Current Insights on the Photoprotective Mechanism of the Macular Carotenoids, Lutein and Zeaxanthin: Safety, Efficacy and Bio-Delivery.

Ocular health has emerged as one of the major issues of global health concern with a decline in quality of life in an aging population, in particular and rise in the number of associated morbidities and mortalities. One of the chief reasons for vision impairment is oxidative damage inflicted to photoreceptors in rods and cone cells by blue light as well as UV radiation. The scenario has been aggravated by unprecedented rise in screen-time during the COVID and post-COVID era. Lutein and Zeaxanthin are oxygenated carotenoids with proven roles in augmentation of ocular health largely by virtue of their antioxidant properties and protective effects against photobleaching of retinal pigments, age-linked macular degeneration, cataract, and retinitis pigmentosa. These molecules are characterized by their characteristic yellow-orange colored pigmentation and are found in significant amounts in vegetables such as corn, spinach, broccoli, carrots as well as fish and eggs. Unique structural signatures including tetraterpenoid skeleton with extensive conjugation and the presence of hydroxyl groups at the end rings have made these molecules evolutionarily adapted to localize in the membrane of the photoreceptor cells and prevent their free radical induced peroxidation. Apart from the benefits imparted to ocular health, lutein and zeaxanthin are also known to improve cognitive function, cardiovascular physiology, and arrest the development of malignancy. Although abundant in many natural sources, bioavailability of these compounds is low owing to their long aliphatic backbones. Under the circumstances, there has been a concerted effort to develop vegetable oil-based carriers such as lipid nano-emulsions for therapeutic administration of carotenoids. This review presents a comprehensive update of the therapeutic potential of the carotenoids along with the challenges in achieving an optimized delivery tool for maximizing their effectiveness inside the body.

Lutein and zeaxanthin are the two most abundant natural xanthophylls (oxygenated carotenoids) with a linear C40 tetraterpene/isoprenoid lycopene-based backbone.Presence of extensive conjugation (more than 10 double bonds) enable these molecules to act as accessory light harvesting pigments apart from chlorophyll.More importantly, the xanthophylls prevent photobleaching of the pigments and proteins in the Light Harvesting Complex (LHC) by sequestering the excess unutilized blue light and preventing triplet chlorophyll associated formation of Reactive Oxygen Species.In human eye, lutein, zeaxanthin along with mesozeaxanthin constitute the three macular pigments forming the so called "yellow spot" of the macula and are implicated in maintaining the redox balance, homeostasis and normal physiology of the eyes.However, unlike plants, xanthophylls must be acquired from dietary sources such as colored leafy vegetables and egg yolk.Increase in the number of eye diseases in the aging population coupled with insufficient bioavailability of xanthophylls has mandated the industrial production of supplements enriched in xanthophylls.The bioavailability and delivery of xanthophylls can be significantly enhanced by suspension in a blend of extra-virgin olive oil and other vegetable oils.

Efficacy and Safety of Dietary Undenatured Type II Collagen on Joint and Motor Function in Healthy Volunteers: A Randomized, Double-Blind, Placebo-Controlled, Parallel-Group Study.

Prevalence of osteoarthritis (OA) is increasing alarmingly worldwide. Slowing down the progression of OA and diverse locomotive organ disorders is gaining interest in improving the quality of life (QOL) and extending healthy life-span. In a pilot study, intake of a small amount of undenatured type II collagen exhibited suppression of damage to the articular cartilage via oral immune tolerance. It also demonstrated improvement of knee and joint flexibility and mobility with continued intake of undenatured type II collagen (NEXT-II®) derived from chicken sternum cartilage. This randomized, double-blind, placebo-controlled, parallel-group clinical investigation (RCT) evaluated the efficacy and safety of 12 weeks of regular intake of NEXT-II® on joint and motor function in healthy Japanese male and female participants (aged 20 to <75 years).Sixty-four participants were randomized to receive either NEXT-II® (undenatured type II collagen 3.2 mg/d) or placebo over a period of 12 consecutive weeks. Efficacy on joint and motor functions were evaluated measuring knee passive range of motion as the primary outcome; the Japan Knee Osteoarthritis Measure (JKOM), Visual Analog Scale (VAS) for knee discomfort, and motor functions (10-meter walking and stair-climbing test) as the secondary outcomes; and Japan Low back pain Evaluation Questionnaire (JLEQ) and VAS for lower back discomfort as the exploratory outcomes.Fifty-eight participants (placebo = 28; NEXT-II® group = 30) completed the study. In the assessment of knee passive range of motion, significant improvements in "flexion" and "flexible angle (range)" were observed in the NEXT-II® group at 4, 8, and 12 weeks of treatment. NEXT-II® induced significant improvements in JKOM, VAS for knee and lower back discomfort, 10-meter walking test, stair-climbing test, and JLEQ.Results demonstrate that undenatured type II collagen is safe and efficacious in improving knee flexibility and mobility, reducing knee and lower back pain, and enhancing motor function.

