What Essential Oils Are Good For Pain And Inflammation?

What Essential Oils Are Good For Pain And Inflammation
Do They Really Work? – Aromatherapy may also lower your stress levels and how you sense pain, More research is needed to know how helpful essential oils really are. Here’s what some small studies found: Black cumin. Older people rubbed black cumin oil into their achy knees 3 times a day for 3 weeks.

They felt better than the group that only took acetaminophen, Eucalyptus. People who breathed in eucalyptus oil had less pain and lower blood pressure after total knee replacement, Frankincense and myrrh. When used together, these oils eased joint inflammation in rats with arthritis. Experts are looking into how this might help with conditions like rheumatoid arthritis,

Ginger. A group of people with ongoing knee problems reported less pain and stiffness a month after a massage with ginger oil. The group that only got a massage did not. They also showed better physical function overall. Lavender. A massage with lavender oil helped ease musculoskeletal pain and knee pain from osteoarthritis,

What essential oil makes pain go away?

Aromatherapy oils can alleviate various ailments in the body, including pain “Clinical research has shown that some of these aromatherapy oils can alleviate various ailments in the body, including pain” So What Are Essential Oils? Essential oils (AKA aromatherapy oils), derive from the bark, fruit, flowers, seeds and leaves, of a broad spectrum of common plants. Which Essential Oils Are Best For Pain? There is an enormous selection of wonderful aromatherapy oils on the market, so it is important to know which ones are suitable for your pain. You may like to try out several of them to see which ones you prefer, as the more you like the aroma, the more beneficial they are likely to be.

  • Peppermint Oil
  • One of Mother nature’s most powerful painkillers, peppermint oil is frequently used for alleviating pain,
  • Wintergreen Oil

Closely related to peppermint, wintergreen oil boasts similar properties due to its methyl salicylate component. Note: “talk to a doctor if you’re taking blood thinners or other medications, as wintergreen can increase the risk of bleeding”, Eucalyptus Oil With its well known anti-inflammatory benefits, this aromatherapy favourite can promote an analgesic effect in the joints and muscles.

  1. Ginger Oil
  2. Renowned for its anti-inflammatory benefits, a 2016 study showed its positive effect on rheumatoid arthritis patients,
  3. Lemongrass Oil
  4. Lemongrass can be instrumental in reducing inflammation and pain, although at the present time, only limited research with mice has highlighted its noteworthy anti-inflammatory properties,
  5. Lavender Oil

Known as the star of aromatherapy, one clinical review of lavender oil indicates that it: “can help alleviate headaches and muscle pain. Such benefits may transfer to back pain as well”, Roman/German Chamomile Oils These popular oils are known to help reduce overall inflammation and muscle spasms. Rosemary Oil Boasting clinically proven benefits, rosemary oil has been shown to reduce pain in rheumatic disorders. Moreover: “such anti-inflammatory and analgesic effects may also be helpful for back pain”, Using Essential Oils “Essential oils may be inhaled in aromatherapy or diluted and applied to your back topically or used in a bath” Although essential oils are made from natural ingredients, they still need to be treated with caution, just like any other chemicals.

  1. To that end, you must ensure that you do not have any contra-indications that could cause serious side-effects or skin irritation.
  2. Topical Application If you want to massage your back or any other part of your body with essential oil, then the latter must be diluted in a carrier oil.
  3. This can be purchased ready made, or mixed at home.

Instructions “Mix up to 6 drops of your selected oil with 6 teaspoons of a carrier oil, such as jojoba or olive oil. Do a patch test of your diluted oil on a small area of skin. After 24 hours, if no irritation develops, it’s likely you can safely use the oil on a larger area of skin, such as your back”,

  • Inhalation
  • If you are suffering from back, or any other form of pain, and/or are experiencing inflammation: inhaling aromatherapy oil via a diffuser can have a very calming de-stressing effect,
  • Instructions

Place a few drops of pure essential oil in an electric diffuser filled with water, and leave it running in your office or home, so you can enjoy the fully benefits. Then, simply add more water or oil to the diffuser as and when required, Diffusers are readily available from Amazon and Ebay. Reference, Cherney, Kristeen (2019). “Treating Back Pain and Inflammation with Essential Oils.” HYPERLINK “https://www.healthline.com/health/essential-oils-for-back-pain” \l “essential-oils-for- : Aromatherapy oils can alleviate various ailments in the body, including pain

What oil is good for inflammation and pain?

– Each of the following oils boast being able to treat pain, tension, and swelling — talk about a triple threat! For added relief, consider mixing two or three of these oils together to create a custom blend. You can also try out different combinations to see which oils work best for you:

  • Peppermint oil, Peppermint contains menthol, which has a cooling effect on sore, achy muscles. It also has analgesic, antispasmodic, and anti-inflammatory properties.
  • Helichrysum oil, Helichrysum relieves muscle spasms, inflammation, and pain.
  • Marjoram oil, Marjoram relaxes muscle spasms and tension. It’s known for its ability to ease pain and inflammation.

Which oils are pro inflammatory?

Omega-6s are found in oils such as corn, safflower, sunflower, soy and vegetable and products made with those oils. Excess consumption of omega-6s can trigger the body to produce pro-inflammatory chemicals, and the American diet tends to be very high in omega-6s.

Do essential oils really work for pain?

Lavender – According to a 2013 study, lavender essential oil may help treat pain in children after a tonsillectomy, Children who inhaled the scent of lavender were able to reduce their daily dose of acetaminophen, or Tylenol, post-surgery. Researchers in a 2015 study found that lavender essential oil can be an effective pain reliever and anti-inflammatory.

  • When diluted lavender essential oil was applied topically during one test, it provided pain relief comparable to that of the prescription medication tramadol,
  • This suggests that lavender could be used to help treat pain and any associated inflammation.
  • Another study in 2012 tested lavender essential oil’s ability to reduce pain in people who experience migraines.

Results showed that inhaling the scent of lavender was effective in lessening the severity of migraine headache symptoms.

What is a natural pain reducer?

Many people have found that natural pain relief options are a great way to help supplement medical treatments for chronic pain. The following natural pain relief options are generally easy to try, inexpensive, and have the potential for reducing your pain. Watch: Video: 4 Little-Known Natural Pain Relievers Try a few of the following options and see what works best for you.

  1. Release your inner endorphins. Endorphins are the natural pain killers produced by your body. They work by binding to the opioid receptors in your brain to block the perception of pain. Spurring increased production of these natural hormones can substantially help reduce your pain, as well as produce profound feelings of pleasure and satisfaction.1 While any activity that gets your blood pumping for a sustained period will release endorphins into your system, check with your doctor before starting a new exercise program. See How a Physical Therapist Can Help with Exercise
  2. Find support and understanding. Unlike a broken leg or other obvious sign of injury, chronic pain is usually unseen. It is a profoundly personal—and often lonely—experience. If this is the case for you, we encourage you to find people who can be supportive and understanding. There may be a chronic pain support group in your local community or hospital. Or you may prefer to interact online. You may get started with a local or online forum seeking help, and then go on to find that you have a lot to contribute—and helping others is also a way to help yourself. Visit our Back and Neck Pain Support Group on Facebook to find online support
  3. Enjoy the outdoors.10 to 15 minutes of sun exposure a day can help the body produce vitamin D. While some studies have found vitamin D supplementation can help reduce chronic pain 2, other studies have not and more research is needed.3 Regardless, being out in the sunshine can boost your mood and may also promote better immune function.4 See Calcium and Vitamin D Requirements
  4. Soak in warm water. Soaking your body in warm water can alleviate many forms of muscle pain and muscle spasm, as well as various types of arthritis. There are many options for a warm soak, including a deep bathtub, whirlpool tub, or warm pool for water therapy. See Water Therapy Exercise Program


