Glycative stress and Anti-aging

Issues and prospects of glycative stress countermeasures

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Issues and prospects of glycative stress countermeasures

Glycative Stress Research Center, Faculty of Life and Medical Sciences, Doshisha University
Supervised by Professor Masayuki Yagi

Historical background of glycation research

More than 100 years have passed since French scientist Louis-Camille Maillard shed light on the phenomenon of food browning as a non-enzymatic reaction between amino acids and sugar 1). Currently, the glycation research fields range from food chemistry to medicine, pharmacy, agriculture, science, and nutrition, etc. Additionally, glycative stress is known as one of the risk factors for aging, but there have been discoveries that show that reduced glycative stress in the living body, thus anti-glycation leads to the prevention of aging and chronic diseases 2). The historical background of basic research from the discovery of glycation reaction to around 2000 has already been summarized in detail by Rabbani et al. (Fig. 1)3). Here, we summarized the historical background up to 2016 regarding industrial applications, which focus on the effects of glycation in the body and anti-glycative effects.

Fig. 1 History of glycation research between 1900 and 2000
(Rabbani N, et al. (2012)3))

Historically, glycation research has progressed step by step, mainly focusing on: browning mechanisms due to glycation, glycated proteins with diabetics, glycation reaction inhibitors and the glycation effects on aging.

In glycation research up until around 1950, the browning mechanism of amino acids and proteins had been the focus of the research and what was brought to light. Then, in 1958, Allen et al. discovered abnormal hemoglobin in the blood of diabetics, which led to hemoglobin A1c (HbA1c) analysis in 1968 4). In Japan, the small blood glucose meter “EYETONE” (Kyoto Daiichi Kagaku) was released in 1968, and an automatic HbA1c analyzer “AUTO A1c HA-8110” (Kyoto Daiichi Kagaku) was released in 1981. The significance of blood sugar as well as glycated protein for diabetes treatment was defined and their measurement environment had been established. In 1992, the automatic glycoalbumin analyzer “GAA-2000” (Kyoto Daiichi Kagaku) was released. Consequently, the significance in measuring glycated protein in blood as an evaluation index of previous blood glucose levels based on the protein life in the living body became clearer 5).

On the other hand, it was found that the development of diabetic complications is affected by the glycation of tissue proteins, and a large-scale clinical evaluation test for the glycation reaction inhibitor “aminoguanidine (drug name: Pimagenine)” (Alteon) was conducted in the US in 1994 6). In Japan, OPB-9195 ((+/-)-2-isopropylidenehydrazono-4-oxo-thiazolidin-5-yla cetanilide) was developed as a drug that may inhibit the glycation reaction in the body even at a dose of 1/10 of aminoguanidine by Otsuka Pharmaceutical Co., Ltd. It was expected to be a savior in the treatment of diabetes 7). However, the development of aminoguanidine was discontinued in 1998 and OPB-9195 was discontinued in 1999 because of problems such as low effectiveness in improving diabetic nephropathy, side effects and safety aspects. So, we currently don’t have an approved glycation reaction inhibitor.

According to the diabetes fact-finding survey, which is continuously conducted by the Ministry of Health, Labor and Welfare, the number of “potential diabetic patients” has increased every time: 13.7 million in 1997, 16.8 million in 2002, and 22.1 million in 2007. In particular, the percentage of “potential diabetic patients” over the age of 50 exceeded 20%, and the impact of increasing medical expenses has became a major issue alongside trying to improve the health of the Japanese people. These are counted as a factor that raises the interest in preventive medicine.

The idea that was adopted after entering the 2000s was that anti-aging was not aimed at rejuvenation and/or perpetual youth and longevity, but at prevention of aging/diseases and the extension of the healthy life expectancy.  In 2001, the Japanese Society of Anti-Aging Medicine (formerly, the Academy of Anti-Aging Medicine Japan) was established. From around this time, the significance of glycation inhibition in anti-aging has become gradually recognized. Regarding ingredients to limit glycation reaction, “horse chestnut extract” and “plantago major extract” (ICHIMARU PHARCOS Co., Ltd.) were released as cosmetic ingredients, and a “mixed herb extract” (product name: AG herb MIX) (ARKRAY) was released as a food ingredient. The keyword “glycation is aging” has been widely recognized due to the launch of these ingredients and anti-glycation products. Among them, the cosmetic series “episteme” (ROHTO Pharmaceutical Co., Ltd.) and “B.A cream” (POLA), which were launched based on the concept of anti-glycation in the fall of 2009, have rapidly raised awareness of glycation through beauty and health information magazines.

