La Roche Posay Vitamin C Percentage

La Roche Posay Vitamin C Percentage

La Roche Posay Review

The story behind La Roche Posay

La Roche Posay is a dermatological skincare brand that formulates products with high efficacy and safety standards to address skin needs, even sensitive skin.

Recommended by tens of thousands of dermatologists across the globe, La Roche Posay has become known for delivering highly effective skincare solutions that target a variety of skin concerns, from acne to aging and more.

What are the Best Skin Care Products of 2021?

 In this La Roche Posay review, we'll discuss a few of the best-selling La Roche Posay skincare products, formulation details, La Roche Posay reviews from real customers, and an alternative for you to consider.

What Are The Best Selling La Roche Posay Skincare Products?

La Roche Posay is committed to making dermatological skincare accessible to those who need it and offers consumers products for each step of their skincare routine.

For instance, you can browse through La Roche Posay cleansers , moisturizers, masks, toners, serums, BB and CC creams, sunscreens, and more. But if you don't have time to look through the extensive range of products, we're going to cover three of the best-selling La Roche Posay skincare products, including Effaclar Duo, Lipikar Balm AP+, and Anthelios Invisible Fluid Facial Sunscreen SPF 50+ .

La Roche Posay Effaclar Duo is a dual-action acne spot treatment that is said to reduce the number and severity of acne blemishes, as well as help to clear blackheads and whiteheads. In addition, this treatment is claimed to reduce 60% of acne in just 10 days.

La Roche Posay Lipikar Balm AP+ is a body cream moisturizer for dry skin to extra dry skin that is clinically shown to reduce dry, rough skin. In addition, this cream is accepted by the National Eczema Association and is suitable for babies as young as two weeks.

Anthelios Invisible Fluid Facial Sunscreen SPF 50+ is a non-greasy, fast-absorbing sunscreen for the face and body that provides broad-spectrum SPF 60 sun protection. This sunscreen is water-resistant for up to 80 minutes.

Inside La Roche Posay Skincare

Did you know that one of the key ingredients used inside La Roche Posay skincare dates back to the 14th century? We're talking about La Roche Posay Thermal Spring Water , unique selenium-rich water at the heart of the brand.

In the 14th century, a French nobleman named Bertrand Du Guesclin discovered the thermal springs of La Roche Posay when he stopped to drink the water. According to legend, his horse, who suffered from severe eczema, submerged itself into the water and was cured of its skin disorder.

 In the 17th century, Pierre Milon, a doctor to Henry IV and Louis XIII, went to the spring to conduct an analysis of the water. He discovered that the thermal water was naturally rich in selenium, an essential trace mineral, which provided the water with its unique soothing and antioxidant skincare properties.

Throughout history, hospitals were built at the site of the spring to treat patients who came from all over the world.

 Fast forward to 1975 and the La Roche Posay Laboratoire Dermatologique was created with its mission being to use this special thermal water to develop skincare products that would provide dermatologists with effective skincare solutions for their patients, especially those with sensitive skin.

In addition to the Thermal Spring Water, we'd like to discuss a few of the key ingredients you can find in the best-selling La Roche Posay skincare products mentioned above.

La Roche Posay Effaclar Duo

One of the key ingredients in La Roche Posay Effaclar Duo is 5.5% benzoyl peroxide . Benzoyl peroxide can effectively kill P. acnes bacteria by destroying the cell walls.

Additionally, benzoyl peroxide can exfoliate and help peel the outer layer of skin. One drawback of benzoyl peroxide is that while it does effectively kill P. acnes , it also kills the good bacteria that keep your skin healthy.

This can leave skin feeling dry and irritated, which for some people can make skin look worse. The formula also features lipohydroxy acid, a derivative of salicylic acid with skin-renewing, exfoliating, and acne-treating properties.

La Roche Posay Lipikar Balm AP+

La Roche Posay Lipikar Balm AP+ features key ingredients such as shea butter, niacinamide , and mineral oil . Shea butter is a very rich occlusive emollient that forms a barrier on the skin to lock in moisture and leave the skin feeling soft.

Niacinamide is a form of vitamin B3 that has been shown to help repair the skin barrier and improve skin moisture, it may also help to reduce inflammation, blotchiness, hyperpigmentation, acne, and wrinkling.

Mineral oil is a clear, odorless oil that functions as an emollient. After application, mineral oil forms a protective film on the surface of skin that helps to prevent evaporation of the skin's natural moisture.

Anthelios Invisible Fluid Facial Sunscreen SPF 50+

Anthelios Invisible Fluid Facial Sunscreen SPF 50+ is formulated with Cell-Ox Shield technology, offering broad spectrum SPF 60 protection plus an antioxidant complex.

 This formula contains the antioxidants tocopherol (vitamin E) and Cassia alata leaf extract. Unfortunately, both of these antioxidants are present in concentrations lower than 1%. We know this by looking at the ingredient list (which is in order of descending concentrations). Both ingredients are listed after the preservative phenoxyethanol, which as our resident biochemist Elle MacLeman discusses, cannot be used in concentrations more than 1%.

Where Are La Roche Posay Skincare Products Sold?

La Roche Posay skincare products are sold online through the company website and third-party e-commerce sites. Consumers can also find this brand in drug stores such as Walgreens and CVS, as well as beauty supply stores like Ulta.

What Is The La Roche Posay Return Policy?

The La Roche Posay return policy states, "If for some reason you're not happy with your order, you can return it in its original packaging with a receipt and receive a full refund."

La Roche Posay Reviews

La Roche Posay reviews on the brand's website are overall very positive, with each of the best-selling La Roche Posay skincare products mentioned above receiving an average of at least 4.5 out of 5 stars.

Many La Roche Posay reviews for Effaclar Duo confirm that this is an effective spot treatment for acne. However, there are quite a few reviews that mention this product can cause skin dryness.

The majority of the La Roche Posay reviews for Lipikar Balm AP+ point out that this is a really great moisturizer for dry skin that doesn't leave a greasy feel on the skin.

La Roche Posay reviews for the Anthelios 60 Melt-In Sunscreen Milk mention that it is easy to use, effective, and non-greasy.

What Are Some Alternatives To La Roche Posay?

After discussing a few of the best-selling La Roche Posay skincare products, it's clear that La Roche Posay uses highly effective ingredients to formulate its products.

The brand particularly stands out when it comes to body moisturizers and sunscreens. However, we didn't have a chance to discuss La Roche Posay's anti-aging products.

Take the La Roche Posay Hyalu B5 Pure Hyaluronic Acid Serum. The third highest concentration ingredient in this serum (after water and glycerin) is denatured alcohol. Denatured alcohol has been associated with skin dryness, irritation, and even disruption of the skin barrier, especially when used in high concentrations. If you're looking for a brand that formulates anti-aging skincare products, we recommend Formulyst .

Instead of La Roche Posay's Hyaluronic Acid Serum, try Formulyst's Super Hyaluronic Water Moisturizer. This lightweight formula features hyaluronic acid , the key molecule involved in skin hydration.

Hyaluronic acid helps boost your skin's natural nourishing powers by attracting atmospheric moisture and transforming it into intense hydration for your skin.

Formulyst Super Hyaluronic Water Moisturizer also contains jojoba oil and apricot kernel oil to balance oil production. Both of these botanical oils are non-comedogenic, so you don't have to worry about clogged pores or breakouts.

There's no need to compromise when it comes to finding skincare that is effective and safe. Carrot & Stick takes a tough-love approach to skincare, perfectly balancing the power of plants with cutting-edge science. Not only is Carrot & Stick good for your skin, it is also good for the environment as the formulas skip harmful chemicals commonly found in skincare products.