  1. Try dry heat therapy. If a soak isn’t for you, or you want to apply heat more often, there are plenty of other heat therapy options for your pain. Consider applying a heat pack or an adhesive back wrap that provides continuous low-level heat. Just remember to carefully read directions before using a heat therapy product to reduce the risk of a burn or other injury. See Benefits of Heat Therapy for Lower Back Pain
  2. Enjoy essential oils. Essential oils have long been valued for their analgesic effects in many cultures. There are many ways to benefit from essential oils—some people inhale them (aromatherapy), others include several drops in their massage oil and enjoy as part of a therapeutic massage. When combined with traditional treatment therapies, several oils, in particular, are thought to have an analgesic effect, including peppermint oil, rosemary, and lavender, but more research is needed.5 Before choosing an essential oil, read its label for intended use to reduce the risk for complications, such as toxic or allergic reactions.
  3. Try massage therapy. A high-quality therapeutic massage spurs blood flow, reduces muscle tension, and boosts feelings of wellness. Massage therapy is defined as the manipulation of soft tissues—muscles, tendons, and ligaments—through hands-on massage by a qualified massage therapist. Like many complementary therapies, there is no substantial agreement in terms of how much massage therapy can help reduce pain, or which type of massage is best for which type of pain, so you may need to try more than one approach to find what works best for you. See Massage Therapy for Lower Back Pain
  4. Stretch and loosen up. Almost everyone can benefit from stretching the soft tissues (the muscles, ligaments, and tendons) in and around the spine. Your back is designed for movement, and if your motion is limited because you are in pain, it can make your back pain worse. See Stretching for Back Pain Relief
  5. Meditate and relax. Meditation comes in many varieties, some complex, others simple. One common approach is just to find a sound that is pleasing to you but may or may not have a meaning (like “som” or another sound), close your eyes, sit still and comfortably, and repeat the sound in your mind. When your thoughts wander, notice that they have wandered and return to your sound. If you feel your pain, notice the pain and return to your sound. Start with a few minutes, and gradually lengthen to 30 minutes if you find meditation to be enjoyable.
  6. Imagine yourself in a better place. This form of meditation, called guided imagery, involves hearing and internalizing therapeutic suggestions to help you feel better and devalue pain signals. In one study of 28 women with osteoarthritis pain, half of the women listened to a 10- to 15-minute recorded script twice daily that guided them through muscle relaxation techniques. Women in the guided imagery group showed statistically significant improvements in their pain levels and mobility within 12 weeks, versus women in the control group who did not see any improvements.6 Guided imagery can be learned with a practitioner or on your own using online videos, phone apps, or CDs. See How to Stop Your Pain with Your Mind
  7. Swap more anti-inflammatory foods into your diet. Anti-inflammatory foods—such as fruits, vegetables, unsaturated fats, whole grains, beans, nuts, and fish high in omega-3 fatty acids—may play a role in reducing pain for some people. The Mediterranean diet and DASH diet both contain lots of anti-inflammatory foods and have been linked to reduced disease risk and better heart health. While some research suggests that an anti-inflammatory diet may help lower pain related to obesity 7 and osteoarthritis, 8 more research is needed. See Food for Thought: Diet and Nutrition for a Healthy Back
  8. Laugh more often. One study showed that social laughter increases pain tolerance.9 Laughing along with others was shown to have the highest positive impact. Laughter has many positive effects, including increasing blood circulation and oxygen intake, and raising your body’s level of endorphins (the body’s natural pain killers). There is a whole movement, called laughter yoga, which helps people enjoy the many benefits of laughter without having to need a reason to laugh—it just focuses on laughing for its own sake.


  1. Get enough restorative sleep. Getting enough sleep is critical to managing pain and promoting healing, so it’s important to employ a variety of sleep aids to help get a healthy amount of sleep. Regular exercise that physically exhausts the body helps promote deep sleep. Visualization, meditation, and other psychological techniques can also help you get to sleep and stay asleep. See Sleep Aids for People with Chronic Pain
  2. Ice it. Using ice and/or a cold gel pack can on the painful area help reduce inflammation and numb pain by slowing your nerve impulses. In general, limit ice therapy to 15 or 20 minutes with a rest of at least 2 hours between applications—and keep a layer between the ice and your skin to reduce the risk for skin damage. See How to Use Ice Massage Therapy for Back Pain

These natural pain-relieving tips are intended as ideas for you to consider on your personal journey with managing chronic pain. If your pain worsens or affects your ability to perform everyday tasks, talk to your doctor for medical treatment options.

Is peppermint oil good for pain and inflammation?

Peppermint oil is promoted for topical use (applied to the skin) for problems like headache, muscle aches, joint pain, and itching. In aromatherapy, peppermint oil is promoted for treating coughs and colds, reducing pain, improving mental function, and reducing stress.

Does peppermint oil reduce inflammation?

Studies demonstrate that peppermint oil (menthol) possesses anti-inflammatory activity.

What can I rub on inflammation?

Topical anti-inflammatory creams can reduce swelling and pain caused by many health conditions and injuries. Voltaren (diclofenac) gel, capsaicin cream, and menthol cream are common topical anti-inflammatory medications.

Which oil is best for arthritis?

Olive Oil – High in monounsaturated fats and anti-inflammatory and antioxidant compounds, olive oils are among the best-studied fats, with many known health benefits. Extra virgin olive oil, the least refined type, is pressed mechanically rather than processed with heat or chemicals that change its properties.

  • It contains biologically active compounds – such as the polyphenols, oleocanthal, oleuropein, hydroxytyrosol and lignans – that have been linked to reduced joint damage in RA.
  • Itchen tips: “Extra virgin oil has a low smoke point, so it’s best for finishing foods or for dressings,” Haas says.
  • The smoke point of virgin olive oil is a little higher, making it a better choice for cooking.” Olive oil doesn’t need to be refrigerated, but lasts longer away from heat and fluctuating temperatures and even longer in the fridge.

Once opened, it will keep for about six months on the shelf and up to a year in the refrigerator.

What is the main cause of inflammation in the body?

Causes of an inflammation – Many different things can cause inflammations. These are the most common:

Pathogens (germs) like bacteria, viruses or fungi External injuries like scrapes or damage through foreign objects (for example a thorn in your finger) Effects of chemicals or radiation

Diseases or medical conditions that cause inflammation often have a name ending in “-itis.” For example:

Cystitis: an inflammation of the bladder Bronchitis: an inflammation of the bronchi Otitis media: an inflammation of the middle ear Dermatitis: a disease where the skin is inflamed

What is the most powerful essential oil?

Frankincense – Often referred to as the “king of oils,” frankincense or Boswellia is one of the most potent and medicinally useful essential oils on the planet. It’s main benefit is boosting the immune system by stimulating its activity and killing germs that cause infection.

Perhaps the most touted health benefit of frankincense is its ability to fight cancer, Additionally, a 2011 clinical trial found that frankincense was more effective than steroidal treatment, the conventional method, in reducing brain swelling after radiation of cancerous brain tumors, making it a good adjunct treatment for this dangerous complication of brain radiation.

On top of its cancer fighting powers, frankincense oil might improve memory, increase male fertility, ease digestion and help you sleep. My favorite way to use frankincense oil (other than by diffusing it) is using it in a skin cream along with lavender or pomegranate oil.

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Does frankincense reduce inflammation?

