Information

Is Hypericum Perforatum (St. John's wort) a C3 or C4 plant?


I've been curious as to which photosynthetic cycle St. John's wort uses to synthesise glucose and more complex carbohydrates. I know it probably won't be the crassulacean acid metabolism (CAM) cycle seeing how that applies mostly to plants that require a limited amount of water as opposed to St. John's wort which tends to prefer moisture. I suspect the answer to my question will be C3 seeing how they dominate in the plant kingdom and I know that St. John's wort requires relatively cool climates to grow successfully -- a trait characteristic of C3 plants seeing how high temperatures tend to cause excessive and wasteful photorespiration.


It's C3, according to the Illinois plant information network. (See CO₂ fixation on this page.) This is the best I could find.


Supplement and Herb Guide for Arthritis Symptoms

Learn about popular supplements for arthritis and how they may help with symptoms.

Interested in supplements to ease arthritis symptoms but don&rsquot know where to start? You&rsquove come to the right place. This guide provides thorough research of the most popular supplements and herbs used for arthritis to help you figure out what&rsquos right for you. While some of these supplements may help treat arthritis symptoms, nothing can substitute doctor-prescribed medications, a healthy diet and exercise. And remember &ndash always talk to your doctor before adding a new supplement to your regimen. Just because something is &ldquonatural&rdquo doesn&rsquot mean it&rsquos without side effects or safe to mix with your medication. For more tips on choosing safe supplements, read this article.

Avocado Soybean Unsaponifiables (ASU)

Origin: A natural vegetable extract made from one-third avocado oil and two-thirds soybean oil.

Claims: Slows the progression of osteoarthritis (OA).

What we know: ASU blocks pro-inflammatory chemicals, prevents deterioration of synovial cells (which line the joints) and may help regenerate normal connective tissue.

Studies: ASU has been studied extensively studied in Europe, where it is routinely used to treat OA. A 2003 study published in the Journal of Rheumatology reported ASU inhibited the breakdown of cartilage and promoted repair. A 2008 meta-analysis found that ASU improved symptoms of hip and knee OA and reduced or eliminated use of nonsteroidal anti-inflammatory drugs (NSAIDs). A large, three-year study published in 2013 in the BMJ showed that ASU significantly reduced progression of hip OA compared with placebo.

Dosage: Softgel take 300 mg daily.

*The French government has tracked ASU&rsquos safety record for more than 15 years and has yet to find any significant problems.

Black Currant Oil

Ribes nigrum see GLA

Origin: Black currant seed oil is obtained from seeds of the black currant. Black currant seed oil contains 15 to 20 percent gamma-linolenic acid (GLA). Do not confuse with black currant berry.

Dosage: Liquid and capsules typical dosage ranges from 360 mg to 3,000 mg daily.

*Black currant oil may increase immune response in the elderly.

Borage Oil

Borago officinalis see GLA

Origin: Oil from the seeds of the borage plant. Borage seed oil contains about 20 to 26 percent GLA.

Dosage: Liquid and capsules take 1,300 mg (oil) daily.

Boswellia Serrata

Frankincense, Boswellia, Boswellin, Salai Guggal also known as Indian Frankincense

Origin: Gum resin from the bark of the Boswellia tree found in India.

Claims: Reduces inflammation and treats rheumatoid arthritis (RA), osteoarthritis (OA) and bursitis symptoms. It&rsquos also used to treat symptoms of ulcerative colitis and Crohn&rsquos disease.

What we know: Boswellic acids, especially one called AKBA, are powerful anti-inflammatories. They block an enzyme called 5-lipoxygenase (5-LOX) that breaks down polyunsaturated fatty acids in foods into leukotrienes, inflammatory molecules that attack joints and other tissues. Boswellia may also help reduce cartilage damage in arthritis. It also shows promise as a cancer treatment.

Studies: A 2014 Cochrane Review found that a three-month course of 100 mg a day of enriched (AKBA) Boswellia reduced OA pain by nearly 20 points and improved function by eight points (on 100-point scales) without serious side effects. In a 2018 systematic review of 20 OA supplements, Boswellia extract was among the standouts, providing significant short-term relief for knee, hip and hand pain. The Natural and Alternative Treatments database, which contains over 12,000 clinical studies, also gives Boswellia high marks for OA pain relief.

Dosage: Capsule or tablet 100 mg daily for OA between 1200 to 3600 mg for RA. Look for 5-Loxin and AKBA-enriched Boswellia and for products with ingredients that enhance absorption like black pepper (piperine) or lecithin.

*Boswellia may increase side effects of other drugs, including antidepressants, anti-anxiety medications, ibuprofen and immunosuppressants.

*There&rsquos also some concern that Boswellia may stimulate the immune system, so use caution if you have RA or lupus.

Bromelain

Pineapple, Ananas comosus

Origin: A group of enzymes found in pineapple that break down protein.

Claims: Decreases pain and swelling of rheumatoid arthritis (RA) and osteoarthritis (OA) and increases mobility.

What we know: There is some evidence that enzymes like bromelain have pain-relieving and anti-inflammatory effects comparable to NSAIDs.

Studies: A 2005 German study showed that bromelain activated immune cells that fight infection. A 2004 review published in Evidence-Based Complementary and Alternative Medicine found that bromelain showed promise as a treatment for OA but said more studies were needed.

Dosage: Capsules and tablets take 500 mg to 2,000 mg three times a day between meals.

*Bromelain can cause stomach upset and diarrhea and should be avoided if you are allergic to pineapples.

* It can also increase the effect of blood-thinning medicine.

Capsaicin

Capsicum frutescens

Origin: The highly purified, heat-producing component in chili peppers.

What we know: Applied as a topical cream, gel or patch, capsaicin activates specific nerve receptors causing local heat, stinging and/or itching sensations. Prolonged activation of these receptors causes them to lose their ability to function properly (and process pain signals) for extended periods of time. Capsaicin must be used regularly to keep the nerve receptors from working properly and processing pain signals.

Studies: Many studies have shown that capsaicin effectively reduces pain from osteoarthritis (OA), rheumatoid arthritis (RA) and fibromyalgia. In a 2010 German study, joint pain decreased nearly 50 percent after three weeks' use of 0.05 percent capsaicin cream.

Dosage: Most capsaicin products &ndash such as Zostrix, Zostrix HP, Capzasin-P and others &ndash contain between 0.025 to 0.075 percent concentrations. Apply regularly three times daily.

*Capsaicin can cause burning and irritation. Avoid applying it near your eyes or on sensitive skin.

Cat's Claw

Uncaria tomentosa

Origin: The bark and root of a woody vine that grows in parts of South and Central America.

Claims: Believed to have anti-inflammatory properties may stimulate the immune system.

What we know: Cat's claw is an anti-inflammatory that inhibits tumor necrosis factor (TNF), a target of powerful rheumatoid arthritis (RA) drugs. It also contains compounds that may benefit the immune system.

Studies: In 2002, the Journal of Rheumatology published a randomized double-blind study of cat&rsquos claw for the treatment of RA. Researchers found that in 40 people with RA, the supplement reduced joint swelling and pain by more than 50 percent compared to placebo.

Dosage: Capsules, tablets, liquid and tea bags 250 mg to 350 mg capsule daily for immune support. Buy only products that contain uncaria tomentosa at least a dozen unrelated plants are also called &ldquocat&rsquos claw.&rdquo Look for a brand that is free of tetracyclic oxindole alkaloids (TOAs).

*Cat&rsquos claw can cause headache, dizziness and vomiting, and can lower blood pressure, so don&rsquot use if taking an anti-hypertensive medication or blood thinner. *Do not use if you have tuberculosis or are taking drugs that suppress the immune system.

Cannabidiol

Origin: One of numerous compounds known as cannabinoids in the cannabis plant. Unlike THC, it does not have an intoxicating effect.

Claims: Reduces pain and inflammation eases anxiety and depression aids sleep

What we know: Evidence is promising that CBD may have pain-relieving properties and that it may help ease anxiety and help sleep, but more research is needed.

Studies: Animal studies indicate that CBD may help temporary reduction in pain and inflammation, but these findings have not been validated in human studies. One study showed that a synthetically derived CBD product was effective for osteoarthritis knee pain.

Dosage: CBD products come in topical, edible and inhalable forms, with pros and cons to each. Many products are derived from hemp, a form of cannabis containing 0.3% or less of THC, the psychoactive compound in cannabis. While there are no established clinical guidelines, experts advise starting with a product containing a few milligrams of CBD in a sublingual form (under the tongue) twice a day. If needed, increase in small increments over several weeks, and depending on the laws in your state, discuss with your doctor whether to try a form containing small amounts of THC. It may interact with some arthritis drugs. Learn more in the Arthritis Foundation&rsquos CBD Guidance for Adults With Arthritis.

Chondroitin Sulfate

Origin: Chondroitin is a component of human connective tissues found in cartilage and bone. In supplements, chondroitin sulfate usually comes from animal cartilage.

Claims: Reduces pain and inflammation, improves joint function and slows progression of osteoarthritis (OA).

What we know: Believed to enhance the shock-absorbing properties of collagen and block enzymes that break down cartilage. Helps cartilage retain water and may reverse cartilage loss when used with glucosamine.