OUR TOP SKINCARE PICKS

1

Carrot & Stick

Carrot & Stick takes a tough love approach to skincare. What does this mean? It perfectly balances the power of plants with innovative science, offering the best of both worlds.Read More

2

Formulyst

The philosophy of Formulyst is long-term and effective skincare, rather than covering up any imperfections. The comprehensive line of products tackles everything from wrinkles to dark spots and dry skin. While some brands rely on unnecessary fillers and scents, Formulyst focuses on ingredients that help create results, such as vitamins, minerals and antioxidants.Read More

La Roche Posay SKIN CARE PRODUCTS

La Roche Posay Vitamin C Serum Review

La Roche Posay Vitamin C Serum Review

The Story Behind La Roche Posay La Roche Posay is a dermatological skin care brand that formulates products with high efficacy and safety standards to address skin needs, even sensitive skin. Recommended by tens of thousands of dermatologists across the globe, La Roche Posay has become known for delivering highly...

La Roche-Posay Effaclar

La Roche-Posay Effaclar

When it comes to acne solutions that go beyond your traditional one-step treatment, the La Roche-Posay Effaclar system is the one that dermatologists recommend. Although the name of this innovative acne system sounds a bit intimidating, the La Roche-Posay Effaclar system is designed to treat acne in just three simple...

La Roche Posay Hyalu B5 Serum Review

The La Roche Posay Hyalu B5 Serum offers users an extremely cosmetically elegant hydrating serum that helps to repair and replump the skin without an overly complex ingredients list. La Roche Posay is a french pharmacy brand that has been known for decades for their effective, yet minimalistic approach to skincare formulation. Their products feature high powered ingredients, but concentrate on a select few in order to address skin concerns without causing sensitivity. The Hyalu B5 Serum follows this approach as well, as a hyaluronic acid serum with a little bit of a twist. Alongside hyaluronic acid, this product also includes vitamin B5 and madecassoside which help to nourish the skin, replenish the skin's natural moisture barrier and soothe irritated skin. Because of this, the La Roche Posay Hyalu B5 Serum would be a great option for those of you who are looking for hyaluronic acid serum for sensitive skin. ... Read More

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La Roche Posay Lipikar Balm Review

The La Roche Posay Lipikar Balm is a slight twist on a classic staple. This creamy body moisturizer was designed to suit extremely dry and sensitive skin types specifically but can be easily incorporated into your body care routine regardless of your skin type. The formula behind this product contains a combination of humectants, emollients and occlusives to throughout repair the skin, alongside a unique prebiotic complex to encourage healthy function within the skin microbiome. La Roche Posay has been a skincare staple in french pharmacies for years, and for very good reason. Like the Lipikar balm, all of their products are carefully formulated to include only what you need, and none of what you don't. So you won't find an extreme amount of ingredients or excess fillers in this formula. Instead, you have a combination of key ingredients that aim to hydrate the skin and seal in moisture in order to protect the integrity of your skin's natural moisture barrier. ... Read More

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La Roche Posay Active C10

The La Roche Posay Active C10 combines all of the expertise of this well known french pharmacy skincare staple along with some of the most powerful active ingredients to create a uniquely formulated cream serum. This product features 10% pure vitamin C in the form of ascorbic acid, along with hyaluronic acid and antioxidants. Each of these ingredients perform different tasks within the skin in order to improve its overall condition and appearance. Starting off with the star of the show, vitamin C is typically a very difficult ingredient to formulate with. Because it oxidizes quickly in the presence of oxygen and water, formulators must be very careful and methodical when constructing the rest of the formula around it. In this particular product, this cream base and air tight tube allow for minimal exposure to stimuli which can diminish the formulas integrity. Additionally, vitamin C acts as incredible skin brighter as well as an antioxidant to protect and preserve the skin. Paired with ingredients like hyaluronic, vitamin C can be an excellent option for those who are looking for a hydrating, lightweight yet powerful serum for their morning routine. ... Read More

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La Roche Posay Anthelios Ultra Light Mineral Sunscreen Spf 50 Review

The La Roche Posay Anthelios Ultra Light Mineral Sunscreen SPF 50 is an excellent option for a mineral sunscreen for those who hate mineral sunscreens. For many of us, the concept of mineral or physical sunscreens conjures up memories of harsh white casts alongside sticky, greasy or tacky residues. But thanks to innovation within the skincare industry, more recent formulas have been able to do away with those drawbacks. Looking at the La Roche Posay Anthelios Ultra Light Mineral Sunscreen SPF 50, we can see how this brand has been able to create such a pleasant feeling product that disappears into the skin. This sunscreen has a fluid like consistency which allows it to be swiped and blended into the skin effortlessly, leaving a completely matte finish. Their Cell-Ox Shield Technology features broad spectrum protection thanks to zinc oxide and titanium dioxide as well as antioxidant blend to prevent oxidative or free radical damage which can also affect the health of your skin. ... Read More

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La Roche Posay Toleriane Hydrating Gentle Cleanser Review

For those with dry or sensitive skin, finding the right second step cleanser can be a huge challenge. Thankfully, in the last few years, there has been a significant trend towards non-foaming, hydrating and sensitive skin friendly cleansers. But if you're looking for a no fuss non-foaming cleanser that has consistently been the top recommended product in this category, there's no better place to start than the La Roche Posay Toleriane Hydrating Gentle Cleanser. Like the name would suggest, this product works to cleanse the skin thoroughly without disrupting your skin's natural moisture barrier or dehydrating the skin. Instead this minimalistic formula contains hydrators like glycerin and ceramides which help to prevent dryness, restore the skin and encourage essential skin processes. Additionally, since this product is so gentle and so carefully formulated, you don't have to worry about this cleanser altering the pH of your skin. ... Read More

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La Roche Posay Purifying Foaming Cleanser Review

There are few brands that embody the french pharmacy skincare as much as La Roche Posay. Known for their simple, streamlined and sensitive skin friendly approach to skin care, this brand offers exceptionally well formulated workhorse products that are effective and budget friendly. One of their more well known product offers that continues to be a best seller for the brand is the La Roche Posay Purifying Foaming Cleanser. This cleanser strikes the perfect balance between purifying the skin and restoring the moisture barrier, thanks to the thoughtful formulation. The ingredients list includes gentle surfactants that remove dirt, debri and impurities from the skian while ceramides replenish moisture and help to maintain the skin's nature moisture barrier. The result is a cleanser that is easily removed and leaves behind a clean but comfortable feeling on the skin. ... Read More

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La Roche-Posay Toleriane Riche Review

If you're looking to try out some french pharmacy classics, there's almost no better brand to check out then La Roche Posay. Like many french pharmacy brands, La Roche Posay excels at creating products that work for all skin types, even those with extremely sensitive skin. Products like the La Roche Posay Toleriane Riche offer those with extremely sensitive skin a comforting and protecting cream that moisturizes without causing irritation or clogged pores. In order to work well with all skin types, this product has an extremely minimalistic ingredient's list, with only a small handful of hydrating and moisturizing ingredients. The formula mainly includes humectants, emollients and occlusives. This blend of ingredients, which includes things like shea butter and glycerin, help to hydrate, moisturize and soften the skin, which helps to maintain balance at the surface of the skin. With all this in mind, we'd recommend this type of product to those of you with highly reactive skin types who struggle to find a moisturizer that doesn't cause any sensitivity or irritation. Additionally, this product makes an excellent moisturizer for all skin types, especially if you're looking for a workhorse moisturizer without any active ingredients that could interact with other actives in your skincare routine. ... Read More

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La Roche-Posay Hydraphase Intense Riche Review

