Life Extension Magazine®

Issue: Dec 2014

Boswellia, Chlorophyllin, Zinc, and Ribogen

Boswellia, Chlorophyllin, Zinc, and Ribogen

By Life Extension.

Boswellia serrata: an overall assessment of in vitro, preclinical, pharmacokinetic and clinical data.

Non-steroidal anti-inflammatory drug (NSAID) intake is associated with high prevalence of gastrointestinal or cardiovascular adverse effects. All efforts to develop NSAIDs that spare the gastrointestinal tract and the cardiovasculature are still far from achieving a breakthrough. In the last two decades, preparations of the gum resin of Boswellia serrata (a traditional ayurvedic medicine) and of other Boswellia species have experienced increasing popularity in Western countries. Animal studies and pilot clinical trials support the potential of B. serrata gum resin extract (BSE) for the treatment of a variety of inflammatory diseases like inflammatory bowel disease, rheumatoid arthritis, osteoarthritis and asthma. Moreover, in 2002 the European Medicines Agency classified BSE as an ‘orphan drug’ for the treatment of peritumoral brain oedema. Compared to NSAIDs, it is expected that the administration of BSE is associated with better tolerability, which needs to be confirmed in further clinical trials. Until recently, the pharmacological effects of BSE were mainly attributed to suppression of leukotriene formation via inhibition of 5-lipoxygenase (5-LO) by two boswellic acids, 11-keto-b-boswellic acid (KBA) and acetyl-11-keto-b-boswellic acid (AKBA). These two boswellic acids have also been chosen in the monograph of Indian frankincense in European Pharmacopoiea 6.0 as markers to ensure the quality of the air-dried gum resin exudate of B. serrata. Furthermore, several dietary supplements advertise the enriched content of KBA and AKBA. However, boswellic acids failed to inhibit leukotriene formation in human whole blood, and pharmacokinetic data revealed very low concentrations of AKBA and KBA in plasma, being far below the effective concentrations for bioactivity in vitro. Moreover, permeability studies suggest poor absorption of AKBA following oral administration. In view of these results, the previously assumed mode of action—that is, 5-LO inhibition—is questionable. On the other hand, 100-fold higher plasma concentrations have been determined for b-boswellic acid, which inhibits microsomal prostaglandin E synthase-1 and the serine protease cathepsin G. Thus, these two enzymes might be reasonable molecular targets related to the anti-inflammatory properties of BSE. In view of the results of clinical trials and the experimental data from in vitro studies of BSE, and the available pharmacokinetic and metabolic data on boswellic acids, this review presents different perspectives and gives a differentiated insight into the possible mechanisms of action of BSE in humans. It underlines BSE as a promising alternative to NSAIDs, which warrants investigation in further pharmacological studies and clinical trials.

Clin Pharmacokinet. 2011 Jun;50(6):349-69

Frankincense (Ru xiang; boswellia species): from the selection of traditional applications to the novel phytotherapy for the prevention and treatment of serious diseases.

Frankincense (Ru xiang; Bowes-llia species), the resinous extract from the trees of the genus Boswellia, has been used for centuries in cultural ceremonies, as a cosmetic agent, and as a traditional medicine to treat a variety of ailments, especially inflammatory diseases including asthma, arthritis, cerebral edema, chronic pain syndrome, chronic bowel diseases, cancer, and some other illnesses. Boswellic acids are the active compounds of frankincense and AKBA (3-O-acetyl-11-keto-b-boswellic acid) is the most important and effective acid among them. Some studies have shown that the use of frankincense can also improve the learning and enhance the memory in animals and human beings. It seems that frankincense might have a potential ability to be used as an alternative natural medicine not only for chronic and inflammatory diseases but also for brain and memory disorders.

J Tradit Complement Med . 2013 Oct;3(4):221-6

Boswellia serrata, a potential antiinflammatory agent: an overview.

The resin of Boswellia species has been used as incense in religious and cultural ceremonies and in medicines since time immemorial. Boswellia serrata (Salai/Salai guggul), is a moderate to large sized branching tree of family Burseraceae (Genus Boswellia), grows in dry mountainous regions of India, Northern Africa and Middle East. Oleo gum-resin is tapped from the incision made on the trunk of the tree and is then stored in specially made bamboo basket for removal of oil content and getting the resin solidified. After processing, the gum-resin is then graded according to its flavour, colour, shape and size. In India, the States of Andhra Pradesh, Gujarat, Madhya Pradesh, Jharkhand and Chhattisgarh are the main source of Boswellia serrata. Regionally, it is also known by different names. The oleo gum-resins contain 30-60% resin, 5-10% essential oils, which are soluble in the organic solvents, and the rest is made up of polysaccharides. Gum-resin extracts of Boswellia serrata have been traditionally used in folk medicine for centuries to treat various chronic inflammatory diseases. The resinous part of Boswellia serrata possesses monoterpenes, diterpenes, triterpenes, tetracyclic triterpenic acids and four major pentacyclic triterpenic acids i.e. b-boswellic acid, acetyl-b-boswellic acid, 11-keto-b-boswellic acid and acetyl-11-keto-b-boswellic acid, responsible for inhibition of pro-inflammatory enzymes. Out of these four boswellic acids, acetyl-11-keto-b-boswellic acid is the most potent inhibitor of 5-lipoxygenase, an enzyme responsible for inflammation.

Indian J Pharm Sci . 2011 May;73(3):255-61

In vitro metabolism, permeation, and brain availability of six major boswellic acids from Boswellia serrata gum resins.

Boswellia serrata gum resin extracts (BSE) revealed potent anti-inflammatory actions in preclinical and clinical studies. In 2002 BSE was assigned an orphan drug status by the European Medicines Agency (EMA) for the treatment of peritumoral edema. In the past pharmacological effects of BSE were mainly attributed to 11-keto-b-boswellic acid (KBA) and 3-acetyl-11-keto-b-boswellic acid (AKBA). Therefore pharmacokinetic and pharmacodynamic studies focused mainly on these two boswellic acids (BAs). However, other BAs, like b-boswellic acid (bBA), might also contribute to the anti-inflammatory actions of BSE. Here, we determined the metabolic stability, permeability and brain availability of six major BAs, that is, KBA, AKBA, bBA, 3-acetyl-b-boswellic acid (AbBA), a-boswellic acid (aBA), and 3-acetyl-a-boswellic acid (AaBA). For permeability studies, the Caco-2 model was adapted to physiological conditions by the addition of bovine serum albumin (BSA) to the basolateral side and the use of modified fasted state simulated intestinal fluid (FaSSIF) on the apical side. Under these conditions the four BAs lacking the 11-keto moiety revealed moderate permeability.Furthermore the permeability of AKBA and KBA was improved compared to earlier studies. In contrast to Aa- and AbBA, bBA and aBA were intensively metabolized after incubation with human and rat liver microsomes. Finally, the availability of all six major BAs could be confirmed in rat brain 8h after oral administration of 240mg/kg BSE to rats showing mean concentrations of 11.6ng/g for KBA, 37.5ng/g for AKBA, 485.1ng/g for aBA, 1066.6ng/g for bBA, 43.0ng/g for AaBA and 163.7ng/g for AbBA.