Studies: In 2016, a large multinational trial found combined glucosamine and chondroitin to be as effective at reducing pain, stiffness and swelling in knee OA as the NSAID celecoxib, without the cardiovascular and GI side effects of celecoxib. But another 2016 analysis of eight trials of glucosamine and chondroitin found equal numbers of studies showing positive effects and neutral effects. A 2011 study showed a significant improvement in pain and function in patients with hand OA using chondroitin alone. Benefits of chondroitin and glucosamine remain controversial, but the supplements appear extremely safe.

Dosage: Capsules, tablets and powder. Often combined with glucosamine. Phillip Barr, MD, a physician at Duke Integrative Medicine in North Carolina recommends 500 mg to 3 grams of glucosamine and chondroitin a day in divided doses. He also advises patients to stay on the

supplements for at least a month to see if they take effect. After a month, they may stop taking them if they don&rsquot see any improvement.

*Some chondroitin tablets may contain high levels of manganese, which could be problematic with long-term use.

*Chondroitin taken with blood-thinning medication like NSAIDs may increase the risk of bleeding.

* If you are allergic to sulfonamides, start with a low dose of chondroitin sulfate and watch for any side effects. Other side effects include diarrhea, constipation and abdominal pain.

*Because chondroitin is made from bovine products, there is the remote possibility of contamination associated with mad cow disease.

Curcumin

Curcuma longa also known as Turmeric

Origin: The main chemical in turmeric, the root of a plant related to ginger. Turmeric is a staple of traditional Chinese and Indian (Ayurvedic) medicine and a prime ingredient in curries.

Claims: Reduces pain, inflammation and stiffness related to rheumatoid arthritis (RA) and osteoarthritis (OA) treats bursitis.

What we know: A potent anti-inflammatory, curcumin blocks inflammatory cytokines and enzymes, including 5-LOX and cyclooxygenase-2 (COX-2), the target of the drug celecoxib.

Studies: In 2016, an industry-sponsored systematic review of randomized controlled trials found that 1,000 mg a day of curcumin reduced OA pain and inflammation as well as nonsteroidal anti-inflammatory drugs (NSAIDs) like diclofenac and ibuprofen. Another 2016 study suggests curcumin might help prevent bone breakdown in people with RA.

Dosage: Choose curcumin extract &ndash whole turmeric is often contaminated with led. 500 mg capsules twice daily. Curcumin makes up only a small percentage of turmeric and can be hard to absorb. Be sure to check the standardized amount of curcumin when looking for a supplement, and choose brands that use phospholipids (Meriva, BCM-95), antioxidants (CircuWin) or nanoparticles (Theracurmin) for better absorption.

*High doses of turmeric can act as a blood thinner and cause upset stomach.

*Avoid turmeric/curcumin if you take blood thinners such as warfarin (Coumadin), are about to have surgery, are pregnant or have gallbladder disease.

Devil's Claw

Harpagophytum procumbens also known as Devil&rsquos Claw Root, Grapple Plant or Wood Spider

Origin: A traditional herb used in South Africa.

Claims: Relieves pain and inflammation. May help lower uric acid levels in people with gout. Acts as a digestive aid and appetite stimulant.

What we know: Harpagoside, the active ingredient in devil&rsquos claw, appears to reduce pain and inflammation in joints. Some studies suggest stomach acid may counteract benefits, so take the supplement between meals when less stomach acid is released.

Studies: In 2002, Phytomedicine published a study of 227 people with non-specific low back pain or osteoarthritis (OA) of the knee or hip treated with devil&rsquos claw extract. After eight weeks of taking 60 mg daily, between 50 and 70 percent of people reported improvement in pain, mobility and flexibility.

Dosage: Capsules, tincture, powder and liquid take 750 mg to 1,000 mg three times a day.

*Do not take devil&rsquos claw if you are pregnant, have gallstones or ulcers, or are taking an antacid or blood thinners.

*It can affect heart rate and may interfere with cardiac, blood-thinning and diabetes medication.

*It may also cause diarrhea.

Dehydroepiandrosterone

Origin: An androgen steroid hormone naturally produced in the body by the adrenal glands. Do not confuse 7-Keto DHEA with DHEA.

Claims: Helps control lupus flares increases the blood level of DHEA.

What we know: Natural DHEA levels have been found to be low in people with rheumatoid arthritis (RA) and lupus, particularly in postmenopausal women, as well as men with ankylosing spondylitis, perhaps due to corticosteroid use. DHEA may help regulate the immune system and control inflammation.

Studies: In people with lupus, DHEA treatment may reduce disease activity and flares. Studies found it allowed doctors to lower women&rsquos corticosteroid dosages. DHEA also appeared to counteract bone loss caused by medication and increase bone density. Long-term safety, overall effectiveness and appropriate dosages have not been established.

Dosage: Capsule and tablets available both as prescription (200 mg) and non-prescription (10-, 15- or 25-mg) products typically 200 mg for lupus. Do not take doses higher than 25 mg without advice from a physician. Effects of long-term use are unknown.

*DHEA side effects include stomach upset, abdominal pain and high blood pressure, as well as acne, facial hair growth, voice deepening, and changes in menstrual pattern.

*It also decreases levels of &ldquogood&rdquo cholesterol (high-density lipoprotein, or HDL).

*DHEA can also increase insulin resistance for people with diabetes and exacerbate liver disease.

*Use is contraindicated in men with prostate cancer and women with uterine fibroids.

Dimethyl Sulfoxide see MSM

Origin: A colorless, sulfur-containing organic by-product of wood pulp processing.

Claims: Relieves pain and inflammation, improves joint mobility in osteoarthritis (OA), rheumatoid arthritis (RA), juvenile idiopathic arthritis (JIA) and scleroderma, and manages amyloidosis (excessive build-up of protein in organs as seen in RA). Increases blood flow to skin.

What we know: Topically, appears to be an anti-inflammatory.

Studies: Controlled studies as a topical application for DMSO and OA have yielded conflicting results. Few human studies.

Dosage: Cream, gel topically, 25 percent DMSO solution take internally, only if prescribed by a physician.

*Side effects of DMSO taken internally include headache, dizziness, drowsiness, nausea, vomiting, diarrhea, constipation and anorexia.

*Topical DMSO also can cause skin irritation and dermatitis.

*Do not use DMSO if you have diabetes, asthma or liver, kidney or heart conditions.

*Never take industrial-grade DMSO. Wash off any lotions or skin products before applying DMSO.

Evening Primrose

Oenothera biennis and other Oenothera species also known as Primrose see GLA

Origin: The seeds of a native American wildflower, containing 7 to 10 percent gamma-linolenic acid (GLA).

Dosage: Capsules, oil and softgel generally five 500-mg capsules per day. For rheumatoid arthritis (RA), 540 mg daily to 2.8 g daily in divided doses. Evening primrose oil may take up to six months to work.

Fish Oil

Origin: Oil from cold-water fish such as mackerel, salmon, herring, tuna, halibut and cod, which are all rich sources of the omega-3 fatty acids, EPA and DHA. Your body can&rsquot make them, so you need to get them from fatty fish or supplements.

Claims: Reduces inflammation and morning stiffness. Treats rheumatoid arthritis (RA), lupus, psoriasis, depression and Raynaud&rsquos phenomenon. Important for brain function and may inhibit RA development.

What we know: Omega-3s block many sources of inflammation, including prostaglandins (pro-inflammatory cytokines) and inflammation-causing leukotrienes. They&rsquore also converted by the body into powerful anti-inflammatory chemicals.

Studies: Fish oil has been extensively studied for dozens of inflammatory and autoimmune disorders. A 2017 systematic review looked at the benefits of fish oil for RA, lupus and osteoarthritis (OA). Of 20 trials involving RA, almost all found that fish oil &ndash in daily doses ranging from 0.2 to nearly 5 grams of EPA and 0.2 to 2.1 grams of DHA &ndash significantly reduced joint pain, stiffness and swelling, and reduced or eliminated patients&rsquo use of NSAIDs. Five of seven studies found fish oil improved lupus disease activity in the short-term. Four studies for OA were inconclusive, but other research has found fish oil helps reduce OA pain. It&rsquos also an effective treatment for gout and may even help prevent gout flares. Some dermatologists recommend fish oil for their patients with psoriatic arthritis because it may help both skin and joint symptoms.

Dosage: Fish, capsules, softgels, chewable tablets or liquid. For general health, two 3-ounce servings of fish a week are recommended, but supplements are the best source to get a therapeutic dose of fish oil. Plus, fish is more likely to be contaminated with mercury and PCBs, which are less common in high-quality supplements. Use fish oil capsules with at least 30 percent EPA/ DHA, the active ingredients. For lupus and psoriasis, 2 g EPA/DHA three times a day. For RA and OA, up to 2.6 g, twice a day. For depression, 6.6 g daily. For psoriasis, at least 1,000 mg a day. Prescription fish oils contain high doses of EPA and DHA you can get the same amounts over the counter, just look for &ldquohigh potency.&rdquo Philip Barr, MD, a physician at Duke Integrative Medicine in North Carolina, recommends the Nordic Naturals brand for potency and purity.

* Check with your doctor if you&rsquore taking blood-thinners because fish oil could increase their effect.

* Due to potentially dangerous levels of mercury, women who are pregnant or hoping to conceive should avoid eating shark, swordfish, king mackerel and tilefish and should eat no more than 8 ounces of albacore tuna each month.

Flaxseed

Linum usitatissimum also known as Flax, Flaxseed Oil, Linseed Oil

Origin: Seed of the flax plant, containing omega-3 and omega-6 fatty acids and lignans (beneficial plant compounds, similar to fiber).

