TEXTUAL SOURCES OF REUSI DAT TON

We may never know what, if any Ancient texts on Reusi Dat Ton may have existed and were lost when the invading Burmese armies destroyed the old Thai capital of Ayutthaya in 1767. Today, the closest thing to an original source text on Reusi Dat Ton is an 1838 manuscript commissioned by Rama III entitled The Book of Eighty Rishis Performing Posture Exercises to Cure Various Ailments. Like other manuscripts of the time, this text was printed on accordion like folded black paper, known in Thai as “Khoi.” This text, popularly known as the Samut Thai Kao features line drawings of the 80 Wat Po Reusi Dat Ton statues along with their accompanying poems. In the introduction, it states that Reusi Dat Ton is a “…system of posture exercises invented by experts to cure ailments and make them vanish away.” (Griswold, 321) This text is housed in the National Library in Bangkok. There are also other editions of this text housed in museums and private collections as well.

The Samut Thai Kao seems to follow an old tradition also found in ancient Indian, Nepali and Tibetan Yoga manuscripts that list 80 to 84 different techniques. The Samut Thai Kao is, however, only a partial collection of all the various Reusi Dat Ton techniques. A 1958 Wat Po publication, The Book of Medicine includes a section on Reusi Dat Ton. While it contains verses based upon the poems at Wat Po, many of the accompanying illustrations depict completely different techniques.

In the Southern Thai town of Songkhla, on the temple grounds of Wat Machimawat is a pavilion known as the “Sala Reusi Dat Ton.” High up on the inside walls of this pavilion is a mural which depicts 40 of the Reusi Dat Ton techniques along with the accompanying poems from the Samut Thai Kao. 

There is a special section devoted to Thai Medical history at the Mahidol University’s Siriraj Medical Museum on the Bangkok Noi campus in Bangkok. There one can view a Reusi Dat Ton display featuring small painted wood Reusi figures that depict over 60 different Reusi Dat Ton techniques. This display is based upon the 1958 Wat Po text The Book of Medicine. 

In Nonthaburi, on the Ministry of Public Health Campus at the Institute of Thai Traditional Medicine, there is the Thai Traditional Medicine Museum. Inside the museum is a small display of Reusi Dat Ton statues. Outside the museum is an artificial mountain upon which have been placed various Reusi statues demonstrating Reusi Dat Ton techniques. Within the mountain is the “Hermit’s Cave” which houses numerous small Reusi statues also depicting Reusi Dat Ton techniques. These statues depict techniques from both the Samut Thai Kao and The Book of Medicine. 

On the outskirts of Bangkok, in the town of Samut Prakan, is the cultural park, the Ancient City or “Muang Boran.” One of the many attractions is a “Sala of 80 Yogi” which features 80 life- sized Reusi statues illustrating various Reusi Dat Ton techniques. There are even depictions of

Reusi Dat Ton techniques not found in either of the two Wat Po texts. While most of these statues are fairly accurate depictions of Reusi Dat Ton techniques, a few actually show Indian Hatha Yoga techniques, which are not part of the Reusi Dat Ton system.

Students of Reusi Dat Ton should bear in mind that while some of the Reusi Dat Don statues, drawings, paintings and poems are beautiful works of art, they were created by artists who were not necessarily all practitioners of Reusi Dat Ton. In fact, a number of images do not illustrate the actual techniques entirely accurately. Even in 1836, there was some uncertainty as to which technique produced which effect and some poems were used for more than one technique. Therefore, students of Reusi Dat Ton should also seek out living teachers who have learned from authentic sources such as actual Reusis, who can teach the techniques in their authentic form.

There are also additional Reusi Dat Ton techniques practiced by Reusis today, which are not found in any text, nor depicted in any sculpture or paintings. These are also traditional techniques, which have been passed down from teacher to student over the centuries. There are close to 300 different exercises and poses, including variations, in the entire Reusi Dat Ton system.

THE BENEFITS OF REUSI DAT TON

In both the Samut Thai Kao and The Book of Medicine, the texts not only describe the techniques, but also ascribe a therapeutic benefit to each pose or exercise. Some poems describe specific ailments while others use Sanskrit Ayurvedic medical terminology.

Some of the ailments mentioned include; abdominal discomfort and pain, arm discomfort, back pain, bleeding, blurred vision, chest congestion, chest discomfort and pain, chin trouble, chronic disease, chronic muscular discomfort, congestion, convulsions, dizziness and vertigo, dyspepsia, facial paralysis, fainting, foot cramps, pain and numbness, gas pain, generalized weakness, generalized sharp pain, headache and migraine, hand discomfort, cramps and numbness, heel and ankle joint pain, hemorrhoids, hip joint problems, joint pain, knee pain and weakness, lack of alertness, leg discomfort, pain and weakness, lockjaw, low back pain, lumbar pain, muscular

cramps and stiffness, nasal bleeding, nausea, neck pain, numbness, pelvic pain, penis and urethra problems, scrotal distention, secretion in throat, shoulder and scapula discomfort and pain, stiff neck, thigh discomfort, throat problems, tongue trouble, uvula spasm, vertigo, waist trouble, wrist trouble, vomiting, and waist discomfort.

Some of the Ayurvedic disorders described in the texts include; Wata (Vata in Sanskrit) in the head causing problems in meditation, severe Wata disease, Wata in the hands and feet, Wata in the head, nose and shoulder, Wata in the thigh, Wata in the scrotum, Wata in the urethra, Wata causing knee, leg and chest spasms, Wata causing blurred vision, Sannipat (a very serious and difficult to treat condition due to the simultaneous imbalance of Water, Fire and Wind Elements which may also involve a toxic fever) an excess of Water Dhatu (possibly plasma or lymph fluids,) and “Wind” in the stomach. Other benefits described in the old texts include; increased longevity and opening all of the “Sen” (There are various types of “Sen” or channels in Traditional Thai Medicine. There are Gross Earth Physical “Sen” such as Blood Vessels. There are also more Subtle “Sen” such as channels of Bioenergy flow within the Subtle Body, known as “Nadis” in Sanskrit. In addition, there are also “Sen” as channels of the Mind.)

In recent years, the Thai Ministry of Public Health has published several books on Reusi Dat Ton. According these modern texts, some of the benefits of Reusi Dat Ton practice include; improved agility and muscle coordination, increased joint mobility, greater range of motion, better circulation, improved respiration improved digestion, assimilation and elimination, detoxification, stronger immunity, reduced stress and anxiety, greater relaxation, improved concentration and meditation, oxygen therapy to the cells, pain relief, slowing of degenerative disease and greater longevity. (Subcharoen, 5-7)

A recent study at Naresuan University in Phitsanulok, Thailand, found that after one month of regular Reusi Dat Ton practice there was an improvement in anaerobic exercise performance in sedentary females. (Weerapong et al, 205)

Here's How Much Yoga It Takes to Truly See Results

The good news: You don't have to be the most flexible person in the room to reap the benefits of practicing. Still, you may wonder what good the occasional yoga class is doing for your body and mind. Well, let out an “om:” You don’t need to hit the yoga studio every day (or even at all) for significant benefits, both physically and mentally.

Reasons to Roll Out the Mat 

Whether you're aiming for the flexibility of a gymnast or just a calmer mind, yoga has immeasurable benefits. Physically, yoga has been shown to improve flexibility, posture, and balance; strengthen bones; and increase muscle strength.

There are plenty of overall health perks too: Research shows that yoga can decrease inflammation, boost immune system function, and improve symptoms associated with chronic health conditions, such as type 2 diabetes, cardiovascular disease, and cancer.The practice can also do wonders for your mental health and mood, reducing depression, stress, and anxiety. But can an average person actually reap these benefits, or do you have to spend half your salary on unlimited access to a yoga studio to see a change?

First things first: “We know from exercise that the more you do, generally the more benefits you get,” says William J. Broad, a science writer and author of The Science of Yoga.

 “Yoga is no different. Practicing once a week is good. Practicing three to four times per week would be better.”

But just like yoga pants, one size doesn't fit all. Loren Fishman, M.D., a back pain specialist who studied yoga and uses it in his rehabilitative practice, believes that even one minute spent in practice can be enough to reset someone's outlook: “One minute in meditation can have a frustrated, angry, terrible-feeling person feeling resourceful, kind, and fun,” he says.

While this way of thinking probably won't lead to Cirque du Soleil-level moves, that doesn’t mean you won’t see—or feel—results.

“Practicing yoga once a week gives you a time every week to focus on your breathing, which in turn, allows you to be present,” says Heidi Kristoffer, a yoga instructor at The Movement. “Being in the present moment gives you a total time-out from the rest of the world and resets your system.”

Physically, a one hour yoga class won’t tout the same calorie-blasting effects as 60 minutes of cardio. But it will increase your blood flow, get your oxygen moving, and, “get any stuck parts of your body ‘unstuck,’” Kristoffer says.

“If you commit to a weekly practice, depending on the class you take, your flexibility will improve over time, leading to fewer injuries, and you will experience toning in all of your muscles,” Kristoffer says. “Not to mention a stronger core, which leads to less back pain.”

It’s clear yoga has mental and physical benefits that can be enjoyed by anyone with just a few minutes. But since there are so many different kinds of yoga, there’s no general rule to determine exactly how much yoga one person needs to see physical results. However, age may play a factor, Broad says.

“I would argue that a 20-something person who is in their prime of life and reasonably good shape needs less yoga to sustain their practice than someone in their 50s or 60s,” Broad says. One study analyzing the effects of yoga on women over 50 found that practicing asanas (yoga postures) even once a week led to an increase in the mobility of spinal joints and flexibility of the hamstring muscles.

Of course, if you want to evolve your practice and nail those mountain-top handstands, you should practice yoga several times per week, says Amanda Murdock, a yoga instructor at YG Studios who specializes in power yoga flow. “If you practice several times a week, you will see longer-lasting benefits, like better range of motion and flexibility, reduction in stress over sustained periods of time, and better posture, to name a few. You’ll also obviously see faster [physical] results.”

While you’ll benefit in the short term (feeling more open, better digestion, better sleep), a single yoga class per month will essentially have you starting from scratch each time you walk on the mat, Murdock says. It can be difficult to listen to your body when you are trying to figure out what you are doing in the class.