Fitoterapia . 2013 Jan;84:99-106

Evaluation of efficacy of flexiqule (boswellia phyto extract) in osteoarthritis of knee

Osteoarthritis is major cause of morbidity and impaired quality of life among the elderly persons. The prolong use of NSAIDs in elderly, is required for constant relief of pain, but they are associated with adverse drug effects. Thus long term use of NSAIDs compromise with its safety profile.The present study was undertaken to evaluate the long term clinical efficacy and safety of Flexiqule(Boswellia phyto extract) , and comparison with Etoricoxib (ETX) as a remedial measure .Boswellia phyto extract ,a herbal extract contains Boswellic acid as active ingredient, which has exhibited anti inflammatory and analgesic effect.Etoricoxib is Cox 2 inhibitor, frequently used NSAIDs,but not without side effects. One hundred patients of either sex, in age group of 50 to 65 years ,with clinical and radiological evidence of Osteoarthritis of knee were selected for trial. Patients were randomly divided into two groups. Group A received capsule Flexiqule 300 per day and group B received Etoricoxib 90 mg per day. All symptoms along with severity and duration were recorded prior to start of drug treatment. Routine hematological and radiological assessment was done before and the end of treatment. The patients were followed up every 04 weekly for six months. Symptomatic assessment score was carried out to determine the clinical efficacy of Flexiqule as compared to Etoricaxib. The minimal level of significance was fixed at 95% confidence limits and a 2-sided p value of <0.05 was considered significant. Clinical Efficacy was assessed by decrease in total sign and symptom scores. Results revealed that the average symptom score for joints involved, before and after the treatment was 7.140 to 1.060 in Flexiqule group while in Etoricoxib group was 6.800 to 1.840. There was reduction in symptom score 86% in Flexiqule group and 82% in ETX group. Thus objective improvement was comparatively better in Flexiqule groups. As for the side effects, No Adverse Reaction(ADRs), was noted in Flexiqule group, While Etoricoxib group experienced side effects -gastrointestinal, neurological or dermatological symptoms, which required due care. Though there was no significant alteration in hematological level, there was significant lowering in SGOT and SGPT levels, which indicated hepatoprotective effect of Flexiqule, and also significant decrease in Creatinine level indicated its renoprotective effect. No Adverse drug side effects were positive factor for prolong usage of Flexiqule as compared to Etoricoxid group. This study indicates that Flexiqule (Boswellia Phyto Extract) has proved better symptom relief in terms of relief of pain and free mobility of joints, equally effective but a safe alternative for long -term use in management of mild to moderate osteoarthritis, in patients unwilling for surgery.

Sch J App Med Sci . 2013; 1(4):226-232

Boswellia serrata extract attenuates inflammatory mediators and oxidative stress in collagen induced arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease which leads to destruction of joints. Current treatment modalities for RA either produce symptomatic relief (NSAIDs) or modify the disease process (DMARDs). Though effective, their use is also limited by their side effects. As a result, the interest in alternative, well tolerated anti-inflammatory remedies has re-emerged. Our aim was to evaluate the antioxidant and antiarthritic activity of Boswellia serrata gum resin extract (BSE) in collagen induced arthritis. Arthritis was induced in male Wistar rats by collagen induced arthritis (CIA) method. BSE was administered at doses of 100 and 200 mg/kg body weight once daily for 21 days. The effects of treatment in the rats were assessed by biochemical (articular elastase, MPO, LPO, GSH, catalase, SOD and NO), inflammatory mediators (IL-1b, IL-6, TNF-a, IL-10, IFN-g and PGE2), and histological studies in joints. BSE was effective in bringing significant changes on all the parameters (articular elastase, MPO, LPO, GSH, catalase, SOD and NO) studied. Oral administration of BSE resulted in significantly reduced levels of inflammatory mediators (IL-1b, IL-6, TNF-a, IFN-g and PGE2), and increased level of IL-10. The protective effects of BSE against RA were also evident from the decrease in arthritis scoring and bone histology. The abilities to inhibit proinflammatory cytokines and modulation of antioxidant status suggest that the protective effect of Boswellia serrata extract on arthritis in rats might be mediated via the modulation of immune system.

Phytomedicine . 2014 May 15;21(6):847-56

Chemoprevention of intestinal adenomatous polyposis by acetyl-11-keto-beta-boswellic acid in APC(Min/+) mice.

Acetyl-11-keto-beta-boswellic acid (AKBA) is a derivative of boswellic acid, which is an active component of the gum resin of Boswellia serrata. AKBA has been used as an adjuvant medication for treatment of inflammatory diseases. In this study, we aimed to evaluate the efficacy of AKBA as a chemopreventive agent against intestinal adenomatous polyposis in the adenomatous polyposis coli multiple intestinal neoplasia (APC(Min/+) mouse model. APC(Min/+) mice were administered AKBA by p.o. gavage for 8 consecutive weeks. The mice were sacrificed and the number, size and histopathology of intestinal polyps were examined by light microscopy. AKBA decreased polyp numbers by 48.9% in the small intestine and 60.4% in the colon. An even greater AKBA effect was observed in preventing the malignant progression of these polyps. The number of large (>3 cm) colonic polyposis was reduced by 77.8%. Histopathologic analysis demonstrated a significant reduction in the number of dysplastic cells and in the degree of dysplasia in each polyp after AKBA treatment. There was no evidence of high grade dysplasia or intramucosal carcinoma in any of the polyps examined within the treated group. More interestingly, interdigitated normal appearing intestinal villi were observed in the polyps of the treated group. During the course of the study, AKBA was well tolerated by the mice with no obvious signs of toxicity. Results from immunohistochemical staining, Western blotting and enzyme-linked immunosorbent assay indicated that the chemopreventive effect of AKBA was attributed to a collection of activities including antiproliferation, apoptosis induction, antiangiogenesis and anti-inflammation. AKBA was found to exert its chemopreventive action through the inhibition of the Wnt/b-catenin and NF-kB/cyclooxygenase-2 signaling pathways. Our findings suggest that AKBA could be a promising regimen in chemoprevention against intestinal tumorigenesis.

Int J Cancer . 2013 Jun 1; 132(11) :2667-81

Boswellic acid suppresses growth and metastasis of human pancreatic tumors in an orthotopic nude mouse model through modulation of multiple targets.

Pancreatic cancer (PaCa) is one of the most lethal cancers, with an estimated 5-year survival of <5% even when patients are given the best treatment available. In addition, these treatments are often toxic and expensive, thus new agents which are safe, affordable and effective are urgently needed. We describe here the results of our study with acetyl-11-keto-b-boswellic acid (AKBA), an agent obtained from an Ayurvedic medicine, gum resin of Boswellia serrata. Whether AKBA has an activity against human PaCa, was examined in in vitro models and in an orthotopic nude mouse model of PaCa. We found that AKBA inhibited the proliferation of four different PaCa cell lines (AsPC-1, PANC-28, and MIA PaCa-2 with K-Ras and p53 mutations, and BxPC-3 with wild-type K-Ras and p53 mutation). These effects correlated with an inhibition of constitutively active NF-kB and suppression of NF-kB regulating gene expression. AKBA also induced apoptosis, and sensitized the cells to apoptotic effects of gemcitabine. In the orthotopic nude mouse model of PaCa, p.o. administration of AKBA alone (100 mg/kg) significantly inhibited the tumor growth; this activity was enhanced by gemcitabine. In addition, AKBA inhibited the metastasis of the PaCa to spleen, liver, and lungs. This correlated with decreases in Ki-67, a biomarker of proliferation, and CD31, a biomarker of microvessel density, in the tumor tissue. AKBA produced significant decreases in the expression of NF-kB regulating genes in the tissues. Immunohistochemical analysis also showed AKBA downregulated the expression of COX-2, MMP-9, CXCR4, and VEGF in the tissues. Overall these results demonstrate that AKBA can suppress the growth and metastasis of human pancreatic tumors in an orthotopic nude mouse model that correlates with modulation of multiple targets.

PLoS One. 2011;6(10):e26943

Effect of Boswellia serrata gum resin on the morphology of hippocampal CA1 pyramidal cells in aged rat.

Experimental evidence indicates that administration of Boswellia resin, known as olibanum or Frankincense, increases memory power. It is reported that beta boswellic acid, the major component of Boswellia serrata gum resin, could enhance neurite outgrowth and branching in hippocampal neurons. We therefore studied whether Boswellia treatment produces morphological changes in the superior region of cornu ammonis (CA1) in aged rats. Sixteen male Wistar rats, 24 months of age, were randomly divided in experimental and control groups. The experimental group was orally administered Boswellia serrata gum resin (100 mg/kg per day for 8 weeks) and the control group received a similar volume of water. The Cavalieri principle was employed to estimate the volumes of CA1 hippocampal field, and a quantitative Golgi study was used to analysis of dendritic arborizations of CA1 pyramidal cells. Comparisons revealed that Boswellia-treated aged rats had greater volumes than control animals in stratum pyramidale and stratum radiatum lacunosum-moleculare. The neurons of CA1 in experimental rats had more dendritic segments (40.25 ± 4.20) than controls (30.9 ± 4.55), P = 0.001. The total dendritic length of CA1 neurons was approximately 20 % larger in the experimental group compared to control. Results also indicated that the aged rats treated withBoswellia resin had more numerical branching density in the apical dendrites of CA1 pyramidal neurons. The results of the present study show that long-term administration of Boswellia resin can attenuate age-related dendritic regression in CA1 pyramidal cells in rat hippocampus.

