioxidant and anti-inflammatory properties compared to the other control treatments. Further, MLS has a stronger antioxidant and anti-inflammatory effects than MRS. Synthesizing these, we rationally infer that shungite could be a one potential agent in such other diseases as well and can also be an alternative to the known applications of pure fullerene. However, the comparison of shungite-treated groups to other potent agents on other skin damage and immune- and oxidative-related diseases such as psoriasis or atopic dermatitis was not part of the study, but could be one of the future study targets. Since the processibility of pure fullerene is very challenging and very expensive, this study suggests that natural mineral shungite, as a novel antioxidant, could provide a new therapeutic insight against oxidative- and inflammatory-related diseases.
Go to:AcknowledgmentsThis work was supported by the Yonsei University Research Fund of 2016.
Go to:Conflicts of InterestThe authors have no conflict of interest to declare.
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