Showing posts with label Oxidative stress. Show all posts
Showing posts with label Oxidative stress. Show all posts

Tuesday, December 12, 2023

Understanding the Concept of “Nutraceutical” - Juniper Publishers

 Pharmacy & Pharmaceutical Sciences - Juniper Publishers


Abstract

Nutraceuticals, a portmanteau of “nutrition” and “pharmaceutical,” represent a rapidly growing and evolving field in the realm of health and wellness. This term refers to products that combine the benefits of both nutrition and pharmaceuticals, as they aim to promote health and prevent or manage diseases. Nutraceuticals encompass a wide range of substances, including vitamins, minerals, herbs, dietary supplements, functional foods, and bioactive compounds. The core concept behind nutraceuticals is their potential to deliver therapeutic effects beyond basic nutrition. They are designed to bridge the gap between dietary insufficiencies and pharmaceutical interventions. Nutraceuticals may enhance overall well-being, support specific physiological functions, or alleviate symptoms associated with various medical conditions.

The appeal of nutraceuticals lies in their natural origins and the premise of fewer side effects compared to traditional pharmaceuticals. These products often contain bioactive compounds with antioxidant, anti-inflammatory, or immune-boosting properties, such as omega-3 fatty acids, probiotics, and plant polyphenols. The nutraceutical industry has witnessed exponential growth due to the increasing awareness of preventive healthcare and the desire for more natural treatment options. However, it also faces challenges related to regulation and quality control, as the boundaries between food and medicine can be blurred.

Keywords: Antioxidants; Disease modifiers; Herbal nutraceuticals; Nutraceutical products; Nutraceuticals, Oxidative stress

Introduction

The original concept in these concepts goes back three thousand years. Hippocrates (460-377 BC), the nicely recognized father of current medication, said “let food be thy medicine and medicine be thy meals” to expect the courting among suitable ingredients for fitness and their therapeutic benefits. The truth in this saying is broadly regarded nowadays. The idea of nutraceuticals is not new, although it has evolved substantially over the years. in the early 1900s, meal producers inside the u.s. started out adding iodine to salt so that you can save your goiter, representing one of the first attempts at creating a functional issue through fortification. these days researchers have identified hundreds of compounds with useful characteristics, and they preserve to make discoveries surrounding the complex advantages of phytochemicals (non-nutritive plant chemical compounds which have protecting or sickness-preventative houses) in meals. In Japan, England, and other countries, nutraceuticals have already got grown to be a part of the nutritional landscape. Client hobby in the relationship between food plan and fitness has elevated the demand for data on nutraceuticals. speedy advances in technological know-how and era, growing fitness care prices, modifications in meals legal guidelines affecting label and product claims, a growing older population, and a rising hobby in reaching wellness via food regimen are among the elements fueling U.S. interest in nutraceuticals. Credible scientific research indicates many capacity fitness benefits from meal components. These blessings should expand the fitness claims now accredited to be diagnosed using the food and Drug administration [1-3].

Approximately 2000 years ago, Hippocrates efficaciously emphasized “let meals be your medication and remedy be your meals”. presently there is an improved worldwide interest due to the recognition that “nutraceuticals” play a major position in fitness enhancement. The term “Nutraceutical” was coined by combining the terms “vitamins” and “Pharmaceutical” in 1989 via Dr. Stephen De Felice, Chairman of the inspiration for Innovation in medicinal drugs. “Nutraceutical” is a marketing term evolved for a nutritional complement that is offered to treat or save your sickness and thus has no regulatory definition. Therefore a “nutraceutical” is any substance that can be taken into consideration as food or part of meals and affords scientific or fitness benefits, encompassing, the prevention and treatment of sicknesses. Such merchandise might also vary from isolated vitamins, nutritional dietary supplements, and diets to genetically engineered “clothier” ingredients, herbal products, s and processed ingredients which include cereals, soups, and liquids. Currently, over 470 nutraceutical and functional food products are to be had with documented health advantages. “Nutraceuticals and useful foods have received huge interest due to their presumed safety and capability dietary and therapeutic results”. The nutraceutical and practical meals industry is in a unique position to capitalize on clients’ hobbies. Be it a multinational pharmaceutical organization, a nutritional organization, a large meals multinational, or a small diet-promoting company, all of them apprehend the converting tendencies and are aware of the extra health-seeking consumer fashion. there may be, hence, a proliferation of this cost-delivered merchandise aimed toward no longer only maintaining oneself wholesome but also prevention/remedy of numerous illnesses ranging from coronary heart illnesses to cancer [4,5].

As referred to in advance, purposeful foods contain larger profit margins than conventional ingredients (30 to 500 percent better). the worldwide marketplace size is anticipated between 30 and 60 billion US$, with Japan, US, and Europe occupying the most important share. By way of 2010, the nutraceutical call is forecast to touch $197 billion. There are lots of misunderstandings regarding the terminologies like “nutraceuticals”, “functional meals”, “nutritional supplements” “clothier meals”, “scientific foods”, “pharma foods”, “phytochemicals” etc. There appears to be a thin dividing line in their interchangeable utilization using oneof- a-kind humans on one-of-a-kind events. “Prescription drugs” can be taken into consideration as tablets used especially to treat sicknesses, even as “nutraceuticals” are the ones which might be intended to save you from sicknesses.

The above difference between prescription drugs and nutraceuticals is lovable, however superficial and inaccurate. prescription drugs are materials that have (or have had) patent protection because of expensive trying out to conform to the specifications of respective Governments. However, many vitamins may in no way get hold of authorities’ approval on account that no person could justify the cost of testing requirements for materials that cannot be blanketed via patent laws. Each prescription drug and vitamin can cure and save your ailment(s), but the best prescription drugs have governmental sanctions [6-8]. Many prescription drugs have their origin in plant life and animals and aren’t any much less “natural” than vitamins. A classic instance of vitamins is synthetic vitamins. Nutraceuticals from time to time referred to as “functional ingredients”, have induced heated debate because they blur the conventional dividing line between meals and medication. When food is being cooked or prepared with the usage of “medical intelligence” without or with the know-how of the way or why its miles getting used, then the meals are known as “functional meals.” for that reason, purposeful food provides the body with the required quantity of nutrients, fat, proteins, carbohydrates important for healthful survival. whilst practical meals aid within the prevention and/or remedy of ailment(s)/ ailment(s) other than deficiency conditions like anemia it’s miles called a “nutraceutical”. Consequently, useful food for one patron can act as a nutraceutical for any other. Examples of nutraceuticals include fortified dairy products (milk as such is a nutrient and its product casein is a pharmaceutical) and citrus result (orange juice is a nutrient and its constituent ascorbic acid is a pharmaceutical).