Anat Sci Int. 2014 Feb 11

The therapeutic effect of the aqueous extract of Boswellia serrata on the learning deficit in kindled rats.

BACKGROUND: It has been reported that epilepsy is a disorder of the central nervous system that causes memory impairment. This study examines the role of the aqueous extract of Boswellia on the learning disability of the pentylenetetrazol (PTZ)-induced kindled rats. METHODS: In this experimental study, 64 male rats were used. Kindling seizures were induced by three injections of 25 mg/kg of PTZ every 15 min. Control animals received normal saline instead. To evaluate the therapeutic effect of Boswellia extract on the PTZ-induced cognitive deficits, the aqueous extract (0, 0.1, 0.5 or 1 g/kg, i.p.) were administrated to all animals for three consecutive days. At 24 h later, passive avoidance learning of animals was examined using shuttle box apparatus, respectively. The time required for the animal stepping through the dark chamber was determined as step-through latency (STL). Data were subjected to the t-test and analysis of variance and followed by Tukey’s test for multiple comparisons. RESULTS: The STL of the kindled rats was significantly reduced compared with control ones (22/375 ± 4/19 for kindled and 295 ± 15/71 for control groups, respectively). Aqueous extract of Boswellia improved passive-avoidance learning ability in both control and PTZ-kindled animals (P < 0.05). CONCLUSIONS: The results can be stated that the Boswellia extract is offset by harmful effects of seizures on cognitive function and consumption of Boswelliaextract increases the learning ability in epileptic animals.

Int J Prev Med. 2014 May;5(5):563-8

Inhibition of fried meat-induced colorectal DNA damage and altered systemic genotoxicity in humans by crucifera, chlorophyllin, and yogurt.

Dietary exposures implicated as reducing or causing risk for colorectal cancer may reduce or cause DNA damage in colon tissue; however, no one has assessed this hypothesis directly in humans. Thus, we enrolled 16 healthy volunteers in a 4-week controlled feeding study where 8 subjects were randomly assigned to dietary regimens containing meat cooked at either low (100°C) or high temperature (250°C), each for 2 weeks in a crossover design. The other 8 subjects were randomly assigned to dietary regimens containing the high-temperature meat diet alone or in combination with 3 putative mutagen inhibitors: cruciferous vegetables, yogurt, and chlorophyllin tablets, also in a crossover design. Subjects were nonsmokers, at least 18 years old, and not currently taking prescription drugs or antibiotics. We used the Salmonella assay to analyze the meat, urine, and feces for mutagenicity, and the comet assay to analyze rectal biopsies and peripheral blood lymphocytes for DNA damage. Low-temperature meat had undetectable levels of heterocyclic amines (HCAs) and was not
mutagenic, whereas high-temperature meat had high HCA levels and was highly mutagenic. The high-temperature meat diet increased the mutagenicity of hydrolyzed urine and feces compared to the low-temperature meat diet. The mutagenicity of hydrolyzed urine was increased nearly twofold by the inhibitor diet, indicating that the inhibitors enhanced conjugation. Inhibitors decreased significantly the mutagenicity of un-hydrolyzed and hydrolyzed feces. The diets did not alter the levels of DNA damage in non-target white blood cells, but the inhibitor diet decreased nearly twofold the DNA damage in target colorectal cells. To our knowledge, this is the first demonstration that dietary factors can reduce DNA damage in the target tissue of fried-meat associated carcinogenesis.

PLoS One. 2011 Apr 25;6(4):e18707

Natural chlorophyll inhibits aflatoxin B1-induced multi-organ carcinogenesis in the rat.

Chemoprevention by chlorophyll (Chl) was investigated in a rat multi-organ carcinogenesis model. Twenty-one male F344 rats in three gavage groups (N = 7 rats each) received five daily doses of 250 microg/kg [(3)H]-aflatoxin B(1) ([(3)H]-AFB(1)) alone, or with 250 mg/kg chlorophyllin (CHL), or an equimolar amount (300 mg/kg) of Chl. CHL and Chl reduced hepatic DNA adduction by 42% (P = 0.031) and 55% (P = 0.008), respectively, AFB(1)-albumin adducts by 65% (P <0.001) and 71% (P < 0.001), respectively, and the major AFB-N(7)-guanine urinary adduct by 90% (P = 0.0047) and 92% (P = 0.0029), respectively. To explore mechanisms, fluorescence quenching experiments established formation of a non-covalent complex in vitro between AFB(1) and Chl (K(d) = 1.22 +/- 0.05 microM, stoichiometry = 1Chl:1AFB(1)) as well as CHL (K(d) = 3.05 +/- 0.04 microM; stoichiometry = 1CHL:1AFB(1)). The feces of CHL and Chl co-gavaged rats contained 137% (P = 0.0003) and 412% (P = 0.0048) more AFB(1) equivalents, respectively, than control feces, indicating CHL and Chl inhibited AFB(1) uptake. However, CHL or Chl treatment in vivo did not induce hepatic quinone reductase (NAD(P)H:quinone oxidoreductase) or glutathione S-transferase (GST) above control levels. These results are consistent with a mechanism involving complex-mediated reduction of carcinogen uptake, and do not support a role for phase II enzyme induction in vivo under these conditions. In a second study, 30 rats in three experimental groups were dosed as in study 1, but for 10 days. At 18 weeks, CHL and Chl had reduced the volume percent of liver occupied by GST placental form-positive foci by 74% (P < 0.001) and 77% (P < 0.001), respectively compared with control livers. CHL and Chl reduced the mean number of aberrant crypt foci per colon by 63% (P = 0.0026) and 75% (P = 0.0004), respectively. These results show Chl and CHL provide potent chemoprotection against early biochemical and late pathophysiological biomarkers of AFB(1) carcinogenesis in the rat liver and colon.

Carcinogenesis. 2007 Jun;28(6):1294-302

Chlorophyllin abrogates canonical Wnt/b-catenin signaling and angiogenesis to inhibit the development of DMBA-induced hamster cheek pouch carcinomas.

BACKGROUND: Chlorophyllin, a water soluble semi-synthetic food-grade derivative is reported to exhibit a wide range of beneficial health effects. We investigated the effect of chlorophyllin supplementation on Wnt/b-catenin and vascular endothelial growth factor (VEGF) signaling in the 7,12-dimethylbenz[a]anthracene

(DMBA)-induced hamster buccal pouch (HBP) carcinogenesis model. METHODS AND RESULTS: Hamsters were divided into 4 groups. The right buccal pouches of group 1 and 2 hamsters were painted with 0.5 % DMBA for 14 weeks. Group 2 animals received in addition chlorophyllin (4 mg/kg bw) in the diet. Group 3 animals received chlorophyllin alone and group 4 animals served as control. mRNA and protein expression of components of Wnt, VEGF, and PI3K/Akt signaling pathways were analyzed by RT-PCR and Western blot analysis. Dietary chlorophyllin administration suppressed the development of HBP carcinomas by altering the expression of several components of the Wnt/b-catenin signaling pathway. This was associated with inhibition of angiogenesis as evidenced by decreased expression of the proangiogenic factors HIF-1a, VEGF, and VEGFR2. Chlorophyllin administration also downregulated the expression of histone deacetylases involved in epigenetic regulation of tumor angiogenesis. CONCLUSION: Dietary chlorophyllin that abrogates Wnt/b-catenin and VEGF signaling by targeting a multitude of key signaling molecules is an attractive candidate for preventing tumor progression.

Cell Oncol (Dordr) . 2012 Oct;35(5):385-95

Chlorophyllin e4 is a novel photosensitizer against human bladder cancer cells.