Frankincense and myrrh suppress inflammation via regulation of the metabolic profiling and the MAPK signaling pathway 1 Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine; Nanjing University of Chinese Medicine, Nanjing 210023, PR China 2 Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing 210023, PR China Find articles by 1 Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine; Nanjing University of Chinese Medicine, Nanjing 210023, PR China 2 Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing 210023, PR China Find articles by 1 Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine; Nanjing University of Chinese Medicine, Nanjing 210023, PR China 2 Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing 210023, PR China Find articles by 1 Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine; Nanjing University of Chinese Medicine, Nanjing 210023, PR China 2 Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing 210023, PR China Find articles by 1 Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine; Nanjing University of Chinese Medicine, Nanjing 210023, PR China 2 Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing 210023, PR China Find articles by 1 Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine; Nanjing University of Chinese Medicine, Nanjing 210023, PR China 2 Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing 210023, PR China 3 Basic Medical College, Nanjing University of Chinese Medicine, Nanjing 210023, PR China Find articles by 1 Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine; Nanjing University of Chinese Medicine, Nanjing 210023, PR China 2 Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing 210023, PR China 1 Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine; Nanjing University of Chinese Medicine, Nanjing 210023, PR China 2 Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing 210023, PR China Find articles by 1 Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine; Nanjing University of Chinese Medicine, Nanjing 210023, PR China 2 Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing 210023, PR China Find articles by 1 Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine; Nanjing University of Chinese Medicine, Nanjing 210023, PR China 2 Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing 210023, PR China 1 Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine; Nanjing University of Chinese Medicine, Nanjing 210023, PR China 2 Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing 210023, PR China Find articles by 1 Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine; Nanjing University of Chinese Medicine, Nanjing 210023, PR China 2 Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing 210023, PR China Find articles by Received 2015 Mar 31; Accepted 2015 Aug 3. © 2015, Macmillan Publishers Limited This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit Frankincense and myrrh are highly effective in treatment of inflammatary diseases, but lacking of the therapy mechanisms. We undertook this stuty to evaluate the effects on Adjuvant-induced Arthritis (AIA) rats and to explore the underlying mechanisms by analyzing the metabolic profiling and signaling pathway evaluated by expression of inflammatory cytokines, c-jun and c-fos and corresponding phosphorylation levels. The results stated the elevated expression levels of TNFα, PGE 2, IL-2, NO, and MDA in serum and swelling paw of AIA rats were significantly decreased after treatment, which exerted more remarkable inhibitive effects of combined therapy. The metbolic profiling of plasma and urine were clearly improved and twenty-one potential biomarkers were identified. Moreover, the inhibited effects of five bioactive components on cytokine transcription in PHA stimulated-PBMC showed the MAPK pathway might account for this phenomenon with considerable reduction in phosphorylated forms of all the three MAPK (ERK1/2, p38 and JNK) and down regulation of c-jun and c-fos. Rheumatoid arthritis (RA) is a globally prevalent chronic autoimmune inflammatory disease characterized by a hyperplastic synovial membrane, which can destroy adjacent articular cartilage and bone in joints,,, Pathologic features of the affected joints include hyperplasia of synovial tissue composed of proliferating synoviocytes and infiltrating leukocytes, including T cells and B cells, which are likely activated by autoantigens, Inflammatory cytokines play pivotal role in the pathophysiology of RA. The activated leukocytes and synovial fibroblasts in the joint tissue secrete several proinflammatory mediators such as TNF- α, IL-1, IL-6, IL-8, PGE 2, INF- γ etc. to cause inflammation and joint degradation, Both the autoreactive T and B cells play essential roles in the autoimmune responses which cause tissue inflammation, autoantibody production and clinical onset of RA,,, T cell-derived cytokines, including IL-1, IL-2, TNF- α, IFN- γ, LT, IL-3, and IL-6, exert important and distinct function in RA pathogenesis,, Elevated serum levels of both PGE 2 and leukotriene B 4 (LTB 4 ) have been reported to correlate with the severity of RA, Besides, reactive nitrogen species (RNS), such as nitric oxide (NO) have been involved in the development of joint destruction in RA, However, the concrete pathogenesis of RA is still unclear completely, even though viral infection and genetic predisposition might be the possible reasons,, The extent of metabolic changes and types of metabolites could be applied as good markers of cytokines-mediated inflammatory processes in RA. A novel system approach to assess metabolic changes in disease is metabolomics, which indicates the overall physiological status coresponding to pathological stimuli, such as genetic, environmental, or lifestyle factors, Changes of metabolites with low molecular weight often mirror the end result of genomic and protein perturbations in disease, and they are closely associated with phenotypic changes. Besides, the pathogenesis of diseases and the action mechanisms of therapy could also be elucidated by identifying biomarkers, analyzing metabolic pathway, discovering drug- target interactions, and so on. Therefore, metabolic profiling has attracted an interest for investigating the RA disease and evaluating therapeutic effects of drugs,,, Currently, the first-line therapies for RA are focused on alleviation of inflammation, pain and joint damage by using glucocorticoids, disease-modifying antirheumatic drugs (DMARDs) and non-steroidal anti-inflammatory drugs (NSAIDs),, But they are limited by a number of well-characterized clinical side-effects, such as cushing syndrome, and diabetes for glucocorticoids, hepatotoxicity, blooddyscrasias, and interstitiallung disease for DMARDs,, and gastrointestinal and cardiac toxiceffects for NSAIDs. Though the use of TNF- α and IL-1 β antagonist have shown substantial efficacy, high cost of medications, and hypersensitivity to infections also can not be ignored. Consequently, there has been a great demand for new antirheumatic agents caplable of acting on multiple cytokines or mediators of inflammation, with fewer toxic or side-effects. Fortunately, effective treatments for rheumatism were available from practitioners of traditional Chinese medicine. Therefore, many researchers have aimed at developing potent therapies and drugs from Chinese medicines with fewer side-effects on RA patients. Ruxiang (Frankincense) is dried gum resin of Boswellia carterii or one of 43 species in the genus Boswellia of the family Burseraceae. It has been commonly used to reduce swelling and alleviate the pain of inflammatory diseases or tumors,, and to invigorate the circulation of blood in China and as an antiarthritic in Ayuredic medicine in India for thousands of years, Moreover, it is also used as dietary supplements for patients with arthritis or other inflammation and pain related disorders in USA, Previous studies in vitro have shown that the boswellic acids isolated from Frankincense exhibited potential immunomodulatory effects,, Myrrh, as a traditional natural medicine, is an aromatic gum resin, which was the plant stem resinous exudate of Commiphora myrrha (Nees) Engl. (Burseraceae) and various other different species of Commiphora family. It has many medicinal powers and has been used widely in clinic for treatment of pain and inflammatory diseases, such as stomach complaints, skin infections, ache, dysmenorrhea, chest ailments, and so on, in India, China, Rome, and Greece,,, Especially, the myrrh was a common analgesic and has been used to clean wounds and sores for more than 2000 years, until the European discovered the morphine. Pharmacological studies also have showed that myrrh possesses multiple activities, including anti-inflammatory, cytotoxic, anesthetic, and antimicrobial effects, In China, Frankincense and Myrrh are often used together on clinic in order to obtain a synergistic effect for relieving pain and activating blood circulation, and especially to treat inflammatory diseases (e.g., RA). However, the evaluation of the effects and mechanisms are lack. So, in this study, the adjuvant-induced arthritis (AIA) as immune-mediated rat model was used to assess the anti-arthritic efficacy of individual and combined extracts of Frankincense and Myrrh resin. The levels of TNF- α, and IL-2, PGE 2, NO, and MDA were determined. To elucidate the mechanism, the metabolic changes in plasma and urine from AIA rats based on UPLC/Q-TOFMS were investigated. The potential biomarkers and metabolic pathway were also identified. Furthermore, the actions of five bioactive compounds (chemical structures see ) drived from Frankincense (compounds 1, 4, and 5 ) and Myrrh (compounds 2 and 3 ), respectively, were investigated on TNF α, IL-1 β, IL-2, IL-10, IL-12, INFγ, and c-jun and c-fos expression in PHA stimulated-PBMC (peripheral blood mononuclear cell) to explore the possible signaling pathways. These data would be useful to further develop and improve the anti-inflammatory agents. The chemical structures of five bioactive compounds drived from Frankincense (compounds 1, 4, and 5) and Myrrh (compounds 2 and 3) (1.3-hydroxylanosta-8,24-dien-21-oic-acid; 2.2-Methoxy-5-acetoxy-fruranogermacr -1(10)-en-6-one; 3. abietic acid; 4. elemonic acid; 5. Acetyl elemolic acid). At beginning of the experiment, i.e. day 0, no significant differences were found in rat hind paw volume (HPV) among all the groups ( P  > 0.05). A significant increase in HPV was observed for the adjuvant injected group on day 13, 20, 24, 27 and 30 day compared to the healthy control rats (P < 0.001). Meanwhile, the paw edema volume was significantly reduced in group treated with standard drug of indomethacin (IMT), individual extract of Frankincense or Myrrh, and combined extracts treated groups which showed significant difference when compared with the arthritis group (P < 0.001 or P < 0.01 or P < 0.05) (See ). Effects of extracts of C. myrrha, B. carterii. and their combination on rat hind paw swelling in adjuvant-induced arthritis model.