Safety assessment of a novel, dietary pyrroloquinoline quinone disodium salt (mnemoPQQ®).

Pyrroloquinoline quinone (PQQ), a potent coenzyme antioxidant naturally occurring in foods, has been demonstrated to protect brain cells by enhancing the expression of nerve growth factors (NGF) and NGF receptors, and suppressing the fibril formation and aggression of amyloid ß. We developed mnemoPQQ®, a novel PQQ disodium salt and assessed its safety in GLP compliant toxicity studies. Acute toxicity studies of mnemoPQQ® in Wistar rats revealed that its LD50 was 1825- and 1410 mg/kg body weight (bw) in male and female rats, respectively, whereas its acute dermal LD50 was >2000 mg/kg bw. mnemoPQQ® was found to be nonirritant to the skin of rabbit in an acute dermal irritation/corrosion study, and classified mnemoPQQ® as a nonirritant to the eye of rabbit in an acute eye irritation/corrosion study. Ames bacterial reverse mutation assay and in vitro Mammalian cell gene mutation test exhibited its non-mutagenic potential. In mammalian in vivo erythrocyte micronucleus test, mnemoPQQ® was classified as non-clastogenic and non-mutagenic. A 90-day sub-chronic toxicity study, conducted at and up to the highest daily dose of 600 mg/kg body weight, revealed no evidence of systemic toxicity. All rats survived the treatment without any significant abnormal clinical signs and alterations in hematology, clinical chemistry, neurological evaluation, thyroid functions, reproductive hormone levels, sperm evaluations, vaginal cytology, endocrine functions, organ weight and gross and microscopic pathology findings. No observed adverse effect level (NOAEL) of mnemoPQQ® was found to be greater than 600 mg/kg body weight. These studies affirm that mnemoPQQ® has broad spectrum safety for human consumption.

Effect of Dietary Pyrroloquinoline Quinone Disodium Salt on Cognitive Function in Healthy Volunteers: A Randomized, Double-Blind, Placebo-Controlled, Parallel-Group Study.

BACKGROUND: Cognitive dysfunctions are increasing alarmingly around the world, and researchers are exploring preventive measures for improving brain performance. Pyrroloquinoline quinone (PQQ), a naturally occurring coenzyme in foods, exhibits potent antioxidant activity, and improves diverse functions which include mitochondrial activation, growth, repair, protection of nerve cells by increased expression of nerve growth factor (NGF) and NGF receptors; and suppression of fibril formation and aggregation of amyloid ß. OBJECTIVE: This randomized, double-blind, placebo-controlled, parallel-group clinical investigation (RCT) evaluated the efficacy and safety of PQQ disodium salt powder (mnemoPQQ®) for improved cognitive function after 12 weeks of supplementation in healthy Japanese male and female (age 40 to <80 Y). METHODS: 64 healthy subjects were randomly assigned to receive either mnemoPQQ® (PQQ disodium salt: 21.5 mg/day) or a placebo over a period of 12 weeks. The efficacy of mnemoPQQ® on cognitive performance (memory, attention, judgment, and cognitive flexibility) was examined using Cognitrax as the primary outcome (primary endpoint), and forgetfulness questionnaire (DECO: Deterioration Cognitive Observee) and Mini-Mental State Examination-Japanese (MMSE-J) as the secondary outcome (secondary endpoint). RESULTS: A total of 58 subjects (placebo = 31; Age = 70.91 ± 3.06 Y; mnemoPQQ® group = 27; Age = 72.10 ± 3.77 Y) completed the study over a period of 12 weeks of supplementation. Significant improvements were observed on the Cognitrax's cognitive function domain score on "composite memory", "verbal memory", "reaction time", "complex attention", "cognitive flexibility", "executive function", and "motor speed" in the mnemoPQQ® group as compared to the placebo group. The DECO and the MMSE-J scores were also significantly improved in the mnemoPQQ® group. No adverse events were observed. CONCLUSIONS: Study demonstrates that supplementation of PQQ disodium salt is useful in improving memory, attention, judgment, and cognitive function, in middle-aged to elderly population, who feel they have become more forgetful because of aging.

An overview of a novel, water-soluble undenatured type II collagen (NEXT-II).