The aim of the study was to investigate the photodynamic effect of the novel photosensitizer chlorophyllin e4 against human bladder cancer cells. T24 and 5,637 bladder cancer cell lines were incubated with chlorophyllin e4 and irradiated with a 650-nm laser light. The controls included cells treated with chlorophyllin e4 but without light as well as cells exposed to laser light without chlorophyllin e4. Photocytotoxicity was monitored with MTT assay and apoptosis was measured by flow cytometry. In addition, confocal laser scanning microscopy was used to assess the subcellular localization of chlorophyllin e4. Chlorophyllin e4 exhibited significant photocytotoxicity in both T24 and 5,637 cells, which resulted in a maximum of 82.43 and 85.06% cell death, respectively. Treatment with chlorophyllin e4 or laser light alone did notinduce cytotoxicity. In addition, chlorophyllin e4-mediated PDT induced a significantly higher percentage of apoptosis in T24 and 5,637 cells compared to the control groups (p<0.01). Moreover, confocal laser scanning microscopy revealed that chlorophyllin e4 co-localized with mitochondria in both cell lines. In conclusion, the remarkable photocytotoxicity, natural abundance and inexpensive composition of chlorophyllin e4 suggest that this compound may be a novel, effective photosensitizer for the treatment of human superficial bladder cancer.

Oncol Rep . 2012 May;27(5):1455-60

Photodynamic therapy with the novel photosensitizer chlorophyllin f induces apoptosis and autophagy in human bladder cancer cells.

Background and Objec-tives Our group recently synthesized a new, low-cost photosensitizer, chlorophyllin f. In this study, the effects of chlorophyllin f-mediated photodynamic therapy (PDT) and its potential mechanisms were examined in human bladder cancer cells. Materials and Methods MitoTracker ® Green probe and LysoTracker® Green probe were used to label mitochondria and lysosomes, revealing the intracellular localization of chlorophyllin f in 5,637 and T24 cells by confocal laser scanning microscopy (CLSM). The cells were treated with chlorophyllin f-mediated PDT; the photo-cytotoxicity of chlorophyllin f was monitored using the Cell Counting Kit-8 assay, and apoptosis was measured by Annexin V-FITC/PI dual staining. Western blotting, transmission electron microscopy (TEM), and staining with Cyto-ID® Autophagy Detection dye, monodansylcadaverine (MDC) and acridine orange were performed to assess autophagy. The role of autophagy was examined by measuring cell viability and apoptosis in both cell lines pretreated with the autophagy inhibitor 3-methyladenine (3-MA). Results Chlorophyllin f showed affinity for mitochondria and lysosomes. It exhibited significant photocytotoxicity, resulting in a maximum of 86.51% and 84.88% cell death in 5,637 and T24 cells, respectively. Additionally, chlorophyllin f-mediated PDT (f-PDT) also induced a significantly higher percentage of apoptosis in treated cells compared to the control groups (P < 0.05). Moreover, the expression of Beclin1 protein and the proportion of LC3-II:LC3-I in both cell lines significantly increased after f-PDT. Autophagy, characterized by an increase in the formation of Cyto-ID® Autophagy Detection dye-labeled autophagosomes, MDC fluorescent dye-labeled autophagic vacuoles and acridine orange-labeled acidic vesicular organelles (AVOs), was observed in f-PDT-treated cells. TEM also revealed double-membrane autophagosome structures 1 hour after f-PDT. Most importantly, when pretreated with 3-MA, the two cell lines showed more significant photo-cytotoxicity and apoptotic cell death compared to those exposed to f-PDT alone (P < 0.05). Conclusion Chlorophyllin f-mediated PDT exerts anti-tumor activity by inducing apoptosis and autophagy, and most importantly, autophagy inhibition enhances f-PDT-mediated apoptotic cell death. These results suggest that chlorophyllin f is a new, effective photosensitizer and that the combination of f-PDT with autophagy inhibitors may be an attractive therapeutic strategy against human non-muscle invasive bladder cancer.

Lasers Surg Med. Apr 2014;46(4):319-34

In vitro bioavailability of iron from the heme analogue sodium iron chlorophyllin.

The use of heme analogues from vegetable origin could provide an alternative iron source of potentially high bioavailability. Sodium iron chlorophyllin is a water-soluble semisynthetic chlorophyll derivative where the magnesium in the porphyrin ring has been substituted by iron. We have used an in vitro model that combines gastric and intestinal digestion followed by intestinal iron uptake in Caco-2 cells to determine the bioavailability of iron from sodium iron chlorophyllin. Our results demonstrate that sodium iron chlorophyllin is stable under simulated gastrointestinal conditions and is able to deliver bioavailable iron to Caco-2 cells. Similar to the heme, the bioavailability of iron from sodium iron chlorophyllin is dependent on the food matrix, and it was inhibited by calcium. Potentially, sodium iron chlorophyllin could be used as an iron fortificant from vegetable origin with high bioavailability.

J Agric Food Chem . 2010 Jan 27;58(2):1327-32

Chlorophyll, chlorophyllin and related tetrapyrroles are significant inducers of mammalian phase 2 cytoprotective genes.

Plant chlorophylls and carotenoids are highly colored, conjugated polyenes that play central roles in photosynthesis. Other porphyrins (tetrapyrroles), such as cytochromes, which are structurally related to chlorophyll, participate in redox reactions in many living systems. An unexpected new property of tetrapyrroles, including tetramethyl coproporphyrin III, tetrabenzoporphine, copper chlorin e4ethyl ester, and of carotenoids including zeaxanthin and alpha-cryptoxanthin is their ability to induce mammalian phase II proteins that protect cells against oxidants and electrophiles. The capacity of these compounds to induce the phase II response depends upon their ability or that of their metabolites to react with thiol groups, a property shared with all other classes of phase II inducers, which show few other structural similarities. Pseudo second-order rate constants of these inducers are correlated with their potency in inducing the phase II enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) in murine hepatoma cells. One of the most potent inducers was isolated from chlorophyllin, a semisynthetic water-soluble chlorophyll derivative. Although chlorophyll itself is low in inducer potency, it may nevertheless account for some of the disease-protective effects attributed to diets rich in green vegetables because it occurs in much higher concentrations in those plants than the widely studied ‘phytochemicals’.

Carcinogenesis. 2005 Jul;26(7):1247-55

Evaluation of the effects of chlorophyllin on apoptosis induction, inhibition of cellular proliferation and mRNA Expression of CASP8, CASP9, APC and b-catenin

Chlorophyllin is a semi-synthetic derivative of chlorophyll with antioxidant and antimutagen properties that controls the enzymes involved in the metabolism of xenobiotics and in the induction of apoptosis. In this study, we evaluated the effects of chlorophyllin on apoptosis induction, inhibition of cell proliferation, and gene expression in the human colorectal adenocarcinoma cell line HT29. Chlorophyllin significantly reduced cell survival after 48 h at 100 :g/mL and after 24 h at 500 or 1,000 :g/mL, respectively based on both MTT cytotoxicity and cell proliferation kinetics assays. These effects were dose dependent. Chlorophyllin did not induce apoptosis after 24 h at any concentration. Chlorophyllin downregulated the cell cycle genes APC and b-catenin (CTNNB1) but did not affect the expression of apoptotic induction genes in the extrinsic pathway (CASP8) or the intrinsic pathway (CASP9). At the studied concentrations, the inhibitory effect of chlorophyllin on cell growth was directly related to the regulation of b-catenin gene expression and not to APC expression, because APC was mutated and inactive. The studied concentrations suggest no potential for chlorophyllin as an apoptosis inducer based on cytomorphological changes or gene expression changes of the studied caspases.

Current Research Journal of Biological
Sciences
. 2012;4(3):315-322

Effects of chlorophyll and chlorophyllin on low-dose aflatoxin B(1) pharmacokinetics in human volunteers.

Chlorophyll (Chla) and chlorophyllin (CHL) were shown previously to reduce carcinogen bioavailability, biomarker damage, and tumorigenicity in trout and rats. These findings were partially extended to humans, where CHL reduced excretion of aflatoxin B(1) (AFB(1))-DNA repair products in Chinese unavoidably exposed to dietary AFB(1). However, neither AFB(1) pharmacokinetics nor Chla effects were examined. We conducted an unblinded crossover study to establish AFB(1) pharmacokinetic parameters among four human volunteers, and to explore possible effects of CHL or Chla cotreatment in three of those volunteers. For protocol 1, fasted subjects received an Institutional Review Board-approved dose of 14C-AFB(1) (30 ng, 5 nCi) by capsule with 100 mL water, followed by normal eating and drinking after 2 hours. Blood and cumulative urine samples were collected over 72 hours, and 14C- AFB(1) equivalents were determined by accelerator mass spectrometry. Protocols 2 and 3 were similar except capsules also contained 150 mg of purified Chla or CHL, respectively. Protocols were repeated thrice for each volunteer. The study revealed rapid human AFB(1) uptake (plasma k(a), 5.05 + or - 1.10 h(-1); T(max), 1.0 hour) and urinary elimination (95% complete by 24 hours) kinetics. Chla and CHL treatment each significantly impeded AFB(1) absorption and reduced Cmax and AUCs (plasma and urine) in one or more subjects. These initial results provide AFB(1) pharmacokinetic parameters previously unavailable for humans, and suggest that Chla or CHL co-consumption may limit the bioavailability of ingested aflatoxin in humans, as they do in animal models.