Claims: Eases symptoms of rheumatoid arthritis (RA), lupus and Raynaud&rsquos phenomenon. Lubricates joints and lessens stiffness and joint pain. Lowers total cholesterol and reduces risk of heart disease and some types of cancer. Improves hot flashes and dry skin.

What we know: Flaxseed is high in alpha-linolenic acid (ALA), a type of omega-3 fatty acid that can be converted to EPA and DHA (the active ingredients in fish oil). Flaxseed is a good source of fiber.

Studies: Lack of studies on whether flaxseed improves symptoms of RA, but omega-3 fatty acids are known anti-inflammatories. Mixed results about whether flaxseed or flaxseed oil can accelerate or slow progression of prostate cancer.

Dosage: Whole seeds, ground meal or flour, capsules or oil. Whole seeds must be ground into meal or flour 30 g (1 ounce) daily. Capsules, available in 1,000 mg to 1,300 mg, no typical dosage. Oil, 1 to 3 tablespoons daily.

*Fiber in flaxseed can impair absorption of some medications.

*Flaxseed acts as a blood thinner, so beware when taking blood thinners, aspirin or other NSAIDs.

*Avoid flaxseed if you have hormone-sensitive breast or uterine cancer.

* Use with caution if you have high cholesterol and are taking cholesterol-lowering drugs.

Ginger

Zingiber officinale

Origin: The dried or fresh root of the ginger plant.

Claims: Decreases joint pain and reduces inflammation in people with osteoarthritis (OA) and rheumatoid arthritis (RA). Increases circulation in people with Raynaud&rsquos phenomenon.

What we know: Ginger has been shown to have anti-inflammatory properties similar to ibuprofen, the COX-2 inhibitor celecoxib and even TNF blockers. It also switches off certain inflammatory genes, which may make it an effective pain reliever and a potential treatment for autoimmune disorders and cancer.

Studies: Studies have confirmed that a daily dose of 500 to 1,000 mg of ginger extract can modestly reduce pain and disability in hip and knee OA. In animal models, larger daily doses of ginger extract &ndash the equivalent of up to 4 grams in humans &ndash had strong anti-inflammatory effects in RA. Recent research suggests that ginger extract containing both gingerols &ndash the active part of ginger &ndash and ginger essential oils may be more effective for chronic inflammation than gingerols alone.

Dosage: Powder, extract, tincture, capsules and oils, up to 2 g in three divided doses per day or up to 4 cups of tea daily. In studies, 255 mg of Eurovita Extract 77 (equivalent to 3,000 mg dried ginger) twice daily. ConsumerLab recently found that only half of reviewed ginger products met quality standards. Philip Barr, MD, a physician at Duke Integrative Medicine in North Carolina suggests medical-grade supplements by Thorne Research and Pure Encapsulations.

* Ginger can interfere with medications for blood thinning.

* It should not be used if you have gallstones.

Ginko

Ginkgo biloba

Origin: Leaf of the ginkgo biloba tree, native to East Asia.

Claims: Increases blood flow and circulation in Raynaud&rsquos phenomenon and claudication (pain in legs or arms caused by reduced circulation due to blocked arteries).

What we know: There is no strong evidence that ginkgo improves mood, menopause symptoms, memory or fatigue.

Studies: Results from a 2002 double-blind, placebo-controlled trial of ginkgo for the treatment of Raynaud&rsquos phenomenon showed that the supplements could reduce the number of disease flares by 56 percent.

Dosage: Liquid, tablet, softgel and capsule and extract typically 120 mg to 240 mg extract daily. Choose supplements standardized to 5- to 7-percent terpene lactones and 24-percent flavonol glycosides, the active ingredients in ginkgo.


*Ginkgo&rsquos side effects include stomach upset, dizziness or headaches.

*Do not take ginkgo if you are taking blood-thinning medication like aspirin, have epilepsy or experience seizures, have diabetes or are scheduled for surgery.

Gamma-Linolenic Acid (GLA)

Origin: A type of omega-6 fatty acid found in evening primrose oil, black currant oil and borage oil.

Claims: Lessens joint pain, stiffness and swelling associated with rheumatoid arthritis (RA). Eases symptoms of Raynaud&rsquos phenomenon and Sjögren&rsquos syndromes.

What we know: Unlike other omega-6 oils, GLA is a potent anti-inflammatory. It blocks enzymes that convert substances from food into leukotrienes (inflammatory molecules) and may stop the body from switching on pro-inflammatory genes.

Studies: In a small 2017 study, RA patients were randomized to receive fish oil supplements, fish oil plus evening primrose oil or no supplements. Disease activity and joint tenderness decreased significantly in the first two groups but not the third. Another trial compared fish oil, borage oil and a combination of

the two in RA patients. All three groups showed significant reductions in disease activity after nine months. In 2011, Australian researchers updated a Cochrane Review of potentially useful herbal therapies for RA. Of 22 studies of different supplements, seven found that GLA from evening primrose, borage or black current seed oil showed potential for relieving pain and disability.

Dosage: Capsules or oil 2 g to 3 g daily in divided doses. Consumer Lab reports that many of the seed oil supplements it tests are rancid. Be sure to check the expiration date.


* May increase bleeding risk avoid if you use blood thinners such as warfarin (Coumadin).

Glucosamine

Glucosamine sulfate, glucosamine hydrochloride, N-acetyl glucosamine

Origin: Major component of joint cartilage. Supplements are derived from the shells of shellfish (such as shrimp, lobster and crab) or from vegetable sources.

Claims: Slows deterioration of cartilage, relieve osteoarthritis (OA) pain and improves joint mobility.

What we know: Glucosamine produced in the body provides natural building blocks for growth, repair and maintenance of cartilage. Like chondroitin, glucosamine may lubricate joints, help cartilage retain water and prevent its breakdown.

Studies: In 2016, a large multinational trial found combined glucosamine and chondroitin to be as effective at reducing pain, stiffness and swelling in knee OA as the NSAID celecoxib, without the cardiovascular and GI side effects of celecoxib. But another 2016 analysis of eight trials of glucosamine and chondroitin found equal numbers of studies showing positive effects and neutral effects. Mayo clinic researchers say evidence supports trying glucosamine sulfate &ndash not glucosamine hydrochloride &ndash with or without chondroitin sulfate for knee OA, especially because the supplements are &ldquoinexpensive and side effects are few and far between.&rdquo

Dosage: Capsules, tablets, liquid or powder (to be mixed into a drink). Often combined with chondroitin. Phillip Barr, MD, a physician at Duke Integrative Medicine in North Carolina recommends 500 mg to 3 grams of glucosamine and chondroitin a day in divided doses. He also advises patients to stay on the

supplements for at least a month to see if they take effect. After a month, they may stop taking them if they don&rsquot see any improvement.

*Glucosamine may cause mild upset stomach, nausea, heartburn, diarrhea and constipation, as well as increased blood glucose, cholesterol, triglyceride and blood pressure.

*Don&rsquot use glucosamine if you are allergic to shellfish.

*People with glaucoma or intraocular hypertension may also have worsening eye pressure if they take a glucosamine supplement.

Green-lipped Mussel

Perna canaliliculus

Origin: Extract from a New Zealand mussel.

What we know: Mussels are rich in omega-3 fatty acids, and because they reduce the production of prostaglandins (pro-inflammatory cytokines) and inflammation-causing leukotrienes, may have the same anti-inflammatory effects as fish oil.

Studies: Results of both human and animal studies have been mixed. A 2006 Clinical Rheumatology review found benefit for both osteoarthritis (OA) and rheumatoid arthritis (RA) in two of five clinical trials. A 2008 review of trials for OA found benefit when green-lipped mussels were added to conventional therapies. Unlike NSAIDs, which can harm the stomach, green-lipped mussel extract may help heal ulcers.

Dosage: Capsules, extract: 300-350 mg three times daily of extract for RA 900-1200 mg daily for OA.

Indian Frankinsense

Melatonin

Origin: A hormone produced by the pineal gland, which is located at the base of the brain.

Claims: Aids sleep and treats jet lag.

What we know: A potent antioxidant, melatonin regulates sleep/wake cycles. It appears to treat insomnia and sleep disturbances related to conditions like fibromyalgia and depression. Aspirin and other NSAIDs can decrease melatonin levels.

Studies: A systematic review of studies shows no evidence that melatonin effectively treats sleep disorders or is useful for altered sleep patterns, such as from shift work or jet lag. However, there is evidence that it is safe with short-term use. Another review of studies showed that melatonin reduced the onset of sleep by four minutes and increased the duration of sleep by nearly 13 minutes another showed that people taking melatonin slept almost 30 minutes longer than people taking placebo.

Dosage: Capsules or tablets 1 mg to 5 mg at bedtime for insomnia, for no longer than two weeks.


*Higher doses or long-term usage require doctor supervision.

*Certain medications interact with it, including NSAIDs, beta-blockers, antidepressants, diuretics and vitamin B-12 supplements.

*Do not take melatonin with alcohol or caffeine.

*Do not use if you have an autoimmune disease or if you have depression, kidney disease, epilepsy, heart disease or leukemia.

Methylsulfonylmethane

Origin: Organic sulfur compound found naturally in fruits, vegetables, grains, animals and humans.

Claims: Reduces pain and inflammation.