A dietary supplement is a product that is intended to supplement the diet that bears or includes one or extra substances like nutrition, mineral, herb, an amino acid or pay attention, metabolite, constituent, extract or mixtures of those. “Medical meals” are a particular class of therapeutic sellers which are meant for the nutritional management of a specific ailment. An example of clinical meals is formulations intended to control sufferers with inborn mistakes in amino acid metabolism. more modern medical ingredients are designed to manipulate hyperhomocysteinemia, pancreatic exocrine insufficiency, inflammatory situations, most cancers cachexia, and other illnesses. The usage of nutraceuticals, as an attempt to accomplish applicable healing outcomes with decreased facet outcomes, compared with different therapeutic dealers has met with amazing monetary success [9,10]. The desire for the discovery and manufacturing of nutraceuticals over prescription drugs is nicely appreciated by pharmaceutical and biotechnology agencies. some popular nutraceuticals consist of glucosamine (for arthritis), lutein (for macular degeneration), ginseng (for cold), echinacea (anti-immune), folic acid, cod liver oil pills, and so on. The most popular functional food and beverage products include omega-3 eggs, omega-three enriched yogurts, calcium-enriched orange juice, and inexperienced tea to mention some. Most of the nutraceuticals do own a couple of healing benefits, however in the gift overview, plenty of attempts have been dedicated to decentralizing them based on their sicknessspecific essential indication. Nutraceuticals was claimed to have a physiological advantage or offer protection in opposition to the subsequent sicknesses (and/or observed to act as):

i. Cardiovascular dealers

ii. Antiobese sellers

iii. Antidiabetics

iv. Anticancer dealers

v. Immune boosters

vi. persistent inflammatory problems

vii. Degenerative sicknesses

viii. Neuro degenerative diseases [11]

Nutraceuticals vs Other Terminologies

There is a lot of bewilderment concerning the terminologies like nutraceuticals, useful meals, dietary supplements, fashion designer foods, clinical foods, pharma foods, phytochemicals, and so on. “Prescribed drugs” can be taken into consideration as pills used specifically to deal with sicknesses, at the same time as “nutraceuticals” are those that are supposed to save you illnesses. inside ecu drugs law a nutraceutical can be described as a remedy for two motives: it may be used for the prevention, remedy, or remedy of a condition or sickness or be administered so that you can restore, correct, or enhance physiological functions in human beings. Each prescription drug and vitamin can treat and save your disease(s), but the best-prescribed drugs have the governmental sanction. capsules are concerned with an approval process before marketing. To be accredited, a drug should exhibit safety and efficacy for its supposed use. Nutraceuticals aren’t capsules without a doubt due to the fact they have no longer long passed via an approval system. Many prescribed drugs have their starting place in plants and animals and are not any much less “natural” than nutrients. A conventional instance of nutrients is synthetic vitamins. “Medical foods” are a specific class of healing agents that can be meant for the dietary control of a selected ailment. An instance of scientific meals is formulations meant to manipulate patients with inborn errors in amino acid metabolism. More recent scientific meals are designed to manage hyperhomocysteinemia, pancreatic exocrine insufficiency, inflammatory conditions, cancer cachexia, and other illnesses. meals are usually diagnosed as secure whereas Nutraceuticals may additionally contain materials that might be “herbal” but won’t be formally recognized as safe [12-14].

Nutraceuticals occasionally referred to as “purposeful foods”, have induced heated debate due to the fact they blur the conventional dividing line between meals and medication. Nutraceuticals barely range from useful foods. When food is being cooked or prepared the usage of “medical intelligence” with or without an understanding of the way or why its miles getting used, the food is called useful food. Hence, functional food provides the body with the specified quantity of vitamins, fats, proteins, carbohydrates, etc. Wanted for its healthful survival. Whilst practical meals aid within the prevention and/or treatment of ailment(s) and/or disorder(s) other than anemia, it is known as Nutraceutical. (Because maximum of the useful foods act in a few ways or the opposite as anti-anemic, the exception to anemia is considered to have a clear distinction between the 2 phrases, functional meals, and nutraceutical.) Examples of nutraceuticals consist of fortified dairy merchandise (e.g., milk) and citrus culmination (e.g., orange juice) and veggies. us nutritional complement fitness and training Act (DHSEA), defined “nutritional supplement” as the usage of numerous criteria. A nutritional supplement is a product (aside from tobacco) that is supposed to supplement the weight-reduction plan that bears or consists of one or greater of the subsequent dietary components: a vitamin, a mineral, an herb, or a different botanical, an amino acid, a dietary substance to be used through man to complement the weight loss plan through increasing the overall daily consumption, or a concentrate, metabolite, constituent, extract, or combos of those substances [15].

A dietary supplement: is meant for ingestion tablet, pill, pill, or liquid shape. isn’t represented to be used as a conventional food or as the sole object of a meal or eating regimen. is labeled as a “nutritional supplement.” consists of products together with an accepted new drug-certified antibiotic, or licensed biologic that was advertised as a dietary complement or food before approval, certification, or license.

for this reason, nutraceuticals vary from nutritional dietary supplements within the following components: Nutraceuticals ought to not only complement the weight-reduction plan but must also resource in the prevention and/or ailment. Nutraceuticals are requested for use as a traditional food or as the sole object of a meal or diet. Nutraceuticals are manufactured to strict requirements and are regulated beneath food safety and satisfactory control regulations [16]. A ray of “treatment desire” in the mind of commonplace patients revolves around nutraceuticals due to their false notion that ‘all herbal medicines are true.” also, the excessive cost of prescription drugs and the reluctance of a few coverage businesses to cowl the expenses of medicine helps nutraceuticals solidify their presence inside the worldwide marketplace of healing procedures and therapeutic marketers [17].

Type of Nutraceuticals

Nutraceuticals are labeled based on various chemical constituents present in herbal flowers. desk 1 suggests the listing of numerous nutraceuticals, their additives, supply, and their capacity blessings. Discussion nowadays, nutraceuticals have obtained high hobbies because of their capacity dietary, and safety profile, aside from healing capability. Pharmaceutical and dietary groups are aware of the converting traits that are due to the advantages of these compounds. most nutraceuticals possess more than one therapeutic advantage. The prevailing examines devoted to a better understanding of nutraceuticals primarily based on their pharmaceutical and therapeutic warning signs. It has to be noted that there might be numerous confusions associated with the terminology of nutraceuticals, including phytochemicals, pharma foods, medical meals, practical foods, nutritional supplements, clothier ingredients, and so forth., there may be a thin dividing line between their interchangeable usage with the aid of distinct humans on one-of-a-kind activities. prescribed drugs are ordinarily taken into consideration as medications that are used especially to treat illnesses, but nutraceuticals are the substances that are often considered to prevent illnesses.

This difference between prescribed drugs and nutraceuticals may be very erroneous and superficial. pharmaceuticals and nutraceuticals each can remedy and save you sickness(s) however, the best pharmaceuticals have governmental sanction. prescribed drugs are compounds that typically own patent safety due to high-priced testing. However, nutraceuticals no longer want those checking out documents.

Scientific foods or medicinal foods are a particular category of healing marketers that might be taken into consideration for the nutritional control of a specific ailment. as an instance, medicinal foods are designed to manage inflammatory situations, most cancers, hyperhomocysteinemia, pancreatic exocrine insufficiency, and other diseases. They also play significant protection against numerous age-related or continual illnesses. Natural drug treatments which are used as a nutrient are considered in this category. Nutraceuticals are in many results and vegetables are answerable for health advantages. because of these health blessings of nutraceuticals, they could often be taken to treat or lessen the risk elements together with excessive LDL cholesterol, excessive blood strain, and diabetes. several of the most popular nutraceutical products marketed these days are botanicals which include ginseng, ginkgo biloba, St. John’s wort, and echinacea. [The list of nutraceuticals being studied is changing usually and reflects ongoing marketplace developments, research, and consumer interest. With rapidly increasing interest in nutraceutical intake, sizeable research is important to warrant the nutraceuticals utilization secure and powerful. The mechanistic actions of nutraceuticals aren’t clean. However, they are probably concerned with a wide type of biological techniques, including activation of signal transduction pathways, antioxidant defenses, gene expression, cellular proliferation, differentiation, and upkeep of mitochondrial integrity

Classification of nutraceuticals: (Table 1-3)

Research Methods

Research on nutraceuticals usually involves a multidisciplinary approach.

Literature review

A thorough review of the controlled articles, including research articles, reviews, and supervisory directions, was conducted to consider the various facets of nutraceuticals.

Data collection

Data were collected through surveys, interviews with specialists, and analyses of nutraceutical production labels and additives. The market dossier was also asked to recognize the current situation.

Data analysis

A qualitative study was used to categorize and compile judgments from brochures, surveys, and interviews. A quantitative dossier, if available, was resolved to recognize the flows and advantages.