Cancer Prev Res (Phila). 2009 Dec;2(12):1015-22

Low-dose dietary chlorophyll inhibits multi-organ carcinogenesis in the rainbow trout.

We recently reported that chlorophyll (Chl) strongly inhibits aflatoxin B(1) preneoplasia biomarkers in rats when administered by co-gavage (Simonich et al., 2007. Natural chlorophyll inhibits aflatoxin B1-induced multi-organ carcinogenesis in the rat. Carcinogenesis 28, 1294-1302.). The present study extends this by examining the effects of dietary Chl on tumor development, using rainbow trout to explore ubiquity of mechanism. Duplicate groups of 140 trout were fed diet containing 224 ppm dibenzo[a,l]pyrene (DBP) alone, or with 1,000-6,000 ppm Chl, for 4 weeks. DBP induced high tumor incidences in liver (51%)

and stomach (56%), whereas Chl co-fed at 2,000, 4,000 or 6,000 ppm reduced incidences in stomach (to 29%, 23% and 19%, resp., P<0.005) and liver (to 21%, 28% and 26%, resp., P<0.0005). Chlorophyllin (CHL) at 2000 ppm gave similar protection. Chl complexed with DBP in vitro (2Chl:DBP, K(d1)=4.44+/-0.46 microM, K(d2)=3.30+/-0.18 microM), as did CHL (K(d1)=1.38+/-0.32 microM, K(d2)=1.17+/-0.05 microM), possibly explaining their ability to inhibit DBP uptake into the liver by 61-63% (P<0.001). This is the first demonstration that dietary Chl can reduce tumorigenesis in any whole animal model, and that it may do so by a simple, species-independent mechanism.

Food Chem Toxicol. 2008 Mar;46(3):1014-24

Oral zinc for the common cold.

CLINICAL QUESTION: Is oral zinc associated with a shorter duration, decreased severity, and reduced incidence of the common cold compared with placebo? BOTTOM LINE: When initiated within 24 hours of symptom onset, oral zinc is associated with a shorter duration of the common cold in healthy people. However, there is no association between oral zinc and symptom severity, and the prevalence of adverse effects with zinc lozenges is high. Given the high heterogeneity of data, these results should be interpreted with caution. Used prophylactically, oral zinc is associated with a reduced cold incidence in children. Prophylactic use has not been studied in adults.

JAMA. 2014 Apr 9;311(14):1440-1

Zinc lozenges as cure for the common cold—a review and hypothesis.

A 7-day reduction in duration of common colds was shown by Eby et al. in 1984 using 23mg zinc gluconate throat lozenges. Over the following 25years, 14 double-blind, placebo-controlled, randomized clinical trials produced widely differing results with about one-half showing success and the remainder showing failure. Positively charged, ionic zinc (iZn), but not bound zinc, is strongly astringent, antirhinoviral, increases interferon-gamma (IFN-gamma) 10-fold, inhibits intercellular adhesion molecule-1 (ICAM-1) and inhibits the release of vasoactive ingredients from mast cell granules. Solution equilibrium chemistry analytical techniques showed lozenge iZn fraction varying from 0% to 100% of total lozenge zinc between trials, with zinc acetate (ZA) releasing 100% iZn, zinc gluconate (ZG) releasing 72% iZn and other zinc compounds releasing much less or none at physiologic pH 7.4. Since only iZn has in vitro benefits, iZn variations are hypothesized to have produced the widely varying clinical results. In support of the iZn hypothesis, lozenge iZn and total daily iZn in trials were found highly correlated with reductions in common cold durations with statistical significance for mean duration (P<0.001) and median duration (P<0.004), while total zinc (iZn plus bound) showed no correlation with changes in duration. Duration reductions (mean 0 days, median 0.43 days) for multi-ligand ZG and ZA lozenges differed significantly from duration reductions (mean 3.37 days, median 2.9 days) for single ligand ZA and ZG lozenges (P<0.001) showing that additive ligands as flavor-masks damaged or eliminated efficacy. Five of 6 trials with lozenges whose zinc compositions had a first stability constant of 1.7 or less succeeded, while only 2 of 9 trials of lozenges with higher stability succeeded (P<0.02). From the strong, multiple statistical relationships found, it is inferred that iZn is the active ingredient in zinc lozenges for colds, as it is in vitro against rhinoviruses, and that solution chemistry analytical techniques used at physiological pH are correct means for lozenge iZn analysis. Zinc lozenges slowly dissolving in the mouth over a 20-30 min period releasing adequate iZn (18 mg) used each 2h are hypothesized to shorten common colds by 6-7 days, which is a cure for the common cold. Due to inadequate lozenge iZn very few of more than 40 different brands of zinc lozenges on the US market are expected to have any effect on the duration or severity of common colds.

Med Hypotheses . 2010 Mar;74(3):482-92

Cubozoan venom-induced cardiovascular collapse is caused by hyperkalemia and prevented by zinc gluconate in mice.

Chironex fleckeri (Australian box jellyfish) stings can cause acute cardiovascular collapse and death. We developed methods to recover venom with high specific activity, and evaluated the effects of both total venom and constituent porins at doses equivalent to lethal envenomation. Marked potassium release occurred within 5 min and hemolysis within 20 min in human red blood cells (RBC) exposed to venom or purified venom porin. Electron microscopy revealed abundant ~12-nm transmembrane pores in RBC exposed to purified venom porins. C57BL/6 mice injected with venom showed rapid decline in ejection fraction with progression to electromechanical dissociation and electrocardiographic findings consistent with acute hyperkalemia. Recognizing that porin assembly can be inhibited by zinc, we found that zinc gluconate inhibited potassium efflux from RBC exposed to total venom or purified porin, and prolonged survival time in mice following venom injection. These findings suggest that hyperkalemia is the critical event following Chironex fleckeri envenomation and that rapid administration of zinc could be life saving in human sting victims.

PLoS One . 2012;7(12):e51368

Ineffectiveness of zinc gluconate nasal spray and zinc orotate lozenges in common-cold treatment: a double-blind, placebo-controlled clinical trial.

BACKGROUND: Zinc gluconate and zinc acetate lozenges have been reported to shorten the duration of common colds in a dose-response manner when the amount of the active ingredient, positively charged zinc ions, is sufficient. OBJECTIVE: To improve results using a zinc gluconate nasal spray with zinc orotate lozenges. DESIGN: Double-blind, placebo-controlled clinical trial. SETTING: Private physician’s clinic in Austin, Texas. PARTICIPANTS: Seventy-seven volunteers, all of whom had 2 or more signs and symptoms of common colds (with at least 1 nasal symptom) on enrollment in the study. METHODS: Zinc gluconate nasal spray (10 mmol) or placebo was used every 15 to 30 minutes, and lozenges were used each several hours to reduce duration and severity of common colds. An intention of treatment was to keep the nasal tissues wet with zinc gluconate solution during wakeful hours. RESULTS: After 7 days of treatment, 10 of 16 (63%) zinc-treated patients were asymptomatic compared to 9 of 17 (53%) placebo-treated patients (P = .57). This treatment caused olfactory region pain in some patients and did not reduce the duration or severity of common colds. Treatment did not produce anosmia, which has been reported in other studies following olfactory region administration of ionic zinc in many species and in humans from zinc nasal sprays and gels. CONCLUSION: We contend that it is unethical to introduce any potentially permanent anosmia-inducing agent such as zinc or other heavy metals into the interior of the nose in a manner that could result in contact with the olfactory region to treat a temporary discomfort such as a common cold or allergy. We found no reason to recommend intranasal zinc gluconate or zinc orotate lozenges in treating common colds.