What we know: MSM is an organic sulfur compound. Sulfur is needed to form connective tissue. MSM also seems to act as an analgesic by lessening nerve impulses that transmit pain.

Studies: A 2006 pilot study of 50 men and women with knee OA showed that 6,000 mg of MSM improved symptoms of pain and physical function without major side effects. No large, well-controlled human studies have been performed.

Dosage: Tablets, liquid, capsule or powder, topical and oral. Typically 1,000 mg to 3,000 mg daily with meals.

*MSM may cause stomach upset or diarrhea. Don&rsquot use MSM if you are taking blood thinners.

Pine Bark

Origin: An extract from the bark of the French maritime pine tree

What we know: Pycnogenol contains procyandin, a powerful antioxidant, and also seems to inhibit pro-inflammatory enzymes, including COX 1 and COX 2.

Studies: A small 2007 University of Arizona study found pycnogenol reduced osteoarthritis (OA) pain by 43 percent and stiffness by 25 percent. Two 2008 studies showed significant improvements in joint pain, stiffness and function that persisted after the supplement was stopped. But a 2012 review concluded that these studies were poorly designed and underpowered.

Dosage: Capsules, tablets: In studies, 100 to 150 mg daily.

Probiotics

Origin: Live bacteria and yeasts that may help prevent and treat disease by keeping your digestive tract and immune system healthy.

What we know: Microbes &ndash bacteria, yeast and viruses &ndash living in your gut, collectively called the microbiome, influence health and disease through complex interactions with your immune system. When these microbial communities get out of whack &ndash through poor diet, antibiotic use, stress or illness &ndash the result can be immune, metabolic and even mental health problems. Probiotics (the live microbes and yeast in supplements) or prebiotics (foods that promote the growth of good microbes, including yogurt, sauerkraut, miso and other fermented foods) may help restore a healthy microbiome and normalize immune function. Scientists believe some types of probiotics are effective for diseases like rheumatoid arthritis (RA), but no one is exactly sure what those are (or even what a healthy microbiome looks like). However, it&rsquos believed that, for most people, consuming a probiotic supplement may have a beneficial effect on health.

Studies: The human microbiome &ndash and by association, probiotics &ndash has been widely studied, but there is still much to learn. One meta-analysis of more than 150 studies involving more than 10,000 people concluded that probiotics successfully treat and prevent a wide range of GI problems, including irritable bowel syndrome (IBS), antibiotic-associated diarrhea, C. difficile infection and H. pylori infection. The most effective probiotic in the analysis was VSL#3, which is

commonly used in clinical trials. Many animal trials, including a Canadian study using VSL#3, have shown that probiotics can quell inflammation in RA. But human studies have been inconsistent, says Veena Taneja, PhD, a researcher at Mayo Clinic in Rochester, Minnesota. Many studies have used a single Lactobacillus or Bifidobacterium species, with only modest success. &ldquoSupplementation with multistrain synbiotic [probiotics plus prebiotics] bacteria in RA patients have shown some benefit,&rdquo she says. In the future, successful probiotic treatments will likely use patients&rsquo own microbes, she adds.

Dosage: Many probiotic bacteria are sensitive to heat and moisture. Refrigerate those that need it, and have online orders shipped overnight with ice. Or look for freeze-dried capsules or tablets in blister packs they don&rsquot need refrigeration. Phillip Barr, MD, a physician at Duke Integrative Medicine in North Carolina, suggests trying a generic probiotic that contains both Lactobacillus and Bifidobacterium along with a probiotic yeast called Saccharomyces boulardii, which has been shown to reduce inflammation and help regulate immune function.

*The latest research shows that the fastest route to a healthier gut may be through your diet. You can shift your microbiome in as little as 24 hours by eating more plants and less meat, sugar and processed food.

Rose Hips

Rosa canina

Origin: The seed pods of roses.

What we know: Rose hips powder &ndash a rich source of vitamin C &ndash appears to decrease inflammation by inhibiting production of inflammatory proteins and enzymes, including COX-1 and COX-2.

Studies: Animal and in vitro studies have shown that rose hips have anti-inflammatory, disease-modifying and antioxidant properties, but results of human trials are preliminary. A 2008 meta-analysis of three clinical trials showed rose hips powder reduced hip, knee and wrist pain by about one-third in nearly 300 osteoarthritis patients and a 2013 trial found that conventional rose hips powder relieved joint pain almost as effectively as an enhanced version. In a 2010 trial of 89 patients, rose hips improved rheumatoid arthritis (RA) symptoms better than a placebo.

Dosage: Capsules, powder: up to 5 g daily in divided doses for capsules. Most research used a standardized form of rose hips from Denmark sold under the names Litozin and Hyben Vital.`

S-adenosyl-L-methionine

Origin: A naturally occurring chemical in the body.

Claims: Treats pain, stiffness and joint swelling improves mobility rebuilds cartilage and eases symptoms of osteoarthritis (OA), fibromyalgia, bursitis, tendinitis, chronic low back pain and depression.

What we know: SAM-e is an effective anti-inflammatory and analgesic for people with OA. Results may be felt in just one week but could take more than a month. SAM-e works closely with vitamins B-12, B-6 and folate, so it is important to get enough of the B vitamins when taking this supplement.

Studies: Over the last two decades, multiple clinical trials involving thousands of people have shown SAM-e to improve joint health and help with the symptoms of OA. A 2002 analysis of 14 SAM-e studies showed it is effective for reducing pain and improving mobility in people with OA. A 2004 University of California, Irvine study found SAM-e equal to the prescription drug celecoxib (Celebrex) and a 2009 study found it comparable to the NSAID nabumetone. Fibromyalgia findings have been mixed, but in two European trials (where it is sold as a drug, not a supplement), SAM-e improved symptoms better than placebo. Extensive research has also found SAM-e effective for depression. In a 2010 Massachusetts General Hospital study, SAM-e combined with antidepressants significantly increased remission rates in patients with major depression who failed conventional therapy.

Dosage: Capsules (preferably in blister pack): 600 to 1,200 mg daily in three divided doses for OA 200 to 800 mg twice daily for fibromyalgia 800 to 1,600 mg twice daily for depression. Because of possible interactions, SAM-e should not be taken without doctor&rsquos supervision.

*High doses of SAM-e can cause flatulence, vomiting, diarrhea, headache and nausea.

*SAM-e may interact with antidepressant medications and should be avoided if you have bipolar disorder or are taking monoamine oxidase inhibitors (MAOIs).

*It may also worsen Parkinson&rsquos disease.

St. John's Wort

Hypericum perforatum

Origin: The yellow flower, leaves and stem of the St. John&rsquos wort plant, is native to Europe and grows wild in the U.S.

Claims: Acts as an antidepressant drug and reduces inflammation and pain.

What we know: St. John&rsquos wort&rsquos mood-elevating properties were believed to be from active ingredients hypericin and hyperforin, chemicals that raise levels of serotonin, a chemical found in the brain. Serotonin levels may be low in people who are depressed and possibly in those who have fibromyalgia. New research, however, suggests the whole preparation (not just the two active ingredients) is more effective.

Studies: No scientific evidence shows that St. John&rsquos wort is effective for reducing inflammation. A Cochrane Review of studies on St. John&rsquos wort for depression showed that current evidence is inconsistent. A study also found that the herb is not effective for social anxiety disorder.

Dosage:
Extract in the form of powder (dried), liquid (10 to 60 drops one to four times per day) or tablet, capsules and tea extract, typically 900 mg daily.


*Although St. John&rsquos wort taken alone is considered safe, it is potentially dangerous if taken with prescription antidepressants.

*St. John&rsquos wort can cause insomnia, restlessness, anxiety, irritability, stomach upset, fatigue, dry mouth, dizziness or increased sensitivity to sunlight.

*Consult your physician before taking St. John&rsquos wort if you are already taking any kind of prescription medications.

*Do not take it if you have Alzheimer&rsquos disease, HIV, depression, schizophrenia, infertility or bipolar disorder.

*It may also reduce effectiveness of oral contraceptives.

Stinging Nettle

Urtica dioica

Origin: The leaves and stem of the stinging nettle plant, a stalk-like plant found in the U.S., Canada and Europe.

Claims: Reduces inflammation, aches and pains of osteoarthritis (OA).

What we know: The antimicrobial, antioxidant, analgesic and anti-ulcer properties of stinging nettle have been studied in Germany and Turkey. Stinging nettle is high in potassium, calcium and magnesium and may be helpful for gout.

Studies: A German study shows that hox alpha, a new extract of stinging nettle leaf, contains an anti-inflammatory substance that suppressed several cytokines in inflammatory joint diseases. In a Turkish study, stinging nettle extract showed antimicrobial effects against nine microorganisms, as well as anti-ulcer and analgesic activity. Stinging nettle root extract combined with sabal fruit extract was shown to be superior to placebo for treating prostate hyperplasia (a precancerous condition) and was well tolerated.

Dosage: Tea, capsule, tablet, tincture, extract or whole leaf capsules, up to 1,300 mg daily tea, 1 cup, three times a day tincture, 1 ml to 4 ml three times a day nettle leaf applied directly to the skin.


*Nettle may interfere with blood thinners, diabetes and heart medications, and lower blood pressure

Thunder God Vine

Tripterygium wilfordii

Origin: Root of a vine-like plant from Asia.

Claims: Reduces pain and inflammation and treats symptoms of RA, lupus and other autoimmune diseases.

What we know: Used in Chinese medicine for years, thunder god vine shows evidence of suppressing the immune system.