Groups Dosage(mg/Kg·d p.o.) Rat hind paw volume in ml ± SD (% inhibition)
Day 0 Day 13 Day 20 Day 24 Day 27 Day 30
Group-I 0.86 ± 0.12 1.03 ± 0.10 1.04 ± 0.07 1.06 ± 0.07 1.16 ± 0.43 0.96 ± 0.15
Group-II 0.863 ± 0.16 1.87 ± 0.15 1.86 ± 0.13 1.86 ± 0.12 1.89 ± 0.12 2.00 ± 0.13
Group-III 10 0.83 ± 0.21 1.73 ± 0.15 1.61 ± 0.13 1.54 ± 0.15 1.47 ± 0.05 1.38 ± 0.06
Group-IV 54.28 0.85 ± 0.23 1.75 ± 0.19 1.61 ± 0.12 1.48 ± 0.08 1.42 ± 0.05 1.38 ± 0.05
Group-V 90.48 0.80 ± 0.20 1.70 ± 0.15 1.64 ± 0.12 1.58 ± 0.11 1.46 ± 0.12 1.36 ± 0.074
Group-VI 33.67 0.82 ± 0.21 1.78 ± 0.16 1.67 ± 0.08 1.55 ± 0.15 1.49 ± 0.11 1.47 ± 0.13
Group-VII 56.12 0.86 ± 0.18 1.88 ± 0.27 1.79 ± 0.20 1.56 ± 0.27 1.49 ± 0.20 1.39 ± 0.13
Group-VIII 46.15 0.87 ± 0.16 1.87 ± 0.17 1.78 ± 013 1.63 ± 0.23 1.53 ± 0.14 1.48 ± 0.09
Group-IX 76.92 0.85 ± 0.15 1.78 ± 0.19 1.69 ± 0.11 1.53 ± 0.15 1.43 ± 0.18 1.36 ± 0.14

On day 30 after adjuvant inoculation, levels of TNF α, PGE 2, IL-2, NO, and MDA in serum were significantly increased ( p  < 0.001) in model rats (group II) than that of control group (group I). After treated with individual extract of Myrrh (46.15 mg/kg·d, 76.92 mg/kg·d p.o., respectively) or Frankincense (33.67 mg/kg·d, 56.12 mg/kg·d, p.o., respectively) and combined extracts (54.28 mg/kg·d, 90.48 mg/kg·d, p.o., respectively), all of cytokines determined in this test were significantly decreased ( P  < 0.001 or P  < 0.01) (see ). What was interesting was the effects of the combined extract at dosage of 90.48 mg/Kg·d and extract of Frankincense extract at dose of 56.12 mg/kg·d exerted remarkablely similar with that of IMT (10 mg/kg·d). The levels of TNF- α, PGE 2, IL-2, NO, and MDA in swelling paw tissue had same trends in model rats than that of control group, and therapeutic trends by test drugs (see ). Effects of Frankincense, Myrrh and combined extracts on the cytokines (NO, MDA, IL-2, PGE 2, TNF α ) levels in rats'serum and right paw swelling of adjuvant-induced arthritia model (, n = 8).

Groups Dosagemg/Kg·d NO ( μ mol/L) MDA (nmol/Ml) IL-2 (pg/mL) PGE 2 (pg/mL) TNF α (pg/mL)
In serum Right paw tissue In serum Rightpawtissue In serum Rightpawtissue In serum Rightpawtissue In serum Rightpawtissue
Group-I 73.72 ± 6.34 83.24 ± 10.86 32.47 ± 3.10 0.61 ± 0.08 48.56 ± 7.93 53.41 ± 13.71 174.05 ± 14.46 84.55 ± 18.74 24.68 ± 3.87 40.49 ± 2.86
Group-II 153.12 ± 10.22 120.08 ± 10.29 41.96 ± 1.76 3.40 ± 0.48 143.10 ± 8.06 110.35 ± 11.77 459.29 ± 22.86 444.19 ± 10.39 110.35 ± 11.78 94.72 ± 25.67
Group-III 10.00 94.99 ± 10.89 67.67 ± 7.49 33.74 ± 2.90 0.80 ± 0.16 79.39 ± 15.33 72.00 ± 25.17 220.76 ± 22.15 123.50 ± 15.48 47.81 ± 5.39 44.40 ± 10.51
Group-IV 54.28 144.61 ± 25.99 78.16 ± 7.93 36.38 ± 4.01 0.95 ± 0.06 97.75 ± 17.33 86.56 ± 10.66 229.56 ± 15.65 303.96 ± 54.17 38.95 ± 11.22 49.35 ± 9.65
Group-V 90.48 92.15 ± 8.39 54.66 ± 5.61 38.24 ± 4.83 2.47 ± 0.57 65.22 ± 8.33 71.40 ± 15.31 345.77 ± 18.36  156.23 ± 18.25 66.42 ± 11.01 35.81 ± 7.49
Group-VI 33.67 86.48 ± 13.52 60.94 ± 3.98 40.20 ± 7.57 2.34 ± 0.48 168.42 ± 13.12 97.52 ± 8.29 342.97 ± 43.92  277.43 ± 25.34 69.44 ± 9.41 55.71 ± 4.58
Group-VII 56.12 114.84 ± 13.52 71.18 ± 14.62 38.53 ± 5.20 2.17 ± 0.48 96.40 ± 10.17 63.30 ± 7.61 354.70 ± 34.30  159.32 ± 26.11 58.93 ± 7.66 37.39 ± 6.11
Group-VIII 46.15 121.93 ± 27.49 73.72 ± 2.52 37.85 ± 4.81 1.91 ± 0.27 b  *** 88.02 ± 8.56 77.11 ± 18.44 350.71 ± 11.39  188.96 ± 20.33 60.68 ± 10.39 44.63 ± 8.20
Group-IX 76.92 62.38 ± 24.23 46.79 ± 6.70 35.11 ± 1.24 1.57 ± 0.30 44.84 ± 11.51 60.52 ± 10.68 304.01 ± 22.97  263.73 ± 33.99 35.87 ± 7.28 31.80 ± 5.98