In 2010, two articles in the February issue of the Japan Diabetes Society Journal proved that postprandial hyperglycemia can be inhibited by ingesting vegetables first 8-9). These have clarified the effects of conventional diet therapy for diabetes, and has since drawn attention as a diet that prevents postprandial hyperglycemia from affecting the body. In 2010, the HbA1c level was added to the diagnostic criteria for diabetes and the significance of glycated protein measurement for diabetes treatment was reconsidered. At the 10th scientific meeting of the Japanese Society of Anti-Aging Medicine (Kyoto), which was also held in the same year, the symposium “Significance of AGEs in aging diseases” regarding the significance of glycation countermeasures was held for the first time. And, the importance of anti-glycation became known in the anti-aging medical field.  In 2011, the Society for Glycative Stress Research was established. It advocates the idea of “glycative stress”, which is a concept that comprehensively captures the stress on the bodies due to the load of reducing sugars and aldehydes, and the subsequent reactions. Since then, glycative stress has come to be considered as one of the risk factors for aging in the anti-aging medical field 2).

From 2011 onward, many anti-glycation ingredients/materials have been produced by a number of food, cosmetics and pharmaceutical (drug) manufacturers, and various foods and cosmetics containing these anti-glycation ingredients/materials have been released. In addition to health and beauty information magazines, programs featuring the glycation mechanism and measures against glycation have been shown on TV, which increased the general publics’ awareness on the issue.

From around 2012, there was a boom in health methods focusing on sugar intake, such as a low-carb diet, eating order diets (carbohydrate-last meal pattern), and ketone body diets. Furthermore, besides dietary fiber intake and the intake of vitamin and mineral supplements, ingesting vegetables actively can inhibit postprandial hyperglycemia. Therefore, more people came to know that it is effective against lifestyle diseases and aging. For this reason, many vegetables have come to be used at fast food stores as well as lunch boxes and sandwiches sold at convenience stores.

In 2015, the vinegar drink “Taberu maeno Ururuzu (vinegar to drink before meals)” (YOMEISHU SEIZO CO., LTD.), which focuses on inhibiting glycation, became available at supermarkets and convenience stores across Japan. On the other hand, the significance of anti-glycation life for aging prevention was featured in “Mom”, a member magazine for customers of the Aeon Group, a major distribution manufacturer. Also, the AGE SOKUTEI SUISHINKYOUKAI has published the “ex AGEs Handbook” and it explains the effects of dietary AGEs intake and how to reduce them. This handbook also draws attention to the effects of dietary exogenous AGEs.

As of 2016, many medical professionals and researchers have advocated contradictory theories about carbohydrate restriction through academic papers, books and health information magazines: some says that there is a risk in it, and others says it’s effective 10-11). In order to correctly convey the effects of glycation and the significance of anti-glycation to the general public, providing accurate information on carbohydrates control, especially on the significance of postprandial hyperglycemia restriction is an important issue. Similarly, there is an issue about organizing information on AGEs accumulation control in the body and the reduction of exogenous AGEs. The awareness level of the terms “glycation” and “anti-glycation” among ordinary consumers has reached about 30%12). In the future, it will be necessary to inform ordinary people about the relationship between glycation and aging as well as various diseases in an easy-to-understand manner, and to provide products and services that can be a countermeasure against glycation (anti-glycation).

Issues of glycative stress countermeasures

    1. Glycative stress measurement and evaluation

Blood glucose is the main causative agent of glycative stress and blood glucose levels are measured via a blood test during a medical checkup, etc. However, the measurement values that are obtained during the medical checkup are fasting blood glucose levels. While postprandial hyperglycemia is what is associated with glycation, ordinary people rarely have the opportunity to know what their postprandial blood glucose levels are. Thanks to the advancements in devices for self-monitoring of blood glucose (SMBG), blood glucose levels can be measured in about 5 seconds by self-blood sampling from the fingertips using a lancet (medical device for blood sampling). Many types of SMBG devices are sold by medical equipment manufacturers in Japan and overseas. Using a SMBG device allows anyone to easily find out their postprandial blood glucose status 13). With that being said, SMBG is classified as a specially controlled medical devices (class III) by the Pharmaceutical Affairs Law. Therefore, advertisements are limited. For this reason, ordinary people that are not diabetic do not have the opportunity to hear about these products and the fact that they can purchase them at pharmacies across the country.

HbA1c, a type of glycated protein in blood, is an index for knowing the blood glucose status of the past 1 to 2 month(s) and is also one of the diagnostic criteria for diabetes. Since HbA1c is listed as a blood test item for medical examinations regarding lifestyle disease prevention as well as specific examinations for people over the age of 35, there are currently more opportunities for ordinary people to know their own measurement values.