Studies: A 2006 review of randomized clinical trials shows that thunder god vine improved symptoms of rheumatoid arthritis (RA) but serious side effects occurred. A 2009 study of 121 RA patients conducted in the United States found that those who received 60 mg three times a day of thunder god vine had better response than those who received 1 g twice a day of sulfasalazine. And a 2014 Chinese study of 207 RA patients found that those who received 20 mg pills three times a day of thunder god vine did not do worse those receiving 12.5 mg per week of methotrexate. Patients who received both the thunder god vine and the methotrexate had the best response of all.

Dosage: Extract up to 60 mg three times a day.

*This root can cause stomach upset, skin reactions, temporary infertility in men and amenorrhea (lack of menstruation) in women.

*It should not be used if you are taking immunosuppressive drugs, like prednisone.

*The leaves and flowers of this plant are highly toxic and can cause death, so preparation should only be made from the root.

Tumeric

Valerian

Valeriana officianalis

Origin: The dried root of the perennial herb valerian.

Claims: Treats insomnia and eases pain has antispasmodic and sedative effects.

What we know: Valerian works as a mild sedative and sleep agent, but it takes two to three weeks to see effect. No known effects on muscle or joint pain and arthritis.

Studies: A randomized, placebo-controlled trial of 184 adults showed two tablets per night for 28 nights produced significant improvements in sleep and quality of life.

Dosage: Capsules, tablets, tincture, softgel or tea 300 mg to 500 mg of valerian extract daily (maximum dose is 15 g of root per day). For insomnia and muscle soreness, take 1 teaspoon of liquid extract diluted in water or a 400 mg to 450 mg capsule, tablet or softgel 30 to 45 minutes before bedtime or as needed. For a milder effect, drink a cup of valerian tea before bed. Avoid powdered valerian root.


*Valerian may cause headache, excitability, uneasiness and insomnia.

*Do not drive or operate machinery while taking it, and do not take with alcohol, barbiturates, tranquilizers or other sedative-type drugs or herbs.

*Do not use valerian longer than one month, or if you have liver disease.


Introduction

Hypericum perforatum (St. John's wort) has been used for centuries in herbal treatment of bacterial and viral infections, respiratory diseases, skin wounds, peptic ulcera, inflammation and mild depression 1 . Hyperforin, isolated from its flowering parts, is the most studied natural component of this plant and has been reported to induce apoptosis in tumor cells 2 and to inhibit tumor cell growth 3 , cancer invasion and metastasis 4 , as well as angiogenesis 5 . In addition, hyperforin is used as “herbal Prozac” to treat mild to moderate depression 6 , reveals antibiotic 7 and antimalarial 8 activity and induces hepatic drug metabolism by activating the cytochrome P450 system via high affinity binding to the steroid- and xenobiotic-sensing nuclear pregnan X receptor (PXR) 9 , making it a critical candidate in drug interaction.

The mechanisms of hyperforin actions are not yet understood, but may include inhibition of 5-lipoxygenase 10 , high affinity binding to the pregnane X receptor 9 , release of Ca 2+ and/or Zn 2+ from intracellular stores 11,12 and affecting of presynaptic and vesicular uptake, storage and release of neurotransmitters such as serotonin, dopamine, norepinephrine, acetylcholine, GABA and glutamate 13,14,15,16,17,18 . Gobbi et al. 14 proposed an impairment of the monoamine storage due to a reserpine-like inhibition of the vesicular monoamine transporter (VMAT). But, whereas reserpine potently targets VMATs in the SLC18 gene family 19 for serotonin, epinephrine, norepinephrine and dopamine, hyperforin additionally affects acetylcholine 18 , substrate of the vesicular acetylcholine transporter (VAChT, also SLC18 gene family) and GABA and glutamate 15,16 , substrates of the vesicular inhibitory amino acid transporters (VIAATs, SLC32 gene family) 20 and the vesicular glutamate transporters (VGluTs, SLC17 gene family) 21 . Storage of neurotransmitters heavily relies on a vesicular electrical and proton gradient, produced by a H + -ATPase, utilizing energy to pump H + continuously into synaptic vesicles. The neurotransmitters can then be concentrated against a gradient in exchange of two protons leaving the vesicle 19 . It has been shown that acidification of isolated synaptic vesicles monitored by acridine orange fluorescence quenching was abolished in the presence of hyperforin 16,22 and it was suggested that dissipation of the H + gradient by a protonophore-like activity eliminates the driving force for neurotransmitter uptake into the vesicles 16,22 .

Not just vesicular, but also plasma membrane transport of neurotransmitters seems to be affected by hyperforin. In this respect, hyperforin was suggested to increase the intracellular sodium concentration, subsequently reducing the sodium gradient required for neurotransmitter (re)uptake from the extracellular space by the presynaptic neurotransmitter transporters 23,24 . Later it was found that hyperforin activates non-selective cation channels in human platelets and rat phaeochromocytoma (PC12) cells 25 and in 2007 hyperforin was introduced as specific activator for the non-selective cation channel TRPC6 (transient receptor potential canonical 6) 26 . TRPC6 is expressed in human platelets 27 and PC12 cells 26,28 and their hyperforin-induced Na + influx, might be due to TRPC6 activation. However, other effects of hyperforin are described which might also participate in its pharmacological actions. For example, hyperforin attenuates various voltage- and ligand-gated ionic conductances in isolated hippocampal neurons or cerebellar Purkinje neurons 29,30,31,32 , it changes the fluidity of membranes 33 and collapses the mitochondrial membrane potential leading to the release of Zn 2+ and Ca 2+ into the cytosol 12 .

In the present study we used fluorescent imaging to monitor intracellular pH, cytosolic sodium changes and neurotransmitter release and the whole-cell patch clamp technique to identify and to characterize the hyperforin-induced conductance in four independent systems, in HEK-293 cells, primary mouse microglia and chromaffin cells and lipid bilayer membranes. Our data show that hyperforin itself acts as a protonophore and thereby mediates a significant proton conductance. This conductance does not require the presence of channel proteins like TRPC6 and its direction depends on the existing driving forces such as membrane potential and pH gradient. In line with these results, accumulation of neurotransmitters is abolished in primary mouse chromaffin cells in the presence of hyperforin.


Introduction

The development of alternative methods to whole plant cultivation for the production of pharmaceutically valuable compounds of commercial interest is an issue of considerable socio-economic importance. Current advancement in biotechnological research has made the plant cell, tissue, and organ culture an attractive alternative to whole plant for the production of biologically important compounds (Rao and Ravishankar, 2002). In particular, root cultures characterized by a high growth rate and active secondary metabolism provides an efficient system to produce great amounts of secondary metabolites highly valued in pharmaceutical industry (Sivakumar, 2006). As an example, root cultures of Morinda citrifolia, Echinacea purpurea, and Panax ginseng were successfully used to produce bioactive molecules such as anthraquinones, rubiadin, phenolics and flavonoids with antioxidative, antibacterial, antiviral, and antifungal properties (Baque et al., 2012). Among medicinal plants, Hypericum perforatum (L.) (Hypericaceae) has received a global attention, owing to its variety of structurally diverse bioactive compounds such as flavonols, naphthodianthrones, and phloroglucinols, which have been reported to have antidepressant activity in different antidepressive model systems (Barnes et al., 2001 Walker et al., 2002 Cirak et al., 2007). Research on H. perforatum has focused primarily on hypericin and pseudohypericin as the major constituents responsible for the antidepressant activity (Walker et al., 2002). Moreover, clinical studies underscored the possible role of flavonoids in several kinds of cancer (Maheep et al., 2011). Recently much attention has been paid to another class of bioactive polyphenols, namely xanthones whose high antifungal activity against human pathogens has been demonstrated (Tocci et al., 2013a,b Simonetti et al., 2015). Root cultures of H. perforatum L. are considered an effective system for the biotechnological production of flavonols, xanthones, essential oils, and other secondary metabolites, with interesting pharmacological activities (Mulinacci et al., 2008 Crockett et al., 2011 Tocci et al., 2011, 2012, 2013b Zubrická et al., 2015). One of the major obstacles to the use of organ cultures for the pharmaceutical industry is the low yield of the metabolites of interest. For this reason, several strategies have been adopted to improve the production of plant-derived secondary metabolites such as two-phase culture system, genetic transformation, metabolic and bioreactor engineering (Georgiev et al., 2012 Tocci et al., 2012 Wilson et al., 2014 Simonetti et al., 2015). Among the various efforts, chitosan elicitation proved to be one of the most effective strategy to enhance the production of bioactive compounds both in in planta and in in vitro root systems (Tocci et al., 2011 Yin et al., 2012). In particular, chitosan treatment has been shown to increase the production of xanthones in H. perforatum root cultures (Tocci et al., 2011, 2012, 2013a).

Xanthone compounds include a group of structurally diverse, biologically active, and synthetically challenging natural products with a wide range of pharmacological properties, e.g., antioxidant, anti-inflammatory, antimicrobial, and cytotoxic activities (Franklin et al., 2009 Naldoni et al., 2009 Al-Shagdari et al., 2013 Nontakham et al., 2014 Zubrická et al., 2015).

It is well-known that the production of bioactive compounds in H. perforatum in vitro roots is greatly affected by various parameters such as inoculum density, culture medium composition, time of culture, type and concentration of growth regulators, and other physico-chemical factors that need to be optimized to maximize the growth of biomass and the production of natural compounds (Cui et al., 2010, 2011 Jin et al., 2012 Zubrická et al., 2015 Valletta et al., 2016).