From the pathologic changes of hind paw and the regulation of pre-inflammation cytokines by the extract of Frankincense and combined extract, it was showed that they slowed the progression of inflammation obviously, accelerated bone resorption, prevented periosteal bone proliferation and cartilage destruction. Typical based peak intensity (BPI) chromatograms of plasma and urine samples, collected from model rats and normal rats in negative modes were shown in, The unsupervised PCA model was used to separate plasma or urine sample into two blocks between model rats and normal rats. A total of 252 ions in plasma samples and 347 ions in urine samples at negative modes were detected from model rats and normal rats. PCA scores plots showed clear clustering of them (). The supervised OPLS-DA divided samples into two blocks and distinguished model rats from normal rats’ cohorts with 100% sensitivity and no less than 95% specificity using a leave one out algorithm, which indicated that the OPLS-DA model was reliable. From the loading plots of OPLS-DA, 36 ions in plasma samples and 43 ions in urine samples at negative modes were deemed discriminatory ( p  < 0.05), and identified as being responsible for the separation between model rats and normal rats (). PCA model results between AIA rats and controls in negative mode. (( A ) 2-D plot of plasma; ( B ) 2-D plot of urine).3D PLS-DA scores plot of LC–MS spectral data (( C ) plasma, R2 = 0.87, Q2 = 0.92; ( D ) urine, R2 = 0.93, Q2 = 0.88). S-plot of OPLS-DA model for AIA vs control group. (( E ) plasma, R2 = 0.86, Q2 = 0.81; ( F ) urine, R2 = 0.96, Q2 = 0.84). PCA analytical results from AIA rats treated with drugs in different groups at negative mode. (( G ) For plasma; ( H ) for urine). In the plasma, ten endogenous metabolites, contributing to the separation between the groups, were identified (). The precise molecular mass was determined within measurement errors (<5 ppm) by Q-TOF/MS/MS. Compared with normal rats, alanyl tryptophan, linoelaidic acid, and docosahexaenoic acid were up regulated ( p  < 0.05), while LysoPC(17:0), LysoPC(15:0), LysoPE(20:2(11Z,14Z)/0:0), LysoPE(20:1(11Z)/0:0), LysoPE(18:1 (9Z)/0:0), LysoPC(18:2(9Z,12Z)), and LysoPC(16:0/0:0) were down regulated significantly ( p  < 0.05). The identified and change trend of the potential biomarkers of AIA rats intervened by frankincense and myrrh.

No. t R /min − m/z metabolites Contentvariance (Group II) Effects of test drugs Pathway (KEGG) Resources
GroupIII GroupV GroupVII GroupIX
1 10.75 568.3625 LysoPC(17:0) Glycerophospholipid metabolism Plasma
2 8.56 540.3321 LysoPC(15:0) Glycerophospholipid metabolism Plasma
3 7.81 564.3312 LysoPE(20:2(11Z,14Z)/0:0) Glycerophospholipid metabolism Plasma
4 9.04 566.3466 LysoPE(20:1(11Z)/0:0) Glycerophospholipid metabolism Plasma
5 7.27 538.3158 LysoPE(18:1(9Z)/0:0) Glycerophospholipid metabolism Plasma
6 6.36 312.0784 Alanyl tryptophan Tryptophan metabolism Plasma
7 7.49 564.3321 LysoPC(18:2(9Z,12Z)) ↓↓ Glycerophospholipid metabolism Plasma
8 13.21 279.2318 Linoelaidic acid Biosynthesis of unsaturated fatty acids Plasma
9 12.50 327.2319 Docosahexaenoic acid Biosynthesis of unsaturated fatty acids Plasma
10 9.75 554.3479 LysoPC(16:0/0:0) Glycerophospholipid metabolism Plasma
11 6.74 160.0394 4,6-Dihydro xyquinoline 5-hydroxytryptophan metabolism Urine
12 5.27 284.0589 Malonyl carnitine Fatty acid metabolism Urine
13 3.15 242.0115 Bicine Urine
14 2.45 230.0109 Homocysteine thiolactone Amino acid biosynthesis Urine
15 1.02 191.0173 Citric acid Citrate cycle (TCA cycle) Urine
16 8.16 268.0634 Isoval eryglutamic acid Urine
17 0.76 191.0183 Glucaric acid Glyoxylate and dicarboxylate metabolism Urine
18 14.35 408.1657 9′-carboxy-gama-tocotrienol Glyoxylate and dicarboxylate metabolism Urine
19 7.08 338.0893 Topiramate Urine
20 13.92 343.0847 Xanthosine Purine metabolism Urine
21 4.51 212.0017 Indoxyl sulfate Tryptophan metabolism Urine