Intern Ther Health Med . 2006 Jan-Feb;12(1):34-8.

Duration and severity of symptoms and levels of plasma interleukin-1 receptor antagonist, soluble tumor necrosis factor receptor, and adhesion molecules in patients with common cold treated with zinc acetate.

BACKGROUND: Zinc lozenges have been used for treatment of the common cold; however, the results remain controversial. METHODS: Fifty ambulatory volunteers were recruited within 24 h of developing symptoms of the common cold for a randomized, double-blind, placebo-controlled trial of zinc. Participants took 1 lozenge containing 13.3 mg of zinc (as zinc acetate) or placebo every 2-3 h while awake. The subjective scores for common cold symptoms were recorded daily. Plasma zinc, soluble interleukin (IL)-1 receptor antagonist (sIL-1ra), soluble tumor necrosis factor receptor 1, soluble vascular endothelial cell adhesion molecule, and soluble intercellular adhesion molecule (sICAM)-1 were assayed on days 1 and 5. RESULTS: Compared with the placebo group, the zinc group had a shorter mean overall duration of cold (4.0 vs. 7.1 days; P < .0001) and shorter durations of cough (2.1 vs. 5.0 days; P < .0001) and nasal discharge (3.0 vs. 4.5 days, P =.02) Blinding of subjects was adequate, and adverse effects were comparable in the 2 groups. Symptom severity scores were decreased significantly in the zincgroup. Mean changes in plasma levels of zinc, sIL-1ra, and ICAM-1 differed significantly between groups. CONCLUSION: Administration of zinc lozenges was associated with reduced duration and severity of cold symptoms. We related the improvement in cold symptoms to the antioxidant and anti-inflammatory properties of zinc.

J Infect Dis. 2008 Mar 15;197(6):795-802

Effect of zincum gluconicum nasal gel on the duration and symptom severity of the common cold in otherwise healthy adults.

BACKGROUND: Previous studies suggest that zinc salts may be effective in treating the common cold. Since rhinovirus infections occur primarily in the nasal cavity, an attempt to arrest the infection at the portal of entry seems logical. AIM: To assess the ability of zinc nasal gel to shorten the duration and reduce the severity of the common cold in healthy adults. STUDY DESIGN: Randomized, double blind, placebo-controlled study. METHODS: Of 1087 patients screened by telephone, 80 patients were enrolled, all presenting within 24-48 h of the onset of illness. They received one dose per nostril of a nasal gel spray containing either 33 mmol/l zincum gluconicum, or an identical placebo four times daily until their symptoms resolved, for a maximum of 10 days. RESULTS: Median duration of cold symptoms in the zinc group was significantly shorter than in the placebo group (median [IQR] 4.3 days [2.5-5.5] vs. 6 days [5-8.5], p=0.002). Nasal drainage, nasal congestion, hoarseness, and sore throat were the symptoms most affected. Significant reduction of total symptom scores started from the second day of the study. Adverse effects (mainly nasal stinging) were similar in both groups. DISCUSSION: Zincum gluconicum nasal gel shortens duration and reduces symptom severity of the common cold in healthy adults, when started within 24-48 h of theonset of illness.

QJM . 2003 Jan;96(1):35-43

Zinc nasal gel for the treatment of common cold symptoms: a double-blind, placebo-controlled trial.

Effective treatment for the common cold have been difficult to develop because so many different types of virus are responsible for this condition. Oral zinc has been studied as a possible means of preventing or alleviating symptoms, with mixed results. We studied a new approach to zinc therapy--an over-the-counter nasal gel formulation (Zicam)—to independently evaluate its efficacy as a treatment for the common cold. Our study was conducted at four sites over a 5-month period. The study group consisted of 213 patients with recent-onset(< or = 24) cold symptoms; 108 patients received zinc therapy, and 105 reviewed placebo. Symptom charts were used to track the duration and severity of each patient’s symptoms. At study’s end, the duration of symptoms was 2.3 days (+/-0.9)in the zinc group and 9.0 days (+/-2.5)in the control group—a statistically significant difference (p <0.05). These results provide evidence that zinc nasal gel is effective in shortening the duration of common cold symptoms off when taken within 24 hours of their onset.

Ear Nose Throat J. 2000 Oct;79(10):778-80, 782

Zinc lozenges: cold cure or candy? Solution chemistry determinations.

Common colds were shortened by 7 days in a 1984 clinical trial using zinc gluconate throat lozenges each 2 h. Between then and 2004, 10 other double-blind, placebo-controlled clinical trials showed widely varying results. This re-analysis of these trials presents solution chemistry methods to elucidate differences in efficacy. Statistically significant correlation was shown between total daily dosages of positively charged zinc species and reductions in median (p = 0.005) and mean duration (p < 0.02) of common colds in these trials.

Biosci Rep. 2004 Feb;24(1):23-39

An open-label, single-center, phase IV clinical study of the effectiveness of zinc gluconate glycine lozenges (Cold-Eeze) in reducing the duration and symptoms of the common cold in school-aged subjects.

Each year, more than 62 million cases of the common cold in the United States require medical attention and more than 80% affect school-aged children. The objective of this prospective, intent-to-treat, phase IV study was to determine the therapeutic and prophylactic effectiveness of zinc gluconate glycine lozenges (Cold-Eeze) for the common cold. Zinc lozenges were administered once daily during the cold season for prophylaxis. For therapeutic purposes, lozenges were given 4 times per day. The primary objective of the study was the treatment effect on cold duration, and the secondary objective was the effect on the number of common colds. A putative control from our previous study was used for comparison. A total of 178 children, ages 12 to 18 years, was enrolled, of which 134 met criteria for efficacy analysis. The average cold duration with therapeutic lozenge use was 6.9 +/- 3.1 days, significantly shorter than the 9.0 +/- 3.5 days found in the control group (P < 0.001). The mean number of colds was 1.28 +/- 1.03 with zinc lozenge prophylaxis versus 1.7 +/- 1.91 without prophylaxis (P < 0.05), a 25% reduction. With prophylaxis, 25% of the subjects did not experience a cold and two-thirds never had a cold or only had 1 cold. There was no antibiotic use for any cold, and there were no adverse events reported. Results of this study are consistent with those from our previous retrospective study showing significantly shorter cold duration and fewer colds with the use of zinc gluconate glycine lozenges. The zinc gluconate glycine lozenges are well tolerated and are an easy-to-administer therapy that has the potential to substantially reduce cold-related school absences and antibiotic use and misuse as well as to provide a cost saving.

Am J Ther . 2003 Sep-Oct;10(5):324-9

Treatment of acute lymphocytic leukemia using zinc adjuvant with chemotherapy and radiation—a case history and hypothesis.

Low blood levels of zinc are often noted in acute lymphocytic leukemia (ALL), but zinc is not administered as part of any modern chemotherapy program in the treatment of ALL. Upon noting low blood levels of zinc in a 3-year-old 11.3 kg girl, zinc at the rate of 3.18 mg/kg body weight/day was administered from the start of chemotherapy through the full 3 years of maintenance therapy. Dosage was split with 18 mg given at breakfast and 18 mg zinc with supper. The result was a bone marrow remission from 95+% blast cells to an observed zero blast cell count in both hips within the first 14 days of treatment which never relapsed. In addition to the reduction of blast cells to an observed count of zero (not a single leukemic or normal blast), red blood cell production and other hemopoietic functions returned to normal at a clinically remarkable rate. There were no side effects from zinc or chemotherapy at any time, and zinc is hypothesized to have improved the patient’s overall ability to withstand toxic effects of chemotherapy. This report identifies zinc treatment as being vital to rapid and permanent recovery from ALL. The extremely broad role of zinc in pre-leukemic adverse health conditions, viral, fungal and tumoral immunity, hemopoietics, cell growth, division and differentiation, genetics and chemotherapy interactions are considered. If a nutrient such as zinc could be shown to strengthen the function of chemotherapy and immune function, then it could be hypothesized that the relapse rate would be lessened since the relapse rate is related to both the rate at which a remission is obtained and the thoroughness of the elimination of leukemic blasts. Identical results also occurred in 13 other children with ALL whose parents chose to treat with zinc adjuvant. Since treatment with zinc and other identified deficient nutrients, particularly magnesium, did not appear injurious in ALL and they appear to be highly beneficial, controlled clinical studies of zinc (3.18 mg/kg body weight/day) with magnesium (8.0 mg/kg body weight/day) as adjuvants to chemotherapy in the treatment of childhood ALL are suggested. Treatment with zinc adjuvant is hypothesized to accelerate recovery from ALL, and in conjunction with chemotherapy, cure ALL.