BACKGROUND: Osteoarthritis, the most common form of arthritis, is a crippling, chronic debilitating bone disease that commonly affects humans, dogs, and horses. Inflammation and inflammatory responses are key factors for causing swelling, redness, pain, and loss of movement in arthritic animals and humans. METHODS AND RESULTS: We developed a novel, water-soluble, undenatured type II collagen (NEXT-II) for osteoarthritis. NEXT-II demonstrated broad-spectrum safety and nonmutagenicity. NEXT-II exhibited significant efficacy in ameliorating pain and inflammation in collagen-induced arthritis in mice. NEXT-II enhanced the proportion of CD4+CD25+T cells, and gene expressions of stimulated dendritic cells induced markers for regulatory T cell such as forkhead box p3, transforming growth factor-ß1, and CD25. Furthermore, NEXT-II was assessed in moderately arthritic dogs receiving either placebo or 10 mg NEXT-II over a period of 150 days. NEXT-II exhibited a significant reduction in overall pain, pain after limb manipulation, and pain after physical exertion compared to the control dogs. Physical health and serum chemistry (alanine aminotransferase, blood urea nitrogen, and creatine kinase) were not altered when these arthritic dogs were treated over a period of 150 days. CONCLUSIONS: These results demonstrate the broad-spectrum safety and efficacy of NEXT-II in ameliorating the symptoms of arthritis. Key Teaching Points: •A novel, water-soluble, undenatured type II collagen (NEXT-II) was developed for osteoarthritis. •The safety studies including acute oral and dermal toxicity, primary dermal and primary eye irritation, Ames' bacterial reverse mutation assay, mouse lymphoma assay, and 150-day long-term safety studies were conducted. •NEXT-II exhibited significant efficacy in ameliorating pain and inflammation in collagen-induced arthritis in mice. •NEXT-II exhibited a significant reduction in overall pain in moderately arthritic dogs without changing physical parameters.

Safety and toxicological evaluation of a novel, fermented, peptide-enriched, hydrolyzed swine placenta extract powder.

Placenta is an important organ that connects the developing fetus to allow nutrient uptake, antibody provisions and gas exchange via the blood supply of the mother. We developed a novel, standardized, stable, water-soluble, peptide-enriched hydrolyzed, Horus fermented placenta powder (HFPEP) from healthy, pathogen-free, swine placenta. Earlier studies demonstrated that HFPEP significantly improves physical fatigue, hepatic functions and repair of muscle fibers. We examined the broad safety of HFPEP in various toxicology models in Good Laboratory Practices-approved laboratories. The acute oral toxicity study was conducted in female Sprague-Dawley rats, and the acute oral LD50 was found to be greater than 5000 mg/kg body weight. Ames' bacterial reverse mutation assay was conducted to determine the ability of HFPEP to induce reverse mutation at selected histidine loci in five tester strains of Salmonella typhimurium viz. TA1535, TA1537, TA98, TA100 and TA102 in the presence and absence of a metabolic activation system (S9) at the doses of 50, 15, 4.5, 1.35 and 0.41 mg/ml. No mutagenic potential was observed. Mutagenic potential was also evaluated using in vivo micronucleus test, and no mutagenic potential of HFPEP was observed. Repeated dose 28-d oral toxicity study was performed in male and female rats with 14-d recovery period at the dose levels of 250, 500 or 1000 mg/kg. No abnormal clinical signs or toxicity were detected. No observed adverse effect level of HFPEP was found to be greater than 1000 mg/kg body weight. These studies affirm that HFPEP has broad spectrum safety for human consumption.

Water-soluble undenatured type II collagen ameliorates collagen-induced arthritis in mice.

Earlier studies have reported the efficacy of type II collagen (C II) in treating rheumatoid arthritis (RA). However, a few studies have investigated the ability of the antigenic collagen to induce oral tolerance, which is defined as active nonresponse to an orally administered antigen. We hypothesized that water-soluble undenatured C II had a similar effect as C II in RA. The present study was designed to examine the oral administration of a novel, water-soluble, undenatured C II (commercially known as NEXT-II) on collagen-induced arthritis (CIA) in mice. In addition, the underlying mechanism of NEXT-II was also identified. After a booster dose (collagen-Freund's complete adjuvant), mice were assigned to control CIA group, or NEXT-II treatment group, to which saline and NEXT-II were administered, respectively. The arthritis index in the NEXT-II group was significantly lower compared with the CIA group. Serum IL-6 levels in the NEXT-II group were significantly lower compared with the CIA group, while serum IL-2 level was higher. Furthermore, oral administration of NEXT-II enhanced the proportion of CD4+CD25+T (Treg) cells, and gene expressions of stimulated dendritic cells induced markers for regulatory T cells such as forkhead box p3 (Foxp3), transforming growth factor (TGF)-ß1, and CD25. These results demonstrated that orally administered water-soluble undenatured C II (NEXT-II) is highly efficacious in the suppression of CIA by inducing CD4+CD25+ Treg cells.