In the urine, detected significant variables in the negative ion mode were summarized in, Eleven endogenous metabolites were tentatively identified by the methods described above. The metabolite of indoxyl sulfate was observed to be up regulated significantly ( p  < 0.05), whereas the other metabolites of 4,6-dihydro xyquinoline, malonyl carnitine, bicine, homocysteine thiolactone, citric acid, isoval eryglutamic acid, glucaric acid, 9′-carboxy-gama-tocotrienol, topiramate, and xanthosine were down regulated obviously ( p  < 0.05). These differences in plasma and urine might denote the potential targeted biomarkers for differentiating RA pathological from normal states. The metabolic pathway analysis with MetPA revealed that the identified biomarkers were critical for the host responding to RA. Five metabolic pathways were disturbed, which included glycerophospholipid metabolism, citrate-cycle- (TCA-cycle), glyoxylate and dicarboxylate metabolism, ascorbate and aldarate metabolism and glycosylphosphatidylinositol (GPI)-anchor biosynthesis. The pathway impact value calculated from pathway topology analysis above 0.1 was filtered out as potential target pathway. In order to elucidate the intervention efficacy of Frankincense, Myrrh, and combined extracts, PCA analysis was carried out to obtain the changes among group I-IX. The variations of plasma and urine metabolic profiling of Frankincense, Myrrh and their combination-treated rats was restored back to the levels more than control-like on the 30 th day (). Furthermore, ten endogenous metabolites in plasma and eleven endogenous metabolites in urine were significantly affected by Frankincense and Myrrh combined extracts ( p  < 0.05 or p  < 0.01), while the IMT regulated eleven endogenous metabolites in urine including topiramate level except for the same metabolites with ones regulated by combined extracts. All of these metabolites were restored back to a control-like level and there was no significant difference between the IMT and combined extract - treated group ( p  > 0.05). There was no obvious effects on metabolites levels of LysoPC(17:0), LysoPC(15:0), and LysoPC(16:0/0:0) (in plasma) for Frankincense extract. The levels of LysoPC(17:0), LysoPC(15:0), and LysoPE(18:1(9Z)/0:0) were also not regulated by Myrrh extract. It was interesting that the metabolites levels of citric acid, isoval eryglutamic acid, glucaric acid, 9′-carboxy-gama-tocotrienol, and xanthosine in urine were neither affected by frankincense extract nor myrrh extract, but by their combination. These changes may not immediately in response to therapeutic effects of Frankincense and Myrrh for the AIA rats, but they were generated from perturbation in organism by administration of Frankincense and Myrrh. The contents of the potential biomarkers in were considered as biomarkers for effect of treatment. To evaluate bioactivitives of five bioactive compounds including 3-hydroxylanosta-8,24-dien -21-oic-acid ( 1 ), 2-Methoxy-5-acetoxy-fruranogermacr -1(10)-en- 6–one ( 2 ), abietic acid ( 3 ), elemonic acid ( 4 ), and Acetyl elemolic acid ( 5 ), a series of dosages were applied on PBMC and MTT assays were carried out (). Then, dosages of 70, 1.0, 1.0, 4.0 and 2.3  μ g/ml were selected for compound 1, 2, 3, 4 and 5 respectively for further analysis on different targets. The results showed that the transcription levels of IL-1 β, IL-2, IL-10, IL-12, TNF α, INF γ could be enhanced in PHA stimulated-PBMC while this tendency could be decreased by treantment of compounds 1–5 (). Especially, compound 5 showed the most marked inhibiting effect for all tested cytokines. ( A ) Analysis of compounds 1–5 on PHA induced proinflammatory cytokine expression in PBMC. PBMCs were induced with PHA (10 ng/ml) for 6 hours and inhibitory effect of them on cytokines expression was studied by RT-PCR analysis. ( B ) Analysis of compounds 1–5 on MAPK. (a) non-phospho ERK, (b) Phospho-ERK, (c) non-phospho JNK, (d) Phospho-JNK, (e) non-phospho p38, (f) Phospho-p38. By Western blotting, the inhibitory effects of them on active forms of MAP kinases were analysed in PHA stimulated PBMC, using antibodies recognizing the phosphorylated and non-phophorylated forms of ERK1/2, JNK and p38 MAPK. ( C ) Analysis of compounds 1–5 on PHA induced c-fos and c-jun expression in PBMC. PBMCs were induced with PHA (10 ng/ml) for 6 hours and inhibitory effect of the five compounds on c-fos and c-jun cytokines expression was studied. Mitogen activated protein kinases (MAPK) pathway is a major pathway accounting for immune responses, including the regulation of cytokine responses, and chemokine responses. PBMC were stimulated with PHA for 3 and 6 h to activate MAPK signaling pathway. The phosphorylation levels of critical kinases, including ERK, JNK and the p38, in PBMC treated by agonist and tested compounds, were assessed by Western blotting (). The phosphorylation levels of ERK, JNK and p38 were significantly enhanced by treatment of PHA, the agonist of MAPK pathway while decreased by treatment of five compounds (). PBMCs treated with five compounds showed marked inhibition of both c-jun and c-fos expression (). The marked reduction in c-jun levels could affect AP-1 levels and thereby the downstream signals resulting in inhibition of inflammatory cytokines. Freund complete adjuvant (FCA)-induced secondary inflammation mimics sub-acute RA, AIA rat model is a useful tool to study the pathology of RA, due to its similarity with human disease sharing common signs and symptoms,, In AIA rat model, swelling of hind paws, increased levels of inflammatory cytokines were indicators of inflammation reaction for immune arthritis. Immunization of SD rats with Mtb not only induces inflammation, but also primes and expands T cells directed against mycobacterial antigens, Activation of T cells following Mtb injection involves the processing and presentation of mycobacterial antigens to specific T cells and subsequent clonal proliferation of the activated T cells. In our study, Frankincense and myrrh, especially their combined extract significantly suppressed arthritis progression as evidenced by reduction of preinflammatory factors. The beneficial effect of frankincense and myrrh on inflammation was previously reported,,,,, Acetone extract of Boswellia carterii gum resin decreased arthritic scores, reduced paw edema and suppressed local tissue TNF- α and IL-1 β in Lewis rats significantly, It is worth mentioning that the combination of Frankincense and Myrrh was more effective in suppressing the intensity of joint inflammation. Furthermore, five bioactive compounds derived from frankincense and myrrh could inhibit the expression of IL-1 β, IL-2, IL-10, IL-12, TNF α, INF γ, c-jun and c-fos in PHA activated PBMC. The phosphorylation levels of ERK, JNK and p38 were significantly inhibited in PHA activated PMBC by compounds 1–5, Especially, the compound 5 showed a more prominent effect against pre-inflammatory factors. Current data could elucidate that five bioactive compounds could exert anti-inflammatory effects via blocking MAPK pathway. Nitric oxide (NO) is a critical biochemical mediator of inflammation and involved in autoimmune mediated tissue damage and inflammation,,, In our study, nitricoxide levels were increased in untreated adjuvant arthritis rats. And it arised from the possibility that excessive nitricoxide production by inducible nitricoxide synthase (iNOS) induced by TNF- α and IL-1 and resulted in the formation of excessive amounts of superoxide (O2−), which reacted with nitricoxide (NO·) to generate peroxynitrite (ONOO−). It had been reported that peroxynitritere acting with tyrosine residues of protein stoproduce nitrotyrosine contributed to rheumatoid arthritis pathogenesis, However, determination of serum nitrotyrosine might provide an evidence for this proposed action mechanism. The decrease in serum nitricoxide levels by Frankincense and Myrrh might be attributed to its inhibition of reactive oxygen species production in the synoviocytes through modulation of TNF- α and IL-1 synthesis. The pre-inflammation cytokines of TNF- α and IL-1 could promote the release of PGs (e.g., PGE 2 causes synovial inflammation), leukotrienes, and oxygen free radical and generate collagenases and neutral protease, which induced the cartilage matrix breakdown, cartilage resorption and bone destruction, MDA was a peroxidation product produced because of lipid attacked by free radicals and the level of MDA represented the intensity of body injury. In our study, Frankincense and Myrrh combined therapy exhibited better effecacy than Frankincense and Myrrh alone for inhibiting the PGE 2 and MDA levels except for TNF- α, This enhanced effect mechanisms between Frankincense and Myrrh still need further to be investigated. The results of plasma metabolomics study of AIA rats stated that endogenous metabolites of LysoPCs and LysoPEs levels were decreased, which led to the metabolic disorder of phospholipid metabolism in inflammation. The decreased levels of glycerophospholipid metabolites, including LysoPC(17:0), LysoPC(15:0), LysoPE(20:2(11Z,14Z)/0:0), LysoPE(20:1(11Z)/0:0), LysoPE(18:1 (9Z)/0:0), LysoPC(18:2(9Z,12Z)), and LysoPC(16:0/0:0), indicated a marked perturbation in the phospholipid metabolic pathways in AIA. Lyso-PC, an important component of oxidized low-density lipoprotein (oxLDL), has been confirmed to be a chemoattractant for T lymphocytes, Lyso-PC also induces antibody formation and macrophage stimulation; therefore, Lyso-PC levels can impact on the inflammation state of an organism. Thus, it can be speculated that the abnormal plasma levels of Lyso-PC in AIA rats increase the progression of AIA. Other differential metabolites, such as Lyso-PE, are important phospholipid synthetic pathway intermediates; abnormalities in these metabolites also reflect the impact of AIA on glycerophospholipid metabolic pathway. The biosynthesis of unsaturated fatty acids metabolic disturbance induced the elevated levels of linoelaidic acid and docosahexaenoic acid, which was metabolized to PGs, thromboxane (TXs) and leukot rienes (LTs) through lipoxygenase and cyclooxygenase pathway and thus regulated inflammation. PGE 2 (prostaglandin E 2 ) is generated from AA via the COX pathway, and it is an important mediator of inflammation, pain, and joint destruction and is found in AIA rats in the synovial. By urine metabolomics research, the glyoxylate and dicarboxylate metabolism, citrate cycle (TCA cycle), and glycosylphosphatidylinositol(GPI)-anchor biosynthesis were disturbed and identified the decreased metabolites. Citric acid is an important intermediate of the tricarboxylic acid (TCA) cycle taken place in mitochondria. TCA cycle is one of the most important energy metabolism. Urinary level of citric acid is used as diagnosis of kidney stones, renal tubular acidosis and bone diseases, The decreased level of urinary citric acid may suggest that the impaired action of citrate synthase in TCA cycle in AIA rats, and probably due to perturbed metabolism in cartilage and chondrocytes. Frankincense, Myrrh and combined extracts could recover this downward trend of citric acid level. Trans fatty acids (TFA) are reported to contribute to inflammation and coronary heart disease. The remarkably increased linoelaidic acid implied the effects on the progress of inflammation degree.4,6-dihydroxyquinoline is downstream metabolite of tryptophan through kynurenine pathway. Tryptophan, an essential amino acid, plays a fundamental role in physiology and biochemistry. Tryptophan metabolism through the kynurenine pathway was considered as one of many mechanisms involved in how immune system continuously modulated the balance between responsiveness to pathogens and tolerance to non-harmful antigens, The level of 4,6-dihydroxyquinoline is significant decreased in urine, and the high level of alanyl tryptophan in plasma, suggested the unbalanced immune response. The regulation to health condition of 4,6-dihydroxyquinoline and alanyl tryptophan level may imply that kynurenine pathway was regulated after the treatment of AIA rats with Frankincense, Myrrh and combined extracts. Indoles endogenous metabolites are usually produced through tryptophan metabolism. Indoxyl sulfate (IS) is metabolized by the liver from indole, which is toxic and produced from tryptophan by intestinal flora. The excretion of IS increased significantly in AIA rats. After 17 days’ therapeutic intervention with Frankincense, Myrrh and combined extracts, the excretion of it went down. These change trends indicated that therapeutic effects of them