Results

Definition and classification of nutraceuticals

Nutraceuticals are products that come from fair beginnings or are fortified with accompanying bioactive compounds that support fitness benefits and provide further basic food. They can be classified into various types, including working snacks, digestive supplements, and herbaceous products.

Key active ingredients

Common life factors of nutraceuticals include vitamins, minerals, antioxidants, probiotics, prebiotics, and phytochemicals. These ingredients are well known for their potential to enhance well-being and prevent illness.

Consumer perspectives

Research shows that purchasers turn to nutraceuticals for a variety of reasons, including common well-being and wellness, ailment prevention, and addressing health concerns. Consumer priorities are frequently affected by perceptions of ease and security.

Regulatory framework

The supervisory foundation of nutraceuticals varies from country to country. In many domains, distinguishing directions and controlling product quality standards guarantee device security and efficacy. Compliance with these requirements is essential for display access.

Market trends

Nutraceuticals have endorsed meaningful developments in recent years, compelled by the growing strength of knowledge among shoppers. Key currents include the rise of working foods, embodied food, and an increasing emphasis on tenable and unaffected sourcing of pieces.

Discussion

Nutraceuticals are complex and are evolving. They maintain a singular slot in food and health activities, contributing to the promise of improved comfort and health support. However, challenges related to experimental confirmation, product quality control, and regulatory agreements remain. Moreover, as manufacturing continues to evolve, it securely and efficiently balances innovation and service choice. Researchers, managers, and industry partners must agree to guarantee that nutraceuticals meet their potential while addressing these challenges.

Conclusion

Many nutraceuticals, functional ingredients, and occurring compounds that had been investigated and reported in diverse research found that these products are extraordinarily active, have a profound effect on cellular metabolism, and frequently have a little unfavorable impact. It’s far natural that human beings’ consciousness is moving to an effective method for the prevention of illnesses to stay healthy. Nutraceuticals are clinical vicinity generated everywhere around the globe. in many cases, nutraceuticals provide a bonus over the synthetic capsules beneath improvement by way of the prescribed drugs enterprise.

It is a novel pharmacological interest which has become exciting in its feasible scientific use and for that reason helps in the prevention and therapeutic of numerous illnesses. maximum of the pharmaceutical companies frequently lacks the motivation to pursue those difficulties in acquiring patents. its miles hope that government groups and research facilities will provide a guide for similar studies in nutraceuticals [18-20].

To Know more about Global Journal of Pharmacy & Pharmaceutical Sciences

To Know more about our Juniper Publishers

Friday, June 24, 2022

Anti-Viral Vaccine Activity of Zinc(Ⅱ) for Viral Prevention, Entry, Replication, and Spreading During Pathogenesis Process - Juniper Publishers

 Biomedical Engineering & Biosciences - Juniper Publishers

Abstract

Anti-viral vaccine activity of Zn2+ ions for viral prevention, pathogenesis processes, and ROS generation causing to oxidative stress have been investigated. AZP is efficient for viral prevention by inhibitions of BSCTV and DNA virus replications. The AZP phenotypes show strongly resistant to virus infection and viral DNA replication could be applied to the prevention of virus infections in humans. ZnOTs exhibit the ability to neutralize HSV-2 virions and blocking HSV-2 attachment activity. Zinc salts can mediate antiviral activity on RSV by altering the ability of the cell to support RSV replication. The effect of zinc sulfate on seroconversion after a simple method vaccination had been identified that accelerated HB vaccination can shorten duration of immunization of this clinical trial for showing its effectiveness. The inhibition of zinc binding activity of hMPV M2-1 protein can lead to the development of novel, live attenuated vaccines as well as antiviral drugs for pneumoviruses. The CCHH zinc finger motif provides potential vaccine candidates for the development of live species-specific attenuated influenza virus vaccines. Chelates zinc ions inhibit HIV-1 replication. The LAIVs are attracting attention as several advantages over inactivated vaccines. Zinc finger reactive compounds also inactivate retroviruses. ZOTEN promoted the presentation of bound HSV-2 virions.

The regulated ZFNs in the presence of HIV-1 Tat may provide a safer and novel genome-editing technology for eradicating HIV-1 proviral DNA from infected host cells. Zinc ions inhibit vaccine virus growth. The ZAP inhibits HIV-1 infection by viral mRNAs degradation. ZAP also inhibits the spreading against XMRV. The treatment of viral infections with viral spreading will be achieved and improved the therapeutic effects by using zinc oxide nanoparticles. ROS and RNS within viruses of viral entry, viral replication, and viral spread are generated in all situations. ROS in virus pathogenesis play an important role in cell signaling and regulate hormone action, growth factors, cytokines, transcription, apoptosis, immunomodulation, and neuromodulation, leading to chronic oxidative stress. Oxidative stress has been occurred in various viral infections. The antioxidant components lead to an excess storage of H2O2, which further increases the hydroxyl radicals and lipid peroxide that signal the cell to undergo a programmed cell death. Thus, zinc-associated vaccine activity mechanism against viruses is indicated that the anti-viral vaccine activity of released Zn2+ ions from zinc solutions and ZnO NPs, may be enhanced by Zn2+ ion-induced Zn2+ ions-coordinated adapted immunity, viral growth regulation, and viral apoptosis and death.

Keywords: AZP/ZOTEN/ZAP/ZFN; Zinc salts and zinc sulfate; ROS; Oxidative stress; HIV/hMPV/ LAV/RSV/XMRV

Abbreviations: APCs: Antigen Presenting Cell; AZP: Artificial Zinc Finger Protein; BSCTV: Beet Severe Curly Top Virus; HIV: Human Immunodeficiency Virus; hMPV: Human Metapneumovirus; HBV: Hepatitis B Virus; HEV: Hepatitis E Virus; HSV2: Herpes Simplex Virus Type-2; IRF3: Interferon Regulatory Factor 3; ISREs: Interferon Stimulated Response Elements; LAIVs: Live Attenuated Influenza Vaccines; LAV: Live Attenuated Virus; NKT: Natural Killer T; PLZF: Promyelocytic Leukemia Zinc Finger; RdRp: RNA-Dependent RNA Polymerase; RNS: Reactive Nitrogen Species; ROS: Reactive Oxygen Species; RSV: Respiratory Syncytial Virus, SSP: Stable Signal Peptide; TALENs: Transcription Activator-Like Effector Nucleases; XMRV: Xenotropic Murine Leukemia Virus related Virus; ZBD: Zinc-Binding Domain; ZAP: Zinc-Finger Antiviral Protein; ZFN: Zinc Finger Nuclease; ZnOTs: ZINC OXIDE TETRAPODS; ZOTENs: Zinc Oxide Tetrapod Nanoparticles

Introduction

Viruses are small protein capsid that harbor genetic information. In the case of enveloped viruses, an additional lipid bilayer surrounds the capsid that enveloped viruses can spread via two distinct routes, either through the cell-free aqueous environment or, alternatively, by remaining cell associated and being passed on by direct cell-cell contact [1].

The other, zinc is the second most abundant trace metal with human body 2-3g, 90% in muscle and bone, and 10% other organs include prostate, liver, the gastrointestinal tract, kidney, skin, lung brain, heart, and pancreas in humans which cellular zinc underlies an efficient homeostatic control that avoids accumulation of zinc in excess [2]. The role of zinc in cell death has apoptosis that the influence of zinc on apoptosis is tissue/ cell type, zinc concentration, and expression of zinc transporters and zinc-binding proteins [2]. Zinc has a rather low toxicity and influences apoptosis by acting on several molecular regulators of programmed cell death, including caspases and proteins from the Bcl and Bax families.