Safety and toxicological evaluation of a novel, water-soluble undenatured type II collagen.

This study was conducted to determine the broad-spectrum safety of a novel, water-soluble undenatured type II collagen (NEXT-II) derived from chicken sternum cartilage. The presence of epitope in NEXT-II was confirmed by using a commercial kit. The acute oral LD50 of NEXT-II was found to be greater than 5000 mg/kg bw in rats, while the single-dose acute dermal LD50 was greater than 2000 mg/kg bw. The primary dermal irritation index (PDII) of NEXT-II was found to be 1.8 and classified as slightly irritating to the skin. In primary eye irritation studies, the maximum mean total score (MMTS) of NEXT-II was observed to be 7.3 and classified as minimally irritating to the eye. Long-term safety studies were conducted in dogs over a period of 150 d, and no significant changes were observed in body weight, heart rate, respiration rate and blood chemistry. NEXT-II does not induce mutagenicity in the bacterial reverse mutation test in five Salmonella typhimurium strains either with or without metabolic activation. Furthermore, two experiments were conducted to assess the potential of NEXT-II to induce mutations with and without metabolic activation at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. No biologically relevant increase of mutants was observed. Also, no dose-dependent toxicity was observed. Furthermore, colony sizing showed no clastogenic effects induced by NEXT-II under the experimental conditions. These studies demonstrated the broad spectrum of safety of NEXT-II.

Safety of oligonol, a highly bioavailable lychee-derived polyphenolic antioxidant, on liver, kidney and heart function in rats.

Oligonol (OLG), derived from lychee fruit, is a novel compound produced from the oligomerization of polyphenols. In this study, the acute effect of OLG treatment was investigated on heart, liver and kidney in rats. OLG treatment at two different doses (15 or 30 mg/kg body weight) and two different time points (1 day or 7 days of treatment) demonstrated that no toxic effects were observed on heart, liver and renal functions. Moreover, OLG did not induce any DNA damage or oxidative stress as measured by 8-hydroxy-2'-deoxyguanosine levels in plasma. OLG supplementation increased the phosphorylation of myocardial endothelial nitric oxide (NO) level (p-eNOS) in both the treatment groups. Even the low dose OLG treatment (15mg/kg b.w) demonstrated an increase in p-eNOS/eNOS ratio after normalization of p-eNOS values with eNOS on day 1 (1.5-fold) and day 7 (2.2-fold) groups as compared to control. The above results suggest that OLG treatment increases endothelial NO levels and may play a role in NO-mediated vasodilatory effects without adverse side effects on cardiovascular function. This endothelial NO production may underlie the beneficial effect of OLG in cardiovascular health.

Vasorelaxant effects of Acer nikoense extract and isolated coumarinolignans on rat aortic rings.

The organic extract of the heartwood of Acer nikoense Maxim. (Aceraceae) showed vasorelaxant activity on rat aorta with or without endothelium. Coumarin [scopoletin (1)] and coumarinolignans [cleomiscosin A (2) and aquillochin (3)] were isolated as major constituents from the organic extract of the heartwood of A. nikoense. Compounds 1-3 exhibited moderate vasorelaxant effects on rat aorta, while 2 and 3 showed vasorelaxant effects in the norepinephrine-stimulated and also in high K+-depolarized preparations.

Inhibitory effects of methyl brevifolincarboxylate isolated from Phyllanthus niruri L. on platelet aggregation.

A platelet-aggregatory inhibitor was isolated from the 50% MeOH extract of Phyllanthus niruri L. leaf. Its structure was determined to be methyl brevifolincarboxylate on the basis of the 1H-, 13C-NMR, and high-resolution mass spectral data. We compared the antiplatelet aggregatory effects of the constituent with adenosine, a well-known inhibitor of platelet aggregation. Platelet aggregation was induced by collagen or adenosine 5'-diphosphate as an activating agent; the extent of inhibition was monitored with a platelet aggregometer employing a laser-scattering method. The inhibitory effects of methyl brevifolincarboxylate were found to be as potent as adenosine that is known to act on an A2A subtype receptor.

Health foods and foods with health claims in Japan.