might base on the regulation of the dysfunction of tryptophan metabolism. Considering the potential linkages, the correlation networks of the potential biomarkers in response to effect of treatment for AIA rats is described in, We speculated how biomarkers either up- or down-regulated implicated in inflammatory and immune responses through the metabolic pathway and literature search. In conclusion, the present studies suggested that combined Frankincense and Myrrh exerted a significant protective effect on HPV, inflammatory cytokines, as well as cytokines expression level. Thus, administration of combined Frankincense and Myrrh suppressed arthritic progression in rats more effective than single drug treatment. These findings might supply beneficial hints for the treatment of rheumatoid arthritis and deserves further clinical investigations. Adult male Sprague-Dawley (SD) rats (200 ± 10 g) were purchased from Nanjing University of Chinese Medicine (rodent license no. SCXK 20080033) and kept at controlled environment conditions at a constant temperature (23 ± 2 °C), humidity (60 ± 10%), and a 12/12 h light/dark cycle. Rats were acclimatized for one week before any experimental procedures and were allowed standard rat chow and water ad libitum. All experimental procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals, and before the animal experiments were carried out, the procedures were approved by the Research Ethical Committee of Nanjing University of Chinese Medicine (Nanjing, China). Freund complete adjuvant (FCA) (100M8717 CAS9007-81-2) was purchased from Sigma-Aldrich (St. Louis, MO, USA). TNF- α, PGE 2, and IL-2 enzyme linked immunosorbent assay (ELISA) kits were procured from Shanghai Westang Bio-tech CO., Ltd., Shanghai, China. Nitrite test kit (Nanjing Jiancheng Bioengineering Institute Co., Ltd., Nanjing, China, no.20081204). Sodium citrate (No.050580052) and Indomethacin (No. H31020148) were obtained from Shanghai Chemical Reagent Company, LTD. Frankincense and myrrh were collected from Guangdong, China, in April 2013 and traded in the market as medicinal resin. They were identified as resin derived from Commiphora myrrha (Nees) Engl. and Boswellia carterii Birdw., respectively, by the corresponding author. The voucher specimens (nos. NJUTCM130123 and NJUTCM130134) were deposited at the Herbarium of the Nanjing University of Chinese Medicine. To induce arthritis, the right hind footpads of male SD rats were sterilized with 70% alcohol and were intradermally injected with 100  μ L FCA (10 mg/ml) suspension of heat-killed Mycobacterium tuberculosis in vehicle (Sigma Aldrich Co-USA) according to the method by literatures,, Control animals were injected intradermally with saline in equal volume. Then the hind paw volumes were measured as the parameter of paw swelling for a period of 13 or 30 days. Chronic inflammation was allowed to progress for 12 days then rats were divided into 9 groups of eight rats each. Urine (in 12 hour intervals) and blood samples were collected on the 0, and 31 th day. We did our best in order to minimize the suffering of experimental animals and to use the number of rats which enables us to generate reliable data. Seventy two rats were randomly allocated into nine groups. Group I (n = 8) served as normal rats which received an equal volume of vehicle control. Group II (n = 8) was arthritic model control treated with vehicle only. Group III (n = 8) was arthritic control which received the indomethacin (IMT) (10 mg/Kg·d p.o.). Groups IV (n = 8) and V (n = 8) were treated with combined extracts of Frankincense and Myrrh (54.28 mg/Kg·d, 90.48 mg/Kg·d, p.o., respectively). Groups VI (n = 8) and VII (n = 8) were treated with extracts of Frankincense (33.67 mg/Kg·d, 56.12 mg/Kg·d, p.o., respectively). Groups VIII (n = 8) and IX (n = 8) were treated with extracts of Myrrh (46.15 mg/Kg·d, 76.92 mg/Kg·d p.o., respectively). The drugs were orally administered through feeding tube daily in 0.02% Tween-80 vehicle for 17 days starting 2 h before injecting FCA on day 13 and were continued up to day 30. The rats were fixed in supine position and anesthetized with 10% chloral hydrate by intraperitoneal injection, blood samples were collected in heparinized tubes on the 31 th day from carotid artery. They were then anti-coagulated in 3.8% natrium citricum (9:1), centrifuged at 3 000 r/min for 10 min and the supernatants were stored at −20 °C until detection of TNF α, NO, MDA, PGE 2, IL-2. Then the animals were sacrificed by cervical dislocation. The hind paw were separated and immersed in physiological saline (tissue : saline, 9:1, g/v) for 24 h, centrifuged at 3 000 ×  g for 10 min, collected supernatant, and stored at −20 °C until detection of TNF α, NO, MDA, PGE 2, IL-2. Urine samples were collected in 12 hour intervals, then centrifuged at 13 000× g for 10 min and stored at −20 °C until analysis. Two hundred microliters of plasma was added to 600  μ L of acetonitrile, and this mixture was vortexed for 30 s and centrifuged at 13 000  g for 10 min to obtain the supernatant. Prior to analysis, the urine samples were thawed at room temperature and centrifuged at 13 000× g for 10 min. The supernatant liquid (1 mL) was added to 3 mL of acetonitrile, vortex mixed for 30s, and centrifuged at 13000× g for 10 min to obtain the supernatant. The plasma and urine supernatants were removed and evaporated to dryness in a 40 °C water bath under a gentle stream of nitrogen. The residues were reconstituted in 200  μ L mobile phase of 70% acetonitrile-water solution, centrifuged at 13 000 ×  g for 5 min and filtered through a 0.22  μ m membrane filter. The filtrates were transferred to an auto-sampler vial and stored at 4 °C. A 5  μ L aliquot of each plasma or urine sample was injected for LC/MS analysis. Chromatography was performed on an AcQuity TM UHPLC system (Waters Corp., Milford, MA, USA) with a conditioned auto-sampler at 4 °C. The separation conditions and mass spectrometric detection methods are listed in, The hind paw volume (HPV) of all animal groups was measured by water displacement plethysmometer at 0, 13, 20, 24, 27 and 30 th day after the injection of FCA emulsion. The inhibited effects of Frankincense and Myrrh were determined by comparing the changes in volumes of hind paws and expressed in milliliter (mL ± SD). The BCA protein assay kit was obtained from Westang Bio-tech Co., LTD (Shanghai, China). Test protocols were listed in, Serum interleukin 2 (IL-2), tumor necrosis factor-α (TNF- α ) and PGE 2 levels were determined by avidin biotin peroxidase complex enzyme-linked immunosorbent assay (ABC-ELISA) kits. All of the ELISA test kits were used according to the manufacturers’ instructions. The procedure were discribed in detail in, NO was measured through the nitric oxide assay kit (nitrate reductase) following the instruction of the manufacturer. Nitric oxide is chemically active and can be rapidly oxidized to nitrite (NO 2 − ) and nitrate (NO 3 − ) in vivo, Nitrite can be further oxidized to nitrate. In this study, NO was determined by colorimetric method based on the nitrate to nitrite conversion through nitrate reductase. Samples preparation and test protocols were shown in, MDA was measured through the MDA kit following the instruction of the manufacturer. MDA is a product of lipid peroxidation and degradation, which can react with thiobarbituric acid (TBA) to form a red product for colorimetric assay (532 nm). Prepare the reagent as guidance of the kit. Obtain the serum and supernatant of tissue described above. Sample solutions and determination discribed in, UPLC/MS data were detected and noise-reduced in both the UPLC and MS domains such that only true analytical peaks were selected for further processing by the software and according to the method discribed in our previous study, The details are listed in, The identities of the potential biomarkers were confirmed by comparing their mass spectra and chromatographic retention times with the available reference standards and a full spectral library containing MS/MS data obtained in the positive and/or negative ion modes. The Mass Fragment application manager (Waters MassLynx v4.1, Waters corp., Milford, USA) was used to facilitate the MS/MS fragment ion analysis through the use of chemically intelligent peak-matching algorithms. This information was then used to search multiple databases and analyzed the potential metabolic pathway using MetPA (See ). Potential biological roles were evaluated by an enrichment analysis using MetaboAnalyst. Peripheral blood was taken from healthy volunteers. Mononuclear cells were isolated in a Ficoll–Hypaque (Pharmacia, Piscataway, NJ) density gradient using standard procedures. The buffy coat containing PBMCs was removed carefully following centrifugation and washed twice in RPMI 1640 medium containing 10% FCS (Sigma). Cells were counted and assessed for viability. Total RNA was isolated from treated PBMCs using Trizol reagent (Sigma, St Louis, MO, USA) following the protocol provided by the manufacturer. Real-time quantitative PCR was performed by using SYBR Green Master mix and Rox reference dye, according to the manufacturer’s instructions. The cDNAs were obtained from the reverse transcription of the RNA from rat brain tissues and astrocyte cells. The primers were listed below. SYBR green signal was detected by Mx3000ptm multiplex quantitative PCR machine. Transcript levels were quantified by using the ΔΔCt value method, Calculation was done by using the Ct value of GAPDH to normalize the Ct value of target gene in each sample to obtain the ΔΔCt value, which then used to compare among different samples. PCR products were analyzed by gel electrophoresis on a 1.5% agarose gel, and the specificity of amplification was confirmed by the melting curves. PBMCs were treated with the optimized doses of compounds 1–5 for the required time points. The cells were lysed with extraction buffer (20 mM HEPES, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM PMSF, 10  μ g/mL leupeptin, and 10  μ g/mL aprotinin). After 30 min at 4 °C, debris was eliminated by centrifugation at 14, 000 rpm for 20 min, and the supernatant was collected. Cell lysates were separated by 10% SDS-PAGE, transferred to polyvinylidene difluoride membranes, and blocked with 0.05% Tween 20 and 5% BSA overnight. The immunoblots were incubated with anti-phospho-specific extracellular signal-regulated kinase (ERK) Ab, anti-ERK Ab, anti-phospho-specific p38 Ab, anti-p38 rabbit Ab, anti-phospho-specific JNK rabbit Ab, or anti- JNK rabbit polyclonal Ab in PBS with 1% BSA for 1 h. Subsequently, the immunoblots were incubated with secondary antibody conjugated with HRP in 1% BSA in PBS, 0.1% Tween 20. After 1 h incubation at room temperature the bands are detected using chromogenic substrate. Statistical analysis Data are expressed as mean ± SEM, and statistical comparisons were carried out using one-way analysis of variance (ANOVA), followed by Student’s t -test. All quantitative data analyses were performed using the SPSS 11.5 software package for Windows. The results were expressed as the mean ± SD. P values less than 0.05 were considered significant. How to cite this article : Su, S. et al. Frankincense and myrrh suppress inflammation via regulation of the metabolic profiling and the MAPK signaling pathway. Sci. Rep.5, 13668; doi: 10.1038/srep13668 (2015). Supplementary Information: This work was supported by the Key Research Project in Basic Science of Jiangsu College and University (No.11KJA360002; 12KJA360002) and the National Natural Science Foundation of China (No.81373889; 81202862, 81102885). This work was also supported by the Construction Project for Jiangsu Key Laboratory for High Technology Research of TCM Formulae (BM2010576; BK2010561), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (ysxk-2014). And the Construction Project for Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization. This work also supported by 2013′ Program for New Century Excellent Talents by the Ministry of Education (Grant NCET-13-0873), 333 High-level Talents Training Project Funded by Jiangsu Province, and Six Talents Project Funded by Jiangsu Province (2012-YY-010). Author Contributions S.S., J.D. and T.C. designed and preformed the experiments.X.H., E.S., J.G. contributed reagents/materials/analysis tools.K.W. helped in taking care of the mice and follow-up.Y.Z. and L.Y. analyzed the microarray gene expression data.S.G., P.L., D.Q. and Y.T. analyzed the metabolic profiling data.S.S. and X.H. wrote the main manuscript text. All authors reviewed the manuscript.