However, the impact of culture conditions on the root metabolism is not limited to single biochemical pathways. Furthermore, the knowledge of biosynthetic pathways of desired compounds in root cultures is still in its infancy, and consequently, the understanding of the regulation of primary and secondary metabolic pathways is required. An omics approach in which a great number of primary and secondary metabolites are identified and quantified is needed to elucidate the function of a whole pathway or intersecting pathways as well as to understand how to increase metabolic fluxes into pathways involved in the production of plant pharmaceuticals (Giddings et al., 2000). With the recent developments in plant metabolomics techniques, it is now possible to detect several hundred metabolites simultaneously and to compare samples reliably to identify differences and similarities in an untargeted manner. On the other hand, the chemical analyses that are based on the whole composition of metabolites, rather than detection of a single constituent, are favored as they cover additionally or synergistically relevant components and can confirm the efficacy of H. perforatum medical preparations (Porzel et al., 2014).

So far the effects of chitosan elicitation and culture time on the whole metabolism of root cultures of H. perforatum have not been completely elucidated. Further studies are desirable to understand the relationships between primary and secondary metabolism with the aim to optimize the biomass growth and the production of bioactive compounds.

Recently, for the first time, a NMR-based metabolomic approach has been applied to study the primary and secondary metabolic changes of H. perforatum in vitro roots after a short period of chitosan treatment (24 and 72 h) and growth in a confined environment (Brasili et al., 2014). This approach has proved useful to demonstrate that root cultures are able to direct the shikimate pathway toward to the biosynthesis of tryptophan and epicatechin in response to a high biomass density and toward the synthesis of xanthones, and epicatechin in response to the chitosan elicitation. The chitosan treatment also stimulated the mevalonate pathway toward isoprenoid intermediate production, such as dimethylallyl-pyrophosphate (DMAPP) and stigmasterol. These latter can function as primary metabolites, participating in essential plant cellular processes, and as secondary metabolites, of which many have substantial commercial, pharmacological, and agricultural value (Vranová et al., 2012). These metabolic variations have been observed after having subjected the roots to two renewals of the culture medium and within 72 h from chitosan elicitation.

In the present study, we have applied a non-targeted NMR-based metabolomics associated to ANOVA simultaneous component analysis (ASCA) to explore the response of primary and secondary metabolism of roots to a longer culture time and a prolonged time of exposure to chitosan, until 192 h, in an attempt to improve the yield of xanthones and to obtain more information about the isoprenoid metabolism. Furthermore, the effect of chitosan treatment on the biomass growth and on the morpho-anatomical features of H. perforatum roots was investigated during the time course.

This metabolomic approach can provide a new platform for global analyses of Hypericum pharmaceuticals and or other phytomedicines and it can be applied to define suitable protocols to produce the desired secondary metabolites with different bioactivities.


Materials and Methods

Plant Materials

Hypericum perforatum L. plants from 2 naturally occurring tetraploids (2n = 4x = 32) and 3 induced tetraploids (2n = 4x = 32) were used for the microarray approaches (Table 1). For the production of induced tetraploids, plants of the diploid sexual line R1 (reselected from the tetraploid apomictic cultivar “Topaz”) were converted to auto-polyploids by colchicine application, as reported by Schallau et al. (2010). Briefly, seeds were imbibed in water for 24 h, placed on filter paper soaked with an aqueous solution of 0.2% colchicine for 24 h and then planted in soil for germination. The C0 plants that survived this treatment were self-pollinated, and their progenies (C1) were screened for tetraploid plants. Sexual tetraploid C1 and C2 plants were used for further crosses with tetraploid apomictic pollinators and were screened for their reproductive phenotype (Schallau et al., 2010 Molins et al., 2014). The reproductive mode of all H. perforatum accessions was estimated by flow cytometric screening of 48 single seeds as described by Matzk et al. (2001).

Table 1. Origins of H. perforatum samples. For each plant accession, the description, the genealogy, the origin, the ploidy and the degree of apomixis is indicated.

Pistils were collected from three apomictic and three sexual plant accessions. In both cases, the developmental stages considered for sampling were bud lengths of approximately 4.0 mm, corresponding to Arabidopsis flower stage 11, and bud lengths of approximately 10.0 mm, corresponding to Arabidopsis flower developmental stage 14 (Galla et al., 2011).

Array Design, cDNA Synthesis and Hybridization

Sequences used for the design of 4x180 arrays (Agilent) were previously deposited at DDBJ/EMBL/GenBank under the accession GBXG00000000. The 60,594 tentative consensus sequences were used to design 60-mer oligonucleotide probes for a microarray analysis. Sequences corresponding to transcripts 𼄀 nt in size were removed to reduce the set of target transcripts to 55,682 sequences. The custom microarray with the design ID 065680 was created using eArray, a free Agilent web-based application that enables the creation of custom microarray designs and oligo libraries (https://earray.chem.agilent.com/earray/). For probe design, the Base Composition Methodology option was employed, and no linker sequence was used. Two different probes were designed for each sequence. A total of 111,200 probes were obtained from eArray and were used for creation of the 4x180K array format. Slides with printed arrays were ordered directly from Agilent Technologies (Santa Clara, CA, USA).

RNA Extraction and Labelling

Total RNA was extracted from collected pistils using the Spectrum™ Plant Total RNA Kit (Sigma-Aldrich) following the protocol provided by the manufacturer. The eventual contamination of genomic DNA was avoided by using optional DNase I (Sigma-Aldrich) treatment. The abundance and pureness of RNAs were assessed using a NanoDrop 2000c UV-Vis spectrophotometer (Thermo Scientific, Pittsburgh, PA). RNA integrity was evaluated with an Agilent 2100 Bioanalyser with RNA Nanochips (Agilent Technologies, Santa Clara, USA). Only samples with RIN values higher than 7.5 were used in the following procedures. The cRNAs were synthesized from 200 ng of total RNA and labeled with cyanine 3 (Cy3)-CTP fluorescent dye according to the manufacturer's instructions (Agilent Technologies). Aliquots of Cy3-labeled cRNAs (1.65 μg) of each sample were used for subsequent hybridization in a H. perforatum custom oligo microarray with the design number 065680 (Agilent Technologies) according to the manufacturer's manual. After hybridization for 17 h at 65ଌ, slides were washed and scanned with an Agilent Microarray Scanner (G2565CA, Agilent Technologies).

Statistical Analysis

Scanned images were transformed into quantified figures using the Agilent Features extraction software (v11.5), and expression data were normalized based on the 75th percentile. Principal component analyses were carried out using T-MeV v4.9.0 software (http://mev.tm4.org) and by using Manhattan distances. Statistical analyses (t-test, unpaired p < 0.01) were carried out using T-MeV v4.9.0 software (http://mev.tm4.org) to define significantly modulated genes between groups of samples. Differentially Expressed Genes (DEGs) were clustered according to their expression patterns in eight groups as reported in Table 2. For each gene and comparison, the fold change (FC) was calculated as the ratio between the average of the normalized expression data measured in three replicates for test and reference samples. For each gene and comparison, the average of the normalized expression data measured for the sexual samples or the average of the normalized expression data measured for the samples corresponding to the earlier developmental stage (floral stage 11) was set as reference (Table 2).

Table 2. Differentially expressed genes.

Bioinformatics𠄺rray Annotation

Annotation of sequences used for the design of 4x180 arrays (Agilent) is described in Galla et al. (2015). Briefly, to annotate all assembled unigenes, a BLASTX-based approach was used to compare the Hypericum sequences to the nr database downloaded from the NCBI (http://www.ncbi.nlm.nih.gov/). The following approach was adopted to improve our efficacy in annotating 18,386 unigenes that did not reach significance for the BLAST results in the nr database. Unigenes were aligned to the Hypericum genome draft HEID 914233 v1 (Altschmied, personal communication) using BLAT (match ≥ 50 base pairs, fident ≥ 0.97). Additionally, high-quality genomic top hits were selected by preventing the existence of long non-aligned segments on the same side for each unigene and genomic contig. Briefly, a genomic top hits was retained only when the alignment fulfilled the two following criteria (hereafter described for the 5𠌮nd of the alignment): (i) 5′ unaligned segment of the genomic contig equal or larger than the 5′ unaligned segment of the unigene only when the 5′ unaligned segment of the unigene was equal or smaller than 10 bp (allowing for an eventually untrimmed adapter sequence at the 5′ end of the unigene) (ii) 3′ unaligned segment of the genomic contig equal or larger than the 3′ unaligned segment of the unigene was allowed only when 3′ unaligned segment of the unigene was equal or smaller than 10 bp (allowing for an eventually untrimmed adapter sequence at the 3′ end of the unigene) or in cases where the unaligned segment of the genomic contig at the 3′ side was smaller than 3 bp. The same criteria were adopted for the 3′ ends of each alignment.

Next, high quality top hits were identified, and genomic sequences upstream and/or downstream of the aligned segments of the genomic top hits with min. 100 and max. 1001 bases of genomic sequence were extracted. All extracted sequences were then used to query the Arabidopsis thaliana TAIR10 proteome with BLASTX (E-value 1e-10 -seg no -culling_limit 1). Top hits for genomic sequences flanking the aligned unigenes were then selected for the following annotations.