The terms 'nutraceuticals' and 'dietary or food supplements' are not very popular in Japan as compared to most of other countries. However, the concept of 'functional foods', which benefits the structure and function of the human body, is known as a result of research studies initiated on the health benefits of foods in 1984. The Ministry of Education organized a national research and development project to evaluate the functionalities of various foods. Researchers from diverse scientific fields succeeded to define new functions of food, successfully incorporating the previously recognized functions of nutrition, sensory/satisfaction and physiological effects of ingredients in foods. Some of the food manufacturers and distributors unfortunately capitalized on such food functionalities to promote 'health foods' by claiming drug-like effects and violating laws. In 1991, the Ministry of Health and Welfare (MHW) now as the Ministry of Health, Labor and Welfare (MHLW) introduced a 'foods for specified health uses' (FOSHU) system, for the control of such exaggerated and misleading claims. The other reason for such enforcement is due to an increase in the population of elderly people and lifestyle-related diseases that include obesity, diabetes mellitus, high blood pressure, cerebro- and cardiovascular diseases and cancer. In 2001, a new regulatory system, 'foods with health claims' (FHC) with a 'foods with nutrient function claims' (FNFC) system and newly established FOSHU was introduced. In addition, MHLW has changed the existing FOSHU, FNFC and other systems in 2005. Such changes include the new subsystems of FOSHU such as (1) standardized FOSHU, (2) qualified FOSHU and (3) disease risk reduction claims for FOSHU. In the present chapter, two guidelines that require good manufacturing practice (GMP) and self-investigative systems for ensuring the safety of raw materials used for products in the dosage forms such as capsules, tablets, etc. have been discussed. Furthermore, issues related to positioning and definition of supplements are also discussed in the light of the enhancement of understanding the beneficial roles that supplements may play for human health in Japan.

Vasorelaxant effects of methyl brevifolincarboxylate from the leaves of Phyllanthus niruri.

Methyl brevifolincarboxylate (1) isolated from the leaves of Phyllanthus niruri L. showed a vasorelaxant effect on rat aortic rings. Compound 1 exhibited slow relaxation activity against norepinephrine (NE)-induced contractions of rat aorta with or without endothelium. The compound did not affect contractions induced by a high concentration (60 mM) of K+, whereas it inhibited NE-induced vasocontraction in the presence of nicardipine. These results suggest that the inhibition of NE-induced vasocontraction by compound 1 is in part attributable to a decrease in [Ca2+]i through receptor-operated Ca2+ channels.

Adenine, an inhibitor of platelet aggregation, from the leaves of Cassia alata.

Adenine was isolated as a platelet aggregating inhibitor from the leaves of Cassia alata by HPLC using a triacontylsilyl silica (C(30)) column. The inhibitory effects of adenine and adenosine (positive control) on the platelet aggregation induced by collagen or adenosine 5'-diphosphate (ADP) as an aggregating agent was evaluated with a platelet aggregometer using a laser-scattering method. As a result, the inhibitory effect of adenine was observed in the platelet aggregation induced by collagen (1.0 microg/ml as the final concentration), but little inhibitory effect was noted in the aggregation induced by ADP (5.0 microM as the final concentration), whereas adenosine exhibited potent inhibitory effects on platelet aggregation induced both by collagen and ADP under the same experimental conditions.

Antiinflammatory activity of heat-treated Cassia alata leaf extract and its flavonoid glycoside.

Antiinflammatory activities of heat-treated Cassia alata leaf extract and kaempferol 3-O-gentiobioside (K3G) isolated from C. alata as an abundant flavonoid glycoside were studied by comparing their activities with the activities of sun-dried C. alata leaf extract. We observed strong inhibitory effects on Concanavalin A-induced histamine release from rat peritoneal exudate cells both in the extracts of heat-treated and sun-dried C. alata leaves. Furthermore, the heat-treated leaf extract exhibited stronger inhibitory effects than the effects of the sun-dried leaf extract at low concentrations in the studies of Concanavalin A-induced histamine release, 5-lipoxygenase inhibition, and also inhibition of cyclooxygenases (COX-1 and COX-2), whereas K3G showed weak inhibitory effects on Concanavalin A-induced histamine release, 5-lipoxygenase, and COX-1. No anti-hyaluronidase effect was detected in any of the materials tested.

HPLC quantification of kaempferol-3-O-gentiobioside in Cassia alata.

Kaempferol-3-O-gentiobioside, the major flavonoid glycoside in Indonesian Cassia alata was quantified in various parts of the plant. The mature leaf was found to contain the highest content of this metabolite. A decrease of the flavonoid content in the juvenile leaf during the period of October through December was also observed. The contents ranged from 2.0 to 5.0% and 1.0 to 4.0% in mature and juvenile leaves, respectively. The other parts studied were flower (sepal and petal), rachis, stem and seed. Kaempferol-3-O-gentiobioside was not detected in the seed.