For a hyaluronic acid treatment that offers long lasting effects, you may want to check out creamier formulations like the La Roche Posay Hydraphase Intense Riche. This unique formula combines the benefits of a lightweight hydrating serum and a more moisturizing cream in order to deliver well balanced nourishment to the skin. For those with drier skin types, hyaluronic acid serums can actually backfire if there are no other ingredients to prevent the moisture from escaping and evaporating, especially in drier climates. In order to prevent this, products like the La Roche Posay Hydraphase Intense Riche contain occlusives, which keep the hydration, delivered through hyaluronic acid, in the skin. Because of this, this product can actually be used in two different ways, depending on your skin type. For those with dry skin, for example, this product can be used underneath a more occlusive moisturizer, while for those with oily skin, this may be enough to be the perfect dose of moisture on its own to finish off your skincare routine. ... Read More

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La Roche-Posay Anthelios AOX Antioxidant Serum SPF 50 Review

La Roche-Posay Anthelios AOX Antioxidant Serum SPF 50 is a lightweight daily face serum with sunscreen that is said to help visibly improve the appearance of fine lines, dark spots, and skin texture. The active sunscreen ingredients in this serum include avobenzone (2.68%), homosalate (9.6%), octisalate (2.88%), octocrylene (5.38%), and oxybenzone (3.46%). All of these ingredients are chemical sunscreen agents, which protect the skin by first absorbing UV light, then transforming that light energy into some other form of energy, such as heat. The combination of these ingredients provides broad spectrum protection, which means the skin is protected from both UVA and UVB rays.

La Roche-Posay Anthelios AOX Antioxidant Serum SPF 50 is formulated with Cell-Ox Shield® technology, which combines these active sunscreen ingredients with antioxidants to further protect the skin. Antioxidants limit oxidative damage caused by free radicals, the unstable molecules that contribute to the formation of premature wrinkles and fine lines on the skin. The antioxidants included in this formula include tocopherol (vitamin E), ascorbyl glucoside (vitamin C derivative), and baicalin derived from Scutellaria baicalensis (Chinese skullcap) root extract. However, both vitamin C and baicalin are used in very low concentrations in this serum, less than 1%. We know this by looking at the ingredient list (which is in order of descending concentrations). Both ingredients are listed after the preservative phenoxyethanol, which cannot be used in concentrations more than 1%. La Roche-Posay Anthelios AOX Antioxidant Serum SPF 50 does contain a decent amount of vitamin E, which not only helps the skin combat free radicals, but is also known to help strengthen the skin's lipid barrier.

... Read More

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La-Roche Posay Toleriane Double Repair Moisturizer with SPF Review

La-Roche Posay Toleriane Double Repair Moisturizer with SPF is a lightweight, fragrance-free, oil-free sunscreen moisturizer for sensitive skin that is said to replenish moisture and help restore the skin's natural protective barrier. This moisturizer features La Roche Posay prebiotic thermal water as the main ingredient. Prebiotics are a type of non-digestible fiber compound that provide indirect benefits by enhancing the growth or activity of beneficial microorganisms that make up the skin's microbiome. Ultimately, this results in healthier looking skin. The active sunscreen ingredients in La-Roche Posay Toleriane Double Repair Moisturizer with SPF include avobenzone (3%), homosalate (5%), octisalate (5%), and octocrylene (7%). All of these ingredients are classified as chemical sunscreen agents, which protect the skin by first absorbing UV light, then transforming that light energy into some other form of energy, such as heat. They offer broad spectrum SPF 30 protection, which means they defend against both UVA and UVB rays.

Another key ingredient in La-Roche Posay Toleriane Double Repair Moisturizer with SPF is niacinamide, a form of vitamin B3 that plays a vital role in cellular metabolism. This means that niacinamide provides skin cells with the energy needed to perform important processes, such as DNA repair and cell turnover. Niacinamide also has the ability to repair the skin barrier and improve skin moisture, as well as reduce inflammation, blotchiness, hyperpigmentation, acne, and wrinkling. La Roche Posay Toleriane Double Repair Face Moisturizer with SPF also contains the skin-replenishing ingredient ceramide NP. Ceramides are lipids naturally produced by the skin and are vital in supporting the skin's barrier function. Without these essential lipids, the skin barrier is weakened, resulting in symptoms such as dry, itchy, or irritated skin. The ceramide NP in this moisturizer will help to maintain healthy, intact, moisturized skin.

... Read More

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La Roche-Posay Anthelios Tinted Mineral Sunscreen Review

La Roche-Posay Anthelios Tinted Mineral Sunscreen contains 100% mineral UV filters in a lightweight, matte finish fluid that is lightly tinted for a healthy glow. The active sunscreen ingredient is titanium dioxide (11%), a mineral sunscreen that works by blocking or deflecting UV light, rather than being absorbed by the skin. It provides broad spectrum protection, which means it defends against both UVA and UVB rays. La Roche-Posay Anthelios Tinted Mineral Sunscreen is formulated with Cell-Ox Shield® technology, which combines titanium dioxide with antioxidants to further protect the skin. Antioxidants limit oxidative damage caused by free radicals, the unstable molecules that contribute to the formation of premature wrinkles and fine lines on the skin. External sources of free radicals include the sun, pollution, blue light, etc. The antioxidants in this sunscreen include tocopherol (vitamin E) and Cassia alata leaf extract. However, Cassia alata leaf extract is present in a very low concentration, less than 1%. We know this by looking at the ingredient list (which is in order of descending concentrations). This ingredient is listed after the preservative phenoxyethanol, which cannot be used in concentrations more than 1%. In fact, Cassia alata leaf extract is almost last on the ingredient list. The formula does contain a decent amount of vitamin E, which not only helps the skin combat free radicals, but is also known to help strengthen the skin's lipid barrier. ... Read More

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La Roche Posay Lipikar Eczema Cream Review

La Roche Posay Lipikar Eczema Cream is a steroid-free, fragrance-free eczema cream that is clinically shown to relieve itchy, irritated skin. It replenishes skin's essential lipids, provides instant and lasting hydration, and helps visibly reduce the signs of eczema. The active ingredient in this eczema cream is colloidal oatmeal (1%). Colloidal oatmeal is a skin protectant that has been used for centuries to soothe and heal the skin, as well as reduce inflammation and redness. Research indicates that the active components in colloidal oatmeal are avenanthramides and beta glucans. Avenanthramides are a group of potent polyphenol antioxidants that provide anti-inflammatory, anti-redness, anti-itch, and antihistamine effects when applied to the skin. Beta glucans have excellent water-binding properties and function as a skin protectant.

La Roche Posay Lipikar Eczema Cream also features the brand's prebiotic thermal water. Prebiotics are a type of non-digestible fiber compound that provide indirect benefits by enhancing the growth or activity of beneficial microorganisms that make up the skin's microbiome. Ultimately, this results in healthier looking skin. Another key ingredient in this cream is niacinamide, a form of vitamin B3 that plays a vital role in cellular metabolism. This means that niacinamide provides skin cells with the energy needed to perform important processes, such as DNA repair and cell turnover. Niacinamide also has the ability to repair the skin barrier and improve skin moisture, as well as reduce inflammation, blotchiness, and more. Lastly, there are many different emollients in the La Roche Posay Lipikar Eczema Cream, including shea butter, mineral oil, castor oil, and more.