The GI identifiers of the best BLASTX hits, with E-values ≤ 1 E-09 and similarities ≥ 70%, were mapped to the UniProtKB protein database (http://www.uniprot.org/) to extract Gene Ontology (GO, http://www.geneontology.org/) terms for further functional annotations. BLAST2GO software v1.3.3 (https://www.blast2go.com/, Conesa et al., 2005) was used to reduce the data to the GOslim level (goslim_plant.obo) and perform basic statistics on ontological annotations as previously reported (Galla et al., 2009).

Annotations for genes involved in plant reproduction were retrieved from Galla et al. (2015). Additionally, a gene ontology-based annotation of the Hypericum flower transcriptome was attempted by querying the Amigo2 database (http://amigo.geneontology.org/) with opportune key words (such as 𠇎gg cell,” �ntral cell,” “synergic,” 𠇊ntipodal cells,” 𠇎mbryo sac,” “gamete,” 𠇎ndosperm,” “meiotic,” and �ll fate”). In a parallel approach, the Amigo2 repository was queried for genes annotated as responsive to hormonal stimuli using the following search criteria: “response to abscisic acid,” “response to auxin,” “response to brassinosteroid,” “response to cytokinin,” “response to ethylene,” “response to gibberellin,” and “response to jasmonic acid.”

Expression Correlation Network

An expression correlation network (Pearson correlation coefficient 𢄠.98 <> Ʈ.98) was created from all differentially expressed unigenes (fold change ≥ 2.0) using the plugin ExpressionCorrelation (http://apps.cytoscape.org/apps/expressioncorrelation) implemented in Cytoscape 3.4.0 (http://www.cytoscape.org/).

Sub-networks were generated from the overall expression correlation network (Pearson correlation coefficient 𢄠.98 <> Ʈ.98 714 nodes, 8367 edges) by selecting nodes (DEGs) according to their annotations. Next, all adjacent edges and nodes connected to these edges were selected and used to infer the sub-network. For each sub-network, GO enrichment analyses were performed with the plugin BinGO with default parameters (p < 0.05, Benjamini & Hochberg's FDR correction with significance level 0.05).

Validation of Sequencing Data by Quantitative Real-Time PCRs

Plant materials were selected according to the genetic and cyto-histological bases of apospory recently described for H. perforatum (Schallau et al., 2010 Galla et al., 2011). Samples were collected separately from a minimum of five plant accessions (Table 1). RNA extractions were performed using a Spectrum™ Plant Total RNA Kit (Sigma-Aldrich). cDNA synthesis was performed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific), following the instructions of the supplier. Primers used in the Real-Time RT-PCR experiments are reported in Table S6. Expression analyses were performed using StepOne thermal cyclers and the 7300 Real-Time PCR System (Applied Biosystems), equipped with 96- and 384-well plate systems, respectively, with SYBR green PCR Master Mix reagent (Applied Biosystems). The amplification efficiency was calculated from raw data using OneStep Analysis software (Life Technologies). Amplification performance expressed as fold change was calculated with the Δ㥌t method using HpTIP4 as a housekeeping gene (Pfaffl, 2001). Error bars indicate the standard error observed among the five biological replicates.

Data Availability

Raw sequences files were made available for download from SRA with the following accession numbers: SRR1646951, SRR1646953, SRR1646955, SRR1646956, SRR1647632, SRR1647633, SRR1647673, SRR1647674, SRR1647677, SRR1647678, SRR1647713, and SRR1647714. Sequences of unigenes investigated by Real-Time qPCR along with unigenes aligned to the BAC sequence HM061166.1 were deposited under the Transcriptome Shotgun Assembly project at DDBJ/EMBL/GenBank under the accession GBXG00000000. The version described in this paper is the second version, GBXG02000000.

The expression data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus (Edgar et al., 2002) and are accessible through GEO Series accession number GSE84768 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE84768).


Introduction

The development of alternative methods to whole plant cultivation for the production of pharmaceutically valuable compounds of commercial interest is an issue of considerable socio-economic importance. Current advancement in biotechnological research has made the plant cell, tissue, and organ culture an attractive alternative to whole plant for the production of biologically important compounds (Rao and Ravishankar, 2002). In particular, root cultures characterized by a high growth rate and active secondary metabolism provides an efficient system to produce great amounts of secondary metabolites highly valued in pharmaceutical industry (Sivakumar, 2006). As an example, root cultures of Morinda citrifolia, Echinacea purpurea, and Panax ginseng were successfully used to produce bioactive molecules such as anthraquinones, rubiadin, phenolics and flavonoids with antioxidative, antibacterial, antiviral, and antifungal properties (Baque et al., 2012). Among medicinal plants, Hypericum perforatum (L.) (Hypericaceae) has received a global attention, owing to its variety of structurally diverse bioactive compounds such as flavonols, naphthodianthrones, and phloroglucinols, which have been reported to have antidepressant activity in different antidepressive model systems (Barnes et al., 2001 Walker et al., 2002 Cirak et al., 2007). Research on H. perforatum has focused primarily on hypericin and pseudohypericin as the major constituents responsible for the antidepressant activity (Walker et al., 2002). Moreover, clinical studies underscored the possible role of flavonoids in several kinds of cancer (Maheep et al., 2011). Recently much attention has been paid to another class of bioactive polyphenols, namely xanthones whose high antifungal activity against human pathogens has been demonstrated (Tocci et al., 2013a,b Simonetti et al., 2015). Root cultures of H. perforatum L. are considered an effective system for the biotechnological production of flavonols, xanthones, essential oils, and other secondary metabolites, with interesting pharmacological activities (Mulinacci et al., 2008 Crockett et al., 2011 Tocci et al., 2011, 2012, 2013b Zubrická et al., 2015). One of the major obstacles to the use of organ cultures for the pharmaceutical industry is the low yield of the metabolites of interest. For this reason, several strategies have been adopted to improve the production of plant-derived secondary metabolites such as two-phase culture system, genetic transformation, metabolic and bioreactor engineering (Georgiev et al., 2012 Tocci et al., 2012 Wilson et al., 2014 Simonetti et al., 2015). Among the various efforts, chitosan elicitation proved to be one of the most effective strategy to enhance the production of bioactive compounds both in in planta and in in vitro root systems (Tocci et al., 2011 Yin et al., 2012). In particular, chitosan treatment has been shown to increase the production of xanthones in H. perforatum root cultures (Tocci et al., 2011, 2012, 2013a).

Xanthone compounds include a group of structurally diverse, biologically active, and synthetically challenging natural products with a wide range of pharmacological properties, e.g., antioxidant, anti-inflammatory, antimicrobial, and cytotoxic activities (Franklin et al., 2009 Naldoni et al., 2009 Al-Shagdari et al., 2013 Nontakham et al., 2014 Zubrická et al., 2015).

It is well-known that the production of bioactive compounds in H. perforatum in vitro roots is greatly affected by various parameters such as inoculum density, culture medium composition, time of culture, type and concentration of growth regulators, and other physico-chemical factors that need to be optimized to maximize the growth of biomass and the production of natural compounds (Cui et al., 2010, 2011 Jin et al., 2012 Zubrická et al., 2015 Valletta et al., 2016).

However, the impact of culture conditions on the root metabolism is not limited to single biochemical pathways. Furthermore, the knowledge of biosynthetic pathways of desired compounds in root cultures is still in its infancy, and consequently, the understanding of the regulation of primary and secondary metabolic pathways is required. An omics approach in which a great number of primary and secondary metabolites are identified and quantified is needed to elucidate the function of a whole pathway or intersecting pathways as well as to understand how to increase metabolic fluxes into pathways involved in the production of plant pharmaceuticals (Giddings et al., 2000). With the recent developments in plant metabolomics techniques, it is now possible to detect several hundred metabolites simultaneously and to compare samples reliably to identify differences and similarities in an untargeted manner. On the other hand, the chemical analyses that are based on the whole composition of metabolites, rather than detection of a single constituent, are favored as they cover additionally or synergistically relevant components and can confirm the efficacy of H. perforatum medical preparations (Porzel et al., 2014).

So far the effects of chitosan elicitation and culture time on the whole metabolism of root cultures of H. perforatum have not been completely elucidated. Further studies are desirable to understand the relationships between primary and secondary metabolism with the aim to optimize the biomass growth and the production of bioactive compounds.

Recently, for the first time, a NMR-based metabolomic approach has been applied to study the primary and secondary metabolic changes of H. perforatum in vitro roots after a short period of chitosan treatment (24 and 72 h) and growth in a confined environment (Brasili et al., 2014). This approach has proved useful to demonstrate that root cultures are able to direct the shikimate pathway toward to the biosynthesis of tryptophan and epicatechin in response to a high biomass density and toward the synthesis of xanthones, and epicatechin in response to the chitosan elicitation. The chitosan treatment also stimulated the mevalonate pathway toward isoprenoid intermediate production, such as dimethylallyl-pyrophosphate (DMAPP) and stigmasterol. These latter can function as primary metabolites, participating in essential plant cellular processes, and as secondary metabolites, of which many have substantial commercial, pharmacological, and agricultural value (Vranová et al., 2012). These metabolic variations have been observed after having subjected the roots to two renewals of the culture medium and within 72 h from chitosan elicitation.