That’s why she recommends getting on your mat at least one day a week to become familiar with your body and to become aware of how you feel after practice versus before practice—which can become a powerful motivator to practice more often. It doesn’t have to happen in a studio (or even on a real mat), but the frequency can help you be in tune with what your body needs at the time.

“Yoga is a lifelong practice,” Murdock says. “That's why yoga is much more than just a workout. It's the mind-body connection and awareness that make a yoga practice powerful, beneficial, and sustainable.”

The verdict's in: Just one class can deliver some of the mind-body benefits of yoga. Still, to truly reap the physical and mental benefits and improve your practice, it's better to block off an hour for class at least once per week. Even if you can't, once you know the fundamentals of the practice, do a little bit every day at home, Broad recommends. “My own personal mantra is, ‘A little bit often is much better than a lot every once in a while,” he says.

Yoga: Fight stress and find serenity

Is yoga right for you? It is if you want to fight stress, get fit and stay healthy.

By Mayo Clinic Staff

Your mobile phone is ringing, your boss wants to talk to you and your partner wants to know what's for dinner. Stress and anxiety are everywhere. If they're getting the best of you, you might want to hit the mat and give yoga a try.

Yoga is a mind-body practice that combines physical poses, controlled breathing, and meditation or relaxation. Yoga may help reduce stress, lower blood pressure and lower your heart rate. And almost anyone can do it.

Yoga — a mind-body practice — is considered one of many types of complementary and integrative health approaches. Yoga brings together physical and mental disciplines that may help you achieve peacefulness of body and mind. This can help you relax and manage stress and anxiety.

Yoga has many styles, forms and intensities. Hatha yoga, in particular, may be a good choice for stress management. Hatha is one of the most common styles of yoga, and beginners may like its slower pace and easier movements. But most people can benefit from any style of yoga — it's all about your personal preferences.

The core components of hatha yoga and most general yoga classes are:

* Poses. Yoga poses, also called postures, are a series of movements designed to increase strength and flexibility. Poses range from lying on the floor while completely relaxed to difficult postures that may have you stretching your physical limits.

* Breathing. Controlling your breathing is an important part of yoga. Yoga teaches that controlling your breathing can help you control your body and quiet your mind.

* Meditation or relaxation. In yoga, you may incorporate meditation or relaxation. Meditation may help you learn to be more mindful and aware of the present moment without judgment.

The health benefits of yoga

The potential health benefits of yoga include:

* Stress reduction. A number of studies have shown that yoga may help reduce stress and anxiety. It can also enhance your mood and overall sense of well-being. 

* Improved fitness. Practicing yoga may lead to improved balance, flexibility, range of motion and strength.

* Management of chronic conditions. Yoga can help reduce risk factors for chronic diseases, such as heart disease and high blood pressure. Yoga might also help alleviate chronic conditions, such as depression, pain, anxiety and insomnia.

Yoga precautions

Yoga is generally considered safe for most healthy people when practiced under the guidance of a trained instructor. But there are some situations in which yoga might pose a risk.

See your health care provider before you begin yoga if you have any of the following conditions or situations:

* A herniated disk

* A risk of blood clots 

* Eye conditions, including glaucoma

* Pregnancy — although yoga is generally safe for pregnant women, certain poses should be avoided

* Severe balance problems

* Severe osteoporosis

* Uncontrolled blood pressure

You may be able to practice yoga in these situations if you take certain precautions, such as avoiding certain poses or stretches. If you develop symptoms, such as pain, or have concerns, see your doctor to make sure you're getting benefit and not harm from yoga.

Getting started

Although you can learn yoga from books and videos, beginners usually find it helpful to learn with an instructor. Classes also offer camaraderie and friendship, which are also important to overall well-being.

When you find a class that sounds interesting, talk with the instructor so that you know what to expect. Questions to ask include:

* What are the instructor's qualifications? Where did he or she train and how long has he or she been teaching?

* Does the instructor have experience working with students with your needs or health concerns? If you have a sore knee or an aching shoulder, can the instructor help you find poses that won't aggravate your condition?

* How demanding is the class? Is it suitable for beginners? Will it be easy enough to follow along if it's your first time?

* What can you expect from the class? Is it aimed at your needs, such as stress management or relaxation, or is it geared toward people who want to reap other benefits?

Achieving the right balance

Every person has a different body with different abilities. You may need to modify yoga postures based on your individual abilities. Your instructor may be able to suggest modified poses. Choosing an instructor who is experienced and who understands your needs is important to safely and effectively practice yoga.

Regardless of which type of yoga you practice, you don't have to do every pose. If a pose is uncomfortable or you can't hold it as long as the instructor requests, don't do it. Good instructors will understand and encourage you to explore — but not exceed — your personal limits.

How to grow garlic

http://youtu.be/rlsnAwoltBM

The Brain, The Big Boss

Introduction

The brain is the most complex part of the human body. This three-pound organ is the seat of intelligence, interpreter of the senses, initiator of body movement, and controller of behavior. Lying in its bony shell and washed by protective fluid, the brain is the source of all the qualities that define our humanity. The brain is the crown jewel of the human body.

For centuries, scientists and philosophers have been fascinated by the brain, but until recently they viewed the brain as nearly incomprehensible. Now, however, the brain is beginning to relinquish its secrets. Scientists have learned more about the brain in the last 10 years than in all previous centuries because of the accelerating pace of research in neurological and behavioral science and the development of new research techniques. As a result, Congress named the 1990s the Decade of the Brain. At the forefront of research on the brain and other elements of the nervous system is the National Institute of Neurological Disorders and Stroke (NINDS), which conducts and supports scientific studies in the United States and around the world.

This fact sheet is a basic introduction to the human brain. It may help you understand how the healthy brain works, how to keep it healthy, and what happens when the brain is diseased or dysfunctional.

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The Architecture of the Brain

The brain is like a committee of experts. All the parts of the brain work together, but each part has its own special properties. The brain can be divided into three basic units: the forebrain, the midbrain, and the hindbrain.

The hindbrain includes the upper part of the spinal cord, the brain stem, and a wrinkled ball of tissue called the cerebellum (1). The hindbrain controls the body’s vital functions such as respiration and heart rate. The cerebellum coordinates movement and is involved in learned rote movements. When you play the piano or hit a tennis ball you are activating the cerebellum. The uppermost part of the brainstem is the midbrain, which controls some reflex actions and is part of the circuit involved in the control of eye movements and other voluntary movements. The forebrain is the largest and most highly developed part of the human brain: it consists primarily of the cerebrum (2) and the structures hidden beneath it (see "The Inner Brain").

When people see pictures of the brain it is usually the cerebrum that they notice. The cerebrum sits at the topmost part of the brain and is the source of intellectual activities. It holds your memories, allows you to plan, enables you to imagine and think. It allows you to recognize friends, read books, and play games.

The cerebrum is split into two halves (hemispheres) by a deep fissure. Despite the split, the two cerebral hemispheres communicate with each other through a thick tract of nerve fibers that lies at the base of this fissure. Although the two hemispheres seem to be mirror images of each other, they are different. For instance, the ability to form words seems to lie primarily in the left hemisphere, while the right hemisphere seems to control many abstract reasoning skills.

For some as-yet-unknown reason, nearly all of the signals from the brain to the body and vice-versa cross over on their way to and from the brain. This means that the right cerebral hemisphere primarily controls the left side of the body and the left hemisphere primarily controls the right side. When one side of the brain is damaged, the opposite side of the body is affected. For example, a stroke in the right hemisphere of the brain can leave the left arm and leg paralyzed.

The Forebrain ------- The Midbrain -------- The Hindbrain

  

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The Geography of Thought

Each cerebral hemisphere can be divided into sections, or lobes, each of which specializes in different functions. To understand each lobe and its specialty we will take a tour of the cerebral hemispheres, starting with the two frontal lobes (3), which lie directly behind the forehead. When you plan a schedule, imagine the future, or use reasoned arguments, these two lobes do much of the work. One of the ways the frontal lobes seem to do these things is by acting as short-term storage sites, allowing one idea to be kept in mind while other ideas are considered. In the rearmost portion of each frontal lobe is a motor area (4), which helps control voluntary movement. A nearby place on the left frontal lobe called Broca’s area (5) allows thoughts to be transformed into words.

When you enjoy a good meal—the taste, aroma, and texture of the food—two sections behind the frontal lobes called the parietal lobes (6) are at work. The forward parts of these lobes, just behind the motor areas, are the primary sensory areas (7). These areas receive information about temperature, taste, touch, and movement from the rest of the body. Reading and arithmetic are also functions in the repertoire of each parietal lobe.

As you look at the words and pictures on this page, two areas at the back of the brain are at work. These lobes, called the occipital lobes (8), process images from the eyes and link that information with images stored in memory. Damage to the occipital lobes can cause blindness.

The last lobes on our tour of the cerebral hemispheres are the temporal lobes (9), which lie in front of the visual areas and nest under the parietal and frontal lobes. Whether you appreciate symphonies or rock music, your brain responds through the activity of these lobes. At the top of each temporal lobe is an area responsible for receiving information from the ears. The underside of each temporal lobe plays a crucial role in forming and retrieving memories, including those associated with music. Other parts of this lobe seem to integrate memories and sensations of taste, sound, sight, and touch.

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The Cerebral Cortex

Coating the surface of the cerebrum and the cerebellum is a vital layer of tissue the thickness of a stack of two or three dimes. It is called the cortex, from the Latin word for bark. Most of the actual information processing in the brain takes place in the cerebral cortex. When people talk about "gray matter" in the brain they are talking about this thin rind. The cortex is gray because nerves in this area lack the insulation that makes most other parts of the brain appear to be white. The folds in the brain add to its surface area and therefore increase the amount of gray matter and the quantity of information that can be processed.

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The Inner Brain

Deep within the brain, hidden from view, lie structures that are the gatekeepers between the spinal cord and the cerebral hemispheres. These structures not only determine our emotional state, they also modify our perceptions and responses depending on that state, and allow us to initiate movements that you make without thinking about them. Like the lobes in the cerebral hemispheres, the structures described below come in pairs: each is duplicated in the opposite half of the brain.