... Read More

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La Roche Posay Anthelios 60 Review

La Roche Posay Anthelios 60 Melt-In Sunscreen Milk is a non-greasy, fast-absorbing sunscreen for face and body that provides broad spectrum SPF 60 protection. The active sunscreen ingredients are avobenzone (3%), homosalate (10.72%), octisalate (3.21%), octocrylene (6%), and oxybenzone (3.86%). All of these ingredients are classified as chemical sunscreen agents, which protect the skin by first absorbing UV light, then transforming that light energy into some other form of energy, such as heat. They offer broad spectrum protection, which means they defend against both UVA and UVB rays. In addition to these active sunscreen ingredients, La Roche Posay Anthelios 60 Melt-In Sunscreen Milk contains butyloctyl salicylate. This multi-functional ingredient helps to solubilize oxybenzone and avobenzone, increases the SPF rating, and improves the spreadability and feel of the product. La Roche Posay Anthelios 60 Melt-In Sunscreen Milk is also said to provide antioxidant protection due to the presence of the antioxidants tocopherol (vitamin E) and Cassia alata leaf extract. However, both of these antioxidants are present in concentrations lower than 1%. We know this by looking at the ingredient list (which is in order of descending concentrations). Both of these ingredients are listed after the preservative phenoxyethanol, which cannot be used in concentrations more than 1%. ... Read More

INGREDIENTS

La Roche Posay Toleriane Double Repair Face Moisturizer Review

La Roche Posay Toleriane Double Repair Face Moisturizer is a lightweight, fragrance-free, oil-free cream for sensitive skin that is said to replenish moisture and help restore the skin's natural protective barrier. This cream features La Roche Posay prebiotic thermal water as the main ingredient. Prebiotics are a type of non-digestible fiber compound that provide indirect benefits by enhancing the growth or activity of beneficial microorganisms that make up the skin's microbiome. Ultimately, this results in healthier looking skin. Another key ingredient in this cream is niacinamide, a form of vitamin B3 that plays a vital role in cellular metabolism. This means that niacinamide provides skin cells with the energy needed to perform important processes, such as DNA repair and cell turnover. Niacinamide also has the ability to repair the skin barrier and improve skin moisture, as well as reduce inflammation, blotchiness, hyperpigmentation, acne, and wrinkling. La Roche Posay Toleriane Double Repair Face Moisturizer also contains the skin-replenishing ingredient ceramide NP. Ceramides are lipids naturally produced by the skin and are vital in supporting the skin's barrier function. Without these essential lipids, the skin barrier is weakened, resulting in symptoms such as dry, itchy, or irritated skin. The ceramide NP in this moisturizer will help to maintain healthy, intact, moisturized skin ... Read More

INGREDIENTS

Pigmentclar Eyes Review

La Roche Posay is one of the most beloved french pharmacy brands on the market. Their products are well known for their simple, sensitive skin friendly formulations and the entire range is consistently recommended by dermatologists for treating all types of skin. Looking at their Pigmentclar Eyes, this product was specifically designed to serve as a lightweight, corrective eye cream. The formula behind this product utilizes two actives including Phe-Resorcinol Caffeine in order to address dark circles and create a more even toned under eye area with long term use. For immediate results, this product also includes light reflecting pigments in order to create a more radiant and bright under eye area upon application. This product also features a cool metal application which helps to soothe and cool down the eye area, which can help to reduce under eye puffiness in the morning. ... Read More

INGREDIENTS

La Roche-Posay Pigmentclar Dark Spot Serum Review

La Roche Posay has been one of the most well loved french pharmacy brands for years. Their Pigmentclar Dark Spot Serum. Like other dark spot and hyperpigmentation serums, this product contains niacinamide, which helps to lighten and brighten age spots, acne scars and other forms of hyperpigmentation. But what sets this product apart is their radiance boosting and light reflecting properties. Even with the most potent formulas, hyperpigmentation can be one of the most time-consuming skin issues, and it may take months in order to fade your dark spots permanently. While you wait to see results, the formula behind the La Roche Posay Pigmentclar Dark Spot Serum includes light reflecting particles, which give the illusive of smoother, brighter, dewier looking skin. Because of this aspect, we'd recommend applying this product as part of your morning routine in order to reap those immediate benefits. ... Read More

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La Roche Posay Cicaplast Baume B5 Review

As one of the most commonly recommended french pharmacy products on the market, the La Roche Posay Cicaplast Baume B5 has achieved cult status over the years. This thick but not overly greasy balm was one of the first sensitive skin friendly, balm-type products to hit the market and has remained at the forefront of this category. This product is often recommended to those who are struggling with dehydrated skin or are having issues maintaining a healthy and well functioning skin barrier. This product is also often recommended for those who are trying to start use high percentage retinols, as it can act as a buffer to offset some of the less appealing aspects of retinol use such as dryness, peeling and hypersensitivity. In comparison to other balm-type products on the market, the Cicaplast Baume B5 has a considerably lighter texture, making it an ideal option for those who are looking for a considerable amount of moisture, but struggle to find products that don't have the potential to clog the pores. Overall, this product is an excellent all-rounder which focuses mainly on maintaining healthy skin without becoming too heavy or suffocating. ... Read More

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La Roche Posay Vitamin C Percentage

Source: https://thedermreview.com/la-roche-posay-review/

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Is Vitamin C Antifungal

Is Vitamin C Antifungal

Future Microbiol. 2016 Dec; 11(12): 1535–1547.

Sodium ascorbate kills Candida albicans in vitro via iron-catalyzed Fenton reaction: importance of oxygenation and metabolism

Pinar Avci, ^{1 , 2 , 3} Fernanda Freire, ^{1 , 4} Andras Banvolgyi, ³ Eleftherios Mylonakis, ⁵ Norbert M Wikonkal,^{* 3} and Michael R Hamblin^{** 1 , 2 , 6}

Pinar Avci

¹Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA 02114, USA

²Department of Dermatology, Harvard Medical School, Boston, MA 02115, USA

³Department of Dermatology, Venerology & Dermato-Oncology, Semmelweis University, Budapest 1085, Hungary

Fernanda Freire

¹Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA 02114, USA

⁴Department of Biosciences & Oral Diagnosis, Institute of Science & Technology, Universidade Estadual Paulista (UNESP), São José dos Campos, São Paulo 12245-000, Brazil

Andras Banvolgyi

³Department of Dermatology, Venerology & Dermato-Oncology, Semmelweis University, Budapest 1085, Hungary

Eleftherios Mylonakis

⁵Infectious Diseases Division, Warren Alpert Medical School of Brown University, Rhode Island Hospital, Providence, RI 02912, USA

Norbert M Wikonkal

³Department of Dermatology, Venerology & Dermato-Oncology, Semmelweis University, Budapest 1085, Hungary

Michael R Hamblin

¹Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA 02114, USA

²Department of Dermatology, Harvard Medical School, Boston, MA 02115, USA

⁶Harvard-MIT Division of Health Sciences & Technology, Cambridge, MA 02139, USA

Received 2016 Mar 23; Accepted 2016 Aug 18.

This article has been cited by other articles in PMC.

Abstract

Aim:

Ascorbate can inhibit growth and even decrease viability of various microbial species including Candida albicans. However the optimum conditions and the mechanism of action are unclear.

Materials/methodology:

Candida albicans shaken for 90 min in a buffered solution of ascorbate (90 mM) gave a 5-log reduction of cell viability, while there was no killing without shaking, in growth media with different carbon sources or at 4°C. Killing was inhibited by the iron chelator 2,2′-bipyridyl. Hydroxyphenyl fluorescein probe showed the intracellular generation of hydroxyl radicals.

Results/conclusion:

Ascorbate-mediated killing of C. albicans depends on oxygenation and metabolism, involves iron-catalyzed generation of hydroxyl radicals via Fenton reaction and depletion of intracellular NADH. Ascorbate could serve as a component of a topical antifungal therapy.