Zinc induced anti-virus activity may be enhanced for Tcell division, maturation and differentiation, lymphocyte response to mitogens, programmed cell death of lymphoid and myeloid origins, gene transcription, and biomembrane function [3]. Nonstructure NS5A is a zinc metalloprotein and zinc coordination is likely required for NS5A function in the hepatitis C replicase that reduced NS5A zinc coordination and zinc binding motif were tolerated for replication [4]. The zinc-finger antiviral protein (ZAP) induced virus during viral infection little is known, however, mutational analysis of the human ZAP promoter revealed that multiple interferon stimulated response elements (ISREs) distal to the transcription start site serve redundantly to control interferon regulatory factor 3 (IRF3)-dependent induction of ZAP trancription [5].

Anti-virus vaccine principle is to viral prevention and devoid serious diseases that live attenuated virus (LAV) vaccines have provided ideal protection from several major diseases. The ability to create a zinc finger nuclease (ZFN) vaccine that can prevent and eliminate persistent viral infections is a long way from being realized, in which the efficacies by available LAV, live attenuated rotavirus, influenza, and varicella zoster vaccines are strong incentive to redouble efforts to improve the safety characteristics of this type of vaccine [6]. Vaccines for numerous infectious diseases have been developed using whole inactivated virions that live attenuated influenza vaccines (LAIVs) are particularly attracting attention as effective strategy due to several advantages over inactivated vaccines [7].

Zinc-binding activity of human metapneumovirus (M21) protein was found to incorporate zinc ions, although the specific roles of the zinc binding activity in viral replication and pathogenesis remains unclear [8]. The pathogenic process, namely viral pathogenesis is the process by which an infection leads to disease that pathogenic mechanisms of viral disease include viral entry, local replication, and spread to organs and shedding of disease site [9]. Accordingly, the zinc induced Zn2+ ion coordinated activity results in regulation of viral growth and may lead to virus death in host cell-virus interaction during pathogenesis process.

In this review, zinc-mediated anti-viral vaccine activities have been investigated for viral prevention, viral entry, replication/ DNA/RNA virus, and spreading to organs during pathogenesis process. Lastly, ROS productions and leading to oxidative stress and virus death are discussed.

Viral prevention

Viral prevention is a major objective in human health. One attractive approach to the prevention is inhibition of virus replication. Artificial zinc finger proteins (AZP) have been targeted to the replication origin of the Beet severe curly top virus (BSCTV), a model DNA virus that the AZP efficiently blocked binding of the viral replication protein, which initiates virus replication, to the replication origin. These results showing to the AZP phenotypes strongly resistant to virus infection and viral DNA replication could be applied to the prevention of virus infections in humans [10]. Inhibition of virus DNA replication by AZP is comparable to viral prevention. Zinc oxide tetrapods (ZnOTs) exhibit the ability to neutralize herpes simplex virus type-2 (HSV-2) virions and blocking HSV-2 attachment activity by ZnOTs can have prophylactic as well as therapeutic applications [11].

Viral entry and replication

Zinc salts was examined in the presence of zinc during preincubation, adsorption, or penetration by the degree of inhibition of respiratory syncytial virus (RSV) replication, the resulting that zinc can mediate antiviral activity on RSV by altering the ability of the cell to support RSV replication [12]. Further, zinc salts did not show any effect on the entry of g-1 hepatitis E virus (HEV) into the host cell, directly inhibit the activity of the viral RNA-dependent RNA polymerase (RdRp), leading to inhibition of viral replication, suggesting their possible therapeutic value in controlling HEV infection [13]. The other, Zinc sulfate has no effect in level of immunity among elderly and zinc may stimulate antibody response in the nonresponders to recombinant hepatitis B vaccine. The effect of zinc sulfate on seroconversion after a simple method vaccination had been identified that accelerated hepatitis B vaccination can shorten duration of immunization of this clinical trial for showing its effectiveness [14]. The inhibition of zinc binding activity of human metapneumovirus (hMPV) M2-1 protein can lead to the development of novel, live attenuated vaccines as well as antiviral drugs for pneumoviruses [8].

The CCHH zinc finger motif provides a critical determinant for virulence in mouse and mutations in the CCHH motif yield potential vaccine candidates for the development of live speciesspecific attenuated influenza virus vaccines [15]. Chelates zinc ions (a bisthiadiazolbenzene-1,2-diamine) from retroviral nucleocapsid zinc fingers inhibit HIV-1 replication [16]. This compound thus acts via a different mechanism than the previously reported zinc ejectors, as its structural features do not allow an acyl transfer to Cys or a thioldisulfide interchange. The cleaved stable signal peptide (SSP) is retained in GPC through interaction with a zincbinding domain (ZBD) in the cytoplasmic tail of G2 [17]. The live attenuated influenza vaccines (LAIVs) are attracting attention as an effective strategy due to several advantages over inactivated vaccines [18]. Zinc finger reactive compounds also inactivate retroviruses by targeting the zinc finger motif in their nucleocapsid proteins. These compounds are effective against respiratory syncytial virus (RSV) that AT- 2-inactivated RSV vaccine may not be enough to produce a highly efficacious inactivated virus vaccine which does not lead to an atypical disease [19]. Intravaginal zinc oxide tetrapod nanoparticles (ZOTEN) promoted the presentation of bound HSV-2 virions to mucosal APCs, enhancing T cell-mediated and Ab-mediated responses to the infection, and thereby suppressing a reinfection [20].

DNA/RNA virus inhibitors

A human papillomavirus type 16 E7 DNA vaccine indicated a significantly stronger E7-specific cytotoxic T-lymphocyte induction and better antitumor protection [21]. Zinc finger nucleases (ZFNs) can specifically and efficiently excise HIV-1 proviral DNA from latently infected human T cells, the resulting from HIV-1infected human primary T cells and latently infected T cells treated with the inducible ZFNs validated that proviral DNA can be excised [22]. The regulated ZFNs in the presence of HIV-1 Tat may provide a safer and novel genome-editing technology for eradicating HIV-1 proviral DNA from infected host cells. ZFNs and transcription activator-like effector nucleases (TALENs) bear sequence-specific DNA-binding modules that recognize HIV DNA sequences [23]. Therapeutic potential of ZFNs and zinc finger arrays (ZFAs) is confirmed with unique specificity to HSV-2 the genome which ZFNs with specificity to HSV-2 genomic DNA are precursors of novel host genome expressed HSV-2 gene-therapeutics or vaccines [24]. Zinc supplementation in cultured HeLa cells inhibits DNA laddering & caspase-3 activity, and inhibitory effect on influenza virus induced apoptotic death can be determined at an early stage of the infection by zinc treatment [25]. Further, zinc ions inhibit vaccine virus growth, and a fraction of RNA which is normally synthesized infected cells, was missing from a proper part of the gradient if the cell were treated with zinc ions within 1 h post infection that a transcriptional step is involved in zinc-caused inhibition of vaccinia virus growth [26]. The zinc-finger antiviral protein (ZAP) inhibits HIV-1 infection by viral mRNAs degradation that ZAP inhibits HIV-1 by recruiting both the 5’ and 3’ mRNA degradation to specifically promote the degradation of multiply spliced HIV-1 mRNA [27].

Higher levels of dietary zinc oxide (ZnO) levels could not provide enhanced protection against porcine reproductive and respiratory syndrome virus (PRRSV) vaccine and infection that have the potential to stimulate or modulate systemic immune responses after vaccination [28]. In the synthesized ZnO tetrapod nanoparticles (ZOTENs), ZOTENs prevent HSV-2 infection and disease that ZOTEN provides a platform for virus capture and presentation of neutralized virions to mucosal antigen presenting cell (APCs), for initiating and boosting adaptive immunity which provides the novel evidence for the protective efficacy of an intravaginal microbicide/vaccine or microbivac platform against primary and secondary female genital herpes infection [29].

Viral spread inhibitor

ZAP inhibits the spreading of replication-competent xenotropic murine leukemia virus-related virus (XMRV) which ZAP inhibits viral infection [30]. The treatment of viral infections with viral spreading will be achieved and improved the therapeutic effects by using zinc oxide nanoparticles [31].