The hypothalamus (10), about the size of a pearl, directs a multitude of important functions. It wakes you up in the morning, and gets the adrenaline flowing during a test or job interview. The hypothalamus is also an important emotional center, controlling the molecules that make you feel exhilarated, angry, or unhappy. Near the hypothalamus lies the thalamus (11), a major clearinghouse for information going to and from the spinal cord and the cerebrum.

An arching tract of nerve cells leads from the hypothalamus and the thalamus to the hippocampus (12). This tiny nub acts as a memory indexer—sending memories out to the appropriate part of the cerebral hemisphere for long-term storage and retrieving them when necessary. The basal ganglia (not shown) are clusters of nerve cells surrounding the thalamus. They are responsible for initiating and integrating movements. Parkinson’s disease, which results in tremors, rigidity, and a stiff, shuffling walk, is a disease of nerve cells that lead into the basal ganglia.

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Making Connections

The brain and the rest of the nervous system are composed of many different types of cells, but the primary functional unit is a cell called the neuron. All sensations, movements, thoughts, memories, and feelings are the result of signals that pass through neurons. Neurons consist of three parts. The cell body (13) contains the nucleus, where most of the molecules that the neuron needs to survive and function are manufactured. Dendrites (14) extend out from the cell body like the branches of a tree and receive messages from other nerve cells. Signals then pass from the dendrites through the cell body and may travel away from the cell body down an axon (15) to another neuron, a muscle cell, or cells in some other organ. The neuron is usually surrounded by many support cells. Some types of cells wrap around the axon to form an insulating sheath (16). This sheath can include a fatty molecule called myelin, which provides insulation for the axon and helps nerve signals travel faster and farther. Axons may be very short, such as those that carry signals from one cell in the cortex to another cell less than a hair’s width away. Or axons may be very long, such as those that carry messages from the brain all the way down the spinal cord.

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Scientists have learned a great deal about neurons by studying the synapse—the place where a signal passes from the neuron to another cell. When the signal reaches the end of the axon it stimulates the release of tiny sacs (17). These sacs release chemicals known as neurotransmitters (18) into the synapse (19). The neurotransmitters cross the synapse and attach to receptors (20) on the neighboring cell. These receptors can change the properties of the receiving cell. If the receiving cell is also a neuron, the signal can continue the transmission to the next cell.

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Some Key Neurotransmitters at Work

Acetylcholine is called an excitatory neurotransmitter because it generally makes cells more excitable. It governs muscle contractions and causes glands to secrete hormones. Alzheimer’s disease, which initially affects memory formation, is associated with a shortage of acetylcholine.

GABA (gamma-aminobutyric acid) is called an inhibitory neurotransmitter because it tends to make cells less excitable. It helps control muscle activity and is an important part of the visual system. Drugs that increase GABA levels in the brain are used to treat epileptic seizures and tremors in patients with Huntington’s disease.

Serotonin is a neurotransmitter that constricts blood vessels and brings on sleep. It is also involved in temperature regulation. Dopamine is an inhibitory neurotransmitter involved in mood and the control of complex movements. The loss of dopamine activity in some portions of the brain leads to the muscular rigidity of Parkinson’s disease. Many medications used to treat behavioral disorders work by modifying the action of dopamine in the brain.

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Neurological Disorders

When the brain is healthy it functions quickly and automatically. But when problems occur, the results can be devastating. Some 50 million people in this country—one in five—suffer from damage to the nervous system. The NINDS supports research on more than 600 neurological diseases. Some of the major types of disorders include: neurogenetic diseases (such as Huntington’s disease and muscular dystrophy), developmental disorders (such as cerebral palsy), degenerative diseases of adult life (such as Parkinson’s disease and Alzheimer’s disease), metabolic diseases (such as Gaucher’s disease), cerebrovascular diseases (such as stroke and vascular dementia), trauma (such as spinal cord and head injury), convulsive disorders (such as epilepsy), infectious diseases (such as AIDS dementia), and brain tumors.

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The National Institute of Neurological Disorders and Stroke

Since its creation by Congress in 1950, the NINDS has grown to become the leading supporter of neurological research in the United States. Most research funded by the NINDS is conducted by scientists in public and private institutions such as universities, medical schools, and hospitals. Government scientists also conduct a wide array of neurological research in the more than 20 laboratories and branches of the NINDS itself. This research ranges from studies on the structure and function of single brain cells to tests of new diagnostic tools and treatments for those with neurological disorders.

For information on other neurological disorders or research programs funded by the National Institute of Neurological Disorders and Stroke, contact the Institute's Brain Resources and Information Network (BRAIN) at:

BRAIN
P.O. Box 5801
Bethesda, MD 20824
(800) 352-9424
www.ninds.nih.gov Top

Prepared by:
Office of Communications and Public Liaison
National Institute of Neurological Disorders and Stroke
National Institutes of Health
Bethesda, MD 20892
 

The health benefits of nutmeg oil

The health benefits of nutmeg oil include its ability to treat stress, pain, menstrual cramps, heart disorders, indigestion, blood pressure, cough and bad breath. The health benefits of nutmeg oil can be attributed to its medicinal properties such as its role as a sedative, stimulant, relaxing, anti-inflammatory, antiseptic, and bactericidal substance.

Nutmeg has the Latin name Myristica fragrans. It is also known by other common names in various countries as mace, muscdier, magic, muskatbaum, noz moscada, myristica, nuez moscada and nux moschata.

The nutmeg tree grows very tall and may reach up to seventy feet in height. Nutmeg oil is obtained from the seed of the nutmeg tree fruit. The fruit, when dried, produces nutmeg (the seed) and mace (the covering). Traditionally, nutmeg was believed to be effective against the plague and hence it was popular during the Elizabethan era.

Health Benefits of Nutmeg Oil

The health benefits of nutmeg oil include the following:

Pain relief: Nutmeg oil is very useful for treating muscular and joint pain as it is an excellent sedative. Nutmeg oil is also anti-inflammatory, so massaging nutmeg oil on the affected area is an effective treatment for arthritis, rheumatism, and lumbago. Nutmeg oil is an essential part of Chinese medicine when it comes to treating abdominal pain and inflammation. It also reduces swelling of the joints. Often, overexertion leads to body or muscle ache, and in such cases, nutmeg oil can be very useful in removing the pain.

Menstrual Cramps:Some women face menstrual irregularities and suffer from menstrual cramps. Nutmeg oil is very helpful for these women, and it can also reduce the associated symptoms of periods like mood swings, depression, and hormone imbalance.

Indigestion: Nutmeg oil is good for digestion and helps in relieving stomach aches and removing gas from the stomach and intestines. Therefore, nutmeg oil is good for indigestion, flatulence, vomiting, and diarrhea. It also encourages an increase in appetite. Care should be taken when consuming nutmeg oil, and it should only be applied internally in low doses.

Blood Circulation: Nutmeg oil is a good stimulant, not only for the mind, but also for the rest of the body. Its relaxing aroma comforts the body, increases blood circulation and therefore helps those who have poor blood circulation.

Respiratory Problems: Nutmeg oil forms an important ingredient in many cough syrups and cold rubs as it helps in relieving congestion and cold symptoms. It is also believed that nutmeg oil can be used for treating asthma.

Brain tonic: Nutmeg oil stimulates the brain and therefore removes mental exhaustion and stress. It is also believed that nutmeg oil improves the quality of your dreams, making them more intense and colorful. It is a good remedy for anxiety as well as depression. Nutmeg oil is often used in homeopathy. In the ancient Greek and Roman civilizations, nutmeg was popular as an effective brain tonic in spite of its high cost and rarity. Nutmeg oil also enhances concentration and increases your overall efficiency at study and work.

Heart Problems: Nutmeg oil can also stimulate the cardiovascular system and is therefore considered a good tonic for the heart.

Bad Breath: The woody aroma of nutmeg oil helps to remove bad breadth. It is also antiseptic in nature and is effective for toothaches and aching gums. As a result, it is also added to numerous toothpastes and mouthwashes.

Liver Tonic: An important health benefit of nutmeg oil is its ability to treat liver disease. The oil is capable of removing toxins from the liver, thereby making it a good liver tonic.

Kidney Health: Nutmeg oil is often recommended for treating kidney infections and kidney diseases. It also helps in dissolving kidney stones and accumulations of uric acid in other parts of the body, like those which lead to gout and joint inflammation.

Other Benefits of Nutmeg and Nutmeg Oil

Spice: The herb is very popular as a spice and is often used in culinary purposes. In cooking, nutmeg is versatile and can be used in potato dishes and meat preparations in Europe, garam masala in India, and as a curry ingredient in Japan.

Incense: Nutmeg is used in various incense sticks due to its woody fragrance. It is also believed that Roman priests used to burn nutmeg as incense.

Flavor: Nutmeg, or Jaiphal as it is called in Hindi, is a very popular flavoring agent in making sweets in India. It is also used in baked goods, sauces, ice cream, and custards. Certain coffee drinks, including cappuccino, are flavored using nutmeg and cinnamon.

Soaps: The antiseptic properties of nutmeg make it useful in the manufacturing of antiseptic soaps. Nutmeg essential oil is used for bathing as well, due to its refreshing nature.

Cosmetics: Since nutmeg oil is antibacterial and antiseptic, it is used in many cosmetics meant for dull, oily or wrinkled skin. It is also used in making after shave lotions and creams.

Room Freshener: Nutmeg oil can be used as a room freshener, again due to its woody and pleasant aroma.

Tobacco: Nutmeg oil is commonly used in the tobacco industry to change the flavor of the tobacco blend slightly.

Blending: Nutmeg oil blends well with many other essential oils including lavender, rosemary, orange, black pepper, clary sage, eucalyptus, ginger, and ylang-ylang oils.

The potential melanogenic effect of compounds from Zingiber cassumunar Roxb

Editor: Devanand Sarkar, Virginia Commonwealth University, UNITED STATES

Received: July 8, 2015; Accepted: October 15, 2015; Published: November 4, 2015

This is an open access article.