Med Hypotheses. 2005;64(6):1124-6

Improved management of primary chronic fatigue syndrome with the supplement French oak wood extract (Robuvit ®): a pilot, registry evaluation.

AIM: The aim of this supplement study was to evaluate French oak wood extract (Robuvit®, Horphag Research Ltd) used as a supplement in association with a defined management plan for chronic fatigue syndrome (CFS) in healthy subjects with CFS, a condition that has, so far, no specific treatment or management standards. METHODS: Robuvit® is a new proprietary and exclusive extract of oak wood with important antoxidant actions. The dosage of the supplementation was 200 mg/day for at least 6 months. The CFS questionnaire and the Brief Mood Introspection Scale (BMIS) questionnaire were used to evaluate mood variations associated with CFS patients. The CFS form includes an analogue scale to record the variations of single symptoms with a score range of 0-10. At inclusion into the registry study, at least 5 symptoms were present. All subjects (age range 35-44; BMI range 24-26) with CFS were tested for oxidative stress: 61 out of 91 subjects had an increased value of oxidative stress. The BMIS scale evaluating mood changes in time was also used. The evaluation was repeated at 3 and 6 months. RESULTS: Out of 91 eligible subjects with CFS, 48 subjects (31 with increased oxidative stress) were accepted as part of the supplement registry study using Robuvit; 43 (30 with increased oxidative stress) were accepted as controls using only the management plan. In the Robuvit® group there were 3 drop outs; also 3 controls were lost. Oxidative stress was increased in 64.58% of subjects that used Robuvit® and in 69.7% of controls. The average values of oxidative stress were expressed for the whole group. The average follow up was 199.3;9.2 days in the Robuvit® group and 202.2;5.5 in the control group with a minimum of 6 months. Considering variations in oxidative stress, there was no significant average change in controls, but a significant decrease from the initial values was observed in Robuvit® subjects after 3 and 6 months. The CFS questionnaire variations in score indicated that there was a significant improvement for most symptoms after 3 and 6 months in the Robuvit® group. Positive variations were also present in controls, indicating the positive effect of an increased attention to CFS. The improvement in signs/symptoms was significantly more valuable in subjects using the oak wood extract considering the main 8 symptoms and the accessory symptoms. Considering the BMIS variations, the totals for positive and negative items were significantly more favourable for Robuvit® subjects. Overall mood evaluation in the oak wood extract group improved from an inclusion average of -6.93;2.1 to +4.32;2.6 at 6 months; in contrast it changed from -6.5;2.5 to -3.4;1.5 in controls. No side effects were observed during the supplementation with Robuvit®. The compliance was optimal with 93% of the capsules correctly used. CONCLUSION: This promising pilot supplement registry study indicates a new opportunity of management for these difficult and often neglected patients. Correlation between oxidative stress and CFS have to be better explored.

Panminerva Med . 2014 Mar;56(1):63-72

Effect of the french oak wood extract Robuvit® on markers of oxidative stress and activity of antioxidant enzymes in healthy volunteers: a pilot study.

We examined in vitro antioxidant capacity of polyphenolic extract obtained from the wood of oak Quercus robur (QR), Robuvit®, using TEAC (Trolox equivalent antioxidant capacity) method and the effect of its intake on markers of oxidative stress, activity of antioxidant enzymes, and total antioxidant capacity in plasma of 20 healthy volunteers. Markers of oxidative damage to proteins, DNA, and lipids and activities of Cu/Zn-superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were determined in the erythrocytes. We have found an in vitro antioxidant capacity of Robuvit of 6.37 micromole Trolox equivalent/mg of Robuvit®. One month intake of Robuvit® in daily dose of 300 mg has significantly decreased the serum level of advanced oxidation protein products (AOPP) and lipid peroxides (LP). Significantly increased activities of SOD and CAT as well as total antioxidant capacity of plasma after one month intake of Robuvit have been shown. In conclusion, we have demonstrated for the first time that the intake of Robuvit® is associated with decrease of markers of oxidative stress and increase of activity of antioxidant enzymes and total antioxidant capacity of plasma in vivo.

Oxid Med Cell Longev . 2014;2014:639868

Absorption, Metabolism, and Effects at Transcriptome Level of a Standardized French Oak Wood Extract, Robuvit, in Healthy Volunteers: Pilot Study.

The consumption of wine and spirits, traditionally aged in oak barrels, exposes humans to roburin ingestion. These molecules belong to a class of ellagitannins (ETs), and their only known source is oak wood. Very little is currently known about roburin bioavailability and biological activity. We reported for the first time human absorption of roburins from a French oak wood ( Quercus robur) water extract (Robuvit®) by measuring the increase of total phenols (from 0.63 ± 0.06 to 1.26 ± 0.18 µg GAE equiv/mL plasma) and the appearance of roburin metabolites (three different glucoronidate urolithins and ellagic acid), in plasma, after 5 days of supplementation. Robuvit® supplementation induced also the increase of plasma antioxidant capacity from 1.8 ± 0.05 to 1.9 ± 0.01 nmol Trolox equiv/mL plasma. Moreover, utilizing a combined ex vivo cell culture approach, we assessed the effect of Q. robur metabolites (present in human serum after supplementation) on gene expression modulation, utilizing an Affymetrix array matrix, in endothelial, neuronal, and keratinocyte cell lines. The functional analysis reveals that Robuvit® metabolites affect ribosome, cell cycle, and spliceosome pathways.

J Agric Food Chem . 2014 Jan 6.

Biological significance of urolithins, the gut microbial ellagic Acid-derived metabolites: the evidence so far.

The health benefits attributed to pomegranate have been associated with its high content in polyphenols, particularly ellagitannins. This is also the case for other ellagitannin-containing fruits and nuts including strawberry, raspberry, blackberry, walnuts, and muscadine grapes. The bioavailability of ellagitannins and ellagic acid is however very low. These molecules suffer extensive metabolism by the gut microbiota to produce urolithins that are much better absorbed. Urolithins circulate in plasma as glucuronide and sulfate conjugates at concentrations in the range of 0.2-20  µ M. It is therefore conceivable that the health effects of ellagitannin-containing products can be associated with these gut-produced urolithins, and thus the evaluation of the biological effects of these metabolites is essential. Recent research, mostly based on in vitro testing, has shown preliminary evidence of the anti-inflammatory, anticarcinogenic, antiglycative, antioxidant, and antimicrobial effects of urolithins, supporting their potential contribution to the health effects attributed to pomegranate and ellagitannin-rich foods. The number of in vivo studies is still limited, but they show preventive effects of urolithins on gut and systemic inflammation that encourage further research. Both in vivo and mechanistic studies are necessary to clarify the health effects of these metabolites. Attention should be paid when designing these mechanistic studies in order to use the physiologically relevant metabolites (urolithins in gut models and their conjugated derivatives in systemic models) at concentrations that can be reached in vivo.

Evid Based Complement Alternat Med . 2013;2013:270418

French oak wood extract (Robuvit®) reduces the wheal and flare response to histamine and decreases capillary filtration in normal subjects

Aim: Objective of the study was to evaluate the effect of supplementation with French oak wood extract (Robuvit®) on the cutaneous histamine reaction and capillary permeability in healthy subjects. Methods: 14 female participants in a control group and 16 females in the test group, supplemented daily with 300 mg French oak wood extract (Robuvit®) for 3 days and received an intracutaneous injection of histamine. After the injection, wheal and redness areas on the skin were evaluated. Time until complete disappearance of flares and skin thickness was measured. Microcirculation flux at the injection site was recorded by Laser Doppler. Capillary filtration in the lower limbs was measured by venous occlusio plethysmography on days 1, 2 and 3 following intake of Robuvit®. Results: In comparison to controls, the reaction to histamin was reduced in the group supplemented with the French oak wood extract: significantly smaller wheal area (-28%), smaller redness area (-13%), less skin thickness (-18%), lower microcirculation flux (-49%). The capillary filtration in supplemented subjects was significantly lower after 3 days (22%) compared to control.
Conclusion: This pilot evaluation indicates an anti-histaminic activity of the French oak wood extract. The reduced capillary filtration suggests a role of Robuvit® in edema prevention.