ROS Generation in Host Cell-Virus Interaction and Leading to Virus Growth Regulation and Virus Death

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) within viruses of viral entry, viral replication, and viral spread are generated in all situations. ROS induce several cellular effects, including cell cycle progression, apoptosis, DNA damage, senescence, and neurodegeneration. ROS in virus pathogenesis play an important role in cell signaling and regulate hormone action, growth factors, cytokines, transcription, apoptosis, immunomodulation, and neuromodulation, leading to chronic oxidative stress [32]. Oxidative stress has been occurred in various viral infections. For human immunodeficiency virus (HIV), the body antioxidant system becomes weaker as HIV progresses that the imbalances inside and outside the cell influence the cell to undergo a programmed cell death [32]. The antioxidant components lead to an excess storage of H2O2, which further increases the hydroxyl radicals and lipid peroxide that signal the cell to undergo a programmed cell death [32]. The influenza virus induces ROS production in host cells that can damage the virus genome which ROS enhance the pathogenesis ability of infectious influenza that leads to the thymus specific elevation of the mitochondrial superoxide, which interferes with the normal functioning of T-cells lymphocyte damage in influenza A virus infections [32]. Influenza A virus infection causes a rapid influx of inflammatory cells, resulting in increased ROS production, cytokine expression, and acute lung injury which inhibition of this activity would restore host cytokine homeostasis following avian influenza virus infection [33]. Promyelocytic leukemia zinc finger (PLZF) controls the ROS levels that in turn PLZF governs the inflammatory function of natural killer T (NKT) cells, in which ROS regulate homeostasis and effector function of NKT cells, both of which are regulated by PLZF [34]. As mentioned above, anti-viral vaccine activities of Zn2+ ions for viral prevention, entry, replication, and spreading with pathogenesis process are exhibited in Table 1. Zinc-associated vaccine activity mechanism against viruses is indicated that the vaccine activity of released Zn2+ ions from zinc solutions and ZnO NPs, may be enhanced by zinc(Ⅱ)-induced Zn2+ coordinated adapted immunity, viral growth regulation, and viral apoptosis and death

Conclusion

AZP can efficiently block binding of the viral replication protein, which initiates virus replication, to the replication origin against BSCTV, DNA virus. The AZP phenotypes show strongly resistant to virus infection and viral DNA replication could be applied to the prevention of virus infections in humans. ZnOTs exhibit the ability to neutralize herpes simplex virus type-2 (HSV-2) virions and blocking HSV-2 attachment activity by ZnOTs can have prophylactic as well as therapeutic applications. Zinc salts can mediate antiviral activity on RSV by altering the ability of the cell to support RSV replication. The effect of zinc sulfate on seroconversion after a simple method vaccination had been identified that accelerated HB vaccination can shorten duration of immunization of this clinical trial for showing its effectiveness. The inhibition of zinc binding activity of hMPV M2-1 protein can lead to the development of novel, live attenuated vaccines as well as antiviral drugs for pneumoviruses. The CCHH zinc finger motif provides a critical determinant for virulence in mouse and mutations in the CCHH motif yield potential vaccine candidates for the development of live species-specific attenuated influenza virus vaccines. Chelates zinc ions also inhibit HIV-1 replication. The LAIVs are attracting attention as an effective strategy due to several advantages over inactivated vaccines. Zinc finger reactive compounds also inactivate retroviruses. ZOTEN promoted the presentation of bound HSV-2 virions to mucosal APCs, enhancing T cell-mediated and Ab-mediated responses to the infection, and thereby suppressing a reinfection.

ZFNs can specifically and efficiently excise HIV-1 proviral DNA from latently infected human T cells, the resulting from HIV-1-infected human primary T cells and latently infected T cells treated with the inducible ZFNs validated that proviral DNA can be excised. The regulated ZFNs in the presence of HIV-1 Tat may provide a safer and novel genome-editing technology for eradicating HIV-1 proviral DNA from infected host cells. Zinc supplementation in cultured HeLa cells inhibits DNA laddering & caspase-3 activity, and inhibitory effect on influenza virus induced apoptotic death can be determined at an early stage of the infection by zinc treatment. Further, zinc ions inhibit vaccine virus growth, and are involved in zinc-caused inhibition of vaccinia virus growth. The ZAP inhibits HIV-1 infection by viral mRNAs degradation that ZAP inhibits HIV-1 by recruiting both the 5’ and 3’ mRNA degradation to specifically promote the degradation of multiply spliced HIV-1 mRNA. Higher levels of dietary ZnO could not provide enhanced protection against PRRSV vaccine that higher levels of dietary ZnO have the potential to stimulate or modulate systemic immune responses after vaccination against PRRSV. ZOTEN provides a platform for virus capture and presentation of neutralized virions to mucosal APCs.

ZAP inhibits the spreading against XMRV which ZAP inhibits viral infection. The treatment of viral infections with viral spreading will be achieved and improved the therapeutic effects by using zinc oxide nanoparticles. ROS and RNS within viruses of viral entry, viral replication, and viral spread are generated in all situations. ROS induce several cellular effects, including cell cycle progression, apoptosis, DNA damage, senescence, and neurodegeneration. ROS in virus pathogenesis play an important role in cell signaling and regulate hormone action, growth factors, cytokines, transcription, apoptosis, immunomodulation, and neuromodulation, leading to chronic oxidative stress. Oxidative stress has been occurred in various viral infections. The body antioxidant system becomes weaker as HIV progresses that the imbalances inside and outside the cell influence the cell to undergo a programmed cell death. The antioxidant components lead to an excess storage of H2O2, which further increases the hydroxyl radicals and lipid peroxide that signal the cell to undergo a programmed cell death. The influenza virus induces ROS production in host cells that can damage the virus genome which ROS enhance the pathogenesis ability of infectious influenza that leads to the thymus-specific elevation of the mitochondrial superoxide, which interferes with the normal functioning of T-cells lymphocyte damage in influenza A virus infections. Zinc-associated vaccine activity mechanism against viruses is indicated that the anti-viral vaccine activity of released Zn2+ ions from zinc solutions and ZnO NPs, may be enhanced by Zn2+ ion-induced Zn2+ ions-coordinated adapted immunity, viral growth regulation, and viral apoptosis and death.

Click here: https://juniperpublishers.com/index.php  

Tuesday, August 17, 2021

In vitro Methods to Substantiate the Antioxidant and Antipollution Claims of a Cosmetic Formulation Enriched with Vitamin C, Thioctic Acid and Ubiquinone - Juniper Publishers

 Organic & Medicinal Chemistry - Juniper Publishers 

Abstract

Skin is the largest organ of the human body with the principal role of first barrier from external stimuli. Exposure to ultraviolet-B (UV-B) radiations and urban pollutants trigger the rapid generation and accumulation of reactive oxygen species (ROS) in skin cells, with the consequent increase in oxidative stress. The development of cosmetics rich in antioxidant compounds is an important strategy to prevent skin-ageing. To substantiate the relative claim, we develop a panel of chemical and cell-based methods to use on a cosmetic formulation, as the ANTIOX SERUM, enriched with functional compounds known to counteract ROS and consequently oxidative stress-related skin-ageing. The antioxidant property of the product was firstly evaluated with the DPPH and Folin-Ciocalteu tests. Using a human keratinocyte cell line, the protective effect against UVB-induced cellular oxidative damage was performed with the DCF-DA assay and the measurement of the ROS levels. The antipollution activity was also investigated using the same experimental method using the benzo[a]pyrene as a stimulus. The obtained results confirm the antioxidant efficacy of the cosmetic product through preliminary chemical screening tests. In HaCaT cells, the cosmetic formulation showed a statistically significant ROS levels reduction after 24 h of treatment with the non-cytotoxic concentrations of the ANTIOX SERUM both in the antioxidant and antipollution in vitro models. Our findings confirmed that the combination of chemical and cell-based antioxidant methods are useful to substantiate the antioxidant and antipollution claims of the cosmetic formulation enriched with functional molecules, such as coenzyme Q10 (ubiquinone), α-lipoic acid (thioctic acid), 3-O-etil-L-ascorbic acid and glutathione.