KEYWORDS : ascorbate, Candida albicans, hydroxyl radicals, oxidative stress, vitamin C

Candida albicans is a major opportunistic fungal pathogen that is associated with a range of clinical conditions. The organism can lead to superficial infections of genital, oral and cutaneous sites as well as systemic infections, especially in hospitalized and/or immunocompromised patients, such as those suffering from AIDS or undergoing chemotherapy [1–4]. Although most superficial infections caused by C. albicans are not life threatening, these infections can have debilitating effects on the patient's quality of life. Several classes of antifungal agents (e.g., azoles, echinocandins) have been developed which are active against Candida infections; however, possible drug interactions and adverse side-effects, emergence of drug-resistant isolates, as well as the high costs of some of these agents underscore the necessity for development of additional treatment alternatives [3,5].

Ascorbate (vitamin C, ascorbic acid) is an essential vitamin and is considered to be a potent antioxidant owing to its ability to act as an electron-donating reducing agent, and a quencher of reactive oxygen species (ROS) [6]. However, there is also increasing evidence that at pharmacologic concentrations, ascorbate can exert pro-oxidant effects via the reduction of transition metal ions, such as iron and copper [7]. Previous studies have shown that high-dose ascorbate exerts selective cytotoxic effects on cancer cells both in vitro and in vivo [6,8]. There have also been several clinical trials in cancer patients, testing the effects of high-dose ascorbate either alone or in combination with chemotherapy, but the results are still controversial [9]. Although the mechanism of action is still not well understood it has been proposed to depend on oxidation of ascorbate. The Fenton reaction and the coupled Haber–Weiss reaction together comprise the following steps: (Equation 1) ferrous ion reacts with oxygen to produce superoxide (Equation 2), which in turn generates H₂O₂ via dismutation (Equation 3), H₂O₂ then reacts with ferrous ion to form HO ^. (Equation 4) ascorbate can reduce Fe³⁺ back to Fe²⁺ to allow the cycle to continue [10].

(1)

(2)

(3)

(4)

A few studies also show that ascorbate could function as a potential anti-infective agent, but the literature is scarce and sometimes conflicting [11–15]. Vilcheze and colleagues in 2013, reported that 4 mM ascorbate could sterilize both drug-susceptible and drug-resistant Mycobacterium tuberculosis, however several weeks of incubation was required for ascorbate to exert its effects in vitro. On the other hand, when cultures were treated with ascorbate in an anaerobic chamber, ascorbate had no effect on M. tuberculosis viability [11]. In another study by Tabaket al. it was shown that 100 mM ascorbate incubated for 48 h was able to decrease viability of Helicobacter pylori by four orders of magnitude in liquid medium under microaerophilic conditions [12]. Ojhaet al. studied the effect of ascorbate on growth and pathogenicity markers of ascorbate on C. albicans [16]. The group demonstrated that upon 24 h of incubation with 0.85 M ascorbate, 80% cytotoxicity could be achieved. Brajtburget al. reported that ascorbic acid enhanced the lethal effects of amphotericin B on C. albicans and Cryptococcus neoformans cells [13]. A recent in vivo study demonstrated that pharmacologic ascorbate concentrations and low-dose amphotericin B was effective in an animal model of recurrent candidemia sepsis [17]. Topically applied ascorbic acid, was not effective in treating acute vaginal candidiasis, but was effective in preventing fungal reinfection following completion of a successful antifungal treatment [18].

Our laboratory has studied photodynamic therapy (PDT) for many years [19,20]. In PDT a nontoxic dye called a photosensitizer (PS) is excited with otherwise harmless visible light, and produces ROS which can kill a broad-spectrum of pathogenic microorganisms and cancer cells [19]. In one of our previous studies, we have reported that addition of ascorbate as an external electron donor could potentiate PDT of HeLa cancer cells [20]. However this effect was concentration, PS and wavelength dependent, such that inhibition of PDT was also observed when the ROS quenching effect of ascorbate predominated [20]. In an attempt to observe whether ascorbate could also potentiate or inhibit antimicrobial PDT (aPDI) of microbial cells, we added sodium ascorbate (ascorbate) and a polycationic functionalized fullerene PS, to a suspension of bacteria or yeast cells in phosphate-buffered saline (PBS), which is the normal medium to carry out aPDT experiments. Taking previous studies into consideration, we used a relatively high concentration of ascorbate and used significantly shorter incubation times expecting that ascorbate alone would have no effect on cell viability. On analyzing the control data we realized that ascorbate did not in fact potentiate the aPDT killing effect, but rather rapidly killed the microbial cells on its own, depending on concentration, incubation time and the level of oxygenation.

This intriguing observation encouraged us to systematically study the ability of ascorbate to kill C. albicans In an attempt to validate previous studies, define the conditions under which C. albicans is either sensitive or resistant to ascorbate and gain more insight into its mechanism of action, we evaluated and compared the effects of PBS and growth media, shaking to allow oxygenation, temperature to regulate level of metabolism, growth phase and addition of a mitochondrial inhibitor or an iron chelator on the killing of C. albicans with ascorbate.

Materials & methods

• Materials

All chemicals were purchased from Sigma-Aldrich (MO, USA) unless otherwise stated. L-ascorbic acid was dissolved in PBS (without Ca^2+/Mg²⁺) (PBS), PBS with D-(+)-glucose (20 g/l), YPD (BD-Difco^TM [NJ, USA], yeast extract 10 g/l; peptone 20 g/l; dextrose 20 g/l) or YPG (yeast extract-peptone [30 g/l; ForMedium Ltd. (Norfolk, UK)], glycerol [38 g/l]) media. Stock solutions of ascorbate were prepared on the same day of experiment and buffered with NaHCO₃ (Fisher Scientific, PA, USA) to bring pH to approximately 7. The final concentration of ascorbate used in all experiments was 90 mM. 2,2′-Bipyridyl (bipyridyl) was dissolved in dimethylacetamide and diluted to 500 μM. Antimycin A (from Streptomyces spp.) was dissolved in ethanol and diluted to 10 μM. 3′(4-Hydroxyphenyl)-fluorescein (HPF; Molecular Probes, OR, USA) was used at 5 μM.

• Cell culture

The C. albicans strain CEC 749 was used in this study [21]. Candida albicans was routinely grown at 30°C on YPD agar and subcultured in liquid YPD medium in an shaking incubator at rpm 120 (New Brunswick Scientific, NJ, USA), at 30°C. Log phase cultures obtained by reculturing stationary overnight cultures were used for all experiments except as follows. In one set of experiments, stationary cultures grown overnight or 4 days and a stationary culture grown for 4 days and refreshed for 4 h were used. The reason for selecting two different time points for stationary phase cultures was because in some studies, C. albicans culture grown overnight or for 48 h was considered to be in stationary phase, while other studies reported the stationary phase to start much later, for example, between 3 and 8 days [22]. All broth cultures were centrifuged at 3200 rpm for 10 min (centrifuge 5417 C; Eppendorf, Hamburg, Germany) and resuspended in PBS. The concentrations were then adjusted by measuring optical density (OD₅₇₀ of 0.65) to give an approximate cell density of 10⁷ CFU/ml. In one experiment, YPG media and in another PBS with glucose, was used as described above.