Abstract 

We investigated the potential melanogenic effect of compounds from Zingiber cassumunar Roxb. Our data revealed that chloroform-soluble extract of Z. cassumunar enhanced mela- nin synthesis in B16F10 melanoma cells. Among the components of the chloroform extract, (E)-4-(3,4-dimethoxyphenyl)but-3-en-1-ol (DMPB) increased melanogenesis in both B16F10 cells and human primary melanocytes. In B16F10 cells, DMPB enhanced the acti- vation of ERK and p38, and the level of tyrosinase. Although the level of microphthalmia- associated transcription factor was unchanged in DMPB-treated B16F10 cells, DMPB increased levels and nuclear localization of upstream stimulating factor-1 (USF1). Consis- tently, DMPB-mediated melanin synthesis was diminished in USF1-knockdown cells. Fur- thermore, DMPB induced hyperpigmentation in brown guinea pigs in vivo. Together, these data suggest that DMPB may promote melanin synthesis via USF1 dependent fashion and could be used as a clinical therapeutic agent against hypopigmentation-associated diseases. 

Introduction 

Melanin, which is synthesized in the melanosomes of melanocytes, serves a number of valuable functions, such as determining the appearance of the skin and protecting it from the harmful effects of ultra violet (UV) radiation (and thus skin cancer), toxic drugs and chemicals [1]. Excessive melanin production occurs in melasma, lentigo, nevocellular nevi and malignant melanoma, whereas the loss of melanocyte function leads to vitiligo. Therefore, it is critical to appropriately control the balance of melanin synthesis in the skin. Melanin synthesis is controlled by various enzymes, such as tyrosinase, tyrosinase-related protein 1 (TRP-1) and tyrosinase-related protein 2 (TRP-2/DOPA, chrome tautomerase). Tyrosinase is a rate-limiting enzyme for melanin synthesis, where it is involved in two distinct reactions: it first catalyzes the hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine (DOPA), and then promotes the oxidation of DOPA to DOPA quinone [2], which undergoes several reactions to eventually form melanin. Therefore, many researchers have sought to control the expression or activation of tyrosinase. The transcription of melanogenic enzymes is regulated by microphthalmia-asso- ciated transcription factor (MITF) [3] and tyrosinase transcription is also regulated by USF (upstream stimulating factor)-1, which is member of the evolutionarily conserved basic-helix- loop-helix family of eukaryotic leucine zipper transcription factors [4]. In the tyrosinase pro- moter, the elements recognized by MITF are also targeted by USF1 [5]. In addition, an essential role of p53, a tumor suppressor protein, in the induction of UV-induced epidermal hyperpig- mentation via direct activation of POMC transcription in keratinocytes [6] and/or regulation of paracrine cytokine signaling, both in keratinocytes and melanocytes, has been reported [7]. 

Numerous studies have sought to identify the factors involved in controlling melanin synthesis. A number of natural products have been reported to inhibit melanogenesis by regulating melanogenic enzymes, including Hoelen extracts [8], sesamol (3,4-methylenedioxyphenol) [9]. In addition, Arthrophytum scoparium extract [10], Caffeoylserotonin [11] and the aqueous fraction from Cuscuta japonica [12] have been shown to inhibit melanogenesis by regulating MITF. These agents have all been used to develop anti-melanogenic agents for the treatment of hyperpigmentation disorders. Several studies have also identified plant extracts that have pro- melanogenic response, including the citrus flavonoid naringenin [13], kavalactones [14], cou- marin [15], and rosmarinic acid [16]. Naringenin upregulates MITF and tyrosinase through wnt/β-catenin pathway. Rosmarinic acid promotes expression of tyrosinase by activating PKA/ CREB pathway. They have been suggested as photo-protecting and pro-melanogenic agents. Therefore, finding a natural product that is capable of regulating melanin synthesis could contribute to treating melanin-dependent diseases. 

Zingiber cassumunar Roxb. (Zingiberaceae) is a tropical ginger that is widely distributed in Southeast Asia [17] and has been used as a traditional herbal medicine for gastrointestinal dis- tress and motion sickness [18]. In addition, two main constituents of Z. cassumunar, phenylbu- tenoids [19–22] and curcuminoids [23], have been reported to exhibit anti-inflammatory [24,25], anti-tumor [21,22] and antioxidant [23] activities. The inflammatory response is believed to be closely related to melanin synthesis [26,27], and Albert et al. have reported that inflammation of the uveal tract is associated with vitiligo [28]. Therefore, the inflammatory response appears to be a cause of hyperpigmentation diseases in human skin. Despite its effects on the inflammatory response, however, little is known about the effects of Z. cassumunar on melanogenesis. Therefore, we herein investigated the effects of Z. cassumunar on melanogenesis. 

Materials and Methods 

Materials and Antibodies 

The polyclonal antibody against tyrosinase and the monoclonal antibodies against phospho- ERK, ERK and β-actin were purchased from Santa Cruz (Santa Cruz, CA, USA). The poly- clonal antibodies against phospho-p38, p38 were purchased from Cell Signaling (Danvers, MA, USA) and the polyclonal antibody against MITF was purchased from Proteintech (Chi- cago, IL, USA). The monoclonal antibody against USF1 was purchased from AbCam (Cam- bridge, MA, USA). The α-MSH and L-DOPA were purchased from Sigma (St. Louis, MO, USA). PD98059 and SB239063 were obtained from Calbiochem (Darmstadt, Germany). 

Cell culture and transfection 

Mouse melanoma cell line B16F10 cells were obtained from ATCC and cultured in Dulbecco’s modified Eagle’s medium (DMEM; WelGene, Daegu, Korea) supplemented with 10% fetal bovine serum (FBS) with gentamicin (50 μg/ml, Sigma) at 37°C in a humidified 5% CO2 atmo- sphere. Primary human epidermal melanocytes were purchased from Lonza (Basel, Switzer- land) and maintained in Melanocyte Growth Medium-4 (Lonza), supplemented with 5% FBS, recombinant human-fibroblast growth factor B, rh-insulin, gentamicin sulfate amphotericin-B, calcium chloride, phorbol 12-myristate 13-acetate, bovine pituitary extract and hydrocortisone, at 37°C in a humidified 5% CO2 atmosphere. Transient transfections of HEK293T cells were carried out using the Vivamagic reagent (Vivagen, Gyeonggi-Do, Korea). Transient transfec- tions of siRNAs were carried out using the Lipofectamine 2000 reagent purchased from Invi- trogen (Carlsbad, CA, USA). 

RNA extraction and reverse transcription polymerase chain reaction (RT-PCR) 

Total RNA was extracted from cells and reverse transcribed, and aliquots of the resulting cDNA were amplified using the following primers: mouse tyrosinase (forward) 5'-CGAGC CTGTGCCTCCTCTAA-3' and (reverse) 5'-CCAGGACTCACGGTCATCCA-3'; mouse MITF (forward) 5’-GGAACAGCAACGAGCTAAGG-3’ and (reverse) 5’- TGATGATCCGATTCACC AGA-3’; and β-actin, (forward) 5'-TGGAATCCTGTGGCATCCATGAAA-3' and (reverse) 5'-TAAAACGCAGCTCAGTAACAGTCCG-3'. After an initial denaturation at 94°C for 5 min- utes, samples were subjected to 30 cycles of denaturation at 94°C for 30 seconds, annealing at 52°C for 30 seconds, and extension at 72°C for 60 seconds. 

RNA interference 

siRNA mediated down regulation of MITF and USF1 was achieved with the MITF specific sequence 5'-GGUGAAUCGGAUCAUCAAG-d(TT)-3' and 5'-CUUGAUGAUCCGAUUCA CC-d(TT)-3' and with the USF1 specific sequence 5'-UGGAAGAUCUCAAGAACAA-d (TT)-3' and 5'-UUGUUCUUGAGAUCUUCCA-d(TT)-3'. Scrambled siRNA (siGEN- OMEnontargetingsiRNA 2) were purchased from Dharmacon (Chicago, IL, USA) and used as a control. 

Western blot 

The cultures were washed twice with PBS and the cells were lysed in lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 10 mM NaF, and 2 mM Na3VO4) containing a prote- ase inhibitor mixture [1 μg/ml aprotinin, 1 μg/ml antipain, 5 μg/ml leupeptin, 1 μg/ml pepsta- tin A, and 20 μg/ml phenylmethylsulfonyl fluoride (PMSF)]. The total cell lysates were clarified by centrifugation at 13,000 Xg for 15 min at 4°C, denatured with SDS sample buffer, boiled, and analyzed by SDS-PAGE. Nuclear extract was isolated using Nuclear extraction kit (Abcam) and lysates were denatured with SDS sample. The resolved proteins were transferred to polyvinylidene difluoride membranes (Millipore; Billerica, MA, USA), probed with the appropriate antibodies, and detected by ECL (AbClon; Seoul, Korea). 

Quantification of melanin 

Melanin contents were measured as described in a previous study [29]. Cells were washed twice with PBS, detached by incubation with trypsin/EDTA, and collected by centrifugation at 1000 Xg for 3 minutes. Thereafter, 5 X105 cells were solubilized in 100 μl of 1 N NaOH-10% DMSO at 80°C for 2 hr. The dissolved melanin was assessed by absorbance at 405 nm, and the melanin content was determined using a standard curve generated with synthetic melanin (Sigma). 

Tyrosinase activity assays 

Active tyrosinase was analyzed as described in a previous study [30]. Cells were lysed in 50 mM sodium phosphate buffer (pH 6.8) containing 1% Triton X-100, 1 μM PMSF, 1 μg/ml aprotinin, and 10 μg/ml leupeptin. The lysates were clarified by centrifugation at 13,000 Xg for 15 min at 4°C. Clarified lysates were reacted with 5mM L-DOPA at 37°C for 2 hr, and tyrosi- nase activity was determined by measuring the absorbance at 470 nm. For analyzing intracellu- lar tyrosinase activity, cells were plated to coverslips in 12-well plates, fixed with 4% paraformaldehyde for 20 min, washed with PBS, and incubated in sodium phosphate buffer with 10 mM L-DOPA for 3 hr at 37°C. The cells were then washed with PBS and the coverslips were mounted on glass slides. 