Keywords: HaCaT cells; Oxidative stress; Antioxidant; UVB radiation

Abbreviations: PAHs: Polycyclic Aromatic Hydrocarbons; ECM: Extracellular Matrix; UVB: Ultraviolet-B; HaCaT: Human Keratinocyte Cell; MMP-1: Metalloproteinase-1; ROS: Reactive Oxygen Species; SD: Standard Deviation; PBS: Phosphate Buffer Saline; OD: Optical Density; FCR: Folin-Ciocalteu Reagent; DHLA: Dihydrolipoic Acid; INCI: International Nomenclature of Cosmetic Ingredients; UV: Ultraviolet

Introduction

Skin is the largest organ of the human body and the first protective barrier from the external stimulus; influencing people's social behaviour through a more youthful and healthy appearance, it presents also an important cosmetic role [1]. Since skin functionality can be compromised by intrinsic and extrinsic factors, finding a way to preserve its integrity is always a current challenge. The first mentioned “intrinsic factor” is referred to the physiological and chronological processes of decay due to genetic factors, which tends to begin after 25 years of age and results in thin, dry skin and fine wrinkles. Extrinsic ageing is engendered by the aggression of external environmental factors as air pollution, smoke and sun exposure that result in coarse wrinkles, loss of elasticity, laxity and rough-textured appearance [2]. In particular, long-term exposure to solar ultraviolet (UV) radiation is the primary cause of extrinsic skin ageing and it is referred to as photo-ageing [3]. UV radiation is divided into three sub-categories based on the wavelength (UVA, UVB and UVC) and it is responsible for about 80% facial ageing through an increase of reactive oxygen species (ROS) and oxidative stress [4]. Both of them can be counteracted by the antioxidant defence system produced by the skin [5]. Antioxidants are chemically stable molecules in the reduced form, capable of yielding electrons or hydrogens to compounds with unpaired electrons to stop the propagation of radical processes. However, when the damage is very extensive, the intrinsic mechanisms are often not enough [6]. For this reason, exogenous antioxidants are used in cosmetic products to support the commercial claim of anti-ageing [7]. The antioxidant property is mainly correlated with the contents of polyphenols, proteins/peptides, vitamins C and E, and a few metals such as selenium [8]. The proposed experimental design was developed using a cosmetic serum, called ANTIOX SERUM, (Matex Lab, Brindisi, Italy), an enriched formulation with four bioactive compounds within the International Nomenclature of Cosmetic Ingredients (INCI), known for their ability to counteract skin ageing: coenzyme Q10, α-lipoic acid, 3-O-etil-L-ascorbic acid and glutathione. Coenzyme Q10, or Ubiquinone, in its reduced form, can release a hydrogen from the benzene group blocking the radical and consequently the oxidative reaction. Furthermore, Coenzyme Q10 exhibits antioxidant activity with a key role in mitochondrial respiration [9]. Even α-lipoic acid, or Thioctic Acid, acts in the reduced form dihydrolipoic acid (DHLA) which is able to limit the development of radicals but also to restore the inactive forms of other antioxidants [10,11]. The 3-O-etil-L-ascorbic acid derives from ascorbic acid, with the addition of an ethyl group on the hydroxyl: this new structure leads to greater protection against ionization of the OH group with the consequent block of oxidation. The molecular modification allows improving the antioxidant activity compared to ascorbic acid, especially in areas exposed to the air such as the skin [12]. Glutathione is one of the main molecules that protect the skin from UV radiation thanks to the activity of the enzyme glutathione peroxidase, which catalyzes the elimination reaction of hydrogen peroxide inside the cell cytoplasm [10]. δ-tocopherol is an isomer of vitamin E, which acts by transferring an hydrogen of the hydroxyl group which blocks O21 and other ROS [11]. The objective of this study is to develop a panel of in vitro methods that can be used to support the antioxidant and antipollution capacities of a selected cosmetic formulation containing functional ingredients. At first, we investigated the free radical scavenging and the phenol content activities using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Folin-Ciocalteu methods, respectively. Moreover, the antioxidant and antipollution activities have been evaluated using an immortalized cell line of keratinocytes stimulated with UVB and urban pollutant, respectively.

Materials and Methods

Materials

Ascorbic acid (code A4403, Merck, Darmstadt, Germany), 2,2-diphenyl-1-picrylhydrazyl (DPPH, code 44150, Thermo Fisher Scientific, Waltham, MA, USA), benzo[a]pyrene (B[a]P, B1760, Merck, Darmstadt, Germany), 2’,7’-dichlorodihydrofluorescein diacetate (H2DCF-DA, D6883, Merck, Darmstadt, Germany), ethanol (Et-OH, code 02860-1L, Honeywell, Charlotte, NC, USA), High Glucose Dulbecco’s Modified Eagle’s Medium (DMEM, code BE12-614F, Euroclone, Milan, Italy), Fetal Bovine Serum (FBS, code ECS0180D, Euroclone, Milan, Italy), Folin-Ciocalteu Reagent (code 1.09001.0100, Merck, Darmstadt, Germany), L-glutamine (code ECB3000, Euroclone, Milan, Italy) antibiotics (107 Units/l penicillin G sodium and 10000 mg/l streptomycin sulfate, code ECB3002D, Euroclone, Milan, Italy), and Sodium Carbonate (Na2CO3, code 0246, VWR International, Milan, Italy).

INCI ingredients

Based on the INCI components, the cosmetic formulation enriched with antioxidant ingredients (Table 1) was taken under investigation.

Cell-free chemical antioxidant assays

Sample preparation: The cosmetic formulation was prepared by making 10% w/v solution in a 70:30 EtOH:H2O, mixed and incubated in the dark for 30 min. Samples were centrifuged at 4000 rpm for 10 min (Mega Star 600R, VWR International, Milan, Italy) and after, supernatants were preserved until use.

DPPH and folin-ciocalteu tests

The radical scavenging potential of the cosmetic product was measured using the DPPH assay. Based on the principles described by Mukesh Singh in 2017 [13], a stock solution of 0.6 mM DPPH was prepared and left in the dark for 2 h. Then, 2 mL of DPPH solution was added to 1 mL of the sample. After 30 min incubation, the absorbance was read at 517 nm (Multiskan Go, Thermo Fisher Scientific, Waltham, MA, USA). The polyphenolic content of samples was determined by the Folin-Ciocalteu method using the mixture of sodium tungstate and sodium molybdate (Folin-Ciocalteu reagent), which is able to oxidize antioxidant molecules and polyphenolic substrates. One hundred µL of the sample were mixed with 1 mL H2O and 0.5 mL of Folin-Ciocalteu reagent (FCR). The solution was vortexed and 1 mL of Na2CO3 20% w/v solution was added to each sample. After 1 h at 95 °C, the absorbance was read at 765 nm (Multiskan Go, Thermo Fisher Scientific, Waltham, MA, USA). A qualitative assessment was also performed for both methods. Internal controls, represented by ascorbic acid and a cosmetic product proved to be without antioxidant activity (CTRL-), were also included.

Cell-based antioxidant assays

Cell culture: HaCaT cells (code BSCL168, Zooprof. Institute of Lombardy e Emilia Romagna, Brescia, Italy) were grown in a complete medium constituted by DMEM containing 10% FBS and 1% antibiotics and L-Glutamine and in conditions of complete sterility in incubation at 37°C with 5% carbon dioxide (CO2) atmosphere.