• Experimental design

The experiments were performed in 35 × 10 mm diameter Petri dishes (BD Falcon NJ, USA) containing approximately 3 × 10⁷ cells in 3 ml growth media, PBS with glucose or PBS. To examine the effects of different media and glucose on ascorbate sensitivity, cells were compared in different growth media; YPD or YPG, or in PBS with glucose. YPD or YPG was used both for initial growth and also for ascorbate treatment. Cells were shaken at 157 rpm at 37°C. At each time point two aliquots (10 μl each) were withdrawn. One was plated on YPD or YPG, the second was added to 90 μl PBS and four additional tenfold dilutions were carried out and plated on agar plates via drop plate method. Plates were incubated between 24 and 48 h in an incubator at 30°C and cell viability was assessed by colony counting. In the next experiment cells were treated with ascorbate in PBS (PBS ascorbate) in a shaker incubator (157 rpm, 4 and 37°C); left at room temperature (25 and 37°C) without shaking, or PBS in a shaker incubator (157 rpm, 4 and 37°C) with no ascorbate. Samples were withdrawn at time points (0, 10, 20, 30, 60 and 90 min).

To determine whether the effect of ascorbate is dependent on the presence of iron, washed cells were resuspended in PBS solution with ascorbate (PBS ascorbate), and bipyridyl (500 μM), an iron chelator that predominantly binds Fe²⁺ [23], was added subsequently, incubated in a shaker (157 rpm, 37°C) and samples withdrawn at time points (0, 10, 20, 30, 60 and 90 min).

For studies using pretreatment with antimycin A (a metabolic inhibitor of the electron transport chain) C. albicans cells were refreshed for 4 h in YPD, with antimycin A (10 μM) added or not after 1 h [24]. Then washed cells were resuspended and kept in a shaker incubator (157 rpm, 37°C) in either PBS or YPD with or without ascorbate and samples withdrawn at time points (0, 10, 20, 30, 60 and 90 min).

Microscopy

Detection of hydroxyl radical generation

HPF fluorescent probe was added to washed cells and kept in a shaker incubator with or without ascorbate in YPD or PBS or kept without shaking at 25°C with ascorbate PBS. Subsequently, cells were spun down (3200 rpm, 3 min) immediately after treatment, supernatant was removed and cells were resuspended in PBS. Images were acquired using a 35 mm 4-chamber glass-bottom petri dish (in vitro scientific).

Fluorescence of HPF was imaged by Olympus FV1000-MPE system with 40× NA0.8 water immersion lens. The HPF was imaged with single photon excitation at 488 nm using multiline argon laser and the fluorescence emission were collected with laser scanning spectral detector at bandwidth 500–545 nm. Brightfield images were acquired simultaneously.

Measurement of intracellular redox status

Intracellular redox status was measured by confocal imaging of the autofluorescence of flavoproteins and NADH using an Olympus FV1000-MPE system with 40× NA0.8 water immersion lens. The flavoprotien autofluorescence was imaged with single-photon excitation at 488 nm using multiline argon laser and the autofluorescence was collected with laser scanning spectral detector at bandwidth 500–600 nm. The autofluorescence of NADH was measured with two-photon excitation at 710 nm using MaiTai Deep See Ti:Sapphire laser (femtosecond) (Spectra-Physics, MaiTai HP DS-OL). The autofluorescence from two-photon excitation was collected with external two-channel photomultiplier detector for NADH (band-pass filter 420–460 nm). Brightfield images were acquired simultaneously.

Transmission electron microscopy

Candida albicans cells were spun down (3200 rpm, 3 min) immediately after treatment, supernatant was removed, cells were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde, and stored overnight at 4°C. After spinning down (1200 rpm, 10 min) and decanting the fixative, 0.1 M sodium cacodylate buffer (pH 7.2) was added to the pellets. After fixation, hot agar was added to each pellet. The solidified cell pellets were then processed routinely for transmission electron microscopy (TEM). The cell pellets were postfixed in 2% OsO₄ in sodium cacodylate, dehydrated in graded alcohol, embedded in Epont812 (Tousimis, MD, USA). Ultrathin sections were cut on a Reichert-Jung Ultracut E microtome (Vienna, Austria), collected on uncoated 200 mesh copper grids, stained with uranyl acetate and lead citrate, and examined on a Philips CM-10TEM (Eindhoven, The Netherlands) at 80 kV.

Statistics

Experiments were repeated at least three times. Data points are means and error bars are standard deviations. Means were compared for significance (p < 0.05) by one-way ANOVA and Bonferroni post hoc test.

Results

• Ascorbate kills C. albicans in PBS but not in growth media, & presence of glucose in PBS decreases killing

As preliminary studies had shown that the ascorbate-mediated killing of C. albicans was pronounced when the cells were incubated in PBS, we compared the time-dependent killing when the cells were shaken at 37°C, suspended either in growth medium (yeast-peptone-dextrose [YPD]) or in PBS. Moreover to test whether the antifungal activity of ascorbate against Candida is altered in different growth media, we compared media containing fermentable carbon source dextrose (YPD) or a nonfermentable carbon source glycerol (yeast-peptone-glycerol [YPG]) [25]. Figure 1 shows that incubation with shaking in PBS ascorbate at 37°C almost eradicated the cells (>5 logs killing) after 90 min, while in growth media there was no killing, but rather modest proliferation (up to fivefold). Growth media containing fermentable or nonfermentable carbon sources made no difference with no killing observed in either case. To further investigate whether presence of glucose in PBS would alter the metabolism or antioxidant capacity of Candida, thus alter killing, we compared incubation with shaking in PBS ascorbate with PBS-glucose-ascorbate at 37°C. Almost two logs of inhibition of killing was observed with added glucose at 60 and 90 min (Figure 1).

Time-dependent killing of Candida albicans with 90 mM sodium ascorbate shaken in phosphate-buffered saline, YPD, YPG or phosphate-buffered saline with 20 g/l glucose at 37°C.

Controls included shaking in PBS or YPD without ascorbate.

*p < 0.05; ***p < 0.001.

PBS: Phosphate-buffered saline; YPD: Yeast-peptone-dextrose; YPG: Yeast-peptone-glycerol.

• Ascorbate requires active metabolism & oxygen to kill Candida in PBS

Since there was a large difference in viability between Candida cells shaken with ascorbate in PBS and shaken with ascorbate in growth media, we asked whether active metabolism could explain this striking difference. Therefore we compared cells that were either shaken with PBS ascorbate at 4°C, or kept in PBS ascorbate without shaking at 25 and 37°C, with cells shaken with PBS ascorbate at 37°C. Control cells were shaken in PBS with no ascorbate at both 4 and 37°C. Figure 2 shows that all three conditions, completely abrogated the killing with PBS ascorbate shaken at 37°C. However, cells exposed to ascorbate without shaking formed colonies at a much later time when compared with nonexposed cells. These findings show the importance of both temperature and sufficient oxygenation levels, implying that active respiration is necessary to achieve killing.

Time-dependent killing of Candida albicans with 90 mM sodium ascorbate shaken in phosphate-buffered saline at 37 or 4°C, and not shaken at 37 or 25°C.

Controls included shaken in PBS alone at 37 or 4°C.

PBS: Phosphate-buffered saline.

• Ascorbate killing is dependent on free iron concentration & is increased with mitochondrial inhibition

The literature suggests that iron-catalyzed Fenton reaction producing HO ^. is involved in the toxicity of ascorbate, thus we tested the effect of a cell-permeable iron chelator, bipyridyl (500 μM) on the PBS ascorbate killing of Candida [26]. There was substantial inhibition of killing (>2 logs) at time-points between 30 and 90 min of incubation in shaken PBS ascorbate (Figure 3A). We hypothesized that mitochondrial respiration played a role in the killing effect, and therefore tested the effect of an inhibitor of Complex III, antimycin A. We reasoned that mitochondrial inhibition could either reduce killing by inhibiting respiration, or alternatively it could increase killing by producing more H₂O₂ [24]. Initial control experiments showed that cells pre-incubated with antimycin A alone were not killed at the concentration employed. Figure 3B shows that the pre-incubation of Candida cells with antimycin A (10 μM) for 3 h increased the subsequent cytotoxic effect of PBS ascorbate.