In the present study, we have applied a non-targeted NMR-based metabolomics associated to ANOVA simultaneous component analysis (ASCA) to explore the response of primary and secondary metabolism of roots to a longer culture time and a prolonged time of exposure to chitosan, until 192 h, in an attempt to improve the yield of xanthones and to obtain more information about the isoprenoid metabolism. Furthermore, the effect of chitosan treatment on the biomass growth and on the morpho-anatomical features of H. perforatum roots was investigated during the time course.

This metabolomic approach can provide a new platform for global analyses of Hypericum pharmaceuticals and or other phytomedicines and it can be applied to define suitable protocols to produce the desired secondary metabolites with different bioactivities.


Current Issue

Functional Plant Biology

Volume 48 Number 7 2021

Special Issue

Diversity of CAM Plant Photosynthesis (Crassulacean Acid Metabolism)

FPv48n7toc Table of Contents

FPv48n7_FO Diversity of CAM plant photosynthesis (crassulacean acid metabolism): a tribute to Barry Osmond

The foreword to the special issue on diversity of CAM plant photosynthesis (crassulacean acid metabolism) highlights some of the key contributions of the Australian plant biologist Professor Charles Barry Osmond to our understanding of the CAM pathway of photosynthesis and provides a brief introduction to the research papers of this issue.

FP20127 Constitutive and facultative crassulacean acid metabolism (CAM) in Cuban oregano, Coleus amboinicus (Lamiaceae)

Low-level constitutive CAM and drought-induced facultative CAM co-occur in leaves of the widely cultivated, aromatic tropical herb Coleus amboinicus (Lamiaceae), highlighting a notable facet of CAM-plant photosynthetic diversity.

FP20247 Does the C4 plant Trianthema portulacastrum (Aizoaceae) exhibit weakly expressed crassulacean acid metabolism (CAM)?

Trianthema portulacastrum, a well-known C4 plant, exhibits CAM-type day-night acid fluctuations in stems, and to a lesser extent in leaves. Although nocturnal acidification is small, Trianthema is only the second genus of vascular land plants in which C4 and CAM have been demonstrated to co-occur in the same plant.

FP20202 Developing Portulaca oleracea as a model system for functional genomics analysis of C4/CAM photosynthesis

Renata Callegari Ferrari, Priscila Pires Bittencourt, Paula Yumi Nagumo, Willian Silva Oliveira, Maria Aurineide Rodrigues, James Hartwell and Luciano Freschi />

Portulaca oleracea has emerged as a model system to answer the intriguing question of how two carbon concentration mechanisms (C4 and CAM) can co-exist within a single leaf. Recent progress has been made with the study of C4 and CAM functional genomics, but similar molecular approaches have not been possible in C4-CAM facultative species. Essential tools for functional gene analysis are now available for P. oleracea, which may accelerate C4-CAM photosynthesis research and the future application of these valuable photosynthetic adaptations within crop biotechnology.

FP20151 Low-level CAM photosynthesis in a succulent-leaved member of the Urticaceae, Pilea peperomioides

Measurements of CO2 gas exchange and titratable acidity revealed features of low-level CAM photosynthesis in Pilea peperomioides. This is the first report of CAM in the family Urticaceae.

FP20305 CAM photosynthesis in desert blooming Cistanthe of the Atacama, Chile

In two Cistanthe species from the Atacama desert, CO2 uptake and leaf acidification patterns were observed that are typical of water-use efficient crassulacean acid metabolism (CAM) photosynthesis. CAM expression in the perennial C. sp. aff. crassifolia was facultative whereas CAM in the annual C. sp. aff. longiscapa was constitutive. Cistanthe becomes the sixth genus known to exhibit CAM within the family Montiaceae.

FP20268 Crassulacean acid metabolism (CAM) supersedes the turgor loss point (TLP) as an important adaptation across a precipitation gradient, in the genus Clusia

By identifying physiological traits more common in drier environments, it is possible to understand the ways in which tropical trees have adapted to deal with drought. By analysing a genus from Central and South America, we were able to test if it is more beneficial to prevent or tolerate water loss. Our results show that preventing water loss has a greater benefit to living in drier niches, which has implications for the ways in which future climates will affect tropical flora.

FP20332 Metabolic profiling of epidermal and mesophyll tissues under water-deficit stress in Opuntia ficus-indica reveals stress-adaptive metabolic responses

To better understand CAM-related metabolites and water-deficit stress responses of Opuntia ficus-indica, comparative metabolic profiling was performed on mesophyll and epidermal tissues collected from well-watered and water-deficit stressed cladodes. A total of 382 metabolites, including 210 (55%) named and 172 (45%) unnamed compounds, were characterised across both tissues. This study revealed a total of 34 unnamed metabolites that accumulated in response to water-deficit stress indicating that such compounds might play important roles in water-deficit tolerance.

FP21087 Leaf water δ 18 O reflects water vapour exchange and uptake by C3 and CAM epiphytic bromeliads in Panama

Monica Mejia-Chang, Casandra Reyes-Garcia, Ulli Seibt, Jessica Royles, Moritz T. Meyer, Glyn D. Jones, Klaus Winter /> , Miquel Arnedo and Howard Griffiths />

The paper defines the niche segregation of C3 and CAM photosynthetic pathways for epiphytic bromeliads along an altitudinal gradient in Panama. Measurement of the leaf water oxygen ( 18 O) stable isotope composition supports transpiration by day or night, since under high humidity water vapour influx resets the leaf water 18 O signal.


Financial Support from the National Institutes of Health (GM073855) and Boston University is greatly acknowledged. We thank Professor Nigel Simpkins (University of Birmingham, UK) for helpful discussions. NMR (CHE-0619339) and MS (CHE-0443618) facilities at Boston University are supported by the NSF. Research at the Center for Chemical Methodology and Library Development at Boston University (CMLD-BU) is supported by NIH grant GM-067041.

This article references 26 other publications.

For reviews on the chemistry and/or biology of PPAPs, see:

For selected examples of the PPAP biological activities and medicinal significance, see:

For select examples highlighting synthetic strategies toward PPAPs, see:

PPAP salts have been prepared to increase the shelf life of the natural products:

For examples of synthetic or semisynthetic PPAP analogs, see:

For examples of natural products as inspiration for DOS libraries, see:

For a recent review highlighting modern strategies in total synthesis, see:

For a recent review on transition metal-catalyzed biomimetic transformations, see:

For an elegant example of dearomatization via allylic alkylation in total synthesis, see:

For Pd-catalyzed annulation with conjunctive reagents analogous to 2, see:

For a review of decarboxylative allylic alkylation, see:

For an example of allyl phenyl ether synthesis via DcA see the following reference and the references therein:

We first attempted intermolecular Tsuji–Trost allylation of 4a but only observed formation of kinetic, dearomatized product 13a which prompted our use of DcA for scaffold synthesis:

In DcA, the nucleophile bearing the carboallyloxy group receives the allylic fragment even in the presence of other nucleophiles:

See the Supporting Information for complete experimental details.

Pd 0 -catalyzed prenylation is often challenging. However, other transition metals and reaction conditions can facilitate prenylation:


Is Hypericum Perforatum (St. John's wort) a C3 or C4 plant? - Biology

a Department of Plant and Environmental Sciences, University of Copenhagen, Bülowsvej 17, 1870 Frederiksberg C, Copenhagen, Denmark

b Department of Pharmacy, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark
E-mail: [email protected]
Fax: +45 35 33 60 30
Tel: +45 35 33 65 57

Abstract

Covering: up to the end of 2013

New mass spectrometry imaging (MSI) techniques are gaining importance in the analysis of plant metabolite distributions, and significant technological improvements have been introduced in the past decade. This review provides an introduction to the different MSI techniques and their applications in plant science. The most common methods for sample preparation are described, and the review also features a comprehensive table of published studies in MSI of plant material. A number of significant works are highlighted for their contributions to advance the understanding of plant biology through applications of plant metabolite imaging. Particular attention is given to the possibility for imaging of surface metabolites since this is highly dependent on the methods and techniques which are applied in imaging studies.


Abstract

The quantitative distribution of several flavan-3-ols was determined using HPLC in a grape (Vitis vinifera) seed extract (GSE) of four cultivars grown in the region of Murcia. Polymer ≥ C4 units made up the largest group of procyanidins in the GSE (90.92%, expressed as HPLC % area). The antioxidant activity of GSE and other reference compounds was investigated by measuring their ability to scavenge the ABTS • + radical cation (TEAC). The most effective compounds were, in order: GSE > rutin > (+)-catechin > diosmin ≥ ascorbic acid. The radioprotective effects of GSE and other reference compounds were determined by using the micronucleus test for anticlastogenic activity, any reduction of the frequency of micronucleated polychromatic erythrocytes (MnPCEs) being evaluated in the bone marrow of mouse exposed to X-rays. The most effective compounds were, in order: GSE > rutin > dimethyl sulfoxide (DMSO) > ascorbic acid > 6-n-propyl-2-thiouracil-6c (PTU) > diosmin. The higher ABTS • + scavenging capacity and anticlastogenic activity of GSE can be explained, structurally, by the high number of conjugated structures between the catechol groups in the B-rings and the 3-OH free groups of the polymeric polyphenolic skeleton and, in addition, by the stability of the aroxyl flavonoid radical generated in the above processes.

Keywords: Vitis vinifera flavan-3-ols procyanidins radical scavenger antioxidant ABTS radical X-irradiation radioprotection anticlastogen

To whom correspondence should be addressed. Tel: 34 968 892512. Fax: 34 968 892656. E-mail: [email protected]


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