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: Frankincense and myrrh suppress inflammation via regulation of the metabolic profiling and the MAPK signaling pathway

Is frankincense an anti-inflammatory?

Abstract – Recently, increasing interest in natural dietary and therapeutic preparations used as dietary supplements has been observed. One of them is frankincense. This traditional medicine of the East is believed to have anti-inflammatory, expectorant, antiseptic, and even anxiolytic and anti-neurotic effects.

The present study aims to verify the reported therapeutic properties of Boswellia resin and describe its chemical composition based on available scientific studies. The main component of frankincense is oil (60%). It contains mono- (13%) and diterpenes (40%) as well as ethyl acetate (21.4%), octyl acetate (13.4%) and methylanisole (7.6%).

The highest biological activity among terpenes is characteristic of 11-keto-ß-acetyl-beta-boswellic acid, acetyl-11-keto-ß-boswellic acid and acetyl-α-boswellic acid. Contemporary studies have shown that resin indeed has an analgesic, tranquilising and anti-bacterial effects.

From the point of view of therapeutic properties, extracts from Boswellia serrata and Boswellia carterii are reported to be particularly useful. They reduce inflammatory conditions in the course of rheumatism by inhibiting leukocyte elastase and degrading glycosaminoglycans. Boswellia preparations inhibit 5-lipoxygenase and prevent the release of leukotrienes, thus having an anti-inflammatory effect in ulcerative colitis, irritable bowel syndrome, bronchitis and sinusitis.

Inhalation and consumption of Boswellia olibanum reduces the risk of asthma. In addition, boswellic acids have an antiproliferative effect on tumours. They inhibit proliferation of tumour cells of the leukaemia and glioblastoma subset. They have an anti-tumour effect since they inhibit topoisomerase I and II-alpha and stimulate programmed cell death (apoptosis).

What has the best anti-inflammatory properties?

Anti-inflammatory foods – An anti-inflammatory diet should include these foods:

tomatoes olive oil green leafy vegetables, such as spinach, kale, and collards nuts like almonds and walnuts fatty fish like salmon, mackerel, tuna, and sardines fruits such as strawberries, blueberries, cherries, and oranges