Effects of iron chelator and mitochondrial inhibitor on ascorbate-mediated killing of Candida albicans.

(A) Time-dependent killing of Candida albicans shaken in PBS at 37°C with 90 mM sodium ascorbate with or without addition of the iron chelator 2,2-bipyridyl (500 μM). **p < 0.01; **p < 0.001. (B) Time-dependent killing of C. albicans shaken in PBS at 37°C with 90 mM ascorbate, or in PBS or YPD with 90 mM ascorbate following treatment with complex III inhibitor antimycin A (10 μM) or cells pretreated with antimycin and shaken in PBS at 37°C in PBS as control.

PBS: Phosphate-buffered saline; YPD: Yeast-peptone-dextrose.

• Ascorbate killing is dependent on growth phase

It is known that stationary phase cells of most microorganisms have higher resistance to many stresses including ROS, however, exact timing that cells go into stationary phase is still not clearly defined in the literature [22]. We compared the susceptibility of log phase, 1 day (early) and 4 days (late) stationary phase, and in the latter case, compared refreshed and not refreshed cultures to PBS ascorbate shaken at 37°C. Early stationary phase cells (1 day) were the most sensitive, being killed 2–3 logs more than log phase cells (Figure 4). Late stationary phase cells (4 days) were most resistant, and 4 h refreshing provided more killing during the first part of the treatment (<60 min) but not during the later part (>60 min). Therefore 4 h of refreshing had opposite effects, giving more killing with 4 days cultures, but less killing with 1 day cultures.

Time-dependent killing of Candida albicans taken from cultures at different growth phases shaken in phosphate-buffered saline at 37°C with 90 mM sodium ascorbate.

PBS: Phosphate-buffered saline.

• Imaging studies show hydroxyl radical formation, changes in cellular redox state & cell morphology

The literature suggests that HO ^. formation could explain the toxicity of ascorbate [11]. Thus, we used the relatively specific fluorescence probe HPF for imaging of intracellular HO ^. generation [27]. Figure 5A & B showed no HO ^. production (green fluorescence) when cells were shaken at 37°C either in YPD alone or in YPD-ascobate. A slight green fluorescence was observed in Figure 5C when Candida was shaken in PBS without ascorbate at 37°C. More pronounced (but still modest) green fluorescence was observed in Figure 5D when Candida was incubated with PBS ascorbate without shaking at 25°C. However when Candida was shaken in PBS ascorbate at 37°C for 30 min there was an intense green fluorescence visible in every cell indicating a marked increase in HO ^. .

Representative confocal imaging of hydroxyl radical (HO^.) generation in Candida albicans after 20 min of incubation in phosphate-buffered saline or phosphate-buffered saline-ascorbate with or without shaker at 37 and 25°C, respectively (cells were stained with 3′(4-hydroxyphenyl)-fluorescein and images were acquired at 40X-zm-6), scale bar = 5 μM.

Cells incubated in: (A) yeast-peptone-dextrose in shaker 37°C; (B) yeast-peptone-dextrose ascorbate in shaker 37°C; (C) phosphate-buffered saline (PBS) in shaker 37°C; (D) PBS ascorbate without shaker 25°C; (E) PBS ascorbate shaker 37°C.

The cellular redox state can be observed by detecting the blue (420–460 nm) autofluorescence from NADH, using 2-photon excitation and the green autofluorescence (495–540 nm) from flavoproteins excited by 1-photon [28–30]. Healthy cells without oxidative stress have high NADH fluorescence and low flavinoprotein fluorescence, whereas cells that have been subjected to oxidative stress have lower NADH and higher flavoprotein fluorescence.

Figure 6 shows that cells stirred in YPD (Figure 6A & B) for 30 min without ascorbate had significant amounts of NADH fluorescence and no visible flavoprotein fluorescence. Cells stirred in PBS for 30 min with no ascorbate (Figure 6C & D) had slightly reduced NADH and slightly increased flavoprotein fluorescence. Cells kept in PBS ascorbate without stirring at 25°C for 30 min (Figure 6E & F) showed a prominent reduction in NADH fluorescence and a small amount of flavoprotein fluorescence appeared. Cells stirred in PBS ascorbate for 20 min (Figure 6G & H) also showed a prominent loss of NADH fluorescence, while a substantial amount of flavoprotein fluorescence was visible. We have determined that a slight component of NADH fluorescence may correspond to flavoprotein fluorescence due to the minor overlap in their two-photon excitation emission wavelengths [29]. Thus the NADH signal observed in 20 min is likely to be due to the increased autofluorescence of flavoprotein.

Representative confocal imaging of cell redox changes in Candida albicans cells after various treatments.

Simultaneous imaging of autofluorescence of NADH (A, C, E & G) and flavoproteins (B, D, F & H). All images were acquired in phosphate-buffered saline (PBS) at 40×. Scale bar = 5 μM. Cells incubated in: (A–B) YPD in shaker at 37°C for 30 min; (C-D) PBS in shaker at 37°C for 30 min; (E–F) PBS ascorbate, without shaker, at 25°C for 30 min; (G–H) PBS ascorbate in shaker at 37°C for 20 min.

• TEM & bright field microscopy

Fungal vacuoles, resembling mammalian lysosomes are the most acidic compartment of the cell with roles in protein degradation, ionic transport, metabolite storage as well as detoxification [31]. Changes in vacuolar morphology have been reported in response to stress ranging from acute osmotic and ionic shock to long-term nutrient deprivation [31]. TEM images of C. albicans treated with PBS ascorbate for 30 min in a shaker incubator revealed vacuole overgrowth, nuclear condensation as well as loss of organelle identity (Figure 7C–D). Consistent with these findings, bright field microscopy images showed a similar pattern resembling a peripheral ring with a central hole (Figure 7H). Same morphology was also observed in bright field images of PBS ascorbate-treated cells without shaking (Figure 7G).

Transmission electron microscopy images of Candida albicans.

(A–D) Transmission electron microscopy images of Candida albicans cells following 30 min incubation at 37°C in:(A) yeast-peptone-dextrose in shaker; (B) phosphate-buffered saline (PBS) in shaker; (C–D) PBS ascorbate in shaker. (A–C) were imaged at 9800× direct magnification, (D) was imaged at 3800×. Scale bar = 500 nm. (E–H) Brightfield microscopy images of C. albicans cells following 30 min incubation in: (E) yeast-peptone-dextrose in shaker at 37°C (F) PBS in shaker at 37°C; (G) PBS ascorbate without shaker at 25°C; (H) PBS ascorbate in shaker at 37°C. Scale bar 5 μM.

Discussion

Candida albicans is a dimorphic yeast that is both a commensal organism and an opportunistic pathogen of humans. It can cause serious infections in immunocompromised patients [2]. Ascorbate, owing to its pro-oxidant properties at high concentrations, has been proposed to be used as an antifungal agent [6]. Ojhaet al. demonstrated the concentration-dependent effects of ascorbate on growth and pathogenicity markers (e.g., proteinase secretion, yeast to hyphal transition) of C. albicans [16]. In their study, 250 mg/ml (1.42 M) ascorbate reduced proteinase secretion and at 85 mM there was an arrest of hyphal transition. Moreover, when varying concentrations of ascorbate were added to C. albicans cells in growth media and incubated for 24 h, cytotoxicity was found to be 33% at 57 mM and 80% at 0.85 M. In a subsequent study, ascorbate exerted pro-oxidant effects on catalase, decreased glutathione and enzyme activities of glutathione peroxidase, glutathione reductase and glutathione-S-transferase [32]. We tried to further elucidate the mechanism of action and investigated the conditions under which ascorbate is cytotoxic toward C. albicans. We propose that the antifungal effect of ascorbate on C. albicans depends on active metabolism, oxygenation, depletion of reduced NADH, liberation of free iron and Fenton-mediated production of HO ^. , that act together as the key elements in its pro-oxidant activity [33]. Ascorbate, as a reducing agent can recycle Fe³ to Fe²⁺, and facilitate subsequent Fenton cycles (Figure 8).