Glycation reaction intermediates are not measured during medical checkup. Blood 3DG can be measured via a research test at some clinical testing centers (SRL). 3DG, glyoxal, and methylglyoxal are measurable via the prelabeled HPLC method, and their measurements are performed at universities and research institutes as well 14). However, there are problems with sample storage stability after labeling and sample pretreatment conditions. For methylglyoxal, measurement kits (Cosmo Bio Co., Ltd.) using the ELISA method are commercially available.

In regards to AGEs, blood pentosidine measurement is covered by insurance as a diagnostic marker for early nephropathy. Its measurement is possible at the Clinical Trial Business Center (LSI Medience Corporation). In addition, measurement kit products to measure pentosidine, CML and CEL via the ELISA method are sold for research by multiple companies (FUSHIMI Pharmaceutical Co.,Ltd.; Cosmo Bio Co., Ltd.; BML, Inc., NIKKEN SEIL Co.,Ltd. etc.). Some toxic AGEs (TAGE: glyceraldehyde-derived AGEs) are measured by some research institutes (TRANS GENIC INC., etc.) on a contract basis. Although most of the AGEs are measured via the ELISA method, the measurement values differ depending on the difference in the antibody specificity that is used and the sample pretreatment conditions, even with the same AGEs measurement values. In particular, there is an issue where there are differences between blood pentosidine measurement values due to the different pretreatment methods of the ELISA method and the HPLC method 15).

The amount of AGEs that accumulate in the skin can be measured via the AGE Reader (DiagnOptics).  The AGE Reader is a very simple device that optically and non-invasively measures fluorescent AGEs. However, this measurement device’s measurement value calibration function is poor and the correction of differences between devices is an ongoing issue. Currently, several home appliance manufacturers in Japan are striving to develop this type of device, and Sharp Corporation plans to release the first domestically produced device in the fall of 2016 16).

In order to use AGEs for glycative stress evaluations, it is necessary to clarify which AGEs are associated with which disease and/or each phenomenon of aging. As of today, papers and such have often reported on the measurement results of the AGEs pentosidine and CML. But, in many cases, these AGEs are selected because of the simplicity of the required measurement methods, and it is not clear whether or not they are important AGEs in the body, compared to other AGEs. In the future, accurate and simple measurement methods for many kinds of AGEs should be researched and developed to evaluate the effects of glycative stress on the living body. Then, the disclosure of the measurement methods and the spread of an inexpensive yet rapid measurement services is an issue that needs to be tackled.

    1. Anti-glycation ingredients/materials

Inhibiting postprandial hyperglycemia is one of the countermeasures against glycative stress. Therefore, ingredients/materials that have the effects of lessening sugar absorption during meals and/or inhibitory effects on a digestive enzyme that decomposes oligosaccharides in carbohydrates into monosaccharides can be deemed to be anti-glycation ingredients/materials 17). “Indigestible dextrins” (Matsutani Chemical Industry Co., Ltd.), a soluble dietary fiber that is already contained in many foods on market, has the effect of lessening sugar absorption during meals and inhibiting the rise in postprandial blood glucose levels. In addition, “Guava leaf polyphenols” (Bizen Chemical Co., Ltd.), fermented black beans extract (Japan supplement foods Co., Ltd.), “Wheat albumin” (Nisshin Pharma Inc.), L-arabinose (UNITIKA LTD.), etc. have the effects of limiting the rise in postprandial blood glucose levels by inhibiting the carbohydrate decomposition via amylase and α-glucosidase in digestive juices. Moreover, organic acids such as vinegar (acetic acid) and citric acid have the effect of delaying digestion/absorption by prolonging the retention time of food in the stomach, so ingesting the acids before meals can lessen the sugar absorption during meals. These ingredients are also used as an ingredient of food for specified health uses to take countermeasures against blood glucose levels.