Cytotoxicity assay (MTT test): As a preliminary assay, the cytotoxicity was evaluated following the method describes by Mosmann [14]. HaCaT cells were homogeneously seeded into 96-well plates at a density of 2 x 104 cells per well. After 24 h, cells were treated with different concentrations of the cosmetic formulation, starting from 40 mg/mL, prepared directly in a culture medium and following serial dilution (1:2). Untreated cells were used as control. After 24 h of treatment, the culture medium was then removed and cells were incubated with 50 µL of MTT solution (1 mg/mL) at 37 °C for 2 h. Subsequently, the solution was removed and replaced with 100 µL of isopropanol. Absorbance was determined at 560 nm wavelength using a microplate reader (GloMAx®, Promega Corporation, USA). Cell survival was calculated by measuring the difference in optical density (OD) of the tested cosmetic formulation with respect to control (untreated cells).

Cell viability (%) = [OD570nm tested product/OD570nm control] x 100

Measurement of intracellular ROS: Intracellular ROS levels were measured using the fluorescent probe H2DCF-DA, as described by Boulton and colleagues [15]. Briefly, HaCaT cells were seeded in a 96-black well plate at 37 °C with 5% CO2. After 24 h, cells were treated for a subsequent 24 h with the tested product, choosing the two highest non-cytotoxic concentrations (5 and 10 mg/mL) with the best solubility, selected by the preliminary MTT assay. Ascorbic acid was also included as an internal control being a known molecule able to protect from free radical damage. At the end of the treatment, HaCaT cells were washed with PBS and incubated with 20 mM DCF-DA solution at 37 °C with 5% CO2. Cells were then washed with phosphate buffer saline (PBS) and exposed to UVB lamp (CAMAG® UV Lamp 4, wavelength 302 nm, Muttenz, CH) with 2.5 mJ/cm2 dose. At the end, HaCaT cells were analysed using a fluorimeter (GloMAx®, Promega Corporation, USA) with excitation of 475 nm and emission at 500-550 nm. Relative ROS production was expressed as the ratio of fluorescence of the treated samples over the response in the appropriate controls:

(fluorescence treatment/fluorescence control) × 100.

Antipollution activity (ROS reduction): The antipollution activity of the cosmetic formulation was evaluated through its ability to decrease ROS formation in HaCaT cells after stimulation with a known pollutant (B[a]P) followed by DCF-DA protocol, as previously described. HaCaT cells were seeded in a 96-well black plate at 37 °C with 0.5% CO2. After 24 h, cells were co-treated for 24 h with the pollutant (B[a]P) and with the product at the two non-cytotoxic concentrations (5 and 10 mg/mL). Untreated cells were used as control. Cells treated with B[a]P were used as positive control while treatment with ascorbic acid was used to confirm the reduction of the oxidative stress in the cellular environment. At the end of the treatment, HaCaT cells were washed with PBS and incubated with the DCF-DA solution as previously described.

Statistical analysis

Data are presented as mean ± standard deviation (SD) of at least three independent experiments performed in duplicate. Statistical significance was calculated in all experimental sets using the One-way ANOVA with Bonferroni’s LSD multiple comparisons as post-test. p < 0.05 was considered a statistically significant difference compared to the relative controls. Statistical analysis was performed using GraphPad Prism version 9.0.0 (GraphPad Software, San Diego, CA).

Results

Radical scavenging potential by DPPH and Folin-Ciocalteu tests

The DPPH and Folin-Ciocalteau tests are used to determine the antioxidant activity and the total contents of polyphenols and ascorbic acid, respectively, directly performed on the finished cosmetic formulation. Table 2 shows the spectrophotometric results, expressed as OD (mean ± SD). In the DPPH experiments, the OD measured for the cosmetic formulation was equal to 0.265 and it was very close to the OD obtained with the ascorbic acid (0.104). It was possible to note that after adding the DPPH reagent, the purple colour was turned to yellow, whereas no change colour was instead observed in the cosmetic product without antioxidant activity, used as negative control (CTRL -) (Figure 1A). In the Folin-Ciocalteau method, the OD measured for the cosmetic formulation and the ascorbic acid were equal to 0.797 and 1.527, respectively, together with the visible blue colour of the samples. Unaltered colour, correlated to the absence of phenolic content, was observed, as expected, in the negative control (Figure 1B).

Evaluation of the cytotoxicity by MTT assay

The cytotoxicity test is performed only on the cosmetic formulation, the ANTIOX SERUM, demonstrated to have an antioxidant activity by the DPPH and Folin-Ciocalteu as preliminary assays. To evaluate the cell viability and to identify the appropriate concentrations, an MTT test was performed. Figure 2 shows the results of HaCaT viability after 24 h treatment (range tested 0.313 to 40 mg/mL for 24 h) with the cosmetic formulation under examination. The obtained data demonstrated cytotoxic activity only with the highest concentrations tested equal to 20 and 40 mg/mL with a percentage of cell viability of 69.82 and 28.15%, respectively, compared to untreated cells. Therefore, 5 and 10 mg/mL were selected for further in vitro studies.

Evaluation of antioxidant activity after UVB radiation

To investigate the protective antioxidant effects of the cosmetic formulation in UVB-irradiated HaCaT cells, the intracellular ROS levels were evaluated using a fluorescent probe H2DCF-DA. As shown in Figure 3, UVB significantly increased the ROS levels compared with untreated cells (p ˂ 0.0001). In the presence of UVB, the cosmetic formulation significantly reduced the ROS level in a concentration-dependant manner. Particularly, it is possible to observe a highly significant reduction (p ˂ 0.0001) of 19.23 and 23.89% after treatment with 5 and 10 mg/mL, respectively. Moreover, ascorbic acid was confirmed as good internal control, capable to reduce ROS levels of about 20%.

Evaluation of antipollution activity

To investigate the potential antipollution effect of the cosmetic formulation, the intracellular ROS levels were evaluated using a fluorescent probe H2DCF-DA in HaCaT cells treated with B[a]P. As shown in Figure 4, cells treated with B[a]P significantly increased the ROS generation compared with those in the untreated cells (p ˂ 0.001). Therefore, we evaluated the effect of the product after 24 h treatment in the presence of B[a]P, used as an urban pollutant. It can be seen that the treatment of the tested product at both concentrations selected led to a strong reduction in ROS levels when compared with HaCaT cells treated only with the pollutant B[a]P: 26.81 and 42.39% less with 5 and 10 mg/mL, respectively (p ˂ 0.0001). Ascorbic acid is confirmed to be a good positive control, which reduces the ROS levels of about 23%.

Discussion

The skin is the first external barrier of our body and it is heavily exposed to environmental stressors. All of them act directly on skin cells through the generation of free radicals and especially reactive oxygen species (ROS) with the consequent stimulation of skin ageing-related signalling pathways, as the matrix metalloproteinase-1 (MMP-1) [16]. Clinically, the biological consequences of an overloading amount of free radicals include wrinkling, laxity, and a leather-like appearance [17]. To counteract the adverse changes induced by ROS levels, the skin is equipped with an effective defence mechanism, consisting of an interacting network of antioxidants, that can be further improved with the daily use of cosmetic products containing functional antioxidant compounds [7,17]. The use of in vitro methods to highlight the antioxidant and antipollution activities is therefore a precise approach to support the relative claims. Currently, many analytical methods allow the determination of the antioxidants’ levels in plant materials and foods [18], but not all of them are appropriate for cosmetic products, which are a mixture of compounds with different physicochemical properties [19]. In our work, we want to present a panel of in vitro tests useful to demonstrate the antioxidant activity of the selected cosmetic formulation, the ANTIOX SERUM, produced by MatexLab (Brindisi, Italy) containing coenzyme Q10, α-lipoic acid, 3-O-etil-L-ascorbic acid and glutathione. Therefore, we decided to perform chemical and cell-based assays making a comparison with a cosmetic product without antioxidant activity. Among the chemical ones, the DPPH and Folin-Ciolcalteu assays were carried out, obtaining a good antioxidant activity for only the ANTIOX SERUM, as expected. The use of these chemical assays provides preliminary data that could be used as screening, considering that both methods are simple, efficient and relatively inexpensive. These assays, however, do not consider the cellular mechanisms. Thus, to better assess the antioxidant capacity of the only ANTIOX SERUM in a more physiological way, experiments were performed in a cellular setting using human keratinocyte cell lines (HaCaT) and ultraviolet-B (UVB) lamp as the irradiation source. 