Immunofluorescence analysis 

B16F10 were plated to 12-well plates containing coverslips, and treated with DMPB (30 μM) for 3 hr. Cells were fixed with 3.5% paraformaldehyde and permeablized with 0.5% triton X- 100 in PBS. The cells were then washed with PBS, blocked with 0.5% BSA and incubated over- night with the anti-USF1 antibody at 4°C. After a further wash with PBS, the cells were incu- bated with Texasred conjugated goat anti-rabbit antibody (Invitrogen, Carlsbad, CA, USA) for 1 hr at 25°C. The coverslips were mounted on glass slides with mounting solution containing 4',6-diamidino-2-phenylindole (DAPI), and the results were observed by fluorescence microscopy (Carl Zeiss, Oberkochen, Germany). 

Cell proliferation assay 

Cell proliferation was measured using the MTT [3-(4,5-dimethythiazol-2-yl) 2,5-diphenylte- trazolium bromide] assay. In brief, B16F10 cells were harvested with 0.05% trypsin/EDTA and seeded to 96-well plates at 5X103 cells/well. After incubation, medium containing 0.5 mg/ml MTT (100 μl; Sigma) was added to each well, and the cells were incubated for 1 hr. The medium was then removed and 100 μl of acidic isopropanol (90% isopropanol, 0.5% SDS, 25 mM NaCl) was added to each well. The mean concentration of absorbance at 570 nm in each sample set was measured using a 96-well microtiter plate reader (Dynatech; Chantilly, VA, USA). 

Guinea pig model experiments 

This study was performed in compliance with the Principles of Laboratory Animal Care and was approved by the Institutional Animal Care and Use Committee (IACUC) of the Asan Institute for Life Sciences, Asan Medical Center (Seoul, Korea). Brownish Kwl:A1 guinea pigs were purchased from Central Lab Animal (Seoul, Korea), anesthetized weekly with a mixture (1:4) of xylazine (Rompun; Bayer Korea, Korea) and Zoletil (Zoletil 50; Virbac, France) given intramuscularly, and shaved. The dorsal skin was separated into three areas (1 cm x 1 cm each), and 100 μM of DMPB, 350 μM of DMPB, or DMSO alone (50 μl each) was topically applied to the center of the allocated area 12 times over 3 weeks. On day 35, skin specimens were obtained by 5-mm punch biopsies, embedded in Tissue-Tek OCT compound (Sakura Fine Technical; Tokyo, Japan), and quickly frozen. The frozen specimens were cut to 10-μm thickness and fixed in ice cold acetone for 10 min. Melanin pigment was visualized with stan- dard Fontana—Masson staining. Image analysis was performed on a representative area of three randomly selected fields using the ImageJ program (http://rsb.info.gov/ij/). For melanocyte counting, sections were stained with Hematoxylin and Eosin, and random fields were examined under a microscope (x400). Dermatologists counted the melanocytes within four dif- ferent fields from a 0.5-mm length of epidermis. All animals were sacrificed with CO2 inhala- tion after study. 

Statistical analysis 

Data are presented as the means from three independent experiments. Statistical analyses were performed using the unpaired Student’s t test. A p-value less than 0.01 or 0.05 was considered statistically significant. 

Results 

(E)-4-(3,4-Dimethoxyphenyl)but-3-en-1-ol from Z. cassumunar enhances melanin synthesis 

The methanol extract of Z. cassumunar was partitioned with hexanes, chloroform, and butanol, subsequently, as described previously [20] and then compared the melanin contents of B16F10 mouse melanoma cells in the presence or absence of these extracts (20 μg/ml) for 48 hr. We found that chloroform extract, but not hexane or butanol extracts, enhanced melanin synthesis in B16F10 mouse melanoma cells (Fig 1A). From the chloroform fraction, we isolated three com- pounds [20]: (E)-4-(3,4-dimethoxyphenyl)but-3-en-1-ol (DMPB), (E)-4-(3,4-dimethoxyphenyl) but-1,3-diene (DMPBD) and (E)-4-(3,4-dimethoxyphenyl)but-3-en-1-yl acetate (DMPBA). As shown in Fig 1B, we repeated the above assay to determine which compound affected melano- genesis, and found that only DMPB increased melanin synthesis in B16F10 cells (Fig 1B), show- ing a dose-dependent effect that was maximum among cells treated with 30 μM (Fig 1C) for 48 hr (Fig 1D). These effects were found to be comparable (though slightly lower) than those of α- melanocyte stimulating hormone (α-MSH) (Fig 1E). Together, these data suggest that DMPB extracted from Z. cassumunar enhances melanin synthesis in B16F10 melanoma cells. 

DMPB enhances tyrosinase expression but not tyrosinase activity 

Since DMPB increased the levels of melanin in our system, we next investigated whether it could affect the expression of tyrosinase, which plays a critical role in melanogenesis. As shown in Fig 2A, Western blotting and RT-PCR analyses revealed that the expression levels of tyrosi- nase were significantly up-regulated in B16F10 cells treated with 30 μM of DMPB for 48 hr. Similarly, tyrosinase activity showed that total tyrosinase activity increased in response to DMPB treatment (Fig 2A). L-DOPA staining showed that the level of intracellular tyrosinase activity was increased in DMPB-treated B16F10 cells (Fig 2B), confirming the abovementioned increase in total tyrosinase activity among DMPB-treated cells. However, when we adjusted the amount of tyrosinase in the reaction mixture, the tyrosinase activity was comparable in B16F10 cells with and without DMPB treatment (Fig 2C). This suggests that the increase of tyrosinase 

Fig 1. (E)-4-(3,4-Dimethoxyphenyl)but-3-en-1-ol from Z. cassumunar enhances melanin synthesis. (A) The methanol extract of Z. cassumunar was partitioned with hexanes, chloroform, and butanol (top panel). B16F10 cells were treated with three fractions of Z. cassumunar (BF: Butanol fraction, CF: Chloroform fraction, HF: Hexane fraction; 20 μg/ml, 48hr). The melanin contents were analyzed by measuring the absorbance at 405 nm (bottom panel). DMSO was used as a control. The mean percentages of melanin content are shown. **, p < 0.01 versus DMSO treated cells. (B) B16F10 cells were treated with the indicated compounds extracted from Z. cassumunar (30 μM each) for 48 hr, and the melanin contents were determined. *, p < 0.05 versus DMSO treated cells. (C,D) B16F10 cells were treated with either various concentrations of DMPB for 48 hr (C) or with 30 μM of DMPB for the indicated times (D), and the mean percentages of melanin content are shown. (E) B16F10 cells were treated with of 30 μM of DMPB or 1 μM of α-MSH for 48 hr. The mean percentages of melanin content are shown. **, p < 0.01 versus DMSO treated cells. 

activity in DMPB-treated B16F10 cells was due to increased tyrosinase levels rather than increased tyrosinase activity. Since increased cell numbers might affect the total tyrosinase activity, we used a colorimetric assay to investigate whether DMPB affected the proliferation of B16F10 cells, but found that cell number increased similarly over time in the presence or absence of DMPB (Fig 2D), suggesting that this agent does not affect the proliferation of B16F10 cells. Together, these findings support our contention that DMPB increases melanin synthesis by up-regulating tyrosinase expression. 

MAP kinases are involved in DMPB-mediated melanogenic control 

The mitogen-activated protein kinase (MAPK) signaling pathway is known to be involved in regulating melanin synthesis by modulating the expression of tyrosinase. UV irradiation and Fig 2. DMPB increases tyrosinase expression but not tyrosinase activity. (A) B16F10 cells were treated with 30 μM of DMPB for 48 hr, and mRNA level of tyrosinase was analyzed by RT-PCR (top panel). Total cell lysate was extracted and tyrosinase levels were measured by Western blot analysis. The relative density of tyrosinase(TYR) was quantitated using Image Studio software (middle panel). The mean percentages of tyrosinase density ± SD are shown *, p < 0.05 versus DMSO treated cells. DMPB-treated B16F10 cells (30 μM, 48 hr) were lysed. Cell lysates (100 μg) were reacted with L-DOPA at 37°C for 2 hr, and tyrosinase activity was determined at 470 nm (bottom panel). The mean percentages of tyrosinase activity ± SD are shown **, p < 0.01 versus DMSO treated cells. (B) DMPB-treated B16F10 cells (48 hr) were reacted with L-DOPA at 37°C for 30 min. Bright-field microscopic images are shown. Scale bars = 50 μm. (C) Cell lysates (20 μg and 40 μg) from B16F10 cells treated with the indicated concentrations of DMPB were subjected to Western blot analysis using an anti-tyrosinase antibody (top panel) or reacted with L-DOPA at 37°C for 2 hr to determine tyrosinase activity (bottom panel). The mean percentages of tyrosinase activity ± SD are shown. (D) B16F10 cells were incubated with various concentrations of DMPB for the indicated time periods, and cell viability was determined by MTT assay. Percentage values were compared between treated and untreated (control). Data are expressed as mean ± SD for three independent experiments. 

α-MSH have been shown to activate p38 MAPK, subsequently up-regulating the expression of tyrosinase [4,12,31]. In addition, ERK activation phosphorylates cAMP response element bind- ing protein (CREB), which binds to the CRE consensus motif in the MITF promoter to up-reg- ulate MITF gene expression [32]. Therefore, we next investigated whether DMPB affects the MAPK signaling cascade. In particular, we compared the activity levels of ERK and p38 using Western blotting with phospho-specific antibodies. At 3 hr after treatment, compared with untreated control cells, DMPB-treated cells increased the activity of ERK and p38 kinase (Fig 3A) but not Jnk (data not shown), suggesting that DMPB-mediated melanogenesis is closely related to increases in MAPK activity. Consistently, the activity of ERK and p38 kinase remained increased for 48 hr (Fig 3B). When B16F10 cells were pretreated with PD98059 and U0126 (a specific inhibitor of MEK), we observed decreases in various DMPB-induced effects, including ERK phosphorylation, tyrosinase expression and melanin synthesis (Fig 3C). Simi- larly, SB239063 and SB203580 (a specific inhibitor of p38) alleviated DMPB-induced p38 phos- phorylation, tyrosinase expression and melanin synthesis in B16F10 cells (Fig 3D). These findings indicate that both ERK and p38 seem to be involved in DMPB-mediated melanogenic control. 