Minerva Biotecnologica . 2013 Dec;25(4):199-205

Epstein-Barr virus infection induces indoleamine 2,3-dioxygenase expression in human monocyte-derived macrophages through p38/mitogen-activated protein kinase and NF-kB pathways: impairment in T cell functions.

Epstein-Barr virus (EBV) infection has been observed in tumor-infiltrated macrophages, but its infection effects on macrophage immune functions are poorly understood. Here, we showed that some macrophages in the tumor stroma of nasopharyngeal carcinoma (NPC) tissue expressed the immunosuppressive protein indoleamine 2,3-dioxygenase (IDO) more strongly than did tumor cells. EBV infection induced mRNA, protein, and enzymatic activity of IDO in human monocyte-derived macrophages (MDMs). Infection increased the production of tumor necrosis factor alpha (TNF-a) and interleukin-6 (IL-6), whereas the neutralizing antibodies against TNF-a and IL-6 inhibited IDO induction. EBV infection also activated the mitogen-activated protein kinase (MAPK) p38 and NF-kB, and the inhibition of these two pathways with SB202190 and SN50 almost abrogated TNF-a and IL-6 production and inhibited IDO production. Moreover, the activation of IDO in response to EBV infection of MDMs suppressed the proliferation of T cells and impaired the cytotoxic activity of CD8(+) T cells, whereas the inhibition of IDO activity with 1-methyl-l-tryptophan (1-MT) did not affect T cell proliferation and function. These findings indicate that EBV-induced IDO expression in MDMs is substantially mediated by IL-6- and TNF-a-dependent mechanisms via the p38/MAPK and NF-kB pathways, suggesting that a possible role of EBV-mediated IDO expression in tumor stroma of NPC may be to create a microenvironment of suppressed T cell immune responses. IMPORTANCE: CD8(+) cytotoxic T lymphocytes (CTLs) play an important role in the control of viral infections and destroy tumor cells. Activation of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) in cancer tissues facilitates immune escape by the impairment of CTL functions. IDO expression was observed in some macrophages of the tumor stroma of nasopharyngeal carcinoma (NPC) tissue, and IDO could be induced in Epstein-Barr virus (EBV)-infected human monocyte-derived macrophages (MDMs). NPC cells and macrophages have been found to produce IDO in a gamma interferon (IFN-g)-dependent manner. Instead, EBV-induced IDO expression in MDMs is substantially mediated by IL-6- and TNF-a-dependent mechanisms via the p38/MAPK and NF-kB pathways, which suppressed the proliferation of T cells and impaired the cytotoxic activity of CD8(+) T cells. This finding provides a new interpretation of the mechanism of immune escape of EBV and shows the immunosuppressive role of EBV-mediated IDO expression in tumor stroma of NPC.

J Virol. 2014 Jun;88(12):6660-71

Epigenomic profiling of young and aged HSCs reveals concerted changes during aging that reinforce self-renewal.

To investigate the cell-intrinsic aging mechanisms that erode the function of somatic stem cells during aging, we have conducted a comprehensive integrated genomic analysis of young and aged cells. We profiled the transcriptome, DNA methylome, and histone modifications of young and old murine hematopoietic stem cells (HSCs). Transcriptome analysis indicated reduced TGF-b signaling and perturbation of genes involved in HSC proliferation and differentiation. Aged HSCs exhibited broader H3K4me3 peaks across HSC identity and self-renewal genes and showed increased DNA methylation at transcription factor binding sites associated with differentiation-promoting genes combined with a reduction at genes associated with HSC maintenance. Altogether, these changes reinforce HSC self-renewal and diminish differentiation, paralleling phenotypic HSC aging behavior. Ribosomal biogenesis emerged as a particular target of aging with increased transcription of ribosomal protein and RNA genes and hypomethylation of rRNA genes. This data set will serve as a reference for future epigenomic analysis of stem cell aging.

Cell Stem Cell. 2014 May 1;14(5):673-88.

Ribosomal protein mRNAs are primary targets of regulation in RNase-L-induced senescence.

The endoribonuclease RNase-L requires 2’,5’-linked oligoadenylates for activation, and mediates antiviral and antiproliferative activities. We previously determined that RNase-L activation induces senescence; to determine potential mechanisms underlying this activity, we used microarrays to identify RNase-L-regulated mRNAs. RNase-L activation affected affected a finite number of transcripts, and thus does not lead to a global change in mRNA turnover. The largest classes of downregulated transcripts, that represent candidate RNase-L substrates, function in protein biosynthesis, metabolism and proliferation. Among these, mRNAs encoding ribosomal proteins (RPs) were particularly enriched. The reduced levels of four RP mRNAs corresponded with a decrease in their half lives and a physical association with an RNase-L-ribonucleoprotein (RNP) complex in cells, suggesting that they represent authentic RNase-L substrates. Sequence and structural analysis of the downregulated mRNAs identified a putative RNase-L target motif that was used for the in silico identification of a novel RNase-L-RNP-interacting transcript. The downregulation of RP mRNAs corresponded with a marked reduction in protein translation, consistent with the roles of RP proteins in ribosome function. Our data support a model in which the RNase-L-mediated degradation of RP mRNAs inhibits translation, and may contribute to its antiproliferative, senescence inducing and tumor suppressor activities.

RNA Biol . 2009 Jul-Aug;6(3):305-15. Epub 2009 Jul 23

Epstein-Barr virus (EBV) microRNAs: involvement in cancer pathogenesis and immunopathology.

The Epstein-Barr virus (EBV), which infects over 90% of adults, appears to have evolved to exploit the normal biology of B-cell development in order to persist as a life-long asymptomatic infection. However, EBV can contribute to oncogenesis. It has become evident that alterations in the expression of microRNAs (miRNAs) from the host cell and EBV can also contribute to cancer pathogenesis. MicroRNAs function by inhibiting translation of select groups of mRNA transcripts containing imperfect annealing sequences in their 3’ untranslated regions (3’ UTRs) and less frequently through other regions of the transcript. A number of studies have demonstrated that profiles of miRNA expression could establish phenotypic signatures of different cancer types where viruses have been evolved with highly sophisticated gene silencing machinery to disturb the host-immune response. Based on current review, it is possible that a specific virus miRNA may be involved in cancer pathogenesis.

Int Rev Immunol . 2013 Jun;32(3):271-81

Mechanistic insight into the ribosome biogenesis functions of the ancient protein KsgA.

While the general blueprint of ribosome biogenesis is evolutionarily conserved, most details have diverged considerably. A striking exception to this divergence is the universally conserved KsgA/Dim1p enzyme family, which modifies two adjacent adenosines in the terminal helix of small subunit ribosomal RNA (rRNA). While localization of KsgA on 30S subunits [small ribosomal subunits (SSUs)] and genetic interaction data have suggested that KsgA acts as a ribosome biogenesis factor, mechanistic details and a rationale for its extreme conservation are still lacking. To begin to address these questions we have characterized thefunction of Escherichia coli KsgA in vivo using both a ksgA deletion strain and a methyltransferase-deficient form of this protein. Our data reveal cold sensitivity and altered ribosomal profiles are associated with a DeltaksgA genotype in E. coli. Our work also indicates that loss of KsgA alters 16S rRNA processing. These findings allow KsgAs role in SSU biogenesis to be integrated into the network of other identified factors. Moreover, a methyltransferase-inactive form of KsgA, which we show to be deleterious to cell growth, profoundly impairs ribosome biogenesis-prompting discussion of KsgA as a possible antimicrobial drug target. These unexpected data suggest that methylation is a second layer of function for KsgA and that its critical role is as a supervisor of biogenesis of SSUs in vivo. These new findings and this proposed regulatory role offer a mechanistic explanation for the extreme conservation of the KsgA/Dim1p enzyme family.

Mol Microbiol. 2008 Dec;70(5):1062-75

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