Ascorbate monoanion (AscH⁻) is a one-electron reducing agent that can reduce ferric iron (Fe³⁺) to ferrous (Fe²⁺) iron producing ascorbate radical (Asc^-.).

Fe²⁺ generated via this reaction, together with free Fe²⁺ that has been possibly released by disruption of iron sulfur clusters in the mitochondria and ferrous polyphosphate stores in the vacuole, Fe²⁺ reacts with O₂ reducing it to superoxide radical (O₂ ^-. ) and producing Fe³⁺ [6,34]. O₂ ^-. undergoes dismutation leading to generation of H₂O₂ and O₂ [6]. Then H₂O₂ reacts with the Fe²⁺ and generates Fe³⁺ as well as hydroxyl radical (HO ^. ) by the Fenton reaction. Depending on the concentration of the elements, HO ^. could also be produced by a partly different pathway, via the Haber–Weiss reaction. The presence of ascorbate allows the recycling of Fe³⁺ back to Fe²⁺, which in turn allows the cycle to continue.

Oxygen and intracellular redox-active iron seem to play a significant role in ascorbate-mediated cell death. Verraxet al. [35] demonstrated that pre-incubation with deferoxamine mesylate, a cell-permeable iron chelator, had a protective effect against ascorbate toxicity in cancer cells, which is also in agreement with our results. In contrast, two different cell-impermeable iron chelators failed to protect against ascorbate toxicity, emphasizing the importance of intracellular iron as compared with extracellular iron [36]. The Collins laboratory concluded that cellular respiration is necessary for bactericidal antibiotics to exert their full effects [37]. O₂ has only limited solubility in liquid media, thus it must be constantly shaken (aerated) to provide enough dissolved O₂. The explanation of the high level of HO ^. generation and killing observed in cells treated with ascorbate in PBS with shaking (aeration), and the lack of killing` without shaking may be attributed to this requirement for oxygenation.

In microbiology, dormancy often refers to a state in which cells are not able to form a colony when plated on an agar medium. However, when conditions are more favorable they can revert to a state of colony-forming competence [38]. It is possible that C. albicans cells exposed to ascorbate in PBS without shaking may have moved toward a state of 'early apoptosis' as there was no overt loss of viability by CFU determination, but only a delayed formation of colonies [39].

In an attempt to determine whether in vitro antifungal activity of ascorbate depends on cellular metabolism, temperature was altered. Cells exposed to ascorbate in PBS with shaking were killed at 37°C, whereas no killing was detected at 4°C. To date, C. albicans stress responses have mainly been investigated on cells cultured on rich, glucose-containing media and this environment differs significantly from mammalian host microenvironments that are often glucose limited [40]. To examine the effects of different media, ascorbate sensitivity was tested in YPD, YPG and PBS media. Metabolism contributes to stress adaptation by generating antioxidants such as glutathione for which the cells need both carbon and nitrogen. Therefore both YPD and YPG could exert dramatic effects upon the stress resistance of C. albicans and protect against ascorbate [40,41]. Subsequently, when glucose as a carbon source was introduced into PBS in the absence of nitrogen, ascorbate killing was partially inhibited.

Based on previous reports cells in stationary phase are generally more tolerant to stress conditions than are cells in the logarithmic-growth phase [42]. In our study, cells in PBS were found to be more sensitive to ascorbate when they were in early stationary phase or in log phase than when they had come from late stationary phase. These findings are in agreement with Uppuliruet al.'s study which reported higher levels of expression of genes involved in resistance against oxidative stress, DNA repair, repair and maintenance of the mitochondrial genome at >3 days [22].

Intracellular autofluorescence is usually dominated by the reduced NADH, and the oxidized flavoprotein, and mitochondrial NADH autofluorescence can be used as an indicator of cellular respiration [28,43]. Elevations in oxidized flavoprotein autofluorescence were shown to be correlated with markers of apoptosis [44]. Increased oxidative stress is most likely responsible for decreasing the redox potential of Candida, thus the intense autofluorescence of NADH in control samples was attenuated by addition of ascorbate. NADH-cytochrome b5 reductase enzyme may reduce the ascorbate radical to ascorbate monoanion, which would in turn draw electrons from NADH [6]. H₂O₂ and HO ^. generated via ascorbate cycle may also decrease the intracellular concentration of NADH because ROS-scavenging enzymes require NADH [28,45]. Fungal vacuoles, containing metal ions, such as iron [31], have a significant role in the response to various stresses, and the vacuoles subsequently change their morphology. Upon exposure to ascorbate in PBS, both with or without shaking, we observed a large central depression with a peripheral ring representing the large electron lucent vacuole. This is likely to be caused by the stress generated by ascorbate, and is consistent with the reduced NADH levels.

Inhibition of cytochrome bc1 (Complex III) with antimycin A increases mitochondrial levels of ROS [24]. Our findings demonstrated that, pretreatment with antimycin A increased the sensitivity of cells to ascorbate treatment in PBS with shaking. This is in agreement with Ojhaet al.'s study, in which ascorbic acid reduced C. albicans tolerance to oxidative stress that had been induced by incubation of cells with H₂O₂ [16].

Future perspective

Taken together, our results demonstrate that with oxygenation C. albicans can be rapidly eradicated by pharmacologic concentrations of ascorbate. However environmental distinctions, such as variations in the level of tissue perfusion and oxygenation in different organ systems will probably affect its toxicity toward Candida. Alterations in concentration of metallic ions as well as accessibility to nutrition will also enhance or reduce the potential killing effect of ascorbate. Understanding these environmental distinctions may provide unambiguous conditions to compare ascorbate's effects on C. albicans. Although NADH levels are altered due to the pro-oxidant effects of ascorbate, Fenton reaction mediated HO ^. generation plays a major role in its cytotoxicity as an active participant in oxidative damage to cells. Unlike commonly used antifungals, ascorbate acts via generation of ROS, therefore can be used in combination with these agents to potentiate their effects. These points should be taken into consideration; when ascorbate is employed as a component of antifungal agents such as antifungal creams for Candida infections and dermatophytes. Further studies are needed to examine whether this approach applies to other microbial species.

EXECUTIVE SUMMARY

• Ascorbate at pharmacological concentrations can kill Candida albicans.

• Ascorbate sensitivity of C. albicans depends on temperature, oxygenation, availability of energy sources, growth media and growth phase.

• Killing is potentiated by a mitochondrial inhibitor and inhibited by an iron chelator.

• Alterations are observed in the morphology of the vacuole, and the NADH concentration.

• Intracellular hydroxyl radicals are produced.

• Ascorbate exerts its pro-oxidant effects on C. albicans via Fenton reaction mediated reactive oxygen species generation.

• Environmental conditions and the mechanism of action should be taken into consideration in both the evaluation of in vitro and in vivo studies and future drug development.

Footnotes

Financial & competing interests disclosure

Funded by US NIH grant R01AI050875. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

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Is Vitamin C Antifungal

Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5131655/

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