 

 DMPB enhances melanogenisis is an upstream stimulating factor- 1-dependent fashion 

It has been reported that tyrosinase gene transcription is regulated by several transcription fac- tors including MITF, p53 and USF1 [4,33,34]. MITF is a basic helix—loop—helix leucine zip- per (bHLH-LZ) transcription factor that binds the tyrosinase gene promoter region to activate tyrosinase gene expression [3]. Therefore, we speculated that MITF might be involved in DMPB-mediated melanogenic regulation. We used Western blotting to analyze the levels of MITF in B16F10 cells treated with DMPB. However, DMPB did not affect MITF expression (Data not shown) and nuclear translocation of MITF (Fig 4A), suggesting that DMPB does not affect MITF expression. To further investigate the potential involvement of MITF in the regula- tion of DMPB-mediated melanin synthesis, we used unique siRNA sequences targeted against MITF to knock down the expression levels of MITF. B16F10 transfected with the siRNA 

Fig 3. MAP kinases are involved in DMPB-mediated melanogenic control. (A) B16F10 cells were treated with 30 μM of DMPB for the indicated time periods, and the phosphorylation of p38 and ERK and levels of tyrosinase were analyzed by Western blot analysis. (B) B16F10 cells were treated with the indicated amounts of DMPB for 48 hr, and the phosphorylation of p38 and ERK and levels of tyrosinase were analyzed by Western blot analysis. (C) B16F10 cells were preincubated with (+) or without (-) the inhibitor (1 μM of PD98059, 10 μM of U0126) for 1 hr, then treated with 30 μM of DMPB for 48 hr, and Western blot analysis was performed with the indicated antibodies (top panel). The melanin contents were analyzed by measuring the absorbance at 405 nm (bottom panel). The mean percentages of melanin content are shown *, p < 0.05 versus DMSO treated cells. (D) B16F10 cells were preincubated with (+) or without (-) p38 inhibitors (5 μM of SB239063 for 30 min, 10 μM of SB203580 for 1 hr) and treated with 30 μM of DMPB for 48 hr, and Western blot analysis was performed with the indicated antibodies (top panel). The melanin contents were analyzed by measuring the absorbance at 405 nm (bottom panel). The mean percentages of melanin content are shown *, p < 0.05 versus DMSO treated Fig 6. DMPB enhances hyperpigmentation in brown guinea pigs. (A) The dorsal skins of guinea pigs were topically treated with DMSO (control), 100 μM DMPB, or 350 μM DMPB (50 μl each) 12 times in 3 weeks. On day 35 (after the first treatment), skin specimens were obtained by a 5-mm punch biopsy. (B) Frozen skin specimens were cut at 10- μM thickness, and melanin pigment was visualized by Fontana-Masson staining. Original magnification, X200. (C) Bar graph showing the mean percentage of melanin ± SD in the epidermis. Fontana-Masson-stained melanin in three randomly selected fields was measured with the ImageJ program. *, p < 0.05 versus DMSO treated skins. 

 

MAPK pathway results in the phosphorylation of MITF on serine 307 and the subsequent upregulation of MITF target genes in osteoclasts [43]. In parallel with increased melanin synthesis, we found that the phosphorylation levels of ERK and p38, but not Jnk, were significantly enhanced after DMPB treatment, suggesting that the DMPB-mediated melanogenic effects may occur via MAPK-mediated pathways (Fig 3A and 3B). Consistent with this notion, the MAPK-specific inhibitors, PD98059, U0126, SB239063 and SB203580, reduced the DMPB- mediated increase of melanin synthesis (Fig 3C and 3D). However, DMPB did not affect the level of MITF (Fig 4A and 4D). Therefore, DMPB seems to regulate melanin synthesis by altering tyrosinase expression in an MITF-independent manner. 

Another bHLH-LZ transcription factor, USF1 is regulated by various signaling pathways, including ERK1/2 and p38 MAPK. In HepG2 cells, activation of ERK mediated by HGF phos- phorylates of USF1 [44]. In response to UV stress, activation of p38 MAPK/USF1 appears essential for protecting skin through enhancement of melanogenesis [45]. Both ERK1/2 and p38 MAPKs phosphorylate at Ser153 site of USF1 [46]. USF1 stimulates melanin synthesis as a  transcription factor of tyrosinase, MC1R and POMC [4, 45], and maintains genomic stability via its involvement in DNA repair [47]. Therefore, it is highly possible that USF1 plays a role in DMPB-mediated melanogenesis. Our data clearly showed that USF1 regulates DMPB-medi- ated melanogenesis as a transcription factor of tyrosinase (Fig 4). DMPB enhanced the level of USF1 (Figs 4A and 5E) and stimulated its translocation to the nucleus (Fig 4A and 4E). In addi- tion, siRNA knockdown of USF1, but not MITF (Fig 4C), led to significant inhibition of tyrosi- nase-mediated melanogenesis by DMPB (Figs 4F and 5F). 

Based on these results, we propose that DMPB promotes melanin synthesis through increasing USF1-mediated tyrosinase expression. 

In this study, we found new pro-melanogenic agent, DMPB. This DMPB clearly enhances melanin synthesis by increasing tyrosinase expression, as shown in cultured melanocyte- derived cells in vitro and in guinea pig skin in vivo. These results provide early evidence that DMPB could act as a pigmenting agent in vivo, and might be useful for treating hypopigmenta- tion-related disorders. Further studies are needed to directly examine the effect of DMPB on skin pigmentation under physiologically relevant conditions. 

Acknowledgments 

National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP), grant number:2012R1A5A1048236 (URL:www.nrf.re.kr); the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea, grant number:HI12C0050 (URL: http://www.mw.go.kr); and the Ministry of Science, ICT and Future Planning (NRF2012M3A9C4048761). The funders had roles in study design, data collection and analy- sis, decision to publish, or preparation of the manuscript. 

Author Contributions 

Conceived and designed the experiments: JP HC SHB ARH EKS SEC DHK ESO. Performed the experiments: JP HC SHB SEC ESO. Analyzed the data: JP HC SHB ARH EKS SEC ESO. Contributed reagents/materials/analysis tools: JP HC SHB ARH EKS SEC ESO. Wrote the paper: JP HC ESO. 

Zingiber cassumunar blended patches for skin application: Formulation, physicochemical properties, and in vitro studies

Asian Journal of Pharmaceutical Sciences

July 2015, Vol.10(4)

* Jirapornchai SuksaereeLaksana CharoenchaiFameera MadakaChaowalit MontonApirak SakunpakTossaton CharoonratanaWiwat Pichayakorn

Abstract

Our work was to study the preparation, physicochemical characterization, and in vitro characteristic of Zingiber cassumunar blended patches. The Z. cassumunar blended patches incorporating Z. cassumunar Roxb. also known as Plai were prepared from chitosan and polyvinyl alcohol with glycerin as plasticizer. They were prepared by adding all ingredients in a beaker and homogeneously mixing them. Then, they were transferred into Petri-dish and dried in hot air oven. The hydrophilic nature of the Z. cassumunar blended patches was confirmed by the moisture uptake, swelling ratio, erosion, and porosity values. The FTIR, DSC, XRD, and SEM studies showed revealed blended patches with amorphous region that was homogeneously smooth and compact in both surface and cross section dimensions. They exhibited controlled the release behavior of (E)-4-(3′,4′-dimethoxyphenyl) but-3-en-l-ol (compound D) that is the main active compound in Z. cassumunar for anti-inflammation activity. However, in in vitro skin permeation study, the compound D was accumulated in newborn pig skin more than in the receptor medium. Thus, the blended patches showed the suitable entrapment and controlled release of compound D. Accordingly, we have demonstrated that such chitosan and polyvinyl alcohol formulated patches might be developed for medical use.

1. Introduction

Topical and transdermal drug delivery systems are intended for external use. They are often dermatologic products such as sunscreens, local anesthetics, antiseptics and anti-inflammatory agents intended for localized action on one or more layers of the skin. Conversely, some transdermal drug delivery systems are designed for percutaneous route of drug delivery in which case skin is not the target. In such case, the drug must be absorbed across the skin which is made up of dermis and epidermis, especially the stratum corneum barrier including sweat glands, sebaceous glands, and hair follicles [1], and pass into deeper dermal layers to reach the systemic blood circulation. Generally, substances intended for transdermal delivery systems are low molecular weight (100-500 Da), potent non-irritation and non-allergenic [2], [3] and [4]. The delivery system can be categorized as either i) drug in adhesive or ii) drug in matrix systems. The drug is dispersed or dissolved in a polymer matrix and attached to an adhesive layer that contacts the skin. In some cases, the polymer matrix can act as the adhesive layer. Polymer matrix layers and/or the added adhesive layer act as a control of the rate of delivery [5] and [6].

Thai traditional medicines (herbal medicines) are popular for the treatment of various symptoms and diseases and to promote good health. Although the Western modern medicines are increasingly popular, Thai traditional medicines are still widely used especially among the rural Thais. Herbal medicines may contain variations of active ingredients parts of plants, other plant materials, or combinations that included herbs, herbal materials, herbal preparations, and finished herbal products. Zingiber cassumunar Roxb., also known as Thai name “Plai”, is a medicinal plant widely cultivated in Thailand and tropical Asia. It is frequently used as an ingredient in marketed phytomedicines [7] and [8]. The rhizome of Z. cassumunar Roxb. has an anti-inflammatory activity. It has been the source of Thai traditional herbal remedies and extracts for topical application to alleviate inflammation [9], [10] and [11]. The chemical composition of the rhizome oils of Z. cassumunar Roxb. has been previously reported [7], [10], [12], [13], [14], [15] and [16]. The major constituents of the crude oils are terpinen-4-ol, α- and β-pinene, sabinene, myrcene, α- and γ-terpinene, limonene, terpinolene, sabmene, and monoterpenes [12] and [17]. (E)-4-(3′,4′-dimethoxyphenyl) but-3-en-l-ol (compound D) is the main active compound in Z. cassumunar that exhibits anti-inflammatory [11], [15] and [18], analgesic and antipyrectic [11], [15] and [16] activity in experimental models. It is also used as topical treatment for sprains, contusions, joint inflammations, muscular pain, abscesses, and similar inflammation-related disorders [19], [20] and [21]. Thus, this work used the compound D as the marker compound for in vitro study.