Many food/cosmetic ingredients have been developed as ingredients that inhibit the formation of glycation reaction intermediates and AGEs. Food ingredient products include “mixed herb extract (product name: AG Herb MIX)” (ARKRAY, Inc.), “Shigiku flower” (UNIAL Co.,ltd.), “SAKURA EXTRACT” (Oryza Oil&Fat Chemical Co. ,Ltd.), “Mangosteen extract” (Nippon Shinyaku Co., Ltd.), “Trapa japonica peel extract” (Hayashikane Sangyo Co., Ltd.), “mixed herbal tea extract” (AntiAging Communication), etc. These products have the effects of inhibiting glycation reactions with complex multi-pathways at various points 18). It has been confirmed that these ingredients inhibit the production/accumulation of AGEs in blood and skin when ingested orally. When it comes to cosmetic ingredients, there are “Horse chestnut extract” and “plantago major extract” (ICHIMARU PHARCOS Co., Ltd.), “Rose extract” (Nichirei Corporation), “Centella asiatica extract” and “Pterocarpus marsupium bark extract” (Matsumoto Trading Co.,Ltd.) and more. These products have a function to inhibit the glycation of skin proteins. In many cases, ingredients that are contained in cosmetics and inhibit glycation reactions have been independently researched and developed by each manufacturer as their own product(s). These products include “Rosa rugosa flower extract” (Sansho Pharmaceutical Co., Ltd.), “Arusaji (composite material of plant extract and amino acid)” (ROHTO Pharmaceutical Co., Ltd.), and “Shiso extract” (Shiseido Japan Co., Ltd.). The glycation reaction inhibitors are considered to prevent the formation of AGEs as a preventive measure against glycation stress.

On the other hand, there are also anti-glycation ingredients/materials that are expected to decompose already accumulated AGEs. Regarding the POLA brand ingredient “Mugwort Extract” contained in the cosmetics B.A cream (launched by POLA in 2009), it may have the potential function to decompose and repair the cross-linked structure formed via the AGEs generation in proteins. Similarly, it was confirmed that “Rooibos extract” and “Astragalus sinicus extract” (POLA) also have the same effect. It has been reported that these cosmetic ingredients decompose AGEs generated on/in collagen gel. Therefore, the continuous use of cosmetics that contain these ingredients reduces AGEs in the stratum corneum19). Besides, a similar function wan found with the cosmetic ingredient “peony extract” (ICHIMARU PHARCOS Co., Ltd.). Researchers say that ingredients such as tea, herbs and vegetables also have a function that decomposes cross-linked AGEs. Researchers also said that pomegranate fruit was found to have an effect on decomposing the cross-linked structure of AGEs, which is affected by the trihydroxybenzene structure of ellagitannins (Fig. 2)20). However, the effectiveness when orally ingested by humans is unknown. Since the function to decompose cross-linked AGEs is expected to be effective in decomposing/excreting already accumulated AGEs, it can be expected to eventually be a countermeasure against glycative stress.

Fig. 2 Ingredients to decompose cross-linked AGEs in pomegranate
(Adapted from Yagi M et al. (2015)20))

Recently, as an enzyme that may affect the decomposition of glycated proteins and AGEs in the body, the application of oxidized protein hydrolase (OPH) has been considered. OPH identifies modifying groups (acyl groups, acetyl groups, etc.) in proteins and peptide N-termimus, and releases/decomposes the modifying groups 21). It also acts on the decomposition of CML-modified proteins 22). OPH also exists in the stratum corneum and its activity decreases due to aging 23). On the other hand, CML in the stratum corneum increases due to aging. Many kinds of teas, herbs, etc. have been listed as active ingredients that enhance OPH activity. In the future, it is expected that the mechanism of OPH action will be analyzed and that the enhancement of OPH activity will promote the decomposition of AGEs.

The key to anti-glycation ingredients/materials are thought to be the combination of actions and ingredients/materials. As for the actions, it is necessary to take measures that act on the entire glycative stress by combining measures against blood glucose, inhibition of AGEs production, and decomposition of AGEs. Measures are also required for factors that accelerate glycative stress such as oxidation, ultraviolet rays, excess lipids, smoking, alcohol (drinking) and stress. Also, it is important to take measures from inside and outside the body, so measures that use both cosmetics and food are necessary (Fig. 3).

Fig. 3 Action evaluation points of glycative stress inhibitor materials
The red and blue lines show examples of evaluation values for each item.
Inhibition of postprandial hyperglycemia: amylase inhibitory action, α-glucosidase inhibitory action
Anti-glycation activity: inhibitory action of fluorescent AGEs production
Inhibition of each AGEs production: 3DG production inhibitory action, CML production inhibitory action, pentosidine production inhibitory action
AGEs decomposition: cross-linked AGEs cutting action
Activation of OPH: OPH activity enhancement effect

Clinical evaluation (human clinical trial)  Postprandial hyperglycemia: postprandial blood glucose measurement
Glycation reaction intermediate: blood 3DG measurement
AGEs: measurement of CML in blood, measurement of pentosidine in blood
AGEs in the stratum corneum: measurement of CML in the stratum corneum
Accumulated skin AGEs: fluorometry from AGEs in the skin

To take measures against glycative stress in order to reduce AGEs in the enitre body, combining many substances to limit the glycation reaction systems and prevent AGEs production/accumulation as well as approaching many types of proteins might be effective.

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Glycative stress and Anti-aging