UVB-irradiation is indeed one of the main causes of increased ROS, capable to induce MMPs expression and the degradation of collagen and elastin fibers, the main structural proteins of the dermal extracellular matrix (ECM) [16,20,21]. These sets of skin alterations could contribute to photo-ageing development. Our results demonstrated the protective effect of the cosmetic formulation against UVB irradiation, with the ROS levels reduced of about 20% after 24 h treatment with the two non-cytotoxic concentrations (5 and 10 mg/mL) in HaCaT cells. We then wondered whether the cosmetic formulation could also have the ability to protect against pollutants. It is well known that environmental pollutants contribute to the increase of oxidative stress and premature skin ageing and the benzo[a]pyrene is one of the most important investigated [22]. It belongs to the polycyclic aromatic hydrocarbons (PAHs), capable to induce the increase in ROS levels in different cell lines, including HaCaT cells [23,24]. In our experimental model, the cosmetic formulation showed a strong ROS level reduction, of about 27% and 42% after treatment with 5 and 10 mg/mL, respectively. Overall, our findings confirmed the good antioxidant activity and the discovery an antipollution effect of the ANTIOX SERUM that can be used to support the relative claims.

Conclusion

The results of our study show an experimental design, composed of chemical and cell-based assay, to prove and support the antioxidant activity of different cosmetic formulations, as demonstrated by testing the product ANTIOX SERUM. Chemical assays have the significant advantage of requiring less time, instrumentation readily available in any laboratory and providing a reliable preliminary analysis. The cellular experiments permit not only to confirm the antioxidant activity in a living environment such as cell cultures but also to find out an antipollution effect that can be used to substantiate the cosmetic product claim.

To Know more about Organic & Medicinal Chemistry 



To Know more about our Juniper Publishers

Click here: https://juniperpublishers.com/index.php  


Wednesday, May 6, 2020

The Effects of Free Radicals on Aging Process - Juniper Publishers

Biomedical Engineering & Biosciences - Juniper Publishers

Abstract

Aging is a process that brings many changes in the body and free radicals are increasingly remarked responsible for most of these changes. A theory which is called "free radical theory of aging" assumes that accumulation of reactive oxygen species (ROS) caused aging. Additionally, aging is a multifactorial process and this process can not reduce to any one single situation. This review aims to summarize various studies about the role of free radicals on aging process.
Keywords: Free radicals; Aging; Oxidative stress.

Introduction

The aging is a very multifactorial and complex biological process. It can be defined as the morphological, functional and structural modifications realized between birth and death [1]. On top of genetic factors, external parameters, such as smoking, nutrition, environmental conditions, alcohol, can promote to its expected appearance. A detailed relevance in this perspective has been paid to the effects of free radicals, particularly to reactive oxygen species (ROS), which are causing “oxidative stress” [2].
There are so many theories about aging process but not a single theory is acceptable. Actually, it is unreliable that only a single theory can describe all the aging mechanisms. "The free radical theory of aging” gives hope among the all of these theories, today. This theory was first suggested by Harman in 1956 [3], and it blamed for the aging process and death of organisms. "The free radical theory of aging” is based on the free radical reactions and their prominent and ubiquitous presence in organisms. The theory supposes that free radical reactions cause production of the aging changes, diseases and the main aging process [4].

Free Radicals and Oxidative Stress

A free radical is a chemical species such as an atom, a molecule or an ion which contains an uneven number or unpaired electrons. There are many sources of free radicals in biological systems. There are some ways in that free radicals can be generated, but their most prevalent source is the mitochondria that uses 90% of the O2 in the human body [5]. Moreover, minor amounts of free radicals are produced by electron transport chain located in endoplasmic reticulum, nuclear and plasma membranes. Free radicals which found in biological systems were supposed to be included in aging and various pathological processes [6].
Reactive oxygen species (ROS) are most common free radicals. The ROS value control is provided not only by ROS generation, also by elimination. The production and elimination of ROS are closely related. Biological systems have complicated and multiplex antioxidant systems which minimize negative effects of ROS or eliminate them [7]. Even so, under certain conditionss there is a balance between these two processes but sometimes this balance can be disturbed. Unquestioning, increase in stable state ROS value, that results from imbalance between production and elimination of ROS, can affect many physiological processes such as aging, carcinogenesis DNA- damage and for initiation of many diseases [2].

Effects of Free Radicals

ROS can cause various damages to biologic macromolecules, such as nucleic acids, proteins and lipids. Especially, polyunsaturated fatty acids of membranes are the most sensitive macromolecules as oxidation targets for ROS. Membrane fatty acids are so sensitive due to lipid peroxidation (LPO) process, because once LPO is initiated, a harmful chain reaction begins [8]. Nucleic acids such as DNA and RNA are also very sensitive to ROS attack. Oxidation of these nucleic acids causes mutations in both mitochondrial and nuclear genomes [9]. Nearly, all amino acids in a protein or in an enzyme can be oxidized by ROS. And these oxidation can cause modifications which lead to losses of function [10].
Many different cellular macromolecules can be attacked by ROS but certainly damage to DNA is most considerable for aging, particularly in postmitotic cells for example neurons. Especially, mitochondrial DNA (mtDNA) is nearby to the position of ROS generation in mitochondria. Mutations such as deletions and point mutations in mtDNA, take part with aging processes in postmitotic organs and tissues like as brain [11] and attain high values in aged individuals, particularly in the control region that is answerable for mtDNA transcription and replication [12,13]. The altered mtDNA expands towards superiority while aging in these postmitotic cells.
Additionally to formation of ROS, the other property of long-lived animals interrelates oxidative stress with aging: the level of fatty acid unsaturation of biological cell membranes. Unsaturated fatty acids especially polyunsaturated fatty acids are the most sensitive macromolecules of cells against free radical damages, in connection with the existence of unstable electrons. These electrons attack double bonds of unsaturated fatty acids. Their susceptibility to LPO increases exponentially by the double bonds number of per molecule. For this reason, decreased value of fatty acid unsaturation will reduce oxidative stress in cells. Several previous studies have determined that the maximum longevity is actually negatively correlated with the level of fatty acid unsaturation of cells and tissues of mammalian [14,15].
In conformance with a considerable function for ROS in nervous system especially in brain aging [16], various studies point out ROS level and neurodegenerative diseases of neural cells. It is specified that ROS can cause death of astrocytes [17] and neurons [18] by both necrosis and apoptosis. Also, evidences containing ROS in main degenerative disorders is consistent with their function in brain and neuronal aging. There are lots of studies which are agreement with that oxidative stress plays a role in degeneration of dopaminergic cells in Parkinson's disease [19]. Many obtained results specify that oxidative stress is one of the first observed processes in Alzheimer's disease [20]. Considering the studies done up to this time, we can say that there is a strong relationship between free radical caused oxidative stress and aging.

To know more about Juniper Publishers

How Does the Multi-Disciplinary Team Impact Chronic Kidney Disease Management? - Juniper Publishers

  Urology & Nephrology - Juniper Publishers Abstract Chronic kidney disease (CKD) is a condition where a gradual kidney function loss le...