Herbal patches are adhesive patches that incorporate the herbal medicines or extracted herb. When applied to the skin the active compound is released at a constant rate. Such patches are recommended for smoking cessation, herbal body detox foot patch, relief of stress, to increase sexuality, as insect repellants, as male energizer, to improve sleep, to postpone menopause, for rheumatoid arthritis, as herbal plasters patches, etc. [22].

The aim of the current study was to prepare a Z. cassumunar containing product incorporating the crude Z. cassumunar oil in blended patches that consisted of chitosan and polyvinyl alcohol (PVA) polymer matrix combination using glycerin as plasticizer. Similarly prepared blended patches without crude Z. cassumunar oil served as control. The patches were evaluated with regard to the physicochemical properties as moisture uptake, swelling ratio, erosion, porosity, Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), X-ray diffraction (XRD), scanning electron microscope (SEM), and in vitro release and skin permeation studies.

2. Materials and methods

2.1 Materials

The Z. cassumunar rhizome powder was purchased from Charoensuk Osod, Thailand. The Z. cassumunar powder was extracted in 95% ethanol and filtered through a 0.45 μm of polyamide membrane to obtain crude Z. cassumunar oil. Chitosan (degree of deacetylation = 85%, mesh size 30) was purchased from Seafresh Industry Public Co., Ltd, Thailand. PVA and glycerin were purchased from Sigma–Aldrich, USA. All organic solvents were analytical grade obtained from Merck KGaA, Germany.

2.2 Analytical method

An Agilent 1260 Infinity system (Agilent Technologies, USA.) was used for this experiment with detection at 260 nm, a 4.6 mm × 250 mm diameter, 5 μm particle size C18 column (ACE 5, DV12-7219, USA.), a flow rate of 1 ml/min, and injection volume of 10 μL. The mobile phase was a gradient elution of 2% acetic acid in ultrapure water (A) and methanol (B) of 60 to 50% of A, 50 to 30% of A, 30 to 20% of A, 20 to 50% of A, 50 to 60% of A, and 60% of A for 0–5 min, 5–15 min, 15–25 min, 25–30 min, 30–32 min, and 32–40 min, respectively [23]. The HPLC validation method of compound D provided a limit of detection of 0.20 μg/ml, the limit of quantification of 0.80 μg/ml, good accuracy (95.38–104.76%), precision (less than 2%CV), and linearity with good correlation coefficient (r2) > 0.9999 in the required concentration range of 2–40 μg/ml. The separation method and validation method of compound D from crude Z. cassumunar oil was described in previous publication [23] and [24].

2.3 Z. cassumunar blended patches preparation

The chitosan was dissolved in 1% acetic acid in distilled water in concentration of 3.5%w/v. The PVA was dissolved in distilled water in concentration of 20%w/v. The blank blended patches were prepared by 2 g of 3.5%w/v chitosan were mixed together with 5 g of 20%w/v of PVA, and homogeneously mixed with 2 g of glycerin as plasticizer to obtain clear polymer blended solution. The Z. cassumunar blended patches were prepared as 3 g of the crude Z. cassumunar oil completely dissolved in absolute ethanol and continuously mixed in polymer blend solution. They were transferred into Petri-dish and dried in hot air oven at 70 ± 2 °C for 5 h. Finally, they were peeled from Petri-dish and kept in desiccator until used.

2.4 Evaluation of blank and Z. cassumunar blended patches

2.4.1 SEM photography

The surface and cross section of blank blended patches and Z. cassumunar blended patches were placed onto copper stub and then coated with gold in a sputter coater. They were photographed under SEM equipment (model: Quanta 400, FEI, Czech Republic) with high vacuum and high voltage of 20 kV condition, with Everhart Thornley detector (ETD).

2.4.2 FTIR study

The FTIR study employed the Attenuated Total Reflectance – FTIR (ATR-FTIR) technique for the chitosan film, PVA film, crude Z. cassumunar oil, blank blended patches, and Z. cassumunar blended patches. They were scanned at a resolution of 4 cm−1 with 16 scans over a wavenumber region of 400 – 4000 cm−1. The FTIR spectrometer (model: Nicolet 6700, DLaTGS detector, Thermo Scientific, USA.) was used to determine IR transmission spectra and record the characteristic peaks.

2.4.3 DSC study

A DSC instrument (model: DSC7, Perkin Elmer, USA) was used to investigate the endothermic transition of the substances that also confirmed the compatibility of each ingredient. The 1 – 10 mg of sample was weighted in DSC pan, hermetically sealed, and run in the DSC instrument at the heating rate of 10 °C/min under a liquid nitrogen atmosphere from 20 °C to 350 °C.

2.4.4 XRD study

The XRD (model: X'Pert MPD, PHILIPS, Netherlands) was employed to study the compatibility of the chitosan, PVA, blank blended patches, and Z. cassumunar blended patches. The generator operating voltage and current of X-ray source were 40 kV and 45 mA, respectively, with an angular of 5 – 40° (2θ), and a stepped angle of 0.02° (2θ)/s.

2.4.5 Moisture uptake, swelling ratio, and erosion studies

For determination of moisture uptake, swelling ratio and erosion, 1 cm × 1 cm patch specimens were used. For moisture uptake determination, the patch specimens were weighed for their initial value (W0), then moved to a stability chamber (model: Climate Chamber ICH/ICH L, Memmert GmbH + Co. KG, Germany) which controlled the temperature at 25 ± 2 °C and 75% relative humidity environment. The specimens were removed and weighed until constant (Wu). The percentage of moisture uptake was calculated by Equation (1) [25]

equation

1

The swelling ratio and erosion study were also determined by drying patch specimens at 60 ± 2 °C overnight. Then, they were weighed (W0) and immersed in 5 ml of distilled water and moved to stability chamber (model: Climate Chamber ICH/ICH L, Memmert GmbH + Co. KG, Germany) which controlled the temperature at 25 ± 2 °C and 75% relative humidity environment for 48 h. After removal of excess water, the hydrated patches were weighed (Ws). They were then dried again at 60 ± 2 °C overnight, and weighed again (Wd). The percentage of swelling ratio and the percentage of erosion were calculated by (2) and (3), respectively.

equation

2

equation

3

2.4.6 Porosity determination

After the patch specimens were equilibrated in water, the volume occupied by the water and the volume of the membrane in the wet state were determined. The porosity of patch specimens was obtained by Equation (4).

equation

4

where W1 and W2 = the weights of the membranes in the wet and dry states (g), respectively, dwater = the density of pure water at 20 °C, and w, l, t = the width (cm), length (cm), and thickness (cm) of the membrane in the wet state, respectively [26] and [27].

2.5 The determination of compound D in patches

The blended patches were cut into 2 cm × 2 cm specimens from different sites. Each Plai patch sample was soaked with ethanol in 10 ml volumetric flask, and sonicated at 25 °C for 30 min. Then, the solution was sampled for 0.5 ml and transferred into 100 ml volumetric flask and adjusted to volume of 100 ml with ethanol. The solution was filtered through a 0.45 μm filter and analyzed with HPLC method.

2.6 In vitro release study of compound D

The modified Franz-type diffusion cell having effective diffusion area of 1.77 cm2 was used for in vitro release and skin permeation study of compound D from the Z. cassumunar blended patches. The receptor medium was 12 ml of isotonic phosphate buffer solution pH 7.4: ethanol = 80:20, thermoregulated with a water jacket at 37 ± 0.5 °C and stirred constantly at 600 rpm with a magnetic stirrer. The crude Z. cassumunar oil was applied on the cellulose membrane (MWCO: 3500 Da, CelluSep® T4, Membrane Filtration Product, Inc., USA) which was used as a barrier between the donor compartment and the receptor compartment. The Z. cassumunar blended patch preparations were cut and placed directly on the donor cells. The 1 ml of receptor solution was withdrawn at 0, 0.5, 1, 2, 3, 4, 6, and 24 h intervals, and immediately replaced with an equal volume of fresh receptor medium. The compound D content in these samples was determined by an HPLC method.

2.7 In vitro skin permeation study of compound D

The in vitro skin permeation of the compound D from the Z. cassumunar blended patches was also carried out using a modified Franz-type diffusion cell [28], and pig skin with hair removed was an applied partitioning membrane [29] and [30]. The newborn pigs of 1.4–1.8 kg weight that had died by natural causes shortly after birth were freshly purchased from a local pig farm in Chachoengsao Province, Thailand. The full thickness of flank pig skin was excised, hair was surgically removed, and the subcutaneous fat and other extraneous tissues were trimmed with a scalpel, cleaned with isotonic phosphate buffer solution pH 7.4, blotted dry, wrapped with aluminum foil and stored frozen. Before permeation experiments, this isolated skin was soaked overnight in isotonic phosphate buffer solution pH 7.4, and mounted on the modified Franz-type diffusion cell with the stratum corneum facing upward on the donor compartment. The crude Z. cassumunar oil and Z. cassumunar blended patches were laid onto the isolated skin in the same way as for the release study. The receptor compartment was 12 ml of isotonic phosphate buffer solution pH 7.4: ethanol = 80:20 and stirred constantly at 600 rpm by a magnetic stirrer, at a constant temperature of 37 ± 0.5 °C. A 1 ml of the receptor solution was withdrawn at 0, 0.5, 1, 2, 3, 4, 6, and 24 h intervals and an equal volume of fresh receptor medium was immediately replaced. The compound D content in these samples was determined by the HPLC method.

All in vitro release and skin permeation studies were performed in triplicate and the means of all measurements calculated. The results were presented in terms of cumulative percentage release or skin permeation as a function of time using the following formula:

equation

5

where Dt was the amount of compound D released or permeated from the Z. cassumunar blended patches at time t and Dl was the amount of compound D loaded into the Z. cassumunar blended patches.

3. Results and discussion

3.1 Evaluation of blank and Z. cassumunar blended patches

Generally, the Z. cassumunar rhizomes were of deep yellow color possessing a strong camphoraceous smell, warm, spicy, and bitter taste [12], [17] and [31]. The extraction of the Z. cassumunar rhizome powder yielded a clear, high viscosity, yellow-orange crude Z. cassumunar oil. The solvent extraction of plant materials likely produced oleoresin, which contained not only the volatile compounds but also waxes and color pigments [32]. In addition, Sukatta et al. 2009 reported two pathways for Z. cassumunar rhizome extraction-hydro distillation and hexane extraction. They confirmed that hydro distillation produced the yellowish, low viscosity crude Z. cassumunar oil, while the crude Z. cassumunar oil from the hexane extraction was yellow-orange in color and had high viscosity. Commonly, our work could confirm from its appearance that crude Z. cassumunar oil was obtained. Therefore, when crude Z. cassumunar oil was added in blank blended patches, it produced the dark yellow patches referred to as Z. cassumunar blended patches. The photographs of blank blended and Z. cassumunarblended patches were shown in previous reports by our research group [33].

The SEM technique was used to photograph the high resolution morphology of the surface and cross section of blank blended patches and Z. cassumunar blended patches (Fig. 1). The surface of blank blended patches was homogeneously smooth and dense with no visual pores (Fig. 1A). The surface of Z. cassumunar blended patches became rough and uneven as a result of widely distributed conglomeration and aggregation in the matrix of Z. cassumunar blended patches (photographed by digital camera and presented in previous publication [33]) (Fig. 1B).

Fig. 1. Surface (×500 (A), ×1000 (B), and ×1500 (C)) and cross section morphology (×1000 (D), ×1500 (E), and ×5000 (F)) of blank blended patches (upper) and Z. cassumunar blended patches (bottom) under SEM technique.

Recorded spectra are shown in Fig. 2. For the chitosan film, the absorption peaks of stretching vibrations of –OH groups broadly overlapped the stretching vibration of N–H ranging from 3750 to 3000 cm−1. The broad stretching vibrations of C–H bond were observed at 2920–2875 cm−1. The bending vibrations of methylene and methyl groups were also absorbed at 1375 cm−1 and 1426 cm−1, respectively. The spectrum bands in the range of 1680–1480 cm−1 were identified as vibrations of carbonyl bonds of the amide group and vibrations of protonated amine group. The vibrations of CO group occurred in the range from 1160 cm−1–1000 cm−1. In addition, the spectrum band located at around 1150 cm−1 related to asymmetric vibrations of CO in the oxygen bridge resulting from deacetylation of chitosan. The spectrum bands at 1080–1025 cm−1 were attributed to –CO of the ring COH, COC, and CH2OH. Finally, the small spectrum peak at ∼890 cm−1 corresponded to wagging of the saccharide structure of chitosan [34]. Furthermore, the spectrum of acetic acid were found at 3050, 1720, and 1432 related to–OH bond in carboxylic acid, C–O bond, and C–O bond, respectively. In addition, the PVA spectrum showed both O–H stretching and C–O stretching at 3449 and 1637 cm−1, respectively [35] and [36].

Fig. 2. FTIR spectra of chitosan, PVA, blank blended patches, and Z. cassumunar blended patches.

The chitosan film, blank blended, and Z. cassumunar blended patches weighed 1.662, 8.747, and 8.821 mg, respectively. They were run with DSC instrument to study the thermal behavior. The thermogram of chitosan film, blank blended, and Z. cassumunar blended patches showed an initial broad peak at 70.33 °C with 231.35 J/g of enthalpy of peak (ΔH), 99.34 °C with 188.307 J/g of ΔH, and 92.37 °C with 78.76 J/g of ΔH, respectively, which was attributed to evaporation of moisture and represented the required energy to vaporize water present in their samples. Moreover, the degradation DSC peak of chitosan film broadly occurred at 323.67 °C with 127.30 J/g of ΔH. In addition, the blank blended patches and Z. cassumunar blended patches revealed high broad endothermic peaks at 257.00 °C with 363.24 J/g of ΔH and 261.00 °C with 606.41 J/g of ΔH, respectively. Although the observed endothermic peaks in blank blended patches and Z. cassumunar blended patches were slightly changed, there were no new exo- or endo-thermic peaks in any experimental ranges indicating compatibility of all ingredients (Fig. 3).

Fig. 3. DSC thermograms of chitosan, blank blended patches, and Z. cassumunar blended patches.

The XRD technique was used to identify and characterize crystalline and amorphous form of chitosan film, PVA film, blank blended patches, and Z. cassumunar blended patches that had been studied in range of 5–40° (2θ values) (Fig. 4). The X-ray diffraction profile of chitosan film showed peaks at ∼10° and ∼23° (2θ). The intensity result of PVA film was 19.69° representing their semi-crystalline characters because of the strong intermolecular interaction between PVA chains through intermolecular hydrogen bonding [37]. Thus, the chitosan and PVA film exhibited the semi-crystalline characteristics, but the XRD patterns of blank blended patches and Z. cassumunarblended patches had broad diffraction halo of amorphous region.

Fig. 4. XRD patterns of chitosan, PVA, blank blended patches, and Z. cassumunar blended patches.

From above experimentals, the FTIR, DSC and XRD results showed that there were no chemical interactions between any components in blank blended patches or Z. cassumunar blended patches.

Limpongsa and Umprayn (2008) reported that moisture uptake, swelling ratio, erosion, and porosity values play important roles for the release behavior of active compound in matrix type patches [38]. Thus, this research evaluated these variables as show in Fig. 5. We found that the moisture uptake, swelling ratio, erosion, and porosity of blank blended patches were 28.85 ± 4.17, 21.01 ± 5.38, 2.39 ± 0.41, and 1.92  ±  0.22%, respectively. When crude Z. cassumunar oil was added in blank blended patches, the moisture uptake, swelling ratio, erosion, and porosity of blank blended patches were 28.51 ± 0.78, 20.93 ± 5.88, 2.42 ± 0.98, 1.86  ±  0.24%, respectively, which were not significantly different from blank blended patches. These results are due to the fact that hydrophilic parts of ingredients could be dissolved and eroded from the blended patches. The chitosan and PVA could swell and immediately had the hydrated blended patches contents. The chains mobility of chitosan and PVA increased, therefore, increasing the hydrodynamic volume of the polymer compact.

Fig. 5. The moisture uptake, swelling ratio, erosion, and porosity values of blank blended patches and Z. cassumunar blended patches.

3.2 In vitro release study of compound D

In vitro release of the crude Z. cassumunar oil released compound D calculated as cumulative percentage release 90.43 ± 19.28% after 24 h (Fig. 6). The almost 100% release of compound D in 24 h might be due to rapid diffusion in the receptor medium as a fast, initial burst during the first 6 h.

Fig. 6. In vitro release of compound D content from crude Z. cassumunar oil and Z. cassumunar blended patches and in vitrorelease kinetics of zero order model (A), first order model (B), and Higuchi's model (C).

The amount of compound D in the Z. cassumunar blended patches was 2.19 ± 0.16 mg/cm2. When the Z. cassumunar blended patches were studied in in vitro, the cumulative percentage release of compound D was 81.49 ± 10.92% after 24 h (Fig. 6). The release behavior was similar to the compound D release behavior from crude Z. cassumunar oil that had a fast initial burst release during the first 6 h. This behavior was likely due to the compound D on the surface of patches might be rapid diffusion. However, the effect may be attributed to the moisture uptake, swelling ratio, erosion, and porosity whereby the patch could absorb the moisture, and create a space and a large free volume within the blended patches that enhanced compound D diffusion [38]. Moreover, Guo et al. 2011 reported enhanced drug diffusion with amorphous matrix type patches [39] which supports our results in in vitro study. The in vitro release kinetics model of compound D provided a better fit to first-order model than to the zero-order and Higuchi's model (Fig. 6).

3.3 In vitro skin permeation study of compound D

The in vitro skin permeation study was carried out in a modified Franz-type diffusion cell using newborn pig skin as a partition membrane. The mean cumulative amount of compound D permeated from crude Z. cassumunar oil and Z. cassumunar blended patches were 38.55 ± 18.48% and 36.72 ± 11.29% after 24 h, respectively (Fig. 7). Although another publication reported that glycerine could enhance drug permeability [40] and [41], the Z. cassumunar blended patches contained only a small amount of glycerin as plasticizer which was unlikely to affect drug permeation. Moreover, compound D was only slightly detected in the receptor medium. Because of its structure, compound D exhibits less hydrophilicity than hydrophobicity [11], [13] and [15]. The in vitro skin permeation kinetics model of compound D provided a better fit to a first-order model than zero-order and Higuchi's model (Fig. 7).

Fig. 7. In vitro skin permeation of compound D content from crude Z. cassumunar oil and Z. cassumunar blended patches and in vitro skin permeation kinetics of zero order model (A), first order model (B), and Higuchi's model (C).

Thus, the newborn pig skins were removed from modified Franz-type diffusion cell apparatus. They were cut into small pieces and homogenized, and then were extracted in absolute ethanol. These solutions were analyzed for the remaining compound D content by HPLC method. They contained 60.54 ± 39.55% and 46.77 ± 17.93% compound D content in crude Z. cassumunar oil and Z. cassumunar blended patches, respectively. Thus, the compound D was highly accumulated in newborn pig skin layer minimum permeation into receptor medium. However, the underlying mechanisms for this effect was never reported and will be further studied.

4 Conclusion

In the current work prepared the Z. cassumunar blended patches made from chitosan and PVA polymer blends incorporating the crude Z. cassumunar oil. The surface and cross section were photographed for morphology study under SEM technique and the physicochemical properties evaluated by FTIR, DSC, XRD, moisture uptake, swelling ratio, erosion, and porosity. The results revealed compatible, homogeneous, smooth, and compact blended ingredients. The blended patches could absorb the moisture that resulted in swelling of blended patches. They were eroded which increased the number of porous channels homogenously to pass compound D from Z. cassumunar blended patches. The blended patches provided a controlled release and skin permeation of compound D when studied by modified Franz-type diffusion cell apparatus. Thus, the blended patches could be suitably used for herbal medicine application.

Acknowledgment

The authors reported no declaration of interests. The authors are thankful to the Faculty of Pharmacy and the Research Institute of Rangsit University (Grant No.74/2555) for financial supports.